PRESENTED TO Marine Biological Library w ITH THE COMPLIMENTS OF The Williams & Wilkms Company BALTIMORE 2, MARYLAND Dick M. Hoover THIRD EDITION SEX AND INTERNAL SECRETIONS VOLUME I VOLUME I CONTRIBUTORS A. Albert David W. Bishop Kichard J. Blandau K. K. Burns A. T. Cowie John W. Everett S. J. 1 olley Thomas II. Forbes J. W. (iowen Koy O. Greep A. M. Guhl Joan G. Hampson John L. JIampson Frederick L. llisaw Frederick L. llisaw, Jr. James II. Leathem Daniel S. Lehrman Margaret Mead John W. Money Helen Padykula Dorothy Price Herbert D. Purves Ari van J'ienhoven Claude A. Villee H. (iuN Williams- Ashman (ieorge B. Wislocki William C. Young M. X. Zarrow Baltimore • 1961 THIRD EDITION SEX AND INTERNAL SECRETIONS W^i^- Edited by William C. Young, Ph.D. :^ Professor of Anatomy, University of Kansas, Lawrence Foreword by George W. Corner, M.D., D.Sc. Director Emeritus, Department of Embryology, Carnegie Institution of Washington The Williams & Wllkins Co Publication was supported in part by Public Health Service Research Grant M-464S from the National Institute of Mental Health, Public Health Service. Copyright ©, 1961 The Williams & Wilkins Company Made in the United States of America Library of Congress Catalog Card Number 60-12279 COMPOHKI) AND PRINTKD BY THE WAVrCRLV PRKSS, INC. BAL'I'IMOUFC 2, MARYLAND, U.S.A. To the Memory of Robert Mearns Yerkes pp* ■'»<^ "^ CONTENTS J oliime I Foreword. George W. Corner i^ Edgar Allen. William C. Youny xiii Preface to Third Edition x\i Preface to First Edition xxiii section a Biologic Basis of Sex 1. Cytologic and Genetic Basis of Sex. /. W. Gowen 3 2. Role of Hormones in the Differentiation of Sex. R. K. Burns 76 section b The Hypophysis and the Gonadotrophic Hormones in Relation TO Reproduction o. Morphology of the Hypophysis Related to Its Function. Herbert D. Purves . ... 161 4. Physiology of the Anterior Hypophysis in Relation to Reproduction. Roy b. Greep ' .240 section c Physiology of the Gonads and Accessory Organs 5. The Mammalian Testis. .4 . Albert 305 (). The Accessory Reproductive Glands of Mammals. Dorothy Price and H. Guy Williams- Ashman 366 7. The Mammalian Ovary. William C. Young , 449 8. The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms. John W. Everett .^ 497 9. Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates. Frederick L. Hisaw and Frederick L. Hisaw, Jr 556 10. The Mammary Gland and Lactation. A. T. Cowie and S. J. Folley 590 1 1 . Some Problems of the IMetabolism and Mechanism of Action of Steroid Sex Hor- mones. Claude A . Villee 643 12. Nutritional Effects on Endocrine Secretions. James H. Leathern 666 \ olume II section d Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, AND Pregnancy 13. Biology of Spermatozoa. David W. Bishop 707 14. Biology of Eggs and Implantation. Richard J. Blandau. 797 15. Histocliemistry and Electron Microscopy of the Placenta. George B. Wislocki and Helen Padykida 883 16. Gestation. M. X. Zarroiv ''^^^^ section e Physiology of Reproduction in Submammalian Vertebrates 17. Endocrinology of Reproduction in Cold-blooded Vertebrates. Thomas R. Forbes. . 1035 18. Endocrinology of Reproduction in Birds. Ari van Tienhoven 1088 vii 79220 viii CONTENTS section f Hormonal Regulation of Reproductive Behavior 19. The Hormones and Mating Behavior. William C. Yoimg 1173 20. Gonadal Hormones and Social Behavior in Infrahuman Vertebrates. .4. AI. Guhl . . 1240 21. Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals. Daniel S. Lchrman 1268 22. Sex Hormones and Other Variables in Human Eroticism. John W. Money .... 1383 23. The Ontogenesis of Sexual Behavior in J\Ian. John L. Hampson and Joan G. Hampson 1401 24. Cultural Determinants of Sexual Behavior. Margaret Mead 1433 Index 1481 FOREWORD George W. Corner, M.D., D.Sc. DIRECTOR EMERITUS, DEPARTMENT OF EMBRYOLOGY, CARNEGIE INSTITUTION OF WASHINGTON Publication of the third edition of Sex and Internal Secretions signalizes the ac- complishment of about a half century's intensive work by investigators of many countries, among whom those of the United States have been notably active. Any such burst of discovery as this rests, of course, upon a long preceding period of more gradual progress. Taking as landmarks Reg- ner de Graaf's recognition that the "female testis" of mammals is an egg-producing organ comparable to the ovaries of birds (1672) and Leeuwenhoek's description of the spermatozoa (1674), we can trace the continuous development of knowledge about the reproductive system down to our own times. Discovery of the actual mammalian ovum by Karl Ernst von Baer in 1827 accelerated the progress of research on the origin of the germ cells, the de^'elopment and discharge of the Graafian follicle, transport and fertilization of the ovum, and implanta- tion and development of the embryo. Such studies inevitably drew attention to the cyclic aspects of reproductive function, par- ticularly from students of animal breeding and from faunal naturalists, who acquired a great deal of information about the estrous cycles of wild as well as domestic animals and those of the laboratory. The work of the English leaders in this kind of in- vestigation, Walter Heape and F. H. A. Marshall, reached fruition in the latter's well known "Physiology of Reproduction," published in 1910. At this same period (1890-1910) gynecologists, especially in Ger- many and Austria, were putting their spe- cialty on a scientific basis. Becoming aware of the current advances in knowledge of embryology and the biology of reproduction of mammals in general, they were seeking similar clues to the explanation of the human menstrual cycle, ostensibly so dif- ferent from the estrous cycle of domestic animals. European workers, notably Hitsch- mann and Adler, Robert Schroedcr, and Robert Meyer, from about 1900 to the beginning of the first World War, put to- gether from operating-room material a histo- logic description of the human cycle that became more and more clear as the em- bryologists related it to their understanding of the general mammalian cycle. The young sciences of psychology, psychiatry and an- thropology also joined the concerted attack upon the problems of sex and reproduction. Since about 1870 European psychiatrists, led by such men as von Krafft-Ebing and Forel, had been studying sex psychology, with the aim of understanding behavior of a kind that was considered abnormal or con- ducive to social difficulties such as those created by prostitution and homosexuality. The way was thus opened for psychology to investigate the biologic basis of normal sex behavior. European and American an- thropologists had begun to document and analyze the sex attitudes of primitive races and distant nations, and even of their own peoples. Nor must we forget the influence of the Women's Rights movement, with its fight against all forms of bondage of women and its emphasis on standards of sex be- havior equally applicable to both sexes. All these new sciences and new social move- ments called for better understanding of basic sex physiology, which only biologists could provide. Thus at the beginning of the 20th century and during the next decades investigation in this field became more intense. Na- turalists, animal breeders, histologists, em- bryologists and gynecologists gradually came to understand each other's problems, and began a period of rapid advance not yet ended nor even slowed down, in which scarcely a year has passed without major contributions. American zoologists were already prepared by their embryologic studies to take part FOREWORD in this exploration, and the rapid devc^lop- ment of medical research in the United States in the early part of the 20th century provided speciahsts in human embryology, pathologists, physiologists, and biochemists who were ready to join the biologists in such investigations. When in 1922 the Na- tional Research Council was called upon by influential groups centered in the American Social Hygiene Association, to bring to- gether existing knowledge and to promote research upon human sex behavior and reproduction, our nation already possessed a corps of competent investigators who rallied to the call of Robert M. Yerkes and Frank R. Lillie, forming the Committee for Research in Problems of Sex. This Com- mittee, with financial support from the Rockefeller Foundation, successfully under- took to encourage research on a wide range of problems of sex physiology and behavior. The younger readers of this book will hardly be a])le to appreciate the full sig- nificance of such an alliance between bi- ologists, psychologists, and physicians on one hand, and social philanthropists on the other. It represented a major break from the so-called Victorian attitude which in the English-speaking countries had long im- peded scientific and sociologic investigation of sexual matters and had placed taboos on open consideration of human mating and childbearing as if these essential activities were intrinsically indecent. To investigate such matters, even in the laboratory with rats and rabbits, required of American scientists, including some of the contribu- tors to the first edition of Sex and Internal Secretions, a certain degree of moral stamina. A member of the Yerkes Committee once heard himself introduced by a fellow scientist to a new ac(}uaintance as one of the men who had "made sex respectabl(\" Ccrlaiiily the prestige of lh<^ Committee and the successes of American in\'(>stigators working with and without its assistance helped to bring about a more I'ealistic attitude toward sex research, although reactions from some ciuarters to such important recent work as that of the late Alfred C. Kins(\v show that the battle is even yet not fully won. Ten years after its formation tiie Com- mittee for Reseai'ch in Problems of Sex, proud of the achievements it had helped to foster, sponsored the first edition of Sex and Internal Secretions. The information thus brought together in 1932 came largely from research in genetics, cytology, em- bryology, and endocrinology, almost ex- clusively utilizing morphologic methods of study. In contrast to the present situation as reflected in the third edition, biochemistrv of the sex glands was still in an elementary stage, having barely achieved the pre- liminary chemical identification of the ovar- ian, placental, and testicular hormones; and the psychology of sex behavior was only beginning to develop its experimental methods. The sum total was, however, a deeply impressive record of progress that drew many new workers into this fi(^ld of research. It would be difficult to ascribe priority in this achievement to any one of the biologic disciplines. Genetics and cytology had provided one of the major clues by C. E. jMcClung's discovery in 1902 of the sig- nificance of the accessory chromosome, and his brilliant conjecture that this minute fragment of protoplasm is related to the determination of sex. Coming just before an outburst of discoveries concerning the chromosomal mechanism of heredity, based largely on the fruit fly Drosophila, the concept of genetic determination of sex gave rise to an immense amount of in- vestigation and theorizing about the way in which a developing individual is caused to become either male or female. Three decades after the publication of McClung's hypoth- esis, enough information on this (luestion was in hand to fill two ci'owded chapters in the first edition of Si.r and I idcriud Secretions. The contribution of cmbryolog}' to our sul)ject goes back to the ISth and 19th centuries and in particulai' to the (h^scrip- tion of the early stages of (lc\('loi)nient of the internal reproductive system, with which the names of Kaspar Fried rich Wolff' and Johannes Aliiller are ind(>libly associated. In this field, too, a period of acliv(> in- vestigation began early in the 20tli century, with th(^ aid of improxcd methods of microt- omy and the apj^lication of pr(H'is(> histo- loiiic staininti to eiubi'vonic tissues. The FOREWORD development of the gonads and the meta- morphosis of the Wolffian and Miillerian ducts into the secondary internal reproduc- tive organs was rapidly worked out in animals of every vertebrate order, by an army of in\'estigators too numerous to mention in a short resume. The story of the first appearance of primordial germ cells and their migration through the tissues of the embryo to the newly forming gonads, adumbrated in the 1880's by the work of Semon, was confirmed and extended to several species of mammals. If in these latter creatures and in the human species, th(> complete line of descent from the fertilized ovum to the first appearance of the germ cells is till not as clear as in many lower forms, enough at least was discovered within a few decades to indicate an essential similarity in the history of the germ cells in all vertebrates. To the embryologists of Europe and America w^e owe in large part also the successful analysis of the mammalian repro- ductive cycle that has been achieved during this half century. In order to procure mammalian embryos of known age the time of ovulation and fertilization had to be related to the outward manifestations of the estrous cycle. Comparati^'e description of the cycles of the various mammals for this purpose was climaxed by the discovery, or rather rediscovery and practical application of cyclic changes in the vaginal epithelium by C. R. Stockard and G. X. Papanicolaou. The vaginal smear method, thus introduced to the experimental laboratories, made pos- sible a wide range of investigations on the physiology and biochemistry of the cycle and the ovarian hormones. As apphed to the white rat by Herbert M. Evans and J. A. Long it became a basic tool in such studies. Another influence which also greatly for- warded investigation of the ovarian cycle has already been mentioned. This was the efTort of the gynecologists and especially gynecologic pathologists to interpret cychc events in the human ovary and uterus. One of the most notable American discoveries, that of the influence of the corpus luteum in decidua formation by Leo Loeb, stemmed from his familiarity with the German studies on the human cvcle. Much of our knowledge of th(^ corpus luteum and its hormone, progesterone, was in fact won by investiga- tors who approached the problem through gynecology. Had it not b(>en for the first World War, moreover, European gynecologic experi- menters might have attained clear knowl- edge of the estrogenic hormones, for even before 1900 Emil Knauer and Josef Halban of Vienna had demonstrated in a preliminary w^ay the endocrine dominance of the ovaries over the uterus, and by 1913 various in- vestigators, notably Henri Iscovesco of Paris and Otfried Fellner of Vienna, had pre- pared crude extracts which we now know contained estrogens. It remained, however, for the American zoologist-anatomist Edgar Allen and his biochemical colleague E. A. Doisy, e(iuipped with the vaginal smear method of testing ovarian hormone action, to isolate an estrogen from the fluid of the Graafian follicles, thus starting an era of ovarian endocrinology which has ultimately resulted in clear definition and discrimina- tion of estrogens and progestins and their respective effects upon the uterus and other organs of the reproductive system. Applying this new knowledge to the complexities of the human reproductive cycle, the zoologists, embryologists, gynecologists and endocri- nologists, among them several distinguished contributors to the first edition of this work, have combined forces to work out a clear account of the endocrine basis of menstruation and the implantation of the primate embryo. The parallel story of the hormones of the testis can be read in the successive editions of Sex and Internal Secretions. Berthold's proof, published in 1849, that in fowls the testis presides over the development of the cock's comb, wattles, and spurs ultimately led to the isolation of the first known androgen in F. C. Koch's laboratory at Chicago, and thence to the development of a great body of knowledge about androgenic steroids. The more complex history of the hor- mones of the hypophysis, becoming some- what clearer in each successive edition of this work, well illustrates a main theme of this introductory essay, namely the de- pendence of scientific advance upon the FOREWORD intermingling of ideas from various fields. It is not merely by chance that among the American workers on the hypophysis two names stand out, those of a surgeon, Harvey Gushing, and a zoologist-anatomist, Philip E. Smith. The central achievement of this half century of intensive work can be summarized in one sentence. It was, first, recognition, description and explication of the reproduc- tive cycle of mammals and man; and, second, identification of the chemical sub- stances that serve to integrate the cycle and preside over gestation. Those who took part in these investigations recognized, of course, that in due time their work must be ex- tended in two directions, downward to the domain of molecular chemistry and ul- timately of ionic physics in order to un- derstand the basic nature of hormone action, and upward to the field of animal and human behavior, where sex gland hormones join with other forms of bodily and mental integration in directing the life and be- havior of the organism. Once the histologists and embryologists had identified the sex gland hormones it was inevitable that further investigation of these remarkable substances should be taken over by biochemists, as can be seen from the successive editions of this book. The chief unsolved problems now demanding atten- tion relate largely to the sites of action of the hormones and the precise molecular effects which they exert upon their target organs. Recent indications that estrogens take part in hydrogen transfer in the citric phase of carbohydrate metabolism, and that progesterone affects uterine muscle cells by altering their permeability to potassium ions, show clearly that there is hope of understanding th(^ action at molecular level of these remarkably specific and powerful substances, which were bai'ely beginning to be known when the first edition of this book appeared in 1932. As the investigators of the future leai'n exactly where the sex gland hormones exert their chemical action, and just what they do to the fundamental elements of th(> cells of their target organs, we may con- fidently expect ever-increasing knowledge even of the most complex sexual and repro- ductive activities. Running over the chapter headings of this third edition of Sex and Internal Secretions, we see indeed that al- most every author concerns himself with one or another aspect of sex and reproduction in the light of what is already known about endocrine regulation. Chapter after chapter deals with cyclic events determined by sex gland hormones. Phenomena which in the first edition could be explained only sup- positionally on a hormonal basis, for ex- ample the "free martin" state in domestic cattle, and menstruation in primates, are now much better understood. Others which seemed hardly within the scope of endo- crinology are now seen to be in some degree influenced by hormone action, and thus to call for discrimination between endocrine effects and other types of regulation such as gene action and control through the nervous system. The experimental embry- ologists, for example, seek to understand the respective effects of genes and hormones in determining the development of the internal accessory sex organs; students of animal psychology likewise are beginning to discriminate between the action of hor- mones and neuro-psychologic factors in de- termining the patterns of sex behavior. Among the subjects discussed in this book, only psychiatry and anthropology are as yet not greatly influenced by our recently acquired stores of endocrinologic informa- tion. The complex, high-level patterns of human thought and behavior with which these sciences deal are presumably far less subject to chemical regulation than to the integrative control of the nervous system as it affects learning, memory, and racial tradition. Yet when we consider the extent to which daily life and ethnic customs are bound up with the sexual and reproductive activities of mankind, we are prepared to find this edition of Sex and Internal Secre- tions not only advancing greatly beyond its predec(>ss()rs in the study of animal be- havioi-, but also looking forward, through exploi'niory chaptcn-s on psychiatric and anthi'opologic asix'cts of liuninn s(\\ Ix^- liavior, to a time when we shall more fully understand the interrelations of all the con- trolling factors even of these most complex activities, upon which the continuation and renewal of life dcp(>nd. EDGAR ALLEN 1892-1943 Soon after his untimely death, February 3, 1943, many of the important details of Edgar Allen's life were recorded by col- leagues who were close to him. Separated from these memorials, however, was Sex and Internal Secretions, understandably the most permanent and tangible memorial. It is appropriate, therefore, that in this long-delayed third edition, much of the material in those records of his life should be combined with the review of the field in which his substantive contributions and directive thought were so important. With the permission of Doctors George W. Corner and William U. Gardner, portions of their biographical sketches have been used here. A few minor errors have been corrected and supplementary information has been added when it was felt that the picture of Edgar Allen would thereby be enlarged and sharpened. For much of the latter, in- debtedness is expressed to Doctor Charles H. Danforth, a long-time friend and senior colleague at Washington University, and to Doctor J. Walter Wilson, with Allen as a graduate student at Brown University. Doctor Allen ("Ed" to his many friends, and "The Skipper" in his department at Yale) was born at Canyon City, Colorado, May 2, 1892. He was the son of a physician about whom little seems to be known. The Allen family moved to Providence when he was very young. His early training was obtained in the public schools of Providence and at Brown University. Immediately after his graduation in 1915 he started graduate study in biology. This was interrupted two years later by World War I, but was sufficient to fulfill the not too rigorous requirements for a master of arts degree. His record during this period does not seem to have been impressive and nothing that has been learned about it foreshadowed his later distinguished accomplishments. How- ever, one hitherto unrecorded experience. mentioned on one occasion to the present biographer, may have had unusual sig- nificance in the years that followed. Doctor Albert Davis Mead, to whom editions 1 and 2 were inscribed, was Professor of Biology and instructor in the course in vertebrate embryology. At that time and for many years later the uteri, tubes, and ovaries of pregnant sows were collected from the local slaughterhouse and dissected by the class. Allen, probably as an assistant in the course, visited the slaughterhouse where his attention was attracted by the numerous large follicles in most of the ovaries. The curiosity thus engendered seems to have been the extent of his interest in reproductive physiology while he was at Brown, but it could have been instrumental in directing his attention to the ovary a few years later in St. Louis, and it could have prepared him to seek the sow's ovaries as a source of follicular fluid when he was desirous of obtaining large quantities of it for his first tests on spayed mice. In May, 1917, he volunteered for service in the Brown University Ambulance Unit. Later he transferred to a mobile unit of the Sanitary Corps with which he served in France. When he was discharged in Feb- ruary, 1919, he held a commission as second lieutenant. Before leaving for France in 1918 he married Marian Pfeiffer, a fellow student enrolled in Pembroke College, the Women's College in Brown University. Throughout the balance of his life she was his devoted companion. She too died as a relatively young woman and did not long survive him. • There are two daughters. For a man in his position, he lived modestly. It is easy to imagine that he valued the warmth and affection of his family and friends and his boat abo\T other luxuries he might have had. When he returned to civilian life he had EDGAR ALLEN no permanent po.sition in sight, but he must have sought the help of Mead, for during the summer of 1919 he was an investigator in the laboratory of the U. S. Fish Com- mission at Woods Hole. Doctor H. C. Bumpus, an older colleague of Mead's, had been Director of the Biological Laboratory in the Fish Commission at Woods Hole and summer appointments, paying two or three himdred dollars, were a source of help to the graduate students at Brown in that period. The summer seems to have been important, not because of any research Allen did, but because it was then that Charles Danforth, who was at Cold Spring Harbor for the summer and had heard Doctor FL E. Walter speak highly of Allen, wrote him and called his attention to the instructorship then open in Washington University School of Medicine. Danforth suggested that Allen communicate with Doctor Robert J. Terry, head of anatomy in that institution. He must have done so promptly and been accepted, with the un- derstanding that graduate study would hv continued. Danforth's recollection of his first sight of Allen is repeated: "When I returned to St. Louis in the fall and went up to the anatomy department I saw a man in the hall whose white liair and impressive bear- ing led me to suspect that he was prob- ably a distinguished alumnus returning for a visit. I soon learned, however, that this was Mr. Allen, who had been appointed instructor in anatomy and was already installed in an office on the third floor." Little time seems to have been lost in starting his work for the Ph.D., although the circumstances under which the choice of a problem was made ar(^ somewhat ohscuiv. Doctor H. H. Willier who met Allen for- the first time that sunnner (prob- ably on "stony beach") does not remembei- that he mentioned any special interest in the physiology of sex and reproduction. Danforth, who saw nuich of him from this time on, l;elie\-(>s that two cii'cunistaiices may lia\-e licen important. His oflice was on the floor with that of Doctoi' I.co Locb, always a stimulating p(M-soii, and in the animal quarters above was a colony of mice which had been developed for use in what was perhaps the first course in em- bryology to be based exclusively on mam- malian material — gametogenesis, follicular growth, ovulation, fertilization, cleavage, etc. He probably discussed problems with Loeb and he must have read the recent paper of Stockard and Papanicolaou in which changes in the vaginal epithelium in the guinea pig were correlated with the o^'arian cycle. Whether he was sensitive to the generally increasing interest in reproduc- tive phenomena or was influenced more by the fact that the mouse had been in- adequately studied and was right at hand and ready is not known. The latter pos- sibility would have been consistent with his temperament and the way he worked. On the other hand, the fact that the first of the three "purposes" stated in his thesis was "to make possible a more efficient mating for the collection of embryological material" may indicate that the larger im- portance of what he was about to start was not yet apparent to him. With the double responsibility of teach- ing and doing the research for a thesis, he must have worked incredibly long hours. But the rewards were great. The observa- tions recorded in his thesis, "The oestrous cycle in the mouse," ignited the fire that was to burn and to be spread during the remaining years of his life, and to eradicate fore\'er the diffidence which characterized him during his earlier graduate years at Brown. Briefly, he observed that large follicles were present in the proestrous and cstrous stages of the cycle, but that ovula- tion had occurred by the time of the metestrum. Regressive changes were noted in the uterus and these were analogized with menstruation in lower primates and the human female. The reference on page 111 to Hobin.son's belief that a secretion tVoni the follicle causes estrous changes rcxcals how close he was to the hypothesis that was to rccciNc gencn-al acc(>ptance only a few years latci'. It is clear, how(>\-ei', that he was not rcad^' for this simple and direct conclusion. Instead, he started by rejecting the suggestion that 1 h(^ growth stimulus EDCAR ALLEN to the genital tract comes from the corpus hiteum and then, after noting that "the follicles are the only remaining ovarian possibility," continued, "the presence of maturing ova in large follicles is the cause of the prooestrum and oestrus" and "the renewal of the ova at ovulation (or their atresia if this fails to occur) is the primary cause of the degenerative changes of the metoestrum." Except for the addition of the active role of the estrogenic substances contained in the follicular fluid, demonstrated by him- self and Edward A. Doisy less than two years later, the pattern of Allen's thought for the next 20 years was contained in his thesis — the cyclic origin of ova from the germinal epithelium, the primacy of the ovum, the growth effects of estrogens, the consequences of their withdrawal, a dis- counting (the word used in his thesis) of the importance of the hormone of the corpus luteum in the regulation of reproduc- tive phenomena. Rarely has so much of the important conceptualization of a pre-eminent scientist been "roughed in" in his thesis. Also of interest is the place of the thesis in the history of American anatomy. It was written at a time when the emphasis was shifting from structural anatomy to functional anat- omy. Rightly or wrongly, there were then, as there are now, those who feel that this trend could go too far. Allen seems to have been caught in this controversy. At all events, he must have felt compelled to enlist the help of friends at Brown, for it was the men there with their orientation toward biology who seem to ha^'e been less concerned with the amenities of the time and to have recommended that he be awarded the Ph.D. The omission of any acknowledgment to indi\4duals or to in- stitutions could have been an understandable oversight in his haste to test the action of follicular fluid on the vaginal epithelium of the mouse, or it could have been a device for avoiding any embarrassment. It is a coincidence that 15 years later in an office at Brown when a younger colleague was threatening to look into the problem of the hormonal control of mating behavior, Allen asked in his characteristically friendly way and also somewhat paternally, ". . . why don't you return to your woi-k on the epididymis?" With hurdles of the thesis and its publica- tion iK^iind him, and the conviction that the follicular fluid contains the substance he was seeking, he must have thought of injecting follicular fluid from the large follicles of the sow (he had seen them at Brown) into ovariectomized mice and ex- amining the vaginas for the sequence of changes he had described in intact mice. It is clear that the idea was not suggested to him by anything he read. As he often said jocularly, we did the work first and looked up the literature later. The published statement to this effect (J. Biol. Chem., 61: 711-727, 1924) was somewhat qualified but almost as direct, ". . .we paid but little attention to the papers of the various workers who have claimed to have demon- strated active preparations until after our own first definitely positive results were obtained." An unconventional approach, but excuseable perhaps when the hunch is as "logical" as it was to Allen. By the early spring of 1923 he had made promising preliminary tests and a few days before Charles Danforth left Palo Alto for the Anatomists' meetings in Chicago, he received an exultant telegram saying that he (Allen) had succeeded in inducing estrus in a spayed mouse by injecting follicular fluid. Others had come close to the dis- covery, but the reason they failed was that no one had had a clear-cut practical test. Danforth is the authority for saying that the ideas back of all this were Allen's, but in order to obtain a purified product of the active hormone in the liquor folliculi he was using, he enlisted the cooperation of Edward A. Doisy whose laboratory was in an adjoining building. Time has made it clear that Doisy was undoubtedly the most capable collaborator Allen could have found anywhere for this kind of research. A side of Allen that ingratiated him to all was seen at the time of the first announce- ment of the discovery of the action of follicular fluid on the vagina and is quoted EDGAR ALLEN from Danforth's letter to the present bio- grapher : "On my way to the 1923 meeting of Anatomists I went around by St. Louis and had dinner that night at the Allen home. In the evening Doisy, Allen, and I had a long and animated discussion over how their findings should be re- ported. Allen, sanguine and ebullient, was all for announcing them imme- diately at the Anatomists' meeting, even though there w^as no place for such an announcement on the program. Doisy, no less convinced of the im- portance of the findings, so far as they went, but temperamentally careful and thorough, thought any announcement should be withheld till more extensive data could be presented. Allen did not weaken, and when he was called upon at the meetings to present his paper, "Ovogenesis during sexual maturity," whic-h had been duly submitted and published in the abstracts, he by-passed that paper with only a few remarks and used the available time to tell about the effects of follicular hormone on spayed mice. I think this oral report was the first public announcement, the first published one being the AUen-Doisy paper in the Journal of the American Medical Association, September, 1923." Danforth's letter continues: "When the report was presented, I think it was received on the whole with reservation if not outright skepticism, particularly since no abstract had been published and the topic was unan- nounced and unexpected. George Corner said later that was true in his case. Someone else who was there told me of his own skepticism and said that he thought Allen, relatively unknown and with an unconventional idea, was re- garded as something of an 'upstart.' Stockard is said to have made some very caustic comments, which I don't recall. Herbert M. Evans seems to have been among the few who immediately sensed the significance of the paper. He and 1 returned to California on the same train and over and over he kept saying, '1 think Edgar (he sometimes called him Ezra) Allen has something,' or words to that effect." Cooperation with Doisy continued in a skillful chemical analysis which soon made possible the isolation and chemical identifica- tion of the estrogenic hormones. It is un- likely that this stage of the investigation would have been greatly prolonged had Allen adhered to a 9 to 5 o'clock schedule or a 40-hour wTek, but it is apparent from a letter he wTote to Doctor Carl G. Hartman that the discovery of the Allen-Doisy test for estrogens might ha\'e been delayed had he not made a midnight trip to the lab- oratory. According to Hartman, "It was Saturday night and Ed and his wife had been at the theatre. Would he or would he not make a midnight visit to the animal colony at the University in St. Louis and examine the castrated mice which had been receiving Doisy's extracts? He did and much to his delight found cornificd cells in the vaginal smears, which might not have been there if he had waited till Monday morning I" Beyond this point, most of what happened has been recorded by the earlier biographers. In 1923, and almost certainly before the importance of his work was fully appreciated, Allen was appointed Chairman of the De- partment of Anatomy in the then 2-year University of Missouri School of Medicine. He was made Associate Dean of the School of Medicine in 1929 and Dean the follow- ing year. He was happy at Columbia, "things have worked out so nicely here. . . ," he wrote Danforth, and later he began to think he "was planted definitely in Mis- souri." But April 1, 1933, he wrote, "Dean Winternitz visited Columbia day before yes- terday and asked me to go on to Yale ..." as Professor of Anatomy and Chairman of the Department of Anatomy in \hc ^'ale University School of McHlicine. The unusual extent to which his reseai'ch and lh(> i-esearch he super\is('(l in his de- partment were suggested by his thesis has been indicated. We find, theictoic, investiga- tions pertaining to the problem of oxogenesis. At a time when it was generally assumed that the female mammal possesses a full EDGAR ALLEN quota of ova when she is born, he demon- strated that new ova arise after birth and even after sexual maturity. As Doctor WilHam U. Gardner, a former student and his successor at Yale, has written, his early conviction that the ovum is "the dynamic center of the folHcle" persisted throughout his life; he left two partially completed manuscripts dealing in part with the subject. Less than a year before he died he wrote to Danforth, "I still think of the ovum as a dynamic center for mitosis and there is no reason why the fertilized ovum shouldn't be." Gardner is of the opinion that Allen's interest in ova prompted the collaboration with Doctors J. P. Pratt, Q. J. Newell and L. J. Bland that resulted in securing in 1930 the first human ova from the oviduct, and in pro- viding evidence bearing on the time of ovulation in the human female. Overshadowing these studies of the ova were, of course, Allen's many investigations on the relationship of the ovarian estrogenic hormones to the growth of tissues. His demonstration that removal of the ovaries of the rhesus monkey under appropriate circumstances is followed by a uterine bleed- ing, indistinguishable from that of normal menstruation, led to the formulation of his estrogen-deprivation theory of menstrua- tion. Although challenged and shown to be in need of modification, his statement of this theory stimulated many studies of that phenomenon which is still not under- stood. After moving to Yale, he became in- creasingly involved in investigations of the influence of steroid hormones on carcino- genesis, especially the relationship of es- trogens to malignant transformation of the uterine cervix. His interest in the growth- stimulating capacity of the ovarian hor- mones was fiu'ther indicated by his use of the mitosis-stimulating and mitosis-arrest- ing drug, colchicine, in studies on the genital tissues. During the relatively short span of his professional life, he and his collaborators published more than 140 original investiga- tions. Of these, most were under joint authorship. This is explained by the fact that he rarely worked alonc^ on a scientific problem. This may have been just as well, for his excitement and enthusiasm reached their peak in team work. In all such re- lationships, but especially when younger colleagues were involved, he gave full sup- port and generous credit. He would have been proud of the large number of th(^ latter who have since worked their way to im- portant posts in anatomy. Were he alive, he probably would still be prodding them to look into this or that problem. Although the number of articles and reviews of which Allen was author or co- author was relatively large, it was small compared with the many which can be attributed to the encouragement and en- thusiasm he inspired among his students and associates, and to the many more which his work inspired in other laboratories throughout the world. In this sense, rather than in the strictest sense of the word, he was a foremost anatomist. At the height of his career, he undertook the editing of the first edition of Sex and Internal Secretions. The editorship of the second edition was shared with Doctors Danforth and Doisy. The former was un- doubtedly over-modest in recalling the parts of the co-editors in the undertaking, but his statement is ciuoted for the information it contains. "I had helped a little with the first edition, especially with Bridges' chapter (Bridges being in Russia at the time), and in the second edition took I'e- sponsibility for Section A, as Doisy did for his group of chapters. I read the entire book in manuscript or in proof (mostly both) as I think Doisy did also, but neither of us, I feel sure, thought of ourselves as co-editors. On Jan. 4, 1939 Allen wrote, T have asked the publishers to make the book, Allen, Danforth and Doisy; as it has been team work all through.' My own reac- tion, and probably Doisy's is expressed in my reply of January 9, 'Instead of writing a long personal letter in reply to yours of January 4 I am going to send a short note by air mail protesting against your suggestion in the last ED(;AR ALLEN paragraph. It would be quite unjust and misleading for you to put Doisy's and my names in any sense coordinate with yours. . . . And to put our names in any Vnit the most subordinate posi- tion would he to give credit that is not due.' " Allen's services were not unrecognized. Three of the universities with which he was associated, Vale, Brown, and Washington University, awarded him honorary degrees. Had he lived a few months longer, he would have received an honorary doctor of law from the University of IVIissouri. In 1987 he was awarded the Legion of Honor in l-*aris where he was guest of the Fondation Singer-Polignac at a colloquium on the sexual hormones. In 1941 he was honored by the Royal College of Physicians of London when they conferred upon him the Baly Medal for researches on the female sex hormones. In the last year of his life he was president of the two national societies most closely representative of his field of work, the Association for the Study of Internal Secretions (now the Endocrine So- ciety) and the American Association of Anatomists. As Corner, Danforth and Stone wrote in their memorial for the American Associa- tion of Anatomists, "Allen was a striking figure personally, for his broad shoulders, ruddy face and snow-white hair made him conspicuous in any group. He made friends (luickly and was always the first to en- coiu'age and to admire good work by others. His boyish frankness and his good will contributed more perhaps than any other factor to the effective cordiality which has prevailed among the American workers in the physiology of reproduction." The writer of this skc^tch recalls that in any meeting Allen was always one of the first to rise to the floor with a (juestion or lo mak(> a constructive' coniineul . The Viest of the spirit of I he man that we can preserve is containe'd in \\\o form of his own words, written inforinall>- to his fri(Mid, ( harles Danfoith, with whom he coi're- sponded so fre(iuentl>' and xoluminously. A numbei- have been selected for the side of him thev will recall, the enthusiasm and imagination that enabled him to do the amount and quality of w^ork he did. November 6, 1935: "We just had a keen thing happen here. Dr. George AI. Smith returned from Europe with in- formation about a drug which prevents mitotic figures from completing divi- sion. . . . Combining this with theelin stimulation. . . ." Alay 23, year not stated: "Have been working since early morning on the slides from the monkey experiments. They are a corking good lot of evidence and I am in one of those elated, trembly, inspired sort of heavens to which new ideas always bring a fellow. If you were in reach, you would have been dragged over precipitately to my scope, or interrupted to talk things over a dozen times. ... As it is, I must run my ideas through a slow movie instead of talking you to death. . . . Growth in the vaginal wall is almost unbelievable. Growth in the cervical glands is more so. They grow so darned much that the cervix looks like a tumor and the cervical canal becomes a tor- tuous thing like the lower Mississippi." A lot of histological detail, then: "the tubes are a knockout . . . this would seem to make ciliation a growth phase of the nonciliated cells and full ciliation dependent on presence of the hormone. The mammary gland whole moimts are wonders. I am so elated as to be almost damned crazy. Am sure I'd crack one of your ribs if I could get at you." Then, "Yesterday a letter from the National Research Council giving me $800 more for monkeys. . . . Ain't it just too lucky to l)eli(n-e. . . ." And (inallw in closing one of his letters: "Well you darned tool, Allen, it will take him 10 miiuitcs to read your letter now, aren't you e\('i' going to stop writing?" About 10 years belore Allen died he sulTered a >r\r]v coi'onaiy attack, whil(> waiting foi' a train in the ,Jackson\ille, I'lorida, Lnion Station, lie lo\-ed swinuning as well as sailing and, after a sti'enuous and fatiguingxisit with Doctoi' IJobei't M. Verkes EJ)C;AR ALLEN in the heat at Orange Park, had taken time for a long swim in the breakers at Jackson- ville Beach. Miraculously he reached New Haven and recovered sufficiently to return to his work. Nothing would have kept him from it. When war was declared in 1941 he was tired, but he insisted on joining the Coast Guard Auxilliary for a weekly tour of duty on Long Island Sound, as opera- tions officer of a flotilla. It was during one of their patrols that he died of coronary occlusion. There was much that was fitting to such an end of his active life. No tribute could have been more appropriate than that phrased by Gardner in th(^ Edgar Allen Memorial Number of the ^'ale Journal of Biology and Medicine, "The 'Skipper' left with 'all sails filled.' " PREFACE TO THE THIRD EDITION The impact of the first two editions of Sex and Internal Secretions can never be measured, but it must be near the front for books of its kind. Few books seem to have served their purpose better and few, 20 to 25 years after their appearance, seem to be valued as greatly by those who are fortunate to possess copies. It was to be expected, therefore, that pressure would be brought to bear for the preparation of a third edition. Whether the Publisher's at- tempts to find an editor miscarried because of the character of the new order ushered in by World War II, or because discretion was considered the better part of valor may never be known. The odds favored the latter explanation because there was no direction in which a successor to Edgar Allen, Edward A. Doisy and Charles H. Danforth could go except down. Neverthe- less, there were reasons for accepting the challenge and attempting to do for the present generation of reproductive phys- iologists what Allen, Danforth and Doisy and their many colleagues did for theirs. Most of the problems to which they ad- dressed themselves had not been solved, although the need for answers was as urgent as ever, and perhaps more so. The definition of these problems had become obscured, partly by the addition of many contradictory and confusing data to the literature relating to them, and partly by the rising tide of interest in other glands of internal secretion, notably the thyroid and adrenal. Finally, new technologies had pervaded the field and there were many new data and concepts to be evaluated and Avoven into the fabric Allen and his contemporaries had created. In the preparation of the third edition little of the first two editions was to be retained except the title. Sex and Internal Secretions, and the ideals by which the authors of these editions must have been guided. Within a framework of careful scholarship, these were seen to be a resume of the solid facts that had been learned from test and retest, broadly critical discussions, enumeration of the important unsolved prob- lems, and the preparation of lists of ref- erences complete enough for the guidance of any seeker of information, whether his interest was in the extension of basic studies or the application to clinical and agricul- tural problems. Adherence to these ideals has not been easy. Even if ample allowance is made for editorial ineptitude, the period 1958 to 1961 is different from 1932 and 1939. Reviews and symposia are more nu- merous and many are in a style that is alien to the traditions of Sex and Internal Secretions. There are demands on the time of many of us which, 20 years ago, were reserved for only a few, and the volume of published reports has long since outstripped oiu- capacity properly to encompass them. Despite the difficulties and misgivings, an effort has been made to step into the void created by the lapse of the old Sex and Internal Secretions. Both similarities and differences will be noted. Relatively more space has been given over to the role of the gonadal hormones in the control of repro- ductive behavior, and relatively less to the biochemical problems of hormone synthesis, utifization, and metabolism. This "slighting" of the biochemical side, if it is to be so considered, does not reflect any lack of appreciation of the key position occupied by this discipline. It is explained, rather, by the opinion of the biochemists who were consulted that another review, just at this time, would be anticlimactic to a number of the excellent reviews which have recently been published. The chapter by Dr. Villee, therefore, is, in his words, a presentation of the general picture without being an exhaustive citation of the tremendous body of relevant literature. The suggestion made above that some of the shortcomings in this third edition are a reflection of changes in our habits of work- ing is not to be taken as an attempt to PREFACE, THIRD E])ITION excuse any errors that properly belong on the editor's doorstep. He has learned as he has gone along, but finds himself reacting very much as he usually does at the end of a lecture — if it were to be given again, parts of it would be done differently. To the contributors, there is a feeling of the deepest gratitude for the time and thought they have given to the preparation of their chapters. The editor is indebted, too, to the National Institute of Mental Health, Na- tional Institutes of Health, for a research grant, M-4648, which has taken care of a number of the costs of publication. Justifica- tion for this action is believed to have been given by the expansion of the section on the gonadal regulation of reproductive behavior. This development, in turn, would have pleased Robert M. Yerkes. He was sensitive to the need for truly scientific studies of sexual behavior, the mechanisms which par- ticipate in its expression, in the determina- tion of its character, in its regulation, and in its development. But he was equally aware of the importance of investigations that are purely physiologic, biochemical, and morphologic, and in a quiet but effective way did much to encourage many that are recorded in the first two as well as in the present edition of this book. PREFACE TO FIRST EDITION It is the purpose of this book to survey the most important recent researches in problems of sex, especially those concerned with internal secretions, in order that con- cepts already established by experimental evidence may be clearly stated and made readily available. While general principles can in many cases be stated concisely, the recent data have accumulated so rapidly that there has not yet been time for retesting and evaluating much of the evidence. This may account in some chapters for emphasis upon certain work with which contributors may have had personal contacts. Further- more, sex and reproduction show such wide ranges of variation in both the structures and functions involved that major dif- ferences, even among species of higher mam- mals, make generalizations both difficult and dangerous. This whole field has recently undergone such rapid growth that many new ciuestions have arisen to challenge the investigator's curiosity. An attempt will be made to indicate productive approaches to some of these unsolved or only partially solved problems. This book is intended for the reader with a moderate biological background, to whom the less involved technical terminology may not prove a serious handicap. It is not our intent that it should be a "popular book on sex." Instead, it is designed for those in- terested in the progress of research in problems of sex, and those who may be already engaged in investigations thereon or casting about for promising problems for investigation. Physicians who are interested in fundamentals will find much valuable recent material. In suppljnng a biological foundation for education in matters of sex, it should also attract the interest of serious students of sex function in man. Specialization in research has reached the point where any detailed authoritative sur- vey requires a group effort. Conseciuently, the editoi attempted to gather together a group of investigators whose work has es- tablished them in their respective fields. Each contributor has develop(>d his chapter in his own way and assumes full respon- sibility for the content of his section, in- cluding his discussion of the work of othei' investigators. Since it was inevitable that considerable correspondence would be involved, intro- ducing the time-transport factor, choice of the group was restricted to American investigators. Several prominent workers in this field, whom we would have desired as co-authors, have necessarily been omitted because of absence abroad or press of other work. As the Foreword indicates, this project saw its inception in a proposal by Dr. E. V. Cowdry, then Chairman of the Medical Division of the National Research Council, to the Committee for Research in the Prob- lems of Sex. In the publication of many of the contributors to this book, acknowledg- ment will be found to this Committee for support of investigations. In a sense the book will serve as a summary of some of the work accomplished under grants from this Committee. Obviously, however, it was not desirable to limit choice of contributors to investigators who had received grants from the Committee, and that consideration has not determined their choice. Instead, it is probable that the Committee in the first place chose to make grants to these men because of the promise of their work shown by their previous investigations. The editor wishes to acknowledge the support of the Committee for Research in Problems of Sex, which has made provision for the editorial expenses involved in the preparation of the manuscript. He also wishes to commend the cooperation between investigators who, even though they may be competitors in the same field, have col- laborated so well. The cooperation has been completely free from the secretive reserve sometimes encountered among investigators who may be leaders in their particular fields. The editor wishes further to acknowl- xxiv PREFACE, FIRST EDITION edge the assistance and friendly interest of Dr E. V. Cowdry, not only during the initial phases of this project, but through- out its progress and consummation. Dr. F. R. Lillie has counselled wisely in regard to titles and content of some of the sections. Valuable counsel and encouragement has also been received from several of the con- tributors. The editor also wishes to commend the contributors for their team-work ni eliminating or reducing overlaps between sections which is so necessary in the unifica- tion of interlocking material from closely related subjects. Edgar Allen University of Missouri School of Medicine Columbia, Missouri May, 1932. SECTION A Biologic Basis of Sex GENETIC AND CYTOLOGIC FOUNDATIONS FOR SEX John W. Gowen, Ph.D. Department of Genetics, Iowa State University, Ames, Iowa I. Basic Literature II. Mechanistic Interpretations op Sex A. Concept of Sex Determination B. Sex as Associated with Visible Chro- mosomal Differences C. Changing Methods of Cytogenetics D. Chromosomal Association with Sex E. Balance of Male- and Female-Deter- mining Elements in Sex Deter- mination III. Sex Genes in Drosophila A. Mutant Tjqjes B. Major Sex Genes C. Other Chromosome Group Associa- tions: Drosophila americana D. Location of Sex-determining Genes IV. Sex under Special Conditions A. Species Hybridity B. Mosaics for Sex C. Parthenogenesis in Drosophila D. Sex Influence of the Y Chromosome E. Maternal Influences on Sex Ratio F. Male-influenced Type of Female Sex Ratio " G. High Male Sex Ratio of Cienetic Origin H. Female-Male Sex Ratio Interactions V. Sex Determination in Other In- sects A. Sciara B. Apis and Habrobracon C. Bombyx VI. Sex Determination in Dioecious Plants A. Melandrium B. Rumex C. Spinacia D. Asparagus E. Humulus VII. Mating Types VIII. Environmental Modifications of Sex A. Amphibia B. Fish IX. Sex and Parthenogenesis in Birds X. Sex Determination in Mammals... A. Goat Hermaphrodites B. Sex in the Mouse 50 C. Sex and Sterility in the Cat 50 D. Deviate Sex Types in Cattle and Swine 51 E. Sex in Man: Chromosomal Basis 52 1. Nuclear chromatin, sex chro- matin 55 2. Chromosome complement and phenotype in man 5(1 3. Testicular feminization 50 4. Superfemale 57 5. Klinefelter syndrome 58 0. Turner syndrome 59 7. Hermaphrodites 59 8. XXXY + 44 autosome type 01 9. XXV + 66 autosome type 03 10. Summary of types 03 11. Types unrelated to sex 03 F. Sex Ratio in Man 65 XL References 00 I. Basic Literature In the first edition of Sex and Internal Secretions published in 1932, Bridges dis- cussed the closely prescribed problem of the genetics of sex, particularly as it was related to one species, Drosophila melanogaster. The treatment was sharply focused on the advances made, chiefly through his own re- searches, in understanding the functions of the genetic and cytologic factors operating during embryologic development which ul- timately establish the sex types. The em- phasis was on gene action through inter- chromosomal balances as they may aff"cct sex expression. The second edition of 1939 brought this material up-to-date and, at the same time, offered a much broader treat- ment by the inclusion of accumulated evi- dence on how differentiation for sex comes about in other forms. There is no substitute for a careful reading of these two presenta- BIOLOGIC BASIS OF SEX tions as background material for a present day understanding of sex determination. The current presentation makes no attempt, except in the barest outline, to repeat this early material other than in those aspects which bear on the advances that have been made since the printing of the second edi- tion. Immediately following the publication of the first edition of Sex and Internal Secre- tions there was a resurgence of interest in the problems considered in that book. The resulting research led to notable advances in available knowledge. Seven years later the second edition was published. A second wave of accomplished research appeared. In the interim of the past 21 years extensive advances have been recorded, particularly in understanding the mechanisms of sex dif- ferentiation in plants as well as in many animals. Among others, the data on Melan- drium, Asparagus, Rumex, and Spinacia have been of first importance. Further in- formation on Drosophila, Habrobracon, and Xiphophorus has notably broadened our viewpoints. Beginnings have been made to a better understanding of the conditions for sex separation in bacteria, protozoa, bees, birds, goats, mice, and man. To these spe- cific contributions may be added the basic advances in understanding the methods by which genes are transmitted from one gen- eration to the next, accomplish their actions in development, and by which chromosomes reorganize and reconstruct gene groups. The period has also been one of excellent monographic treatments on different phases of the subject. Wilson's The Cell in Devel- opment and Inheritance (1928) and earlier, Morgan's Heredity in Sex (1914), Schra- der's The Sex Chromosomes (1928) and Goldschmidt's Lymantria (1934) retain their pre-eminence. To this list have now been added the publication of extensive tabulations of chromosomes of cultivated plants by Darlington and Janaki Ammal (1945), of animal chromosomes by Makino (1951 ), and the yearly index to plant chro- mosomes beginning with 1956, compiled by a world-wide editorial group and published by the University of North Carolina Press, Chapel Hill, North Carolina. Those volumes give access to the basic chromosomal consti- tutions and their sex relations for far more species than were heretofore available. In- heritance information has been made more accessible and at the same time in more de- tail. The tabulations of mutants observed in particular species as in D. melanogaster (Morgan, Bridges, and Sturtevant, 1925; Bridges and Brehme, 1944) have been fol- lowered by those of corn, mouse, domestic fowl, rat, rabbit, guinea pig, Habrobracon, and many other animal forms, together with similar tabulations for cereals and a number of other plant species. Books of particular interest include those of Hart- mann. Die Sexualitdt (1956), White, Ani- mal Cytology and Evolution (1954), Gold- schmidt, Theoretical Genetics (1955,1, Hartmann and Bauer, Allgemeine Biologic { 1953) , and Tanaka, Genetics of Silkworms (1952), and special reviews in various vol- umes of Fortschritte der Zoologie (1 to 12) (Wiese, 1960). Basic normal development of Drosophila has been presented in con- venient book form in papers by Cooper, Sonnenblick, Poulson, Bodenstein, Ferris, Miller and Spencer, Biology of Drosophila (Demerec Ed., 1950) . Special papers hav- ing a direct bearing on the subject matter include particularly those of Pipkin (1940- 1960) in analyzing the various chromo- somes of Drosophila for sex loci, of Tanaka (1953) and Yokoyama (1959) for their studies and reviews of the genetics of silk- worms, and of Westergaard (1958) on sex determination in dioecious flowering plants. II. Mechanistic Interpretations of Sex The records of search for basic mecha- nisms involved in the determination of sex have been foremost in the writings of man from the beginning of historical record. These ideas have included all forms of mechanisms both intrinsic and extrinsic to the organism. The most constructive ad- vance, although not realized at the time, came through the recognition of cell struc- ture, chromosome maturation and the dis- crete behavior of the inheritance. Differ- ences in clu-omosome behavior were noted but not alwaj^s related to facts of broader significance. The period was one of expand- ing observations and development of ideas as they applied to species in general and FOUNDATIONS FOR SEX also to the further complications which arose with more extended study. These complications included differences not only in one chromosome pair but in several. The significance of a balance between these chromosome types as well as with the en- vironment was grasped by Goldschmidt and particularly by Bridges where his more favorable material brought out sharper contrasts in types and in the chromosome behavior. Ideas related to chromosome bal- ance as they may affect developmental processes were developed. Goldschmidt em- phasized that this balance could include factors in the cytoplasm as well as in the chromatin material. Bridges' observations on the other hand, pointed most strongly to the chromatin elements where changes in chromosome numbers were often accom- panied by sharp differentiation of new sexual types. Concepts of sex determination broadened. They came to include all the chromosomes in the haploid set, a genome, as a whole. The important concept of single chromo- some difference being all important has been replaced with that of a balance be- tween the chromosomes. It has sometimes been emphasized that it is this balance which is the most significant element in sex determination. Present day research seems to be pointing rather to even finer struc- tures than the chromosomes in that the main search is on for the particular genes within the inheritance complex which con- tribute the characteristics of sex. Bridges' work has sometimes been asserted as com- mitted to a particular type of balance in the chromosome. However, his writings 1 1932) make it clear that this balance is, in his judgment, basically due to the genes actually contained within the chromosomes rather than to the chromosomes themselves. This view is further emphasized in the second edition of Sex and Internal Secre- tions. "Sex determination in numerous forms with visible distinctions between the chromosome groups of the two sexes was at first interpreted on a 'quantitative' basis, as due to graded amounts of 'sex-chroma- tin.' But because even more species were encountered in which no visible chromo- some difference was detected, the formula- tion was changed to include also 'qualita- tive differences in sex-chromatin.' Now, sex is being reinterpreted in terms of genes, with investigation by breeding tests pene- trating to detail far beyond the reach of cytological investigation. The chromosomal differences are now treated as rough guides, and chromosomal determination is l)cing resolved into genie determination Hence the difference in sex must be put on the same basis as that of any other organ differentiation in plant or animal, for ex- ample, the difference between legs and wings in birds— both modifications of an- cestral limbs." In contrast to what some have inter- preted. Bridges was not committed to a specific chromosome balance, as for in- stance the X chromosomes to the A chro- mosomes in Drosophila, for all species. Rather he looked on that particular balance as but one mode of gene association in chromosomes that could bring about dif- ferences in gene action on development which would lead to sexual differentiation in its various forms. In that sense his ideas prepared him for, and were in conformity with, the types of sex differentiation which later became established in such forms as Melandrium and in the silkworm. In his concepts of sex determination and in his active interest in making genetics more spe- cific, there can be little doubt that his theory would include those of the present and would also be welcome as refining and more sharply defining the significance of cytologic and genetic elements important to sex differentiation. A. CONCEPT OF SEX DETERMINATION As frequently happens, observations are made often before their significance is more than dimly understood. Background infor- mation may be insufficient for the facts to become clear. This was true when a lone chromosome was discovered as a part of the maturation complex in sperm formation of Pyrrhocoris. The association of the pres- ence of this element with the idea of its be- ing the arbitrator between male and female development in certain species was not made until 10 years later. Henking (1891) observed in Pyrrhocoris 11 paired chromo- 6 BIOLOGIC BASIS OF SEX some elements and one unpaired element which after spermatocyte divisions led to sperm of two numerically different classes, one carrying 11 chromosomes plus the ac- cessory chromosome and the other carrying only the 11 chromosomes. His observations were confirmed by several other investiga- tors and in principle for other insect species over a decade following. The first sugges- tion that this chromosome behavior was re- lated to sex diiTerentiation came from McClung's (1902) observations on the "ac- cessory chromosome" of Xiphidiiim fascia- tum. It is of some interest to examine the steps l)y which these conclusions were reached. "The function exercised by the accessory chromosome is that it is the bearer of those qualities which pertain to the male or- ganisms, primary among which is the fac- ulty of producing sex cells that have the form of spermatozoa. I have been led to this belief by the favorable response which the element makes to the theoretical require- ments conceivably inherent in any struc- ture which might function as a sex determi- nant. "These requirements, I should consider, are that: (a) The element should be chro- mosomic in character and subject to the laws governing the action of such struc- tures, (b) Since it is to determine whether the germ cells are to grow into the passive, yolk-laden ova or into the minute motile spermatozoa, it should be present in all the forming cells until they are definitely es- tablished in the cycle of their development, (c) As the sexes exist normally in about (■(lual projoortions, it should be present in half the mature germ cells of the sex that bears it. (d» Such disposition of the element in the two forms of germ cells, paternal and maternal, should be made as to admit of the readiest response to the demands of en- A'ironment i-egarding tlie ])i'oportion of the sexes, (el It should show variations in structure in accordance with the vai-iations of sex potentiality observable in different species, (f) In parthenogenesis its function would be assumed by the elements of a cer- tain polar body. It is conceivable, in this regard, that another form of polar body miglit function as the non-determinant bearing germ cell." The important fact es- tablished by this reasoning was that a chro- mosome could be the visual differentiator between the fertilized eggs developing spe- cific adult sexual differences. It w^as of little consequence perhaps that for sex itself, in this species, the chromosome arrangement was misinterpreted. The validity of the rules was probed through examinations of the cells of many species by many different observers. B. SEX AS ASSOCIATED WITH VISIBLE CHROMOSOMAL DIFFERENCES Variations in the chromosome complexes contributing to sexual differentiation were soon found. Probably the most frequent type observed in animals and plants was that in which a single accessory chromo- some of the male was accompanied by, and paired with, another chromosome either of the same or of a different morphologic type. This other chromosome, as distinguished from the accessory chromosome now gen- erally called the X, was designated the Y chromosome. For different species the Y ranged in size from complete absence, to much smaller, to equal to, to larger than the X. Besides the X and Y chromosomes, the autosomal or A chromosomes, the ho- momorpluc pairs, complete the species chromosome complement. In this terminol- ogy Sperm (Y + A) + egg (X + A) = XY + 2A cells of lie determinino; tvpe Sperm (X + A) + egg (X + A ) = 2X + 2A cells of female detei'mining tyi)e Variations in numbers and sizes of chro- mosomal pairs making up the autosomal sets of different species are familiar cyto- genetic facts. These variations may extend from one pair to many chromosome pairs depending on the species. Similar variations may occur in chromosomes of eitluM- the X or Y types. The X is commonly a single chromosome but may be a compound of as many as 8 chromosomes in Aiicaris inciirva (doodricli. 1916). The Y mav be lacking FOUNDATIONS FOR SEX entirely in some species or may be redupli- cated in others. Males which form two types of sperm are often called heterozygous for sex, although the term digametic may be better, whereas the females are homoga- metic. In other species the females show the chromosomal differences whereas the males are uniformly homogametic. These types are principally known in the Lepidoptera, more primitive Trichoptera, Amphibia, some species of Pisces, and Aves. The single accessory chromosome is present in the fe- males whereas the males have two. It may l)e unpaired or be with another chromosome of different morphology and size. The sex types may be symbolized by designating the accessory chromosome by Z and its mate W as a means of separating possible differences between them and the X and Y chromosomes. The significance of these dif- ferences is not clearly established with the consequence that some investigators prefer to substitute the XY designation for the fe- males and the XX condition for the males of these species. The zygotic formulae are: Sperm ZA + egg ZA = 2Z + 2A male Sperm ZA + egg WA = ZW + 2A female A third major chromosomal arrangement accompanying sex differentiation came to light as a result of Dzierzon's 1845 discov- ery of parthenogenesis in bees. Chromoso- mal and genetic studies have shown both Apis and Habrobracon of the Hymenoptera to be haploid, N, for each germinal cell in the male complex and diploid, 2N, in the fe- male. N may stand for any number of chro- mosomes, as 16 for Apis or 10 for Habro- bracon. The same chromosomal pattern characterizes, so far as known, the other genera of Hymenoptera. Haploidy vs. dip- loidy is viewed as the most obvious feature predicting differentiation toward the given sex type even though, as in Habrobracon, there is evidence for a particular chromo- some of the N set carrying a locus for sex differentiating genes. The zygotic sex for- mulations are: No sperm + egg N = N male Sperm N + egg N = 2X female In development, as in some other species oi widely diverse origins, chromosome pol- yploidy may take place causing the soma cells to differ from the germ cells in their chromosome coniponents. The common phenotype for plants and lower animals has differentiated sex organs which are combined in the same individual. Plant species seldom depend on any but hermaphroditic types for their reproduc- tion. Lewis' (1942) tabulation for British flora had but 8 per cent of all species de- pend on other than this form of reproduc- tion. Those that had perfected dioecious systems were not all alike in the system adopted, although species with XY + 2A males and XX + 2A females were in high frequency. Dioecious reproduction was rarely the system common to a whole genus. Recent and multiple origins of dioe- cious types are indicated by the irregular distribution of species with bisexual repro- duction within the different genera and families. Methods for preventing inbreed- ing have taken other channels as self- sterility genes reminiscent of fertility or sex alleles found in the older bacteria and protozoa. Animals of the lower phyla are those which are most frequently hermaph- roditic. Within hermaphroditic species chromosomal distinctions are ordinarily ab- sent. The higher forms with sex and chro- mosome differences, on the other hand, may show reversion to the hermaphroditic con- dition from the dioecious or bisexual states. Other less common cytogenetic controls of sex development have become recognized and better understood. Discussion of their gene and chromosomal arrangements will be considered when these cases arise. The al)ove types will be sufficient to furnish a basis for interpreting the newer data. C. CHANGING METHODS OF CYTOGENETICS The earlier studies of sex determination depended on the natural arrangements of chromosomes found in different species and on occasional chromosomal rearrangements occurring as relatively rare aberrant types. Further development of genetics has in- creased the tools for these studies. Mutant genes have been shown to control chromo- some pairing in segregation (Gowen and Gowen, 1922; Gowen, 1928; Beadle, 1930). BIOLOGIC BASIS OF SEX Different drugs such as chloralhydrate and particularly colchicine and various forms of radiant ^energy have made it possible to create new types by doubling the chromo- somes, by chromosome rearrangement and by changing the chromosome number. Bet- ter genetic tester stocks of known composi- tion have been organized, which together with a more exact understanding of the in- heritance structure of the species have made for more critical studies. Techniques, which have improved chromosome differen- tiation and structural analysis through bet- ter methods, better dyes, tracers to mark chromosome behavior (Taylor, 1957), and the use of hypotonic solutions in the study of cells (Hsu, 1952), have removed doubts that were created by the earlier technical difficulties. Instrumentation has improved for the measurement, physically and chemi- cally, of cell components. D. CHROMOSOMAL ASSOCIATION WITH SEX The first genetic linkage groups were as- sociated with the sex chromosomes and were soon shown to follow the patterns of the different chromosome sets observed within the different species. In man, hemo- philia inheritance was observed to follow that which was presumed for the X chro- mosome. Barring in birds and wing-color pattern in Lepidoptera on the other hand were found to follow the chromosomal pat- terns of their species where the male was ZZ and the female Z or ZW for the sex chro- mosomes. The utility of these methods was further probed by Bridges' observations that when chromosome behavior in Dro- sophila resulted in sperm or eggs carrying uncxjx'cted chroiiiosomc combinations there followed ('(nmlly unexpected phcnotypes in the progeny. The cliaractei'istics of these unexpected progeny, in turn, I'ollowt'd those expected if genes for them were carried m the sex chromosomes. The use of these link- age groups as tracers, both as they natu- rally occur and as they may be reorganized through the treatment effects of such agents as radiant energy, open the possibility ol assigning sex effects to, not only chromo- somes, but also to particular i-laces within the chromosomes. Both polyploids and aneuploids, as oc- curring naturally and as marked by tracer genes, give insight into sex differentiation and its dependence on chromosome inherit- ance behavior. True polyploids in a species are formed as a consequence of multiplying the entire genome. The possible types may have a single set of chromosomes and genes in their nuclei (haploid ) , 2 sets (diploid) , 3 (triploid), 4 (tetraploid), and so on, rep- resenting the genomes 1. 2. 3. 4. to whatever level is compatible with life. Multiplying the genomes within the nucleus often in- creases cell size but seldom gives the or- ganism overtly different sex characteris- tics.i Aneuploids, on the other hand, give quite different results, the result being de- pendent upon the particular chromosome that may be multiplied. Particular tri- somies in maize, wheat, and spinach, for example, are distinguishable by marked differences in phcnotypic appearance that is not attributable to cell size but rather to abrupt deviations in particular charac- teristics. The genes in the particular tri- somic set are unbalanced against those of the rest of the diploid sets within the or- ganism. Their phenotypes express these differences. E. BALANCE OF MALE AND FEMALE DETERMINING ELEMENTS IN SEX DETERMINATION The foundations for the basic theory that sex determination rests on quantitative re- lationships of two genes or sets of genes localized in separate chromosomes rests largely on tlie work of Morgan, Bridges and Sturtevant, 1910, with Drosophila and the breeding work of Goldschmidt, 1911, with Lymantria. The work of Goldschmidt soon gave extensive descriptions of diploid inter- sexuality in L]imantria dispar. The details of this work scarcely net'd review because they have \)vvn repealed several times and have been smnmarized recently in Gold- schnu(h's Theoretical Genetics (1955). Fioui (hildschmidt's viewpoint, ''the basic point was that definite conditions between the sexes, that is, interscxuality, could be pro.lnced at will l)y proper genetic combina- , tions (crosses of subspecies of L. dispar) • The notable exception of the Hynienoptera will I )c discussed later. FOUNDATIONS FOR SEX without any change in the mechanism of the sex chromosomes, and this in a typical quantitative series from the female through all intergrades to the male, and from the male through all intergrades to the female, with sex reversal in both directions at the end point. The consequence was: (1) the old assumption that each sex contains the potentiality of the other sex was proved to be the result of the presence of both kinds of genetic sex determiners in either sex; (2j the existence of a quantitative relation, later termed 'balance' (though it is actually an imbalance), between the two types of sex determiners decides sexuality, that is femaleness, maleness, or any grade of inter- sexuality; (3) one of the two types of sex determiners (male ones in female hetero- gamety, female ones in male heterogam- ety) is located within the X-chromo- somes, the other one, outside of them; (4) as a consequence of this, the same deter- miners of one sex are faced by either one or two portions of those of the other sex in the X-chromosomes; (5) the balance system works so that two doses in the X-chromosomes are epistatic to the deter- miners outside the X, but one dose is hypo- static; (6j intermediate dosage (or po- tency) conditions in favor of one or the other of the two sets of determiners result, according to their amount, in females, males, intersexes, or sex-reversal individuals in either direction; (7) the action of these determiners in the two sexes can be under- stood in terms of the kinetics of the reac- tions controlled by the sex determiners, namely, by the attainment of a threshold of final determination by one or the other chain of reaction in early development; while in intersexuality the primary deter- mination, owing to the 1X-2X mechanism, is overtaken sooner or later — meaning in higher or lower intersexuality — by the op- posite one, so that sexual determination finishes with the other sex after this turning point. The last point is, of course, a problem of genie action." Bridges developed his idea of "genie bal- ance" as a consequence of his observations on chromosomal nondisjunction, particu- larly as it illustrated the loss or gain of a fourth chromosome in modifving nonsexual characters. The similarities and contrasts of this view from that of Goldschmidt are indicated by the following quotation (Bridges, 1932) : "From the cytological re- lations seen in the normal sexes, in the in- tersexes, and in the supersexes, it is plain that these forms are based upon a quanti- tative relation between qualitatively differ- ent agents — the chromosomes. However, the chromosomes presumably act only by virtue of the fact that each is a definite collection of genes which are themselves specifically and qualitatively different from one another. There are two slightly differ- ent ways of formulating this relation, one of which, followed by Goldschmidt, places primary emphasis upon the quantitative aspect of individual genes. The other view, followed by the Drosophila workers, em- phasizes the cooperation of all genes which are themselves qualitatively different from one another and which act together in a quantitative relation or ratio. Goldschmidt developed his idea through work with the sex relations in Lymantria and has sought to extend it to ordinary characters. The other formulation, known as 'genie balance,' was developed from the ordinary genetic relations found in characters. Both are crystallizations of fundamental ideas with which the earlier literature was fairly saturated and no great claim to distinctive originality should be ventured for either or denied for one only. Both are physiological as well as genetic — that is, they are formu- lations of the action of genes, not merely statements of the genie constitutions of in- dividuals nor merely studies of the way genes act. The physiological side has been emphasized by Goldschmidt and the ge- netic side by Drosophila workers. But Goldschmidt's tendency to represent the view of genie balance as without, or even as in opposition to, such physiological for- mulation is groundless — as groundless as would be the reciprocal contention that Goldschmidt's theory is only one of 'pheno- genetics.' "A common element in the foundation of both formulations is that if a gene is repre- sented more than once in a genotype the phenotypic effect is expected to be different, though roughly in the same direction as be- 10 BIOLOGIC BASIS OF SEX fore and roughly proportional to the quan- titative change in the genie constitution." Since the sexually different types ob- served by Bridges were accompanied by whole chromosomal differences, he could point to losses or gains of autosomes, with an internal preponderance of genes tending to develop male organs, as balanced by genes of the X chromosomes tending in the direction of producing female organs or of suppressing alternative male organs. The net effect of the X chromosome favoring fe- maleness, and of the set of autosomes fa- voring maleness, terminates in the devel- opment of male or female, according to the ratio of these determiners in the whole genotype. The effect of the X chromosome goes on the basis of whole numbers 1, 2, 3, 4, as does the similar variation in the sets of autosomes. Goldschmidt, since he was dealing with TABLE LI Chromosomal numbers and kinds for the different recognized sex types of Drosophila Type Superfemale . . . Triploid meta- female Female* Female Female Female Female Female Female P'emale Female Female Intersex* Intersex Intersex Male Male Male Male Male* Supermale Chromosomes X Y A 3 2 4 3 4 4 3 3 3 1 3 3 2 3 2 2 2 1 2 2 2 + vS 2 2 2 + yL 2 2 2 2 1 1 3 4 2 3 2 1 3 2 1 2 2 2 3 2 4 3 X/A Balance 1.5 L3 LO LO LO LO LO 1.0 LO LO LO LO .75 . ()7 .()7 .50 .50 .50 .50 .50 .33 * These forms are cited by Bridges from his own observations, from L. V. Morgan (1925) and from Sturtevant (unpublished). As yet but limited studies of these forms, which must be rare, have been published. Fourth chromosomes generally, but not always, equal mimber of the other indi- vidual chromosome gn)U])s. males and females of the diploid type, took a corresponding view for the Z chromo- somes of his moths with this difference. Since the Drosophila chromosome pattern is XY + 2A for the males as contrasted to 2X + 2A for the females and the pattern for Lymantria is ZZ -|- 2A for the males and ZW + 2A for the females, it was neces- sary to use a relation which was reciprocal to that of Drosophila; the male determining element or elements were assigned to the Z chromosomes. Lymantria has a rather large number of different races found in different geographic locations. Within any one of these races this formulation apparently suf- ficed. However, from crosses between races it was soon observed that the progeny showed ranges in sexuality all the way from phenotypic males to phenotypic females al- though these females were actually genetic males. To Goldschmidt, this variation indi- cated different potencies of the male-deter- mining element. Similar differences were at- tributed to the female element which he had first assigned to the cytoplasm but for which he later favored a W chromosome location. In applying this postulate of discrete chromosome contributions to sex according to their number, Bridges made the further assumption that female-producing genes l)redominate in the X and are scattered through it in more or less random fashion as are the genes affecting so-called somatic characteristics as wing shape or bristle pat- tern. The quantitative relations for the dif- ferent chromosomal types, together with their descriptions, are indicated in Table 1.1. In the formulation of Table 1.1, the X and Y chromosomes are counted separately, whereas a set of A chromosomes (auto- somes), is allowed a value of but one even though comjiosed of a 2nd, a 3rd, and a 4th chromosome for the haploid genome. The dii)loid set of autosomes is given a weight of two, and so on. From these data Bridges observed that the presence or ab- sence of a Y chromosome did not affect the sex types. The X chromosomes and auto- somes were, however, important. He held that their importance stood as the ratio of thcii' prcsuiiK'd iM'oducts to each other. The FOUNDATIONS FOR SKX 11 ratio is 1 for the perfect female and 0.5 for the perfect male. Between these values in- tersexual conditions develop. Beyond the value 1, development is overbalanced by- excess female genes resulting in the super- female. Values less than 0.5 create a de- ficiency in the female elements or excess of male elements and a supermale results. Schrader and Sturtevant (1923) proposed another system. Instead of the ratio of X clu-omosomes to autosomes, they suggested that a straight difference between the prod- ucts of the female determining elements of the sex chromosomes and the male effects of the autosomes causes the sex changes. ]3ridges criticized this system on the basis of the fact that progressive polyploidy did not change the sex type or ratio between the X and autosomes, whereas the numerical difference between them would be progres- sively increased. While keeping the X/A ratio as descriptive of the ultimate effects of the genes in these chromosomes, he modi- fied their proposed weight from 1 : 1 for X:A to 1 for the X and 0.80 for the A. All formulations for explaining sexual differences are beset with a lack of an un- biased quantitative scale by which these differences can be measured. The estimates of the changes in sexuality are left to the insight of the observer. It seems reasonable to suppose that the quantitative relations between the male and female sex determin- ing elements should have intermediate values when the specimens under observa- tion show a mixture of organs of either sex. This agrees with Bridges' considerations of this problem. It is not so clear, however, that the so-called supersexes^ really are what the names may connote to many readers. The superfemales, with their three X chromosomes and two sets of autosomes, are quite inviable; small in size, wings re- duced and irregularly cut on the margins, ovaries developed to only the early pupal stage, and reproductive tracts much re- duced in size.^ The supermales with one X - Recently termed metafemales bj' Stern (1959b). '^ Further development of the ovaries is able to take place in normal XX + 2A hosts. Larval XXX + 2A ovaries transplanted into fes/fes hosts IM-oduce eggs in the recipient host which on fer- tilization are capable of developing into adult ima- goes. These imagoes show that there is a low per- chromosomc and three sets of autosomes are described as resembling males but are sterile. The wings arc somewhat spread and bristles less in size. They are late emerging and poorly viable. Neither type can be re- ferred to as superior to normal female or normal male in anatomic develoi)ment or physiologic functioning. A new type, re- cently described by Frost (1960j, empha- sizes this difficulty. Females, called triploid metafemales, Table 1.1 had 4X chromosomes and 3 second, 3 third, and 2 fourth auto- somes. Viability was greater than superfe- males but still low. Fertility was about 10 per cent. Progeny per female about 10. The flies were like triploids in bristles, eye and wing cells large, sex combs absent. They showed characteristics of superfemales in rough eyes, narrow wings without inner margins, and smaller body build. The dis- tribution of these different Drosophila sex types (Table 1.1) showed that optimal development comes when the X/A values are 1.0 and 0.5. Any deviation away from these values tends to make the sex system less rather than more efficient. In normal Drosophila sex differences are probably expressed in every cell making up their bodies. These differences are made visible to us only under special conditions. In adult organ differentiation, the sexual differences are manifest through such things as the body size of the males being about three-fourths that of the females, differences in coloration of the tergites, the appearance of sex combs, the development of the gonads into ovaries and testes, and the formation of a secondary reproductive system composed of several glands and ducts. The origin of these last elements is of particular interest. The ovaries can be distinguished from the testes as early as the second instar through their size and position within the fat body. They are lo- cated about two-thirds the larval length back from the mouth parts. The sex combs, on the other hand, take their origin from imaginal discs which are located in the head region, possibly one-third back from the mouth parts. The secondary reproduc- centage of crossing over and high nondisjunction rate in the XXX + 2A ovaries and a high mortality rate in the offspring (Beadle and Ephrussi, 1937). FOUNDATIONS FOR SEX 13 larger sex combs. Some 95 per cent of the variation in sex comb teeth has been ac- counted for by this equation. The above equation results when the ef- fect of the sex chromosomes and autosomes is considered as operating on a simple addi- tive basis. It is interesting to consider these effects on the basis of the ratio of sex chro- mosomes to autosomes as utilized by Bridges. As is customary, the male geno- type is given a weight of 0.50, the intersex 0.67, the female 1.00, the superfemale 1.50, and the triploid female 1.00. With these values the data on the sex combs are fitted by the equation Sex combs = 13.40 - 6.38 X/A The fit of this equation to these data shows control of less of the variation in the sex comb teeth. Only 76 per cent of the variation is accounted for by these methods whereas 95 per cent is accounted for when the effects of the X and A chromosomes are considered as additive. If it is agreed that the condition of the sex combs is a good unbiased measure of the degree of sexuality of the Drosophila, it follows that it would be more probable that the genes in the X chromosomes oper- ate additively with those of the autosomes. III. Sex Genes in Drosophila A. MUT.\XT TYPES Bridges' concept of sex determination turned on the action of sex genes located more or less fortuitously throughout the inheritance complex of the species. In Dro- sophila it happens that the major female determining genes seem to be located in the X chromosome and the male determining genes in the autosomes, whereas the Y chromosome seems essentially empty of sex genes. In support of this concept limited data are cited on specific genes affecting the reproductive system or its secondarily differentiated elements and two cases where genes affected the primary reproductive system as a whole. During the interim be- tween 1938 and the present, the numbers of these genes and the breadth of their known effects have been notably increased. Again the genes as a whole affect every phase of sexuality, morphology, fertility, and physiology. Single genes may occa- sionally alter both sexes or may frequently affect only male or only female phenotypes. Single genes may appear to influence two or more distinct characteristics observable in the developing flies, although this multi- ple phenotypic expression may go back to a gene action which is controlling a single event in development. Genes affecting the structural development of either male or female organs frequently are accompanied by sterility of various degrees. A very large category of genes is known only through its effects on sterility of either or both sexes. Experience has shown that when properly analyzed anatomic changes are probably basic to the sterility. In this sense genes for sterility should be considered genes for sex characters. Berg (1937) fur- nished data on the relative frequency of sterility mutations in the X chromosome as against those in the autosomes. 12.3 per cent mutations in which the males were sterile were found in the X chromosome against 4.5 per cent found in the second chromosome. These results show that the X chromosome has many gene loci occu- pied by genes capable of mutating to ste- rility genes which affect males. Sterility is also common for the females but requires more testing. The loci for these genes are widely distributed both within and among the chromosomes. Genes affecting sex morphology are found in all Drosophila chromosomes. Of 17 which have been recently studied; 6 were in the 1st chromosome, 5 in the 2nd, 5 in the 3rd, and 1 in the 4th. Insofar as can be determined these genes are no different than those affecting other morphologic traits. They may be dominant, they may be re- cessive, and a limited number of them may show partial dominance. They affect a vari- ety of sex characteristics and do not always involve sterility of one or the other sex. The loci occupied by these sex genes may have several alleles, some of which may lead to sterility, others not. Most affect characters like size and development of the ovaries, the characteristics of the eggs, duct development, spermathecae, ventral recep- tacles, parovaria, paragonia, sex combs, po- sition of genitalia, and so on. ^^ FOUNDATIONS FOR SEX 15 this gene in the second chromosome. Price ( 1949j placed the gene within the second chromosome near the locus of "brick" by inducing crossing over in males by expos- ing them to x-rays. Newby (1942) exten- sively studied the embryologic sequence in development of the organs of these inter- sexual types. The Ix^ gene did not affect the males, XY 4- 2A, but did change the fe- males XX + 2A into intersexes when it was in the heterozygous condition. The intersex- uals had 9 tergites; the first 6 were like those in the normal female and the last 3 were small and irregularly formed. There were 6 sternites, the first 5 being normal, but the 6th malformed. Anal valves were lateral as in the male but a third small valve was also present at the ventral side of the anus. The plates forming the claspers were of irregular pattern and found ventral to the anus. The vaginal plates were often extruded into a genital knob and were be- low the claspers. The knob occasionally be- came heavily pigmented. The internal or- gans ranged from nearly female through those which were of hermaphroditic type containing representative organs of both sexes to individuals almost wholly male. Newby concluded that intersexuality ex- presses itself as a response to the develop- mental pressures of both sexes, not as de- velopment in the one direction followed by a change. Gowen in 1940 established a stock carry- ing the dominant gene Hr which had ap- l)eared as a mutant in one of his cultures of D. melanogaster. This gene affected diploid females of XX + 2A type but not the males of XY -I- 2A constitution. In the presence of the Hr gene the diploid phenotype of the females changed into a sterile type with male secondary reproductive system associ- ated with the female counterpart. The first 6 segments were complete with 6 spiracles. The 7th was small with spiracle. The 8th was small but without spiracle. Sternite forming rudimentary ovipositor was usu- ally protruded. Ninth and 10th segments resembled those of males with large tergal plates. Claspers were abnormal and had a pair of small plates flanking the anus ma- joi-ly in vertical position. Organs formed, although sometimes modified or missing, included: sex combs of 6.9 long slender teeth, gonads distinguishable from those of the ordinary male or female in the 3rd and possibly the 2nd instar, genital ducts male and/or female, male accessory gland, penis deformed, sperm pump, vas deferens, sper- matheca, ventral receptacle often displaced, and occasionally parovaria. The primary gonads were often abnormal ovaries but in rarer instances bore a crude resemblance to testicular tissue. The yellow of the testes was frequently present as material clinging to the ovary. Superfemales with one dose of the Hr gene had sex combs and developed parts of both the male and female external and in- ternal reproductive systems. Sex combs had an average of 5 long and slender teeth. Ab- dominal segment 8 developed as in the fe- male, and formed the vaginal plate. The latter was abnormal in shape, ordinarily becoming a sclerotic protuberance. Seg- ments 9 and 10 developed more as in the male but were incomplete and abnormal. The genital arch did not develop but the inner lobe of tergite 9 showed irregular and abnormal growth as for the claspers. Seg- ment 10 developed, as in the males, into longitudinal plates flanking the anus. The internal genitalia were underdeveloped but consisted of mixtures of male and female organs. Gonads were rudimentary but gen- erally consisted of a pair of ovaries with small traces of yellow pigmentation. The triploid fly with one dose of the Hr gene was largely female with developed ducts, ovaries and eggs, but was sterile. The male characteristics were small sex combs and dark abdominal plates. In superfe- males, sex combs were present and teeth were intermediate between those of the dip- loid female and triploid female. The gene showed a dosage effect in triploids which was less than that observed in diploids and was in relation to the relative balance of the gene with its normal alleles, 1:2 for the triploid and 1 : 1 for the diploid. The developmental effects of Hr as well as the pigment producing potentialities of testes, ovaries and hermaphroditic gonads have been discussed by Fung and Gowen (1957a, b). Hr has been shown to be allelomorphic to a recessive gene, tra, described by Sturte- vant (1945) and known to be located in the 16 BIOLOGIC BASIS OF SEX 3rd chromosome. The location of this allele, tra, is at 44 to 45 or between the genes scar- let and clipped. When homozygous the gene transformed diploid females into sterile males. Heterozygotes showed no detectable differences from normal females of XX con- stitution. Males XY homozygous for tra or heterozygous for it were indistinguish- able from normal males. The homozygotes XX, tra/tra were female in body size, but otherwise were nearly male in appearance. They had fully developed sex combs, male colored abdomens, normal male abdominal tergites, anal plates, external genitalia, genital ducts, sperm pumps, paragonia, and showed the usual rotation of the genital and anal segments through 360 degrees. They mated with females readily and normally. The testes, however, although normal in color, elongated, curved, and attached to the ducts were of small size. Testis size was never that found in normal brothers. The addition of a single Y or two Y's did not alter fertility. The triploid females 3X + 3A homozy- gous for tra, had large bodies, ommatidia and wing cells. They resembled the diploid homozygous tra individuals in having male external genitalia, well developed ejacula- tory ducts, sperm pumps, and accessory glands, testes elongated but narrower than those of normal males, and sex combs aver- aging about 9.6 teeth. They mated with fe- males but were completely sterile. Triploids with one or two doses of tra were like wild type triploids in having no sex combs and being female throughout. In- tersexes having one or two doses of tra were similar to intersexes having only wild type genes in the locus. Sturtevant obtained one superfemale which was homozygous for tra. It had male genitalia and sex combs with only about half the normal number of teeth. This in- dividual argues for a greater balance to- ward the female side of sexual development than either the diploid or triploid females previously discussed. The evidence is, how- ever, contradictory to that furnished by the Hr gene as indicated earlier. A combination of two or more genes, Beaded and various Minutes, having well known phcnotypic effects, has sometimes produced phenotypes which have been in- terpreted as peculiar, low grade types of intersexuality in males (Goldschmidt, 1948, 1949 and 1951). The data showed that the Beaded cytoplasm favors the low grade intersexual male whereas the Minute cyto- plasm favors the reduced male with the hetcrozygote being intermediate. Just how far these types may be related to the other types strongly affected by specific genes is a matter of question, having at least other interpretations (Sturtevant, 1949). In 1950, Milani trapped an inseminated female of D. subobscura w^iich segregated intersexual progenies. Spurway and Hal- dane (1954) studied these intersexual types. A recessive guiding development to- ward these intersexes was located on the 5th chromosome of subobscura. When present it caused the XX homozygous females, ix/ix + 2A, to have sex combs on both the first and second tarsal joints. The numbers of teeth making up the sex combs w^ere re- duced as also were the sizes of the teeth. The illustration in Spurway and Haldane's (1954) paper indicates that the number of teeth was 7 on the first tarsal joint and 5 on the second joint, whereas the sex combs of the males had 11 teeth on the first joint and 9 on the second. A series of changes were observed in the genital plates which graded from those resembling true females to those approaching the male type. C. OTHER CHROMOSOME GROUP ASSOCIATIONS I DROSOPHILA AMERICANA I), aniericana has 4 chromosomes in the female genome and 5 chromosomes in the male genome. As compared with D. virilis the X chromosome is fused with the 4th chromosome and the 2nd chromosome is fused with the 3rd, the 5th and 6th chromo- somes are free in the female, whereas in the male genome the Y chromosome, 4th, 5th and 6th chromosomes are free and the 2nd and 3rd fused. Stalker (1942) has shown that the three female genomes are balanced and lead to triploid females as they do in D. melanogaster. D. aniericana triploids differ from their diploid sisters in having bigger ommatidia, larger wing cells and somewhat larger bodies. When these trip- loids are bred to diploid males they give FOUNDATIONS FOR SEX 17 rise to 6 chromosomally different types of offspring: diploid males, diploid females, triploid females, intersexes, females carry- ing a Y and a male limited 4th chromosome hut otherwise diploid, and tetraploid fe- males. All intersexes cytologically show a Y and a 4th chromosome present. Intersexes without the Y are presumed also w^ithout male limited 4th chromosomes and would be expected to be inviable or very weak. No supermales or superfemales, that w^ould correspond with those found in D. melano- gaster, were observed so are presumed to l)e inviable due to unbalance for the 4th chromosomes. Among 948 progeny of trip- loid females X diploid males there were 9 individuals that were phenotypically abnor- mal females. They had slightly spread, ven- trally curved wings with slightly enlarged wing cells. In 8 of the 9 the first section of the costal vein was shortened so that no junction was made with the first vein at the distal costal break. Heads were large with rough eyes, thoraxes shortened, legs fre- (luently malformed, and abdomens small with unusually wide 7th sternites. Genitalia were apparently normal with well devel- oped ovaries. This type carries three doses of any genes contained in the 4th chromo- some to two doses of the genes in the other chromosomes. Its phenotype represents a trisomic condition. The sex characteristics of the flies ob- served in D. americana seem to follow the same patterns as those of D. melanogaster as judged by the numbers of the X chromo- somes and autosomes. A Y chromosome in the intersex was not observed to affect sex expression. The intersexes could be grouped into six classes ranging from extreme male type to the most female type. The male type showed largely male organs, courted females, and had motile but nonfunctional sperm. The most female type had nearly normal ovipositor plates, well developed uterus, ventral receptacle, spermathecae and oviducts. At least one gonad showed egg strings, although a small patch of orange-red tissue was present at the tip. Two types of chromosomes were observed in the nuclei of these extreme female type intersexes; those like the chromosomes in any normal diploid cells and some which were so swollen as to be almost unrecog- nizable. Such swollen chromosomes were not found in the other classes of intersexes or in diploid or triploid individuals. They are suggestive of some noted by Metz (1959) in Sciara. Most of the intersexes were of the male type, 45 per cent, with de- creasing numbers for each of the other five classes until those in the most female class constituted only about 4 per cent of the total. D. LOCATION OF SEX-DETERMINING GENES The problems of isolating and determin- ing the modes of action of the factors nor- mally operating in sex determination have received extensive study since they were reviewed by Bridges in 1939: Patterson, Stone and Bedichek (19371, Patterson (1938), Burdette (1940), Pipkin (1940- 1942, 1947, 1959), Poulson (1940), Stone (1942), Dobzhansky and Holz (1943), Crow (1946), and Goldschmidt (1955). From his work on Lymantria dispar, Gold- schmidt concluded that sexual differentia- tion was controlled by a major male factor, M in the Z chromosome and a factor F directing development toward the female and at first assumed to be in the cytoplasm but later considered to be in the W chromo- some. The heterogametic female of this species would then be FM and the male be MM. In considering this problem, Gold- schmidt attempted to distinguish between the sex determiners responsible for the F/M balance and modifiers affecting special de- velopmental processes (Goldschmidt, 1955). At the other extreme Bridges' study of trip- loids led him to consider that sex in Droso- phila was determined by the interaction of a number of female tendency genes found largely in the X chromosome and of genes having male bias located largely in the au- tosomes. These numerous genes were con- sidered as being distributed throughout the whole inheritance complex. Search for the more exact locations of these genes within the different chromosomes of Drosophila has largely taken the form of determining the variation in sex types as induced by the addition or deletion of various pieces of the different chromosomes to either the normal male, normal female, or triploid complexes. 18 BIOLOGIC BASIS OF SEX The sections of the chromosomes added were derived from previous translocations generally to the 4th chromosome and were of varying lengths determined through cy- tologic and genetic study. Summaries of these comparisons are found particularly in the papers of Pipkin (1940, 1947, 1959). In their search for a major female sex factor in the X chromosome, Patterson, Stone and Bedichek (1937), Patterson (1938), Pipkin (1940), and Crow (1946) finally were un- able to show that any single female sex de- terminer, located in the sex chromosome, was of primary importance to sex. Evidence for multiplicity of genes with a bias toward female determination was found by Dob- zhansky and Schultz (1934) and by Pipkin (1940). They were able to transform dip- loid intersexes into weakly functioning hy- potriploid females by the addition of long fragments of the X chromosome to the 2X + 3A intersex complement. Short sec- tions of the X chromosome in some cases shifted the sex type in the female direction. Pipkin found that additions of short X chromosome sections, to the 2X -|- 3A chro- mosome sets, although covering in succes- sion the entire X chromosome, were insuffi- cient to make the flies other than of the intersexual type. Longer and longer frag- ments from either the left or right end of the X chromosome caused a qualitatively progressive shift toward femaleness. Weakly functional hypotriploid females re- sulted when either right- or left-hand sec- tions of two translocations with t-lz (17) and Iz-v (W13) breaks were present in the 2X + 3A chromosome complement. These duplication intersexes possessed 1 or 2 sex comb teeth when reared at 22 °C. and up to 5 well developed teeth when reared at 18°C. These facts give support to the multiple sex gene theory of Bridges or at least that quantitative differences in sex potencies exist within the X chromosome. This con- clusion was further strengthened by the hy- pointersexes lacking a short portion of one of their two X chromosomes although pos- sessing three of each autosome, inasmuch as these types were shifted strongly in the male direction. These studies of the X cliro- mosonie show that several parts of this chi-omosomc are concerned witli female dif- ferentiation and that the effects are irregu- larly additive. Similar search of the autosome II and III for genes of male potency showed that small shifts in the male direction were found in hyperintersexes for several short regions of chromosome III but for none of chromosome II (Pipkin, 1959, 1960). Tl^ree slightly different right-hand end regions of chromosome III produced the largest shifts in the male direction in hyperintersexes, but no increase in number of sex comb teeth. These changes were comparable with those produced in the female direction by the addition of very short sections of the X-chromosome to the 2X + 3A intersex complement. On the other hand, none of the seven different hypointersexes lacking a short section of the 3rd chromosome from the 2X + 3A complement showed a shift in the female direction. This is rather surpris- ing as hypointersexes for two short regions in the X chromosome were shown by Pip- kin (1940) to shift the sex type in the male direction as was to be expected. From these results Pipkin (1959) derives the conclu- sion that 3rd chromosome aneuploids as well as those of the 2nd chromosome and X chromosome support the deduction that dosage changes of portions of the X chro- mosome are more powerful than dosage changes of portions of either of the large autosomes in affecting sex balance. This view receives further support through changes of size and number of sex comb teeth as observed in the chromosomal types carrying the gene Hr and reviewed earlier. Influence of the Y chromosome on sexual differentiation has generally been ruled out as XO nondisjunctional flies are male al- though they are sterile (Bridges. 1922). Similarly Dobzhansky and Schultz (1934) ruled out the Y chromosome as an effective influence on sex types of triploid intersexes of D. melanogaster since the mean sex type of Y -(- 2X 4- 3A intersexes did not differ significantly from the sex tyyies of siblings 2X + 3A. " The steps taken in the studies of chro- mosome IV are of interest. Dobzhansky and Bridges in 1928 concluded that the 4th chromosomes i^lay no part in sex determina- tion in D. melanogaster. The evidence was FOUNDATIONS FOR SEX 19 of two kinds. Triploid females were out- crossed separately to males of two diploid stocks. The triploid daughters from these crosses were again crossed to males like their fathers. This repeated outcrossing to the different stocks resulted in a shift in the grade of the intersexes in both cases, in one case to a very high proportion of ex- treme male-like intersexes and in the other to nearly as high a proportion of extreme female-type intersexes. These results were interpreted as showing that the grade of development of the sexual characters was dependent on genetic modifiers. The second experimental test consisted of subjecting a triploid stock to selection toward a line which produced a high proportion of ex- treme male type intersexes and to another line which would have a high proportion of extreme female type intersexes, each being much higher than the original stock. The l)rocedure established a line with a high proportion of extreme male type and an- other line which was not so extreme in its proportions but was definitely higher in fe- male type intersexes than the original stock. Again these results w'ere interpreted as in- dicating the selection of modifying genes of unknown positions within the inheritance complexes. This evidence had been preceded by Bridges' (1921) discussion in which he wrote "the fourth-chromosome seems to have a disproportionately large share of the total male-producing genes; for there are indications that triplo-fourth intersexes are predominately of the 'male-type', while the dijjlo-fourth intersexes are mainly 'female- type'." In 1932, Bridges concluded for Dro- sophila intersexes that, in spite of the fa- vorable genetic checks, in repeated and varied tests, it has been impossible to state with any assurance whether the 4th chro- mosome is or is not a large factor in the variability encountered. In our own work a stock of attached X triploids has for many years consistently produced only male type intersexes. This is in contrast to what we frequently see within other lines of triploids as made up utilizing the cIIIG gene (Gowen and Gowen, 1922). Lines established from these triploids or- dinarily have three intersexual types: male, intermediate, and female. These lines, how- ever, may be subjected to selection in both directions. In our experience, male intersex lines are established rapidly and remain relatively permanent. On the other hand, female intersex lines take many more gen- erations and are less stable. These lines have been extensively examined for their 4th chromosome constitutions (Fung and Gowen, 1960). The male intersex lines sel- dom show more than two 4th chromosomes. On the other hand, the female intersex lines rarely show two 4th chromosomes but gen- erally have more than three, the number sometimes going as high as four. More tests are needed but the evidence would seem to indicate that the fourth chromosome does have sex genes. These genes, contrary to the first notion of Bridges, are more frequently of the female determining type than of the male determining type. This would make the 4th chromosome like the X in that it carries an excess of female influencing genes and is not like the rest of the autosomes which have an excess of male determining genes. These observations are of particular interest in view of Krivshenko's (1959) pa- per. In this investigation on D. busckii, cy- tologic and genetic evidence was presented for the homology of a short euchromatic element of the X and Y chromosome with each other and also with the 4th chromo- some or microchromosome of D. melano- gaster. This conclusion is based on (1) ob- served somatic pairing of the X and Y of D. busckii by their proximal ends in gan- glion cells and the conjugation of the short euchromatic elements of these chromosomes at their centromeric regions in the salivary gland cells; (2) the presence in the short Y chromosomal element of normal allelo- morphs to four different mutant genes of the short X chromosomal element; (3) the presence in the short element of the D. busckii X chromosome of chromosome IV mutants: Cubitus interruptus. Cell and shaven of D. melanogaster. These consider- ations furnish proof for the homology of this X chromosomal element with the 4th chromosome of Drosophila. These observations of Krivshenko sup- port our findings that the 4th chromosome of D. melanogaster has an excess of female 20 BIOLOGIC BASIS OF SEX determining functions. It would further show that autosomes may behave differ- ently with regard to their sex-determining properties according to the chance distribu- tion of sex genes which happen to fall within them as they do in Rumex (Yama- moto, 1938j. The finding that the chromo- some IV has a bias toward female tenden- cies further strengthens Bridges' multiple sex gene theory and weakens the theory of an all-or-none action of the whole X chro- IV. Sex under Special Conditions A. SPECIES HYBRIDITY Hybrid progeny coming from species crosses are apt to represent but a very few of the possible genotypes of the total num- ber that conceivably could come from the gene pool. The hybrid phenotypes may dis- play three kinds of characteristics. The common set is that derived from genes in either or both parents through ordinary meiotic segregations and dominance. The second set shows intermediate develop- ment of the characters found in the two parent species. The third set of characters that complete the animal is new to those observed in either parent species. These new characters may be the loss of a few dorso- central and scutellar bristles, broken or missing cross veins, or abnormal bands in the abdomen as in D. simulans x D. melano- gaster hybrids (Sturtevant, 1920b), extra antigenic substances as found in dove hy- brids (Irwin and Cole, 1936), or more nu- merous characteristics as in the mule. Fre- quent among these new characteristics is sterility. The sterility may extend to either or both sexes and affect the secondary sex ratios. As Sturtevant (1920b) points out, crosses between the domestic cow and male bison give male offspring with humps de- rived from the bison which are so large as to prevent their being born alive. The fe- male hybrids lack these humps and are con- se(iuently born normally. The abnormal sex ratio observed at time of birth is due to causes external to the hybrid itself and at- tributable to the structure of the mothers. A comparable case was found during the study of female sterility in interspecies hy- brids of Drosophila pseudoobscura in which Mampell (1941) showed that in the hybrids of certain strains, the females produced no or few offspring because of interspecies le- thal genes connected with a maternal effect. Comparable cases as well as those depend- ent on other mechanisms are known for other groups. The progeny may also be al- tered to give new sex types, generally inter- sexes. These intersexes often replace either the male or the female sex group. However, despite their apparent relation, the changes in the sex ratio and the appearance of in- tersexes can have different causes. D. sim- vlans X D. melanogaster hybrids emphasize that there may be no relation between the peculiar hybrid sex ratios and the intersexes since extreme differences in sex ratio occur but no intersexual types. Species, however, may have natural dif- ferences in the sex potencies of their X chromosomes and/or their autosomes. In crosses between D. repleta and D. neo- repleta involving a sex-linked recessive white-eyed mutant type of D. repleta Stur- tevant (1946) obtained about 15 per cent fertile matings in 500 mass cultures, a total of 532 females to 635 males. All progeny as expected were wild type in character. The males, however, had long narrow testes and were totally sterile, a condition later shown to be due to a gene in the X chromosome located near the white locus. Females sug- gested intersexuality in having three anal plates instead of the usual two. Mating of Fi hybrid females to white D. repleta males gave 9 per cent fertility, the 179 offspring being distributed as 70 wild type females, 9 white females, 42 wild type males, and 58 white males, although the expectation for the classes was equality. Evidence indicates that some of the 9 white females were in- tersexes as were possibly some of the white males. The wild-type males again had the long narrow testes and sterility of the Fi male progeny. Wild type females were mod- erately fertile. By continued backcrossing to 1). repleta males having white or white- singed, a female line was picked up which continued to have the unusual sex ratios but had more fertility. It was presumed that the D. neorepleta gene responsible for the unusual ratios was originally associated with MHotlier gene that decreased fertility FOUNDATIONS FOR SEX 21 in females largely of D. repleta constitution and that the foundation female for the more fertile line came as a result of a cross- over between an infertility gene and that responsible for the unusual sex ratios. Con- tinued back crosses of females of this line to white D. repleta males have been made. Out of 33 fertile cultures, 16 gave approxi- mately equal ratios of wild-type and white females, wild-type and white males; and 17 gave 472 wild-type females, 5 white fe- males, 63 white intersexes, 482 wild-type males, and 339 white males. The white fe- males presumably represented crossovers between the loci of white and the critical gene in the X derived from D. neorepleta. The intersexes were of extreme type with gonads very small (rudimentary ovaries in those cases where they were found at all). External genitalia were missing or of ab- normal male type. Other somatic character- istics included weakness which prevents emergence and accounts for the loss of about 88 per cent of the flies expected in that class. The intersexual condition was suggested as being caused by an autosomal dominant gene derived from D. neorepleta which so conditions the eggs before meiosis that two D. repleta X chromosomes result in the development of intersexes rather than females. The action of this gene occurs before meiosis and may in fact be absent from the intersexes themselves. This was confirmed by crosses of white brothers of the intersexes to pure D. repleta females when the offspring were normal for both sexes; but when these Fi daughters were mated to D. repleta males only intersexes and males resulted. This last cross further showed that although this gene was derived from D. neorepleta in D. neorepleta cyto- plasm, the D. neorepleta cytoplasm was not necessary for the intersexes to result. The case also has an important bearing on the location of the sex-determining factors, for in this cross the characteristics were only secondarily governed by the cytoplasm through earlier determination by genes of the mothers' nuclei. Significant parallels are found between the autosomal gene of D. neorepleta and the third chromosome Ne gene of D. mela- nogaster (Gowen and Nelson, 1942) de- scribed in the section on high male sex ratio. The D. neorepleta gene caused the cytoplasms of the eggs laid by mothers carrying it to become more male potent. The female potencies of two X chromosome D. repleta zygotes were unable to balance these male elements. Many died late in de- velopment. Those able to emerge became intersexes. The Ne gene also sensitizes the cytoplasms of all eggs of mothers carrying it causing any 2X + 2A, 3X + 3A, 3X + 2A or 2X -h 3A intersexes of female type to die in the eggs at 10 to 15 hours whereas males XY + 2A and male-type intersexes live. Other mechanisms for causing sex and sex ratio changes are known, i.e., Cole and Hollander (1950), but few are as well worked out as that of D. repleta x D. neo- repleta. New mechanisms will certainly be found for the opportunities for genetic anal- ysis of sex in hybrids are many. B. MOSAICS FOR SEX Recent genetic work has emphasized the fact that individual D. melanogaster may be composed of cells of more than one genie or chromosome constitution. The main type of sex mosaic is the gynandromorph com- posed of cells of female constitution on one side, XX + 2A, and male, X + 2A on the other, the loss of the X chromosome coming at an early cleavage (Morgan and Bridges, 1919; L. V. Morgan, 1929; Bridges, 1939). The mosaic areas are large since the cells of each type may be in nearly equal numbers. At the other extreme Stern (1936) has shown that phenotypic mosaics may de- velop as a consequence of the somatic chro- mosome pairs crossing over at late stages in embryologic development. Special genes, Minutes, materially increase the frequen- cies of these crossovers. The proportion of the body occupied by the cross-over type cells is small because crossing over takes place so late in development. Recently another agent in the form of a ring chromosome has been discovered which greatly increases the production of sex mo- saics. Some ring chromosomes are relatively stable whereas others are quite unstable, the instability depending to some extent on aging of the eggs and environmental factors (Hannah, 1955). The instability is manifest 22 BIOLOGIC BASIS OF SEX by frequent gynandromorphs, XO males, and dominant lethals among the rod and ring zygotes. It has been suggested that the instability is due to heterochromatic ele- ments. Hinton (1959) has observed the chromosome behavior of these types in Feulgen mounts of whole eggs that were in cleavages 3 to 8. He found strikingly ab- normal chromosome behavior in these cleaving nuclei. For some cell divisions chromosome reproduction was interpreted as being through chromatid-type breakage fusion bridge cycles. As a result of this be- havior mosaics are formed which are inter- mediate between those of the half gynan- dromorphs and those which occur much later because of somatic crossing over. In terms of volume of cells included, the ab- normal types may include only a few cells of the total organism, a fair proportion of the cells, or a full half of the whole body. These unstable ring chromosome mosaics may be a part of the secondary reproduc- tive system or for that matter any other region of the body. When the mosaic cells are incorporated in the region of sex organ differentiation male or female type organs or parts of organs may develop as governed by the cell nuclei being X, XX or some frac- tion thereof. Gynandromor|)lis appear sporadically and rarely in many species but in some in- stances genes which activate mechanisms for their formation are known. In the pres- ence of recessive homozygous claret in the eggs of D. siniulans, gynandromorphs con- stitute a noticeable percentage of the emerging adults. The gene nearly always operates on the X received from the mother causing it to be eliminated from the cell. The resulting gynandromorphs are similar to those of D. melanogaster. The fact that the claret gene should affect the X and a particular X chromosome is suggestive of the manner in which given chromosomes arc eliminated in Sciara. Other types of sex mosaics will be found in the descriptions of other species, i)articularly in the Hyme- noptci'a. C. PARTHENO(iENESIS IN DROSOPHILA Parthenogenesis is of interest as it changes the sex ratios in families and brings to light new sex types and novel methods for their development (Stalker, 1954), A survey of 28 species of Drosophila showed a low rate of parthenogenesis in 23 species. Adult progeny were obtained for only 3 species. For D. 'parthenogenetica the origi- nal rate was 8 in 10,000 whereas that for D. polymorpha was 1 in 19,000. These rates could be increased by selection of higher rate parents: 151 and 70 per 10,000 unfer- tilized eggs of the first and second species respectively. D. parthenogenetica diploid virgins pro- duced diploid and triploid daughters as well as rare XO sterile diploid males. Triploid virgins produced diploid and triploid fe- males and large numbers, 40 per cent, of sterile XO diploid males. Diploid virgins heterozygous for sex-linked recessive garnet produced homozygous and heterozygous diploid females as well as +/+/g and + /'g/g triploid females. No homozygous wild-type or homozygous garnet triploid fe- males or garnet mosaics were found. Dip- loid females crossed to fertile diploid males produced few if any polyploid progeny or jjrimary X chromosome exceptional types. Of the unfertilized eggs from diploid virgins which started development, 80 per cent died in late embryonic or early larval stages. The i)arthenogenesis in diploid females de- pended on two normal meiotic divisions fol- lowed by fusion of two of the derived hap- loid nuclei to form diploid progeny, or the fusion of three such nuclei to form triploitl progeny. In the triploid virgins similar fu- sions of the maturation nuclei may produce diploid and triploid females but the large number of dii)loid XO sterile males were picsunicd to be the result of cleavage with- out prior nuclear fusion. Such cleavages without fusion in eggs of dijiloid virgins would lead to the production of haploid embryos. They were presumed i-esponsible for the large early larval and embryonic single sex locus. In principle at least, the honey bee could have the Habrobracon scheme of sex de- termination. Rothenbuhler (1958) has re- cently collected the researches which test this possibility. Tests of the multiple allele hypothesis as applied to the honey bee were made by Mackensen (1951, 1955) who in- terpreted evidence for inviable progeny pro- duced by mating of closely related individ- uals as proof that this species as well as Habrobracon juglandis follows the multiple allelic system. The discovery of male tissue of bii)arental origin in mosaic bees from re- lated i)arents was considered as further evi- dence for the multiple allelic theory of sex determination (Rothenbuhler, 1957). Most recent cytologic evidence supports the concept that there are 16 chromosomes in the gonadal cells of the male and 32 in those of the female (Sanderson and Hall, 1948, 1951; Ris and Kerr, 1952; Hachinohe and Onishi, 1952; Wolf, 1960». Hachinohe and Onishi (1952) found 16 chromosomes as characteristic of the meiosis in the drone. Wolf observed a nucleus in both bud and spermatocyte of the only maturation (equa- tional) division. The greatest progress has been made in understanding the mechanisms of sex mosai- cism in the Hymenoptera species. These mosaics, although ordinarily of rather rare occurrence, have a direct bearing on sex determination and development. In Apis, iiolyspermy furnishes the customary basis for their formation. One sperm fertilizes the haploid egg nucleus and another sperm, which has entered the egg instead of degen- erating as it ordinarily does, enters into mitotic cleavage and eventually forms is- lands of haploid cells of paternal origin among the diploid cells derived from the fertilized egg (Rothenbuhler, Gowen and Park, 1952). Evidence showing that genetic influences affect the sperm nucleus toward stimulating its independent cleavage is found to exist in Apis material (Rothen- buhler, 1955, 1958). Tliousands of gynan- dromorphs have been observed in Apis, all but a small number of which have been pro- duced in this manner. This method of initi- ating sex mosaics also exists for Habrobra- con (Whiting, 1943b) but is rather rare. In Habrobracon the frequent mode has a different origin. The gynandromorph is formed from the cleavage products of the normal fertilization of the egg nucleus com- bined with those of a remaining nuclear product of oogonial meiosis. Under these conditions the female tissue is 2N of bi- parental origin and the male tissue is N of maternal origin (Whiting, 1935, 1943b). This type is less frequent in Apis but one specimen has been described by Mackensen (1951). A number of other ways in which sex mosaics may occur are occasionally ex- pressed in these species. Three different kinds of male tissue have been observed in individual honey bee mosaics produced by doubly mated queens — haploid male tissue from one father, haploid male tissue from the other genetically different father, and diploid male tissue of maternal-paternal origin. In other cases, the diploid, biparental tissue was female and associated with two kinds of male tissue (Rothenbuhler, 1957, 1958) . Cases where the haploid portions of the sex mosaics are of two different origins, one paternal and the other maternal, while the female portion is representative of the fertilized egg, are known in Habrobracon (Whiting, 1943b). Similarly, Taber (1955) observed females which were mosaics for two genetically different tissues and which he accounted for as the result of binucleate eggs fertilized by two sperm. Mosaic drones of yet another type were observed by Tucker (1958) as progeny of unmated queens. They were interpreted as the cleav- age products from two of the separate nu- clei formed in meiosis. These cases represent a number of the possible types that arise through meiotic or cleavage disfunctions under particular environmental or heredi- tary conditions. Tucker (1958) studied the method by which impaternate workers were formed from the eggs of unmated queens. For this purpose he used genetic markers, red, char- treuse, ivory, and cordovan. Observations 34 BIOLOGIC BASIS OF SEX Avore nuult' i)ii 237 workers from hotcrozy- gous mothers. For the chartreuse loeus. 12 to 20 per cent were homozygous, for x\w i\ory locus 1.8 per cent, and (he cordovan locus on lesser numbers 0 per t-ent. An egg which is destined to become an automictic worker, a gynandromorph with somatic male tissue or a mosaic male, is retained within the queen for an unusually long time during which meiosis is suspended in anaphase 1. Normal reorientation of first division spindle is i)ossibly inhibited by this aging so that after the egg is laid meiosis II occurs with two second division spindles on sejia- rate axes as Goldschmidt conjectured for rarely fertile rudimentary Drosophila (19")7i. Two polar l)odies and two egg prontich^i are formed. The polar bodies take no fiu'ther part in develoinnent. In most of the unusual eggs the two egg pronuclei unite to form a diploid cleavage nucleus which develops into a female. Rarely the two egg pronuclei develop separately as two haploid cleavage nuclei to form a mosaic male. Two unlike haploid cleavage nuclei, one descending from each of the two sec- ondary oocytes after at least one cleavage di\ision. unite to form a dijiloid cleavage nucleus which develops together with the remaintler of the haploid cleavage nuclei to produce a gynandromorph with mosaic male tissues. The male and female tissues within these unusual gynandromor[ihs or female types were identical with normal drone or normal female tissues so were probably haploid and diploid resiiectively. Genetic segregation observed within the mothers of automictic workers allows the estimation of the distance between the locus of the gene and its centromere. "With random recombination and "central union" Tucker estimates this distance for the chartreuse locus to centromere as 28.8 units and for the ivory to its centromere 3.6 units. Four lines of bees of diverse origin all showed a low percentage of automictic or gynandro- morphic types produced from queens in each line. Various chance environmental condi- tions apparently influence the rate of pro- duction of these types. However, there were some females in two of the lines with higher frequencies inciicating that innate factors may have significant eft'ects on their fre- quencies. Observations on Drosophila spe- cies— I), porthenogeuetica (Stalker, 1956b), I). ))ia)njabeirai (INIurdy and Carson, 1959) and D. nielanogaster (Goldschmidt, 1957) — strongly suppt)i-t this A-iew. The [irohleni of si'x determination in Hal)- rol)i'acon presently stands as a function of multii)le alleles in one locus, the heterozy- gotes being female and the azygotes and homozygotes male. The occasionally diploid males are regularly produced from fertilized eggs in two allele crosses after inbreeding. These diploid males are of low viability and are nearly sterile. Their few daughters are triploids, their sperm being dijiloid. Apis probably follows the same scheme, as a few cases of mosaics with diploid male tis- sue are known and close inbreeding results in a sufficient number of deaths in the egg to account for diploid males which might be formed. ^lormoniella (Whiting, 1958), how- ever, shows that this scheme for sex determi- nation does not hold for all Hymenoptera. In this form diploid males may occur through some form of mutation. They may then develop from unfertilized eggs laid by triploid females. In contrast to Habrobra- con the dijiloid males are highly viable and fertile. Their sperm are diploid and their numerous daughters triploid. Virgin triploid females produce 6 kinds of males, 3 haploid and 3 dij^loid. Similarly ]\Ielittobia has still a different and as yet unexplained form of sex determination. Haploid eggs develop into males. After mating many eggs are laid which develop into nearly 97+ per cent fe- males. The method of reproduction is close inbreeding but no dijiloid males or "bad" eggs are formed. The prol)lem of the sex determining mechanism remains open (Schmieder and Whitiuii, 1947). The silkworm, Bomby.r mori, differs from Drosophila, Lymantria and the species thus far discussed in having a single region in the ^^' chromosome (Hasimoto, 1933; Tazima, 1941, 1952) occupied by a factor or factors of high female potency. The strong female potency has thus far been connnon to all races. The chromosome patterns of the sexes are like those of Abraxas and Lyman- tria: males ZZ + 2A and females ZwV 2A. The diploid chromosome number is 56 in both sexes. Extensive, well executed studies FOUNDATIONS FOR SEX 35 liave revealed no W chruniosome loci for genes expressed as morphologic traits. From radiation-treated material it has been pos- sible to pick up a translocation of chromo- some II to the W chromosome as well as a cross-over from chromosome Z. This chro- mosome together with tests of hypoploids and hyperploids have materially aided in understanding how the normal chromosome complexes determine sex. The sex types re- sulting from different chromosome arrange- ments have been summarized by Yokoyama 1 1959) and are presented in Table 1.3. Whenever the W chromosome was ab- sent a male resulted. Extra Z or A chromo- somes did not influence the result. Similarly with a W chromosome in the fertilized egg a female developed. Again extra Z or A chromosomes did not influence the result. A full Z chromosome was essential to sur- vival. Hypoploids deficient for different amounts of the Z chromosome in the pres- ence of a normal W chromosome all died without regard to the portion deleted. Hy- perploids for the Z chromosome, on the other hand, when accompanied with a W chromo- some all lived and showed no abnormal sex- ual cliaracteristics. Parthenogenesis led to the pioduction of both sexes, although the males were more numerous than the fe- males. Diploidy was necessary for the em- l)ryo to go beyond the blastoderm stage. Triploid and tetraploid cells were often found. High temperature treatments led to merogony (Hasimoto, 1929, 1934). The ex- ceptional males were homozygous for a sex- linked recessive gene and were explained by assuming that the egg nuclei were in- activated by the high temperature and the exceptional males developed from the union of two sperm nuclei. This conclusion was supported by cytologically observed l)olyspermy (Kawaguchi, 1928) and by cytologic observation of the union of two sperm nuclei by Sato ( 1942 ) . Binucleate eggs were also believed to occur, which when fertilized by different sperm may each con- struct half of the future body. This type of mosaicism was influenced by heredity iGoldschmidt and Katsuki, i927, 1928, 1931 ). Polar body fertilization was also be- lieved to occur, one side of the embryo orig- inating from the ordinary fertilized egg nu- cleus and the other side from the union of TABLE 1.:^ Sex in Bombyx iitori (Summarized by T. Yokoyama, 1959.) Sex Chromosome Types and Numbers W z A Male W II.W.ZL w w WW WW zz zz zzz z z zz zzz zz zz AA Male Male Female Female Female Female Female Female AAA AAA AA AA AAA AAAA AAA AAAA nuclei of two of the polar bodies. Similarly, dispermic merogony was noted following the formation of one part of the body from the fertilization nucleus, the other part from the union of two sperm nuclei, the result being a gynandromorph or mosaic. VI. Sex Determination in Dioecious Plants \. MELAXDRUM t LYCHNIS] Over the last 20 years studies on several species of dioecious plants have made not- able advances in unclerstanding the mecha- nisms by which sex is determined. Melandrium album has been shown to have the same chromosome arrangement as Drosophila. The male has an X and Y plus 22 autosomes, whereas the female has XX plus 22 autosomes. Sex-linked inheritance is known for genes borne in the X chromo- somes as well as for genes born in the Y chromosome. The X and Y chromosomes are larger than any of the autosomes with the Y chromosome about 1.6 times that of the X in the materials studied by Warmke ( 1946) . Separate male and female plants are characteristic of the species. By use of colchicine and other methods, Warmke and Blakeslee (1939), Warmke (1946), and Westergaard (1940) have made various l^olyploid types from which they could de- rive other new X, Y and A chromosome combinations from which information was obtained on the location of the sex deter- mining elements. The Y chromosome carries the male determining elements, the X chro- 36 BIOLOGIC BASIS OF SEX mosome the female determining elements. The guiding force of the elements in the Y chromosome during development is suffi- cient to override the female tendencies of several X chromosomes. Data derived by each of these investigators are shown in Table 1.4. From these data Warmke (1946) con- cluded that the balances between the X and the Y chromosomes essentially determined sex with the autosomes of relatively little importance. Where no Y chromosomes were present but the numbers of X chromosomes ranged from 1 to 5, only females were ob- served, even though the autosomes varied in number from two to four sets. When a Y chromosome was present the individual was of the male type unless the Y was balanced by at least 3 X chromosomes when an occa- sional hermaphroditic blossom was formed. TABLE L4 Numhers of X, Y, chromosomes and A, autosome sets and the sex of the various Melandrium plants (Data from H. E. Warmke, 1946; and M. Westergaard, 1953.) Ratio Chromosome Warmlce Westergaard Constitution X/A 4A 5X Female 1.3 4A 4X Female 1.0 Female 4A 3X Female 0.8 4A 2X Female 0.5 3A 3X Female 1.0 Female 3A 2X Female 0.7 Female 2A 3X Female 1.5 2A 2X Female 1.0 Female Bisexual* X/Y 4A 4X Y 4.0 Male 4A 3X Y Malet 3.0 Male 4A 2X Y Malet 2.0 Male 4A X Y Male 1.0 3A 3X Y Malet 3.0 3A 2X Y Malet 2.0 Male 3A X Y Male 1.0 2A 2X Y Malet 2.0 2A X Y Male 1.0 Male 4A 4X YY Malet 2.0 4A 3X YY Male 1.5 4A 2X YY Male 1.0 Male 2A X YY Male 0.5 * Occasional staminate but never carpellate blossom. t Occasional licrina])hr(i(lit ic blossom. When 4 X chromosomes were present to- gether with a Y, the plants were hermaphro- ditic but occasionally had a male blossom. Two Y chromosomes almost doubled the male effect. Two Y chromosomes balanced 4 X chromosomes to give a majority of male plants. Only an occasional plant showed an hermaphroditic blossom. Autosomal sex effects, if present, were only observed when plants had 4 sets and 3 or 4 X chromosomes balanced by a Y chromosome. Warmke used the ratio of the numbers of X to Y chromo- somes as a scale against which to measure clianges from complete male to hermaphro- ditic types. No mention is made of quanti- tative measures of the sex character changes with increasing X chromosome dosages. This is of interest since in many forms changes in chromosome balance are accom- panied by changes of phenotype which are unrelated to sex. That such phenotypic changes do accompany changes in autosomal balance in Melandrium are proven, how- ever, by further observations of Warmke in 4 trisomic types coming from crosses of triploids by diploids. Of 36 such trisomies analyzed, 5 or 6 of them were of different growth habits and morphologic types. These differences did not affect the sex patterns since all were females. Warmke and Blakes- lee in 1940 observed an almost complete array of chromosome types from 25 to 48 in progeny derived from crosses of 3N x 3N, 4N X 3N, and 3N x 4N. Out of about 200 plants studied, only 4 were found to show indications of hermaphroditism. These types were 2XY and 3XY. As noted from the table, even the euploid plants would occa- sionally be expected to have an hermaphro- ditic blossom. Of the 200 plants, all with a Y (XY, 2XY, 3XY) were males and all plants without the Y (2X, 3X, 4X) were females. In an 8-year period up to 1946. Warmke was able to observe only one male trisomic. From these facts he concluded that the autosomes are unimportant in the sex determining mechanism utilized by this species. In their crosses they were unsuc- cessful in getting a 5XY plant, the point at which the female factor influence of the X chromosomes might be expected to nearly equal or slightly surpass that of the single Y. From the j^hysiologic side the obscrva- FOUNDATIONS FOR SEX 37 tion of Strassburger in 1900, as quoted by both Warmke and Westergaard, that the fungus Ustilago violacea when it infects Melandrium will cause diseased plants to produce mature blossoms with well devel- oped stamens (filled with fungus spores) as well as fertile pistils, shows that these fe- males have the potentialities of both male and female development. The case suggests that sex hormone-like substances may be produced by the fungus which acting on the developing Melandrium sex structures cause sex reversions. Should this be true, Melandrium cells would have a parallel with those of fish where sex hormones incor- |)orated in the developing organism in suf- ficient quantities can cause the soma to develop a phenotype opposite to that ex- pected of their chromosomal type. For other aspects see Burn's chapter and Young's chapter on hormones. Westergaard's studies (1940) with Euro- pean strains of Melandrium were in prog- ress at the same time as those of Warmke and Blakeslee. In their broad aspects both sets of data are concordant in showing the l^rimary role of elements found within the Y chromosome in determining the male sex and of elements in the X chromosomes for the female sex. Examination of Table 1.4 shows that the strain used by Westergaard has a Y chromosome containing elements of greater male sex potentialities than the strain used by Warmke. A similar difference appeared in the sex potencies of the autosomes of European strains. Instead of obtaining essentially only male and female plants in crosses in- volving aneuploid types, Westergaard ob- tained from 3N females (3A + 3X) x 3N males (3A + 2XY) 10 plants which were more or less hermaphroditic, 21 females, and 15 males. Studies of the offspring of these hermaphrodites through several generations showed that their sex expression required effects by both the X chromosomes and cer- tain autosome combinations which under special conditions counterbalanced the fe- male suppressor in the Y chromosome. In- creasing the X chromosomes from 1 to 4 increased the hermaphrodites from 0 to 100 per cent in the presence of a Y chromosome. However, in euploids these types would be all males. The significance of the autosomes is further shown by the fact that among 205 aneuploid 3XY plants, 72 were males and 133 were hermaphrodites. As pointed out for Drosophila, quanti- tative studies on the effects of sex chromo- somes and autosomes in Melandrium are handicapped by not having a suitable scale for the evaluation of the different sex types. The data presented by Westergaard and by Warmke make this difficulty become partic- ularly evident. In the interest of quantizing the X, Y, and A chromosome on sex the author has assigned a value of 1 for the male type, 3 for the female type, and 2 when the types are said to be hermaphro- ditic. When the types are mixed, as for ex- ample, in the data of Warmke where he says a particular type is male with a few blos- soms, the type is assigned a value of 1.05 or 1.10, depending on the numbers of these blossoms. His bisexual type which comes as a consequence of Y, 4X and 4A chromo- some arrangement is given a value of 2, al- though possibly the value should be some- what higher as it may well be that the fully bisexuals are further along in the scale to- ward female development than the hermaph- roditic types. The data are treated on the additive scale both as between chromosomal types and within chromosomal type. This is apparently unfair if we examine the work of Westergaard in which it looks as if par- ticular autosomes rather than autosomes in general make a contribution to sex determi- nation. The results, when these methods are used, are as follows: Westergaard in Tables 1 to 5 of his 1948 paper gives information on sex types with a determination of the numbers of their different kinds of chromosomes. Analysis of these data by least square methods shows that the sex type may be predicted from the equation Sex type = - 1.37 Y + 0.10 X + 0.01 A + 2.34 This equation fits the data fairly well con- sidering that the correlation between the variables and the sex type is 0.87. This analysis again shows that the Y chromo- some has a strong effect toward maleness. The X chromosomes are next in importance 38 BIOLOGIC BASIS OF SEX with an effect of each X only about 1/13 that of the Y and in the direction of female- ness, the autosomes have one tenth the effect of the X chromosomes but they too have a composite effect toward femaleness. It is to be remembered that the Y chromosome variation is limited to 2 chromosomes whereas the X chromosomes may total 4, and the autosomes may range from 22 up to 42, so that the total effect of the auto- somes is definitely more than their single effects. These data are for aneuploids. Ex- amining Westergaard's data for 1953 for the euploids and assigning the value of 1.5 for the type observed when there was one Y chromosome, four X, and four sets of auto- somes, we have the following equation: Sex value = -1.29 Y + 0.10 X - 0.01 (autosome sets) + 2.53 In these data, as distinct from those above, the autosomes are treated as sets of auto- somes since they are direct multiples of each other so the value of the individual auto- some is but 1/11 that given in the equation. This equation shows no pronounced dif- ference from that when the aneuploids were utilized. The Y chromosomes have slightly less effect toward the male side. The X chromosomes have practically identical ef- fects but there has been a shift in direction of the autosomal effects on sex, although the value is small. The constants are sub- ject to fairly large variations arising through chance. In Table 10 of Westergaard's 1948 paper he presented data on the chromosome con- stitution and sex in the aneuploids which carried a Y chromosome. These data are of particular interest as the plants are counted for the i)roi)ortions of those which are male to those which are hermaphroditic. The plants with a Y chromosome plus an X are all males. Those which have either one, two or three Y chi'omosomes balanced by two X chromosomes have 89 per cent males. The plants with three X chromosomes and one oi- two Y's have 36 p(T cent males and those which have one or two ^■ chroiiiosoincs and four X chromosomes have no males. The woik iiiv()l\-cd in getting these data is, of course, large indeed and is definitely handicapped by the diiiicuHies in obtaining certain types. Thus the XXYYY and the 4X + 2Y types depend only on one plant. There are eight observations but the fitting of the data for the X and Y constitutions eliminates three degrees of freedom from that number so that statistically the obser- vations are few. The data do have the ad- vantage that the sex differences can be measured on an independent quantitative scale. The equation coming from the results is: Percentage of males = 13.2 Y - 36.4 X + 134.4 These results show that the Y chromosome increases the proportion of males and the X chromosome increases the proportion of intersexes. The data are not comparable with those analyzed earlier as these data are describing simply the ratio between the males and intersexes, instead of the rela- tions betw'een the males, intersexes, and fe- males. The equation fits the observations rather well, as indicated by the fact that the correlation between the X's and Y's and the percentages of males is 0.98, but there are large uncertainties. As a contrast to these data we have those presented by Warmke (1946) in his Tables 2 and 3. These data give the numbers of X and Y chromosomes found within the plants but not the numbers of autosomes, the auto- somes being considered as 2, 3, and 4 gen- omes. Analyzing these data in the same manner as those of Westergaard's Table 1, we find that the Sex type = -1.05 Y + 0.22 X -0.04 A -f2.25 As indicated for Westergaard's data, the A effect is now in terms of the diploid type e(iualing 2, the trijiloid 3, and the tetra- l)loid 4. The Y chromosome has a i)ronounccd ef- fect toward maleness, the effect l)eing some- W'hat less in Warmke's data than that of Westergaard's. The X chromosomes on the other hand, have nearly twice the female infhience in Warmke's data that they do in the phiiits grown by Westergaartl. A differ- ence in sign exists for the effect of the A rhi'oinosnnie genom(\s as well as a difference in [\\v (|uantitati\'e effect. Th(^ values for FOUNDATIONS FOR SEX 39 both sets of data are small and toward the male side. As Westergaard points out, the strains used by these investigators are of different geographic origins. The evolution- ary history of the two strains may have a bearing on the lesser Y and greater X ef- fects on the sex of the American types. Chromosome changes seem to have occurred in the strains before the studies of Warmke and Westergaard and will be discussed. The location of the sex determiners has been studied by both investigators utilizing techniques by which the Y chromosome be- comes broken at different places. These breakages may occur naturally and at fairly high rates in individuals which are Y + 2X + 2A. These facts suggest that the break- age of the Y chromosome occurs in meiosis since the breakage comes in selfed individ- uals of highly inbred stocks where heterozy- gosity is not to be expected, in the Y chromosome which has no homologue thus does not synapse, and in the second meiotic
  • eriments with fowl pox it has been shown that the number of eggs developing parthenogenetically increases considerably following vaccination. The factors leading to parthenogenesis are considered to be the genetic characteristics of the strain of birds and the presence of an activating agent or agents in the blood stream of the hens. The parthenogenetic forms are of particu- lar interest to the problem of sex determina- tion. The females should l)e producing two types of oocytes Z -I- A and W -h A of which presumably the Z + A alone survive since the embryos capable of being sexed are all males. The embryos are also diploids. The 2Z -I- 2A could be derived from a fusion of the Z -H A polav body nuclei as noted earlier or possibly chromosome doubling coming later in the early cleavage. A ge- netic element seems partially to control the parthenogenetic process. Chromosome dou- bling would lead to cells with identical pairs of chromosomes. The gene would be homo- zygous. Inbreeding of poultry leads to a con- tinning and rai^id loss in the vialiility of FOUNDATIONS FOR SEX 49 most strains of chickens. A greater loss would be expected for truly homozygous chickens or poults as birds are known for the large numbers of sublethal genes they carry. In fact, it is surprising that any sur- vive to the adult stage. The doubling of the W and A type would result in individuals lacking the Z chromo- some. From what was observed in Amphibia and fish the WW + 2A, individual if it survived, would be expected to be female. Since this type has not as yet been detected it may be inferred that it is inviable be- cause of loss of certain essential genes in the Z chromosome. X. Sex Determination in Mammals A. GOAT HERMAPHRODITES Goat hermaphroditism as reported by As- dell (1936), Eaton (1943, 1945) and Kondo (1952, 1955a, b) is of particular interest when comjjared with human hermaphrodit- ism as observed by Overzier (1955) and of testicular feminization as reported by Ja- cobs, Baikie, Court Brown, Forrest, Roy, Stewart and Lennox (1959) and others. In each species the phenotypic range in sex- ual development extended from nearly per- fect female to nearly perfect male, with the most frequent class as an intermediate. Ex- ternal appearance of each was partially cor- related with internal structure. When inter- nal female structures as the INIiillerian ducts were present, the external appearance was more female-like. When the male structures AVolffian ducts were developed, the external api^earance was more male-like. The pres- ence of the dual systems within certain of these hermaphroditic types indicates, as in Drosophila, that there is independence of development of each system without a so- called turning point calling for differentia- tion of the female sex followed by that of the male sex or vice versa. In goats the hermaphroditic types were traced to the action of a recessive autosomal gene (Eaton, 1945; Kondo, 1952, 1955a, b). This gene apparently acts only on the fe- male zygote. In homozygous condition the eml)ryos bearing them develop simultane- ously toward the male as well as toward the female types. This development resembles closely that of the Hr gene in Drosophila, because, although Hr is dominant and the one in goats is recessive, they both operate only on the female type and both tend to develop jointly both male and female sys- tems in sexual development. One jarring note comes in relating the cytologic basis for sex determination in goats with that for the intersexes. The sex ratios for the different crosses clearly place the hermaphrodites as genetic females ex- pected to have the XX chromosome con- stitution. The XX constitution would then also agree with that found for human hermaphrodites as discussed later in this paper. Makino (1950) has shown for one case of the intersexual goat that its sex chromosomes were of the male type. Ma- kino's excellent studies with other species made this observation of particular signifi- cance as it was contrary to the other mor- phologic and genetic evidence on these hermaphrodites. The implications were fully realized by Makino when the cytologic ob- servations were made so that as far as pos- sible the observations should be critical on this point. However, there are several sources of cell variation that suggest the desirability of further checks. The chromo- some number of the goat is large, normal mitoses rarely appear in the gonads of the intersexes, and the chromosomes of the goat's spermatogenesis are so small as to make difficult details of structure or identi- fication. Some of the difficulties possibly could be avoided by making tissue cultures and determining the somatic chromosome numbers of their cells. Kondo (1955b) has shown that under the breeding conditions of Japan when the sire was heterozygous, the percentage of inter- sexes actually approached the expected value 7.3 per cent. When the sires were homozygous recessive individual matings showed 14.6 per cent hermaphrodites as was expected. Continued mating of homozygotes should show 25 per cent of the total kids hermaphrodites, or the equivalent of 50 per cent of the female progeny. Hermaphroditism in goats has a further advantage in that the locus is apparently linked closely to the horned or polled condi- tion. The horned condition, in consequence, becomes a valuable indicator marking the presence of the hermaphroditic factor in the 50 BIOLOGIC BASIS OF SEX otherwise indistinguishable male types. With these characteristics the goat types have remarkable advantages over other species for the solution of problems of hermaphroditism. The gene for goat hermaphroditism has even more interest when it is contrasted with that of another gene, tra, discovered by Sturtevant (1945). Tra is recessive wdth no distinguishable heterozygous effect. In the homozygous state it converts the zygotic female into a form with completely male genitalia and internal reproductive tract with no evidence of the female sexual repro- ductive system. The gene effects in Dro- sophila are more extreme than those in goats but are concordant in showing that there are loci in the autosomes which may be occupied by recessive genes having direct effects on phenotypic development of the genotypic female. This evidence indicates the significance of these genes rather than the happenstance of their being in the autosome, X or Y chromosome. B. SEX IN THE MOUSE The mouse has the XY + 38 A chromo- somal arrangement for the males and XX + 38 A for the females. Similar karyotype pat- terns have been reviewed for some Amphibia and fish. Other Amphibia and fish may have their karyotypes reversed as both forms are found in nature or observed in breeding studies. Similar reversals may be made ex- perimentally in the phenotypes even though the genotypes remain unaltered. Birds show the sex differentiating arrangement of ZW for the females and ZZ for the males. Par- thenogenesis seems to lead to males of ZZ type in domestic fowl and turkeys. In an evolutionary sense the mammals could have originated from and perpetuated either of the major karyotype sex arrangements. Mice and men are alike in that the X has female-determining properties and the Y male potencies. How much part the genes in the autosomes have in sex develojoment is not yet clear. Welshons and Russell (1959) have shown that mice of the presumed X() constitution are females and arc fertile. They have 39 as the modal number of chromosomes found in their bone marrow cells, wiiereas the genetically proven XX types have 40 cln'omosomcs. X ('hroinosoinc- linked genes' behavior substantiate the chromosomal constitutions of XO and XX as females and XY as males. These results are further supported by the breeding behavior of the X-linked recessive gene, scurfy (Russell, Russell and Gower,. 1959). This gene is lethal to the hemizygous males before breeding. The genetics of the scurfy females have been analyzed by trans- planting the ovaries to normal recipient fe- males and obtaining offspring from them. In the scurfy stock the XO type occurred as 0.9 per cent of the progeny. The YO progeny w^ere not identified and probably die pre- maturely. Nondisjunction of the X and Y chromosomes in the males could result in sperm carrying neither X nor Y chromo- somes. These sperm on fertilization of the X egg would give an XO + 2A type individ- ual. Because the result is a female, this would support the Y chromosome as of male potency. The mouse arrangement may then be expected to be like Melandrium in which a well worked out series of types is known. Sex ratio in mice is strain dependent over what has thus far proven to be a 10 to 15 per cent range. Weir (1958) has shown that for two strains of mice established by select- ing for low and high pH, the sex ratio figures were 33 and 53 per cent for artificially in- seminated mice and 41 and 52 per cent for natural matings of these respective strains. The differential pH values for the bloods of the low line were 7.498 ± 0.006 and for the high line 7.557 ± 0.007 as of the sixth generation of selection. The parents with the more alkaline bloods tended to have greater percentages of males in their prog- enies. These results direct attention to the genotype dependent phenotypic factor which may be of some importance for variations in sex ratios. C. SEX AND STERILITY IN THE CAT The tortoiseshell male cat has long inter- ested geneticists because it has seemed that by theory it should not be. However, nature has wonderful ways of circumventing best laid hypotheses, sometimes when they are fals(\ sometimes when they have not been probed dee])ly enough. The yellow gene for coat color in cats is sex-linked. This gene operates on an autosomal background of ^(■lu's for black oi' tabby. Tlu^ females may FOUNDATIONS FOR SEX 51 be phenotypically orange as the double dose, 0/0, covers up the effects of the other coat color genes; or tortoiseshell, 0/+; or black or tabby, +/+. The males may be orange, '0/, black or tabby, +/, and the type unex- pected tortoise. The tortoiseshell males are timid, keep away from other males, and are generally sterile. Testes are of much reduced size and solid consistency. Exceptionally, tortoiseshell males may mate and offspring presumed from the matings may be born. Active study of these males commenced as early as 1904. Komai (1952) has offered a unified hypothesis for their origin. Komai and Ishihara (1956) have contributed added information and a review of the literature to which the reader is referred. The cat has 38 chromosomes including an X-Y pair for the males. The tortoise males agree in having this arrangement (Ishihara, 1956) , the X being 3 or 4 times the length of the Y in all cytologic preparations from Japanese cats. Komai (1952) visualizes the cat X chromosomes as composed of a pairing segment containing the kinetochore and gene loci among which is that for the orange gene and a differential segment, not found in the Y chromosome, containing the factor-com- plex for femaleness. The Y chromosome is visualized as having a segment containing the kinetochore and capable of pairing with the X chromosome. This segment may cross over with the X so that it may acquire the locus for orange or its wild type. The Y chromosome is viewed as containing two differential segments. The one carrying the factor complex for maleness is located to correspond with the X differential segment carrying the female sex factor. The second Y differential segment is at the other end of the chromosome and contains the male fertility complex. The tortoiseshell sterile males are interpreted as caused by a Y chromosome crossing over with the X chromosome to in- corporate the male segment and the 0 gene in the resulting Y chromosome but with the loss of the male fertility segment. The gam- ete carrying this modified Y fertilizing an egg with a normal X chromosome containing the wild type instead of the 0 gene develops into the sterile tortoiseshell male. The data show that the probability of these events oc- curring is small. Komai records as reliable ■65 tortoiseshell male cats where the inci- dence of the O gene in the whole population of Japanese cats is 25 to 40 per cent. Of the 65, 3 were apparently fertile. These cases and the few others found in the literature are regarded as caused by those rare occasions when the Y chromosome incorporates the 0 gene but retains the male fertility complex as might occur in double crossing over. The hypothesized factor locations and crossing over arrangements also may explain the un- expected black females which are known to occur in some matings. Although not men- tioned, black males and orange males show- ing the same sterility features as the sterile tortoiseshell males should also be found in the cat poi)ulation. If found they would further strengthen the hypotheses. It is difficult to understand why, even with its low initial frequency, the fertile tortoise- shell male would not establish itself in the Japanese cat population, inasmuch as they are so admired and sought after by all the people if any tortoiseshell males became as fertile as the tortoiseshell male "lucifer" (Bamber and Herdman, 1932) known to have sired 56 kittens. Ishihara's work (1956) seems to close the door on another attractive hypothesis to ex- plain the origin of these unexpected cat types. Tortoiseshell male reproductive or- gans include small, firm testes showing re- duced spermatogonial development. To- gether with the interaction of the 0 gene with the wild type allele they suggest the human types XXY + 2A which may arise from nondisjunction. However, the chromo- some type is shown to be XY -f- 2A = 38 which is fatal to this hypothesis. It is of interest that Komai in 1952 postu- lated the male complex and fertility factors in the Y chromosome of a mammal. The case has a further parallel in the plant Melan- drium in that the work of both Westergaard (1946) and Warmke (1946) indicated the Y chromosomes of this plant to contain such factor complexes although in differing ar- rangements. D. DEVIATE SEX TYPES IN CATTLE AND SWINE As a caution in the mushrooming of cyto- logic interjiretations of sex development, at- tention may be directed to the freemartin types known particularly from the work of 52 BIOLOGIC BASIS OF SEX Keller and Tandler (1916), Lillie (1917), and the researches stimulated by their ob- servations on cattle twins. The freemartin in cattle develops in the same uterus with its twin male. The blood circulations anasto- mose so that blood and the products it con- tains are common to both fetuses during de- velopment. The development of the female twin is intersexual, presumably because of substances contributed by the male twin to the common blood during uterine growth. The freemartin intersexuality may be graded into perfectly functioning fertile females to types with external female genitalia and typically male sex cords except germ cells are absent, vasa efferentia, and elements of the vasa deferentia. The conditions are simi- lar to those discussed for amphibia, fish, and rabbits in which early sex development passes through neutral stages during which it may be directed toward one sex or the other by the right environmental stimuli. Intersexes in swine have been interpreted as owing to similar causes (Hughes, 1929; Andersson, 1956) although the resulting phenotypes may not be quite as extreme. The resulting intersexes for both cattle and swine presumably are not caused by chromo- somal misbehavior but to the right environ- mental stimuli operating on suitable gene backgrounds. The observations of Johnston, Zeller and Cantwell (1958) on 25 intersexual pigs all from one breeding group of York- shires suggest significant inheritance effects. The intersexes were of two types, "male pseudohermaphrodites" and "true hermaph- rodites," but there was some intergrading of their phenotypes suggesting that they may be the products of like causes. Common or- gans between the two groups included uteri, vulvae, vaginae, testes, epididymis, and penis or enlarged clitori. The "true hermaph- rodites" were separated on the basis of no prostates, bulbo-urethral glands, or seminal vesicles as well as having testes or ovotestes with ovaries. A similar case was described by Hammond (1912) but, as in one of the above cases, the supposed ovaries when sec- tioned seemed to be lymphatic tissue. Favor- able nerve tissue^ from 6 of the Yorkshire pigs was examined foi- nuclear chromatin. The cases were found chromatin positive. Phenotypically these cases also have paral- l(>ls in mice and man. E. .SEX-iN man: chromosomal basis A surprise even to its discoverers, Tjio and Levan (1956), came with the observa- tion that the somatic number of chromo- somes in cultures of human tissue was 46 rather than the previously supposed 48. Search for the true number has been going on for more than half a century. In early investigations the numbers reported varied widely. Difficulties of proper fixation and spreading of the chromosomes of human cells accounted for most of this variation and the numerous erroneous interpretations. Among the observations that of de Wini- warter (1912) was of particular interest in showing the chromosome number as 46 autosomes plus one sex chromosome with the Y being absent. This number was also found later by de Winiwarter and Oguma (1926). Observations by Painter (1921, 1923) showed 46 chromosomes plus an X and a Y, a total of 48. This number was subsequently reported by a series of able investigators, Evans and Swezy (1929), Minouchi and Ohta (1934), Shiwago and Andres (1932), Andres and Navashin (1936), Roller (1937), Hsu (1952), Mitt- woch (1952), and Darlington and Haque (1955). As Tjio and Levan indicated, the acceptance of 48 as the correct number, with X and Y as the sex chromosome arrangement, was so general that when Drs. Eva Hanson-Melander and S. Kul- lander had earlier found 46 chromosomes in the liver cells of the material they were studying they temporarily gave up the study. In the few years since 1956, the ac- ceptance of 46 chromosomes as the normal complement of man has become nearly universal. There are 22 paired autosomes plus the X and Y sex chromosomes. The reasons which have warranted this change of viewpoint are no doubt many, but three improvements in technique are certainly significant. The first came as a consequence of simplifying the culture of human somatic cells. The second followed Hsu's (1952) recognition that pretreatment of these cells before fixation with hypotonic solutions tended to better spreads of the chromosomes on the division plates when subsefiuently stained by the squash tech- niciuo. Pretreatment of the cultures with FOUNDATIONS FOR SEX 53 colchicine made the studies more attractive by increasing the numbers of usable cells that were in the metaphase of cell division. Ford and Hamerton (1956) in an inde- pendent investigation, closely following that of Tjio and Levan, observed that the human cell complement contained 46 chro- mosomes. They, too, agreed with Painter and others that followed him that the male was XY and the female XX in composition. A flood of confirming evidence soon fol- lowed: Hsu, Pomerat and Moorhead (1957), Bender (1957), Syverton (1957), Ford, Jacobs and Lajtha (1958), Tjio and Puck (1958), Puck (1958), Chu and Giles (1959), and a number of others. In most instances the results of the dif- ferent investigators were surprisingly con- sistent in showing that the individual cell chromosome counts nearly always totaled 46. This was no doubt due in part to the desirability of single layers of somatic cells for identifying and separating the different chromosomes into distinct units. Chu and Giles' results illustrate this consistency. For 34 normal human subjects, including 29 American whites and 4 American Ne- groes, and one of unknown race, and re- gardless of sex, age, or tissue, the diploid chromosome number of the somatic cells was overwhelmingly 46. In only five indi- viduals were other numbers observed in isolated cells. Out of 620 counts, 611 had 46 chromosomes; two individuals, whose majority of cells showed 46, had 3 cells with 45 chromosomes; three other individuals, the majority of whose cells showed 46, had 6 cells with 47 chromosomes. Average cell plates counted per individual was nearly 20. The only recent observations at variance with these results were those of Kodani (1958) who studied spermatogonial and first meiotic metaphases in the testes from 15 Japanese and 8 whites. In these studies at least several good spermatogonial meta- phases in which the chromosomes could be counted accurately, and secondly at least 15 spermatocyte metaphases in which the structure of individual chromosomes could be observed clearly, were made on each specimen. The numbers of cells studied in metaphase were generally above these num- bers, one reaching 60 metaphases. Some var- iation was noted within individuals. Among individuals, numbers of 46, 47, and 48 were observed. Among 15 Japanese, 9 had 46, 1 had 47, and 5 had 48 chromosomes, whereas among the whites 7 had 46, and 1 had 48. Sixteen of the 23 individuals had 46 chro- mosomes. Karyotype analyses indicated that the numerical variation was caused by a small supernumerary chromosome. On the basis of these observations it would appear that individuals within races may vary in chromosome number and yet be of normal phenotype. However, in view of the exten- sive observations by others, it seems un- likely that the variation between individ- uals is as large as that indicated. It will require much further study to establish any other number than 46 as the normal karyo- type of man. This is particularly true in view of the work of Makino and Sasaki (1959) and Alakino and Sasaki cited by Ford (1960), in which they studied the hu- man cell cultures of 39 Japanese and found without exception 46 chromosomes, and the earlier work of Ford and Hamerton (1956) on spermatogonial material where they, too, found 46 chromosomes in that tissue. The best features of these human chromosome studies will come in the identification of the individual chromosomes making up the human group. The chromosome pairs may be ordered according to their lengths. The longest chromosome is about 8 times the length of the smallest. The chromosomes may be classified according to their centro- mere positions. The chromosomes are said by most observers to be fairly easily sepa- rated into 7 groups. Separation of the indi- vidual chromosome pairs from each other and designation of the pairs so that they can be identified by trained investigators in all good chromosome preparations is not possible according to some ciualified cytolo- gists and admitted difficult by all students. However, standardized reporting in the rapidly growing advances in human cell studies should refine observations, reduce errors, and encourage better techniques. With this in mind, 17 investigators working in this field met in Denver in 1959 in what has come to be called the "Denver confer- ence" (Editorial, 1960). From an examina- tion of the available evidence on chromo- some morphologies an idiogram was set up as a standard for the somatic chromosome 54 BIOLOGIC BASIS OF SEX CD LO 00 CM ^ m CM CM 'li m ^ CM i| m m 8 1^ 9m c £ II CM C30 £ Sg ^ *o 1 «. C^ ►5 '^ ^ 9^ *- -f = O ^ •^ CD in ^ i <7> ^D 4M .£ i -^ CD « « >t J 2 ?: C^ -• « CO cording to not easily ] and Thood( € < CM CM )ered ac ich are H. Tjio C < S s numl tes \vh I of J. n « CM Ill • CD ^ m-m %J7 #c #• *— iO CM in \^ 00 2 >e #c i£ CM s s S c £ £ ^-^i a> d -e LO S J^ £ PI O q; i CO o ^ct v£ Fig. 1.1. Id r number, th chromosom r^ 00 FOUNDATIONS FOR SEX 55 complement of the normal human genome. A reproduction of this standard is presented in Figure 1.1, as kindly loaned by Dr. Theo- dore T. Puck for this purpose. The autosomes were first ordered in re- lation to their size and such attributes as would help in their positive identification. Numbers were given to each chromosome as a means of permanent identification. Bas- ically, identification is assisted by the ratio of the length of the long arm to that of the short arm; the centromeric index calculated from the ratio of the length of the shorter arm to the whole length of the chromosome ; and the presence or absence of satellites. Classification is assisted by dividing the chromosome pairs into seven groups. Groups 1-3. Large chromosomes with ap- proximatel}^ median centromeres. The three chromosomes are readily distin- guished from each other by size and centromere position. Group 4-6. Large chromosomes with sub- median centromeres. The two chromo- somes are difficult to distinguish, but chromosome 4 is slightly longer. Group 6-12. Medium sized chromosomes with submedian centromeres. The X chromosome resembles the longer chro- mosomes in this group, especially chro- mosome 6, from which it is difficult to distinguish. This large group is the one which presents major difficulty in iden- tification of individual chromosomes. Group 13-15. INledium sized chromosomes with nearly terminal centromeres ("ac- rocentric" chromosomes). Chromosome 13 has a prominent satellite on the short arm. Chromosome 14 has a small satellite on the short arm. No satellite has been detected on chromosome 15. Group 16-18. Rather short chromosomes with approximately median (in chro- mosome 16) or sul>median centromeres. Group 19-20. Short chromosomes with ap- proximately median centromeres. Group 21-22. Very short, acrocentric chro- mosomes. Chromosome 21 has a satel- lite on its short arm. The Y chromo- some belongs to this group. Separations of the human chromosome pairs into the seven groups is not as difficult as designating the pairs within groups (Patau, 1960). The svstem is a notable ad- vance in summarizing visually the current information in the hope that availability of such a standard will promote further refine- ments, lessen misclassification, and contrib- ute to a better understanding of the problems by cytologists and other workers in the field. 1. Xuclear Chromatin, Sex Chromatin Sexual dimorphism in nuclei of man (Barr, 1949-59) and certain other mammals may be detected by the observable presence of nuclear chromatin adherent to the inner surfaces of the nuclear membrane. The ma- terial is about 1 /x in diameter. It frequently can be resolved into two components of equal size. It has an affinity for basic dyes and is Feulgen and methyl green positive. Nuclear chromatin can be recognized in 60 to 80 per cent of the somatic nuclei of fe- males and not more than 10 per cent of males. It is known to be identifiable in the females of man, monkey, cat, dog, mink, marten, ferret, raccoon, skunk, coyote, wolf, bear, fox, goat, deer, swine, cattle, and opossum, but is not easily usable for sex differentiation in rabbit and rodents be- cause these forms have multiple large par- ticles of chromatin in their nuclei. The tests can be made quickly and easily on skin biopsy material or oral smears. Extensive utilization of the presence or absence of nuclear chromatin in cell samples of man has been made for assigning the presumed genetic sex to individuals who are pheno- typically deviates from normal sex types. (See also chapters by Hampson and Hamp- son, and by Money.) Numerous studies on normal individuals seem to support the test's high accuracy. However, in certain cases involving sexual modification, ques- tions have arisen which are only now being resolved. In male pseudohermaphroditism, sex, determined by nuclear chromatin, is male, thus agreeing with the major aspects of the phenotype. For female pseudohermaph- roditism, individuals with adrenal hyper- plasia or those without adrenal hyperplasia give the female nuclear chromatin test. For cases listed as true hermaphrodites Grum- bach and Barr (1958) list 6 of the male type and 19 of the female type. For the syndrome of gonadal dysgenesis they list 90 as male and 12 as female among the proved cases and 15 more as female among those that are 56 BIOLOGIC BASIS OF SEX suspected. In the syndrome of seminiferous- tubule dysgenesis where there is tubular fibrosis, 9 are listed as male and 18 female. Where there is germinal aplasia, 15 are listed as male and 1 as female. The seeming difficulties in assigning a sex constitution to some of these types are now being dis- sipated through the study of the full chro- mosome complements which are responsible for these different disease conditions. As ob- servations on different chromosome types have been extended, evidence has accumu- lated to show that the numbers of sex nuclear chromatins, for at least some of the nuclei making up the organism, often equals (n — 1) times the number of X chromo- somes. The majority of male XY nuclei are chromatin negative as are most of the Tur- ner XO type. Female nuclei XX have a sin- gle chromatin positive element as do the XXY and XXYY types. The XXX and XXXY have 14 and 40 per cent respectively with two Barr bodies in cases for which quantitative data are available. However, a child with 49 chromosomes, but whose cul- tured cell chromosomes appear as single heteropycnotic masses making identification of the individual chromosomes difficult, showed 50 per cent of the cell nuclei with three Barr elements (Fraccaro and Lindsten, 1960) . The chromosome constitution of these nuclei was interpreted as trisomic for 8, 11, and sex chromosomes. Sandberg, Cross- white and Gordy (1960) report the case of a woman 21 years old having various somatic changes which does not fit this sequence. The chromosome number was 47 and the nuclei were considered trisomic for the sixth largest chromosome. Two chromatin positive bodies were ])rosent in the nuclei. 2. Chrotnosome Complement und Phenotijpe in Man Experience of the past 50 years has em- phasized that genes and trisomies or other types of aneuploid chromosome complexes may lead to the development of abnormal phenotypes expressing a variety of charac- teristics. Drosophila led the way in illustrat- ing how the different gene or chromosome arrangements may affect sex expression. In- vestigations of human abnormal types, par- ticularly those with altered sex differentia- tion, have reccntlv .^liown that man follow.-^ other species in this regard. The Y carries highly potent male influencing factors. Gene differences often lead to characteristic phe- notypes of unique form. 3. Testicular Feminization The testicular feminization syndrome il- lustrates one of these types. As described by Jacobs, Baikie, Court Brown, Forrest, Roy, Stewart and Lennox (1959), "In complete expression of this syndrome the external genitalia are female, pubic and axillary hair are absent or scanty, the habitus at puberty is typically female, and there is primary amenorrhoea. The testes can be found either within the abdomen, or in the inguinal canals, or in the labia majora, and as a rule the vagina is incompletely developed. An epididymis and vas deferens are commonly present on both sides, and there may be a rudimentary uterus and Fallopian tubes. The condition is familial and is transmitted through the maternal line." A sex-linked recessive, a sex-limited dominant, and chro- mosome irregularities of the affected per- sons have been postulated as mechanisms causing the apparent inheritance of this condition. Chromosome examinations of the cells of affected persons have shown 46 as the total number and X and Y as the sex complement. The karyotype analysis agrees with the Barr nuclear chromatin test in that the cells are chromatin-negative but both are at variance with the sex pheno- types in the sense that aside from sup- pressed testes the patients are so completely female. Genetically, Stewart (1959) has de- scribed two color-blind patients with the testicular feminization syndrome in the first five patients he reported. The limited data from these cases suggest that the genie basis for this condition is either independent or but loosely linked with color blindness. This evidence does not exclude sex-linkage but does make it less probable. The third hy- pothesis of autosomal inheritance may take one of several forms. A recessive gene which affects only the male phenotypes when in homozygous condition is apparently un- tenable because the matings from which these individuals come are of the outbreed- ing type and the ratios apparently do not differ from the one-to-one ratio expected of a heterozygous dominant instead of that re- FOUNDATIONS FOR SEX 57 quired for an autosomal recessive. The hy- pothesis advanced by Witschi, Nelson and Segal (1957), that the presence of an auto- somal gene in the mother converts all her male offspring into phenotypes of more or less female constitution, in a manner com- parable to that of the Ne gene in Dro- sophila (Gowen and Nelson, 1942) which causes the elimination of all the female type zygotes, is also made unlikely by the ratios of normal to testicular feminization pheno- types observed in the progenies of these affected mothers. The evidence favors a simple autosomal dominant, acting only in the male zygotes and perhaps balanced by some genes of the X chromosome, which have sufficient influence on the developing male zygote to guide it toward an inter- mediate to nearly female phenotype. The observations of Puck, Robinson and Tjio ( 1960) indicate that the action of a gene for this condition may not be entirely absent in the female, because in heterozygous con- dition in an XX individual it seemed to delay menarche as much as 8 years. If this delay be diagnostic for the heterozygote, it will further assist in the genetic analysis of this problem. Evidence on this point should be a part of the genetic studies. Cases closely similar to those described by Jacobs, Baikie, Court Brown, Forrest, Roy, Stewart and Lennox (1959) are presented by Sternberg and Kloepfer (1960). The patients show no trace of mas- culinity. They are remarkably uniform in anatomic expression. Except for failure to menstruate due to lack of uteri they un- dergo normal female puberty. Cryptorchid testes, usually intra-abdominal, if removed precipitate menopause symptoms. Four un- related cases were found in this one study with 7 additional cases traced through pedi- gree information. A total of 11 affected in- dividuals was found in 6 sibships having 26 siblings of whom 5 were normal males. In each kindred the inheritance was com- patible with that of a sex-linkecl recessive gene. A chromosomal study of a thyroid tissue culture from one case revealed 46 chromosomes with normal XY male con- figuration. The individuals observed were designated as ''simulant females." 4- Superfemale The human superfemale has been recog- nized by Jacobs, Baikie, Court Brown, Mac- Gregor, Maclean and Harnden (1959) in a girl of medium height and weight, breasts underdeveloped, genitalia infantile, vagina small, and uterocervical canal 6 cm. in length. Ovaries appeared postmenopausal with normal stroma, and as indicated by a biopsy specimen, deficient in follicle forma- tion. Menstruation was thought to have begun at age 14, but was irregular, occurring every 3 to 4 months and lasting 3 days. The last spontaneous menstruation was at 19. Estrogen therapy caused some development of the breasts and external genitalia, vagina, and uterus with slight uterine bleeding. The patient's parents were above 40 years of age, mother 41, at time of her daughter's birth. Examination of sternal marrow cultures showed 47 chromosomes in over 80 per cent of the cells examined. The extra chromo- some was the X, the chromosomal type being XXX plus 22 pairs of autosomes. Buccal smears showed 47 per cent of nuclei contained a single chromatin body and 14 per cent contained 2 chromatin bodies as expected of a multiple XX or XXX geno- type. In comparison, 25 smears from 20 nor- mal women had 36 to 51 per cent chromatin positive cells but none of these contained 2 chromatin bodies. Two chromatin bodies were seen in some cells of the ovarian stro- mal tissue. The patient showed a lack of vigor, mentally was subnormal, was under- developed rather than overly developed in the phenotypic sexual characteristics. Ex- amination of the patient's mother showed her to be XX plus 22 pairs of autosomes, the normal 46 chromosomes. Other cases show that types with XXX plus 22 pairs of autosomes are of female l)henotype but may vary in fertility and development of the secondary sexual char- acteristics from nonfunctional to functional females bearing children ( Stewart and San- derson, 1960; Eraser, Campbell, MacGilli- vray, Boyd and Lennox, 1960). The triplo X condition in man has a greater range of de- velopment and fertility than in Drosophila. In man ovaries may develop spontaneously. In Drosophila they require transplantation 58 BIOLOGIC BASIS OF SEX to a diploid female host where they may at- tach to the oviducts and release eggs for fertilization (Beadle and Ephrussi, 1937). These cases present confirmation of two facts already mentioned for Drosophila. They show that when the X chromosome has primarily sex determining genes, the organism generally becomes unbalanced when 3 of these X chromosomes are matched against two sets of autosomes. The re- sulting phenotypes are female but relatively undeveloped rather than overdeveloped. The second is that the connotations evoked by the prefix "super" are by no means ap- plicable to this human type or to the Dro- sophila type. The characteristics of the patient also suggest that the autosomes may be carrying sex genes opposing those of female tenden- cies as observed in both Drosophila and Rumex genie imbalance. 5. Klinefelter Syndrome In the Klinefelter syndrome there is male differentiation of the reproductive tracts with small firm descended testes. Meiotic or mitotic divisions are rare, sperm are ordi- narily not found in the semen. The type is eunuchoid in appearance with gyneco- mastia, high-pitched voice, and sparse fa- cial hair growth. Seminiferous tubules show- ing an increased number of interstitial cells are atrophic and hyalinized. Urinary excre- tion of pituitary gonadotrophins is generally increased, whereas the level of 17-keto- steroids may be decreased. The nuclear chromatin is typically female. Of the dozen or more cases studied (Jacobs and Strong, 1959; Ford, Jones, Miller, Mittwoch, Pen- rose, Ridler and Sha])iro, 1959; Bergman and Reitalu quoted by Ford, 1960), only one, having but 5 metaphase figures, had less than 47 chromosomes in the somatic cells and XXY sex chromosomes. That case was thought to have typical female chro- mosomes XX + 22 AA. Two other cases were of particular interest as indicating further chromosome aberration. Ford, Jones, Miller, Mittwoch, Penrose, Ridler and Shapiro (1959) studied one patient who displayed both the Klinefelter and Mongoloid syn- dromes. The chromosome number was 48, the sex chromosomes being XXY and the 48tli chromosoinc being small acrocentric. This individual had evidently developed from an egg carrying 2 chromosomal aberra- tions, one for the sex chromosomes and the second for one of the autosomes. The other case, Bergman and Reitalu as cited by Ford (1960), had 30 per cent of its cells with an additional acrocentric chromosome which had no close counterpart in the nor- mal set. Data where the Klinefelter syndrome oc- curs in families showing color blindness (Polani, Bishop, Ferguson-Smith, Lennox, Stewart and Prader, 1958; Nowakowski, Lenz and Parada, 1959; and Stern, 1959a) further test the XXY relationship and give information on the possible position of the color blindness locus with reference to the kinetochore. Polani, Bishop, Ferguson- Smith, Lennox, Stewart and Prader (1958) tested 72 sex chromatin-positive Klinefelter patients for their color vision and found that none was affected by red-green color blindness. Nowakowski, Lenz and Parada ( 1959) tested 34 cases and detected 3 af- fected persons, 2 of whom were deutera- nomalous and one protanopic. Stern (1959a) l^oints out that these cases and their ratios are compatible with the interpretation of the Klinefelter syndrome as XXY. One of the deuteranomalous cases had a deutera- nomalous mother and a father with normal color vision. This case could have originated from a nondisjunctional egg carrying 2 maternal X chromosomes fertilized by a sperm carrying a Y chromosome. The other two cases had normal fathers with hetero- zygous mothers. There are several explana- tions by which the color-blind Klinefelter progenies could be obtained. The hetero- zygotes might manifest the color-blind con- dition. The second hypothesis, which is favored, is that of crossing over between the kinetochore and the color-blind locus at the first meiotic division to form eggs each carrying 2 X chromosomes, one homozygous for color blindness, and the other for normal vision. An equational nondisjunction would form eggs homozygous for color blindness which on fertilization by the Y chromo- somes of the male would give the necessary XXY constitution for the color-blind male which is Klinefelter in phenotype. A third possibiHty is that these exceptions may arise without crossing over as the result of FOUNDATIONS FOR SEX 59 nondisjunction at the second meiotic divi- sion. If the hypothesis of crossing over is ac- cepted, the color-blind locus separates freely from its kinetochore and would suggest that the position of the locus is at some distance from the kinetochore of the X chromosome. A disturbed balance between the X and the Y chromosomes alters the sexual type. A single Y chromosome, contributing fac- tors important to male development, is able to alter the effects of two sets of female influencing X chromosomes. Yet two Y chromosomes in a complex of XXYY plus 44 autosomes seem to have little or no more influence than one Y (Muldal and Ockey, 1960). The locations of the sex-influencing genes in man are thus more like those of the plant Melandrium than of Drosophila in which the male-determining factors occur in the autosomes. The relative potencies of the male sex factors compared with those of the female, however, are much less than those in Melandrium. 6. Turner Syndrome Turner's syndrome or ovarian agenesis further substantiates the female influence of the X chromosomes. The cases occur as the developmental expression of accidents in the meiotic or mitotic divisions of the chro- mosomes. These accidents lead to adults unbalanced for the female tendencies of the X chromosome. The gonads consist of con- nective tissue. The rest of the reproductive tract is female. Growth stimuli of puberty are lacking, resulting in greatly reduced fe- male secondary sexual development. Pa- tients are noticeably short and may be ab- normal in bone growth. In its more extreme form, designated as Turner's syndrome, the individuals may show skin folds over the neck, congenital heart disease, and subnor- mal intellect, as well as other metabolic conditions. Earlier work (Barr, 1959; Ford, Jones, Polani, de Almeida and Briggs, 1959) shows that 80 per cent of the nu- clear chromatin patterns are of the male type. Evidence from families having both this condition and color blindness suggested that at least some of the Turner cases would be found to have 45 chromosomes, the sex chromosome being a lone X (Polani, Lessof and Bishop, 1956). Work of Ford, Jones, Polani, de Almeida and Briggs, (1959) has confirmed this hypothesis and added the fact that some of these individuals are also mosaics of cells having 45 and 46 chromo- somes. The 45 chromosome cells had but one X, whereas the 46 had two X's. This finding may explain the female-chromatin cell type observed in about 20 per cent of the cases having the Turner syndrome. Such mosaics of different chromosome cell types could also be significant in reducing the severity of the Turner syndrome and in increasing the range of symptoms which characterize this chromosome-caused disease as con- trasted with those characterizing Turner's disease. Further cases observed in other investigations, Fraccaro, Kaijser and Lind- sten (1959), Tjio, Puck and Robinson (1959), Harnden, and Jacobs and Stewart cited by Ford (1960) have all shown 45 chromosome cells and a single X chromo- some. As with the XXX plus 44 autosome super females, the Turner type, X plus 44 autosomes, also shows a rather wide range in development from sterility with extensive detrimental secondary effects to nearly nor- mal in all respects. Bahner, Schwarz, Harn- den, Jacobs, Hienz and Walter (1960) re- port a case which gave birth to a normal boy. Other cases have been described (Hof- fenberg, Jackson and jVIuller, 1957; Stewart, 19601 in which menstruation was estab- lished over a period of years. The XO type in man and Melandrium is morphologically female. In Drosophila on the other hand, the XO type is phenotypically nearly a perfect male. It is further to be noted that the X chromosome of Drosophila appears to have a less pronounced female bias than that of man when balanced against its as- sociated autosomes, inasmuch as the XO + 2A type in Drosophila is male as contrasted with the XO + 2A type in man which is female. At the same time it seems that the autosomes in the human may be influential in that the female gonadal development is suppressed instead of going to completion as it does in the XX type. 7. Hermaphrodites Hermaphroditic phenotypes in man, to the number of at least 74 (Overzier, 1955), have been observed and recorded since 1900. Types with a urogenital sinus pre- 60 BIOLOGIC BASIS OF SEX dominated. The uteri were absent in some cases, even when complete external female genitalia were present. Ovotestes were found on the right side of the body in over half the cases; separate left ovaries or testes were about equally frequent ; in three cases separate testis and ovary were indicated. The left side of the body showed a different distribution of gonad types; about one- fourth had ovotestes, another fourth ova- ries, and one-twelfth testes. Unilateral dis- tribution of gonad types was most frequent. The presence or absence of the prostate seemed to have significance because it is sometimes absent in purely female types. In recent literature similar cases have been called true hermaphrodites. This is an ex- aggeration in terms of long established practice in plants and animals where true hermaphroditism includes fully functioning gametes of each sex. Hungerford, Donnelly, Nowell and Beck (1959» have reported on a case of a Negro in which the culture cells had the chromo- some complement of a normal female 46, with XX sex chromosomes. Unfortunately, the possibility that this case may be a chro- mosome mosaic was not tested by karyo- type samples from several parts of the body. Harnden and Armstrong (1959) estab- lished separate skin cultures from both sides of the body of another hermaphroditic type. The majority of the cells were apparently of XX constitution with a total of 46 chromo- somes. However, in one of the 4 cultures established, some 7 per cent of cells had an abnormal chromosome present, suggesting that the case might involve a reciprocal translocation between chromosomes 3 and 4 when the chromosomes were ordered ac- cording to size. All the other cell nuclei were normal. The fact that the majority of the cells in these two cases were XX and with 46 chromosomes seems to predicate against the view that either changes in chromosome number or structure of the fertilized egg are necessary for the initiation of hermaplu'o- dites. Ferguson-Smith (1960) describes two cases of gynandromorphic type in which the reproductive organs on the left side were female and on the right side were male. The recognizable organs were Fallopian tube, ovary with primordial follicles only, imma- ture uterus in one case, none in the other, rudimentary prostate, small testis and epi- didymis, vas deferens, bifid scrotum, phal- lus, perineal urethra, pubic and axillary hair, breasts enlarging at 14 years. Testicu- lar development with hyperplasia of Leydig cells, germinal aplasia, and hyalinization of the tubules was suggestive of the Klinefelter syndrome. Nuclear-chromatin was positive in both cases. Modal chromosome number was 46. The sex chromosomes were inter- preted as XX. The 119 cell counts on one patient showed a rather wide range; 7 per cent had 44 chromosomes, 13 per cent had 45, 62 per cent had 46, and 18 per cent had 47 chromosomes. The extra chromosome within the cells containing 47 chromosomes was of medium size with submedian kineto- chore as generally observed for chromo- somes of group 3. It is surprising that the male differentia- tion in these four and other hermaphroditic cases (Table 1.5) is as complete as it is. Other observations show that the Y chro- mosome contains factors of strong male potency, yet in its absence the hermaphro- dites develop an easily recognized male system. It is not complete but the degree of gonadal differentiation is as great as that observed in the XXY 4- 2A Klinefelter types. The bilateral sex differentiation in hermaphrodites would seem to require other conditions than those heretofore considered. Another case of hermaphroditism is that presented by Hirschhorn, Decker and Cooper (1960). The patient's j:)henotype was intersexual with phallus, hypospadias, vagina, uterus. Fallopian tubes, two slightly differentiated gonads in the position of ova- ries. The child was 4 months old. Culture of bone marrow cells showed that the indi- vidual was a mosaic of two types. About 60 per cent of the cells had 45 chromosomes of XO karyotype, and 40 per cent had 46 chromosomes with a karyotA'^pe XY. The Y chromosome when present was larger than Y chromosomes of normal individuals. The change in size may be related to the association of the XO and XY cells and be similar etiologically to the case discussed by yivtz (1959) in Sciara triploids. There are mosaics in Drosophila formed from the loss by the female in some cells of one of lioi- X chromosomes, as for instance FOUNDATIONS FOR SEX 61 in ring chromosome types, which may dis- play primary and secondary hermaphroditic development. For this to happen the altered nuclei apparently find their way into the region of the egg cytoplasm which is to differentiate into the reproductive tract. As seen in the adults, organ tissue of one chro- mosome type is cell for cell sharply differ- entiated from that of the other chromosome type with regard to sex. These observations indicate that for these mosaics the basic chromosome structure of the cell itself determines its development. In fact most mosaics of this species show this cell-re- stricted differentiation. Several problems arise when these well tested observations are considered in comparison with those now arising in the chromosome mosaics of the sex types in man. It would seem unlikely that the bone marrow cells or for that matter any somatic cells not a part of the reproductive tract would operate to modify the adult sex or a part thereof. Rather the developmental secjuence should start from cell differences within the early developing reproductive tract. Circulatory cells or sub- stances would be of dubious direct signifi- cance from another viewpoint. All cells of the body would ultimately be about equally affected by any cells or elements circulating in the blood. With strong male elements and strong female elements the result ex- pected would be a reduction in sex develop- ment of either sex instead of the sharply differentiated organ systems which are ob- served. This raises the question, are the chromosomally differentiated cell sex mo- saics primary to or secondarily derived from the tissues of the ultimate hermaphro- dites? Study of the cell structure of the sex organs themselves as well as much other information will be necessary to clear up this problem. There are, however, other types of con- trolled sex development, as by various genes, which lead to the presence of both male and female sexual systems. Genes for these phenotypes are relatively rare, but once found are transmitted as commonly expected. The inherited hermaphroditic cases in Drosophila are certainly relevant to the testicular feminization syndrome in man. Are they equally pertinent to the highly sporadic hermaphroditic forms just considered for man? If so, they indicate a genie basis for these types which would probably be beyond the range of the micro- scope to detect. The low frequency of true hermaphrodites in the human, together with lack of information on possible inheritance mitigates against the genie explanation ; al- though genie predisposition acting in con- junction with rare environmental events as occurs in our Balb/Gw mice (Hollander, Go wen and Stadler, 1956 ) could explain the rare hermaphrodites observed in that par- ticular line of mice and a limited number of its descendants. 8. XX XY + U Autosome Type The XXXY -I- 44 autosome type in the human has been studied by Ferguson-Smith, Johnson and Handmaker (1960) and Ferguson-Smith, Johnston and Weinberg (1960). The two cases described were char- acterized by primary amentia, micro- orchidism and by two sex chromatin bodies in intermitotic nuclei. The patients were similar in having disproportionately long legs; facial, axillary, and abdominal hair scant; pubic hair present; penes and scrota medium to well developed ; small testes and prostates; vasa deferentia and epididymides normally developed on both sides of the body and no abnormally developed Miiller- ian derivatives. Testes findings were like those in Klinefelter cases with chromatin- positive nuclei, small testes with nearly complete atrophy, and hyalinization of seminiferous tubules and islands of abnor- mal and pigmented Leydig cells in the hyalinized areas. The few seminiferous tubules present were lined with Sertoli cells but were without germinal cells. Nuclear chromatin was of female type. About two- fifths of the nuclei had double and two- fifths single sex chromatin. The modal chro- mosome count for bone marrow cells was 48, 75 per cent of the cells having this number. Chromosome counts spread from 45 to 49. This type, XXXY plus 44 auto- somes, may be looked upon as a superfemale plus a Y or a Klinefelter plus an X chro- mosome. In either case the male potency of the genes in the Y chromosome is able to dominate the female tendencies of XXX to develop nearly complete male phenotypes. Both cases had severe mental defects but TABLE 1.5 Chromosome kinds and numbers for different recognized sex types in man External Type Male Female Female (rare he- mophilic) Eunuchoid female Female Female Female Female Female Female Male . Male. Male. Male. Male. Male . . . Female. Female . Female . Female . Male. Male. Hermaphrodite Hermaphrodite. Intersex Numbers of Chromosomes chromoso- il rearrange- 1 + X fragment 1 2 2 2 2 Interpreted a.s XX trisomic for Sand 11 or as XXXX 4X + Yl 3X+ Yj 1 3 3 2l /mosaic Missing or Extra Chromo- somes ? Chr mosome 3 orT(X;A) Small auto- some ? Large chromo- some or T(X;A) X or Y (X or Y) +21 46,47,48 cliromo- somes X, Y X or Y -fA set Sex Chromatm Negative Positive Negative Negative Positive Negative Positive Negative Negative Negative Negative Negative ± Negative Positive Positive Triple positi Negative Double posit Double posit Negative Double posit Negative Negative Negative Designating Term Normal male Normal female Pure gonadal dysgenesis CJonadal dysgenesis Testicular feiuinizati Turner Turner type female Tinner? gave birth to boy Turner Turner Klinefelter Klinefelter mongoloid Klinefelter Klinefelter Klinefelter Klinefelter Sujierfemale Superfemale gave birtli to children Testicular deficiency Precocious puberty Triploid Hermaphrodite or inter- sex dei)cnding on defini- tion Investigators* 2 3, 4, 5, 5, 12, 13, 14, 40, 41 41 16, 17, 18, 40, 41 19 40 23, 40 24, 25 41 26 29, 30, 31,. 32, 33 35, 39, 40 34 41 Tjio and Levan, 1956. Nilsson, Bergman, Reitalu and Waldenstrom, 1959. Harnden and Stewart, 1959. Stewart, 1960b. Stewart, 1960a. Elliott, Sandler and Rabinowitz, 1959. Jacobs, Baikie, Court Brown, Forrest, Roy, Stewart and Len- nox, 1959. Stewart, 1959 Lubs, Vilar and Bergenstal, 1959. Sternberg and Kloepfer, 1960. Puck, Robinson and Tjio, 1960. Ford, Jones, Polani, de Almeida and Briggs, 1959. Fraccaro, Kaijser and Lindsten, 1959. Fraccaro, Kaijser and Lindsten, 1960a. 15. Bahner, Schwarz, Harnden, Jacobs, Hienz and Walter, 1960. 16. Jacobs and Strong, 1959. 17. Bergman, Reitalu, Nowakowski and Lenz, 1960. 18. Nelson, Ferrari and Bottura, 1960. 19. Ford, Jones, Miller, Mittwoch, Penrose, Hidlor and Shapiro, 1959. 20. Ford, Polani, Briggs and Bishor), 1959. 21. Crooke and Hayward, 1960. 22. Muldal and Ockey, 1960. 23. Jacobs, Baikie, Court Broun, MacCJregor, Maclean and Harnden, 1959. 24. Eraser, Campbell, .MacCiillivray, Boyd and Lennox, 1960. 25. Stewart and Sanderson, 1960. 26. Jacobs, Harnden, Court Brown, Cohl.stein, Close, Mac- Gregor, Maclean and Strong, 1960. 62 FOUNDATIONS FOR SEX 63 27. Ferguson-Smith, Johnston and Handiiiaker, 196( 28. Book and Santesson, 1960. 29. Harnden and Armstrong, 1959. 30. Hungerford, Donnelly, Nowell and Beck, 1959. 31. Ferguson-Smith, Johnston and Weinberg, 1960. 32. deAssis, Epps, Bottura and Ferrari, 1960. 33. Gordon, O'Gorman, Dewhurst and Blank, 1960 34. Hirschhorn, Decker and Cooper, 1960. 35. Sasaki and Makino, 1960. 36. Bloise, Bottura, deAssis, and Ferrari, 1960, 37. Fraccaro, Kaijser and Lindsten, 1960c. 37a. Fraccaro and Lindsten, 1960. 38. Fraccaro, Ikkos, Lindsten, Luft and Kaijser, 1960. 39. Harnden, 1960. 40. Ferguson-Smith and Johnston, 1960. 41. Sandberg, Koepf, Crosswhite and Hauschka, 1960. 42. Hayward, 1960. it should be remembered that they were sought in institutions for which this is a criterion of admittance. Their mental abil- ity was distinctly less than that of Kline- felter XXY cases which have come under study. The pattern of the XXXY effects on the reproductive tract, however, was com- parable with that observed in the XXY genotypes. The effects of one Y chromosome were balanced by either two or three X chromosomes to give nearly equal pheno- typic effects. 9. XXY + 66 Autosome Type XXY + 66 autosome type was estab- lished by Book and Santesson (1960) for an infant boy having several somatic anom- alies which may or may not be relevant to the sex type. Externally the genitalia were normal for a male of his age, penis and scrotum with testes present in the scrotum. Again the Y chromosome demonstrates its male potencies over two X's even in the presence of three sets of autosomes. The case is of particular significance since fur- ther development may indicate what male potencies an extra set of autosomes may possess. 10. Summary of Types Other types of sex modifying chromo- somal combinations and their contained genes have been observed particularly as mosaics or as chromosomal fragments added or substracted from the normal genomes. No doubt other types will be discovered during the mushroom growth of this period. Time can only test the soundness of the observations for the field of human chro- mosomal genetics and cytology is difficult at best requiring special aptitudes and ex- perience. Mistakes, no doubt, will be made. The status of the subject is summarized in Table 1.5. 11. Types Unrelated to Sex Other cases not related to sex or only secondarily so were scrutinized during the course of these studies. The information gained from them is valuable as it strength- ens our respect for the mechanisms in- volved. The sex types which are dependent on loss or gain of the X and/or Y chromo- somes belong to the larger category of monosomies or trisomies. Numbers of auto- somal monosomic and trisomic syndromes have also been identified in the course of these investigations. Similarly, not all cases that have been studied have turned out to be associated with chromosomal changes. This in itself is important since it lends confidence in those that have, as well as redirects research effort toward the search for other causes than chromosomal mis- behavior. The first trisomic in man was identified through the study of Mongolism. The condition affects a number of primary characteristics but not those of sex, for males and females occur in about equal numbers. The broad spectrum of these ef- fects points to a loss of balance for an equally extensive group of genes in the two sexes. The common association of charac- teristics making up these Mongoloids, to- gether with their sporadic appearance and their change in frequency with maternal age, all suggest the findings which Lejeune, Gautier and Turpin (1959a, b) and Lejeune, Turi)in and Gautier (1959a, b) were able to demonstrate so successfully. They estab- lished that the tissue culture cells of Mongol- oid imbeciles had 47 chromosomes and that the extra chromosome was in the small acrocentric group. Lejeune, Gautier and Turpin (1959a, b) have now confirmed these observations on not less than nine cases. Jacobs, Baikie, Court Brown and Strong (1959), Book, Fraccaro and Lind- sten (1959) and Fraccaro (cited by Ford, 1960) as well as later observers have sub- stantiated the results on more than ten other cases. The well known maternal age effect, whereby women over 40 have a chance of having IMongoloid offspring 10 to 40 times as frequently as those of the younger ages, would seem to point to non- 64 BIOLOGIC BASIS OF SEX disjunction in oogenesis as the most im- portant cause of this condition. Some women who have had previous JMongoloid progeny have an increased risk of having others. This is an important consideration in that genetic factors may materially assist in bringing about nondisjunction in man as they are known to do in Drosophila (Gowen and Gowen, 1922; Gowen, 1928). The prod- ucts of the nondisjunctions approach those expected on random distribution of the chromosomes (Gowen, 1933) so that occa- sionally more than one type of chromosome disjunction will appear in a given indi- vidual. Such a case is that illustrated by Ford, Jones, Miller, Mittwoch, Penrose, R idler and Shapiro (1959) in which the nondisjunctional type included not only that for the chromosome important to Mon- golism but also the sex chromosomes sig- nificant in determining the Klinefelter condition. This individual showed 48 chro- mosomes, 22 pairs of normal autosomes, 3 sex chromosomes XXY, and a small acro- centric chromosome matching a pair of chromosomes, the 21st, within the smallest chromosomes of the human idiogram. The analysis of Mongolism showed the way for the separation of the various human sex types through chromosome analyses. Chromosome translocations furnish an- other means of establishing an anomaly that may then continue on an hereditary basis as either the male or female may transmit the rearranged chromosomes. Po- lani, Briggs, Ford, Clarke and Berg (1960), Fraccaro, Kaijser and Lindsten (1960b), Penrose, Ellis and Delhanty (1960) and Carter, Hamerton, Polani, Gunalp and Weller (1960) have studied Mongoloid cases which they interpreted in this manner. In some cases the rearranged chromosomes have been transmitted for three generations. Several of the translocations were con- sidered to include chromosomes 15 and 21. Another trisomic autosomal type was rc- ])ortcd by Patau, Smith, Therman, Inhorn and Wagner (1960). The patient was fe- male and had 47 chromosomes. The extra chromosome was a medium-sized acrocen- tric autosome belonging to the D group. Despite extensive malformations affecting several organs the patient lived more than a year. Another female iiortraying the same syndrome has since been found, so other cases may be expected. Among the charac- teristics are mental retardation, minor mo- tor seizures, deafness, apparent micro or anophthalmia, horizontal palmar creases, trigger thumbs, Polydactyly, cleft i)alate, and hemangiomata. The third trisomic type was also reported by Patau, Smith, Therman, Inhorn and Wagner (1960). Six individuals have been observed. The characters affected are men- tal retardation, hypertonicity (5 patients), small mandible, malformed ears, flexion of fingers, index finger overlaps third, big toe dorsiflexed (at least 4), hernia and/or dia- phragm eventration, heart anomaly (at least 4), and renal anomaly (3). The sexes were two males and four females. The ex- tra chromosome was in the E group and was diagnosed as number 18. Edwards, Harnden, Cameron, Crosse and Wolff (1960) have described a similar case but they consider the trisomic to be number 17. Ultimate com- parisons of these types no doubt will decide if this is a 4th trisomic or if all the cases belong in the same group. The Sturge-Weber syndrome apparently is caused by another trisomic. Locomotor and mental abilities are retarded. Hayward and Bower (1960) interpret the 3 chromo- somes responsible as the smallest autosomes, number 22, of the human group. Trisomic frequencies should be matched by equal numbers of monosomies. Turpin, Lejeune, Lafourcade and Gautier (1959) have reported polydysspondylism in a child with low intelligence, dwarfing, and multi- l)le malformations of spine and sella turcica. The somatic cell chromosome count was only 45 but one of the smallest acrocentric chromosomes appeared to have been trans- located, the greatest part of this chromo- some being observed on the short arm of one of the 3 longer acrocentric chromosomes. Th(> condition appears to be unique and not likely to be found in other unrelated fami- Vws. However, the phenotyj^ic effects were so severe that all members of the proband's family would seemingly be worthy of care- ful sur\'('y for their chromosome character- istics. The comi)lex pattern of multiple anom- alies renders each syndrome distinct from the otliei's. Chromosome losses or gains from FOUNDATIONS FOR SEX Go the normal diploid would be expected to lead to the complex changes. Mongolism is influenced by age of the mother and prob- ably to some extent by her inheritance. It is to be expected that the other trisomies may show parallel relations. Other trisomies may be expected although, as the chromo- somes increase in size, a group of them will have less opportunity to survive because of loss of balance with the rest of the diploid set. Thus far most of these conditions af- fect the sex phenotypes. This is in accord with the results in Drosophila. Changes in the balance of the X chromosomes are less often lethal than the gain or loss of an auto- some. Other animals show like effects. In plants, loss or gain of a chromosome, al- though generally detrimental, often causes less severe restrictions on life. Harmful ef- fects are observed but do not cause early deaths. This may be because many aneu- ploids are within what are presumably polyploid plant species. Ford (1960) has collected the data on 13 different phenotypes that could come under suspicion of chromosomal etiology as ex- amined by a number of workers. Careful cytologic examination of patients suffering from one or another of these diseases has shown that the idiograms were normal in both number and structure of the chromo- somes. The disease conditions were: acrocephalosyndactyly, arachnodactyly (Marfan's syndrome), chondrodystrophy, Crouzon's disease, epiloia, gargoylism, Gau- cher's disease, hypopituitary dwarfism, juvenile amaurotic idiocy, Laurence-Moon- Biedl syndrome. Little's disease, osteogene- sis imperfecta, phenylketonuria, and anen- cephalic types. To this list Sandberg, Koepf , Crosswhite and Hauschka (1960) have now been added neurofibromatosis, Lowe's syn- drome, and pseudohypoparathyroidism. F. SEX RATIO IN MAN Sex ratio studies on human and other ani- mal populations have always been large in volume. The period since 1938 is no excep- tion. Geissler's (1889) data on family sex ratios, containing more than four million births, have been reviewed and questions raised by several later analysts. Edwards (1958) has reanalyzed the clata from this population and considered these points and reviewed the problems in the light of the following questions: (1) Does the sex ratio vary between families of the same size? (2) Do parents capable of producing only uni- sexual families exist? (3) Can the residual deviations in tlie data be satisfactorily ex- plained? Probability analyses were based on Skellam's modified binomial distribution, a special case of the hypergeometrical. The following conclusions were drawn. The probability of a birth being male varies between families of the same size among a complete cross-section of this 19th century German population. There is no evidence for the existence of parents capable of pro- ducing only unisexual families. With the assumption that proportions of males vary within families, the apparent anomalies in the data appear to be explicable. These studies have a bearing on the variances ob- served in further work dealing with family differences such as that of Cohen and Glass ( 1959) on the relation of ABO blood groups to the sex ratio and that of Novitski and Kimball (1958) on birth order, parental age, and sex of offspring. Novitski and Kim- ball's data are of basic significance, for the interpretations are based on a large volume of material covering a one-year period in which improved statistical techniques were utilized in the data collection, in showing that within these data sex ratio variation showed relatively little dependence on age of mother, whereas it did show dependence on age of father, birth order, and inter- actions between them. These observations have direct bearing on the larger geographic differences observed in sex ratios as dis- cussed by Russell (1936) and have recently been brought to the fore through the studies of Kang and Cho (1959a, b). If these data stand the tests for biases, they are of signifi- cance in showing Korea to have one of the highest secondary sex ratios of any region, 113.5 males to 100 females, as contrasted with the American ratio of about 106 males to 100 females. Of similar interest is the lower rate of twin births, 0.7 per cent in Korea vs. about 1 per cent in Caucasian populations and the fact that nearly two- thirds of these tW'in births in Korean peoples are identical, whereas those in the Cauca- sian groups are only about half that num- ber. The reasons for these differences must 66 BIOLOGIC BASIS OF SEX lie in the relations of the human X and Y chromosomes and autosomes and the bal- ance of their contained genes. Little or nothing is known about how these factors operate in the given situations. Acknowledgment. In formulating and organizing the material on which this paper is based I have been fortunate in the helpful discussions and analytical advice contrib- uted so generously by Doctors H. L. Cai'son, K. W. Cooper, H. V. Grouse, C. W. Metz, S. B. Pipkin, W. C. Rothenbuhler, and H. D. Stalker, and others having primary research interests in this field. To them, and particu- larly to my research associates Doctors S. T. C. 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Microscopic observations of parthenogenetic embryonic tissues from virgin turkeys. J. Hered., 46, 133-134. YoKOYAMA, T. 1959. Silkworm Genetics Illus- trated. Tokyo: Japan Society for Promotion of Science. Zoschke, U. 1956. Untersuchungen iiber die Bestimmung des Geschlechtes beim Spinat unter besonderer Beriicksichtigung der Zuch- tung eines monozischen oder gleichzeitig schossenden Spinats. Ztschr. Pflanzenziicht., 35, 257-296. AL ROLE OF HORMONES IN THE DIFFERENTIATION OF SEX R. K. Burns, Ph.D., D.Sc. ihon.) CA.RNEGIE INSTITUTION OF WASHINGTON, DEPARTMENT OF EMBRYOLOGY, THE JOHNS HOPKINS UNIVERSITY, BALTIMORE, MARY'LAND I. The Hormone Theory of Sex Dif- ferentiation 76 II. Methods of Experimental Analy- sis 78 A. Grafting of Gonads or Gonad Tis- sues in Bird Embryos 78 B. Grafting Experiments in Amphib- ian Embrj'os 79 C. Use of Pure Hormones as Sex Dif- ferentiating Agents 82 D. Sex Differentiation in the Absence of Hormones 82 III. The Bisexual Organization of the IiIarly Embryo as the Basis of Sex Reversal 83 IV. Experimental Reversal of Sex Dif- ferentiation IN the Gonads 83 A. Bisexual Organization of the Gonad and the Physiologic Mechanism of Sex Differentiation 83 B. Sex Reversal in Amphibian Gonads 86 1. Constitutional differences and the character of the reversal process 80 2. Parabiosis and grafting of the gonad or the gonad primordium 87 3. Administration of steroid hor- mones 91 C. Sex Rkv'ersal in Avian (Ionads. 95 1. Organization of avian gonads. . . 95 2. Effects of administering pure hormones 9(i 3. Effects of grafting gonads into the coelomic cavity 99 4. Sex reversal in vitro 100 D. The Problem of Sex Reversal in Mammalian Gonads 100 1. Bisexual potentialities in the embryonic gonads of mammals 100 2. Bisexual potentiality in the em- bryonic ovary of the rat 103 3. Experimental transformation of the testis in the opossum. . . 105 V. The Role of Hormones in the De- velopment OF the Accessory Sex Structures 110 A. Differentiation of the Embryonic Gonaducts Ill 1. The Miillerian ducts 112 2. The male duct system 120 B. Derivatives of the Cloaca and Urinogenital Sinus 121 C. External Genital Structures 127 D. Differentiation of Other Types of Sex Character 129 VI. The Pituitary and the Differen- tiation OF Sex 132 VII. Group Differences in the Rela- tions OF Hormones to Sex 134 VIII. The Organization of the Sex Pri- mordium AND Its Role in the Dif- ferentiation OF Sex 137 A. Constitution and the Morphologic Representation of Sex Primordia 137 B. Constitutional Factors and Physi- ologic Differences in the Organi- zation of Sex Primordia 138 C. Influence of Sex Genotype on the Reactions of Sex Primordia 139 IX. The Time F'.^ctor in the Responses OF Sex Primordia: Receptivity and "Critical Periods" 140 X. Specificity of Hormone Action and the Significance of P.\radoxical Effects 141 XI. Time of Origin and the Source of (ioNAD HoR.MONES 143 XII. A Comparison of the Effects of Em- BRYt)Nic and Adult Hormones in Skx Differentiation 145 .\1I1. IvMBRYONic Hormones and Inductor Substances 148 XIV. References 151 I. The Hormone Theory of Sex Diflfereiitiation The modern era in the study of the pliys- iology of sex differentiation is iisually dated from the sohition of the freemartin iiroblem through the simultaneous but entirely inde- 76 HORMONES IN DIFFERENTIATION OF SEX pendent studies of Lillie (1916, 1917) and Keller and Tandler (1916). The theoretic explanation of the anomaly proposed by these authors was generally accepted and had two far-reaching effects ; it at once pro- vided a simple, functional concept of the na- ture of embryonic sex differentiation which was readily susceptible of experimental test, and it directly stimulated the pioneer ex- periments in the field — the attempt to con- trol sex differentiation in chick embryos by grafting gonad tissue to the chorioallantoic membrane (Minoura, 1921) and the appli- cation of the technique of parabiosis to the problem in amphibian embryos (Burns, 1924, 1925). However, as is usually the case with epoch-making theories, the concept of hormonal control of embryonic sex differen- tiation had roots going far back into the past. The effects of castration in domestic ani- mals and in humans had been familiar since earliest times, and it was long appreciated that in the vertebrates the gonads are neces- sary for maintaining the structural integrity of the accessory organs of reproduction and for the regulation of their functional acti^vi- ties. It was first clearly demonstrated by Berthold (1849) that this control is exer- cised through the agency of a substance (of a nature as yet unknown) produced in the gonads and carried throughout the body in the circulating blood (for the historical background of this experiment see Forbes, 1 949 ) . Thus the conception of a blood-borne agent capable of controlling the growth and activities of distant structures, was estab- lished long before the name hormone was given to such substances. The theory that the differentiation of the genital structures in the embryo is controlled by a hormone, or hormones, produced by the embryonic gonads was a natural outgrowth of this knowledge. This view was first proposed as an hypothesis by Bouin and Ancel in 1903, suggested by the observation of an unusu- ally rich interstitium in the testes of pig embryos during the period of sex differentia- tion. No direct evidence in support of the hypothesis was forthcoming, however, until the hormone theory, in virtually its present outlines, was formulated by Lillie and by Keller and Tandler as an explanation of the freemartin. The freemartin^ had been familiar to breeders of cattle for centuries as a sexually abnormal calf, born as twin to a normal male, and its anatomy had been accurately described by John Hunter in the eighteenth century (for references see Lillie, 1917). The external genitalia and mammary glands are typically female in character and the animal was usually regarded as a female, but in rare cases the clitoris may be greatly enlarged and peniform (Numan, 1843, see Lillie, 1917, Fig. 29; Buyse, 1936). Inter- nally, however, elements of the genital tracts of both sexes are present and frequently well developed, and later investigators were often in disagreement as to the primary sex of the creature. The gonads of the freemartin are rudimentary in form but usually show the histologic structure of an abnormal testis which is almost invariably sterile (for an exception see Hay, 1950, and also Hart, cited by Lillie, 1917, page 417). In many cases, however, the gonads are intersexual, showing varying degrees of agenesis of the ovarian cortex associated with rudimentary tubular structures in the medullary or hilar regions (Chapin, 1917; Willier, 1921). Com- monly a well developed male duct system is present, but the development of the fe- male genital tract is variable and in the more modified cases it may be virtually ab- sent. It is unnecessary to go into the various lines of evidence which were used to es- tablish the fact that the freemartin is zy- gotically a female (Lillie, 1917, 1923) ; the point has recently been demonstrated cyto- logically by the Barr method (Moore, Graham and Barr, 1957). It will be useful, however, to review the circumstances which pointed to the cause of the anomaly. A free- martin is always associated at birth with a male twin (which is normal) and never with another female; in addition, the dizygotic origin of the pair was demonstrated by Lillie in many cases. Furthermore, for the birth of a freemartin it is necessary that the placentas of the twins be united, with the presence of vascular anastomoses (Fig. 2.1). In the absence of such connections the fe- male twin is always normal. There is a cor- relation between the degree of abnormality ^ For a discussion of the origin of this name see Forbes (1946). 78 BIOLOGIC BASIS OF SEX Fig. 2.1. Twin calves removed fiom the uterus, showing chorionic fusion and anastomosis between major vessels; male twin, left; freemartin, right. (After F. R. Lillie, J. Exper. Zool., 23, 371-452, 1917). observed and the extent of the vascular union, and also with the stage of develop- ment at which the anastomosis was pre- sumably established. The constancy with which these conditions appear pointed in- evitably to the conclusion that the abnor- mal development of the female twin is caused by the transfusion, from an early stage of development, of a hormone pro- duced by the gonads of the male twin. The invariable dominance of the male member of the pair was explained provisionally on the basis of histologic studies (Lillie and Bascom, 1922; Bascom, 1923) which indi- cated that the testis is active endocrinologi- cally long before the ovary (rf. Bouin and Ancel, 1903). This conclusion has been sup- ported in recent years by the results of castration in mamnialian embryos (q.i\).- ■Reccnll_\' eNidciice lias come from (|uitc (hl- ferent sources that in most twins or muUiplc biilhs in cattle, placental anastomoses are established at an early stage. Calves, even when of different sex and with other characteristics indicating dizygotic or polyzygotic origin, possess identical comple- ments of l)lood factors (red cell agglutination types), which can only l)e explained on the basis of an early exchange of blood (Owen, Davis and Morgan, 1946). Since erythrocytes are compara- tively short-lived cells, it is indicated in these cases that in-imitive erythroblasts must have been ex- changed early in development and colonized the hemopoietic tissues of the recipients. It has been shown also (Anderson, Billingham, Lampson and Medawar, 1951) that diz3^gotic twin calves of dif- II. Methods of Experimental Analysis The demonstration in tlie case of the free- martin of the probable nature of the trans- forming agent and its mode of transmission at once suggested means of attacking the problem of embryonic sex differentiation ex- perimentally. At first grafting methods were mainly employed, using the embryos of birds and amjihibians. A. CRAFTING OF GONADS OR GONAD TISSUES IX BIRD EMBRYOS Historically, the first experiments were those of JNIinoura (1921) who trans})lanted pieces of adult testis or ovary to the chorio- allantois of chick embryos during the period of sex differentiation. Such grafts become vascularized and are then in communication witli the host embryo by way of the umbili- cal circulation. Various modifications of ferent sex are, with few exceptions, tolerant to grafts of each other's skin. Tliis is true of skin ex- changes in monozygotic twins (as would be ex- jiected) but is never found in other degrees of r(>hitionship. As in tlie preceding case, the ex- ])lanalion is found in an early transfusion of blood l)etwe(ni the twins. The exceptional cases are doubtless to be explained (as in the freemartin study) on the occasional failure of placental anas- tomosis to occur. This evidence is cited for its bearing on tlic point of early exchange of blood; tlie fact that blood cells and other elements are exchanged does not seem to be of significance for the sex hormone theory. HORMONES IX DIFFERENTIATION OF SEX f9 the embryonic genital structures of the host (especially of the Miillerian ducts) were noted and attributed to the influence of the grafted tissues. But later investigators failed to confirm these findings, and it was shown eventually that the anomalies observed by ]\Iinoura were unspecific in character and bore no constant relation to the sex of the grafted tissue (for a review and discussion see AVillier, 1939). Similar modifications were also found after transplantation of various nongonadal tissues, and are appar- ently induced by changes in the physical environment incidental to operation, such as lowering of the temperature and the hu- midity (Willier and Yuh, 1928). Evidently the original experiments had not been ade- quately controlled with respect to such fac- tors. Obviously this type of experiment does not correspond exactly with conditions in the freemartin. The grafted tissue came from adult gonads, and there was no way of determining the hormone output of such grafts or whether, indeed, they produced hormones at all. Furthermore, sexual dif- ferentiation in the host embryos has gen- erally begun before transplantation to the chorioallantois is practicable. This objection was avoided, however, by modifying the ex- l)eriment. Sexually undifferentiated embry- onic gonads were transplanted to the chorio- allantoic membrane of a host embryo already well advanced in sex differentiation. In this case changes might be anticipated in the grafts. However, this experiment, as well as transplantation of the gonad-form- ing region of the blastoderm (Willier, 1927, 1933), also gave negative results. The grafted gonads differentiated to a variable degree, depending on the state of develop- ment of the primordia at the time of trans- plantation, but when sexual differentiation occurred it showed no constant relation to the sex of the host. These failures to obtain evidence that gonad tissues growing on the chorioallantois influence the differentiation of host struc- tures, or are themselves modified by the hormones of the host, raised serious doubt as to the role of hormones in embryonic sex differentiation in birds ; and this feeling was not entirely removed by the demonstration some years later that the sexual differentia- tion of the chick can be readily modified by treatment with pure hormone preparations. It was not until embryonic gonads were transplanted directly into the body cavity of another embryo (Bradley, 1941 ; Wolff, 1946) that unmistakable evidence was ob- tained. The conditions under which this re- sult was achieved — close proximity of the graft to the developing host structures — suggested that the failure to obtain positive results by chorioallantoic grafting was per- haps largely a matter of the quantity or concentration of the hormone. Recently, however, a true "freemartin effect" has been reported in twin chicks of different sex, which developed from an egg with two yolks (Lutz and Lutz-Ostertag, 1958). There was local development of cortex on the left testis of the male twin, and a marked inhibi- tion of the ]Miillerian ducts of the female, effects paralleling those produced by in- tracoelomic gonad grafts. This is the only recorded case of a natural freemartin in birds. B. GR.\FTIXG EXPERIMENTS IX AMPHIBIAN EMBRYOS The principal experimental procedures de- veloped for amphibian embryos are illus- trated diagrammatically in Figure 2.2, which shows the different modes of grafting, and the resulting vascular relationships, as compared with the freemartin (Fig. 2.1). The first experiments actually undertook to reproduce as nearly as possible the situation which arises by chance in the freemartin. The method devised was parabiosis— the grafting together of two embryos in the manner of "Siamese twins" (Burns, 1925) so that in later development there is a common circulation (Figs. 2.2A and 2.9). When members of such a pair happen to be genetically of the same sex, normal sexual differentiation would be expected to follow; but in pairs of different sex opportunity for cross-circulation of sex hormones is pro- vided. Circulatory anastomosis is estab- lished in such pairs long before the begin- ning of sex differentiation in the gonads, and so a favorable situation is provided for testing the possibility of hormone action. The results obtained by this method vary 80 BIOLOGIC BASIS OF SEX A B C Fig. 2.2. Diagram illustrating different modes of grafting in amphibians in order to bring about vascular continuity between individuals, and association of gonads of different sex. A. Homoplastic twins in salamanders. The body cavities are largely separated and vascular com- munications between the gonads are remote. For combination of dissimilar species see Fig. 2.9B. B. Anuran twins, showing side-to-side or head-to-tail union; reversal changes appear only under the first condition, when the gonads are in close proximity. (After E. Witschi, in Sex mid Internal Secretions, The Williams & Wilkins Company, 1932). C. Orthotopic trans- plantation of the gonad primordium by the method illustrated in Fig. 2.4, resulting in two gonads of opposite sex resident in a single individual (Humphrej''s method). greatly, depending on the species under study and on various experimental condi- tions, as will appear later. The grafting of gonads alone (as opposed to the union of whole organisms) can be car- ried out in embryonic stages of development or in early larval life. The latter method was tried first. The gonads, attached to a segment of the mesonephric bodies, were re- moved from young larvae at or soon after the onset of sex differentiation and inserted into the body cavity of older larvae (Burns, 1928) . The development and activity of such grafts depends on the extent to which they become attached and vascularized. When graft and host are of different sex the grafts typically become intersexual, developing the structure of ovotestes; and when a large and well differentiated graft is in close proximity to the gonads of the host the latter may be similarly modified (Fig. 2.3). This method of grafting has the disadvantage, however, that reversal is usually incomplete, and when graft and host gonads show reciprocal modification it is sometimes difficult to de- termine the primary sex of either. The method described above was soon greatly improved upon by the development of a technique for transplanting, at an ear- lier stage, the prospective gonad-forming tissue from one embryo to another (Hum- phrey 1928a, b). At first such grafts were placed in ectopic locations, but later it was found advantageous to place them in the normal (orthotopic) position in an embryo from which the corresponding gonad pri- mordium had been excised (Fig. 2.4). After such an operation the host embryo bears on one side its own gonad and on the other a gonad which, in approximately half of all cases, has come from an embryo of the other sex (Fig. 2.2C). This method has im- portant advantages over those previously described. A single embryo bearing an or- thotopic graft survives better and is more easily reared tlian are parabiotic pairs, and gonads grafted in tlio orthotopic position usually develoj) better than in foreign sur- roundings. Most important of all, the donor embryo may be reared, thus establishing with certainty the original sex of the grafted gonad. This precise method has yielded un- HORMONES IN DIFFERENTIATION OF SEX 81 Fig. 2.3. Transplantation of the salamander gonad in early larval life (Burns, 1928): previously unpublished photographs. A. A large, but somewhat degenerate, grafted ovary lies just anterior to (above) the gonads of the host, which show changes in external form in the vicinity of the graft. B. Cross-section of host's right testis at the level of the white line, showing normal medullary development with well differentiated testis lobules, and periph- erally a strongly developed cortex. A B Fig. 2.4. Diagrams illustrating Humphrey's orthotopic transplantation method. A. Posi- tion of the gonad- and mesonephros-forming area of the embryo (stippled) which is excised and reimplanted in the corresponding position in a host embryo from which the primordium has just been removed. B. Cross-section of host at later stage showing position of the im- planted material (between heavy lines) at the left. In the mesodermal layer a part of the lateral mesoderm lies below, the gonad- and mesonephros-forming material above, with the Wolffian duct at the top. Medial to the Wolffian duct lies the mass of primordial germ cells (more densely stippled). 82 BIOLOGIC BASIS OF SEX equivocal results which have in general con- firmed and extended those obtained by para- biosis. C. USE OF PURE HORMONES AS SEX DIFFERENTIATING AGENTS Early attempts to influence embryonic sex differentiation by the use of crude hor- mone preparations were almost entirely un- successful because of lack of potency, or the toxicity of the extracts. However, the isolation and eventual synthesis of steroid hormones made available a variety of ac- tive and nontoxic substances, and the use of pure hormones largely superseded grafting techniques. Direct administration of stand- ard hormone preparations has the great ad- vantage that dosages can be exactly known and regulated; also the timing of treat- ments is readily controlled and varied. The first successful experiments using pure hormones were carried out on chick embryos. Similar results were obtained at almost the same time by several groups of investigators (Kozelka and Gallagher, 1934; Wolff and Ginglinger, 1935; Dantchakoff, 1935, 1936; Willier, Gallagher and Koch, 1935, 1937j who introduced the hormones, in oily or in aqueous solution, into the in- cubating egg. Striking transformations were produced, involving both the structure of the gonads and the accessory organs of sex. In the best cases reversal of the gonads was histologically almost complete. The effects of crystalline sex hormones have also been investigated in many species of amphibians. Two methods have been utilized. Larvae may be treated individually by repeated injections, or in groups by con- tinuous immersion, the hormone being dis- solved in the water in which the larvae are reared. The latter method is particularly convenient for anuran tadpoles. Treatment can be started very early, the concentration is readily varied, and in many cases com- plete transformations have been obtained with the use of extremely low concentra- tions. In mammalian embryos experimental study of sex differentiation was long delayed by the lack of operative techniques ade- quate for dealing with embryos in utero. The advent of pure hormones made possible the first successful experiments in this field. In i^lacental forms, in spite of a very high mortality, large doses of crystalline hor- mones can be administered to the mother during early stages of pregnane}" with pro- nounced effects on the genital systems of the embryos (for a review of the earlier experi- ments see Greene, 1942, and for a recent summary Jost, 1955) . About the same time, experiments were begun using the pouch young of a marsupial, the North American opossum (Burns, 1939a, b; Moore, 1941). So undeveloped are young marsupials at birth that virtually the entire course of morphologic sex differentiation takes place postnatally, and the embryos in the pouch are directly accessible for experimentation. Hormones were administered by injection (Burns) or by inunction, the application of an ointment containing the hormone to the skin. Except for minor differences attributa- ble to dosage or other experimental factors, the results were similar, and in general agreement with those obtained in placental mammals by treatment during pregnancy. D. SEX DIFFERENTIATION IN THE ABSENCE OF HORMONES Although the evidence obtained by graft- ing techniques and by administration of hormones shows that the differentiation of embryonic genital structures may be pro- foundly modified or even completely re- versed, such evidence is not in itself con- clusive with respect to the central problem, the role of hormones in the normal differ- entiation of sex. The transmissible sub- stances responsible for sex reversal in graft- ing experiments have not been isolated or identified and it may be argued that experi- ments with pure hormones show merely that the differentiating embryonic sex primordia are capable of reacting when hormones are introduced experimentally. Such evidence docs not prove, however, that embryonic gonads actually produce such hormones. For this question the crucial test is the capacity of the embryonic genital structure to de- velop in the absence of gonads or removed from all hormonal influence. Evidence on this point has been forth- coming in recent years and gives strong supi)ort to tiie hormone theory. Two differ- ent experimental approaches have been de- veloped. Early castration of the embryo has HORMONES IN DIFFERENTIATION OF SEX 83 now been achieved in both mammals and birds, and improved methods of culturing embryonic organs in vitro have made is pos- sible to observe for a sufficient time the de- velopment of sex primordia in complete physiologic isolation. Since 1947 castration has been success- fully performed in amniote embryos by two techniques, surgical castration and irradia- tion of the gonad region (for summaries see Jost, 1950; Wells, 1950; Raynaud, 1950; Wolff, 1950; Huijbers, 1951). In all cases serious failures of sexual differentiation fol- low removal or destruction of the gonads. Finally, the cultivation in vitro of indi- vidual sex primordia in virtual absence of hormonal influences has yielded results simi- lar in all respects to those of castration. The results of these experiments will be taken up in detail as they relate to the develop- ment of particular structures. in. The Bisexual Organization of the Early Enihryo as the Basis of Sex Reversal The capacity of vertebrate embryos to undergo a reversal of sex, either spontane- ously, as in various developmental anoma- lies of undetermined etiology ("intersexu- ality," "hermaphroditism"), or as a result of experiment, is based on the fact that every individual, regardless of genie sex constitu- tion, passes in early development through a sexually undifferentiated or ''indifferent" phase. During this period virtually all of the embryonic structures necessary for the development of either sex are laid down morphologically and are present for a cer- tain time as discrete 'primordia. The extent to which the primordia of the genetically recessive sex are developed and the length of time during which they are present vary in different groups and species. This fact is of great importance in the experi- mental transformation of sex. In species in which the structures of the recessive sex are imperfectly represented, or are present for only a brief period in early development, opportunity for sex reversal is correspond- ingly limited; but in other cases the rudi- mentary structures of the recessive sex (as for example Miillerian ducts in males or vestigial prostatic glands in females) per- sist indefinitely and mav even survive in the adults of some species. The existence of a considerable degree of embryonic bisexual- ity in most groups (see Fig. 2.22) provides a definite morphologic basis for experi- mental transformation of sex and for the sporadic occurrence of sex anomalies as well. The derivation of the various embryonic primordia which give rise to the male and female genital systems, and their history in the normal differentiation of sex, have been extensively reviewed by AVillier (1939) and will not be taken up again in detail. The main features of normal development will be outlined briefly when dealing with the experimental behavior of individual structures. It must be remembered, however, that the individual parts of the genital sys- tem have widely different embryonic origins, and are morphologically and physiologically very dissimilar, and at any particular stage of development may vary greatly in their relative maturity and so in their reactivity to hormones. Many of the basic structures (e.g., the embryonic sex ducts, the urino- genital sinus) are taken over bodily from other systems and only secondarily acquire a sexual status. It cannot be expected, there- fore, that all parts of the developing sex complex will be capable at all times of re- sponding harmoniously to experimental con- ditions which are often of necessity rigid and artificial or improperly timed. The rec- ognition of such differences aids in under- standing the variability so frequently en- countered in the reactions of sex structures to hormones, and the importance of such experimental factors as the timing of treat- ment and dosage. IV. Experimental Reversal of Sex Differentiation in the Gonads A. BISEXUAL ORGANIZATION OF THE GONAD AND THE PHYSIOLOGIC MECHANISM OF SEX DIFFERENTI.ATION The sexually undifferentiated gonads of most amphibians exhibit bisexual organiza- tion in a primitive and simple form. In early larval life the gonad, irrespective of its future sex, contains two histologically distinct components in which male and fe- male potentialities are segregated. Inter- nally, there is a hilar or centrally placed 84 BIOLOGIC BASIS OF SEX NDIFFERENT GONAD OVARY TtSTIS Fig. 2.5. Diagrammatic representation of the male and female components of the sexually un- differentiated amphibian gonad and their roles in sex differentiation: the medulla is stippled, the cortex is plain. The broken arrows indicate the mutually antagonistic or inhibitory actions exerted between the two components in the course of sex- ual differentiation (Witschi). mass, the medulla, which has the develop- mental potentiality of a testis. Surrounding the medulla is a peripheral zone, the cortex, which is specifically female in potency. The topographic relationships of medulla and cortex are illustrated diagrammatically in Figure 2.5, and as they appear histologically in male salamanders in Figure 2.QA and B. Male and female potentialities are evidently pre-established in the medullary and corti- cal components at an early stage since final differentiation as a testis or an ovary does not involve the transformation of one sex comj^onent into the other but rather the gradual predominance of one element and the recession of the other. Not only are the two components dis- tinctly segregated in the indifferent gonad, they have separate origins. It has long been recognized that the medulla in both sexes is derived from the mesonephric blastema in the form of a series of cellular strands, the medullary cords or rete cords, which grow into the genital ridge at an early stage. At first similar in appearance in the two sexes, their later differentiation follows very different patterns. In the ovary the cords expand distally, forming a series of saccular cavities, the ovarian sacs. Most of the germ cells are excluded from these sacs and come to lie in a peripheral layer beneath the peri- toneal epithelium covering the gonad. This zone becomes the cortex. In prospective testes the cords branch and proliferate rap- idly, enveloping and incorporating the ma- jority of the germ cells in a compact central mass, the medulla ( Fig. 2.6 ; for a fuller de- scription see Willier, 1939). Thus the rela- tive proportions of cortex and medulla in the sexes depend on the pattern of differen- FiG. 2.6. Sections showing the histologic appearance of cortex and medulla in early larval stages under various conditions. A. Normal testis of an Amby stoma tigrinum larva; note the thin cortical zone, comparable to a germinal epithelium, which covers most of the surface. B. The cortex of an intersexual testis dissected free from the medullary core, from the punc- tatum member of an Ambtjstoma tigrinum, 9 ; A. Punctatum, $ pair (c/. Burns, 1935, plate 4). C. Intersexual testis of the male member of a tigrinum-tigrinum pair, showing tl)e rela- tive development of the medullary and cortical components (see Burns, 1930). HORMONES IN DIFFERENTIATION OF SEX 85 tiation of the medullary cord, which results in a very unequal allocation of the germ cells between the two components. Prob- ably the sex genotype acts primarily by determining the developmental pattern of the medullary cord. The role of the germ cells in the formation of the gonad appears to be a purely passive one since the non- germinal tissues are capable of producing the typical structure of a testis or an ovary in the absence of all germinal elements (for a review of this subject see Burns, 1955b). The origin of the medullary component of the gonad from the mesonephric blastema and its dominant role in gonad formation has been demonstrated in a striking experi- ment by Houillon (1956). Formation of the gonad is dependent on the normal develop- ment of the mesonephros, and this can be repressed or entirely prevented by blocking the development of the primary nephric duct (pronephric ductj at an early stage. In the absence of the nephric duct the meso- nephric blastema is reduced in quantity and delayed in its appearance; typically only a few mesonephric tubules develop and these are poorly differentiated. In consequence of the suppression of the mesonephric blas- tema, medullary cords are lacking or poorly developed, and the result is a vestigial gonad consisting chiefly of a rudimentary cortex. The essential role of the medullary cords in gonadogenesis is also demonstrated in the gonads of toads (Witschi, 1933) in which the so-called "organ of Bidder" corresponds to an anterior segment of the genital ridge in which medullary cords are absent. The proportion of cortex to medulla as laid down in embryonic gonads depends primarily on genie constitution, but the stage of development is a factor in the rep- resentation of the two elements at any par- ticular time, since during the progress of sexual differentiation one component (cor- responding to the genetically determined sex) shows an increasing predominance from stage to stage. Furthermore, the morpho- logic representation of the two sex compo- nents in the indifferent gonad is subject to variation in different groups, species, or races. In some species the recessive com- ponent is weakly represented or virtually absent, even in early development, or when present its existence may be of brief dura- tion. In such cases capacity for sex reversal is reduced or lacking. In other species, in which the recessive sex component is well developed, or when it persists over a con- siderable period of time, capacity for ex- perimental reversal is correspondingly in- creased. The alternative behavior of the cortical and medullary components of the gonad in normal differentiation, as well as their be- havior under experimental conditions, long ago suggested that the physiologic mecha- nism of sex differentiation consists essen- tially of an antagonistic interaction between the two elements, in which the genetically dominant component gradually inhibits its antagonist (Fig. 2.5). This concept of "cor- ticomedullary antagonism" (Witschi, 1932) has been generally accepted as the basic mechanism in the histologic differentiation of the gonad and forms the starting point for the inductor theory of sex differentia- tion.^ A number of seemingly unrelated ex- perimental procedures which are capable of inducing sex reversal all appear to have a common base of action by influencing or controlling this simple mechanism. For ex- ample, sex reversal is in some cases readily induced by external or environmental influ- ences of an unspecific character, which ap- parently produce their effects by depressing or destroying the dominant gonad compo- nent. Classical experiments of this type are the ^ As originally formulated, this theory postulated simply that each gonad component produces a substance which specifically inhibits the differen- tiation of the other. These substances, called med- ullarin and corticin, were considered to be similar in character and to behave like the embryonic in- ductors of earlier development, being transmitted by diffusion and having strictly localized effects. Subsequently the theory was elaborated to allow for stimulatory as well as inhibitory action, with each inductor system assigned a dual role; ac- cordingly, positive and negative factors were as- sumed and so designated, e.g., medullarin* and medullarin'. More recently it has been proposed that interactions between the sex inductors are of the nature of an immunologic reaction (Chang and Witschi, 1956), the positive factor appearing first in the role of an antigen which stimulates the other system to produce an antibody, the inhibi- tory factor (for the development of the inductor theory see Witschi, 1934, 1939, 1942, 1950, 1957). To account for the great taxonomic variability in the action of the sex inductors thej' are assumed to be proteins. BIOLOGIC BASIS OF SEX use of extreme^; of temperature to induce reversal of differentiation in the gonads of anuran larvae (Witschi, 1929; Piquet, 1930; Uchida, 1937 1 . The reversal is due primarily to an unfavorable effect on the dominant component, high temperatures causing cor- tical degeneration in females and low tem- peratures inhibiting medullary development in males. Also, simple surgical interventions or even pathologic injury may have the same effect. Castration in certain cases re- sults in complete reversal of sex through the reactivation and renewed development of a recessive gonad component left behind at operation. In adult male toads removal of the testes permits the organs of Bidder to develop into ovaries, which may become fully functional (Ponse, 1924). A compara- ble case is found in the reversal of sex which takes place in female chicks castrated soon after hatching. Removal of the dominant left ovary is followed by development of the rudimentary right gonad, composed largely or entirely of medullary tissue, into a small testis. Finally, rare cases of partial or com- plete sex reversal in adult hens, which oc- cur as a result of pathologic destruction of the functional ovary, appear to have the same morphologic basis (Crew, 1923; for a discussion see Domm, 1939 1 . But although sex differentiation in most vertebrates ends in the complete dominance of one sex component, remarkable devia- tions from this plan are known in certain groups and species. An extreme is found in the prevalence of hermaphroditism, in vary- ing degree, in many teleost fishes and in cyclostomes (see ch. 17) which may be of the juvenile type and temporary, or may persist in adults. In toads the curious struc- ture known as Bidder's organ is present in adults of both sexes; it represents a local region of the genital ridge in wliich medul- lary cords are never formed and furtlier dif- ferentiation does not occur. Since it corre- sponds morphologically to the cortical component of the gonad it retains through- out life the potentiality of an ovary. This condition is apparently possible in toads because of the very low level of antagonism in this genus. Stranger still is the situation found in the female of certain insectivores (the mole, Godet, 1949, 1950; the desman, Peyre, 1952, 1955j in which the medulla of the adult ovary is testis-like and devel- oped to a remarkable degree. Except during the reproductive period it greatly exceeds the cortex in bulk. Its cords are tubular in form, resembling testis tubules, and a well developed interstitium indicates an endo- crine activity which is reflected in the strong masculinization of the genital tract. The clitoris is large and penis-like, and male ac- cessory glands, absent or rudimentary in the females of most mammals, are well devel- oped. Another species in which the ovarian medulla is highly developed, at least throughout fetal life, is the horse (Cole, Hart, Lyons and Catchpole, 1933). Thus many patterns are found with respect to the persistence of the heterotypic sex compo- nent of the ovary and its final fate. B. SEX REVERSAL IN AMPHIBIAN GONADS 1. Constitutional Differences and the Char- acter of the Reversal Process Modern amphibians, far from being a homogeneous group, are extremely diversi- fied in structure and function and are often highly specialized. Such diversification ob- viously has had a long evolutionary history. Correspondingly, the processes of sex re- versal as evoked experimentally in amphib- ian gonads, often follow very different histo- logic and physiologic patterns in different groups, species, or races. These differences must rest ultimately on genetic constitution ; more immediately they are predetermined, in labile fashion at least, in the structural and physiologic organization of the gonad primordium which is in itself a complex sys- tem. The organization of the early gonad may vary greatly (according to species and ac- cording to sex) with respect to the cortical and medullary elements as laid down histo- logically in the primordium — is the hetero- tyi:)ic sex component of the primordium well represented or is it quantitatively deficient fiom the beginning? The subsequent be- liavior of the heterotypic component is also important— does it jiersist and regress slowly over a considerable period of time or is its existence transient? Furtliermore. how does it react when the normal balance of the dif- HORMONES IN DIFFERENTIATION OF SEX 87 ferentiation process is experimentally dis- turbed— does it respond readily by growth and differentiation or is it relatively inert and refractory? In particular cases the ca- pacity of a gonad for reversal under experi- mental conditions obviously depends on which of the various situations prevails. An- other variable concerns the humoral activity of the gonad, as regards the time of onset and the factors of quantity or rate of pro- duction. Species differ widely in this respect and marked sex differences are also found. Presumably all such characteristics exist as predispositions within the gonad primor- dium, but they are not as a rule irreversibly determined. In addition the process of reversal, as seen histologically, may be influenced by experi- mental conditions such as the procedure em- ployed, the stage of development at which reversal is initiated and the duration of the experiment. If conditions are favorable at the beginning of sex differentiation, reversal may take place directly without leaving ob- vious histologic traces, i.e., an individual of one sex may adopt the developmental pat- tern of the other virtually from the start. If, on the other hand, transformation is not initiated until sex differentiation is well ad- vanced, various stages of intersexuality will appear in the transforming gonads, until one sex component finally establishes com- plete dominance and the other disappears. The first situation commonly occurs when larvae are reared in optimal concentrations of hormone dissolved in the aquarium wa- ter; exposure to the hormone is continuous from a stage long antedating the appearance of morphologic sex differentiation, and all embryos regardless of sex genotype develop as one sex. However, the same result may occur also in grafting experiments (parabio- sis or gonad transplantation) in which het- eroplastic combinations of different species assure decisive predominance of one sex from the beginning by virtue of great in- equality in size and in rate of development. The second situation is encountered when the conditions of the experiment do not lead to establishment of dominance at an early stage. Reversal sets in late, intersexual stages may be prolonged, and complete transformation mav never occur. 2. Parabiosis and Grajting of the Gonad or the Gonad Primordium Experiments of this kind involve the in- teraction of embryonic or larval gonads through the agency of substances of a hu- moral nature but of unknown chemical con- stitution. In some species, or under certain experimental conditions, the effects may be limited and highly localized, appearing only when the interacting gonads are in contact or in close proximity. Transport of the hu- moral agent takes place apparently by dif- fusion through the intervening tissues (Figs. 2.25 and 2.3 j . In other cases the effects are exerted over great distances and the sub- stances must of necessity be carried in the blood. This does not necessarily mean, how- ever, that different substances are involved in the two cases. As will be shown, hor- mones in low concentrations may have only local effects and, given a sufficient concen- tration in the blood stream, there is no rea- son to suppose that the so-called "inductor substances" could not act at a distance. The mode of transport does not seem to be cru- cial for the definition of these substances (for discussions see Willier, 1939, page 134, and Burns 1949, 1955b). In most species of amphibians which have been investigated the male is the dominant sex. In grafting experiments, whether the method is parabiosis or transplantation of embryonic gonads, testes as a rule induce sex reversal in ovaries without being greatly modified themselves. In some cases domi- nance of the testis is so extreme that no real reversal of the ovary occurs, only an almost complete suppression and sterility. This type of response is seen, for example, in parabiotic pairs of the wood frog (Witschi, 1927) and in the newt Triturus (Witschi and McCurdy, 1929), and is probably cor- related with a constitutional inadequacy of the medullary component in the embryonic ovaries of the species in question. In other species, on the other hand, a severe initial repression of the ovary is followed by a de- layed reversal, which may have a prolonged course but which eventuall}^ may be quite complete. In parabiotic pairs of certain spe- cies of Ambystoma (Fig. 2.7) there is a se- vere inhibition of the ovary before active transformation is initiated, and when rcver- 88 BIOLOGIC BASIS OF SFA' Fig. 2.7. An extreme degree of inhibition and reduction in certain regions of an ovary under the dominance of a well developed testis (Humphrey, 1942). A. Level showing sterile medulla above, with degenerate cortical zone below. B. Medulla with rete cord and a single germ cell above, small cortical remnant below. C. Region showing complete atrophy. (From Biological Symposia, Vol. IX, Jacques Cattell Press, Lancaster, Pa.) *^-V: •4 Fig. 2.8. Sections Uuoiifiii a tian.sforniing o\;ny in an older case. Tlir cortex is extremely reduced and the medidlary area is well differentiated as a testis. In B, except for the cortical renmant, the histologic picture is that of a normal testis of intermediate development, with well defined lobules (Humphrey, 1942; cf. Fig. 2.7). sal begins it may be confined to local re- gions of the ovary. Transformation may set in independently at several sites, resulting in localized masses of testicular tissue which are, however, histologically normal (Fig. 2.8). Ultimately all renmants of cortex dis- ai)i)ear and transformation is complete. Such individuals are capable of breeding as males (this depends on the new testis establishing proper connections with the duct system) HORMONES IX DIFFERENTIATION OF SEX 89 notwithstanding they have the genotype of the opposite sex (for a discussion see Hum- phrey, 1942). To insure invariable predominance of the ovary in sex reversal it is usually necessary to provide a marked advantage in size and rate of development in favor of the female. This can be done experimentally by resort- ing to heteroplastic combinations (Fig. 2.9). In parabiotic pairs composed of two species of very different size {e.g., Amhystoma ti- grinum-Amhy stoma maculatum) and with a corresponding difference in growth rate, when the members are of different sex the larger species is almost invariably domi- nant (Fig. 2.11; Burns, 1935). When the large partner is a female the ovaries are enormously larger than the testes of the male and are always normal. The testes in some cases undergo reversal almost from the beginning of differentiation, and toward metamorphosis are represented by very small ovaries which contain a few well de- veloped ovocytes. However, in most indi- viduals transformation sets in after consid- erable differentiation has occurred, the testis cords becoming hollowed out to form ovarial sacs (Fig. 2.11) while the cortex persists and grows rapidly. At metamorphosis males are either completely transformed or the process is far advanced. Complete transformation of this type has also been reported by Wits- chi (1937) in A. tigrinum-A. jefjersonianum pairs. A similar result is obtained when single gonad primordia are transplanted hetero- plastically by Humphrey's method (Fig. 2.4) . In individuals bearing gonads of differ- ent sex, when the ovary is of the larger spe- cies it is dominant regardless of whether it belongs to the host organism or was derived from the graft. Histologically the reversal process is the same as in parabiotic pairs (Humphrey, 1935a, b). For fuller discus- sions of species and racial differences as they affect physiologic sex dominance and the capacity of the gonads to undergo re- versal in different species see Witschi (1934, B Fig. 2.9. Heteroplastic combinations uniting diiierent species of salamander {Amhystoma tigrinum and A. punctatum) which differ greatly in eventual size and rate of growth. A. Ven- tral view of paired embryos just after operation, showing fusion in the cervical region (punc- tatum member at left). B. A pair after metamorphosis showing the great difference in size; the larger animal is the tigrimnii member. 90 BIOLOGIC BASIS OF SEX 1957). Humphrey (1942), and Gallien (1955). The progress of sex reversal, and the mechanism by which the transformation is effected, can be analyzed histologically only when it takes place as a secondary process, after a certain amount of sex differentiation has previously occurred. In this case both histologic components are distinctly repre- sented but the normally recessive compo- nent seeks to become dominant; ovaries become testes by regression of the differenti- ated cortex accompanied by growth and dif- ferentiation of the medullary element (Figs. 2.8 and 2.10), and testes are converted into ovaries by the reverse process (Figs. 2.6C and 2.11A-D). The mechanism is flexible, however, and there is much variability, even among individuals in the same experiment, with respect to the stage at which reversal sets in, its progress, and its final outcome. In some cases removal of the dominant go- nad after reversal is far advanced may be followed by a second reversal toward the original sex (Humphrey, 1942). However, transformation is not always a secondary process, set in action only after a certain amount of differentiation has al- ready occurred; as noted above, when the ciuantitative disparity between the interact- ing gonads is sufficiently marked reversal may proceed from the earliest stages of dif- ferentiation. In this case the term "reversal" is less apt since there were no previous his- tologic steps to be retraced. Transformation is indicated chiefly by the unbalanced sex ratios at the end of the experiment; but in certain cases it is confirmed by character- istic histologic peculiarities (Burns, 1935, Fig. 28; Witschi, 1937, Fig. 39). In the het- eroplastic grafting experiments of Hum- phrey, on the other hand, direct proof is available since in many cases the sex of the grafted gonad, although conforming with that of the host, differs from the sex of the donor animal which is reared to provide a direct control. Although primary reversal of sex differ- entiation, as described above, occurs more readily when a marked disparity in size leads to early dominance, it may also occur under conditions which greatly retard de- H^iH^ Fig. 2.10. Two views of an ovary of Atnbystoma tigrinuin uudcigoing reversal under the influence of the testes of a male partner. The cortical zone, witli characteristic early ovo- cytes, is still prominent; however, medullary development is i)roceeding and in tlie region rei^resonted in B, testis lobules are forming. (From l^ K. Buiiis, J. Exper. Zool., 55, 123-129, 1930; 60,339-387, 1931.) HORMONES IN DIFFERENTIATION OF SEX 91 ^*V^ .^v- _^' A S-.-' / V "-^~ .\:!^> Fig. 2.11. Stages in the transformation of testis to ovary in the male (punci.ii luii ) imni- ber of an Ambystorna tigrinum-A. punctatum pair, joined in heteroplastic parabiosis (Burns, 1935). yl to D show, at successive levels in the same gonad, the degeneration of the medulla by vacuolation of the rete canals and lobules, accompanied by persistence and growth of the cortex. E shows a section through one of the dominant and entirely normal tigrinum ovaries; a gross picture of these ovaries is seen in F. (From R. K. Burns, Anat. Rec, 63, 101-129, 1935.) velopinent and delay the beginning of sex differentiation. This appears to be the ease in the first parabiosis experiments (Burns, 1925) in which the method of joining inter- fered with feeding and resulted in a severe retardation of growth. Such pairs developed so slowly that sex differentiation was de- layed for weeks and in many cases for months. When it eventually took place members of a pair were almost invariably of the same sex (c/. Humphrey, 1932) al- though there was great variation in the size and stage of differentiation of the gonads. In this experiment the usual physiologic dominance of the male was also disturbed, male-male and female- female pairs appear- ing in nearly equal numbers.^ The manner ^ The author's interpretation of the results in this experiment has often been questioned by Witschi. However, all but a small part of this ma- terial was subsequently re-examined by Humphrey (1932) whose study confirmed the original conclu- sions except for minor details in\-olving at the most only eight pairs. In Humphrey's opinion there were certain histologic indications that these in which extremes of temperatures induce sex reversal in tadpoles through a differen- tial inhibitory effect on the medullary or the cortical component of the embryonic gonad has been referred to earlier. The above re- sult may have a similar physiologic basis if, under prolonged repression, the usually dominant male gonad should prove to be more susceptible to unfavorable conditions than the female. 3. Administration of Steroid Hormones Since sex hormones of adult type became available in pure form their effects on the differentiation of sex have been tested in many species of amphibia. They are readil}' administered in two ways, by injecting di- rectly into the body cavity or, in aqueous so- lution, by adding them to the water in which the larvae are reared. The results on the whole are striking; in certain species there pairs were originally heterosexual, although trans- formation had proceeded to the point where a com- plete reversal was imminent. 92 BIOLOGIC BASIS OF SEX is complete transformation of ovary to testis or of testis to ovary, and in some cases the transformed individuals have been proved functional and capable of breeding. In other si)ecies, however, negative, equivocal, and in many cases paradoxical results have been obtained by the use of the same substances. A hormone that completely transforms all individuals of the opposite sex in one species may have only a weak or impermanent ef- fect in another, or no effect at all in a third. Obviously the gonads of different species differ greatly in their responses to steroid hormones. There is also a correlation with sex. In some species the gonad of one sex undergoes reversal with relative ease whereas that of the other is difficult or im- possible to transform, although increased dosages are sometimes effective. In certain species in which the sex chromosome com- plex is known it is the homogametic sex that is readily reversible (Gallien, 1955). It is also clear that experimental conditions strongly influence the result. The time fac- tor, that is to say the stage of differentiation at which treatment is initiated, is obviously important; in general, the most complete transformations are obtained when the hor- mone has been present from the beginning of the differentiation process. Dosage is like- wise of great importance and the optimal dosage varies greatly from one species to another. A negative response at one dosage may become positive when the dosage is in- creased; on the other hand, strong "para- doxical effects" (stimulation of the charac- ters of one sex by the hormone of the other) are often encountered with high dosages which are absent at lower levels. At present it is not possible to give consistent explana- tions for all such contradictory results ; how- ever, better understanding is gained when they are classified into convenient cate- gories. The effects of male hormones on sex dif- ferentiation in frogs. On the whole, the most successful reversals obtained by the use of steroid hormones have been in frogs of the family Ranidae, of which some six species have now been studied with similar results. Both male and female hormones induce re- versal of sex in young tadpoles, but male hormones are more effective and far more consistent in their effects. In Rana tempo- raria treatment with testosterone propionate transforms all genie females into males. The transformation is complete and permanent; moreover, transformed individuals are capa- ble of functioning in their new capacity (Gallien, 1944). A similar transformation has been obtained in Rana sylvatica, a phe- nomenally low concentration of the hor- mone (1/500,000,000 parts dissolved in the aquarium water) inducing complete histo- logic transformation (Mintz, 1948). Com- parable results have been reported after use of the male hormone in several other species of this genus (Table 2.1). In the case of Rana catesbiana it was found necessary to "prime" the gonads by simultaneous treat- ment with gonadotrophin, otherwise they were unresponsive; when so treated, how- ever, complete transformation is obtained. The gonadotrophic substance alone initiates a precocious differentiation of sex but with- out any tendency to transformation, serving only to precipitate the normal differentia- tion process. Complete and permanent mas- culinization of females by testosterone pro- pionate has also been reported recently in a tree frog, Pseudacris (Witschi, Foote and Chang, 1958), and a virtual transformation at the age of metamorphosis in Rhacophonts (Iwasawa, 1958), indicating that other anu- ran families may resemble the Ranidae in their reactions to the male hormone. In marked contrast, male hormone is without effect on gonad differentiation in the toad (Chang, 1955). The effects of female hormones in the Ranidae. These are variable and less con- clusive. The results frequently depend on dosage and the effective dose may vary greatly in different species. Estradiol benzo- ate in weak doses has a slightly feminizing action in male tadpoles of Raiia temporaria (Gallien, 1941), but stronger doses produce complete feminization in an "undifferenti- ated race" (one in which sex differentiation of males occurs relatively late) of the same species, all tadpoles at metamorphosis be- ing females (Gallien, 1940, 1955) . It is note- worthy that in this case both genie constitu- tion (race) and the dosage of the hormone may be factors in the result. In other ranid species estradiol, administered in low dos- ages during the period of sex differentiation, has a completely feminizing effect ; at meta- HORMONES IN DIFFERENTIATION OF SEX 93 TABLE 2.1 The ejects of synthetic male and female sex hormones on the differentiation of the gonads in various species of amphibians. The cases listed are those in which a complete, or near complete, and histologically norma transformation was achieved by the age of metamorphosis. In some species the reversal wa permanent and functional. For details see text. ACTION OF MALE HORMONE ON FEMALES SPECIES INVESTIGATORS RESULT COMMENT RANA TEMPORARIA GALLIEN 1938, 1944 COMPLETE TRANSFORMATION PERMANENT AND FUNCTIONAL RANA SYLVATICA MINTZ 1948 COMPLETE TRANSFORMATION AT METAMORPHOSIS DOSAGE 1/500 000 000 IN ACQUARIUM WATER RANA PIPIENS FOOTE 1938 COMPLETE TRANSFORMATION- TREATMENT FOR 65 DAYS RANA CATESBIANA PUCKETT 1939, 1940 COMPLETE TRANSFORMATION AT METAMORPHOSIS ADMINISTERED WITH GONADOTROPIN RANA CLAMITANS MINTZ, FOOTE 8 WITSCHI 1945 COMPLETE TRANSFORMATION- TREATMENT FOR 95 DAYS SOME PRODUCED SPERM RANA AGILIS (DALMATINA) VANNINI 1941, PADOA 1947 COMPLETE TRANSFORMATION PSEUDACRIS NIGRITA WITSCHI, FOOTE 8 CHANG 1958 COMPLETE TRANSFORMATION EFFECT PERMANENT RHACOPHORUS SCHLEGELII IWASAWA 1958 TRANSFORMATION ALMOST COMPLETE AT THE AGE OF METAMORPHOSIS ACTION OF FEMALE HORMONE ON MALES PLEURODELES WALTLII GALLIEN 1954 COMPLETE TRANSFORMATION PERMANENT AND FUNCTIONAL XENOPUS LAEVIS GALLIEN 1953 COMPLETE TRANSFORMATION PERMANENT AND FUNCTIONAL RANA TEMPORARIA GALLIEN 1941, 1944 COMPLETE TRANSFORMATION EFFECT NOT PERMANENT RANA ESCULENTA PADOA 1938, 1942 COMPLETE TRANSFORMATION AT METAMORPHOSIS AT LOW DOSAGES ONLY RANA SYLVATICA WITSCHI 1952, 1953 COMPLETE TRANSFORMATION AT METAMORPHOSIS AT LOW DOSAGES ONLY RANA CATESBIANA PUCKETT 1939, 1940 COMPLETE TRANSFORMATION AT METAMORPHOSIS ADMINISTERED WITH GONADOTROPIN BUFO AMERICANUS CHANG 1955 COMPLETE TRANSFORMATION AT METAMORPHOSIS EFFECT NOT PERMANENT j morphosis all tadpoles are females (Table 2.1). This result has been reported in R. esculenta (Padoa, 1942), in R. sylvatica (Witschi, 1951, 1952, 1953), and Puckett (1939, 1940) obtained the same effect in R. catesbiana when a gonadotrophin was given simultaneously with the estrogen. However, such transformations, although histologi- cally complete, are not in all cases perma- nent (Gallien, 1955) ; moreover, the effects of high dosages of the same hormone may be quite different, as wdll be shown. For other species only partial transformations have been found, as in R. clamitans (jMintz, Foote and Witschi, 1945) and in R. pipiens (Foote, 1938). A similar type of incomplete transformation by the female hormone has recently been reported in the tree frog, Pseudacris (Witschi, Foote and Chang, 1958) , again suggesting that in their pattern of response to sex hormones the Hylidae re- semble the Ranidae. Various other anuran species have yielded divergent results. In the primitive frog, Xenopus laevis, complete and functional transformation of males is effected by an aqueous solution of estradiol benzoate (Gal- lien, 1953), and in the toad {Bufo ameri- canus, Chang, 1955) low doses of estradiol completely transform testes into ovaries although high doses have little effect. Fi- nally, in Discoglossus the effect of estradiol is purely feminizing but the transformation is incomplete, the males exhibiting all de- grees of intersexuality without obvious re- lation to dosage (Gallien, 1955). Paradoxical ejfects. Thus far emphasis has been placed chiefly on cases in which sex hormones have acted in a sex-specific manner, each type of hormone directly or indirectly promoting the development of structures of the appropriate sex, while in- hibiting or behaving in neutral fashion to- ward those of the other. Reference has been made more than once, however, to the fact that in other cases the effects are just the reverse of theoretical expectation and op- posed to the concept of hormones as specific 94 BIOLOGIC BASIS OF SEX sex-differentiating agents. Anomalous or paradoxical results of this kind have ap- peared in experiments with both types of hormone, and involve not only the gonads but other sex structures as well. Such a result was first reported by Padoa (1936) who found that a crystalline form of female hormone (Crystallovar) had a strong masculinizing effect on the sexual differen- tiation of tadpoles of Bana esculenta, all gonads developing as testes. This unex- pected result (the so-called "paradoxical effect") was confirmed by others and has been found to be usually associated with the use of high dosages. In the course of time it was shown in this and in two other spe- cies [Rana temporaria, Rana sijlvatica) that the same hormone (estradiol) may have diametrically opposite effects when administered in different dosages. As was emphasized earlier, low doses have a proper feminizing action, producing all female in- dividuals according to theoretical expecta- tion; with high dosages, on the contrary, only males are obtained, and at intermedi- ate levels all individuals become intersexual (Padoa, 1938, 1942; Gallien, 1941, 1955; Witschi, 1952, 1953). Indeed, identical amounts of the same substance may have opposite effects when different solvents are employed. Administered in oil the effect on the gonads is feminizing but in aqueous so- lution complete masculinization occurs (Gallien, 1941). This also would appear to be a dosage effect since in aqueous solution the rate of uptake is presumably much faster than in oil. There are no histologic indications in the above experiments as to how the paradoxical effect is mediated. But, although such effects are found in various ranid species, they do not occur in Dis- coglossus regardless of dosage, thus empha- sizing the importance of species differences in the phenomenon (Gallien, 1955). Male hormones also produce paradoxical effects on the gonads and, although the doses employed have generally been high, it again appears that the result often de- pends on the species tested. The same dose of the same substance may have opposite effects in different species. Testosterone or cthinyl-testostcrone in large doses have strong feminizing effects on the testes of the salamander Pleurodeles, whereas the devel- o])ment of the ovaries is retarded but other- wise unaffected, a typical paradoxical effect (Gallien, 1950, 1955). Ethinyl-testosterone has the same effect in Discoglossus, but in Rana temporaria this hormone has only the expected masculinizing action. Such differ- ences in response may arise from differences in sensitivity on the part of the gonads or gonad components; a dose which is rela- tively large for one species may not be so in the case of another. The effects of sex hormones in urodele am- phibians. In urodele amphibians the effects of sex hormones on the differentiation of the gonads are perhaps even more variable; however, as opposed to the situation in the Anura, it is the female hormones which are more effective in producing reversal than the male (Gallien, 1955). In only one in- stance, the newt Pleurodeles, has a func- tional transformation of sex been achieved (Gallien, 1954). In this species prolonged treatment with estradiol benzoate com- pletely reverses the differentiation of all males, some of which become capable of laying eggs. Varying degrees of transforma- tion have been reported in other urodele genera after shorter periods of treatment, in Amby stoma (Burns, 1938a; Ackart and Leavy, 1939; Foote, 1941) and in Hynobius (Hanaoka 1941a). There was great varia- tion in the timing of treatment and in the dosages employed in these experiments; the incomplete character of the reversal may be due in part to such factors, but the role of species variability must also be great. On the other hand the male hormone, in marked contrast to its dominating role in the Anura, has but a limited transforming action in Urodeles. Indeed, it frequently, but not always, produces paradoxical effects icf. Burns, 1939c; Foote, 1941; Bruner, 1952). In Pleurodeles, in which the males arc completely transformed by estradiol, the effect of testosterone is limited to a severe inhibition, which affects the gonads of both sexes but is more extreme in males (Gallien, 1955). Medullary development is almost completely suppressed, and after an interval of recovery the vestigial gonads give rise almost exclusively to rudimentary ovaries, a result mentioned pi'e\-i()usly in discussing paradoxical effects. In tliis case, however, there is clear histologic evidence as to how HORMONES IN DIFFERENTIATION OF SEX 95 the effect is mediated. Reversal is caused in- directly by a severe inhibition of mesoneph- ric development. Since the sex cords which give rise to the medulla of the testis are de- rived from the mesonephric blastema, in- hibition of this tissue prevents their forma- tion. In the absence of proper medullary development, the cortical rudiment of the testis eventually becomes active to produce an ovary. This, apparently, is another ex- ample of spontaneous differentiation of the heterotypic gonad component when released from domination. A summary of the effects of steroid hor- mones in am'phihians. Sex hormones of adult type, such as testosterone and estradiol, have effects which vary greatly in different taxonomic groups of amphibians and also according to experimental conditions, such as dosage, timing, and duration of treat- ment. In many species their effects are spe- cific to a degree, closely simulating the effects expected of natural hormones. Histo- logically complete and in some cases func- tional transformations of the gonads have been produced in a number of species, in- volving two orders and several families (Table 2.1). Nevertheless, in other species only partial or temporary reversals are ob- tained, and negative or even paradoxical re- sults have come from use of the same hor- mones. Constitutional differences between taxonomic units obviously underlie some of the conflicting results. Sex genotype is also involved, because in a particular species reversal may proceed easily in one direction whereas in the other it is difficult or im- possible to produce. Following in part Gal- lien (1955), the results may be tentatively grouped as follows: a. In the higher anurans of the family Ranidae, and perhaps also in the Hylidae, the male hormone induces complete, and in many species a permanent reversal of sex. The action of female hormones on the con- trary is highly variable; transformation may be incomplete or unstable, and with high dosages paradoxical or masculinizing effects often appear. On the other hand, loiv doses of the same hormone have in many cases proper feminizing effects in the same species. In one species (Rana catesbiana) complete reversal of sex in both directions has been obtained. b. In certain urodeles and lower anurans, female hormones induce a transformation of male gonads which may be complete and stable, as in Pleurodeles and Xenopus, or partial, as in Discoglossus and various spe- cies of Ambystoma. The extent to which partial reversals are attributable to particu- lar experimental conditions is uncertain. Male hormones in general are much less ef- fective, and are prone to induce a paradoxi- cal inhibition or a feminization of the testis. These effects have in some cases been shown to be mediated indirectly, through an in- hibition of nephrogenesis which suppresses the differentiation of the medullary sex cords. The paradoxical effects of female hor- mones, on the other hand, are in many cases a matter of high dosages; how such effects are exerted is unknown but the action is probably indirect. This point will be dis- cussed elsewhere in connection with the problem of paradoxical effects on other sex structures. C. SEX REVERSAL IN AVIAN GONADS 1. Orgariization of Avian Gonads In avian as in amphibian gonads a spe- cific morphologic basis for sex reversal ex- ists during early development in the form of medullary and cortical components which have the usual potentialities. In birds, how- ever, the situation is complicated by the peculiar lateral asymmetry which affects in some degree the entire genital system and which is especially pronounced in the fe- male of most species (Fig. 2.12). The sum- mary which follows is based primarily on the chick (for a fuller account see Willier, 1939). In the left embryonic ovary the pre- ponderance of the cortex is great, even in the early stages, whereas in the rudimentary right ovary the cortex is essentially absent, being briefly represented by a transient germinal epithelium which disappears even earlier than that of the testis (Wolff, 1948) . In fact, the right ovary virtually ceases to develop at a stage when only medullary tissue (primary sex cords) has been laid down. In the male the asymmetry is mor- phologically less marked but it is expressed nevertheless in the better development and longer survival of the germinal epithelium (potential cortex) on the left testis. These 96 BIOLOGIC BASIS OF SEX INDIFFERENT STAGE mwim hwwvm RIGHT GONAD LEFT GONAD EFFECTS OF ? HORMONE ON MALE EFFECTS OFd* HORMONE ON FEMALE i005mg Mlko.M Omg \l'^\i\i\i\llll/l/yf\ LEFT TESTIS IWA RIGHT OVARY LEFT OVARY Fig. 2.12. Diagrams sliowing lateral differences in gonad organization in the chick embryo with respect to the representation of cortical and medullary elements; and differences in reaction to sex hormones based on these differences. (After N. T. Spratt, Jr., and B. H. Willier, Tabulae Biologicae, 17, 1-23, 1939). Note the stronger representation of the cortical component in the left gonad in both sexes, and its influence on the responses of gonads to hormones in relation to dosage. For details see text. structural differences are correlated with different capacities for sex reversal under experimental conditions, as will appear.^ The experimental study of sex differentia- tion in birds has been limited largely to two domestic species, the chick and the duck. Histologic sex differences first appear in the gonads of these species around the seventh and the ninth days of incubation, respec- tively, but the future pattern of develop- ment is essentially determined much earlier. This is shown by the fact that when the sexually indifferent gonads of chicks are transplanted at the genital ridge stage to the choi'ioallantoic membrane of another em- bryo, they continue in most cases to develop independently, in accordance with genotype, giving rise to typical testes or ovaries, and in the case of gonads of female constitution to characteristic right or left ovaries as well ° The spontaneous reversal of sex that frequently follows removal of the dominant left ovary in the young female chick is an example, and the basis for this phenomenon lies in the predominantly medullary ciharacter of the rudimentary right ovary as described earlier. (Willier, 1933, 1939j. The same capacity for self-differentiation has been demon- strated under the more radical conditions of isolation. Histologically undifferentiated gonads of either species when cultured in vitro differentiate into testes, or into right and left ovaries of characteristic structure (Wolff and Haffen, 1952a). In some cases there is injury to the germinal tissue under culture conditions; the gonads may show a reduction in the number of germ cells, or in some cases complete sterility, but other- wise the structure is normal (Fig. 2.13). Duck gonads seem to be more hardy under conditions of culture than those of the chick, and in general show better growth and liisto- logic differentiation. 2. Effects of Administering Pure Hormones As noted earlier, sex reversal in the gon- ads of birds was not demonstrated experi- mentally until \)uve hormones became avail- able. The first successful experiments were those of Kozelka and Gallagher (1934) ; Wolff and Ginglingcr (1935) ; Willier, Gal- HORMONES IN DIFFERENTIATION OF SEX 97 Fig. 2.13. Histologic difYerentiation in the gonads of duck embryos developing in vitro, after isolation just at the beginning of sexual differentiation (Wolff and Haffen, 1952a). A. Normal form and histologic differentiation of the testis in comparison with an ovary develop- ing under the same conditions (B). C and D show, respectively, the structure of these gonads under higher magnification. (From Et. Wolff and K. Haffen, J. Exper. Zool., 119, 381-404, 1952.) lagher and Koch (1935, 1937) ; and Dant- chakoff (1935, 1936) who introduced steroid hormones into incubating eggs before the beginning of sex differentiation. The results vary in detail but are consistent in the main outlines; they may be stated briefly, follow- ing chiefly the reports of Willier, Gallagher and Koch and of Wolff and Ginglinger. Female hormones (estrone or estriol) do not significantly affect the differentiation of embryonic ovaries but testes are highly transformed. Because of the better develop- ment and longer survival of the germinal epithelium the left testis is more amenable to reversal than the right. Relatively low doses convert it into an ovotestis. The distal ends of the medullary cords become hol- lowed out into tubular structures like the medullary cords of the ovary (Fig. 2.14.4) ; at the same time a zone of cortex develops peripherally, arising as a proliferation of the germinal epithelium. A small, unchanged medullary mass usually persists at the hilus. But with larger doses even this may dis- appear and the cortex becomes much thicker. Such cases are practically indistinguishable from ovaries. The right testis, however, is more difficult to transform. In the above ex- periments it was not greatly modified at lower dosages ; even when the left testis was almost completely transformed into an ovary the right never entirely lost its tes- ticular character. Because of the poor de- velopment of the germinal epithelium its capacity to produce cortex is limited. How- ever, Wolff (1948) made a special study of the right gonad in both sexes, assuming that stimulation of the gonad at an earlier stage, before regression of the germinal epithelium can be detected in either sex, might reveal a greater capacity for cortical differentia- tion. To insure rapid action a water soluble form of the hormone was used. In this way a considerable differentiation of cortex was obtained. The importance of a persistent search for the proper experimental condi- tions is again demonstrated. Male hormones, on the other hand, are less effective in transforming the embryonic ovaries of birds. Again lateral differences in 98 BIOLOGIC BASIS OF SEX B^\^ Fig. 2.14. Hi.Ntolojiic m'X rraiisl'ormation in the te.sles uf cluck eiiil)iyu« lrcate(i with fe- male sex hormones. A. Ovotestis produced from a left testis by treatment with a dose of 0.2 mg. of estriol. Note the presence of a thick cortex peripherally, and the reduction of the testicular tissue to a hilar mass of medullary cords. The intervening highly vacuolated tissue is characteristic of ovarian medullary cords. B. Section through the cortex and medullary region of a left tcslis coinplolcl}- transfoinied into an ovary by a dose of 2.0 mg. of estrone. Note the thick cdilrx (aboxc) coxcicd \>y a liciniinal epithelium, and the loss of structure in the medullary ii'gioii Ixlow. (From B. H. W'illior, in Sex and Internal Secretio7is, 2nd ed., The Williams & Wilkins Co., 1939.) reaction are found due to the different liisto- logic constitution of the right and left pri- mordia, and the results also differ when different forms of the male hormone are em- ployed. After treatment with testosterone the cortex of the left ovary is reduced in thickness and shows degenerative changes; however, it does not entirely disappear. At the same time there is hypertrophy of the medulla and some of its cords acquire the solid structure of testis cords. The result is an ovotestis. Because of its predominantly medullary constitution the rudimentary right ovary is converted superficially into a testis-like gonad. The medullary mass hypertrophies and some of the cords arc transformed into testis cords. In general, larger amounts of hormone are required to transform ovaries than for the conversion of testes (for summaries see Willier, 1939; Wolff, 1950). After hatching there is a tend- ency for experimentally modified gonads to revert toward the original sex (Wolff, 1938) . Similar conditions of reversal have been produced in the embryonic gonads of ducks by hormone treatment (Lewis, 1946) . The problem of the jiaradoxical action of hormones presents itself again in the case of a^•ian gonads. Certain male hormones of ui'inary origin (androsterone, dehydro-an- drostcrone) have a marked feminizing ef- fect, like that produced by t'cnuile hormones. In relatively large doses both substances induce cortical differentiation in testes, es- pecially the left, which may be transformed into an" ovotestis (Willier, 1939; Wolff 1938). Other androgenic substances have like ef- fects, but again it has been shown that they ai-e not jiroduced by low dosages. However, as the concentration of the hormone is raised HORMONES IX DIFFERENTIATION OF SEX 99 the degree of intersexiiality and the number of intersexual gonads steadily increase (Wolff, Strudel and Wolff, 1948). Since various accessory sex structures also show paradoxical reactions the problem will ap- pear again. 3. Effects of Grafting Gonads into the Coe- lomic Cavity The demonstration that steroid sex hor- mones are capable of inducing sex trans- formation in avian gonads led to a reinvesti- gation of earlier failures to obtain reversal by means of chorioallantoic grafting. Even- tually it was shown that the difficulty was largely a matter of the method. When gonad primordia are transplanted directly into the coelomic cavity of a host embryo of different sex, varying degrees of transformation, or even a virtual reversal, are obtained. The first experiments of this type were only a partial success (Bradley, 1941). Em- bryonic gonads of the chick and the duck, isolated at 96 to 120 hours of incubation, were inserted into the body cavity of host embryos through a small slit in the somato- pleure, using both homoplastic and hetero- plastic host-graft coml)inations. In the case of the chick, host embryos were always con- siderably younger than donors. In all cases the grafts underwent primary sex differenti- ation in accordance with genotype, and only a small minority showed specific modifica- tions. The results were rather inconclusive because in no case were the changes of a conspicuous character, and there was great variability in the growth and differentiation of the grafts, making it difficult to assess the significance of the modifications. In some cases changes of the same type ap- peared in the gonads of the host embryo. The modifications noted by Bradley fall into three main classes. (1) Vacuolation of the medullary cords of testes growing in fe- male hosts (in a few cases) caused them to resemble the hollow medullary cords of ovaries. (2) In some cases ovaries growing in male hosts developed solid medullary cords of male type. (3) Rudimentary right ovaries (always testis-like in character) had a tendency to become enlarged when grow- ing in male hosts. A similar effect was some- times seen in the right ovaries of hosts bear- ing testis grafts. No ready explanation was available for the inconstant occurrence of these effects or for their quantitative varia- bility, because no clear correlation was found between the degree of modification and the relative proximity of the interacting gonads. Finally, similar changes appeared in a few cases when the host-graft combina- tions involved the same sex; consequently the specific character of the modifications was left in doubt. The matter was clarified by the experi- ments of Wolff (1946) who used a modifica- tion of the method with better results. Grafts taken from older embryos (6 to 11 days) were implanted into hosts of about 50 hours of incubation. Under these conditions a striking transformation of gonad differentia- tion was obtained in the host, and in addi- tion the developing gonaducts [q.v.) were strongly modified. Ovaries grafted into male hosts induced differentiation of cortex on the left testis to such an extent that it some- times approached the structure of an ovary. The right testis (which was usually more distant from the graft) was less modified but its growth was inhibited. On the con- trary, implantation of a testis in the same manner produced no important effect on the differentiation of the ovaries of the host but the development of the Miillerian duct was strongly inhibited indicating that the graft is endocrinologically active. In their histo- logic character the effects of gonad grafts are similar to those produced by crystalline hormones, differing only, as a rule, in being more localized in relation to the position of the graft. The demonstration in this experi- ment that, physiologically, the ovary is the dominant gonad in birds is consistent with the earlier observation that relatively larger doses of pure hormones are required for the transformation of ovaries than for testes. These positive results after so many fail- ures suggested that the ineffectiveness of chorioallantoic grafts in the earlier experi- ments was possibly a matter of hormone production, failure of the graft to maintain a sufficient level of the hormone in the blood. This view is substantiated by the later ex- periments of Huijbers (1951) who showed that multiple grafts of well differentiated testes on the chorioallantois have marked 100 BIOLOGIC BASIS OF SEX effects on the accessory sex structures of the host, similar to those induced by intra-em- bryonic grafts. 4. Sex Reversal in Vitro More recently the technique of culture in vitro has been employed by Wolff and his collaborators with great success to study the development of embryonic gonads, and it has been possible to produce typical reversal of sex differentiation in vitro by two meth- ods. Prospective ovary and testis of the chick or duck, isolated at the very beginning of sex differentiation and placed in close contact in the culture dish,^ become firmly fused, facilitating the transmission of hu- moral influences. As in the case of gonad grafts in the coelomic cavity, the ovary un- der such conditions proves to be the domi- nant gonad (Wolff and Haffen, 1952b). It readily induces cortical differentiation on the testis, which becomes an ovotestis, and may even approximate closely the structure of a normal ovary of the same age (Fig. 2.15). The same type of transformation oc- curs in testes after introduction of estradiol benzoate into the culture medium. With respect to the histologic character of the reversal process, the resemblances be- tween the effects of gonad grafts implanted in the body cavity, crystalline hormones in- jected into the whole organism, and the re- sults of the same procedures applied to iso- lated gonad primordia in vitro are extremely close. The in vitro studies demon- strate again the autonomous character of the differentiation process, and its flexibility in the presence of extraneous hormones is shown to be independent of the organism as a whole. Hormones in vitro evidently act directly on the gonad mechanism. 1). TIIK PROBLEM OF SEX REVERSAL IN MAMM.\LIAN GONADS 1. Bisexual Potentialities in the Emhrijoiiic Gonads of Mam^nals In marked contrast with the striking ef- fects of steroid sex hormones on the differ- entiation of the gonads of birds and various " Combinations of gonads in the culture dish must initially be made at random but the other gonad of each donor is cultured separately in order to establish its sex. species of amphibians has been the failure thus far to ol)tain comparable effects in mammalian embryos with the exception of a single species, the North American opos- sum, a marsupial. Essentially negative re- sults have been reported for a number of species of placental mammal, in which preg- nant females were treated with relatively large dosages of sex hormones during the period of sex differentiation. Experiments of this type were carried out in the rat by Greene, Burrill and Ivy (Greene, 1942), and in the guinea pig (Dantchakoff, 1936, 1937), the mouse (Turner, 1939, 1940; Raynaud, 1942j , the rabbit (Jost, 1947 a) , the hamster (Bruner and Witschi, 1946; White, 1949), and the monkey (Wells and van Wagenen, 1954). With the single exception of the opossum (to be described later) the modi- fications induced are minor in character and are of three types: (1) a general retardation of growth and development of the gonads, without obvious signs of sex reversal, which occurs in both ovaries and testes and may be produced by either type of sex hormone;'^ (2) a variable degree of hypertrophy of the medullary elements of ovaries after treat- ment with male hormone, reported in only a few cases (Dantchakoff, 1939; Jost, 1947a; Wells and van Wagenen, 1954) ; and (3) the occasional persistence of localized patches of germinal epithelium on the surface of well differentiated testes, a condition which sometimes appears after treatment with either type of sex hormone. IVIinor changes of this character have not generally been accepted as convincing evidence of sex re- versal. Notwithstanding this array of negative findings, the failure of the embryonic gon- ads of placental mammals to respond defi- nitely to sex hormones can hardly be at- tributed to an inherent lack of bisexual potentiality. During the early stages of their development they show, histologically, the same evidences of bisexual structure as the gonads of other vertebrates, although typi- cally the bisexual phase is of relatively brief duration and the recessive sex com- " This effect is of common occurrence and is best explained as a depression of the gonadotrophic function of the anterior pituitary, a mechanism whicli is well established in adult organisms (Moore and Price, 1932). HORMONES IX DIFFERENTIATION OF SEX 101 .»■ c'tMi Fig. 2.15. Sex transformation in the testis of the duck, isolated in vitro at the beginning of sexual differentiation and cultured in close contact with an embryonic ovary (Wolff and Haffen, 1952b). A. The ovary of such a combination, showing the thick covering germinal epithelium and the vacuolated condition of the medullary region. This young ovary is es- sentially normal in structure. B. Intersexual condition induced in the testis under the in- fluence of the ovary. The heavy germinal epithelium representing the cortex is as well developed as in a normal ovary ; the meduUaiy region retains largely the compact structure of a testis, but signs of vacuolation are appearing. This gonad is an ovotestis. C and D represent, respectively, the ovary and the completeh' transformed testis in another experi- ment. The two gonads in this case show almost identical structure, featuring the thick cortex and vacuolated medulla of an ovary. In these experiments the other testis, cultured alone, developed normal testicular structure. (From Et. Wolff and K. Haffen, Arch. x\nat. microscop. et Morphol. exper., 41, 184-207, 1952.) 102 BIOLOGIC BASIS OF SEX Primary sex cords Rete cords Germinal epithelium A Germinal epithelium Medullary cords (primary sex cords) Cortical cords ^Germinal epithelium (secondary sex cords) Fig. 2.16. Diagrams illustrating schematically the main features of gonad differentiation in amniote embryos with reference to the origin of the medullary and cortical components. A. Origin of the primary sex cords (medullary cords) from the germinal epithelium. B. Gonad at the indifferent stage of sexual differentiation ; the well developed primary sex cords i-epresent the male or medullary component, whereas the germinal epithelium represents, potentially, the cortical component. C. Differentiation of a testis consists in the further development of the primary sex cords, and the reduction of the germinal epithelium to a thin, serous membrane, accompanied by development of the tunica albuginea. D. Differen- tiation of an ovary consists in reduction of the primary sex cords to medullary cords of the ovary, whereas the cortex is formed by continued development of cortical cords from the germinal epithelium. poneiit is often weakly represented (Fig. 2.16). In the ovary, medullary cords repre- senting the male component are present but tend to become vestigial in most species.^ In the embryonic testis the germinal epi- '^ Notable exceptions should bo mentioned in the case of certain species such as the mole (Godet, 1950) and the desman or "water shrew" (Peyre, 1955) in which, as a normal condition, the ovarian medulla is so strongly developed as to resemble a testis, and so active physiologically as to pro- duce strong masculinization of many parts of the genital tract. A somewhat similar development and hypertrophy of the medullary component also occurs in the fetal ovary of the horse (for the lit- erature on this unusual condition see Cole, Hart, Lyons and Catchpole, 1933; Parkes, 1954). thclium usually disapi)ears early, and with its involution the potentiality for cortical development is permanently lost. On the other hand, hermaphroditismus verus not in- fi'ec|iu>ntly occurs as a developmental anom- aly iu many mammalian species (including man), indicating the existence of a basic bipotentiality. As an example, an extensive literature dealing with this subject in ro- dents has recently been summarized by Hol- lander, Gowen and Stadler (1956) and Kirk- man (1958). Also it must be remembered that the classsical example of an embryonic gonad transformed by the action of a sex hormone is found in the freemartin. In some HORMONES IX DIFFERENTIATION OF SEX 103 freemartin gonads morphologic transforma- tion may be extreme, although the resulting testis-like structure is histologically abnor- mal and is almost invariably sterile (Wil- lier, 1921 ).» 2. Bisexual Potentiality in the Embryonic Ovary of the Rat One of the best known cases illustrating a well marked capacity for bisexual differ- entiation in a mammalian gonad is provided by the embryonic ovary of the rat. The gon- ads of rat embryos have been isolated at various stages, both before and during the period of histologic differentiation, and transplanted to various locations in adult hosts of both sexes, normal and castrate, beneath the capsule of the kidney (Buyse, 1935; Mclntyre, 1956), subcutaneously (Moore and Price, 1942), to the omentum (Holyoke, 1949) and into the anterior cham- ber of the eye (Torrey, 1950). In general, differentiation of the transplanted gonad proceeds without reference to the sex or the hormonal status of the host (certain minor exceptions will be noted later) ; however, there is a great difference in the behavior of testis and ovary after transplantation with respect to their capacity for autonomous differentiation. There is virtual agreement among all investigators that the testis pri- mordium from the beginning of its develop- ment possesses a remarkably stable organi- zation, and develops more or less normally in the various foreign environments, even when isolated before the beginning of histo- logic sex differentiation.^*^ The case of the ovary is entirely different; its organization appears to be extremely labile and it is in- capable of fully autonomous development until a relatively late stage of differentia- tion, after a well formed cortex is present. Before this stage (w^iich according to Tor- rey is reached about the 17th day of devel- opment) ovaries in a high percentage of cases either do not develop at all, or are ^ For an important exception in which a free- martin testis is well supphed with germ cells and essentially normal in appearance see Hay (1950). " Torrey considers that the self-differentiating capacity of the testis probably dates from the lay- ing down of the early gonadal blastema, i.e., the material of the primary sex cords, which occurs as early as the eleventh day of development. prone to undergo spontaneous reversal, due apparently to incapacity of the prospective cortical component to develop effectively in abnormal tissue environments. On the other hand, the medullary component of the trans- planted ovary suffers no such handicap and frequently assumes the lead in development. The embryonic ovary of the rat thus pro- vides a flexible system for the study of the morphogenetic capabilities of the cortical and medullary components at different stages of development and under different experimental conditions. When transplanted early in development prospective ovaries may give rise (Buyse, 1935) to structures of four types: (1) poorly developed grafts of indeterminate sex, (2) atypical ovaries of retarded development, (3) ovotestes in which both sex components are readily identifiable, and (4) rudimen- tary testes. As a group, ovaries are ad- versely affected by transplantation. Some fail entirely to develop the specific struc- ture of gonads (type 1, above) and those that do give rise to ovaries that are greatly retarded (type 2). On the other hand, the medullary component of the prospective ovary resembles the testis in possessing con- siderable powers of self-differentiation. Thus in many cases the two components de- velop together, resulting in an ovotestis; in still others the cortical element fails com- pletely to survive and the medulla alone develops, giving rise to a rudimentary testis. The development of types 3 and 4 is favored by the fact that cortical differentiation is almost always severely repressed. This may have the effect of releasing the medullary element from an inhibition normally im- posed by the dominant cortex. In all cases, however, the phenomenon of reversal was found to be unrelated to the sex of the host] it seems to occur spontaneously, as it were, in consequence of a disturbance in the nor- mal balance between cortical and medullary systems. A similar behavior is seen wdien entire re- productive tracts of rat embryos, including the gonads, are transplanted subcutaneously into hosts of various ages, male or female, and into castrate hosts of both sexes (Moore and Price, 1942). Again it was found thai the sex or the hormonal status of the host has no apparent influence on the result. 104 BIOLOGIC BASIS OF SEX Testes develop normally except that in cas- trate hosts there is some hypertrophy of the interstitial tissue, presumably in response to the gonaclotrophin of the host (a phe- nomenon also reported by Jost, 1948b I. Again many prospective ovaries give rise to gonads in which both cortex and medulla are well differentiated, the hypertrophied med- ullary cords sometimes approaching the structure of testis tubules. Cells resembling the interstitial cells of the testis are also found around these transformed medullary cords in grafts developing in castrate hosts. On the whole the ovarial cortex is better developed than in the experiments of Buyse, because perhaps the gonads were usually older and better differentiated at the time of transplantation. Similar forms of develojiment are found when embryonic gonads are transplanted to the omentum of adult hosts (Holyoke, 1949). Testes develop in a virtually normal manner regardless of the sex of the host; this author, however, describes certain ef- fects which appear relatively late, after the testis has acquired its characteristic tubular structure. These are: (1) repression of tu- bule growth in some cases, with degenera- tive changes, a condition which was ob- served only in grafts growing in female hosts; (2) an increase in the amount of in- terstitium present, similar apparently to that reported by Moore and Price. Trans- planted ovaries display the same variability as in the foregoing experiments; cortical development is adversely affected and some- times fails altogether. In all cases in which cortical structure could be well identified there was also more or less hypertrophy of the medulla, sometimes to the point where the gonads were classified as ovotestes. This latter condition was reported only in male hosts, and this is perhaps the only change that might be interpreted as a reversal of sex conditioned by the sex of the host. On this point the findings differ from those of Buyse, of Moore and Price, and of Torrey, all of whom reported similar changes but without relation to the host's sex. A further study of the problem was made by Torrey (1950). In his experiments the embryonic gonads were transplanted to the anterior chamber of the eye, using hosts of various ages and of both sexes. He con- firmed the main conclusions of Buyse and of Moore and Price (Holyoke's study was not then available for consideration), namely, that regardless of the stage at which the primordium is isolated, testes are capable of an autonomous and virtually normal development, irrespective of the type of host in which they develop, whereas ovaries vary greatly in the state of differen- tiation attained, depending on the stage of development at which they are isolated. Torrey paid particular attention to the importance of developmental age in the fate of grafted ovaries. When transplanted before the appearance of a definite cortical zone (zone of secondary sex cords), prospective ovaries show little capacity for development of cortex; on the contrary (as found by l^revious workers), there is a marked tend- ency to hypertrophy of the medullary com- ponent, leading in some cases to testis for- mation. This tendency is not influenced by the sex of the host; rather, it seems to be inherent in the state of organization of the primordium at the time of transplantation. In the young ovary the medullary compo- nent (primary sex cords) is already in existence; the cortex does not appear as a discrete tissue until much later, and only after a well defined cortex is present is the gonad capable of development as an ovary. The fate of the transplanted ovary appears, then, to be primarily a iiiatter of the self- differentiating capacities of the elements already formed and present in the primor- dium at the time of its isolation. The de- velopment of these elements is influenced also by the temperature of the eye chamber, a point not directly involved in the present discussion. In the foregoing experiments it has been emjihasized that the hormonal environment provided by the host seems to have no im- portant influence on the sexual differentia- tion of the transplanted gonads (for a par- tial exception as noted above see Holyoke) . The question thus arises whether the hor- mones of embryonic gonads might be more effective. An answer to this question was sought by Mclntyre (1956) who trans- planted the eml)ryonic testis and ovary to- gether beneath the capsule of the kidney of adult castrate hosts of both sexes. The gon- ads were ])laccd in close contact in order HORMONES IN DIFFERENTIATION OF SEX 105 to determine whether hormones or other diffusible substances might be produced, capable of modifying the differentiation process. As a control procedure, ovaries were transplanted alone, or in association with nongonadal tissues, into noncastrate male hosts. The results in most respects correspond with those already described. The testis was found to develop normally regardless of the sex of the host or the presence of a contigu- ous ovary. The behavior of grafted ovaries differed, however, according to whether they were associated with an embryonic testis, or developed alone or with other tissues (control operations). In the jEirst case the ovaries were strongly modified along the lines previously described. Some differenti- ated poorly or hardly at all, others showed fairly good development of the cortex with some primary follicles, but in the medullary area tubular structures resembling testis tubules were found. Still others had a few well formed follicles in the cortex, but again tubular structures were present in the me- dulla which sometimes contained ovocytes. The two last mentioned categories would seem to correspond to the "ovotestes," or ovaries with "transformed medullary cords" described by previous writers. In contrast, however, ovaries grafted alone, or with non- gonadal tissues, were found to differentiate in an almost normal fashion. It is at this point that the findings depart from those of other investigators. The conclusion was reached that the modifications observed in ovaries associated with embryonic testes are due to a substance produced by the testis, and considered to be of the nature of a medullary inductor. However, both with respect to the sever- ity of cortical inhibition and the degree of masculinization of medullary structures, these modified ovaries do not appear to differ significantly from those described by previous investigators when ovaries were grafted alone. The significance of the results rests then upon failure to obtain similar changes in the control ovaries. The age and state of differentiation of the control gonads at the time of transplantation is important. They were from donor fetuses of 15 days development, and although older than any used by Buyse they were still within the period during which Torrey found the ovary to be extremely labile in its differentiation. According to Torrey only a small proportion of ovaries aged 15 to 16 days developed as such (5 of 17 cases) whereas at an age of 17 days — after the cortical zone is estab- lished— ovaries are obtained almost without exception (10 of 11 cases). Further experi- ments are needed to clarify this matter. In all of the foregoing experiments there was general agreement (for a partial ex- ception see Holyoke) that the hormonal en- vironment provided by adult hosts of both sexes, intact or castrate, has no important influence on the sexual differentiation of the transplanted gonads, even in the case of ovaries which are still in a labile state. The reason for this is not clear. Possibly it is a matter of insufficient concentration of the host hormone (compare the case of the chick, p. 99), or it may be that after trans- plantation a certain interval, perhaps a crit- ical one, elapses before vascularization makes the graft accessible to the hormones of the host. On the other hand, it has been pointed out that in the embryos of placental mamtnals pure hormones thus far have pro- duced no significant changes in the dif- ferentiation of the gonads. The cjuestion remains whether there is an essential differ- ence between the sex hormones of adults and the hormones or sex-differentiating sub- stances elaborated by embryonic gonads. 3. Experimental Transjormation of the Tes- tis in the Opossum Up to now the clearest experimental dem- onstration of sex reversal in the gonad of any mammal, and the only one to be pro- duced by a steroid hormone, has been ob- tained in the gonads of young opossums (Didelphis virginiana) . In this species the embryonic testes, if taken in time, are read- ily transformed into ovotestes or even into "ovaries" of remarkably normal histologic structure by the action of estradiol dipro- pionate (Burns, 1950, 1955a, 1956b). The hormone is administered at short intervals, beginning at a stage of development corre- sponding to stage B in Figure 2.16. This is the condition found at birth in litters born at stage 34 of McCrady's series (McCrady, 1938). It is characterized by the presence of well developed primary sex cords which 106 BIOLOGIC BASIS OF SEX are just in process of separation from the overlying germinal epithelimii. The germi- nal epitheliimi, however, is still present as a layer of low, columnar cells and at this stage represents, potentially, the female compo- nent of the young testis. Normally the ger- minal epithelium does not survive long after birth. In the course of the first day of post- natal life irreversible changes occur which lead to its rapid involution. It is the pres- ence at stage 34 of a viable germinal epi- thelium which makes possible the subse- quent conversion of the testis into an ovary, since it is this layer which must produce the cortical zone of the transformed gonad by the proliferation of secondary sex cords. In so doing it plays precisely the same role as the corresi)onding layer in the develop- ment of the ovary. Treatment with estradiol dipropionate has been carried out over varying periods up to an age of 30 days postpartum or somewhat longer, after which survival becomes diffi- cult (Burns, 1939b). At the present time 46 male fetuses have been studied histologi- cally, comprising all the surviving males of 13 litters. Without exception, every speci- men shows histologic modifications of the type described below, the stage of transfor- mation attained varying only with the length of treatment. The process of trans- formation consists at first of a gradual in- hibition and suppression of testicular differ- entiation, accompanied by persistence of the germinal epithelium. At the same time the differentiation of the interstitial tissue is severely repressed (compare A and B, Fig. 2.17). Atrophy of the interstitial tissue has also been described by Raynaud (1950) in the testes of mouse embryos treated with estrogen. After an interval (which varies in different experimental litters and is appar- ently influenced by dosage) the germinal e})it helium again becomes active, producing secondary sex cords which form a cortical zone of varying thickness depending on the length of treatment (Fig. 2.181. The first essential in obtaining transfor- mation of the testis is the timing of the first treatment, which must not be later than Fig. 2.17. The effects of female hormone on differentiation of the testis in young opossums. A. Normal testis about 10 days after birth. Note tlie thin, .serous character of the epithehum covering the testis (originally the germinal epithelium), the presence of a distinct tunica albuginea, the prominent testis cords (prospective tubules), and the richly developed inter- stitium. B. Testis (at somewhat higher magnification) of a young male aged 14 days, modi- fied by the action of the female hormone estradiol dipropionate. Note the greatly reduced condition of the testis cords and interstitial tissue, the thick, spongy character of the tunica albuginea, and especially the survival of the germinal epithelium long after the stage at which it normally undergoes involution. HORMONES IX DIFFERENTIATION OF SEX 101 Fig. 2.18. A. Testis of an opossum aged 20 days, converted into an ovotestis by the action of estradiol dipropionate. Internally the reduced and disorganized medullary region is seen, separated from the external cortical zone by the well defined, fibrous tunic layer. The large, irregular ca\ity in the upper center, lined by a heavy eiuthelium, is the rete testis. B. De- tail at higher power of the cortex, showing the structure of the cortical cords (which are sterile) and the highly developed germinal epithelium. stage 34 if the germinal epithelium is to be preserved. In earlier experiments, in which treatment was begun after stage 35, there were no significant effects on the differen- tiation of the gonads, even in cases where almost complete transformation of the ac- cessory sex structures had occurred. It is now evident that in these experiments the first application of the hormone came too late to prevent involution of the germinal epithelium, thus precluding development of a cortex. Also of importance is the dosage, which must be kept at a low level to secure a good result. This point is crucial for the survival of germ cells in the developing cor- tex. High dosages always result in com- plete sterilitij of the cortical zone, even when this layer is otherwise well developed (Fig. 2.18). With lower doses, however, germ cells are found in limited numbers in the cortex, sometimes sparsely scattered, sometimes in small groups, and not infrequently these cells display the cytologic characters of young ovocytes (Fig. 2.20). Often there is a considerable growth of the cytoplasm and well formed primordial follicles are seen (Fig. 2.20B). Treatment with relatively low doses of estradiol (of the order of 0.2 to 0.3 fxg. per day) from birth to an age of 20 days pro- duces a remarkable transformation of the testis, which retains hardly any normal fea- tures (Burns, 1956b). Rather, it presents the appearance of a somewhat atypical ovary (Fig. 2.19B). The only remnant of testicular structure is a small, central nodule at the junction of the rete canals, and the massive cortical zone is covered externally by a thick germinal epithelium. The cortex of the transformed testis contains germ cells in considerable numbers, including a few large ovocytes. Views of cortical areas in gonads of this group are shown in Figure 2.20.4 and B. In more recent experiments, using still lower doses and a longer period of treatment (thus far the longest experi- ment has extended to an age of 33 days post- partum ) , the result is even more striking, the structure of the cortex in many cases ap- proximating that of normal ovaries. Always, however, certain remnants of testicular structure ]icrsist in the medullary region (Fig. 2.21, compare A and B). The number of ovocytes in the cortex is enormously 108 BIOLOGIC BASIS OF SEX Fig. 2.19. A. The testis of a normal male opossum aged 20 days for comparison with that of another male, B, treated for 20 days with a low dosage of estradiol dipropionate as de- scribed in the text. Only a remnant of testis structure survives as a nodular mass in the medullary region, representing straight tubules at the point of junction with the rete canals. Note the well developed cortical zone witli numerous germ cells and a heavy germinal epi- thelium. greater than in the preceding experiment. It is not clear whether this is due mainly to the lowered dosage or to what extent it is a result of multiplication of ovogonia present in smaller numbers in the younger gonads (see Fig. 2.195). In any case, dosage in some manner influences the survival and multiplication of the gonia. Although the cortex of the transformed testis is always well developed, there is great variability in the extent to which testicular structures have survived in the medullary zone. Some gonads exhibit well preserved male sex cords and present the picture of a typical ovotes- tis, whereas in others (Fig. 2.21B} only traces of the male elements remain in the form of degenerate sex cords and patches of fibrous tissue. In these cases transforma- tion is all but complete. Since this work is still in progress inter- pretation must be tentative. It seems that the primary effect of the female hormone is a strong inhibition of the testis, affecting both the primary sex cords and the inter- stitium. Both influences are apparent at an early stage (Fig. 2.17). Inhibition of testicu- lar differentiation i:)resumably i-)ermits sur- vival of the germinal epithelium, to be followed later by a renewal of activity pro- ducing secondary sex cords and the cortex. This course of events may simply be the result of release from an inhibition nor- mally imposed by the differentiating testis. Counterparts are seen, for example, in the spontaneous development of the medullary component in transplanted rat ovaries when cortical differentiation is interfered with, in the development of the rudimentary cortex in Pleurodeles after the medulla has been suppressed by testosterone (p. 94), or in the development of the heterotypic sex com- ponent after castration in the toad or the newly hatched chick. In the light of cur- rent knowledge regarding the secretory capacity of the embryonic testis (for dis- cu.ssions see Jost, 1953, 1957; Burns, 1955b, and later in this chapter) it is proba- ble that the primary condition for survival of the germinal epithelium is suppression of the interstitial tissue; cortical differentia- tion is presumably the consequence of es- cai)e from an inhibition normally exerted HORMONES IX DIFFERENTIATION OF SEX 109 fel!t".^*.Z '" WS *3i?^~ k' l't%^ .♦3^"* *A-. \"%. ^Mj Fig. 2.20. A. View at higher magnification (X 1000) of the cortex of ;i traiisiorme.l te.stis containing gonia, and other germ cells showing the early meiotic prophase stages of young ovocytes. B. Cortex of another transformed testis of the same experimental group showing the formation of primordial follicles (X 1000). by the testis hormone. For this there is no direct evidence; however, in typical ovo- testes, with a well developed cortex, the tubular elements may also at times be very well preserved but the interstitium is de- generate. This interpretation does not re- quire positive stimulation by the female hormone to promote cortical differentiation, but it does not exclude the possibility that this may occur. The germinal epithelium of transformed testes is strongly hypertrophied in comparison with that of normal ovaries of the same age (Figs. 2.18B, 2.195, and 20) , a condition which is seen also in the ovaries no BIOLOGIC BASIS OF SEX Fig. 2.21. A. The normal ovary of a young female aged 30 days; note the highly developed cortex and the absence of conspicuous structures in the medullary area. B. Transformed testis of a young male treated with estradiol dipropionate for a period of 33 days ; for details see text. Observe the remarkable development of the cortex associated, however, with dis- tinct remains of testicular structure in the medulla. of females receiving estradiol. Also, preco- cious growth of a certain number of follicles commonly occurs in the cortex of trans- formed testes (Figs. 2.195 and 2.20). This effect, however, may be exerted indirectly in response to gonadotrophic stimulation. It should be noted that in the only other case of an embryonic mammalian gonad transformed by hormone action, that of the freemartin, the reversal involves the con- version of ovary to testis. In the opossum the situation is reversed. In those cases of comjjlete, or near complete, transformation in amphibians, in which the sex chromosome complex has been determined, it appears that it is always the homogametic sex that is readily transformed (Gallien, 1955). This generalization would seem to apply also in birds and in the case of the freemartin. The opossum, however, is certainly an exception; the male in this species is heterogametic (Painter, 1922; Tijo and Puck, 1958; Gra- ham, 1956). In certain fishes at least sex genotype is apparently of no conseciuence .-^ince functional sex reversal proceeds eciually well in either direction (Yamamoto, 1953, 1958). V. The Role of Hormones in the Development of the Accessory Sex Structures The heterogeneous character of the vari- ous structures comprising the genital com- plex of the embryo has been previously emphasized as providing a basis for great variability in their behavior under experi- mental conditions. On the basis of embry- onic origin and morphologic relationships the accessory sex structures fall into three principal groups: (1) the embryonic sex ducts and related structures which are taken over from the primitive nephric system; (2) derivatives of the cloaca or the urinogenital sinus, derived at an early stage from the primitive gut; and (3) external organs of sex. Because of the great diversity of the so-called secondary sex characters in verte- brates, and because as a rule they become sexually differentiated only in jiostpubertal life, these structures can be considered only in special cases. Two distinct stages can be recognized in the development of the accessory sex struc- tures: an early phase which is independent of sex, and wliich follows a \-ii-tuallv identi- HORMONES IN DIFFERENTIATION OF SEX 111 cal course in all individuals, and a later phase, the period of sexual differentiation, which is chiefly hormone conditioned. Dur- ing the first phase the primordial structures necessary for the development of both sexes are laid down and develop in similar or identical fashion up to a certain point, at which stage each embryo possesses, morpho- logically and for a certain time, the capacity to develop into an individual of either sex (Fig. 2.22A). In this early, indifferent phase of development the sex primordia show lit- tle reactivity to hormones, capacity for re- sponse evidently requiring a certain degree of maturation in the reacting organs or tis- sues. The onset of sexual differentiation of the accessory structures follows the appear- ance of sexual differentiation in the gonads, and this is the phase of development in which control by hormones is predicated. A. DIFFERENTIATION OF THE EMBRYONIC GONADUCTS A complete account of the origin and nor- mal development of the embryonic sex ducts has been given by Willier (1939) and Burns (1955b) . In the embryos of most vertebrates both sex ducts are present and equally de- veloped throughout the sexually undifferen- tiated period. In many amphibians this primitive condition is retained throughout larval life or indefinitely ; the male, or Wolf- fian ducts, function as nephric ducts in both sexes and in females they are permanently retained in this capacity. The Milllerian ducts persist throughout life in the males of VAS DEFERENS MES0NEPHR05\5 EPIDIDYMIS VAGINAL CANAL BULBAR GLAND 1 H PHALLUS V i URINOGENITAL ^'i-*^ SINUS |1 > BULBAR GLAND POUCH YOUNG - ± 10 DAYS FEMALE a MALE-±35DAYS Fig. 2.22. Early (io\ clopnicnt and sexual differentiation in the genital tracts of young opossums. A. The lusrxu.il stage of development in a female embryo ±10 days of age, show- ing the paired gonaducts of both sexes, the sexually indifferent stage of the urinogenital sinus, and the undifferentiated genital tubercle or phallus. B. Male and female at about 35 days, when sexual differentiation is far advanced, showing the structures which develop from the primitive sex ducts, and dimorphic development of the sinus region. The phallus shows chiefly a difference in size, without marked morphologic divergence. (From R. K. Burns, Survey Biol. Progr., 1, 233-266, 1949.) 112 BIOLOGIC BASIS OF SEX many species as complete if somewhat rudi- mentary canals. In amniote embryos, on the other hand, the ducts of the genetically re- cessive sex are typically transient struc- tures. Sexual differentiation consists in the retention of one sex duct with development of its derivative structures, whereas the other either disappears completely or sur- vives only in a more or less vestigial condi- tion (Fig. 2.22B). Under these circum- stances experimental reversal of sex, to be successful, must be properly timed with re- spect to the state of development of the heterotypic duct. Once regression has been determined it is impossible to preserve the duct. 1. The Miillerian Ducts: the Effects of Fe- male Hormones The effects of female hormones, whether produced by grafted ovarian tissue or ad- ministered in pure form, may be stated gen- erally as follows: in female subjects as a rule they accelerate sexual differentiation, inducing a precocious hypertrophy of the Miillerian ducts which with large doses may become extreme. In males female hormones cause persistence of the Miillerian ducts fol- lowed by differentiation in varying degrees depending on timing of treatment, dosage, and the special status of the duct in the species under consideration. There are many deviations from this pattern, however, aris- ing in part from basic group or species dif- ferences and in part from the many experi- mental variables." In most amphibians the IVIiillerian ducts of both sexes respond readily to sex hor- mones during larval life. In Triturus {Tri- ton), after castration, both sex ducts remain indefinitely in a more or less undifferenti- ated condition, thus providing an ideal basis for sex reversal. Grafting gonads into cas- trates of either sex readily induces differen- tiation of the appropriate duct (de Beau- mont, 1933). Furthermore, in the males of various species which have undergone com- plete sex reversal the later development of " For reviews and references to a large literature covering amphibians, birds, and mammals see Humphrey, 1942; Wolff, 1938; Willier, 1939; Rav- naud, 1942; Greene, 1942; Moore, 1947; Jost, 1947a, 1948a, 1955; Ponse, 1949; Burns, 1949, 1955b; Stoll, 1950. the ]\liillerian ducts is always in accordance with the altered sex of the gonad, and the ducts may eventually become completely functional (see e.g., Humphrey, 1942; Ponse, 1949; Gallien, 1955). The reaction of the Miillerian ducts to female hormones (estra- diol, estrone) varies in different species and is greatly influenced by the stage at which treatment occurs and by dosage as well. In Ambystoma the ducts show a marked hy- pertrophy in females and the response in males is almost as great. The backward growth of the incomplete ducts is also ac- celerated (Burns, 1938a; Foote, 1941). Large doses, paradoxically, may arrest the backward extension of the duct (as do male hormones, q.v.) but the part already laid down becomes greatly hypertrophied (for a summary see Gallien, 1955). The effects of the female hormone in bird embryos are particularly striking (Wolff, 1938, 1950; AVillier, 1939; Gaarenstroom, 1939; Stoll, 1948). In male embryos both oviducts persist and hypertrophy as does also the right duct of the female, which nor- mally undergoes involution (Figs. 2.23 and 2.12). Once established these effects are per- manent, development continuing even after hatching (Wolff, 1938). However, the period of susceptibility to the hormone is limited. Retention and permanent development can be assured only by treatment up to the seventh day of incubation (this is the so- called "stabilization effect" of Wolff) ; later treatment is without effect for the preserva- tion of the ducts, irreversible changes having occurred which determine their regression with finality (Wolff, 1953b). The hormone of the embryonic ovary has the same effects. Ovaries grafted into the body cavity of male embryos cause persistence and devel- opment of\he oviducts (Wolff, 1946). The effect of the hormone appears to be a direct one since it occurs independently of any ef- fect on the gonads. In mammalian embryos the effects are in general similar, but marked species differ- ences have been found. Female hormones cause accelerated development of the Miil- lerian duct derivatives in females, and with high dosages oviducts, uteri, and vaginal canals all show great hypertrophy. In male embryos the ducts are frequently retained and also differentiate regionally into ovi- HORMONES IN DIFFERENTIATION OF SEX 113 A B Fig. 2.23. The effects of sex hormones on development of the sex ducts in chick embryos. A. Normal female embryo of 18 days, showing development of the left oviduct with shell gland, and retrogression of the right. B. Genetic male embryo, 18 days, treated with 2.0 mg. estrone. Both oviducts are present and greatly hypertrophied. Compare with C for the nor- mal condition. C. Normal male embryo at 17 days of incubation. Note the paired Wolffian ducts and absence of both oviducts. D. Genetic female embryo at 17 days, after treatment with 1.0 mg. androsterone, showing absence of both oviducts and extreme hypertrophy of the Wolffian ducts. For the normal female anatomy compare with A, and for normal size of male ducts see C. (From B. H. Willier, in Sex and Internal Secretions, 2nd ed., The Williams & Wilkins Co., 1939.) duct, uterine tube, and vaginal canal (Greene, 1942; Burns, 1939b, 1942a and b; Moore, 1941; Raynaud, 1942; Jost, 1947a). Details of structure depend on the state of development of the rudimentary Miillerian ducts in the males of the species in question. The vagina may be defective or absent en- tirely in males of certain species, as in the opossum, in which the duct is usually incom- plete, without a connection to the urino- genital sinus. In other species the effects are slight or lacking entirely (Raynaud, 1950, the field mouse; White, 1949, the hamster; Davis and Potter, 1948, man). The effects of male hormones. The effects of male hormones on the ]\liillerian ducts are more variable; strong inhibitory effects are obtained in many species and under proper experimental conditions; but with large doses stimulating or "paradoxical ef- fects" often appear, such as have been de- scribed in the case of the gonads. The time of administration of the hormone is important. Both in chick embryos and in larval amphibians treatment with androgens before the appearance of the duct, or during the formative period, may result in total suppression.^^ In amphibians, in which the ]\Iiillerian duct develops slowly, a particu- larly interesting situation is found. Early administration of testosterone propionate prevents development entirely (Burns, 1939c; Foote, 1941, Amhystoma) or may leave only the ostial rudiment (Gallien, 1955, Pleurodeles) ; however, treatment dur- ing the period of formation may result in suppression of the unformed portion of the duct, while the part already laid down per- sists and with large doses may even be strongly hypertrophied (Mintz, 1947). Here is a striking paradox in which different re- gions of the same structure (which are, how- ever, developmentally of different age) " See, for example, for amphibians, Burns, 1939c ; Foote, 1941; Hanaoka, 1941b: for the chick, Wil- lier, 1939; Wolff, 1938, 1950; Gaarenstroom, 1939; Stoll, 1948; Huijbers, 1951. Exceptions must be noted, however, in a few cases: the field mouse (Raynaud, 1950); the hamster (Bruner and Wits- chi,'l946, White, 1949); man (Davis and Potter, 1948) in which no clear effects were observed, whether because of true species differences or other experimental variables is not clear. 114 BIOLOGIC BASIS OF SEX react in an opposite way to the same treat- ment. In chick embryos also, early treatment with male hormone may completely sup- press development of the Miillerian ducts (Figs. 2.23 and 2.12; see also Gaarenstroom, 1939; Stoll, 1948, 1950) and a similar effect is produced by grafts of the embryonic testis (Wolff, 1946; Huijbers, 1951). Again, however, suppression of the ducts depends on certain rather precise conditions, the dose must be adequate and the hormone must act at the proper stage of development. They can be suppressed completely before the 6th or 7th day of development (Stoll, 1948; Huijbers, 1951) but later treatment is ineffective. Thus (as was found also for the ''stabilizing effect" of female hormone on the Miillerian ducts of male embryos) there is a limited period of development during which the ducts are susceptible to inhibition by male hormones. In contrast with these clear-cut results, however, a paradoxical hypertrophic effect of certain male hormones (androsterone, dehydro-androsterone and related compounds) has been reported on the ^Miillerian ducts of chick embryos after rather large doses (Willier, 1939; Wolff. 1938; Wolff, Strudel and Wolff, 1948). In the embryos of mammals effective in- hibition of the Miillerian ducts by male hor- mones has not been found, but suppression of regional parts of the duct sometimes oc- curs. The ostial portion is suppressed in the hedge-hog (Mombaerts, 1944) and the vagi- nal segment (the last part to be laid down) is frequently inhibited in female opossums (Burns, 1942a, b) and in mice (Raynaud, 1942). In the mouse and in the rabbit fail- ure of the posterior ends of the ducts to unite to form vagina and corpus uteri has been reported (Raynaud, 1942; .lost, 1947a). In female opossum embryos treated with testosterone propionate the vaginal canals are absent in about half of all cases and arc always absent in males (in which, as noted earlier, the terminal portion of the Miillerian duct is lacking) . However, a para- doxical stimulation of the Miillerian duct and its derivatives also takes place in opos- sums of both sexes when large doses (25 to 100 ^g. per day) are employed (Fig. 2.24; see also Moore, 1941, 1947; Burns, 1939a. 1955b) an effect which completely disa]^)- pears when the dose is lowered to ±5 /xg. or less (Burns, 1942a, b). In contrast with the failure of androgenic hormones to inhibit effectively the Miillerian ducts of mammalian embryos it is known that they are normally inhibited by the hormone of the fetal testis. In castrated male fetuses of the rabbit the ducts persist instead of regressing and develop almost as well as in normal females; conversely, the embryonic testis when grafted into a female fetus inhibits the Miillerian duct in the vicinity of the graft (Jost, 1953, 1955). On the other hand, a crystal of testosterone pro- pionate implanted in the same manner lacks this inhibiting power. Testosterone also fails to inhibit development of the Miillerian ducts in castrate males although in all other respects it fully compensates for the ab- sence of the testis (Jost, 1947b, 1953, 1955). This discrepancy has led to the suggestion (Jost) that in mammals another substance may be required for the inhibition of the Miillerian ducts. In the fetal rat, on the other hand (Price, 1956), neither testoster- one nor the presence of the fetal testis in- fluences the differentiation of the ]\Iiillerian ducts in genital tracts when isolated at an age of 17.5 days and cultured in vitro. In this case, however (as suggested by Price), it is likely that development of the Miille- rian ducts has already been irreversibly de- termined before the time of explantation. This question will come up again in a dis- cussion of the stage at which irreversible determination occurs in the rat. The effects of castration on development of the Miillerian ducts. Although the effects of steroid hormones on the development of the Miillerian ducts are on the whole con- sistent with theory (failure of the ducts of mammalian embryos to be inhibited by male hormone is a notable exception) such results do not constitute evidence that sex hor- mones are present and active in the normal differentiation of sex. A direct test of this question is provided by castration of the eni])ryo. The effects of castration in am- phibian larvae have been previously men- tioned (p. 112); after removal of the gon- ads both sex ducts fail to differentiate fui'tluM-, ]X'rsisting indefinitely in the condi- tion in which they were at operation. In re- cent years this difficult operation lias been HORMONES IN DIFFERENTIATION OF SEX 115 B D Fig. 2.24. Diagrammatic representation of the effects of relatively large doses of testos- terone propionate, administered from birth to an age of 50 days, on the development of the sex ducts in young opossums. A. Sex ducts as they appear in a normal male at 50 days; the vas deferens (Wolffian duct), epididymal tubules and remnants of mesonephric tubules are shown in black; the atrophic Miillerian duct is unshaded and the testis stippled. B. The ef- fects of the male hormone on the male duct system appear throughout; however, large dosages also induce a paradoxical growth and development of the uterine and tubal regions of the Miillerian duct, but the vaginal segment is absent as in the normal male. C. The sex ducts as they appear in a normal female at 50 days — breakdown and disappearance of the Wolffian duct and associated structures, regional development of the Miillerian duct into tubal, uterine, and vaginal segments. The contribution of the urinogenital sinus to the vaginal canal is indicated in stipple. D. The effects of the male hormone in a female subject : note the preservation and great hypertrophy of the male duct system which is not, however, as large or as well differentiated as in the treated male; note also the striking paradoxical effect of a large dosage of androgen on the female genital tract which, at the same dosage level, is far greater than in the treated male (B). These paradoxical effects of androgen on the Miillerian duct dorivativos disappear entirely at lower dosages. carried out in a number of species of birds and mammals. ^'^ Early castration of avian cml)ryos is fol- lowed by persistence and development of Miillerian ducts in both sexes (the chick and the duck, Wolff and Wolff, 1951; Huij- bers, 1951). After total castration in males both ducts persist and develop, but partial castration results in regression as usual. In females, in which the right duct normally regresses, both ducts persist and are well developed. ^^ Thus, in the absence of the gon- ads the Miillerian ducts follow the same pattern of development regardless of sex (Fig. 2.25). It is clear that the testes are 'Mu the rabbit (Jost, 1947b); the mouse (Ray- naud and Frilley, 1947) and the rat (Wells, 1946, 1950); in the chick and the duck embryo (Wolff, 1950; Wolff and Wolff, 1951; and Huijbers, 1951). " In birds involution of the right Miillerian duct of the female is normally conditioned in some manner by the ovaries, and it has been shown fur- ther that the presence of either ovary is sufficient (Wolff and Wolff, 1951). The exact nature of the inhibitory factor in this interesting case is not known. necessary for normal inhibition of the ducts in male embryos, and it was pointed out earlier that a graft of the embryonic testis has the same effect in females (Wolff, 1946). The ovaries, on the contrary, have no posi- tive role in the development of the Miille- rian ducts, but actually inhibit the right duct; in their absence both ducts develop without hormonal conditioning. Among mammalian embryos the case of the rabbit is best known (Jost, 1947b). Cas- tration of the female again has no important consequences; the Miillerian ducts continue to develop and shortly before birth are only slightly smaller than in normal females. In male castrates, on the other hand, the situa- tion is very different; if the operation is performed early enough the Miillerian ducts, instead of regressing, persist and develop, becoming practically indistinguishable from those of castrate females (Table 2.2, Fig. 2.26). Thus, as in birds, castrates of either sex follow identical patterns of development. Nevertheless, if castration is delayed be- 116 BIOLOGIC BASIS OF SEX MALE CASTRATE TYPE FEMALE Fig. 2.25. Diagrams summarizing the effects of castration on the development of the syrinx (top row) oviducts, and genital tubercle (below) in bird embryos (chick and duck). The left column shows the normal condition in the male : note the complete retrogression of the Mlillerian ducts (broken lines) under the influence of the embryonic testes (black). In the right column, the normal female condition showing the retrogression of the right oviduct. The arrows indicate the inhibitory action normally exerted by the ovaries on the syrinx, right oviduct and genital tubercle. The castrate type, which appears regardless of genetic sex, is seen in the center. In castrates both oviducts persist in complete form and are as well developed as the normal left oviduct ; the syrinx and genital tubercle, however, assume the male form in the absence of the inhibition exerted by the ovaries. Note the extreme asym- metry of the male syrinx and tubercle, as compared with the primitive symmetrical form retained in the normal female. (After Et. Wolff and Em. Wolff, J. Exper. ZooL, 116, 59-97, 1951.) TABLE 2.2 Effects of castration in male rabbit fetuses studied at the age of 2S days (After A. Jost, Recent Progr. Hormone Res., 8, 379-418, 1953.) AGE AT CASTRATION MULLERIAN DUCTS WOLFFIAN DUCTS AND DERIVATIVES PROSTATIC GLANDS EXTERNAL GENITALIA 19 DAYS (2 coses) PERSISTENT 20-21 DAYS (llcoses) PERSISTENT CAUDAL REMNANTS (in 3cases) VENTRAL BUDS PRESENT 22-23 DAYS (Scases) UTERO-VAGINAL SEGMENTS PERSISTENT CAUDAL REMNANTS HYPOSPADIC 23 DAYS (4 cases) ABSENT- SMALL ABSENT (=NORMAL) SEMINAL VESICLES PRESENT WELL DEVELOPED 24 DAYS (3 coses) ABSENT ( = NORMAL) WELL DEVELOPED Unilateral castration is followed by normal development. Castration effects are prevented by Testosterone propionate given at operation. yond a certain stage (about the 22nd day rather limited period during which involu- of gestation in the rabbit; Jost, 1947b, c) tion of the oviducts is determined, as was the ducts subsequently undergo involution shown also for the chick in the case of male as usual (Table 2.2). Evidently there is a hormones administered experimentally. HORMONES IN DIFFERENTIATION OF SEX A -m B 11^ ^'# ms^sL-^^; ^m& Fi(j. 2 2b Ihc ctlLit^ ul e i^lialiou uu llif dux uluiniuul uf the sex ducts iii the rabbit. A. Persistence ot the Mullenan duct (uterine level) above, and the vaginal canal, below, in a castrate male. B. The same structures seen in a castrate female as compared with tlie condi- tion in the noimal female shown in C . Note almost complete involution of the male duct except for remnants (CW) in the castrate male, and compare with the castrate and the normal female. Castration of the female (S) has little effect on the pattern of development which follows; on the other hand, castration of the male results in involution of the Wolffian duct and development of the Mlillerian duct, a reversal of the normal pattern. (From A. Jost, Arch. Anat. microscop. et Morphol. exper., 36, 271-315, 1947.) Once conditioning has occurred the effect is irreversible. In view of the decisive role of the em- bryonic testis in the development of the Mlillerian ducts, as shown by castration experiments, it is significant to note the con- dition of the genital tracts in certain "lateral gynandromorphs" of genetic origin in mice (Hollander, Gowen and Stadler, 1956) which have an ovary on one side of the body and a testis on the other. Both gonads are usu- ally small and hypoplastic. Without excep- tion, however, on the side of the testis the Mlillerian duct derivatives are absent en- tirely and the male duct system is developed. Contralaterally, a female genital tract is always found although it shows great varia- tion in size. An almost identical condition has recently been described in a gynandro- morphic hamster (Kirkman, 1958). The manner in which the influence of the testis tends to be limited to its own side of the body in these cases is of special interest, and will be considered later in dealing with the localized character of hormone effects. It is undoubtedly related to the early stage at which the testis begins to exert its effect and probably also to its reduced size in nearly all cases. The development of the Mullenan ducts after isolation in vitro. At this point, in the case of mammalian embryos, an obvious question presents itself. Is development of Mlillerian ducts in male castrates in fact a purely autonomous process, due to release from an inhibition normally exerted by the testes or, in the absence of the gonads, does some other humoral factor intervene to as- sure their development? It is known that in various species of mammals estrogens are present in the placenta and fetal fluids in considerable amounts (for references see Price, 1947; Parkes, 1954), and the pos- sibility arises that development of the ducts after castration may be maintained under the influence of an estrogenic substance of nongonadal or of maternal origin. This ques- tion has been answered, in the negative, by experiments designed to test the self- differentiating capacity of the ducts under conditions of physiologic isolation. When explanted in vitro (with pieces of the associ- ated mesonephric bodies) the Mlillerian ducts of rat embryos, regardless of the sex of the donor embryo, survive and continue their development in the same manner as in castrate fetuses (Jost and Bergerard, 1949; Jost and Bozic, 1951). In these ex- periments the ducts were isolated at ages of 15 to 16 days, and 16 to 17 days re- 118 BIOLOGIC BASIS OF SEX spectively. Similar behavior is seen in grafts to the eye chamber of castrate hosts (the guinea pig, Bronski, 1950) and after trans- plantation of the entire embryonic genital tract into castrate and noncastrate hosts of various ages (the rat, Moore and Price, 1942). Although the experimental environ- ments in all of these cases cannot be con- sidered hormone-free in the strict sense (Jost and Bozic, 1951), the results indicate that the development of the female sex ducts when removed from the influence of the em- bryonic testis is a matter of autonomous differentiation. The above experiments show that in rat embryos the fate of the JMlillerian ducts is undetermined up to an age of 16 to 17 days at least (Jost and Bozic), since at this age these ducts in male embryos still retain their capacity for autonomous development when removed from the inhibiting influence of the testis. Experiments on the same spe- cies (Price, 1956; Price and Pannabecker, 1956 1 indicate that involution is irreversibly determined at about the age of 17 days. The genital tracts of male fetuses were removed at 17.5 days of gestation and cultured in vitro. At this stage the first signs of involu- tion can be detected in the region of the ostium but the posterior extremities of the ducts are still growing. After explantation the ducts continue to regress regardless of whether the testes are included in the ex- plant or not, whereas those of female fetuses under the same conditions develop nor- mally. It seems that the fate of the Miil- lerian ducts in the male becomes irre- versibly fixed within the brief period of a day by exposure to the testis hormone. This question has })een investigated in some detail in the chick. It had been shown earlici- that after transplantation to the cliurioalhintois, Miillerian ducts from em- bryos of either sex differentiate completely if isolated before the beginning of sex dif- ferentiation. But if transplanted later than the 10th day of incubation the ducts of male embryos invariably degenerate; beyond tliis age their involution has been finally de- termined (Wolff and Ostertag, 1949). Mak- ing use of the technique of culture in vitro the analysis has been carried fartliei' (Wolff and Lutz-Ostertag, 1952). When isolated before the appearance of sex differentiation in the gonads, again the Miillerian ducts of both sexes undergo a complete differentia- tion, as in castrate embryos; but if isolated after the 9th day the ducts of male em- Ijryos i^romptly undergo involution. How- ever, if the Miillerian ducts of female em- bryos are cultured in the presence of an embryonic testis, a typical involution takes place (although at the same time the ad- jacent Wolffian ducts develop normally). Furthermore, when testosterone propionate is added to the medium it has the same effect as the embryonic testis (Wolff, Lutz-Oster- tag and Haffen, 1952; for a summary see Wolff, 1953b). These results hardly leave the role of the male hormone in doubt. It follows also that the action of the hormone in vitro must be direct. Evidence has been obtained to show that the process of involu- tion of the ducts is of the nature of an autolysis produced by the action of pro- teolytic enzymes which are presumabh' ac- tivated l)v the male hormone (Wolff, 1953b). Summary and conclusions. On the basis of present knowledge, the following gen- eral statements may be made concerning the role of sex hormones in the differentia- tion of the Miillerian ducts. a. Female hormone, in most species, stim- ulates precocious growth and development of the Miillerian ducts and their derivative structures in embryos of either sex when given in adequate dosages. Administered at an early stage it prevents the normal in- volution of the ducts in male embryos after which development may continue without further treatment. In certain mammalian species, however (hamster, field mouse, man), negative results have been reported, which may possibly be related to the level of estrogen prevalent during gestation in these species or to dosage. b. Male hormone, if administered early, has an inliibitory action on the Miillerian ducts in the embryos of birds and am- phibians. In some species the primary de- \-el()l>nient of the duct is entirely suppressed; in other cases only a partial inhibition oc- curs. In mammals, the hormone of the em- bryonic testis ]M-o(lnccs involution of the (hicts in males at tlic beginning of sex dif- HORMONES IN DIFFERENTIATION OF SEX 119 ferentiation, and testis grafts inhibit the Miillerian ducts of females; on the other hand, male hormones of adult type fail to inhibit the ducts, or produce only a limited and localized involution in a few species. c. The time factor is critical in the hor- monal control of the jNIiillerian ducts. To suppress primary differentiation of the ducts in birds and amphibians, male hormone must be administered before or during the formative stage of development. However, the fully formed duct remains sensitive to hormones up to the onset of sex differentia- tion. Female hormone administered before this stage insures retention and develop- ment of the ducts in males, and during the same period they are subject to inhibition by male hormone or by the embryonic testis. Beyond this point the final development or the involution of the ducts has been irre- versibly determined and hormones are with- out effect. d. In the absence of previous hormonal conditioning, as after early castration or early isolation in vitro, the Miillerian ducts of either sex are capable of an autonomous differentiation, comparable to development in normal females; this development can be prevented, however, and involution in- duced, by introduction in vitro of an em- bryonic testis, or (in birds) by the ad- dition of male hormone to the medium. e. In amniote embryos the testis hormone is the controlling factor in the differentiation of the Miillerian ducts. The involution of the ducts in males is conditioned by the testes, whereas in the absence of gonads there is an almost normal development of the ducts in embryos of either sex. This does not mean, however, that the embryonic Miillerian ducts are insensitive to female hormone; in sufficient concentration it pro- duces hypertrophic de^'elopment in both sexes. f. The evidence from cultivation in vitro shows that the effects of hormones on the Miillerian ducts are exerted directly and are independent of the organism as a whole. 3. The Male Duct System The male sex duct, or Wolffian duct, de- velops first as the duct of the pronephros (primary nephric duct) and subsequently serves as the mesonephric duct in both sexes. As the mesonephros is replaced by the metanephros in amniote embryos, the Wolf- fian duct loses its excretory function and its fate in the two sexes is different. In the fe- male it disappears entirely or survives only as vestiges, but in males it acquires a new status as the male sex duct. At the same time a certain number of mesonephric tubules, which connect the duct with the rete canals of the testis, become a part of the epididy- mis, and the seminal vesicle develops as a diverticulum of the duct near its junction with the urinogenital sinus. During its early history as the duct of the mesonephros the Wolffian duct is unresponsive to sex hor- mones, but with the onset of sex differentia- tion it becomes responsive to the male hor- mone. The role of male hormone in the differen- tiation of the Wolffian ducts. In the female of amphibians experimental transformation of ovaries into testes is followed by hyper- tro])hy and masculinization of the AVolffian ducts (Humphrey, 1942), and transplanta- tion of testes into larval castrates of either sex has the same effects (de Beaumont, 1933). Administration of male hormones produces marked hypertrophy of the Wolff- ian ducts in larval amphibians of either sex [e.g., Burns, 1939c; Foote, 1941; for summary see Gallien, 1955). A similar re- sponse occurs in the embryos of birds and has also been reported in many species of mammals (Figs. 2.23, 2.24). ^^ This is the case in female embryos as well as in males. Development of epididymal tubules (from the epoophoron) also takes place in females (Fig. 2.24D) which also commonly develop seminal vesicles (Greene, 1942; Raynaud, 1942; Wells and van Wagenen, 1954 L With higher dosages all these structures undergo extreme hypertrophy. Female hormones, on the contrary, appear to have little influence on the development of the Wolffian ducts, although in a few cases a jiartial involution of the ducts has ^" This effect has been widely reported : see Wil- lier (1939), Wolff (1938, 1950) for the chick; Greene (1942) for the rat; Raynaud (1942), Turner (1940) for the mouse; Jost (1947a) for the rabbit; Godet (1949) for the mole; Burns (1939a and b, 1945a); and Moore (1941) for the opossum. 120 BIOLOGIC BASIS OF SEX been reported (Raynaud, 1942; Greene, 1942). It should be noted that the latter observations are in mammalian embryos (mice and rats) in which the hormone of the testis (see below) is essential to insure re- tention of the ducts. Partial involution in males under the influence of a female hor- mone is possibly only a result of inter- ference with the normal activity of the tes- tis. On the other hand, a paradoxical action of female hormone, causing partial retention and hypertrophy of the male duct, has been occasionally reported (e.g., Greene, 1942; Moore, 1941). Although the method of ad- ministration in these cases does not permit accurate estimation of the dosage it was evidently rather large. With these excep- tions, the effects of sex hormones on the male duct system are consistent with theory. The effects of castration on the develop- ment of the male sex duct are striking and agree with the results of hormone adminis- tration. They show that in all species the presence of the embryonic testis is necessary to induce sexual differentiation of the duct, and to insure its retention in mammalian embryos. In amphibian larvae (Triturus, syn. Triton) the Wolffian ducts persist after castration in their capacity as nephric ducts, but remain in a sexually undifferentiated condition (de Beaumont, 1933). In bird embryos also there is no significant altera- tion of the Wolffian ducts after castration. It is in mammals that the ducts become de- pendent on the testis and its hormone for survival as well as for sexual differentiation. In rabbit embryos castrated before the 22nd day of gestation (Jost, 1947b) the ducts in both sexes completely regress, following the female pattern of development (Fig. 2.26) ; involution in castrates is prevented, however, and normal development is main- tained by prompt administration of male hormone. A more variable atresia of the ducts also occurs in fetal rats after castra- tion (Wells and Fralick, 1951). In mice Raynaud reports a difference in reaction in the two sexes. After castration the Wolffian ducts of males undergo a complete involu- tion but they may be partially retained in females. It is suggested that in this species the ovaries may play a positive role in the involution of the duct in females (Raynaud, 1950). The development of the Woffian ducts in vitro. Further evidence that retention and sexual differentiation of the Wolffian ducts and associated structures (epididymal tu- bules and seminal vesicles) are dependent on the testis and its hormone is provided by the behavior of the male duct system after isolation, using the technique of organ cul- ture. Development in isolation provides a parallel to development in the castrate fe- tus, with exclusion, however, of possible in- fluence by hormones of maternal or pla- cental origin or from some extragonadal source in the fetus. When the mesonephric bodies of rat em- bryos, including long segments of the gona- ducts, are removed at 15 to 16 days of gesta- tion and cultured without the gonads, the Wolffian ducts of both males and females degenerate, but at the same time the Miille- rian ducts survive and develop normally. The degeneration of the Wolffian ducts can- not, therefore, be due to unfavorable condi- tions in the medium (Jost and Bergerard, 1949) . The same result was obtained using slightly older fetuses of ±16.5 days (Jost and Bozic, 1951). However, the most com- plete study of this question is that of Price and Pannabecker (Price, 1956; Price and Pannabecker, 1956) who explanted male genital tracts of 17.5-day rat fetuses under various conditions designed to test the role of the embryonic testis and the male hor- mone. When both testes are included with the explant, development of the male duct system proceeds normally up to an age of 21.5 days (approaching term for the normal fetus I and the seminal vesicles develop as usual. Normal development ensues also when only one testis is left with the implant. But if one testis is removed and the lateral halves of the genital tract are spread widely apart on the surface of the medium, de- velopment is normal only on the side where the testis is present ; on the other side serious defects appear; the duct is thin and weakly developed, and the seminal vesicles are small or even lacking. Finally, if both testes are removed the Wolffian ducts regress com- pletely. However, the addition of male hor- mone to such a prci^aration fully compen- sates for the al)scncc of the testes and development of the male duct system is again normal. HORMONES IN DIFFERENTIATION OF SEX 121 Fig. 2.27. The effects of castration on the development of the prostatic glands in the rab- bit. A. The sinus region in a male fetus, aged about 27 days, castrated before the 20th day of gestation; above is the canal of the urinogenital sinus, below it the dark, bilobed struc- ture represents the vaginal cord as it unites with the wall of the sinus. No sign of prostatic buds is seen. B. The sinus region in a young male of the same age castrated at about 21 days (20 days, 20 hours). Two large prostatic buds are seen ventral to the vaginal cord which were present at the time of castration. No further development has occurred. C. The sinus region in a male fetus of 28 days, castrated at the age of 23 days. Castration at this age is followed by essentially normal development. (From A. Jost, Arch. Anat. microscop. et Morphol. exper., 36, 271-315, 1947.) Fig. 2 2S Hi-told.i In k lu ( - be twcin noimal h i i i i I , i i \ternal genitalia {B) m i.ihlut htu^i^ aitcM -e\ual diffeientiation Mk uiinoj-nut il uu itu^ i-^ larger in the femuiioundcd b^ the pieputial fold, ■wliith maik'- off the glans clitoridis from the surrounding tissues. In the male the urethral cleft is narrower and com- pletely enclosed within the preputial fold. The paired erectile bodies are seen above the urethral cleft. Castration at an early stage alwaj^s results in genitalia of female type (A) re- gardless of the sex of the castrated embrvo. (From A. Jost, Arch. Anat. microscop. et Mor- phol. exper., 36, 271-315, 1947.) Altogether, the evidence clearly indicates that the male hormone is the essential de- termining factor in the survival and sexual differentiation of the male sex ducts and seminal vesicles. Notwithstanding the minor exceptions noted above, the female hor- mone evidently has little role. The reason for the insensitivity of the Wolffian ducts to the female hormone may possibly be found in their long phylogenetic history as nephric ducts in both sexes, in which ca- pacity they must be retained in some groups beyond the period of sex differentiation or even permanently. B. DERIVATIVES OF THE CLOACA AND URINOGENITAL SINUS Sexual dimorphism of the amphibian clo- aca chiefly takes the form of special cloacal glands which in males become highly de- veloped at the breeding season, causing the prominent swelling of the cloacal region so conspicuous in males. In the females of vari- ous species they may be absent, present in a rudimentary state, or in some cases dif- ferently specialized (Noble, 1931). For their development and maintenance these glands depend almost entirely on the testis. After experimental transformation of sex, the sub- sequent differentiation of the cloacal glands 122 BIOLOGIC BASIS OF SEX TABLE 2.3 Effects of hormones on derivatives of the urogenital sinus and the external genitalia in mammalian embryos ACTION OF MALE HORMONE ON FEMALES SUBJECT OF EXPERIMENT FORM OF SINUS VAGINAL DEVELOPMENT SINUS EPITHELIUM PROSTATE FORM OF GENITALIA OPOSSUM MALE TYPE SUPPRESSED IN 50% OF CASES HYPERTROPHIC* BUT NOT CORNIFIED H 1 G H LY DEVELOPED MALE TYPE RAT MALE TYPE SINUS PORTION SUPPRESSED HIGHLY DEVELOPED MALE TYPE MOUSE MALE TYPE SINUS PORTION SUPPRESSED WELL DEVELOPED MALE TYPE RABBIT MALE TYPE SUPPRESSED WELL DEVELOPED MALE TYPE MONKEY MASCULINIZED SINUS PORTION SUPPRESSED STRATIFIED SQUAMOUS LARGE OR VARIABLE MASCULINIZED- PENIS-LIKE ACTION OF FEMALE HORMONE ON MALES OPOSSUM FEMALE TYPE SINUS PORTION HYPERTROPHIC VAGINAL TYPE- HIGHLY 60RNIFIED COMPLETELY SUPPRESSED FEMALE TYPE RAT FEMALE TYPE SINUS PORTION WELL DEVELOPED SUPPRESSED FEMALE TYPE MOUSE FEMALE TYPE VAGINAL CORD WELL DEVELOPED STRATIFIED SQUAMOUS- METAPLASTIC SUPPRESSED FEMALE TYPE » r/7/5 effect appears only with large dosages corresponds to the altered sex of the gonad. Castration of mature males results in retro- gression of the glands and after early castra- tion the cloaca remains sexually undifferen- tiated in both sexes (de Beaumont, 1933). However, testis tissue grafted into castrates (de Beaumont) , or treatment with male hormone {e.g., Burns, 1939c), readily in- duces development of male cloacal glands in individuals of either sex (for a review see Humphrey, 1942). In the development of mammalian em- l)ryos the urinogenital sinus is separated at an early stage from the cloacal region of the hind-gut by formation of the perineal sep- tum. In its primitive condition the sinus is a short canal, extending from the neck of the bladder to the exterior, with a meatus at the base of the genital tubercle. The paired gonaducts open into it near the neck of the bladder (Fig. 2.22). Sexual differenti- ation in females chiefly involves anatomic and histologic changes associated with de- velopment of the vagina, to which the urino- genital sinus makes an important contribu- tion; at the same time the male sex ducts regress and largely disappear (Fig. 2.22B). In placental mammals fusion of the poste- rior ends of the Miillerian ducts as they ap- proach the urinogenital sinus gives rise to the unpaired, median vagina, but in marsu- pials the ducts remain separate and paired lateral vaginal canals are formed (Fig. 2.22B). In male embryos the main features of sinus differentiation are the involution of the ]\Iiillerian ducts with absence of vaginal development, and the differentiation of elab- orate prostatic glands. Sex hormones show a high degree of specificity in their effects on the sinus structures, inducing development of typically male or female forms. Results are available for a number of species be- longing to several orders of mammals,^'' and are in agreement except for minor details (for some representative species see Table 2.3). The histologic aspects of the differentia- tion of the sinus are well illustrated in young opossums. The effects of male hormone (testosterone propionate) in male and fe- male pouch young are compared in Figure 2.29. The effect of the hormone in males is ^\See Greene (1942) for the rat: Raynaud (1942), Turner (1940) for the mouse; Jost (i947a) for the rabbit; Godet (1950) for the mole; Wells and van Wagenen (1954) for the monkey; Burns (1939a, b) and Moore (1941) for the opossum. Simimaries for these and other species are to be found in Colloques Intcrnationaux : La Differencia- lion Sexuelle choz les Vertebre.*, Masson et Cie., Paris, 1951. As an exception, the effects in the hamster are rather slight (Bruner and Witschi, 1946; White, 1949). HORMONES IN DIFFERENTIATION OF SEX 123 m^M ^\'»^'^ :::^rHi. Fig. 2.29. The effects of testosterone propionate on the development of the urinogenital sinus and prostatic glands in young opossums. A. Extreme hypertrophy of the sinus and prostate in a male aged 50 days, treated from birth ; compare with the condition in a normal male of the same age (C). The effect of the same dose of hormone in a littermate female is shown in B. Female opossums normally never develop prostatic rudiments, as shown in D and E, representing cross-sections through the urinogenital sinus somewhat below (D) and at the point of junction {E) of the lateral vaginal canals (c/. Fig. 2.225). Note the great difference in the volume of prostatic tissue in the treated male (A) as compared with the treated female (B), although dosage and other conditions were the same. merely to exaggerate the normal processes of development. With large doses there is a moderate hyperplasia of the sinus epithe- lium in males and a tremendous hyper- trophy of the prostatic glands. But in fe- males a striking deA'elopment of prostatic glands also occurs (although normally the female possesses no prostatic rudiments) to- gether with a change in form, resulting in a sinus that is typically male. Quantitatively, these effects are proportional to dosage, but with the same dosage an interesting sex dif- ference is constantly observed with respect to the magnitude of the response. The pros- tate (Fig. 2.29.4, B) is invariably more strongly developed in male subjects than in females. This difference in size apparently depends on an inherent difference in growth capacity in homologous tissues of different sex genotype when exposed to the same intensity of stimulation (Burns, 1942b, 1956a). This effect appears regularly in the case of many other sex structures of the opossum as will be seen. The induction of prostatic glands and a male form of sinus is of regular occurrence in female mam- malian embrvos exposed to male hormones (Table 2.3).' Two special points concerning prostatic differentiation in young opossums are of in- terest. Brief treatment of female embryos with androgen, just at the time when the prostatic buds are appearing in males, is sufficient to induce buds which are then capable of continued differentiation after the hormone is withdrawn (Moore, 1945 ». This is an unusually clear case of permanent conditioning of a sex structure by brief ex- posure to a hormone at a critical stage in development. Also of interest is the fact that by gradually reducing the dosage of male hormone a level is reached, at approximately 5 /xg. per day, which induces prostatic buds in young females which are identical in size and appearance with those of normal males of the same age (Burns, 1942a) . With- 124 BIOLOGIC BASIS OF SEX out attempting to allow for the constitu- tional sex factor mentioned above, this amount of androgen would appear to be roughly equivalent to the hormonal activity of the embryonic testes at this period. Female hormone has opposite effects on the urinogenital sinus and its derivatives in young opossums. Estradiol dipropionate completely suppresses prostatic differentia- tion in males and transforms the sinus epi- thelium into a stratified squamous epithe- lium of vaginal type (Fig. 2.30) ; in fact the histologic picture is one of intense prolifera- tion and cornification like that of the adult vagina at estrus. Moreover, a single dose of estrogen administered during the 15th day of pouch life, shortly before the prostatic buds would normally appear, results in com- plete suppression of the prostate, an effect which is also permanent (Burns, 1942a, b, c) . Thus, there is a relatively short period dur- ing which induction and continued develop- ment of ])rostatic glands in females, or their permanent suppression in males, is wholly conditioned by the presence of the appropri- ate hormone. Quantitatively as well as qual- itatively the reactivity of the embryonic sinus epithelium to estradiol is remarkable. Again a sex difference, as measured by growth and proliferation is seen, this time in favor of the female. Transformation to a typical vaginal epithelium in the estrous phase can be induced in very young male embryos, long before the time of appearance of prostatic buds (Fig. 2.31; Burns, 1942c). It is hardly surprising that an epithelium of this type permanently loses all capacity to produce prostatic tissue. The effects of castration on the develop- ment of the urinogenital sinus and its de- rivatives in mammalian embryos follow the pattern previously described for the sex ducts. The male form of sinus is incapable of developing in the absence of the testes, whereas morphogenesis of the female form is not significantly affected by castration B Fig. 2.30. The effects of estradiol dipropionate on the development of the urinogenital sinus and prostate in young opossums. A. The normal sinus of a young male aged 30 days, showing the condition of the prostatic buds, for comparison with a normal female of the same age (fi). Note the bilobed form of the sinus canal in the male as compared with the typical pentangu- lar form in the female. C. The effect of the female hormone in a male littermate of the same age, treated from the time of birth. Complete suppression of the prostatic glands has occurred, the form of the sinus canal is typically female, and the sinus epithelium has been transformed into a thick stratified squamous epithelium, like that of the adult vagina, in a state of pronounced keratinization and desquamation. The effect of an identical dose in a female subject is similar but much more intense. HORMONES IN DIFFERENTIATION OF SEX 125 Fig. 2.31. The effects of a stronger dosage of estradiol dipropionate on the urinogenital sinus at a much earher stage. A. Young male treated from birth to an age of about 12 days, for comparison with the normal male sinus (inset, B) of the same age. Compare the charac- ter of the sinus epithelium with that in the older specimen shown in Figure 2.30C This condition of the sinus epithelium is induced at an age which precedes by se^■eral days the normal appearance of the prostatic buds, which never develop. (Table 2.2; Jost, 1947b; Raynaud and Fril- ley, 1947; Wells, 1950). In castrate males prostatic differentiation is prevented and development of a vagina (correlated with persistence of the Miillerian ducts in male castrates) results in a sinus of female type (Fig. 2.27 A). Male and female castrates are morphologically very similar, and both closely resemble the normal female. Again it is clear that the embryonic testis is the es- sential factor in male development, whereas the female pattern is independent of hor- monal conditioning and also of sex consti- tution, since in the absence of the gonads it develops spontaneously in castrates of either sex. The factor of time is again of paramount importance and sharply limits the effective- ness of castration. This holds for the devel- opment of other accessory structures (Table 2.2) but is particularly clear in the case of the prostate which will serve to illustrate. In male rabbit embryos castrated on or after the £3rd day of gestation there is only a slight effect on prostatic development, which continues in a practically normal manner; but if the operation is performed a day ear- lier there is a distinct reduction in size. In fetuses castrated from the 20th to the 21st day only small ventral buds are found which are already formed at the time of operation, and after castration earlier than 20 days prostatic buds are absent altogether (Fig. 2.27; Jost, 1947b, c). The period from 20 to 21 days, then, is critical for the appearance of the prostatic buds and their further differ- entiation for which the embryonic testis is essential. However, absence of the testis is fully compensated for by male hormone ; in castrates receiving androgen the develop- ment of all male parts proceeds normally. A similar result has been obtained in the fetal rat (Wells, Cavanaugh and Maxwell, 1954). Late castration has little effect, but castration on day 18 results only in buds which do not undergo branching. Earlier cas- tration has not proved feasible in this spe- cies. 126 BIOLOGIC BASIS OF SEX Fig. 2.32. The effects of sex hormones on the sex type of tlic copulalory stniciur(-s in young opossums. A. The appearance of the phallus, or genital luljerclc, ui ;i normal male (left) and female opossum aged 20 days. Sex is difficult to distinguish by form alone but the phallus is somewhat larger in the male. Sex is readily distinguished at this age, however, by HORMONES IN DIFFERENTIATION OF SEX 127 C. EXTERNAL GENITAL STRUCTURES Copulatory organs homologous with those of higher vertebrates are not found in am- phibians. They are developed to an extent, however, in certain birds and reptiles and become highly specialized in mammals. The copulatory organ in amniote embryos devel- ops from a simple primordium, the genital tubercle, which is common to both sexes. It becomes specially developed as the penis in the male but in females it persists in a more or less rudimentary form, known in mam- mals as the clitoris. The genital tubercle of birds arises as a small, conical protuberance just within the cloacal orifice. In chickens it is not highly developed, although larger in the male than in the female, but in the males of ducks, geese, and certain other birds it be- comes considerably larger and more modi- fied, constituting a penis (Fig. 2.25). In the embryos of mammals (except the Mono- tremes) the genital tubercle is external in position, arising as an eminence near the ventral rim of the urinogenital meatus. The developing copulatory organs of birds and mammals react readily to sex hormones and are extremely sensitive to castration. In birds the clearest experimental results have been obtained in duck embryos, because of the more pronounced sexual dimorphism in this species. Treatment with female hormone (estradiol benzoatej before the 12th day of incubation completely arrests development of the penis in males, and the rudimentary clitoris of the female may be even smaller than normal (Wolff, Em., 1950 ) ; beyond this age, however, the hormone is no longer ef- fective, the form of the prospective penis having been finally determined. The effects of the male hormone are less precise and it is not essential for normal development (see the effects of castration below) . Testosterone proprionate produces great hypertrophy but the structure is not entirely normal, the characteristic spiral form of the penis being imperfectly developed (Wolff, Em., 1950). This abnormality is perhaps a result of over- dosage as the dosages used were undoubt- edly very large. Although in the chick the dimorphism of the genital tubercle is less pronounced than in the duck, it reacts in the same way ; male hormones stimulate and fe- male hormones inhibit growth and morpho- logic differentiation (Reinbold, 1951). In mammalian embryos the sex type of the developing genital tubercle, or phallus, is easily controlled by sex hormones (Table 2.3) ; in fact, this structure is unusually sus- ceptible to modification and may be com- pletely transformed. Typical are the results in the rat (Greene, 1942), the mouse (Ray- naud, 1942; Kerkhof, 1952), the hamster (Bruner and Witschi, 1946) and in pouch young of the opossum (Burns, 1939a, b; Moore, 1941). The rat and the mouse are similar in their behavior. Male hormone does not affect the development of the penis in males except to produce hypertrophy, but in females the genital tubercle is greatly en- larged and assumes the character of a pe- nis.^' Female hormone has opposite effects; females differentiate normally but in males the tubercle fails to enlarge and a hypo- spadic condition frequently appears. In young opossums the form of the copu- latory structures is completely controlled in accordance with the type of hormone given, with results which are identical in the two sexes except for a difference in size (Fig. 2.32). The basis for the transformation of the phallus has been analyzed histologically (Burns, 1945b). The various histologic con- ^' Strangely enough this is not tyi^ically the case in freemartins. The chtoris as a rule is not greatly modified (Lillie, 1917). There are, however, some striking exceptions (Buyse, 1936; Numan, 1843, illustrated in Lillie, 1917, Fig. 29). the scrotal sac in males and the presence of the pouch folds and mammary rudiments in females. B. The typical form produced by male hormone (left) and female hormone in specimens treated from birth to an age of 20 days; the normal condition at this age is shown above. This striking difference in form is produced without regard to the sex of the subjects, which in this case are both male (note the scrotal sacs). C. The result of administer- ing male hormone (testosterone propionate) from birth to an age of 50 days, in a female subject (left; note the pouch) and a male littermate. Observe the identity in form but dis- tinctly greater size of the penis in the male. D. Comparison of the effects of estradiol dipro- pionate, given from birth to an age of 30 days, in a female subject (left) and a male litter- mate. In both C and D it is shown that the hormones produce genitalia of typical male or female form, regardless of the sex of the subject. 128 BIOLOGIC BASIS OF SEX stituents of the organ respond to the appro- priate hormone in a highly specific manner. The erectile bodies, the development of which largely determines the form and the size of the penis, are strongly stimulated by male hormone and almost entirely inhibited by female hormone (Fig. 2.33j. Qualita- tively, these responses are independent of sex constitution, but with identical dosages marked differences in size, such as were noted previously in the case of the prostate, the sinus epithelium and derivatives of the sex ducts (Figs. 2.29 and 2.24) , are again ob- served in the two sexes. Female hormone, in addition to inhibiting the erectile tissue, in- duces an extreme hyperplasia of the vulvar and periurethral connective tissues (Fig. 2.335). It is this response which produces the gross swelling of the vulvar region, so conspicuous in estrogen treated embryos of .,*m^''- Fig. 2.33. The effects of male and female hormone^ on the differentiation of the histologic constituents of the phallus in young opossums. A. The effects of androgen in a male, aged 30 days, treated from birth onward. There is great hypertrophy of the erectile bodies but otherwise structiue is normal; at the top, the paired corpora cavernosa are imited at the mid-line ; below, the urethral canal, with the bulbo-urethral glands on either side ; laterally, the large bulbs of the corpora spongiosa, with their muscular investments. B. The effects of estrogen in another male littermate. The erectile bodies are almost completely suppressed and there is an enoimous hyperplasia of the periurethral connective tissue. The urethral canal (urinogenital sinus) is greatly enlarged, as in a female, and the sinus epithelium is transformed into stratified scjuamous epithelium like that of the fully developed vaginal canals. The sex of the subject makes no difference in the character of these responses. HORMONES IN DIFFERENTIATION OF SEX 129 both sexes (Fig. 2.32). With increasing dos- ages all these effects are accentuated. The phallic structures of mammalian em- bryos react to castration according to the pattern already established for the sex ducts and the prostate (Jost, 1947b; Raynaud and Frilley, 1947). In both sexes castration is followed by development of external geni- talia of female type (Fig. 2.28; Table 2.2) ; the male type of differentiation is depend- ent on the testis whereas the female form is capable of developing without hormonal conditioning, in a somatic or asexual man- ner. At this point it will be useful to recapitu- late for mammalian embryos the effects of castration, or of early isolation, on the de- velopment of the genital system as a wdiole. It has been shown that in the absence of the gonads, or of any hormonal conditioning, the embryonic sex primordia collectively fol- low the female pattern of development. In all castrates, regardless of sex, the external genitalia and the derivatives of the urino- genital sinus are of female type, the INIiil- lerian ducts persist and continue to develop in a virtually normal fashion, whereas the Wolffian ducts undergo involution. Thus castrates of either sex toward term have fe- male genital systems which are anatomically complete and almost as well developed as in normal females. It is noteworthy that the pattern of de- velopment observed in castrate fetuses cor- responds closely with a condition in human subjects known clinically as gonadal dys- genesis. Individuals presenting this anomaly either lack gonads entirely or show evidences of gonadal atresia at an early stage of de- velopment. Regardless of chromosomal sex as established by the Barr test (Barr, 1957) they possess external genitalia of female type and female genital tracts which, how- ever, are of infantile proportions. Recent evidence indicates that some individuals of this type may lack the Y-chromosome, being of XO constitution (Ford, Jones, Polani, de Almeida and Briggs, 1959; chapter by Gowen) . In bird embryos the effects of castration on the genital tubercle are similar except that the sex relation observed in mammals is reversed ; in this group the male form of the organ corresponds to the asexual condition, which develops without hormonal condition- ing in castrates of both sexes (Fig. 2.25; Wolff and Wolff, 1951). This is not an ex- ceptional finding; it corresponds with the behavior of various other avian sex charac- ters, such as the syrinx {q.v.) , the spurs, and the sex plumage in species such as domestic fowl. The transposed relationship seen here is in line with the dominant role played by the grafted ovary and the greater potency shown by the female hormone in producing sex reversal in the gonads of the chick. The developmental behavior of the genital tubercle after isolation in vitro has been studied in the duck, with results which cor- respond with those of castration. Isolated at 7 to 9 clays of incubation, before the be- ginning of sex differentiation in the gonads, primordia of the genital tubercle always as- sume the male form as in castrates, regard- less of the sex of the donor. By the 10th day, however, the sex type has become fixed, and when isolated after this stage differentiation always follows the sex genotype (Wolff and Wolff, 1952b). D. DIFFERENTIATION OF OTHER TYPES OF SEX CHARACTER Two further examples will be considered as illustrations of the role of hormones in the development of sex characters of quite different type, the mammary glands, and the syrinx of birds. The mammary glands of field mice have been extensively studied by Raynaud (for a summary see Raynaud, 1950). The rudiments of the glands first ap- pear as bud-like ingrowths of the epidermal epithelium which penetrate the underlying mesoderm but retain a connection with the epidermis by a constricted neck (Fig. 2.34.4, B). This phase of development follows the same course in both sexes. Toward the 16th day of gestation differences appear in males which coincide with the beginning of mas- culinization of the female genital tract; the mammary buds lose their connection with the epidermis and remain as isolated epithe- lial nodules in the mesenchyme (Fig. 2.34C) . In females, on the contrary, the buds retain their attachment to the epidermis, and as development continues a circular fold ap- pears surrounding the mammary rudiment, which leads to elevation of the nipple. 130 BIOLOGIC BASIS OF SEX SS^M •K^« ;'^ vVi^^#-p^ r-^i -^^-"i^^^^f^^ 7, ^^^^s^^^^i^^ss ^^^^^^^^^^^^ Fig. 2.34. The normal development of the mammary rudiments in embryos of the field mouse, and the effects of sex hormones (for a summary see Raynaud, 1950). A. Early appear- ance of the mammary thickenings in the female (left) and the male (right). B. Later stage, showing growth of the mammary primordia and penetration into the mesenchymal layer. C. Stage of sexual differentiation: in females the mammary rudiment remains attached to the epidermis and the nipple later develops at this point; in males the rudiment becomes de- tached from the epidermis and persists as a small epithelial nodule in the underlying mesenchyme. For the effects of hormones and of castration on this pattern see text. D. Nip- ple development in a male embryo induced by treatment of the mother with estradiol di- propionate. For details see text. (After A. Ravnaud, Arch. Anat. micro.scop. et Morphol. cx- per., 39, 518-569, 1950.) HORMONES IN DIFFERENTIATION OF SEX 131 Treatment with sex hormones during the latter half of gestation shows that the proc- esses described above are readily controlled or reversed experimentally. In female fe- tuses of mothers injected with male hor- mone, development of the mammary gland follows the male pattern; the mammary buds separate from the epidermis and per- sist only as nodular rudiments, the nipple fold does not appear and nipples fail to de- velop. The female pattern of differentiation is converted completely to the male type. The use of female hormones, on the other hand, leads to a somew^hat paradoxical re- sult ; there is an inhibition of the mammary buds rather similar to that exerted by an- drogen, but nipple development on the con- trary is strongly stimulated (Fig. 2.34D). The dosages were large, however, and the effects of female hormones under more phys- iologic conditions have not as yet been de- termined. With respect to development of the nipple similar results have been reported in the laboratory mouse (Greene, 1942); male hormone completely inhibits the nijv ples in females whereas female hormone in- duces typical development in males. Of particular interest are the effects of castration on mammary development in mice. When the embryonic gonads are de- stroyed by irradiation the mammary glands continue to develop in both sexes according to the normal female pattern (Raynaud, 1950). This pattern obviously does not de- pend on the ovary but represents the asexual or anhormonal type of development. Its ap- pearance in castrate males indicates that the regression of the mammary glands in the male is normally determined by the testis. This is in agreement with the results of ad- ministering male hormone, as described above. Once more the predominant role of the male hormone in mammalian sex differ- entiation is demonstrated. An entirely different type of sex character, and one which exhibits sexual dimorphism in a striking way, is the syrinx of birds. This organ has received special study in the duck (Wolff, Em., 1950) . The syrinx makes it ap- pearance as a vesicular dilatation at the junction of the trachea and bronchial tubes, and at first it is small and symmetrical in form in both sexes. This is the permanent condition of the syrinx in the female but in males a pronounced asymmetry soon ap- pears, involving an enlargement of the left side of the vesicle with corresponding modi- fications of the cartilaginous rings. By the 10th day of incubation the asymmetry is extreme (Fig. 2.25). The appearance of di- morphism follows closely the beginning of sex differentiation in the gonads. Experimental studies have shown that the dimorphism of the avian syrinx is condi- tioned by the ovary, or by the female hor- mone (Wolff, Em., 1950). Estradiol ben- zoate, introduced into incubating eggs, inhibits the development of the male syrinx and the female form appears. However, if large doses are used, or if treatment is too long delayed, a paradoxical result appears, consisting in the development of atypical and intermediate forms (Lewis and Domm, 1948). ]Male hormone (testosterone propio- nate) in moderate dosages has little effect on the syrinx (a slight enlargement may occur) but with large doses a paradoxical tendency is again found; the male syrinx is inhibited and may actually be reduced in size, resem- bling somewhat the female form. Castration again reveals the dominant role of the female hormone in birds. In cas- trates of both sexes the form of the syrinx is male, both in size and in its asymmetry (Fig. 2.25) ; absence of the ovary is critical but the presence or absence of the testis is of no consequence in the sexual differentiation of this structure. In its response to castration the syrinx thus behaves like the genital tu- bercle. The differentiation of the syrinx has also been studied in vitro (Wolff and Wolff, 1952a; Wolff, 1953a) with similar results. When explanted before the onset of sex dif- ferentiation, the result is the same as after castration; in an anhormonal environment the male form develops without regard to the sex of the donor. When explanted after the beginning of sex differentiation, how- ever, development proceeds always in ac- cordance wuth genotype. At this stage the form of the female syrinx has already been irreversibly determined. The syrinx develop- ing in vitro responds directly to sex hor- mones introduced experimentally. Addition of estradiol benzoate to the culture medium 132 BIOLOGIC BASIS OF SEX has the expected result; regardless of sex constitution, only the female type of syrinx develops. But male hormone (testosterone proprionate) under the same conditions pro- duces, paradoxically, organs of female or of intermediate form. The dosages employed, however, were extremely large (5 mg. and 40 mg. per cc. solvent) ^^ and in the light of the effects of large doses of androgen on the gonads and other structures the anomalous result is not surprising. VI. The Pituitary and the Diiferentiation of Sex It does not appear that the anterior lobe of the pituitary is concerned in the primary differentiation of sex, i.e., in the morpho- genesis of the gonads themselves. Early hy- pophysectomy does not interfere with their histologic differentiation, up to the stage at least where they are fully characterized as ovaries or testes; neither is the process of differentiation appreciably delayed. Later, however, deficiencies of a secondary order may appear in the genital tracts of hypo- physectomized animals; the development of various accessory sex structures may be considerably retarded but without any es- sential change in character. This effect is explainable as the result of diminished se- cretory activity on the part of the gonads through lack of adequate gonadotrophic stimulation. The question of when the func- tional interrelation between the gonads and the anterior pituitary is established has been reviewed by Willier (1952, 1955) with spe- cial reference to the chick, and .lost (1953, 1955) has dealt with this problem in mam- malian embryos and fetuses. In each case gonadotrophic activity is evident shortly after the period of gonad differentiation and covers the period when the sex ducts and other accessory structures are differentiat- ing. Some of the evidence on which these statements are based will be briefly re- viewed. In amj)hibian embryos or early larvae, ^* Under the conditions of culture the exphints develop in close contact with droplets of the hor- mone solution, and may actually be exposed to extremely high concentrations. Culture with the oil solvent alone, however, shows that the solvent is not the disturbing factor. hypophysectomized long before the time of sex differentiation, the development of ova- ries and testes proceeds normally and with- out appreciable delay until toward the end of the larval period (Smith, 1932, p. 752; Chang and Witschi, 1955; Chang, 1955). It is also well known that during larval life the gonads are capable of responding readily to gonadotrophic substances by rapid growth and precocious maturation of the germinal elements (e.g., Burns and Buyse, 1931; Burns, 1934). During this period pituitary stimulation merely accelerates the normal processes of development and matu- ration. It has been shown previously that in many amphibian species administration of steroid sex hormones during the larval period induces transformation of the gon- ads; however, an interesting case is known in which sex hormones appear to be without effect unless a gonadotrophin is also adminis- tered (Puckett, 1939, 1940). The tadpoles of a so-called "undifferentiated race" of Rana catesbiana all have gonads w^iich structurally resemble young ovaries until late in larval life, when differentiation of the males occurs rather abruptly. The ad- ministration of gonadotrophin alone to the undifferentiated tadpoles initiates sex dif- ferentiation precociously, the two sexes ap- pearing in the usual 50:50 ratio. The ad- ministration of sex hormones of either type to tadpoles during the indifferent period is without effect; the gonads are apparently incapable of responding at this stage of de- velopment. However, when the sex hor- mone and gonadotrophin are administered concurrently a striking response occurs; not only is sex differentiation precipitated, as when gonadotrophin was administered alone, but a complete transformation of sex takes place, resulting in all males or all fe- males, according to the type of sex hormone employed. The gonadotrophin is evidently necessary to initiate sexual diff(>rentiation but the type of differentiation which follows is determined by the type of sex hormone. In chick embryos "hypophysectomy" be- fore the onset of sex differentiation has been accomplished in two ways, by partial de- capitation, in which the forebrain area is remov(>d surgically after 33 to 38 hours of incubation (Futio, 1940), and bv irradiation HORMONES IX DIFFERENTIATION OF SEX 133 of the hypophyseal region (Wolff and Stoll, 1937) . After excision of the forebrain, histo- logic differentiation of the gonads proceeds normally. Later, however, the interstitial tissue of the testis fails to develop or is de- ficient in quantity, and the cortex of the left ovary remains rather thin through fail- ure of secondary sex cords to develop in the usual numbers. The development of the gonaducts, on the other hand, is normal in both sexes. Wolff and Stoll reported that, after destruction of the hypophysis by ir- radiation, differentiation of the gonads con- tinued in a normal manner to the end of incubation and again the gonaducts were found to develop normally. Such embryos, moreover, undergo sex reversal in the usual manner when treated with sex hormones (Wolff, 1937). The available experimental evidence indicates, then, that the hypophy- sis has no appreciable part in the primary differentiation of sex, a conclusion which is supported by van Deth, van Limborgh and van Faassen (1956). In mammalian embryos and fetuses, hy- pophysectomy has been carried out by pro- cedures similar to those described for the chick, namely, partial or total decapitation and irradiation with x-rays. The former method was used on embryos of the rat (Wells, 1947, 1950) and the rabbit (Jost, 1947d, 1950, 1951a), and the latter on the embryos of the mouse (Raynaud and Fril- ley, 1947; for a summary see Raynaud 1950) . In the case of the rabbit and the rat, the operation was not performed early enough to affect the primary differentiation of the gonads; in the mouse, however, ir- radiation on the 12th day of gestation, just at the beginning of differentiation, was without effect except for a certain reduction in the number of germ cells when the hy- pophysis was entirely destroyed. The re- sults differed sharply, however, with refer- ence to the condition of the genital tracts in hypophysectomized male rabbit fetuses, as opposed to those of the rat and the mouse. In the two latter species no significant changes in the development of the accessory genital structures were observed. It may be that in these species the entire process of sexual differentiation is independent of pi- tuitary function, although the possibility is perhaps not excluded that an extraneous gonadotrophin, of maternal or placental ori- gin, may be substituted. In the rabbit, on the other hand, definite defects were found in the development of certain accessory genital structures, resembling those which follow embryonic castration but somewhat less severe. However, if gonadotrophin is administered following decapitation these deficiencies do not appear; they may there- fore be ascribed to lack of gonadotrophic activity (Jost, 1951a, 1953). The defects observed vary in severit}^ according to po- sition ; the anterior regions of the gonaducts and the epididymides, which are near the testes, develop normally, whereas distant structures such as the prostatic glands and external genitalia may be almost as se- verely affected as in castrate fetuses. This observation indicates that the testis is act- ing in an intermediary capacity. In the ab- sence of the pituitary its humoral activity is diminished to a level adequate for normal differentiation of nearby structures but in- sufficient to maintain the development of more distant parts. The point has been pre- viously established that after decapitation there is a decrease in the amount of inter- stitial tissue. The problem of the time of onset of gon- adotrophic function in its relation to gonad secretion and the dift'erentiation of the geni- tal tract has been studied in the rabbit by Jost (1951a). By examining the genital tracts of decapitated male fetuses at short intervals to determine when the first signs of abnormal development appear, and by vary- ing also the age at which decapitation was performed, he was able to define rather closely the beginning of gonadotrophic func- tion and the period during which it is criti- cal for normal development of the genital structures. Following decapitation on the 19th day of gestation no marked defects in the genital structures appear until about the 22nd day, after which abnormalities be- come more and more pronounced until the 24th day. If decapitation is delayed until the 24th day no important anomalies are subsequently found. It should be recalled that the latter date coincides with the stage after which castration likewise has no effect on development. The interval from the 22nd 134 BIOLOGIC BASIS OF SEX to the 24th clay of development thus falls within the period during which testis ac- tivity is most essential for normal morpho- genesis (p. 125). In the light of these results the anterior hypophysis was studied cytologically for direct evidences of secretory activity, using the MacManus periodic acid-Schiff (PAS) test (Jost and Gonse, 1953; Jost and Ta- vernier, 1956). PAS-positive cells are first seen in small numbers, and faintly stained, on the 19th day of development. Thereafter they increase in numbers and in staining reaction, reaching a maximal development during the 22nd and 23rd days; on the 24th day these cells abruptly decrease in number and stainability and almost disappear. The peak of gonadotrophic activity, as indicated by the cytologic evidence, falls again dur- ing the 2-day period when the secretory ac- tivity of the testis is at its height, as judged by the conseciuences of castration, a re- markable example of endocrine correlation (for a fuller account see Jost, 1953, 1955). It was once widely believed that in hu- man anencephalic monsters the pituitary is absent or vestigial in character ; more recent studies have revealed, however, that al- though difficult to identify grossly, anterior lobe tissue can usually be demonstrated by careful histologic examination {e.g., Ange- vine, 1938). Nevertheless, cases are known in which apparently no anterior lobe tissue is present, and such cases are pertinent to the present discussion. Barr and Grumbacli (1958, and personal communication of Dr. Grumbach) have described such a case in a newborn male infant, in which no malforma- tion of the genital system was evident ex- cept that the testes were somewhat smaller than usual. They were not otherwise abnor- mal, however, and interstitial tissue was present. In a similar case, also a male, re- ported by Blizzard and Alberts (1956), the external genitalia were small but normal in structure. The testes also were small and undescended, lying in a pelvic position, the tubules were somewhat atrophic and no interstitial tissue was present. No other ab- normalities were noted. It is perhaps sig- nificant in this case that absence of the interstitial cells is correlated with under- development of the external genitalia and failure of the testes to descend. In two cases of congenital absence of the hypophysis, a male and a female (Brewer, 1957), develop- ment of the gonads and genital system was apparently normal. Cases in which complete absence of the pituitary has been demonstrated are un- fortunately few but of great value since they represent in humans the closest ap- proach to hypophysectomy in experimental animals. The consequences of the deficiency and the conclusions to be drawn in the two cases are similar; the primary differentia- tion of the gonads is evidently independent of the pituitary but secondary defects may appear later, both in the gonads and in the genital tract. The testes may be underde- veloped, the tubules may show secondary atrophy or degenerative changes, and the interstitial tissue may be reduced or lack- ing, but in some cases it appears to be well develoj^ed. There is need for a careful cor- relation of the status of the interstitial tis- sue in such cases with the presence or ab- sence of defects of the accessory organs. The consequences to the gonad of absence of the pituitary may hinge on whether a secondary source of gonadotrophin is avail- able to the fetus (Jost, 1953). This is a matter which may be expected to vary in different groups or species. As yet too few species have been studied to clarify the point. VII. Group Differences in the Relations of Hormones to Sex Differentiation The extensive experimental data reviewed in the foregoing pages show clearly that the embryonic gonads produce sex specific sub- stances which must be regarded as hormones and which act as physiologic agents in the differentiation of sex, controlling not only the development of the various accessory sex structures but in many cases the differ- entiation of the gonads themselves. That the substances are hormones in the usual sense is shown by the fact that they regulate the development of distant structures in such fashion that they can only be distributed by way of the circulating blood. This, however, does not preclude a sharply localized action under jiroper circumstances. That they are ('hib<)iat('(l in the gonads is demonstrated bv HORMONES IX DIFFERENTIATION OF SEX 135 many types of grafting experiments, and above all by the results of embryonic cas- tration. At the same time, steroid hormones of the adult type are also capable in many cases of reproducing closely the effects of the embryonic hormones, thus suggesting a basic similarity. This is not to say, however, that in all groups and species the role of hormones in the differentiation of sex is the same, either with respect to their effects on individual structures or their part in the differentiation process as a whole. Along with ontogenetic processes in general, hor- monal relationships have evolved differently in the different vertebrate groups. Amphibians. In amphibians both sex hor- mones seem to have active and essentially coordinate roles in sexual differentiation. With respect to gonad differentiation, grafted gonads of opposite sex induce trans- formation of both testes and ovaries by acting selectively on the appropriate gonad components. In many cases the interacting gonads are reciprocally modified, both be- coming strongly intersexual ; when this re- ciprocal effect does not occur it is appar- ently due to the decisive predominance of one member. Moreover, the gonad com- ponents in many cases react in a similar or identical manner to both natural and syn- thetic hormones. Male hormones induce precocious differentiation of the male duct system and the cloacal glands in individuals of either sex and when administered early may also completely suppress the develop- ment of the jMiillerian ducts; conversely, female hormones stimulate differentiation of the ]\Iiillerian ducts but are without effect on such male structures as Wolffian ducts and cloacal glands. But if such evidence demonstrates beyond question that the lar- val sex structures are capable of reacting specifically to hormones of the proper type, the role of hormones in the normal differ- entiation of sex is more directly demon- strated by the effects of larval castration. In castrates of either sex the gonaducts and other sex accessories remain indefinitely in an undifferentiated or slightly differentiated condition. The sexually neutral type in am- phibians thus tends to be morphologically intermediate between the sexes; however, either sex type may be readily obtained from the castrate type by transplanting an ovary or a testis (p. 112). The positive role of both hormones in sex differentiation is apparent. Birds. In bird embryos also steroid sex hormones stimulate precocious growth and differentiation of the appropriate sex pri- mordia, and in general have inhibitory ef- fects on structures of the other sex. There is a high degree of specificity in the interaction between hormone and end organ, and from the results of hormone administration alone it might be inferred that the two hormones have coordinate roles in sex differentiation. However, the results of castration show clearly that the roles of the two hormones are different and unequal. The Miillerian ducts persist and continue to develop in a similar manner in castrates of both sexes (p. 115). The male hormone is evidently the decisive factor in their differentiation since the presence of the testes causes in- volution of the ducts in males whereas the ovaries are not essential for their develop- ment in females. On the other hand the sex- type of the genital tubercle and the syrinx is conditioned by the ovaries. Both structures are normally developed in castrate males, for which male hormone is evidently not essential, whereas in castrate females also they closely approach the male condition in both form and size. It is the inhibitory action of the ovary, therefore, that deter- mines the dimorphism of the syrinx and the genital tubercle. ^^ These conclusions are confirmed by the fact that when isolated and cultured in vitro the Miillerian ducts, and the genital tubercle and syrinx, behave exactly as in castrate embryos; however, addition of male hormone to the culture medium causes involution of the Miillerian ducts, and female hormone prevents male differentiation of the tubercle and syrinx. Significant differences thus appear in the reactions of the accessory sex structures in birds as compared with amphibians. In the '^This i.s true also of such striking sex charac- ters as phunage type and spurs in adult fowl, whereas the head furnishings are under the con- trol of the male hormone. There is, however, much ■\-ariation in the relationships of secondary sex characters and gonad hormones in birds (Domm, 1939). 136 BIOLOGIC BASIS OF SEX latter positive stimulation by the proper hormone is necessary to induce final sexual differentiation. In birds, the role of the sex hormones appears to be primarily inhibi- tory; in the absence of gonads Miillerian ducts develop normally in castrates of either sex and genital tubercle and syrinx spon- taneously assume the male form. No posi- tive stimulus is necessary, only release from the inhibitory influence of the opposing gonad. Thus, the castrate type in birds is not undifferentiated or intermediate in type, but is rather a mosaic in which certain char- acters are typically male, others typically female. Again, however, both hormones are essential for the realization of normal sex differentiation but with the female hormone having the major role with respect to the number of structures controlled. Mammals. In mammals still another pat- tern appears in the relation of the accessory sex structures to the gonads and their hor- mones. Administration of pure steroid hor- mones demonstrates again that most of the genital primordia, regardless of sex consti- tution, are capable of reacting to the ap- propriate hormone, and if excessive dosages are avoided the responses are in most cases specific. To summarize, administration of male hormone does not significantly affect the development of male embryos except to accelerate the rate of differentiation; in fe- males, on the other hand, it induces differ- entiation of male structures whereas fe- male primordia are inhibited or fail to respond. In like manner, female hormones have a feminizing action on male embryos. Again, it might be assumed from the re- sults of hormone administration that the two hormones have comparable roles in nor- mal differentiation. In reality, what is dem- onstrated is the capacities of the sex primor- dia to respond to hormones experimentally introduced; the true role of hormones in normal differentiation is disclosed only when the embryonic genital tract is required to develop in the absence of the gonads or other hormonal influences. Castration of mammalian embryos reveals that normal differentiation depends chiefly if not ex- clusively on the male hormone. Castrated embryos, regardless of genie sex, develo):) female cliaracters (Miillerian derivatives, female sinus form and external genitalia, mammary glands) which are almost as well differentiated in castrates as in normal fe- males (Figs. 2.26-2.28, and Table 2.2). Any possible influence of a maternal hormone in this result appears to be excluded (at least for the sex ducts and their derivatives) by studies of development in vitro. Culture of the isolated gonaducts results in persistence and development of the ]\Iiillerian ducts and involution of the Wolffian ducts, re- gardless of the sex of the donor embryo (pp. 117, 121) providing always that isolation is carried out before irreversible determination has occurred. In mammalian embryos, then, the testes and the male hormone are all imjiortant for the normal differentiation of sex. Moreover, the role of the male hormone is a dual one; its presence is essential to insure retention and development of male parts and at the same time to prevent the differentiation of female structures, which are capable of de- veloping autonomously, regardless of the presence or absence of female hormone. The latter apparently has no essential role in primary sex differentiation. At this point it should be recalled that such a conclusion had in fact been forecast much earlier on the basis of castration of the newborn rat (Wiesner, 1934, 1935; see Burns, 1938b). At birth morphogenesis of the genital structures of young rats is far advanced and profound modifications after castration are not to be expected ; however, it was found that in cas- trate males a marked atrophy promptly ap- peared in such sex accessories as the seminal vesicles and external genitalia, suggesting that a hormonal influence had been re- moved, whereas in castrate females develop- ment proceeded more or less normally until the approach of puberty. These results were confirmed and extended by LaVelle (1951) in the ncnvboi'n hamster. Wiesner (1934, 1935) proposed that sex differentiation might be explained on the basis of one hor- mone, the male, the presence or absence of which would account for the two types of development, an hypothesis now confirmed ill a striking way by the results of castra- tion during embryonic and fetal develop- ment. The pre-ciiiiiiciit role of the male hormone HORMONES IN DIFFERENTIATION OF SEX 137 in mammalian sex differentiation stands in contrast with the situation in birds, in which the female hormone has the major role. The parallel with the well known difference in the sex-chromosome complex has been noted but this provides no immediate explanation and may be merely coincidence. On the other hand, another explanation may be found in the special physiologic conditions incidental to the evolution of intra-uterine development in mammals. A situation in which the female hormone has an active role in sex differentiation might present a serious difficulty with male embryos con- stantly exposed during development to the influence of the mother's hormones (the presence of considerable amounts of mater- nal estrogen during pregnancy is an estab- lished fact in many species (Price, 1947; Parkes, 1954). Elimination of the role of the female hormone coincident with the evo- lution of viviparity would then be advan- tageous. The problem of female develop- ment has apparently been met by a change in the status of the female sex primordia which, as amply shown by the results of castration and cultivation in vitro, do not require positive stimulation but develop autonomously unless inhibited l)y the male hormone. VIII. The Organization of the Sex Priniordiuni and Its Role in the Differentiation of Sex The role of hormones as specific condi- tioners of sexual differentiation is, however, only one aspect of the problem. Of far greater complexity, and fundamental to the selective character of the differentiation process, are the special attributes of the in- dividual sex primordia which predetermine their reactions to the presence, or absence, of a particular hormone. Each primordium possesses a complex organization, not only as to sex type and morphologic character, but also with respect to such detailed phys- iologic properties as the timing of receptiv- ity, the thresholds at which responses occur, and definite capacities for growth. It is ob- vious that specificity of hormone action does not exist independently of specificity of re- sponse. This organization of the primordium derives ultimately from the genotype of the species, operating through the same proc- esses of ontogeny that prescribe the special characteristics of other embryonic parts and systems; it is intrinsic as opposed to the conditioning activities of hormones and other modifying agencies which, with re- spect to the primordium, are external and secondary. Thus, sex primordia may be ex- pected to show variations in behavior to- ward hormones which will be peculiar to and integrated with the patterns of develop- ment characteristic of particular groups or species. A. CONSTITUTION AND THE MORPHOLOGIC REPRESENTATION OF SEX PRIMORDIA It has been pointed out that even in the bisexual or undifferentiated period of devel- opment many variations are found among different species in the extent to which the structures of the recessive sex are repre- sented morphologically, i.e., are laid down in the form of discrete primordia. The ab- sence or the deficient representation in one sex of certain heterotypic primordia may be normal for particular species, whose pattern of development thus places special limita- tions on sex reversal. In some amphibians reversal is difficult or impossible to in- duce experimentally because of the weak or transient representation of the reces- sive sex component in the gonad; there is no real transformation, merely a severely inhibited, or vestigial gonad. Certain acces- sory structures of the recessive sex may also tend to be abortive or imperfectly devel- oped. This is the case for the Miillerian ducts in the males of various species. In young male opossums, for example, the iNIiillerian duct rarely completes its devel- opment to the point of union with the urinogenital sinus, or the connection if formed is quickly lost, so that the terminal segment of the duct is lacking. Conse- quently, it has never been possible to induce vaginal development in male opossums by treatment with female hormones, although the uterus and Fallopian tube are present and highly developed. Moreover, the tend- ency which leads to absence of this region of the duct in male embryos appears to be present also, but more weakly expressed, in the female. Although the terminal segment 138 BIOLOGIC BASIS OF SEX of the duct is always present, it is suscepti- ble to inhibition by male hormone while the tubo-uterine portion is never so affected (Burns, 1942b). Thus, specific morphologic defects which appear in experimental re- sults can often be directly related to devel- opmental peculiarities of the species in question and in final analysis are an expres- sion of constitutional factors. However, constitutional differences which control the morphologic representation of sex primordia do not as a rule involve sim- ply the presence or the absence of a part, but more commonly have a quantitative ex- pression, affecting the extent to which the structures are developed or the length of the period during which they are present and capable of responding. An example is found in the gonads of birds, in which there are marked lateral differences between right and left sides (p. 95). In consequence, the effects of hormones on the right and the left gonads may be different, not qualitatively but in degree. The left ovary, with its strongly developed cortical component (Fig. 2.12), is only moderately affected by doses of male hormone which almost completely transform the rudimentary right gonad (Willier, 1939). The morphologic differences between right and left testes are less marked, but consistently the germinal epi- thelium is better developed and tends to survive longer on the left side than on the right. Consequently, female hormone read- ily transforms a left testis into an ovotestis, and with stronger dosages into an almost normal ovary, but the right testis is but slightly affected except when the dosage is very large. It appears also from studies of the effects of graduated dosages that thres- hold differences for the two sides may be involved, thus a physiologic as well as a morphologic factor is introduced. It is not held that experimental failures or anomalies are always directly traceable to specific morphologic deficiencies; however, the fre- quency with which such correlations appear indicates the importance of underlying structural variations in modifying the re- sponses of sex primordia under experimental conditions. B. CONSTITUTIONAL FACTORS AND PHYSIOLOGIC DIFFERENCES IN THE ORGANIZATION OF SEX PRIMORDIA In the foregoing cases obvious morpho- logic differences provide, at least in part, a basis for observed differences in the experi- mental behavior of sex primordia. It is un- likely that morphologic differences of this order exist without an underlying physio- logic differentiation. On the other hand, un- der experimental conditions, physiologic dif- ferences often become apparent which have no visible morphologic expression, as in the inhibition of the vaginal canals of female opossums cited above. Certain accessory sex structures in birds exhibit lateral differences in sensitivity to hormones which are evi- dently a reflection of the general tendency to asymmetrical development in this group. In normal females only the left Miillerian duct develops into a functional oviduct ; the right, although originally well developed, regresses at an early stage. After castration, however, both ducts develop equally (Fig. 2.25) as is also the case when the ducts are isolated in vitro. Involution of the right oviduct, then, is conditioned in some way by the ovaries and it has been shown that either the right or left ovary alone is ef- fective (Wolff and Wolff, 1951). Evidently the ovaries exert an inhibitory action on the right oviduct which is not effective on the left. Presumably a threshold difference is involved.^*' Other examples may be cited. The syrinx and the genital tubercle remain small, sym- metrical, and essentially undifferentiated in females, but in males they become large and highly asymmetrical (Fig. 2.25). Unlike the paired structures previously dealt with, these organs are single and median in posi- tion and the asymmetry of the male form is due to unequal development of the lateral halves of the organs. It is well established in the case of eacii that the female hormone ""It seems unlikely tliat the inhibitoiy factor in this case is the female hormone since the introduc- tion of estrogenic hormones into incubating eggs causes persistence of the right oviduct. However, the concentration or dosage may be a factor in this curious effect. HORMONES IN DIFFERENTIATION OF SEX 139 inhibits the male type of development (pp. 127, 131) which, on the other hand, develops spontaneously in both sexes after castration (Fig. 2.25) or after isolation in vitro, with- out hormonal conditioning. A difference in susceptibility to inhibition by the female hormone apparently masks the inherent dif- ference in growth potential and the primary symmetry of the female structure is pre- served. The marked asymmetries of the genital system in birds thus appear to rest on lat- eral differences involving such physiologic characteristics as growth potentials and reaction thresholds. These in turn are ap- parently correlated with a more extensive asymmetry involving the whole organism. Lateral growth differentials are established in the blastoderm of chick embryos as early as the head-process stage. In testing the organ- forming potencies of regional pieces of the blastoderm it was found (Willier and Rawles, 1935; Rawles, 1936) that when cor- responding pieces of the same size from right and left halves of the blastoderm are trans- planted to the chorioallantoic membrane they consistently show marked differences in capacity for growth and self-differentia- tion, which are manifested both in the size attained by the graft (growth capacity) and in the quality of the histologic differentia- tion. Regardless of the particular tissues or organs dealt with, grafts from the left half of the blastoderm are consistently larger and better differentiated than those from the corresponding pieces of the right half. Lillie (1931) also postulated critical differences in growth rate and threshold to sex hor- mones on the two sides of the body in explaining the occurrence of "gynandromor- phic" plumage in adult fowls and its dis- tribution. He pointed out that the sharply defined difference in plumage on the two sides of the body is usually accompanied in gynandromorphs by gross bodily asym- metry (hemihypertrophy) favoring the left side. It would seem that lateral differences in the morphologic and physiologic proper- ties of the sex primordia of birds are not peculiarities of sexual differentiation ; rather they are an aspect of the general pattern of somatic organization in this group. In the normal differentiation of sex as well as in experimental studies these differences are exploited by hormones whose effects serve merely to exaggerate or to obliterate tend- encies inherent in the organization of the individual primordia. C. INFLUENCE OF SEX GENOTYPE ON THE REACTIONS OF SEX PRIMORDIA Another example of the way in which constitutional factors operate to modify or set limits to hormone action is seen in the influence of the sex genotype on the re- sponses of sex primordia to hormones, as il- lustrated especially in young opossums (Burns, 1942b, 1956a). In comparing the effects of identical doses of the same hor- mone (whether male or female) in embryos of different sex, it was found in the case of many structures that the amount of growth induced was influenced by the sex of the individual. Under identical experimental conditions the effect of a male hormone on the growth of a particular male structure was always greater in male embryos than on the homologous structure in females, and vice versa. This result is well illustrated by the reactions of the genital tubercle or phal- lus in male and female littermates which received identical doses of testosterone pro- pionate. The transformed phallus of the fe- male cannot be distinguished anatomically or histologically from the male organ, ex- cept for a constant and considerable dif- ference in size (Fig. 2.32). Such an effect was cited earlier in the case of the prostate (Fig. 2.29) and it occurs for various other male structures such as the vas deferens I Wolffian duct) and the epididymis (Fig. 2.24). After treatment with female hormone corresponding differences are observed in the response of female structures. The Miiller- ian ducts of male embryos hypertrophy and undergo a typical differentiation into ovi- duct and uterus; nevertheless, in size these organs do not approach those produced by the same dosage in females (Fig. 2.24). The same is true in the case of the hyperplastic reaction of the sinus epithelium and for other structures. Differences in size can be 140 BIOLOGIC BASIS OF SEX detected at an early stage and increase throughout the period of treatment. To account for the constancy of these dif- ferences it seems necessary to assume that sex constitution in some way conditions the reactivity of the primordia, placing certain limitations on rate of growth. When sex constitution and type of hormone adminis- tered are the same there is, in effect, a sum- mation of the two factors, but when they are different a conflict occurs. It might seem more simple to suppose that the embryonic gonads and their hormones are involved in this result, rather than to assume a differ- ential reactivity on the part of the sex pri- mordia; hormones of the same type would in one instance reinforce each other whereas in the other case unlike hormones oppose each other. However, this simple hypothe- sis cannot be sustained. Although the pres- ence of a hormone from the embryonic testis may be safely accepted, there is as yet no evidence in mammals that the ovary pro- duces significant amounts of hormone at this period, thus no supplementary factor can be assumed in the case of females receiving female hormone. In the male, moreover, his- tologic studies reveal that the size differ- ences observed are much too great to be ac- counted for by the secretory activity of the embryonic testis ; for example, the difference in size between the male and the female prostate after treatment with male hormone (Fig. 2.29) is many times greater than the volume of the normal prostate, which may be taken as the measure of normal testis activity. The conclusive argument against participation of embryonic hormones in this phenomenon has come, however, from ex- amination of the embryonic testes of ex- perimental animals (Burns, 1956a). There is a great reduction in the size of the testis (and the ovary as well) and histologic study shows complete suppression of the inter- stitial tissue, the intertubular spaces being filled with a dense, nonstaining connective tissue of mucoid type. In contrast, the nor- mal testis of the same age has a rich inter- stitium which is well developed as early as the 10th day of pouch life (Fig. 2.17.4 ». Evidence to be summarized later points strongly to the embryonic interstitial tissue as the source of the testis hormone, and in the absence of this tissue it does not seem that the testis can be a factor in the result. IX. The Time Factor in the Responses of Sex Primordia: Receptivity and "Critical Periods" Of special importance is the factor of de- velopmental age as it relates to the appear- ance of receptivity and the timing of de- terminative changes in sex primordia. This becomes apparent when the reactivity of a primordium to sex hormones, or its capacity for independent differentiation after isola- tion, is tested at successive stages of devel- opment. Typical studies of the second type are the experimental analyses of the appear- ance of sex-specific organization in the geni- tal ridge of chick embryos (Willier, 1933) and in the differentiating gonads of the rat (Torrey, 1950; see pp. 103, 104). Such stud- ies show that the organization of embryonic gonads with respect to sex type and capacity for self-differentiation is acquired gradu- ally, leading step by step to changes which are stable and irreversible. Such transitions coincide in some cases with distinct morpho- logic events. In Willier's study of chick gon- ads fixation of sex-type, with capacity for autonomous differentiation, coincides with the appearance of a distinct germinal epi- thelium on the genital ridge. In rat gonads (Torrey) the sexes differ greatly in this re- spect; differentiation of prospective testes becomes an autonomous process from the first laying down of the medullary blastema, whereas the ovary has but little capacity for self-differentiation until much later, after the appearance of a distinct cortical zone. The fact that at certain stages of develop- ment changes of an irreversible nature can be demonstrated has led to the recognition of so-called "critical periods," during which rather abrupt transitions occur from a state of lability to one of complete autonomy. Thereafter, hormones or other extraneous factors no longer have decisive effects. Such stages have been demonstrated for various types of sex primordia and are often nar- rowly limited in time. In chick embryos continued development of the Miillerian ducts, or their involution, depends normally on the ty{)e of gonad present, but at a cer- HORMONES IX DIFFERENTIATION OF SEX 141 tain stage their fate can be permanently conditioned by hormones administered ex- perimentally (Wolff, 1938; Stoll, 1948). In male embryos involution of the ducts is pre- vented by administration of female hor- mone at the proper stage (p. 112), and once "stabilized" in this manner their sub- sequent development is assured without fur- ther treatment. Male hormones adminis- tered before this stage induce involution of the ducts in the living embryo or in vitro, but beyond this point have no effect. The genital tubercle of the female duck shows a critical stage in relation to the em- bryonic ovaries during the 9th day of in- cubation (Wolff and Wolff, 1952b). When isolated in vitro before this stage the tu- bercle always develops the male form, which is also the condition found in castrated em- bryos. Isolation after the 9th day, however, results always in a structure of female type. About the 9th day of development, then, its future character becomes fixed after which differentiation proceeds without further need of hormonal conditioning. Similar re- sults were obtained in the case of the syrinx. If isolated before the stage of final deter- mination the sex type of both j^-imordia can be readily controlled in vitro by addition of hormones to the medium. The Wolffian ducts of mammalian em- bryos behave similarly. In this instance the male hormone is necessary at a certain stage to insure retention of the ducts. Castration of male rabbit embryos before the 22nd day is followed by involution but later castra- tion has little effect; changes of an irre- versible nature have occurred which insure continuation of development regardless of hormonal conditioning. A critical period of brief duration also exists for the prostate glands of young opossums, involving the response to both types of sex hormone. Estrogens permanently suppress prostate development in males if a single dose is ad- ministered just before the stage when the buds should appear. Male hormones, on the other hand, induce prostatic glands in fe- males at this stage which thereafter con- tinue to develop without further treatment. The effects of castration on the prostate are like those described for the Wolffian ducts. In rabbit embryos the critical period falls from the 22nd to the 23rd day of gestation after which the operation has but slight effect (Table 2.2). It appears from much evidence of this kind that sex primordia typically pass through developmental phases which are crucial with respect to the origin, the sur- vival or the future mode of differentiation of the structure in question. At such stages, and for brief periods, formative or suppres- sive, trophic or involutionary, responses are readily induced by hormones. However, the physiologic status of the primordium itself prescribes the specific quality of the re- sponse and the timing as well. X. Specificity of Hormone Action and the Significance of Paradoxical Effects Perhaps the objection most frequently urged against steroid hormones as specific agents in sexual differentiation is the com- mon occurrence of paradoxical effects, in which a hormone of one type stimulates the differentiation of structures of the other sex, sometimes in a striking manner. Such responses have been encountered in all ma- jor groups thus far investigated, and prac- tically every type of sex character may be involved. The frequency with which this phenomenon is associated with high dosages has been noted, with emphasis on the fact that in low concentrations the effects are usually sex specific. Some apparent excep- tions to this general rule may, indeed, be due to difficulty in defining a low dose in particular cases in view of the efficiency of extremely low concentrations in certain spe- cies (e.g., Mintz, 1948). Specificity of action obviously implies that male hormones stim- ulate development of male characters in em- bryos of either sex, whereas female pri- mordia are inhibited or give no positive response; in like manner, female hormones should induce differentiation of female pri- mordia while inhibiting the development of male structures. Convincing examples of specific action in this sense are found in the complete and even functional transforma- tions obtained in various amphibian species with low concentrations of hormones (Ta- ble 2.1) ; in the maintenance of normal dif- ferentiation after castration by treatment 142 BIOLOGIC BASIS OF SEX with a crystalline hormone; and in the manner in which many accessory sex struc- tures or their primordia respond to the ap- proiH'iate hormone, both in vivo and in vitro. It should be emphasized further that para- doxical effects seldom appear to the ex- clusion of normal responses, but usually as accompanying phenomena. For example, large doses of testosterone propionate pro- duce strong hypertrophy of the entire male genital system in opossum embryos as ex- pected; however, they also cause hyper trophy of the Miillerian duct derivatives, a response that disappears at low dosages al- though the effect on male structures remains. In the interpretation of paradoxical ef- fects the problem of direct vs. indirect ac- tion arises. It may be argued tentatively that in sufficient concentration hormones of either type stimulate the primordia of the other sex by direct action ; both sets of pri- mordia are capable of responding but at different thresholds, those of heterotypic structures being very high. Such a situation would permit selective action at ordinary or "physiologic" levels and at the same time account for the appearance of paradoxical effects at higher concentrations. As a mat- ter of fact, the dosages which elicit para- doxical responses are as a rule so far above physiologic levels as to have doubtful sig- nificance for normal differentiation. Evi- dence favoring the thesis of direct action has been cited and in one case at least a typical paradoxical effect has been produced in vitro. Large doses of testosterone propionate have a strong feminizing (i.e., inhibitory) action on the syrinx of the duck in vitro (Wolff and Wolff, 1952a). However, the presumption that the paradoxical action must have been exerted directly does not establish its nature. The high concentration of male hormone obviously has an adverse effect, resembling the inhibitory effect of the female hormone, but the inhibition is possibly of a general nature rather than specific. First attempts to culture the syrinx on a simple synthetic medium also resulted in atypical differentiation (Wolff, Haffen and Wolff, 1953) due apparently to nutri- tive deficiencies. High concentrations of hormones ?>? vitro mav onlv create general conditions unfavorable for normal growth and differentiation. On the other hand, paradoxical effects are certainly in some cases not mediated di- rectly but are of secondary origin. The fem- inization of the testes that occurs in certain amphibians after treatment with male hor- mones (p. 94; for a summary see Gallien, 1955) is an example. An early disturbance of mesonephric development interferes sub- sequently with differentiation of the medul- lary sex cords, thus preventing testicular development. After metamorphosis, when the hormone is withdrawn, a certain recov- ery occurs and development is resumed, but in the virtual absence of the medullary component only the cortical rudiment de- velops. It should be noted, however, that during the hormone phase of this experi- ment there is inhibition of the cortex as well as suppression of the medulla, and it is only after the male hormone is withdrawn that development of the cortex is resumed. Al- though the paradoxical effect on the medulla, is pronounced it is indirect, and it does not occur alone but in conjunction with a par- tial atresia of the cortex. Thus the picture is more complicated than first appears. On the other hand, the correlation be- tween the appearance of paradoxical effects and the use of high dosages suggests other possibilities as to the manner in which such effects are mediated. It is a familiar fact in endocrine physiology that prolonged treat- ment with sex hormones disturbs the nor- mal endocrine balances and may influence the activity of other glands. It is possible that certain paradoxical effects may origi- nate in this way. As yet there is no direct evidence of this in embryonic organisms but it is well known that under abnormal or pathologic conditions both the gonads and the adrenal glands of adult animals are capable of producing the hormones of the other sex. This is the case for certain tu- mors of the gonads and adrenal cortex, and it is characteristic of the adrenal hyper- plasias which produce the adrenogenital syndrome in fetal and postnatal life. It is also well established that under abnormal physiologic conditions ovaries may produce considerable amounts of androgen (cf. Hill, HORMONES IN DIFFERENTIATION OF SEX 143 1937a, b; Deanesly, 1938; for further dis- cussion see Ponse, 1948). A striking case of adrenal disturbance induced by a sex hor- mone appears in frog tadpoles completely inasculinized by large doses of estradiol. Histologically, the change takes the form of a massive hyperplasia of the adrenal cortical or interrenal tissue (Padoa, 1938, 1942; Witschi, 1953; Segal, 1953) which may attain 10 times the normal volume. In this remarkable case, however, the mas- culinization of the ovaries is not caused by the adrenal hyperplasia, because in hypo- physectomized tadpoles the hyperplasia does not occur but the paradoxical mascu- linizing effect still persists. Nevertheless, when excessive doses of sex hormones can induce glandular disturbances of this order, the possibility remains that they may be only secondarily involved in the appearance of paradoxical effects.-^ Perhaps the simplest explanation of the paradoxical effects of high dosages lies, however, in the possibility that, when pres- ent in excess, a hormone may be trans- formed in the organism into one of opposite type. In this event two hormones are in fact acting simultaneously and the specificity of the administered substance is not in question. This possibility was first sug- gested by findings in the adults of several mammalian species, including man. Treat- ment with large amounts of testosterone may be followed by excretion of consider- able quantities of estrogen in the urine, which disappears when the male hormone is withdrawn (for the older literature see Burrows 1949, Ch. VI). This may occur in normal males, in castrates or in eunuchoid types. In female subjects the estrogen thus produced is sufficient to stimulate female characters or functions, e.g., a marked hy- perplasia of the vaginal epithelium appears. Without the knowledge that estrogen is be- ing produced this would be regarded as a typical paradoxical effect. Conversion of testosterone to estrone or estradiol also -^ For fuller discussions of various forms of para- doxical effects and their interpretations see Gallien (1944, 1950, 1955), Wolff (1947), Ponse (1948), Padoa (1950), Jost (1948a) and Burns (1949, 1955b). takes place in ovariectomized and adrenal- ectomized women (West, Damast, Sarro and Pearson, 1956). It is evident that neither gonads nor adrenals are necessary for such conversions, which may even occur in vitro (Baggett, Engel, Savard and Dorfman, 1956; Wotiz, Davis, Lemon and Gut, 1956). Finally, it has been established that the in- jected male hormone is the actual source of the estrogen by the use of testosterone la- beled with C'^ (Baggett, Engel, Savard and Dorfman, 1956; Wotiz, Davis, Lemon and Gut, 1956; Heard, Jellinek and O'Donnell, 1955; for a recent review of this subject see Dorfman, 1957). Although it may be tech- nically difficult to demonstrate such con- versions in embryonic organisms, there are no grounds for supposing that they cannot occur. XI. Time of Origin and the Source of Gonad Hormones Evidence that the embryonic gonads be- gin to produce their hormones early in the course of sexual differentiation comes from many sources. In the more strongly modi- fied freemartins, conditions indicate that the hormone of the male twin must have been active at an early stage. The ovaries of the female are severely inhibited with almost complete suppression of cortical differentia- tion (Willier, 1921). Lillie (1917) suggested that in such cases the first action of the male hormone might be to produce, in ef- fect, a "castration" of the female twin by suppression of the ovarial cortex. It now ap- pears from the results of actual castration experiments that this point is probably not significant as there is nothing to indicate that the ovary is active endocrinologically at such an early stage (Bascom, 1923) . In amphibians, either after parabiosis or transplantation of the gonad priraordium, changes in the gonads can be detected very early in relation to the onset of sex differen- tiation, and in some circumstances reversal occurs by direct differentiation as a gonad of opposite sex. The gonads involved are as a rule widely separated and the hor- monal nature of the transforming agent in these cases is beyond question. Similar indications are found in birds. Embrv- 144 BIOLOGIC BASIS OF SEX onic ovaries grafted into the coelomic cavity induce cortical differentiation in the testes of male hosts at a very early stage, and dur- ing the same period testis grafts inhibit the difTerentiation of the IMiillerian ducts. A similar effect is observed when histologically undifferentiated gonads of duck embryos are cultured in vitro in close contact (Wolff and Haffen, 1952b). When ovaries and tes- tes are thus associated the latter exhibit typical reversal changes from the very be- ginning of sexual differentiation. In mammals the activity of the testis hormone in the early stages of sex differen- tiation is revealed by the promptness with which castration effects appear. In the ab- sence of the testes, changes in certain ac- cessory sex structures are evident as soon as sexual differentiation can be observed. In the case of the prostate the hormone is actually necessary for the appearance of the primary buds. Conversely, implantation of an embryonic testis into a female rabbit embryo inhibits the Miillerian ducts and initiates development of male accessory structures (Fig. 2.35; for a more detailed summary see Willier, 1955). The source of the hormones produced by Ostium of Mullerian Duct fted Testl Fig. 2.35. Localized effects of an embryonic testis grafted to the broad ligament of a fe- male rabbit embryo (Jost, 1947b). The inhibitory effect of the grafted testis has caused a great reduction in the size of the host ovary and has suppressed the Mullerian duct (un- shaded) in the vicinity of the graft. These structures are normal on the opposite side. Con- versely, the testis has induced complete retention of the Wolffian duct and epididymis (stippled) on the side of the graft and a partial retention on the other side. The influence of the graft is strongest in its immediate vicinity and beyond a certain distance disappears, indicating that the hormone spreads locally by diffusion rather than through the circulation. The results also suggest that the stimulatory action on the male structures is stronger than the inhibitory effect on the Miillerian duct, since it extends further. Such a situation prob- ably results from threshold differences in reactivity of the two end-organs to the testis hor- mone. HORMONES IN DIFFERENTIATION OF SEX 145 the embryonic gonads is a matter which can be discussed with some assurance only in the case of the mammahan testis. In the testes of adult mammals the interstitial tis- sue has long been recognized as the source of the male hormone and, as was pointed out in the beginning, the marked develop- ment of this tissue in the testes of pig em- bryos led to the first suggestion that a hormone might be involved in sexual differ- entiation (Bouin and Ancel, 1903). In con- nection with the freemartin studies, an examination was made of the gonads of nor- mal calf embryos and fetuses (Bascom, 1923) which pointed to the well developed interstitial cells as the probable source of the male hormone. This provided a plausible explanation of the invariable dominance of the male twin, because in fetal ovaries no in- dication of internal secretion could be found until relatively late in gestation ; in the tes- tis, on the contrary, interstitial tissue was seen in increasing amounts from practically the beginning of sex differentiation. It is unnecessary to multiply cases in which the presence of interstitial tissue in the embryonic testis coincides with indica- tions of hormone activity. On the other hand, instances in which a reduction in tes- tis activity (as evidenced by the condition of the sex accessories) can be correlated with the status of the interstitial tissue are pertinent. Decapitation of rabbit embryos (Jost, 1951a) is followed by definite re- tardation in the development of certain male accessory structures, although the growth of the embryo as a whole is normal. Exam- ination of the testes in these specimens showed a reduction in size and in the num- ber of interstitial cells; whether there was also cytologic abnormality has not been as- certained. The defects of the sex accessories in the decapitated fetuses resembled those which appear after incomplete or unilateral castration. Structures near the defective tes- tes (epididymides, vasa deferentia) were virtually normal but more distant structures (sinus derivatives, external genitalia) showed failures of development comparable to those produced by complete castration. Inadequacy of the testes to maintain nor- mal development in these cases is appar- ently due to a quantitative deficiency of the interstitial tissue and the male hormone. A similar reduction of the interstitial tis- sue occurs in decapitated rat fetuses (Wells, 1950) and in this instance cytologic changes in the interstitial cells were also seen. In this species, however, no clear effects were observed on the accessory sex organs, as is also the case in fetal mice hypophysecto- mized by irradiation (Raynaud and Frilley, 1947; Raynaud, 1950). Negative findings in these cases may be attributable to species differences as to the stage at which the in- terstitial tissue becomes active ; however, it is more probable that the different result is due simply to the longer period of observa- tion in the rabbit, allowing more time for the deficiencies to appear.-- Also pertinent in this connection is the behavior of the interstitial tissue of the em- bryonic rat testis transplanted between the lobules of the seminal vesicle of a castrate adult; the interstitium of the grafted testis undergoes a considerably hypertrophy and the epithelium of the host's seminal vesicle gives a corresponding response (Jost, 1951b). But when the host is also hypo- physectomized such grafts are deficient in interstitial cells and there is little or no re- sponse by the seminal vesicle (Jost and Co- longe, 1949). The correlation between the state of development of the interstitial cells and the evidences of hormonal activity in these cases is direct and striking (for a re- cent review of this subject see Jost, 1957). XII. A Comparison of the Effects of Emhryonic and Adult Hormones in Sex Differentiation A problem has long existed as to whether the hormones or hormone-like substances produced by the embryonic gonads are es- sentially similar in character to adult sex hormones. When the effects of the two types of hormone on the development of embry- onic sex primordia are studied under com- parable conditions the resemblances are in "It should be noted that treatment with go- nadotrophins prevents the reduction in the inter- stitial tissue after decapitation, and may even pro- duce hypertrophy of the interstitial cells (Wells, 1950, Jost, 1951a). In one instance also a graft of the fetal hypophysis had the same effect (Jost). 146 BIOLOGIC BASIS OF SEX many cases extremely close; moreover, some of the apparent discrepancies have since been found to be due not to fundamental differences in the two types of hormone but to differences in experimental conditions as regards such factors as timing and dos- age. The most important objections to ster- oid hormones of adult type as controllers of sex differentiation have been noted previ- ously in various connections. However, a brief recapitulation is in order: they are (1) the frequent occurrence of paradoxical effects; (2) the failure of male hormones to inhibit effectively the Miillerian ducts of mammalian embryos; and (3) their failure in nearly all cases to have significant ef- fects on the differentiation of mammalian gonads. The first objection has been dealt with (p. 141) and will not be disscussed fur- ther. In the other cases the failure is not absolute ; moreover, it is confined to a single group, the mammals, and is not of general application. To an extent, group or species differences may be involved. In the hedge- hog, for example, the funnel region of the oviduct is inhibited by male hormone (Mombaerts, 1944), and in female opos- sums the vaginal portion is suppressed on one or both sides in about 50 per cent of all individuals (p. 114). Furthermore, in mouse and rabbit embryos male hormone prevents the union of the posterior ends of the Miil- lerian ducts to form the vaginal canal (Ray- naud, 1942) and corpus uteri (Jost, 1947a). These partial effects in themselves require explanation. Failure of steroid hormones to reproduce more fully the effects of the em- bryonic hormone may lie, at least in part, in experimental conditions other than the type of hormone. On the other hand it is possible, as suggested by Jost (1953, 1955), that in mammals a special substance is required for the inhibition of the Miillerian ducts other than the ordinary testis hormone. The failure of steroid hormones to modify the gonads of placental mammals (even when the accessory sex structures are pro- foundly transformed) is in marked contrast with the striking results obtained in many lower vertebrates and in the opossum. It is also at variance with the strong modifica- tions usually found in freemartin gonads, and this has often been cited as proof that different types of hormone are involved. However, the freemartin is still almost unique among mammals as an example of gonad transformation induced by another embryonic gonad, and may yet prove to be a special case of a type peculiar to the bo- vine family.^^ The possibility must be con- sidered that in placental mammals gonad differentiation (as opposed to the differen- tiation of the accessory sex structures) has come to be under direct genotypic control; nevertheless, the demonstration, after many earlier failures, of a thoroughgoing trans- formation of the testis in the opossum sug- gests that certain essential experimental conditions have perhaps not been fully re- alized. In any case, it may be a difficult matter to determine whether the refractori- ness of mammalian gonads to steroid hor- mones is indeed due to a fundamental dif- ference in the character of the hormones themselves or to a change in the status of the embryonic gonads affecting their reac- tivity to hormones. In many other situations it appears that embryonic and adult sex hormones are in- terchangeable without observable differ- ences in the results. Testosterone propionate or methyl-testosterone, administered to cas- trated rabbit embryos at the time of opera- tion, prevent the usual castration changes in all male structures, in this respect fully re- placing the embryonic testis (Jost, 1947b, 1950, 1953), although they do not inhibit the Miillerian ducts. A similar effect of tes- "'' There is, in fact, a notable scarcity of free- martins in a strict sense in other groups in which, on the grounds of placental fusion, the phenome- non might be expected to occur at least occasion- ally. For the literature on scattered cases inter- preted as freemartins, see Willier (1939) and Witsclii (1939); and for a case (the marmoset, Wislocki) in which no freemartin effect was found although the essential conditions seemed to be present, see Witschi (1939). It is possible that the piesence of the hormone is not the only factor to be considered ; lack of reactivity on the part of the gonad may be the principal factor and one which may vary in different groups, correlated perhaps with the presence of maternal or placental estro- gens during pregnancy. There is still a surprising lack of information for many groups; for exam- ple, the freemartin condition in sheep (at least as regards sterility) may occur more frecjuently than lias been supi)oyed (sec Stormont, Weir and Lane, 1953). HORMONES IN DIFFERENTIATION OF SEX 147 tosterone propionate in maintaining tlie male accessory glands has been shown in castrated rat embryos (Wells, 1950; Wells and Fralick, 1951 ) . In opossum and other mammalian embryos synthetic androgens readily induce in females prostatic glands which are histologically indistinguishable from those of normal males although the latter are known to be conditioned in cer- tain species by the hormone of the embry- onic testes. Also, with proper timing, syn- thetic androgens induce involution of chick Miillerian ducts, either in vivo or in vitro, in a manner not histologically distinguish- able from the effects of the embryonic testis (p. 114). Examples of this kind could be multiplied. Furthermore, the female hor- mone estradiol controls the sex type of vari- ous avian sex primordia, even when admin- istered in vitro, closely simulating the normal action of the embryonic ovary. Wlien it is added to the culture medium, testes are transformed into ovotestes in the same fash- ion as when cultured in association with an embryonic ovary (Wolff and Haffen, 1952b) , and it produces a typical female syrinx or genital tubercle in vitro regardless of the sex of the donor embryo (Wolff and Wolff, 1952a; Wolff, 1953a). Conversely, an example of an embryonic hormone substituting for an adult sex hor- mone is seen in the effect of a graft of the embryonic testis on the epithelium of the seminal vesicle in an adult castrate rat (Jost, 1948b, 1953; Jost and Colonge, 19491. Within a few days the vesicle epithelium in the vicinity of the graft is completely re- stored. Although the interstitial tissue of the grafted testis is somewhat hypertrophied under the influence of the host's hypophysis, it is improbable that a radical change in the character of the testicular secretion could be induced so quickly. That the hor- mone of the graft must be attributed to in- terstitial cells which cytologically are like those of the adult testis is also significant. It should be noted further that both estro- gens and androgens (which can be detected by standard methods of assaying adult sex hormones) have been extracted from chick embryos in the latter half of the incubation period (Leroy, 1948) and from fetal mam- malian gonads as well {e.g.. Cole, Hart, Ly- ons and Catchpole, 1933). If special em- bryonic hormones are necessary for the control of sex differentiation, it would seem that hormones of adult type are also being produced during the same period. Many minor inconsistencies can be pointed out in comparing the effects of the two types of hormone, but experimental conditions are usually too dissimilar to justify such detailed comparisons. It is note- worthy that the most "normal" results from the use of steroid hormones have been ob- tained under conditions which most closely approach the ideal, as when larval amphib- ians absorb the hormone continuously but in low concentration from the surrounding water. In many such experiments involving many species (Table 2.1) all individuals develop in accordance with, the type of lior- mone used, and without obvious histologic abnormalities. Under similar conditions, however, if the concentration is increased, all gonads become intersexual and very strong doses may actually produce effects exactly opposite to those obtained at very low levels (p. 94). It is nevertheless too simple to suppose that all difficulties may be avoided simply by empirically arriving at the proper dose. What constitutes the optimal dose is not easy to determine from one species to an- other for, regardless of absolute concentra- tion, the hormone level in the internal en- vironment of the experimental organism may be greatly affected by such factors as the rates of absorption, utilization, and in- activation. These are factors which vary widely with different hormone preparations, different methods of administration, and also no doubt from one organism to another. In the second place, it is difficult or impos- sible to adjust the dosage accurately and flexibly from stage to stage, to correspond with the changing conditions of develop- ment and the state of the reacting struc- tures; however, a continuing equilibrium is doubtless more nearly approached when doses are relatively low and the hormone enters continuously through the gills or by infusion from a graft. In comparison with normal development experimental condi- tions must always be arbitrary and inflexi- ble; a dose which permits a normal re^^jionsc 148 BIOLOGIC BASIS OF SEX in the case of one structure may be quite discordant for others, resulting in serious disharmonies or even in paradoxical reac- tions. Nevertheless, much can be learned analytically of the processes involved in sex differentiation without requiring per- fectly integrated results. XIII. Embryonic Hormones and Inductor Substances According to generally accepted theory, the normal differentiation of the gonads is the result of an antagonistic interaction be- tween the cortical and medullary compo- nents, in which one element (as prescribed by sex genotype) gradually becomes pre- dominant while the other retrogresses. It has been previously emphasized that in ex- perimental sex transformation no new prin- ciples or processes are involved ; the normal mechanism is simply set in reverse. The transformation process as it appears at vari- ous stages presents essentially the same his- tologic picture, whether the impulse to re- versal comes from a developing gonad of opposite sex or from an administered hor- mone. It has been shown further that steroid hormones are capable in many cases of re- directing the differentiation process from its inception, leaving but slight histologic traces of reversal. Thus the processes of sex differ- entiation in the gonads are amenable to control by hormones at least over a consid- erable range of the developmental period. The cjuestion then arises as to the manner in which the antagonistic interactions be- tween cortex and medulla are mediated in normal development, and the nature and relationships of the physiologic agents in- volved. On this subject differences of opin- ion have long existed. The well known the- ory of Witschi^ postulates special inductor substances elaborated by the cortical and medullary tissues. Because the special sphere of the inductor substances is presumed to be the regulation of gonad differentiation, their field of action is thus topographically restricted; when at later stages the gonads begin to exercise control over the develop- ing accessory sex structures, frequently over considerable distances, the action of embry- onic hormones is presumed. However, since steroid hormones also may influence, or even completely control, the mechanism of gonad differentiation, it is important to know whether such control is exerted sec- ondarily, i.e., by regulating the inductor sys- tems, or whether hormones are capable of playing the role of inductors. It must be re- membered that the inductor substances have not as yet been isolated or directly identi- fied; their existence and their character are postulated from the nature of the effects attributed to them. Consequently, this prob- lem can only be approached indirectly by comparing the effects of sex hormones un- der as many conditions as possible with those ascribed to the inductor substances. Although the activities of the inductor substances are ordinarily confined to the gonads, it is held that under favorable con- ditions their influence may extend some- what further, but only within a limited range. This view was originally based on observations in certain parabiosis experi- ments (Witschi, 1932) and involves the manner in which inductor substances are supposedly transported. In parabiotic pairs of frogs, so closely united that the gonads lie within in a common body cavity, gonads of different sex do not influence each other significantly except when they are in con- tact or in very close -proximity. The action of the inductor substances, that is to say the intensity of their effects, seems to be roughly proportional to the distance be- tween the interacting gonads (Fig. 2.2B). This observation suggested that the induc- tor substance is transmitted only by dif- fusion through the tissues, the concentration declining steadily with distance from the point of origin. Failure to be effective at greater distances presumably indicates that the agent is not distributed through the blood in the manner of a hormone. This, however, may mean only that in early stages of development the humoral sub- stances, whatever their nature, are not pro- duced in sufficient quantity to reach or maintain an adequate level in the blood- stream. In parabiotic salamanders, on the other hand, typical sex reversal also occurs, although the interacting gonads as a rule are widely separated (Fig. 2.2.4). In this case the inducing agent must be blood- borne; nevertheless, the changes in the re- HORMONES IN DIFFERENTIATION OF SEX 149 versing gonads occur at the same time and are of the same histologic character as those attributed to inductor action. Evidently the effects of gonads acting from a distance through the agency of blood-borne hor- mones are not distinguishable from those attributed to inductor substances when the interacting gonads are in close juxtaposi- tion. Furthermore, in other experimental situ- ations where hormones are almost certainly involved, effects of a strongly localized character can be observed under proper conditions. When sexually differentiated gonads are grafted into the coelomic cavity of chick embryos (Wolff, 1946) ovaries have a transforming effect on the testes of male hosts which varies according to their rela- tive proximity; and at the same stage of development testis grafts modify the adja- cent gonaducts. A similar response appears when well differentiated larval gonads of Amhystoma are transplanted into the body cavity of another larva (Fig. 2.3) and in this case the effects are reciprocal. Also, after unilateral castration of male rabbit embryos (Jost, 1953), the sex accessories on the two sides of the body may show distinct differences in reaction. On the unoperated side normal differentiation of the sex ducts occurs, but on the operated side the Miil- lerian ducts are not completely inhibited and the Wolffian ducts are only partially preserved (c/. also Price and Pannabecker, 1956). It would seem that the remaining testis produces enough hormone to insure normal development of nearby structures, but at this early stage its output is insuffi- cient to maintain proper development of structures at greater distances. A compara- ble result was obtained when an embryonic testis was implanted in a female embryo in close proximity to one ovary of the host (Jost, 1947b, 19531. The ovary on the side of the grafted testis was inhibited and atrophic whereas the other was normal (Fig. 2.35), and duct development followed dif- ferent patterns on the two sides. On the side of the testis graft the Wolffian duct and epididymis persisted and developed but the IMiillerian duct was suppressed in the vi- cinity of the graft. On the side of the nor- mal ovary these relationships were reversed. Evidently the pattern of development is de- termined by proximity to the grafted testis. The same situation as regards the differ- entiation of the sex ducts and accessory structures is often encountered in so-called "lateral gynandromorphs" which occur spo- radically in many mammals. In such cases, where embryonic hormones are clearly in- volved, the localized aspect of their action often resembles closely the postulated ef- fects of inductor substances. It is interesting to compare the results of the experiments cited above with conditions found in a type of lateral gynandromorphism of doubtful etiology which occurs in a certain genetic strain of mice (Hollander, Gowen and Stad- ler, 1956). These gynandromorphs have an ovary on one side and a testis on the other, but without relation to laterality. Typically both gonads are small and underdeveloped, with the testes as a rule more severely af- fected. It is the condition of the accessory sex structures in these cases that is of spe- cial interest. On the side of the testis, de- velopment of the gonaducts without excep- tion follows the male pattern (24 cases), a vas deferens and epididymis are present, and Miillerian duct derivatives are lacking (Fig. 2.36). This condition corresponds ex- actly to the role of the testis as the condi- tioner of male duct development and the in- hibitor of the Miillerian duct, as revealed by the results of castration and culture in vitro. The development of the seminal vesicles is variable and the external genitalia, although usually underdeveloped, are nearly always of male type. These conditions in turn can be correlated with the size of the testis and the factor of distance from a gonad which is probably subnormal in its secretory ac- tivity. On the side of the ovary the oppo- site picture prevails; the Miillerian deriva- tives are always present, although variable in size, whereas the male accessories are either imperfectly developed or in most cases altogether lacking. A similar condi- tion has recently been reported in a gy- nandromorphic hamster (Kirkman, 1958) . It may be noted that lateral differences of this kind are not infrequently met with in cer- tain human intersexes (Jost, 1958; Wilkins, 1950). Evidence from other types of experiment 150 BIOLOGIC BASIS OF SEX Fig. 2.36. Composite drawing illustrating the condition of the genital systems in a group of "gynandromorphic" mice, which have an ovary on one side of the body and a testis on the other, after Hollander, Gowen and Stadler (1956). On the side of the testis (which as a rule was partially or entirely descended and is shown as dissected out) a complete male duct system with seminal vesicle is present; the female genital tract is absent on this side. On the side of the ovary a complete female genital tract is found, although it varied greatly in size in different cases. On this side the male duct system was usually absent, but appeared in whole or in part in about one third of all cases. Compare with conditions induced by a uni- lateral testis graft shown in Figure 2.35. Abbreviations: Bl., bladder; Ep., epididymis; O, ovar}^; Ovd., oviduct; R, rectum; S.V., seminal vesicle; T, testis; U, uterus; V.D., vas deferens. bears on this point. When the pituitary is absent the normal secretory activity of the embryonic testis may be materially reduced. In male rabbit fetuses deprived of their hyi)ophyses by decapitation, although the testes are present, an apparent decrease in endocrine activity has local effects which reseml)le those of castration (Jost, 1951a, 1953). In their lowered state of activity, the influence of the testes on the accessory sex structures is graduated according to dis- tance. Structures near the gonads, such as the vas deferens and epididymis, are nor- mally developed but the more distant sinus derivatives and external genitalia are of fe- male (i.e., castrate) type. Here again a level of activity adequate to maintain normal development of nearby structures is ineffec- tive at greater distances, and the result is not compatible with the view that the hor- mone is distributed only thi'ough the blood stream. Approaching the ciuestion from yet an- other direction, a clear demonstration of local action by a hormone appears in an ex- periment cited previously, in which an em- bryonic testis is engrafted between the lob- ules of the seminal vesicle of a castrate host; there is complete cytologic recovery of the atrophic epithelium in lobules con- tiguous with the graft, but the effect di- minishes rapidly with distance and soon dis- appears. Greenwood and Blyth (1935) have also described a sharply circumscribed ef- fect on the feathers of capons. A very small dose of female hormone injected subcutane- ously changes the pigmentation of growing feathers at the site of injection, but beyond a very short distance it has no effect. On the other hand, after local implantation of hor- mone pellets, both localized and more dis- tant effects may be registered at the same time, indicating that both modes of distri- bution are simultaneously effective [cf. HORMONES IN DIFFERENTIATION OF SEX 151 Robson, 1951; Grayhack, 1958). A survey of a considerable literature on the local action of sex hormones (Speert, 1948) indicates that, with due consideration for such factors as vascularity and method of application, dual action in the above sense is chiefly a matter of dosage. Larger doses may have strong local effects accompanied, however, by a definite "systemic" action on distant structures. With small doses only local ef- fects appear. Finally, it should be empha- sized that localized activity has been dem- onstrated under suitable conditions in the case of many other endocrine glands and their hormones.^^ Thus numerous parallels and resem- blances may be adduced with respect to the behavior of the hypothetical inductor sub- stances and the local effects of sex hor- mones. On the basis of their histologic ef- fects on gonad differentiation, their mode of distribution and range of action, and the period during which they operate, it seems that no clear or final distinctions can be drawn. Although theoretically the possibil- ity cannot be excluded that hormones act indirectly on gonad differentiation by con- trolling the existing inductor systems, there are obvious advantages in postulating a single humoral agency; theory is simplified and hypothetic substances are replaced by known entities (for further discussions of this problem see Wolff, 1947; Jost, 1948a, 1953, 1955; Ponse, 1949; Burns, 1949, 1955b; Witschi, 1950, 1957). XIV. References AcKART, R. J. AND Leavy, S. 1939. Experimental reversal of sex in salamanders by the injec- tion of estrone. Proc. Soo. Exper. Biol. & Med., 42, 720-724. Ander.son, D., Billingh.am, R. E., L.mvip.son, G. H., AND Medaw.ar, p. B. 1951. The use of skin grafting to distinguish between monozygotic and dizygotic twins in cattle. Heredity, 5, 379-397. Angevine, D. M. 1938. Pathologic anatomy of hypophysis and adrenals in anencephalv. Arch. Path., 26, 507-518. =*See Etkin (1936) Etkin and Huth (1939) Gorbman (1950) foi- the pituitary-thyroid relation- ship; Kaltenbach (1953a and b) for the thyroid hormone; Katsch, Gordon and Charipper (1948) and Weinstein, Schiller and Charipper (1950) for adrenal hormones as they affect certain genital tissues. Baggett, B., Engel, L. L., Savard, K., and Dorfman, R. I. 1956. The conversion of testosterone- 3-C" to C"-estradiol-17/3 by human ovarian tissue. J. Biol. Chem., 221, 931-941. Barr, M. L. 1957. Cytologic tests of chromo- somal sex. In Progress in Gynecology, \o\. 3, pp. 131-141. New York: Grune and Stratton. B.ARR, M. L., AND Grumbach, M. M. 1958. Cy- tologic tests of chromosomal sex in relation to sexual anomalies in man. Recent Progr. Hormone Res., 14, 225-344. Ba.scom, K. F. 1923. The interstitial cells of the gonads of cattle, with especial reference to their embryonic development and significance. Am. J. Anat., 31, 223-259. Berthold, a. a. 1849. Transplantation der Ho- den. 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Artificial induction of func- tional sex-reversal in genotypic females of the medaka {Oryzias latipes). J. Exper. Zool., 137, 227-260. SECTION B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduce tion MORPHOLOGY OF THE HYPOPHYSIS RELATED TO ITS FUNCTION Herbert D. Purves, M.Sc, M.B., Ch.B. DIRECTOR, ENDOCRINOLOGY RESEARCH OTAGO UNIVERSITY, DUNEDIN, NEW ZEALAND I. Introduction I(i2 II. Embryonic Development 1(52 III. Anatomic Subdivisions of the Hy- pophysis. Definition of Terms. 1(53 A. Anatomic Divisions and Com- ponents 1(53 B. Histologic Divisions of the Hy- pophysis: Functional Parts. . . . KKi C. Zones in the Pars Anterior and Pars Distalis 1(57 IV. A General Statement of the Prob- lems OF THE Special Cytology of the Hypophysis 1(57 V. Secretory Granules 1(58 A. The Nature of Secretory Granules 1G8 B. The Staining Reactions of Secre- tory Granules by Modern Meth- ods 1(59 VI. Cell Classes and Cell Types.... 171 A. Classification 171 B. Favorable Species 172 C. Reactivity 172 D. Specificity of Granules 173 E. Changes in Cell Proportions with Alteration in Function 173 VII. The Acidophil Cell Class 174 A. Acidophil Granules 174 B. Lipoid Content of Acidophil Granules 174 C. Sulfydryl and Disulfide Content of Acidophil Granules 175 D. Hormone Content of Acidophil Cells 175 E. Hormone Content of Acidophil Granules 176 F. Acidophil Cell Types 176 G. Secretory Functions of Acidophil Cell Types 178 H. Acidophil Cells in Relation to Somatotrophin Secretion 180 VIII. The Basophil Cell Class 181 A. Basophil Granules 181 B. Hormone Content of Basophil Cells 183 C. Hormone Content of Basophil Granules 183 D. Does the Periodic Acid-Schiff Re- action Demonstrate the Hor- mone Content of Basophil Cells? 183 E. Differential Staining of Basophil Cells by Resorcin-Fuchsin, Kresofuchsin, Aldehyde-Fuch- sin, and Alcian Blue: (3-Cells and 5-Cells 184 F. The Nomenclature of Basophil Cells 187 G. Specific Basophil Cell Types... 187 H. The Three Fimctional Types of Basophil Cell in the Rat Hy- pophysis 188 I. The Pars Anterior of the Bat with Special Reference to Tw^o Types of Gonadotrophs 194 IX. Corticotrophin: the Problem of Its Origin 195 A. Basophil Cells and Corticotro- phin 195 B. Hypophyseal Responses to Adre- nal Ablation 196 C. Hvpophvseal Responses to Stress . 197 D. The Sixth Cell Type 198 X. The Pars Intermedia and Inter- medin Secretion 198 XI. The Pars Tuberalis 201 XII. Cytologic Changes Accompanying Secretory Responses Concerned WITH Reproductive Function... 201 A. Sexual Maturation in the Rat. . . 201 B. Sexual Maturation in Other Ani- mals 202 C. Seasonal Breeding 202 D. Induced Sexual Maturation in the Female Rat 202 E. The Active Breeding Phase in the Female 203 F. Pseudopregnancy and Pregnancy. 204 G. Pregnancy Changes in the Human Hj'pophysis 204 161 162 HYPOPHYSIS AND GONADOTROPHIC HORMONES H. Lactation 205 XIII. The Human Hypophysis 206 A. Structure 206 B. Specific Basophil Types in the Human Hypophj'sis 206 C. Differential Staining of Basophil Cells in the Human Pars Dis- talis 207 D. Functions of the Basophil Cells of the Human Pars Distalis 209 1. The 5-cell 209 2. The blue /3-cell 209 3. The 7-cell 209 E. The Amphophil Cells of Russfield. 209 F. The Purple /3-Cell in the Cushing Syndrome 210 G. Crooke's Cell Changes Produced by Corticosteroid or Cortico- trophin Administration 211 H. Changes in the Purple /3-Cells in Addison's Disease 212 I. The Pharyngeal Hypophysis 212 XIV. Electron Microscopy of the Adenohypophysis 212 A. Endoplasmic Reticulum 213 B. Golgi Region or Zone 213 C. Palade Granules 222 D. Secretory Granules 222 E. Microvilli 223 XV. The Neurohypophysis and Neuro- hypophyseal Secretion 223 A. Neurosecretory Phenomena in the Hypothalamus and Neurohy- jiophysis 223 B. The Chemical Nature of the Stain- able Neurosecretory Material . . 227 C. The Physical Form of Neurosecre- tion in the Neurohypophysis: the Secretory Granule 227 D. The Hormone Content of Nemo- secretory Granules 228 E. The Distribution of Oxytocin and Vasopressin 228 F. Inferences Concerning Rate of Secretion from Cytologic and Histochemical Studies 229 XVI. References 229 I. Introduction The aim of this chapter is to present cer- tain morphologic characteristics of the hy- pophysis. Emphasis is given to those aspects of the structure which are, in the light of modern knowledge, of importance in eluci- dating the functions of this organ. Although the study of morphologic features does not by itself provide clear answers to questions concerning the endocrine functions of the hypopliysis, it is evident that the morpho- logic peculiarities of the gland are related to its functions, and that the consideration of morphologic data in conjunction with the evidence derived from physiologic observa- tions does assist in the construction of ac- ceptable hypotheses concerning these func- tions. 11. Embryonic Development The adenohypophysis is derived from Rathke's pouch, a process of epithelial tis- sue derived from the buccal ectoderm. The distal portion of this process forms a hollow structure which lies in close contact with the infundibulum. The infundibulum is an outgrowth of neural tissue from the floor of the third ventricle, and differentiates into the neurohypophysis. The work of Burch (1946) suggested that contact of the epithelial element with the neural element was necessary for the differ- entiation of the adenohypophysis from the former. He found in the frog {Hyla regilla) that translocation of the infundibulum or of the epithelial anlage, at a stage in develop- ment before these elements made contact, prevented the differentiation of the epithe- lial element. The pars intermedia did not develop, nor did acidophil and basophil cells appear in the epithelial cell mass. These re- sults must, however, be reconsidered in view of the later findings of Etkin (1958). Etkin succeeded in transplanting the epithelial anlage at the earliest possible stage, and found that this does not prevent the differ- entiation of a pars intermedia. The devel- opment of normal pigmentation showed that the pars intermedia so differentiated func- tioned in its transplanted position and, in the later tadpole stages, pigmentation was greater than normal, indicating hyperf unc- tion of the pars intermedia such as develops in the differentiated pars intermedia which is subsequently transplanted in the frog. Etkin's experiments were done with the wood frog, Bana sylvatica, but it is unlikely that the differences are due to species differences. Etkin observed differentiation of a jmrs dis- talis, but no chromophil cells were present at the stages examined. These chromophil cells usually do not appear until a later stage in normal animals. It has, therefore, not yet been demonstrated that a full differentiation of a functional pars distalis can occur with- out continued contact with the infundibu- HYPOPHYSEAL MORPHOLOGY 163 It should be noted that Etkin's results do not show that contact with neural tissue is unnecessary for the initiation of the differ- entiation of the adenohypophysis from its epithelial anlage, because the buccal ecto- derm is in contact with neural tissue before the development of either Rathke's pouch or the infundibulum. They do show, how- ever, that the development of the adenohy- pophysis can proceed without continued contact with the neural element. According to Eakin and Bush (1957), the pars nervosa develops without any consist- ent departure from normality in frogs (Hyla regilla) adenohypophysectomized at the limb-bud stage. Regeneration of a pars anterior often occurred giving albino tad- poles which metamorphosed normally. In one case a black nonmetamorphosing tad- pole was found to have an adenohypophys- eal fragment composed exclusively of cells similar to those of the normal pars inter- media. The first appearance of granulated cells in the developing adenohypophysis presents some features of endocrinologic interest. Jost and Danysz (1952) found glycoprotein granules in basophil cells in the rabbit fetus after 20 days gestation. In the rat (Jost and Tavernier, 1956) glycoprotein granules ap- pear in the ventral portion of the pars an- terior after 15 days and in the central por- tion after 17 days gestation. In both species the granules appear when the first evidence for gonadotrophin and thyrotrophin is ob- served. In the human fetus basophil cell granules appear in the developing hypophysis much earlier than acidophil cell granules. Baso- phil cell granules are seen after 8 weeks ges- tation, whereas the first definite acidophil cell granules appear only at the 19- to 20- week stage (Pearse, 1953; Romeis, 1940). Romeis identified these early-appearing basophil cells as /3-cells (purple ^-cells) in the specific sense in which he uses this term. Inasmuch as intermedin appears in the hu- man fetal hypophysis about the same time as these basophil cells (Keene and Hewer, 1924) , it is possible that the ^-cells of Ro- meis in the human hypophysis are inter- medin-secreting cells and correspond to the cells of the pars intermedia of other mam- mals. More convincing evidence in support of this hypothesis is presented in the section on the human hypophysis. III. Anatomic Subdivisions of the Hypophysis. Definition of Terms The hypophysis is very variable in form in different vertebrates. Even among the mammals there is considerable variation. The comparative anatomy of the hypophy- sis has been reviewed by Romeis (1940), Green (1951), and Herlant (1954a). The nomenclature is in a state of considerable confusion and ill adapted to the needs of the comparative morphologist. Confusion arises from the great numbers of synonyms in use, the use as synonyms of terms that are not strictly synonymous, and the use of the same term for structures that are not strictly homologous. Because of the importance of a rigid and consistent system of nomenclature, it seems advisable for the purpose of this chapter to adoi^t such a system and to use a single term for each structure, avoiding all use of syn- onyms except where it is necessary to quote from, or to refer in specific terms to, the publications of other authors. I shall make no attempt to list the various synonyms or near synonyms in use, or to eciuate my usage with the varying usages which appear in past and current literature. A. ANATOMIC DIVISIONS AND COMPONENTS The mammalian hypophysis (and that of most terrestrial vertebrates) may be di- vided into three parts (Fig. 3.1). (1) The median eminence, forming the floor of the third ventricle of the brain and contiguous with the hypothalamus. (2) The hypophys- eal stalk, an attenuated connection between the other two divisions. (3) The lobular hy- pophysis, an expanded structure enclosed in the pituitary fossa, the latter being a cavity or sac formed by dura and bone. Each division consists of an adenohypo- physeal component derived by way of Rathke's pouch from the buccal ectoderm and a neurohypophyseal component derived from the neural tissue in the floor of the third ventricle. These components will be named by the addition of prefixes to tlif names characterizing the three division-, thus: 164 HYPOPHYSIS AND GONADOTROPHIC HORMONES DIVISIONS PARTS Lobes neural component- pars enninens pars nervosa (•,•) adeno component- ^^ (epit-heliGJ ) pars tuberalis pars intermedia pars anterior Fig. 3.1. Diagrams representing sagittal sections of a conventionalized mammalian hypo- physis showing the divisions, components and parts. The adeno-eminence and adenostalk together form the pars tuberalis. The adenolobe is almost completely divided by the hypo- physeal cleft into an anterior lobe and an intermediate lobe. In many mammals the anterior lobe is entirely composed of pars anterior tissue and the intermediate is entirely composed of pars intermedia tissue as shown here. For further details refer to the text. Adeno-eminonce Neural eminence Adenostalk Neural stalk Adenolobe Neural lobe It must be added here that the division between eminence and stalk is in some spe- cies indefinite and cannot always be made; the stalk is variable in extent, and in some species it does not exist as a junctional re- gion between the other divisions. Clearly the division into eminence and stalk is of de- scriptive value only and has no functional significance. In most mammals, and in most other ter- restrial vertebrates with the exception of birds, Rathke's pouch adheres to the neural component during development. The cavity of Rathke's pouch persists in the adult as a flattened cavity — the hypophyseal cleft (re- sidual lumen) — which allows an easy sepa- ration of the adenolobe into two parts (Fig. 3.2) . The portion of adenolobe which is ad- herent to the neural lobe is the intermediate lobe. The composite structure formed by the adherent neural and intermediate lobes is called the posterior lol)e. The remaining por- tion of the adenolobe is the anterior lobe. Hypophyseal hormones are commonly ex- tracted from the separated anterior and pos- terior lobes. The anterior lobe hormones are somatotrophin, prolactin, corticotrophin, thyrotrophin, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), and are the products of the pars anterior tissue which the anterior lobe contains. Posterior lobe hormones are intermedin, which derives HYPOPHYSEAL MORPHOLOGY 165 from tlie pars intermedia tissue in the inter- mediate lobe, and the neurohypophyseal hormones, oxytocin and vasopressin. In whales (Wislocki and Geiling, 1936) and porpoises (Wislocki, 1929) and in the armadillo (Oldham, 1938), manatee (Old- ham, McCleery and Geiling, 1938) , elephant (Wislocki, 1939), pangolin (Herlant, 1954a), beaver (Kelsey, Sorensen, Hagen and Clausen, 1957) , and the whole class of birds (DeLawder, Tarr and Geiling, 1934; Rahn and Painter, 1941), the cavity of Rathke's pouch is obliterated during devel- opment so that there is no hypophyseal cleft (Fig. 3.3). Moreover, in all these animals there is no adherence of the adenolobe to the Figs. 3.2, 3.3, 3.4, and 3.5. Diagrams of sagittal sections of the hypophyses of rat, cat, ox, and blue whale showing some of the variations encountered. The left side of the diagram is anterior, the right, posterior. (Modified from B. Romeis, in Handbnch der mikroskopischen Anatomie des Menschen, Vol. 6, Part 3, JuHus Springer, Berlin, 1940.) Fig. 3.2. The rat hypophysis is of the conventional form but rotated to bring the anterior lobe below and the posterior lobe above the axis of the gland. Fig. 3.3. The hypophysis of the blue whale. In this form the adenolobe is not divided by a cleft and is not in close contact with the neural lobe, being separated from it by a fold of con- nective tissue (D) continuous with the dura. The adenolobe is filled with pars distalis tissue which combines the functions of the pars anterior and the pars intermedia of the more usual mammalian form. In these respects the hypophyses of birds, porpoises, the armadillo, the ele- phant, the pangolin, and the beaver resemble the hypophysis of the whale. Fig. 3.4. In the hypophysis of the ox the pars intermedia is less extensive than the inter- mediate lobe and the remainder of the intermediate lobe is occupied by typical pars anterior tissue. The segment of pars anterior tissue which is in the intermediate lobe and is separated from the bulk of the pars anterior by the hypophyseal cleft is known as the cone of Wulzen (Wulzen, 1914). Fig. 3.5. In the cat hypophysis the neural lobe is deeply embedded in the adenolobe. Par? intermedia tissue occupies the entire intermediate lobe and extends into the anterior IoIk- particularly in the caudal region. 166 HYPOPHYSIS AND GONADOTROPHIC HORMONES neural lobe, and these two structures can be easily separated from one another. This is not a separation into anterior lobe and pos- terior lobe, which terms are not applicable to this type of hypophysis. The relation between the adenolobe and neural lobe in man is unique and is described in the section on the human hypophysis. B. HISTOLOGIC DIVISIONS OF THE HYPOPHYSIS: FUNX'TIONAL PARTS For the purpose of correlating structure with function, it is necessary to separate the hypophysis into parts on the basis of inter- nal structure or of function, a separation in- dependent of variations in gross anatomy. It matters not in theory whether the separa- tion is based on fine structure or on function, because one determines the other. In prac- tice the exact delimitation of the functional partition is determined by methods which are histologic or cytologic. For the determi- nation of the homology between the histo- logically distinct parts of the hypophysis in different species, function must be the de- termining criterion. Histologically distinc- tive parts in different species can be consid- ered homologous and be given the same name only if they are equivalent in function. Nature has set a trap for those who are care- less in this respect. The partition of the adenhypophysis into functional parts is var- iable. In terrestrial vertebrates the neurohy- pophysis consists of two functionally distinct parts (Green, 1951). Although following Green in this partition of the neurohypoph- ysis, I must, however, reject his terminol- ogy, which used the anatomic terms "median eminence" and "neural lobe" in a modified sense. I propose, therefore, to use for these parts the names "pars eminens'' and "pars nervosaJ^ The two parts of the neurohypophysis are: (1) Pars eininens — consists of the neu- ral eminence and neural stalk and the con- tinuation of this tissue in the neural lobe to the point where it becomes the pars nervosa. Characterized by a vascularization in com- mon with the adenohypophysis. Its venous effluents form portal vessels which pass into and arc the main blood supply of the pars anterior or the pars distalis of the adenohy- pophysis. (2) Pars nervosa — that part of the neural lobe whose venous effluent passes to the systemic veins directly. There are two other differences between the pars eminens and the pars nervosa which characterize them. First, the arteries and veins of the pars eminens are confined en- tirely to the surface, where they connect with a rich vascular network. The interior of the pars eminens is relatively sparsely supplied with capillary loops — simple hair- pin loops in small animals, more complex vascular spikes in larger animals — which penetrate it from the surface network. In contrast, the arteries and veins of the pars nervosa ramify within its substance and con- nect with a capillary network of the usual form. Second, in histologic sections the pars eminens is characterized by a low content, the pars nervosa by a high content, of stain- able neurohypophyseal secretion. In most mammals, and in most other ter- restrial vertebrates except birds, the parts of the adenohypophysis (usual form) are three. ( 1 ) Pars tuberalis — consists of the adeno-eminence and adenostalk. Continues distally with the pars anterior, from which it is distinguished by the absence of the chromophil cells which characterize the latter. (2) Pars intermedia — a part of the adenolobe adjacent to and adherent to the neural lobe. Histologically it is character- ized by the presence of cells of uniform ap- pearance. These contain basophil granules, in quantities that vary greatly in different species. In some species granules appear to be absent. Functionally the pars intermedia is ciiaracterized by the presence of inter- medin and of no other known hormone. The pars intermedia in some species is co-exten- sive with the intermediate lobe. In some, e.g., the sheep and the cow (Fig. 3.4), it is less extensive than the intermediate lobe, and the remainder of this lobe consists of pars anterior tissue (Wulzen, 1914). Such a detached portion of the pars anterior is known as a cone of Wulzen. In some species, sucli as tlie cat (Fig. 3.5). the pars inter- media is more extensive than tlie interme- diate lobe, and extends somewhat into the anterior lobe. (3) Pars anterior — this tissue occupies the part of the adenolobe which is not pars intermedia. It is characterized by HYPOPHYSEAL MORPHOLOGY 167 the presence of a mixed cell population in- cluding several types of chromophil cells. In man, whales, porpoises, armadillo, manatee, elephant, pangolin, beaver, and in the whole class of birds, there is no pars in- termedia (Fig. 3.3). The adenolobe contains throughout a mixed population of cells re- sembling that of the pars anterior. This structure differs from the pars anterior in containing intermedin in addition to the an- terior lobe hormones, and in all probability contains an additional cell type correspond- ing to the pars intermedia cells. This struc- ture I will call pars distalis to indicate that it is not strictly homologous wth the pars anterior as previously defined (Geiling, 1935). In these species, therefore, the parts of the hypophysis are two: (1) Pars tuber- alis — as in other species. (2) Pars distalis — the whole adenolobe containing a mixed cell population throughout its extent. It may be noted here that, although in mammals the absence of a pars intermedia is correlated with the absence of the hypo- physeal cleft, in lower vertebrates a pars in- termedia may be present despite the absence of the cleft (Green, 1951). C. ZONES IN THE PARS ANTERIOR AND PARS DISTALIS The different cell types in the pars ante- rior or pars distalis are not uniformly dis- tributed. The inequalities in the distribution of the acidophil cells, although not more marked than those of other cell types, are more conscipuous because of the larger num- ber of these cells and their conspicuous stain- ing. Zones rich in acidophil cells are called acidophil zones, zones poor in acidophil cells are called basophil zones. The zona tuberalis in the pars anterior adjacent to the junction of the latter with the pars tuberalis is a baso- phil zone. This zonation, seen in many pituitaries, aids in the distinction between specific cell types and functional variants. When two cell groups with different stainability are shown to have different distributions char- acteristic for each, the groups so differen- tiated may be accepted as types. When there is no difference in the distribution of cells with minor differences in coloration, etc., the possibility remains that these different appearances are those of a single cell type in different functional states. IV. A General Statement of the Problems of the Special Cytology of the Hypophysis Nine distinct hormones are secreted by the hypophysis. Some simplification of the problem of relating the secretion of each hormone to specific cytologic details results from the fact that 2 of these hormones are secreted from the neurohypophysis and 1 from the pars intermedia, leaving only 6 to be secreted from the pars anterior. A com- plete revolution in the physiology of neuro- hypophysis has recently resulted from the discovery of methods for staining neuro- secretion in this structure. It is indeed a source of wonder and delight that so much new and fascinating knowledge should re- sult from the application of a simple techni- cal discovery. The last 20 years have also seen great advances in the cytology of the pars anterior. Indeed, the progress here can be claimed to be more extensive, if less spec- tacular, than in the neurohypophysis. For all this the author, constrained to review the comparative cytology of the pars anterior, approaches his task with foreboding. A pe- culiar difficulty arises here which is not en- countered in the neurohypophysis. The staining reactions of the granules of func- tionally equivalent cells are not consistent from species to species. This variation is eas- ily understandable. The hormones them- selves vary in chemical composition from species to species, even to the extent of the hormone from one mammalian species being inactive in another species. The secretory granules of pars anterior cells contain the hormones in association with hormonally in- active proteins no less variable than the hormones themselves. Functionally equiv- alent cells in different species may therefore have no specific component common to both despite the fact that they serve an identical function in the two species. In this situation generalizations concern- ing the cytology of the pars anterior are diffi- cult and hazardous. The treatment of the subject given here seems to be consistent with the recorded observations in a numl)cr of species. Without doubt further in\'o>tiL:;t- 168 HYPOPHYSIS AND GOXADOTROPHIC HORMONES tion, especially in additional species, will reveal its inadequacies. V. Secretory Granules A. THE NATURE OF SECRETORY GRANULES Many of the cells of the pars anterior contain characteristic secretion granules stainable by a variety of procedures which indicate that the granules are composed mainly of characteristic proteins. The se- cretion of the different hormones is inti- mately related to the formation and dis- charge of secretory granules in different cells, and the nature of the hormone secreted by any cell is intimately related to the chem- ical nature of the secretory granules in that cell. The special cytology of the hypophysis, which deals with the differences between cells with different functions, is practically confined to the study of the secretory gran- ules, these being the only truly specific char- acters that can be made visible by current techniques. Granules similar to those in the cells of the pars anterior are seen in other tissues where the cells secrete proteins which act as hormones or enzymes. Examples are the granules of the hormone-secreting a- and ^-cells of the pancreatic islets, and the zymogen granules of the enzyme-secreting acinar cells of the pancreatic parenchyma. The nature of zymogen granules of the pan- creas and the relationship of the granule to the secretory process have been clarified by the epoch-making researches of Palade (1956 », and of Siekevitz and Palade (1958a, b). As seen in electron micrographs, the zy- mogen granules in the guinea pig pancreas are spherical bodies about 600 m/x in di- ameter. At certain stages in their formation a smooth membrane becomes visible at the periphery. It may be assumed that zymogen granules are bound by a smooth-surfaced membrane throughout their intracellular existence, and that, when in the fully formed granule an enclosing membrane is not visible in electron micrographs, it is be- cause it is closely applied to the homo- geneous content of equally high density. Secretion of zymogen into the ducts after feeding the previously fasted guinea pig re- sults in the disappearance of membrane-en- closed granules from the apical pole of the cell and the appearance of the dense content of the granule without enclosing membranes in the lumen of the duct. Apparently the enclosing membranes are left behind at the cell border and therefore presumably coa- lesce with the cell membrane during the passage of the granule through this struc- ture. Restoration of the cell content of enzymes and enzyme precursors occurs rapidly af- ter secretory discharge. These products are formed in the basal part of the cell in which the synthesizing system is located. This sys- tem is revealed in electron micrographs as a closely packed arrangement of rough-sur- faced membranes forming part of the endo- plasmic reticulum. In light microscopy it is revealed by the cytoplasmic basophilia due to the ribonucleic acid component of the system. It is especially to be noted that the formation of the secretory product is not accompanied by any formation of mem- brane-enclosed zymogen granules, but dense material in a more or less diffuse form ap- pears in the cavities of the endoplasmic re- ticulum. The filling of relatively large vesi- cles in the Golgi zone with dense material and the accumulation of membrane-en- closed zymogen granules in the apical re- gion of the cell occur relatively late in the secretory cycle and seem to result from in- tracellular transport and enclosure in mem- branes of products synthesized earlier in the basal region. One implication of these observations is that a hormone could be formed and re- leased by an endocrine cell without being stored in the form of granules. This is pos- sible inasmuch as the granule does not play any part in the formation of the secretion. Electron microscopy of the rat hypophy- sis indicates that the granules are similar in structure to zymogen granules of the pan- creas. The enclosing membrane is easily visible in the basopiiil granules of the pars anterior and in the neurosecretory granules of tlic pars nervosa as an electron-dense nienibrane enclosing a less dense content. The acidophil granules of pars anterior cells, like the zymogen granules of the pan- creas, seem to contain solid protein of a density that does not permit the demon- stration of an enclosing membrane except in HYP0PHY8EAL MORPHOLOGY 169 thoye graniilcs tliat are only ixartially filled (Farquhar and Wellings, 1957). There is no necessity for secretory gran- ules to contain a single substance. The zy- mogen granules of the pancreas contain a mixture of enzymes and enzyme precursors. It is certain that some granules in the hy- pophysis contain a mixture of materials, some hormonal and others not hormonal. The staining reactions of specific granules may therefore be due to a nonhormonal con- stituent. This seems to be true of acidophil granules in the pituitary, inasmuch as the characteristic staining is due to a protein which is highly insoluble over a wide range of pH, whereas the hormones characteristic of these cells, growth and lactogenic hor- mone, are more soluble and can be extracted by procedures which do not dissolve the characteristic protein. Similar considera- tions apply to the neurosecretion in the nerve fibers of the neural lobe, because the characteristic hormones are octapeptides, whereas the characteristic staining is due to a protein with high cystine content presum- ably identical with the van Dyke protein I van Dyke, Chow, Creep and Rothen, 1942) . The granules of basophil cells contain glyco- protein, and the characteristic staining reac- tions seem to be due to the glycoproteins because they are similar to the staining reac- tion seen in other sites containing glycopro- tein. In the basophil cells, however, it is not known whether the glycoproteins responsible for the staining reactions are identical with the hormones or whether they exist in the vesicular glands in association with hormo- nally active molecules themselves not stain- able or not made visible by staining reac- tions because of their low concentration. Parallelism between intensity of staining and hormone content, which has been used to support the claim that it is the hormone product itself of these cells which is stained, does not in fact contribute to the solution of this problem. Staining reactions demon- strate the numbers of granules present, and parallelism between staining and hormone content indicates that the hormone is like- wise contained within the granule. Chemi- cal investigation of the nature of the iso- lated hormones and of the protein materials responsible for the staining reactions is the only source of evidence which might show that the staining reactions are due to a product distinct from the hormonally ac- tive product. This may be regarded as established for acidophil cell granules, neu- rosecretory granules, and for the glycopro- tein granules of the pars intermedia cells, but has not been established for glycopro- tein-containing cells of the basophil cells of the pars anterior. In this site, therefore, it is still possible that the staining reactions arc due to the hormonally active product itself and not to any associated but hor- monally inactive protein. B. THE STAINING REACTIONS OF SECRETORY GRANULES BY MODERN METHODS The recognition of a diversity of cell types in the pars distalis as reported by Schonemann (1892) dei)ends on the pres- ence of distinctive proteins in different cells. Although at one time theories which made the different cell types stages of a secre- tory cycle had a certain popularity, we now know that the different cell types made visible by staining methods have different secretory functions. This was first demon- strated by the observations of Smith and Smith (1923a, b). This momentous experi- ment showed a diversity of endocrine func- tion in the anterior lobe and a correlation between function and type of cell. It also destroyed the hypothesis that different cell types could be merely different phases of a secretory cycle. All these results followed from the demonstration that in the bovine hypophysis there is constantly present a zone situated anteromedially which is poor in acidophil cells and, therefore, is mainly comprised of basophil cells and chromo- phobes. The tissue from this zone im- planted into tadpoles had a predominantly thyroid-stimulating and consequent meta- morphosis-inducing effect, whereas tissue rich in acidophils promoted body growth and was poor in thyroid-stimulating action. It is clear, then, that staining reactions in pituitary cells may be related to the nature of the hormone produced, and we may, therefore, consider in what manner the staining reactions are linked to the specific function. Although it is reasonable to as- sume that differences in the product of dif- ferent cells arise from variations in their enzvme content, the slight differences in 170 HYPOPHYSIS AND GONADOTROPHIC HORMONES the enzymes which would produce these ef- fects are not, with present technicjues, de- monstrable by staining reactions. It is there- fore not possible at present to stain different cell types in the pituitary, if by this phrase we mean the demonstration of differences in the cytoplasmic make-up of cells with different secretory functions. Differences in staining, which are observed with more or less ease, are due to the staining reactions of the products of hormone synthesis within the cell ; they do not demonstrate differences in the synthesizing mechanisms directly. One consequence of this is that specific cell types are only differentiated w^hen there is, within their cytoplasm, an adequate amount of their specific product in the form of granules. Specific staining of granules requires methods which stain granules while leaving other cytoplasmic components unstained. Simple basic dyes such as methylene blue are not useful for this purpose. The baso- philia of the basophil granules is weak, the binding power is weaker than that of ribo- nucleic acid. The capacity for dye absorp- tion is great at high pH, so that cells with heavy accumulations of basophil granules appear densely stained, and it is this density of staining in densely granulated cells, rather than any exceptional strength of binding, that justifies the use of the term basophil for a certain class of cells in the adenohypophysis (Peterson and Weiss, 1955). Simple acid dyes do stain certain secretory granules specifically and find a use as counterstains to other procedures. Modern methods of staining allow clear- cut differentiations of specific granules by methods that do not depend on the distinc- tion between acidophilia and basophilia. The results of histochemical procedures may be expected to show a more consistent relation to function than the results of empiric staining methods. The McManus (194G» periodic-acid Schiff (PAS) reaction, which under certain conditions is specific for i)rotein-carbohydrate complexes in sec- tions of animal tissues, allows a partition of the granules into two classes, which may have a consistent functional significance. An intense red or magenta color produced by this procedure in certain granules indi- cates that they contain glycoprotein. These glycoprotein-containing granules are baso- phil granules, showing the same character- istic basophilia wherever they occur in all cells of all species. Their staining reactions to acid dyes are, however, variable. The PAS reaction can be combined with counter- stains which demonstrate the granules com- posed of simple proteins. The latter gran- ules are acidophil granules. Inasmuch as both the acidophil class and basophil class are composite, additional methods must be applied to effect a further separation of each class. Methods of gen- eral usefulness for this purpose are: (1 1 Staining by a mixture of acid dyes in such methods as Heidenhain's azan or Cross- mon's (1937) modification of Mallory's stain. Granules may be stained orange, red, or blue, or a purple color from a mixture of red and blue dye. The coloration in each case depends on acidophilia; it has no rela- tion to basophilia. The varying colors de- pend on variations in the strength and the quality of the acidophilia. Acidophil gran- ules, like other strongly acidophilic struc- tures, are stained either orange or red. Basophil granules may be orange, red, pur- ple, or blue. (2) The use of elastic tissue stains such as kresofuchsin, resorcin-fuchsin, or Gomori's (1950) aldehyde-fuchsin, of which only the latter is in present-day use as a stain for hypophyseal granules. With these stains it is possible to stain some baso- phil (glycoprotein) granules while leaving others unstained. By combining the results of the above methods of staining, at least 10 distinctly different staining reactions have been ob- served in specific granulation in the adeno- hypophyses of vertebrates. To date, how- ever, no more than 4, or possibly 5, distinct staining reactions have been observed in the pars anterior of any one species. There is no possibility, therefore, of any constant relationship between staining and function. Before progressing to the subject of cell classes and cell types, the problem presented by the existence of cytoplasmic ribonucleic acid should be discussed. Ribo- nucleic acid occurs in the cytoplasm of aci- dophils and basophils. It is a component of the Palade granules (Palade, 1955) which are described in the section on electron microscopy of the adenohypophysis. These HYPOPHYSEAL MORPHOLOGY 171 granules are quite distinct from the secre- tory granules. At one time the basophilia of basophil granules was ascribed to ribonucleic acid. It is now clear both from electron micros- copy and from specific staining responses that cells of the basophil class generally contain less cytoplasmic ribonucleic acid tlian do acidophil cells and that ribonucleic acid is not a constituent of basophil gran- ules. The cytoplasmic basophilia demonstrated by the red staining by pyronin in the py- ronin-methyl-green stain shows an entirely different distribution from the basophilia revealed by neutral toluidine blue or by the Gram stain and it appears probable that this pyronin-staining is specific for cyto- jilasmic ribonucleic acid. Pyronin basophilia is observed in all classes of cells of the an- terior lobe and is increased in conditions where rapid secretion rather than storage is thought to be taking place. It is notably increased in the large degranulated cells observed in the hypophysis of pregnant ani- mals and in animals treated by estrogen (Herlant, 1943). Treatment for a short time with a ribo- nuclease preparation will remove the py- ronin basophilia without removing the Gram-positive reaction or the violet meta- chromasia with toluidine blue in the baso- phil granules (Foster and Wilson, 1951; Pearse, 1952). Prolonged exposure to bile salts (Foster and Wilson, 1951) or ribo- nuclease preparations (Pearse, 1952) will remove the basophilia of the basophil gran- ules, as well as the cytoplasmic basophilia. This does not indicate that the basophilia of the basophil granules is due to ribonu- cleic acid. Pearse (1952) has demonstrated the absence of ribonucleic acid in specific basophil granules by means of the coupled tetrazonium reaction. Acetylation for 8 hours blocks the reaction in the basophil granules but does not block the reaction of nucleolic and cytoplasm. VI. Cell Classes and Cell Types A. CLASSIFICATION Present knowledge has diminished some- what the number of distinct adenohypo- physeal hormones, and it seems possible that no more than six may have to be ac- commodated in the pars anterior. These are somatotrophin or the growth hormone, prolactin or the lactogenic hormone, cortico- trophin, thyrotrophin, FSH, and LH. It seems probable from recent developments that six cell types may be present in the pars anterior. It is, therefore, profitable to discuss pituitary cytology and its relation to pituitary function on the assumption that each hormone may be the single product of a specific cell type. This may, or may not, be true; a system of classification must not be too rigid. The differentiation of the cells of the anterior pituitary into acidophils, basophils, and chromophobes must be regarded as a differentiation of an unknown number of specific cell types into three classes. The cells are classified by, and take their names from, the staining reactions of their specific granules. Even the smallest detect- able amount of granulation serves for this purpose, provided there is no confusion with mitochondria or cytoplasmic basophilia. It may be noted here that it is usual to say that cells are stained by a reagent when they contain a large number of uniformly distributed stained granules. This usage is dangerous unless the convention is adopted and rigidly adhered to that all staining re- actions of cells, unless explicitly stated to be otherwise, refer to the staining of specific granulation. It is particularly important when recording the absence of staining to note and state if there are granules that are unstained by the reagent. An absence of staining when there is an absence of gran- ules is not at all of the same significance. The failure to differentiate between the staining of granules and the staining of other cytoplasmic components is a common error. Cell types of the acidophil class contain specific granules which have similar prop- erties and, therefore, react alike to the cruder staining methods. In the same way the specific granulation of the cell types of the basophil class have certain properties in common which are to be ascribed to a simi- larity in the chemical nature of the secre- tions which enable them to be recognized as a distinctive class. Chromophobic cells whicli lack specific granulations of cither HYPOPHYSIS AND GONADOTROPHIC HORMONES the acidophil or basophil cell classes will include all cells in a resting or inactive phase which temporarily do not contain spe- cific granulation, and will also include any specific chromophobe type of cell which does not contain stainable granules at any time. The characteristic of the acidophil cells is that they contain specific granules which are of a solid protein nature of high insolu- bility, easily preserved by any method of fixation. These acidophil granules have a strong affinity for acid dyes of all sorts, their degree of acidophilia being somewhat less than that of the hemoglobin of the red blood corpuscles. The cells of the basophil class are characterized by the presence of specific granulation in the form of droplets of glycoprotein which are not highly re- fractile and whose contents are freely solu- ble at physiologic pH. Their retention in the cell depends on the impermeability of the membranes, and they are released or dis- solved when the cytoplasmic integrity is destroyed by cytolytic agents. The glyco- proteins of the basophil cells have affinities for acid dyes which vary among the spe- cific types in any one species and also from species to species. For a rigid division of chromophil cells into the acidophil and baso- phil classes, appeal must be made to an in- vestigation of the chemical nature of the granulation by the PAS reaction. The sub- sequent subdivision of these classes into specific cell types is at present only par- tially achieved by tinctorial methods, and then only in favorable cases. It depends on empiric staining procedures, the results of which are not consistent from species to species. B. FAVORABLE SPECIES The fii'st feature that would be noticed by anyone who applied standardized stain- ing procedures to pituitaries of a number of manrmalian species would l)e the greatly differing results obtained. These differences depend not only on the relative proj^ortions of the different cell types, but also on the intensity of coloration and quality of the staining reaction. Thus some mammalian species provide pituitaries whose anterior lobes do not at first sight appear promising, because basophil cells occur only in small numbers and with weak staining reactions not conducive to their differentiation into specific types. Acidophil cells may seem to be all of one type and all give the same staining reactions, either because only one type is present, or because two types are present but with staining reactions too close to be separated by the standardized arbi- trary procedui'e used. Other species pro- vide pituitaries which, from the first inspec- tion, show themselves favorable for detailed study in that a wider variety of different cell types with strong and distinctive stain- ing reactions is revealed. Examples of mammalian i)ituitaries with staining properties favorable to the differ- entiation of the specific types are those of the bat (Herlant, 1956a,), dog (Purves and Griesbach, 1957a), monkey (Dawson, 1954bj, and cat (Herlant and Racadot, 1957). In these species the presence of five distinctive cell types in the pars distalis can be inferred from the results of the staining procedures. In other species the number of cell types is presumably similar, but the staining reactions of the granules are less favorable for the purjioses of tinctorial dif- ferentiation. C. REACTIVITY In si)ecies whose pars anterior cells con- tain granules with favorable staining reac- tions, subsequent experimentation will show whether the cell types so revealed will favor the experimenter by marked variations in their appearances in correlation with changes in their secretory activity. In some sj^ecies the variations in appearance, in con- ditions producing widely different levels of secretion of specific hormones, are so small and inconspicuous that much time and care- ful measurement may be necessary to detect the morphologic equivalent of the secretory change. In other species the most striking alterations of cell size, contour, and gran- ule content occur as accompaniments of changes of secretion rate, including the ap- pcarance of new forms and substances which are rarely to be seen in the "normal" pitui- tary. Examples of such changes are the ap- pearance of thyroidectomy cells and castra- tion cells in the rat i)ituitary under conditions producing rapid secretion of thy- rotrophin or gonadotrophins respectively. HYPOPHYSEAL MORPHOLOGY 173 Reactivity in this sense, by enabling the significance of tinctorial or morphologic differences to be tested, is of primary im- portance both in the verification of parti- tions based on tinctorial or morphologic dif- ferences and in the identification of cell types as secretors of specific hormones. Only those differences which are correlated with a difference in reactivity, and which can be related to the production of specific hor- mones, can be regarded as of specific signifi- cance. Variations in the size and shape of the cell or its nucleus, the amount of cyto- plasmic basophilia, the form of the Golgi apparatus, and the amount of granulation may be related to variations in the state of activity in the single cell type and are, therefore, of functional significance but not of specific value. D. SPECIFICITY OF GRANULES One implication of the method of classi- fication formulated here is that all the se- cretory granules within any one cell will be of a single type. This seems to be true of the majority of mammals, judging from my own observations. Admittedly it is possible with some techniques to obtain different colors in granules in the one cell, just as it is possible to stain erythrocytes two dif- ferent colors in the one blood vessel, but such color differences are not significant. Apart from this, there is the possibility, since secretory granules in general contain a mixture of proteins and peptides and not a single substance, that granules vary in quality, due to differing proportions of the several components at different times as a result of different rates of secretion or from other effects. If this is so, differing shades of color may be obtained with some proce- dures in granules containing the same hor- mone. E. CHANGES IN CELL PROPORTIONS WITH ALTERATIONS IN FUNCTION The modifications in secretory activity, which occur as the result of environmental influences, or after experimental interven- tion, produce in some species distinct dif- ferences in the appearances of stained sec- tions of the pars anterior. These differences are often referred to as "changes in the ]iroportions of chromophil cells." Since in many instances such changes in the propor- tions of different cell types occur rapidly and without any proportionate number of mitoses, changes in the proportions of cells have, in the past, been ascribed to the trans- formation of cells of one type into cells of another type. It cannot be said as yet that transformation of one cell type to another does not occur, but, if it does, it must be limited. The impossibility of telling what appearance any given cell would have shown at some time other than the time at which it was taken from the animal pre- cludes a direct examination of transforma- tions. The zonation phenomenon, however, is of the greatest assistance in setting limits to transformations that may occur. The stability of the acidophil and basophil zones in certain mammals shows that basophil cells and the chromophobes of the basophil zone are not transformable into acidophils, nor are acidophils transformable into baso- phils. Moreover, the fact that the anterior lobe hormones have been shown in some species to be not uniformly distributed within the anterior lobe, but to have char- acteristic distributions, different for the dif- ferent hormones, suggests that each hor- mone is the product of a specific cell type not transformable into cells with a different function. If, therefore, basophils cannot be trans- formed into acidophils or acidophils into l^asophils, if for the most part each hormone is produced by its own specific cell type which is not transformable into another type with a different function, how can the apparent difference in the proportions of acidoi^hils, basophils, and chromophobes be explained? Three effects are operative. First, in many species a large proportion of the cells of the pars anterior are classified as chromophobes. Most of these cells are cells in a temporarily inactive phase and contain either no specific granulation or granulation in amounts too small to give decided stain- ing reactions. With an alteration in secre- tion rate large numbers of chromophobe cells may accumulate granules. The trans- formation, however, is only from a specific cell low in granule content to one with high granule content, and not the production of a specific cell type from an undifferentiated cell. Second, the size of individual cells 174 HYPOPHYSIS AXD GOXADOTROPHIC HORMONES greatly influences the proportionate count obtained from the examination of thin sec- tions. The larger cells not only show a greater area of cross section in the sections in which they appear, but they also appear more frecjuently owing to their greater ex- tension in the direction perpendicular to the plane of section. The third effect, which is also related to the size of the cell as well as to its granule content, is the ease of recogni- tion. When the cytoplasm is scanty the true nature of the cell may be missed and it may be classed as a chromophobe, whereas with equal granule density but with an increased width of cytoplasm it would be classed as a chromophil. These three effects are responsi- ble for most of the variations in cell propor- tions reported in experimental studies of the |)ars distalis. It should be noted here that, although marked variations in apparent cell pro])or- tions occur in some species, the hypophyses of other species are remarkably uniform in appearance despite extreme variation in secretory function. Thus the bovine hy- pophysis shows little change in response to thyroxine deficiency, thyroxine administra- tion, castration, or administration of sex hormones. The tendency for experimental work to be concentrated on those species in which functional changes are accompanied by marked cytologic changes has obscured the fact that such obvious cytologic changes are not a necessary accomjianiment to changes in a secretion rate. VII. The Acidophil Cell Class A. ACroOPHIL GRANULES Acidojihil granules consist of a membrane (enclosing solid contents. The presence of the membrane has been demonstrated by elec- tron microscopy (Farquhar and Wellings, 1957). The contents include an insoluble protein, phospholipid, and hormones. In size acidophil granules range from 0.1 to about 1.0 jx. In species where two types of acidophil cells can be seen, it is usual for the granules in one type to have a larger aver- age size than those in the other. In species with large granules, the fact that tliere is a variation of size in the granules within in- dividual cells can be determined with the light microscope. The granule size varies with the species, being relatively^ large in man, dogs, and cats, but fine in guinea pigs, mice, and sheep. The acidophil granules are highly refrac- tile in the fresh state. In living cells, under dark-ground examination, they are seen to be in constant rapid motion and show a re- fractility and luminosity greater than that of any element in basophil or chromophil cells. Their high refractility is a conse- quence of their solid nature. In typical acidophil cells a high concentration of gran- ules is present in the cytoplasm, and this confers on acidophil-containing areas an opacity which enables them to be distin- guished in the fresh state from zones in which acidophil cells are absent (Smith and Smith, 1923b). In rats, almost total degranulation of the acidophil cells follows complete thyroxine deficiency, and in such hypophyses the nor- mal opaque appearance of the anterior lobe changes to the translucent semitransparent api)earance normally seen in the acitlophil- free areas of the bovine hypophysis (Purves and Griesbach, 1946). Acidophil granules are quite different from mitochondria. Specific staining meth- ods for the demonstration of mitochondria leave the granules unstained (Severinghaus, 1932; 1939). When what may be termed ordinary methods of fixation and staining are used, the staining reactions of mito- chondria and acidophil granules are similar. The acidophil granules, like mitochon- dria, are readily released when the cells are disrujited in water, saline solutions, or su- crose solutions. In suspension they have the appearance of spherical highly refractile bodies of varying size. They have a higher density than any other element, but sedi- ment more slowly than nuclei and erythro- cytes because of their smaller size. On pro- longed centrifugation acidophil granules make their way through the layer of nuclei and erythrocytes and api)car on the bottom of the centrifuge tube. Tiiey differ from mitochondria in not swelling up and disin- tegrating in saline solutions or distilled wa- ter, and in not collai)sing on drying. H. L1I>()1I) COX'l'K.N r OK A('n)()PHlL GRANULES The acidophil granules isolated by cen- trifugation (Herlant, 1952a) are positively HYPOPHYSEAL MORPHOLOGY 175 stained by the Baker acid hematein test for phospholipid. In sections the same test pro- duces a dark coloration of acidophil cells which is attributable to, in the main, stain- ing of the granules (Rennels, 1953). There is some conflict of opinion as to whether this staining indicates the presence of phos- pholipid or is the result of some property of the granule protein. Herlant (1952a) and Elftman (1956) consider the reaction not to indicate phospholipid, but the evidence on which this conclusion is based is contradic- tory to the observations of Racadot (1954) and Ortman (1956) . The value of the hema- tein staining as a specific stain for acidophil granules is diminished by the positive stain- ing of mitochondria and of phospholipid and other materials in basophil cells. C. SULFHYDRYL AND DISULFmE CONTENT OF AcmopHiL c;ranules Ladman and Barrnett ( 1954) described the staining of acidophil cells in the rat by the Barrnett-Seligman technique which is l)resumed to be specific for sulfhydryl and disulfide groups. This staining in acidophil cells is obtained only after fixation in Zen- ker's fluid. After fixation in acid alcohol and a number of other fixatives the cells do not stain. This observation could be interpreted to indicate the presence in acidophil gran- ules of a sulfur-rich protein soluble in acid alcohol and preserved only by fixation in Zenker's fluid. Because of the possibility of formation of mercury compounds by the ac- tion of the fixative, and because such com- pounds might also react with the reagent used to demonstrate sulfhydryl groups, these results should be interpreted with cau- tion. An increased staining after Zenker fixation, in comparison with acid alcohol fixation, is common to many animal tissues (Barrnett, 1953; Barrnett and Seligman, 1954). It has not been shown that treatment with Zenker's fluid after previous fixation in acid alcohol does not induce this type of staining. Adams and Swettenham (1958) showed that staining of the acidophil granules in the human pars distalis by the Barrnett-Seligman reaction was not due to cystine, because it was not blocked by oxidation of the sulfide linkages to sulfonic acid groups by performic acid. It should also be noted that the acidophil granules did not react positively to other histochemical tests for cystine. D. HORMONE CONTENT OF ACIDOPHIL CELLS An indication of the probable nature of the hormonal secretions of cells of the acido- phil class is obtainable from the assay of portions of the acidophil and basophil zones of the pars anterior. Experiments of this sort were first made by Smith and Smith (1923a, 1)), who found that the central basophil zone of the bovine pituitary stimu- lated metamorphosis in hypophysectomized tadpoles with accompanying stimulation of the thyroid gland; the outer acidophil-rich portions caused an unusual stimulation of growth without causing either metamorpho- sis or activation of the thyroid. These re- sults indicate the probability that growth hormone is present in high concentration in parts of the gland which are rich in acido- phil cells. Azimov and Altman (1938) and Friedman and Hall ( 1941 ) found a differ- ential concentration of prolactin in the pe- ripheral acidophilic zones of the bovine hypophysis. The problem of the distribution of the corticotroi)hin in the predominantly acido- jihilic and basophilic zones was apparently decided by the consistent results obtained by Smelser ( 1944) and Giroud and Martinet ( 1948) , who found that the adrenal weight- increasing action was predominantly situ- ated in the basophil zone. Later discoveries concerning the unreliability of the adrenal weight increase as a measure of adreno- corticotrophic activity made these results of less significance than was formerly believed. Some observations suggested that the adre- nocorticotrophic hormone may, in fact, be present in high concentration in the acido- phil zone. Chiti and Zinolli (1952) found that both the acidophilic and basophilic zones of the pig hypophysis have an action on the guinea pig adrenal cortex, and that there is a difference in the response to the two zones. The response to the basophilic zone was more marked in the fasciculata of the adrenal cortex, whereas the tissue of the acidophilic zone caused an enlargement of the reticularis. Desclaux, Soulairac and Cha- neac (1953) using beef pituitaries found that implants of the basophil zone placed in con- tact with the adrenal of the rat did not mod- 176 HYPOPHYSIS AND GONADOTROPHIC HORMONES ify the lipid content of the adjacent adrenal cortex, whereas similar implants from the acidophilic zone produced a discharge of lipid followed by a re-accumulation. The response, therefore, to the local presence of tissue from the acidophilic zone was simi- lar to that produced by injections of cortico- trophin. Inasmuch as Halmi and Bogdanove (1951) have shown that rat pituitaries which have lost their acidophil granules after total thyroidectomy still contain nor- mal amounts of corticotrophin, it would seem that corticotrophin is not a constituent of the acidophil granules in the rat. My own assays of the basophil and acido- phil zones of the pig pars anterior, using the ascorbic acid depletion assay, showed that both zones contained high concentrations of corticotrophin, but that there was a higher concentration in the basophil zone than in the acidophil zone. In this species, as in the rat and bovine, corticotrophin is not asso- ciated with acidophil granules, and although a different situation may be found in other species, I would ascribe Herlant's (1952b, 1953a ) results to the absorption of the hor- mone by the granules or other particles sed- imenting with them. The results of implan- tation experiments are explicable by the fact that corticotrophin is present in acidophil and basophil zones in about the same amount; apparently the histologic response is modified by the presence of other mate- rials, different for each zone. Inasmuch as somatotrophin and prolactin are the two hormones which are in higher concentration in the acidophil zones of pig and beef anterior lobes, it is concluded that these hormones are in the acidophil cells. The two hormones are not necessarily pro- duced in the same cell, because in many mammals two types of acidophil cells can be seen. Purves and Sirett (1959) have shown that prolactin and somatotrophin have dif- ferent disti'ilnitions in the anterior lobe of the wallaby hyjiophysis, indicating a sepa- rate origin for each hormone. !■:. HORMONE CONTENT OF ACIDOPHIL GRANULES Acidophil granules prepared by the differ- ential centrifugation of the susi)ension pre- pared by disintegrating the anterior lobes of sheep, pig, and ])ovin(> hypophyses in hyjicr- tonic sucrose solution, were examined for their hormone content by Herlant (1952a, b; 1955). The easily sedimented granules contain prolactin and somatotrophin, whereas thyrotrophin and gonadotrophins remain in the supernatant, from which they can be thrown down only by centrifuging at much higher speeds. The granules also con- tain corticotrophin, but in view of the well known tendency of this hormone to become firmly bound to proteins, this finding is of doubtful significance. Only if it were shown that corticotrophin added to a suspension of granules did not become bound to them could the association of this hormone with the granules be regarded as indicative of its in vivo location. The results of Brown and Hess (1957) are not in conflict with Herlant's findings, although they interpreted them as showing somatotrophin in the mitochondrial frac- tion. Having experimented with both sheep and beef pituitaries, I am of the opinion that Brown and Hess underestimated the rate of sedimentation of acidophil granules, and that there is no likelihood of sediment- ing the mitochondria in this material and leaving the acidophil granules in the super- natant. Combining the evidence obtained from assays of tissue and of separated granules, it can be concluded that cells of the acido- phil cell class produce somatotrophin and prolactin, and that these hormones are stored in their granules. F. ACIDOPHIL CELL TYPES In some species an easy differentiation of the acidophil cells of the pars anterior into two si)ecific types is obtained by staining methods. The differentiation depends on the tendency of the granules in one cell type to be stained orange with Orange G, whereas the other type is stained a red color with azocarmine in the azan method, or with acid fuchsin in Crossmon's (1937) method, or with erythrosin in the Cleveland-Wolfe method used by Wolfe, Cleveland and Campbell (1933)^. It will be convenient for the purposes of discussion to adopt the term ''carminophil" used by Friedgood and Dawson (1940) for red-stained cells, and the term ''orange- ophil" used by Lacour (1950) for orange- HYPOPHYSEAL MORPHOLOGY 17' stained cells. These terms are expressive of the relative affinity of the two types of granules in a species in which such a dif- ferentiation is obtained. The term "orange- ophil cell" means the cell whose granules show the greater tendency to orange staining; the term "carminophil cell" means the cell whose granules show the greater tendency to be stained red, whether by azocarmine, acid fuchsin, or erythrosin. The equivalence of the three methods was demonstrated in the dog by Hart- mann, Fain and Wolfe (1946), and by Goldberg and Chaikof! (1952a). I find that Crossmon's method gives in the human pi- tuitary an equivalent differentiation to that obtained by Romeis (1940) with a modified azan method (Fig. 3.6). The results are most convincing when a differentiation is obtained by Crossmon's method, because in this method the differentiation appears spontaneously on staining the slide in an acidified mixture of orange G and acid fuchsin. The ease with which the differentia- tion is achieved varies in different species. In the sheep the Crossmon method gives two shades of red-orange or orange-red that are distinguished from each other only with difficulty, whereas in the dog the colors are pure orange and pure red. The azan method permits of modifications both in the solu- tions and the sequence which allows it to be accommodated to the varying staining pro]X'rties in different species. The azan method, therefore, allows differentiations to be obtained that are not possible by simpler methods. For this versatility a price must be paid in the form of a tendency to in- consistent and variable results. An orangeophil-carminophil distinction has been reported in the hypophyses of the dog (Wolfe, Cleveland and Campbell, 1933; Hartmann, Fain and Wolfe, 1946) ; arma- dillo (Oldham, 1938) ; rabbit and cat (Daw- son and Friedgood, 1937, 1938a; Dawson, 1939; Friedgood and Dawson, 1940); man (Romeis, 1940); bovine (Gilmore, Petersen and Rasmussen, 1941); opossum (Dawson, 1938); ferret (Dawson, 1946); monkey (Dawson, 1948) ; l)at (Herlant, 1956a) ; and wallaby (Ortman and Griesbach, 1958). In vertebrates other than mammals two types of acidophils have been recognized in the hypophyses of birds (Rahn, 1939; Rahn and Painter, 1941; Payne, 1942. 1943); snakes (Hartmann, 1944; Cieslak, 1945); newts (Copeland, 1943); fishes (Scruggs, 1939) ; and frogs (Green, 1951). It is not certain that the two types of cells observed in some of the above investigations were both acidophils. Mikami (1957) con- siders the cells stained orange by the azan Fig. 3.6 (/(,;/ ) >( i i nm <.i i Ik p.-u- anterior of the dog showmti iwti i\ |i<- <>[ ,i< iiLiplnl cell as seen after Crossmon staining. (1) Orangeophils; {2) carminopliils. Crossmon, X 820. Fig. 3.7 (right). The same section as Fig. 3.6 decolorized and restained by PAS. The orangeophils (1) are pale, whereas the carminophils (2) are distinctly stained. Other cell- which are more intensely colored by PAS than by Crossmon, are basophils. PAS, X 810. 178 HYPOPHYSIS AND GOXADOTROPHIC HOPtMOXES method in the rostral zone of the pars dis- talis of the fowl to be basophils because their granules are intensely stained by the PAS reaction. Of different significance are the definite but relatively weak colorations observed in the carminophil cells of the dog (Purves and Griesbach, 1957a), and frog (Ortman, 1956a). The amount of color formed must be considered in relation to the amount of protein present. In the fully granulated acidophil cell there is a large amount of protein. With close packing of the granules, as much as 50 per cent of the cytoplasmic space can be occupied by solid protein granules. The amount of specific protein in basophil cells is usually very much less. Any nonspecific staining of pro- teins, such as by a weak iodine solution, or by the methods used for staining paper electrophoresis strips, will show the high protein content in the fully granulated acidophil. Calculations based on the color produced by PAS in the mixed proteins of blood plasma (approximately 7 per cent solution of protein containing 1 per cent of sugar by weight) make it evident that ciuite a strong color would be produced in acido- phil cells if there were but one reacting group per molecule of protein. It is only when the color is intense in relation to the amount of protein reacting that the pres- ence of many reacting groups and, there- fore, probably the presence of polysaccha- ride, may be inferred. The color produced in the granules of carminophil cells is not of this intensity. In order to resolve the doubt concerning the significance of moderate PAS colora- tions in heavily granulated cells, Purves and Griesbach (1956) used a jireliminary buffer extraction before fixation and found that the carminophil granules in the dog were insoluble, and were still stainable by PAS after extraction at pH 7.5, a treatment that dissolved all the basophil granules. They, therefore, defined basophil granules as granules containing soluble glycopro- teins. This definition enables one to bypass the question 'Ts the PAS reaction in car- minophil granules sufficiently strong to in- dicate the presence of glycoprotein?", the answer to which is a matter of opinion, and to substitute for it a practical solubility test which is easy to carrv out and intevDi'ct. When a sensitive PAS sec[uence is used^ acidophil cells generally show a weak color which is easily masked by counterstaining (Fig. 3.7). The cytoplasm of chromophobes shows a similar weak coloration; against this background the acidophils may appear to be unstained. The granules of the car- minophil cells of the sheep, dog, frog, wal- laby (Ortman and Griesbach, 1958) , and cat (Herlant and Racadot, 1957), give re- actions distinctly stronger than this. The observation of Pearse (1951) that the car- minophil cells of the rabbit do not contain glycoprotein does not exclude the possibility of a weak reaction in this species, because he used the Hotchkiss (1948) modification of the PAS sequence, which suppresses weak reactions and is allegedly more specific for glycoproteins. Herlant and Racadot inferred that the carminophil cell in the cat was a basophil, but because the color produced by the PAS reaction is weak compared to that in typical basophils, I would regard this as a case to be submitted to the solubility test before a definite decision is made. G. SECRETORY FUNCTIONS OF ACIDOPHIL CELL TYPES In view of the evidence presented earlier, which indicates that acidophil cells secrete somatotrophin and prolactin, it can be ex- ])ected that in species in which two types of acidophil can be distinguished, one type will secrete somatotrophin, the other pro- lactin. The reactivities of the cell types in mammals, in which two types are distin- guishable, show that this is indeed the case and the point is discussed at some length in the section on acidophil cells and somato- trophin secretion which follows. In general one of the two specific cell types is reactive in relation to the repro- ductive cycle and shows marked fluctua- tions in activity, which can be correlated with the secretion of prolactin at times when its luteotrophic, mammotrophic, or lacto- genic action is apparent. The second type is relatively stable in relation to the repro- ductive cycle, and is assumed to secrete so- matotrophin. Evidence in support of this as- sumption is available in some species. Manimals in wliich the cai-iniiiophil cells HYPOPHYSEAL MORPHOLOGY 179 are active during pregnancy and lactation are cat (Dawson, 1946; Herlant and Raca- dot, 1957), rabbit (Pearse, 1951), monkey (Dawson, 1948), and bat (Herlant, 1956a). In the rabbit the discharge of luteinizing hormone which causes ovulation occurs within 1 hour after coitus. Pearse (1951) found at this time a discharge of granules from basophil cells which presumably were secreting luteinizing hormone. For the first 3 to 4 hours after coitus the carminophil cells were accumulating granules ; thereafter there was a slow discharge of granulation during the next 10 hours. These changes in the carminophil cell are consistent with a luteotrophic function for the secretion of these cells. The responses of the carminophil cell of the cat (Dawson, 1946) are similar to those of the rabbit. They are consistent with an accumulation of granules at estrus and for a time after mating, with a secretion with luteotrophic action immediately after ovu- lation, with a further accumulation of gran- ules during the later stages of pregnancy when a luteotrophic action by the hypophy- sis is not necessary, and with a phase of strong secretory activity with a lactogenic action, beginning at parturition and con- tinuing for the first 3 weeks of lactation. It should be noted here that Herlant and Racadot (1957), while confirming the luteo- trophic action of carminophil cell secretion, consider that in the cat this cell secretes the luteinizing hormone. They relate lacto- genesis to the secretion of a hormone, pre- sumably prolactin, by chromophobe cells, which are rich in cytoplasmic ribonucleic acid. I am inclined to think, from the simi- larity in the responses of carminophil cells in cat and rabbit, that they serve the same function in both species, but admit the ne- cessity for further investigation in the cat. Because of its unusual breeding cycle, the bat (Herlant, 1956a) presents peculiar ad- vantages for the correlation of specific cell types with specific secretion. In this species the carminophil cells show two phases of secretory activity during the breeding cycle. The first phase begins at the time of ovula- tion and terminates during the latter half of pregnancy; the second phase begins a little before parturition and continues throughout lactation. In animals wliich do not lactate. these cells involute rapidly after parturition. The responses indicate secretion of prolactin with luteotrophic and lactogenic actions at appropriate times. In two mammalian species the carmino- phil cell is the stable type, and the orangeo- phil the reactive type during the breeding cycle. These are: (1) Human hypophysis. The orangeophil cells are active during pregnancy and contain orangeophil granula- tion towards the end of pregnancy. These cells have been called pregnancy cells by Erdheim and Stumme (1909). Romeis (1940) calls the orangeophil cells seen in small numbers in the hypophyses of males and nonpregnant females "e"-cells, and the numerous orangeophil cells seen in late pregnancy "7/"-cells. Any differences be- tween the two are presumably due to dif- ferent states of activity. The granules of both show the same staining reactions, and only one type of orangeophil cell is seen in any one hypophysis. (2) The rat (Lacour, 1950; Dawson, 1954a). Purves and Gries- bach (1952) inferred the separate existence of somatotroi)hin and prolactin-secreting cells from the differential effects of estrogen and a deficiency of thyroxine on the acido- phil cell population, but could not achieve a tinctorial differentiation. The orangeophil staining achieved by Lacour was not repro- ducible. Dawson's (1954a) modification of the azan stain has usually given an orangeo- phil reaction in the active-looking acidophils of the pregnant rat pituitary, and in the sim- ilar cells appearing after estrogen adminis- tration but the staining is sometimes un- successful. Obviously the staining affinities of the two types of granulation in the rat are very similar. In all tliese species except the rat, the reactive acidophil type, whether it be the carminophil cell in the cat, rabbit, mon- key or bat, oi- the orangeophil cell in the human hypophysis (Floderus, 1949), has a different distribution from that of the nonreactive type, and the marked increase in the number of visible cells at certain times is due to the appearance of granules in previously nongranulated, and, there- fore, chromophobic cells. In the rat the orangeophil staining appears to be the re- sult of a change in the nature of the gran- ules in a jiroportion of the acidophils, which 180 HYPOPHYSIS AND GONADOTROPHIC HORMONES in the normal nonpregnant animal are car- minophil. It appears, then, that in a nmiiber of mammals the secretion of somatotrophin and the secretion of prolactin are the func- tions of two distinctive acidophil types, not transformable into one another, and with variable staining reactions in different spe- cies. In most of the species studied thus far, the prolactin-secreting cell is the more carminophil type. In view of the variations in staining affinities which make the dif- ferentiation of two types by staining meth- ods easy in some species and difficult in others, it is possible that in some species two types of acidophil occur whose gran- ules stain alike, and the human hypophysis is presumably one in which the prolactin- secreting cell is the more orangeophil. The variability in the nature of the hormones in different species (the variability of soma- totrophin is particularly well attested) makes this variability in the staining reac- tions of cells serving the same functions in different species credible. Some recent observations indicate that more than two acidophil cell types may be present in some species. Herlant (personal communication) has found four distinct acidophil cell types in the pars anterior of the mole {Talpa europanaea), and Ortman and Griesbach (1958) have evidence for four acidophil cell types in the wallaby {Wallabia nifogrisea). Purves and Sirett (1959) found prolactin concentrated in the rostral portion and somatotrophin concentrated in the caudal portion of the anterior lobe of the wallaby hypophysis. Somatotrophin, therefore, is se- creted by orangeophil cells in the caudal zone of the pars anterior, and it is probable that the carminophil cells of the rostral zone are the source of the prolactin. II. ACIDOPHIL CELLS IN RELATION TO SOMATOTROPHIN SECRETION The association of the acidophil cells with the production of growth hormone was first deduced from the observations of acido- phil cell adenomas in the hypophyses of patients showing the symptoms of acro- megaly or gigantism, and, as Gushing and Davidoff stated in 1927, no one today can have anv reasonable doubt that the sul)- stance which provokes the overgrowth is a product of the acidophil cells. The absence of acidophil cells in the pars anterior of the dwarf mouse (Smith and MacDowell, 1930) is often cited as evidence for the origin of a growth-regulating hor- mone secreted by the acidophil cells. Smith and MacDowell's report indicated that there may be deficiencies in cell types other than the acidophils, but Ortman (1956b) finds that basophils, both /3-cells and 8-cells, are present and appear similar to those present in normal litter mates. It seems, therefore, that there is a specific deficiency in growth hormone secretion associated with a de- ficiency of acidophil cells in these animals. However, the dwarf mice also show evi- dence of deficient thyrotrophic, gonado- trophic, and adrenocorticotrophic secretion and growth may be stimulated in them by thyroxine administration (Nielson, 1952) as well as with growth hormone. Treatment with both hormones is necessarj^ to produce the appearance of full-grown mice. The lack of acidophils in the dwarf mouse hypophysis cannot be due to degranulation of these cells by thyroxine deficiency, because thyroxine administration does not cause regranula- tion. Hewer (1943) described a case of human dwarfism in which the hypophysis was markedly deficient in acidophil cells. It should be noted that the cessation of growth in the rat after hypophysectomy is due to the loss of thyrotrophin and conse- quently of the thyroid secretion as well as the loss of somatotrophin. This view is necessitated by the observations of Ge- schwind and Li (1952) who showed that either thyroxine or somatotroi)hin produces growth in hypophysectomized rats. Changes in the hypophysis resulting from thyroxine deficiency accompany the arrest of growth which occurs in totally thyroxine-deficient rats. Zeckwer, Davison, Keller and Livin- good (1935) pointed out that the disappear- ance of acidophil cells occurs in the rat hy- pophysis after thyroidectomy and related this to the cessation of growth in such ani- mals, because in partial thyroxine deficiency resulting from incomplete ablation of the thyroid, the acidophil cells were retained and growth continued. The relation of the acidophil cell (Icgvanulation to thvroxine HYPOPHYSEAL MORPHOLOGY 181 supply was studied by Purves and Gries- bach (1946) who showed that, although 2.5 /xg. of DL-thyroxine were required to pre- vent thyroidectomy changes in the baso- phils of the rat hypophysis, the acidophils were protected from degranulation by 0.5 ^g. per day and the effect of even smaller quantities in retaining some of the acido- phil cells was observed. The rate of growth observed in these animals was related di- rectly to the content of acidophil granules. Degranulation of the acidophil cells oc- curs in rats treated with potent goitrogenic agents. As judged by the acidophil cell re- sponse, thiourea is much less effective than thiouracil in suppressing thyroid secretion because the administration of 0.25 per cent of thiourea in the drinking water does not cause the loss of acidophil cells. Using the regranulation of the acidophil cells as a sensitive indicator of small amounts of thyroxine, Purves and Gries- bach (1946) demonstrated that the ad- ministration of iodide in relatively high dosage (1 mg. per day) produced in thy- roidectomized rats an extrathyroidal syn- thesis of material with thyroxine-like activ- ity in amounts which were of physiologic significance. The partial regranulation of the acidophils on high iodide intakes in totally thyroidectomized animals is asso- ciated with continued growth at a subnor- mal rate. They concluded that from injec- tions of 1.3 mg. of potassium iodide per day, approximately 0.12 /xg. of L-thyroxine might be produced. Hum, Goldberg and Chaikoff (1951) showed that injections of iodide (1 mg. per day or more) caused re- granulation of acidophil cells in rats whose thyroid tissue had been destroyed by ad- ministration of radioactive iodine and con- sidered that the effect was due to extrathy- roidal synthesis of thyroxine. Marine, Rosen and Spark, (1935) re- ported almost total loss of acidophils from the hypophyses of thyroidectomized rab- bits, but overlooking changes in the baso- phil cells, they related the acidophil cell changes to the production of thyrotrophic hormone. In the thyroxine-deficient dog, changes in the acidophil class of cell are apparently limited to the a-cell. Goldberg and Chai- koff (1952b) observed in dogs with com- plete thyroid destruction produced by radio- active iodine, a complete degranulation of the a- (orangeophil) cells whereas the e- (carminophil) cells were well preserved and full of granules. The animals were adult and the effect on growth was not observed. The selective degranulation of the a-cells is, however, consistent with the view that these cells are concerned with somatotrophin se- cretion. VIII. The Basophil Cell Class A. BASOPHIL GRANULES In the light of modern knowledge basophil granules are defined as secretory gran- ules containing soluble glycoproteins. The ]\IcjManus (1946) PAS reaction serves as a group reaction for the identification of all granules of the basophil class, as it imparts to them an intense red or magenta color in- dicative of a high content of protein-bound carbohydrate. Glycogen and certain lipoid inclusions are two other intracellular sub- stances which can be colored intensely by the PAS reaction. In paraffin sections pre- treated with diastase these substances will be absent. Insoluble substances of an un- known nature which give a color with the PAS reaction occur in some acidophil cells, and should be sought for in material sub- mitted to extraction before fixation. Suitable extraction methods are: (1) Freeze and thaw the tissue on the stage of a freezing microtome three times before fix- ation. Basophil granules will dissolve in the mixture of intracellular and extracellular fluids. (2) Place small pieces of tissue in cold alcohol or acetone for 30 minutes, then in buffer for 1 hour before fixation. This al- lows some control of the pH at least in the peripheral portions of the tissue. (3) Perfuse the animal by way of the aorta with buffer which is saturated with ether at 37°C., or to which desoxycholate has been added as a cytolyzing agent. This method allows the assay of the extracted tissue for content of unextractable hormones. In the other meth- ods the granule contents, although in solu- tion, still remain to a large extent inside the tissue. It should be pointed out here that me- chanical damage resulting from the handling of the delicate tissues of the hypophyses of 182 HYPOPHYSIS AND GOXADOTROPHIC HORMONES smaller animals causes a loss of the gran- ules from basophil cells. Such hypophyses are best fixed in situ. Basophil granules show a high capacity, but only a weak binding power, for basic dyes (Peterson and Weiss, 1955) . This baso- philia does not serve for the characteriza- tion of basophil granules because of the prevalence of cytoplasmic basophilia and the staining of acidoi)hil granules by basic dyes. There has in the past been some confusion between the basophilia of basophil granules and that of ribonucleic acid. Dempsey and Wislocki (1945) pointed out that the stain- ing of basophils with aniline blue was not concerned with cytoplasmic ribonucleic acid, and suggested that the aniline blue stained an acidophil substance which repre- sented the true secretory granules of the cells. The nature of the specific granules in basophil cells was clarified by the researches of Herlant (1942), who observed a violet metachromatic staining of the basophil granules in the human pituitary by neutral solutions of toluidine blue. This metachro- masia is observed only in undehydrated sec- tions and demonstrates a specific type of basophilia quite distinct from that due to cytoplasmic ribonucleic acid. This type of basophilia, which is common to many sites containing polysaccharide material, was considered by Herlant to indicate that the specific granules of the basophil cells were composed of glycoprotein. After a short treatment with alcohol to cytolyze the cells, the glycoprotein material was found to be extractable by water, and the resultant ex- tract was rich in FSH. Herlant demon- strated the glycoprotein nature of the baso- phil granules by the Bauer method for polysaccharide, and the observations were subsequently confirmed by the more satis- factory McManus (1946) method for gly- coprotein (Herlant, 1949). Other observa- tions confirming the glycoprotein nature of basophil gramdes have been reported by Catchpole (1947, 1949), Pearse (1948), and Purves and Gricsbach (1951a I. The Herlant metachromasia is observed ill the basophil granules and indeed in all P.\S stainable structures in rat, dog, and litiinan hypophyses. This metachromasia docs not serve to characterize basoi)liil gran- ules, inasmuch as the carminophil cells of the dog and of the wallaby are also meta- chromatically stained by toluidine blue. Basophil granules vary considerably in size in different species. In the rat they are seen in electron micrographs as spherical bodies of a maximal size of 150 mix. The in- dividual granules are not seen by the light microscope, and the flocculent "granules" seen in stained sections are the result of the uneven distribution of the granules and their aggregation during fixation and de- hydration. In the human hypophysis the basophil granules are much larger, and are singly visible in lightly granulated cells in stained sections. They vary greatly in size and the largest among them are as large as, or larger than, acidophil granules. In fresh tissue the basophil granules do not show the high refractility of the solid acidophil granules, and are therefore pre- sumed to be vesicular, consisting of a mem- brane enclosing a solution of glycoprotein. The staining procedures most generally used in the study of the cytology of the adenohyphophysis are various trichrome procedures using mixtures of acid dyes. As has been said before, by these methods baso- phil granules may be stained orange, red, purple, or blue. By such methods it is, of course, not in general possible to distin- guish acidophil granules from basophil gran- ules. This was not realized until recently because of a premature assumption that only two types of chromoj^hil cells are pres- ent in the pars anterior of mammals, and because of a widespread erroneous belief that all blue dyes are basic dyes. In the literature, the term "basophilic" is con- stantly applied to structures stained blue by aniline blue in variations of the tri- chrome staining procedures and is, there- fore, used as a synonym for blue rather than as an indication of staining affinity. It may be stated here, as regards tlie mul- tiplicity of basophil cell tyi)es included in the basophil cell class, that the pars inter- media cells are basophils, and that there are in addition three specific types of baso- phil cells distinguishable in the pars anterior of certain favorable mammalian species. The immediately succeeding sections are concerned with the basophil granules and the basophil cell types of the pars anterior. HYPOPHYSEAL MORPHOLOGY 183 B. HORMONE CONTENT OF BASOPHIL CELLS Evidence of the hormonal content of baso- phil cells can be obtained by the assay of portions of tissue rich in basophil cells and comparison with the results obtained from adjacent portions rich in acidophil cells. In- vestigations of this type have been made by Smith and Smith (1923bj, Voitkevitch (1937a, bj,Herlant ( 1943), Smelser (1944), and Giroud and Martinet (1948). These in- vestigators agree that tissue rich in baso- phil cells has a high content of thyrotrophin and gonadotrophin. It seems that the gon- adotrophic effects include both follicle-stim- ulating and luteinizing actions. Cortico- trophin in both pig and bovine anterior lobes is also somewhat more concentrated in the basophil zone. Because of the preva- lence of chromophobes as well as basophils in the basophil zones, it cannot be inferred that all these hormones are in the basophil cells. Moreover, Smelser has shown that the ratio of the potencies of the two zones is different for thyrotrophin, corticotrophin, and gonadotrophin, so that these hormones seem to be stored in different cells. Inasmuch as three distinctive types of basophil cell can be seen in the pars an- terior of some other species, it is likely that a similar diversity occurs in the bovine hypophysis. This species is, however, not favorable for the discernment of specific basophil cell types. C. HORMONE CONTENT OF BASOPHIL GRANULES The basophil granules are sensitive struc- tures, and lose their glycoprotein content when the cells in which they are contained are damaged, as by freezing or mechanical distortion. It appears that this is the result of the instability of the enclosing membrane to solutions of low osmolar content. Thyrotrophin, FSH, and LH are, like the the glycoproteins of basophil granules, solu- ble in water at neutral pH, and are com- pletely extractable from anterior lobes after acetone drying or mechanical disintegra- tion. These hormones seem to be located in membrane-enclosed structures in living cells, because they can be centrifuged down from suspensions of anterior lobe tissue dis- integrated in 0.88 M sucrose. McShan and Meyer (1952), and McShan, Rozich and Meyer (1953), using the anterior lobes of castrate rats, were able to recover as much as 75 per cent of the gonadotrophin in a small granule fraction sedimented by cen- trifuging at 20,400 X g for 1 hour. There was evidence that grinding increased the amount of hormone in solution, presumably by disruption of granules. The hormone was extracted from the granules by isotonic sa- line, and from this solution could no longer be sedimented. This behavior is accounted for by the enclosure of the hormone in a membrane with properties similar to that of the mitochondrial membrane and, there- fore, stabilized by high sucrose concentra- tions. Herlant (1952a) worked with the anterior lobes of sheep, pig, and beef hypophyses, and obtained a small granule similar to that obtained by McShan and his associates which contained gonadotrophic activity. He found that these small granules gave an intense coloration with the PAS reaction, and thus demonstrated that they were baso- phil granules. It does not seem that the thyrotrophin content of the small granule fraction has been tested except by Brown and Hess (1957) . Their results are not easily interpreted because they sometimes used frozen instead of fresh beef anterior lobes and their fractions are wrongly identified, but it does seem that thyrotrophin can be centrifuged down from material dispersed in sucrose solution (0.25 m in their experi- ments). Although these observations are not by themselves conclusive, when considered in conjunction with the cytologic observations presented in the succeeding section, they provide confirmatory evidence for the view that thyrotrophin, FSH, and LH are made by basophil cells and are contained within their characteristic granulation. The behav- ior of corticotrophin in tissue dispersed in solution needs further investigation. D. DOES THE PERIODIC ACID-SCHIFF REACTION DEMONSTRATE THE HORMONE CONTENT OF BASOPHIL CELLS? The fact that in the rat pars anterior the amount of PAS reacting material in thyro- trophs, FSH cells, and LH cells, parallels the content of thyrotrophin, FSH, and LH, 184 HYPOPHYSIS AND GONADOTROPHIC HORMONES respectively, raises the question as to whether or not the hormones themselves are responsible for the staining reactions. The parallelism between depth of staining and hormone content does not contribute to the solution of this problem, because this par- allelism is a consecjuence of the fact that the hormone is stored in the specific gran- ules and the number of granules present de- termines the depth of staining. This cjues- tion, therefore, resolves itself into two parts. First, are the hormones glycoproteins, and second, are the hormones major constituents of the granule contents? The reported sugar contents of the prepa- rations of thyrotrophin, FSH, and LH pro- duced by the fractionation of extracts from the anterior lobes of domestic animals do not establish the glycoprotein nature of the hormones, because it has never been shown that similar glycoprotein-containing fractions do not result from the fractiona- tion of extracts of tissue which are free from these hormones. Taking advantage of the ease with which rat hypophyses can be ob- tained free from these particular hormones by pretreatment of the animal with thy- roxine and estrogen, I have compared the extracts from hormone-free anterior lobes with those from normal anterior lobes. Simi- lar amounts of protein with similar sugar contents were extracted from both types of gland. Fractionations by salt or alcohol which resulted in potent preparations of thyrotrophin, FSH, and LH, when applied to the extracts of the normal gland, gave a similar partition of the glycoproteins ex- tracted from the hormone-free gland. It is clear that the preparations of the reputedly glycoprotein hormones obtained by frac- tionation of glandular extracts with varying concentrations of salts or alcohol are com- posed mainly of inert materials resulting from the fractionation of a mixture of plasma proteins and soluble cytoj^lasmic constituents. In extracts from castrate rat anterior lobes in which the gonadotrophins are pres- ent at many times the normal level, the FSH fraction contains a small but definitely increased amount of PAS reacting material compared with the corresponding fraction from an equal weight of hormone-free an- terior lobe. There is. therefore, in the l)as()- phil granules a glycoprotein which accom- panies the FSH during fractionation of the extract, and this glycoprotein is present in amounts which would contribute to the staining reactions of the granules. The po- tency of this material is close to that of the highly purified FSH preparations reported by Steelman, Kelly, Segaloff and Weber (1956). Persons observing pars anterior cells un- der the microscope often form impressions which exaggerate the amount of specific granulation present in the pars anterior. They often also have exaggerated ideas about the potency of the separated hor- mones. Taking account of the small per- centage of granulated thyrotrophs in the rat pars anterior and the small fraction of the cytoplasmic content of these cells which is in the form of granules, there must be only about 1 /xg. of specific thyrotroph granulation per mg. dry weight of anterior lobe. Yet the anterior lobe has a potency of 0.2 I.U. thyrotrophin per mg. dry tissue, equivalent to 10 /xg. of the potent prepara- tion of thyrotrophin obtained by Condliffe and Bates (reported by Sober and Peterson, 1958). It seems reasonable to assume that baso- phil granules, like other specific secretory granules, contain a mixture of substances, and it appears likely that, at least in the rat pars anterior, the glycoprotein hormones form a considerable proportion of the gran- ule content and contribute significantly to its staining reactions. E. DIFFERENTIAL STAINING OF BASOPHIL CELLS BY RESORCIN-FUCHSIN, KRESOFUCHSIN, AL- DEHYDE-FUCHSIN, AND ALCIAN BLUE: ^- CELLS AND 8-CELLS The three basoi)hil cell types in the pars anterior of some niainmals can be distin- guished from each otlici' by diagnostic fea- tures which are independent of any specific diffei-ential staining of the granules, but e\-en in these species a differential staining of the sjiecific granulation is an important aid in the study of specific types. In some species the granules of different cell types react differently to acid dyes and ap- pear in different colors after trichrome stain- ing methods. The recognition of the pres- ence of a multiplicity of basophil types was HYPOPHYSEAL MORPHOLOGY 185 delayed not so much by the difficulty of distinguishing between them as by the dif- ficulty of recognizing the basophil nature of the different types in the species in which the different types were highly dis- tinctive. It is for this reason that the first clear recognition of two types of basophil cells came from the discovery of Romeis (1940) that kresofuchsin applied to sections of human pars distalis gave a differential staining of the granules of cell types that by the azan procedure were stained alike. There seem to be four types of basophil cells in the human pars distalis. Two of them which Romeis did not consider suf- ficiently distinctive to warrant separation were designated by him as "/3-cells." The other two types he designated as "y-cells" and "8-cells." Kresofuchsin stained the granules of both types of /?-cells and left the granules of y-cells and 8-cells unstained. The ^- and the 8-cells after azan stain- ing were so much alike that a clear differ- entiation between them could be obtained only by the use of kresofuchsin, but the y- cells were so distinctive that the use of kresofuchsin to distinguish them from ft- cells was not necessary. In this way kreso- fuchsin acquired a reputation for distin- guishing y8-cells from S-cells rather than for distinguishing /3-cells from y-cells and 8- cells. The premature assumption that only two types of basophil cells would be present in the pars distalis of man and the pars an- terior of other mammals led to the wide- spread use of the terms "^S" and "8" to desig- nate cell types or cell groups in species other than man, and thus set the stage for an episode of nomenclatural confusion, the results of which have been exacerbated by a naive assumption that cell types desig- nated by the same Greek letter by different authors in different species ought to have the same functions. The history of the use of elastic tissue stains — kresofuchsin, resorcin-fuchsin, and aldehyde-fuchsin — is interesting and shows the difficulties that are experienced with the use of these materials. Erdheim and Stumme (1909) introduced the use of resorcin- fuchsin as an elective stain for the gran- ules of basophil cells in the human hypophy- sis, and it appears among the staining reactions listed l)v Bailev and Davidoff (1925) as characteristic of basophil cells. The term ''/?" was proposed by these au- thors to replace the term "basophil" which they thought was too specific in meaning to characterize a type of cell which could be stained specifically in several ways, some of which did not appear to involve baso- philia. Berblinger and Burgdorf (1935), in a study of connective tissue in the human hypophysis, used the commercially avail- able kresofuchsin as a stain for elastic tis- sue, and counterstained with orange G — phosphomolybdic acid and aniline blue. They noticed in the pars distalis that, al- though kresofuchin stained the granules of some of the basophil cells, there were other cells which did not differ in any visible re- spect except that their granules were not stained by kresofuchsin and were stained only by the aniline blue. Thornton (personal communication) and I too have observed the same phenomenon with paraffin sections of formalin-fixed human hypophyses stained with aldehyde-fuchsin. According to the age of the aldehyde-fuchsin solution and the duration of staining, the granules of a varying proportion of the basophils are stained with aldehyde-fuchsin, whereas others remain unstained in an erratic and nonreproducible fashion that cannot pos- sibly represent a distinction between differ- ent functional cell types. Rodriguez (1937) used Berblinger and Burgdorf's staining combination in a study of the cells of the anterior lobes of the hypophyses of a num- ber of mammalian species. He found kreso- fuchsin-stainable granulated cells in bovine, horse, sheep, dog, monkey, and rabbit hy- pophyses. He did not find such cells in the guinea pig or rat, although there were in these species cells with a diffuse staining. Using resorcin-fuchsin for the staining of basophil cells, von Soos (1934) apparently ol)tained results similar to those produced by kresofuchsin. He found cells with re- sorcin-funchsin stainable granules in nine mammalian species but not in birds. In most species the number of basophil cells revealed by resorcin-fuchsin was similar to the number shown by Mallory staining of adjacent sections. At the time of the above-mentioned in- vestigations, Romeis was using a combina- 186 HYPOPHYSIS AND GOXADOTROPHIC HORMONES tion of kre^ofuchsin and Heidenhain's azan which, although differing in details from the method of Berblinger and Burgdorf, should have given, as far as the basophils were concerned, equivalent results. His re- sults are published in his magnificent mono- graph (Romeis, 1940) on the hypophysis. Unlike the results of Berblinger and Burg- dorf, kresofuchsin in Romcis's hands gave a clear-cut discrimination between two dis- tinctive groups of cells, those whose gran- ules stained with kresofuchsin and those whose granules stained not with kresofuch- sin but only with the aniline blue of the azan stain. During his investigation Romeis found that the kresofuchsin supplied by the manufacturers differed from that he had ob- tained earlier in that, while still staining elastic tissue, it was no longer useful for staining the hypophysis. He found that resorcin-fuchsin he made himself could be substituted for kresofuchsin with results which were equal in every respect. Romeis examined the hypophyses of a number of mammalian species other than man with results that differed from those of Rodriguez and von Soos in some respects. He found cells with granules stainable by kresofuchsin or resorcin-fuchsin in some species in which these authors had not been able to demonstrate them. As has been stated earlier, Romeis called the cells whose granules stained with kresofuchsin ^-cells, and those whose granules did not stain with kresofuchsin, but were stained with aniline blue, 8-cells. He found both /?- and 8-cells in the bovine hypophysis and in the hy- pophyses of the dog, horse, cat, and rat. He found /3-cells alone in the bat and guinea pig; in the latter they were very scanty. He also noted that kresofuchsin stained strongly the pars intermedia cells in the guinea pig, mouse, and pig. The results that Romeis obtained with resorcin-fuchsin have been duplicated by Ezrin, Swanson, Humphrey, Dawson and AVilson (1958). Despite many attempts, other investigators have not been able to repeat these results using Romcis's method. There is, however, no doubt about the cor- rectness of his observations. Gomori (1950) introduecul ahU^liydo- fuclisin as an elastic tissue stain, and Ilahni (1950, 1951a) used it to differentiate be- tween ^- and 8-cells in the rat and mouse. It has since been used successfully by a number of authors and on a number of spe- cies. The question whether aldehyde-fuchsin gives results equivalent to those once given by kresofuchsin is one which is not easily answered. The results are variable, depend- ing on the fixative and on the treatment of the slide before staining, so that the term "stainable by aldehyde-fuchsin" cannot be given any precise meaning. Moreover, there are in the pars anterior of many, if not all mammals, three types of basophil cells, and according to conditions, the aldehyde-fuch- sin may stain the granules of one, two or all three types, or fail to stain any of them. I think, however, that it may be assumed, that with any particular fixative and prior treatment of the slide, the elements most readily stained by kresofuchsin or resorcin- fuchsin would also be most readily stained by aldehyde-fuchsin. Aldehyde-fuchsin suffers from the defect of variability in staining power from batch to batch, and it often happens, as Romeis found with kresofuchsin, that a batch of this stain will stain elastic fibers but fail to stain basophil granules. Gabe (1953) de- scribed the preparation of an aldehyde- fuchsin which can be kept as a dry powder. To obtain results equivalent to those given by Gomori's preparation it is necessary to dissolve this powder in a concentration of 0.5 per cent in 70 per cent alcohol acidified with hydrochloric acid. Gabe's own proce- dure does not give the same results. Recently Herlant and Racadot (1957) have indicated that Alcian blue (Steedman, 1950) at pH 1 stains the basophil granules in the toad and cat hypophysis that are stained by aldehyde-fuchsin. I find that a 3 per cent solution of Alcian blue in 70 per cent alcohol acidified with hydrochloric acid stains rat and human hypophyses with results equivalent to those obtained with aldehyde-fuchsin except that elastic fibers are not stained. H this procedure proves equally useful in other species it may sup- plant aldehyde-fuchsin and thus eliminate the uncertainty attending the use of this at |)resc>nt indispensable stain. HYPOPHYSEAL MORPHOLOGY 187 F. THE NOMENCLATURE OF BASOPHIL CELLS One difficulty in the use of the term "^" for a basophil cell type whose granules stain with aldehyde-fuchsin and "8" for a baso- phil cell type whose granules do not stain with aldehyde-fuchsin is the presumption that only two types of basophil cells are present. In addition there is the possibility that with a different staining technicjue the staining reactions may be reversed, so that one man's /3-cells could be another man's S-cells. When three easily distinguished cell types are present, it is necessary to show that the granules of only one type are stain- able by aldehyde-fuchsin before the term /? may be allocated. The naming of the re- maining two types will be arbitrary whether one is called 8 or not. The major objection to the use of the terms /3 and 8 according to rules based on staining procedures is the lack of agreement as to which rule and which staining procedure should be used. Herlant (1956a) suggested that the terms 13 and 8 should be transposed in species other than man so that cells whose granules are stained by aldehyde-fuchsin would be called 8-cells, and the cells whose granules are unstained would be called ^-cells. There are in addition a number of usages of the terms /S and 8 to denote cells distinguished by staining reactions other than aldehyde- fuchsin in its several variants. It seems therefore advisable to avoid altogether the application of these terms according to rule until there is agreement as to which rule is to be followed. The usages of Romeis in man, Halmi in the rat and mouse, and Gold- berg and Chaikoff in the dog can best be regarded as arbitrary. In the present account cells whose gran- ules are stained by aldehyde-fuchsin after any specified staining procedure will be stated to be AF-positive and called AF cells after such procedure. Cells whose granules are not stained by aldehyde-fuch- sin after such a procedure will be stated to be AF-negative and called non-AF cells. It is emphasized that cells which are AF- positive after one procedure may be AF- negative after a different procedure. The varying degrees of acidophilia of basophil granules allow in some species a distinction between "purple" basophils and "blue" basophils (Purves and Griesbach, 1957b). The granules of purple basophils retain more or less of the red component of azan or Crossmon or other trichrome stain- ing methods and appear in purplish shades after procedures that result in a blue colora- tion of the granules of blue basophils. Pur- ple basophils can be distinguished from blue basophils by counterstaining PAS stained sections with phosphotungstic acid-orange G (Herlant, 1953). The purple basophils, being relatively acidophilic, retain the or- ange G and become brick red, whereas the blue basophils remain magenta. In sections stained by aldehyde-fuchsin and counterstained by a trichrome method we may distinguish purple AF cells, blue AF cells, purple non-AF cells and blue non- AF cells. Inasmuch as in many species there are in the pars anterior two basophil types which can be distinguished from each other by certain diagnostic features, although their granules stain alike, it is necessary to introduce additional terms to enable each cell type to be given a distinctive name. Such terms can be derived from the dis- tribution of the cells in the pars anterior — "peripheral" or "central," "rostral" or "cau- dal"— or from some other diagnostic fea- ture^"pale" for lightly granulated cells, etc. Such terms have no precise significance; they are used merely to generate a suffi- cient number of terms to enable each dis- tinguishable cell type to receive a distinctive name. Thus, after fixation in formol-sub- limate there are in the rat pars anterior blue AF cells (/3-cells of Halmi, functionally thyrotrophs), and peripheral and central blue non-AF cells (functionally FSH cells and LH cells, respectively). The advantage of these terms is that they are, or should be, free from any implication that cells similarly designated in different species must have the same function. G. SPECIFIC BASOPHIL CELL TYPES It is now established that in the pars an- terior of certain mammals (the rat, Purves and Griesbach, 1951a, 1954; monkey, Daw- son, 1954b; bat, Herlant, 1956a; and dog, Purves and Griesbach, 1957a) three types of basophils can be distinguished. In the rat and bat, species which are favorable for 188 HYPOPHYSIS AND GONADOTROPHIC HORMONES the identification of cell types, the three basophil cell types have been identified as thyrotrophs, FSH cells, and LH cells. It will be noted that it is possible to distinguish cell types by specific staining reactions or by other diagnostic features without identi- fying them. By "identification" is meant here the allocation of specific function or functions to a distinguishable cell type. It is not yet certain that all vertebrates have separate FSH and LH or that the phys- iologic reciuirements of all species would re- quire the two hormones, if present, to be secreted separately and by different cells. In some species there may be only one type of basophil cell with gonadotrophic func- tions. It is likely, too, that in some species the duality of the gonadotrophic basophils is concealed by the fact that the staining reactions of the two types may be so alike that a distinction by tinctorial methods cannot be easily or consistently obtained. The term "gonadotroph" can be used as an inclusive term to denote the two types of cells secreting gonadotrophins and is also applicable in cases where a distinction of two such cell types has not been made. Thyrotrophs and gonadotrophs have been identified in the guinea pig (D'Angelo, 1955), Xenopus (Cordier, 1953; Saxen, Saxen, Toivonen and Salimiiki, 1957), two species of fish, Phoxinus phoxinus (Barring- ton and Matty, 1955) and Caecobarbus geerstii (Olivereau and Herlant, 1954) , and in the pars distalis of the fowl (Payne, 1944, 1949; Brown and Knigge, 1958; Mikami, 1957 ». From observations recorded by Her- R.L. P I. Fig. 3.8. Diagram of a horizontal section through tlie rat hypophysis showing the distribution of three types of basophil cell. P.D., pars distalis; P. I., pars intermedia; P.N., pars nervosa; R.L., hypophyseal cleft or residual lumen ; P.G., follicle- stimulating hormone cells (peripheral gonado- trophs); C.G., luteinizing hormone cells (central gonadotrophs) ; T.. thyrotrophs. lant (1954a) it seems that a differentiation of two kinds of basophil cells presumably thyrotrophs and gonadotrophs is generally observable in amphibians and bony fishes. H. THE THREE FUNCTIONAL TYPES OF BASO- PHIL CELL IN THE R.\T HYPOPHYSIS The identification of three functional types of basophil cells was first achieved in the rat by means which did not depend on specific staining reactions, the shape of the cells, their distribution and other minor features (Fig. 3.8). The pars anterior of the rat hypophysis has certain advantages which helped these identifications. Its hor- mone content and secretory function have been extensively investigated. It contains an adequate number of w^ell granulated basophils. Its small size allows its cell popu- lation to be evaluated under the micro- scope without an inordinate expenditure of time and the use of tedious cell enumeration techniques. Another favorable quality shown by the rat hypophysis is its reactivity. The hor- mone content can be varied over a wide range by experimental procedures, and vari- ations in secretion rate are accompanied by striking changes in the size and granule content of basophil cells. Especially con- spicuous are the hyalinized cells derived from basophil cells. Under conditions of rapid secretion there is an accumulation of a structureless protein solution called hya- line substance in certain small cytoplasmic vesicles. By distension and coalescence of these vesicles one or more large hyaline- filled spaces result. Evidence suggesting that the basophil cell class might consist of two distinct func- tional subdivisions, one concerned with thy- rotrophic activity and one with gonado- ti'ophic, was obtained from the study of the changes occurring after thyroidectomy and castration. In both these conditions an in- crease in the number and activity of cells of the basophil class are observed and the ini- tial cellular hypertrojihy is followed by hya- linization of a proportion of the basophills. The hyalinized cells appearing after thy- roidectomy (Figs. 3.9 and 3.10) are called thyroidectomy cells, those appearing after castration (Figs. 3.11 and 3.12) castration or signet ring cells. In the rat there are a HYPOPHYSEAL MORPHOLOGY 189 Fig. 3.9 {npixr left). Section of the rat pais anterior showing the eaily response of the thyrotrophs to thyroidectomy (6 days after thyroidectomy). The chister of large pale cells is formed bv thyrotrophs which have degranulated but have not vet begun to hyalinize. PAS, X 540. Fig. 3.10 (dipper right). Thyroidrctomy cells in the rat pars anterior 66 days after thyroid- ectomy. Extensive accumulations (if li>;ihne substance distort the cells. Normal specific gran- ulation is absent. The coarse, inteiisrly stained granules (T granules) are characteristic of pro- longed thyroxine deficiency states. PAS, X 780. Fig. 3.11 (loiver left). Section of the rat pars anterior showing changes in follicle-stimulating honuonc cells 10 wicks aft( i- laslration. The cells are numerous and large and a high propor- I Kin lia\ r arciiiiiulaird hyahiir iiiaicrial to form "signet-ring" cells. There is an increase in the i-diitciit (if granule- wliicli fdini cdarse, darkly stained clumps. PAS, X 580. Fi(i. 3.12 {lower right). Section of the rat pars anterior showing changes in luteinizing hor- mone cells 16 weeks after castration. A proportion of the cells have accumulated hyaline ma- terial and formed "signed-ring" cells. Both hyalinized and nonhyalinized cells show varying intensities of staining indicating considerable variation in granule content in different cells. There is often a higher content of granulation within the Golgi body than in the peripheral cytoplasm. The characteristic difference between the appearances of the granulation in fol- licle-stimulating hormone and luteinizing hormone cells is retained after castration. PAS, X 580. large number of consistent differences be- tween thyroidectomy and castration cells. Morphologic differences were observed by Hohlweg and Junkmann (1933), Zeckwer, Davison, Keller and Livingood (1935); Zeckwer (1936, 1937, 1938a, b), and by Giiyer and Clans (1935, 1937). A full ac- count of the thvroidectomv and castration 190 HYPOPHYSIS AND GONADOTROPHIC HORMONES TABLE 3.1 Differences between Castration and Thyroidectomy Cells in the Rat Castration Cells Thyroidectomy Cells Reference Vesiculation gives rise to "signet- Vesiculation less regular Schleidt (1914) ring" cells Vesicles are large, smooth, and well Secretory accumulation less regular Zeckwer et al. (1935) defined Early stages do not resemble the Early stages full of fine foamy vesicles Zeckwer et al. (1935) early stages of thyroidectomy cells coalescing to form larger ones at 14th to 18th day Not vesiciilated at 5 weeks Large amounts of hyaline material at 5 weeks Zeckwer (1938a, b). Uniformly granulated Poorly granulated Zeckwer (1938a, b). Suppressed by estrogen Not affected by estrogen Zeckwer (1938a, b). Generally a large single vesicle Usually multiple vesicles Guyer and Claus (1935, 1937) Cells usuall}' scattered Cells commonly grouped Guver and Claus (1935, 1937) Nuclei not vesiculated Nuclei commonly vesiculated Guyer and Claus (1935, 1937) Golgi body conspicuous and regular Golgi body diffuse Guyer and Claus (1935, 1937) Reese, Koneff and Cytoplasm forms a definite band Partitions between multiple vesicles around the vesicle are very fine and no band of cyto- plasm at the periphery Wainman (1943) Final size not great Final size very great Reese, Koneff and Wainman (1943) Shape compact, oval, or round Shape irregular, polyhedral Only a proportion of the cells hya- Practially all hyalinized Reese, Koneff and linized Wainman (1943) Nuclei and nucleoli not enlarged Nuclei and nucleoli enlarged and ve- Reese, Koneif and sicular Wainman (1943) Mitochondria smaller Mitochondria very large Reese, Koneff and Wainman (1943) cells was given by Reese, Koneff and Wain- man (1943). The recorded differences be- tween the two types of cells in the rat are summarized in Table 3.1. Castration basophils, at first only weakly staining, become more strongly staining in the period from 2 weeks up to 4 months from operation. In long term experiments one of the striking differences between cas- tration cells and thyroidectomy cells is the strong staining by aniline blue of the cyto- plasmic granules of the castration cells whereas the thyroidectomy cells take so little stain that they have been considered by many observers to be chromophobes. Zeckwer, Davison, Keller and Livingood (1935) were the first to trace the differences between these types of cells and to infer front them that these cells were the results of similar changes occurring in two distinct kinds of basophil cell. Guyer and Claus (1935; 1937), while observing differences in the cells, considered they were exjilicable as being different processes occurring in the same cell type. Severinghaus (1939) did not consider the changes produced by thyroid- ectomy different in any fundamental way from those produced by castration. He was no doubt misled by the fact that a certain number of "signet-ring" cells are always present (Wolfe, 1941, 1943) and arc there- fore found in small numl)ers among the thy- roidectomy cells in thyroidectomized ani- mals. The next important clue was pr()\i(l('(l by Reese, Koneff and Wainman ( 1943 ) who ob- sel•^•ed that two classes of basophil cells are normally present in rat hyi)ophyses, one oval or round in section (Figs. 3.13 and 3.14), the other polygonal and angular (Fig. 3.15). They noted that in the early response to thyroidectomy there is an increase in the iiiiiiibci' of cells which are irregular or i)oly- h('(hal, whereas aftei- castration the in- HYPOPHYSEAL MORPHOLOGY 191 Fig. 3.15 (upper Icjt). Section aldehyde-fuchsin (AF). The cells granules to be concentrated at the 1mi,. :;.1:] i '-/'/" - "< '-'idii of the mt pans aiUciiur showiug, iulliclf-.-liiuulating lior- mone cells stained by periodic acid-Schiff (PAS). The cells are ovoid in shape and con- tain clumps of densely stained granules. PAS, X 720. Fig. 3.14 (lower left). Section of the rat pars anterior showing luteinizing hormone cells stained by periodic acid-Schiff (PAS). ■M The cells have rounded con tours and the la basophil granules are more unil'orinly dis- Wa perscd throughout the cytoplasiu than in the ^ other two basophil types. One cell near the center of the field shows a prominent nega- tive image of the Golgi body. The cytoplasm enclosed by the Golgi body has a higher ■* ' granule content that the peripheral cyto- plasm. PAS, X 720. of the rat pars anterior showing thyrotrophs stained by have angular contours and there is a tendency for the periphery. AF, X 720. created number of basophils is due to an in- crease in conijiact cells which are oval or round. The McManus (1946) PAS reaction for the demonstration of glycoproteins in histo- logic sections initiated researches based on the idea that this reaction might demon- strate the hormone content of basophil cells. Herlant (1949, 1951) and Pearse (1949, 1951 ) ascribed the reaction given by baso- phil granules to the presence of gonado- trophin. Catchpole (1949) found in castra- tion cells and in certain basophil cells of normal animals glycoproteins which could have been gonadotrophins. He looked for but did not find glycoprotein with the solu- bility of thyrotrophin in thyroidectomy cells. He considered that the glycoprotein staining did not parallel the staining with aniline blue. Purves and Griesbach (1951a) set out to test the hypothesis that the PAS reaction could. be used to demonstrate the hormones of basophil cells by examining rat hypophy- ses in which thyrotrophin or gonadotrophin or both were absent. Such hypophyses were obtained by treatment of intact rats with thyroxine or estrogen or both thyroxine and estrogen. In hypophyses that contained only thyrotrophin the glycoprotein reaction and the aniline blue-stained granules were con- fined to cells that were polyhedral in shai:)e and were distributed throughout the interior of the pars anterior. In hypophyses contain- ing only gonadotrophins the glycoprotein reaction and the aniline blue-stained grnn- 192 HYPOPHYSIS AND GONADOTROPHIC HORMONES ules were confined to cell^ that were ovoid or spherical. The most conspicuous staining was in cells in the periphery of the pars anterior, especially on the inferior surface and at the anterior margin. In hypophyses which con- tained neither thyrotrophin nor gonado- trophin basophil cells could not be demon- strated by either the glycoprotein reaction or aniline blue. These results showed that the different hormones were stored in the granules of dif- ferent cells and that the hormone content was proportional to the granule content. Any staining method which demonstrated the granules would give the same result and, had the Dempsey and Wislocki (1945) sug- gestion that aniline blue stained the hor- mones been followed up, results similar to those obtained by the PAS reaction would have been obtained. The PAS reaction pro- vided a more specific staining for basophil granules; its more important role, however, was the psychologic stimulus it gave to in- vestigations relating staining to hormone content because there was a chemical reason for supposing that this reaction would dem- onstrate the content of thyrotrophin and gonadotrophins. Purves and Griesbacli < 1951a) gave the name "thyrotrophs" to the cells whose gran- ules contain thyrotrophin and the name "gonadotrophs" to the cells whose granules contain gonadotrophins. Halmi (1951a, b) applied Gomori's (1950) aldehyde-fuchsin to the staining of the i)ars anterior and obtained the dis- tinction between yS-cells and 8-cells that had previously been obtained by Romeis (1940). It was soon established that the ^-cells of the rat pars anterior are thyrotrophs (Purves and (h-iesbach, 1951b; Halmi, 1952a). Restaining iJroccdincs demonstrate that tbe same cytoi)lasmic granules are revealed by aldehyde-fuchsin, ])hosphotungstic acid- aniline l)lue, and the histochemical reaction for glycoprotein. The specific staining of the thyrotrophs is therefore a specific staining of thyrotrophin-containing granules. In rats wliich have been treated with thyroxine the granulation is reduced to such a low level that the thyrotrophs cannot be demon- strated by any of the staining i)roce(hn'cs (Purves and Griesbach, 1951c; Halmi, 19511), 1952b). In rats which have been thy- roidectomized there is a rapid discharge of thyrotrophin which reduces the granule con- tent in the cells to a low level. There is simultaneously a marked increase in the number of functioning thyrotrophs and an increase in the size of the individual cells. The total hormone content of the gland is affected in opposite directions by these two effects, and may not be greatly different from normal despite a low concentration of the hormone in the cell cytoplasm. Under these conditions staining by aldehyde- fuchsin is not obtained although the cyto- plasm and the hyaline substance are still stainable by other methods. The thyro- trophs, therefore, after thyroidectomy are easily confused with 8-cells although they are still cleary differentiated from 8-cells by their characteristic shape (Purves and Griesbach, 1951b). The specific staining of thyrotrophin-con- taining granules by aldehyde-fuchsin con- stitutes a notable advance in the study of the functional aspects of pituitary mor- phology. The differentiation of thyrotrophs from other cells by Halmi's (1951a) method clarifies not only the study of thyrotrophic function, but also that of gonadotrophic functions in the rat pituitary because the two types of basophils concerned with the secretion of the gonadotrophic hormones ap- pear as 8-cells which can be studied with- out confusion with thyrotrophs. It was soon apparent (Purves and Gries- bach, 1952; Si{)erstein, Nichols, Griesbach and Chaikoft', 1954) that the gonadotrophs in the rat pars anterior are of two types. The peripheral gonadotrophs near the sur- face of the anterior lobe and especially con- centrated on the inferior surface and at the anterior border show a consistently different a])pearance from the i)aler rounded cells whicli are scattered throughout the interior and arc the only basophils adjacent to the l)ai's intci'niedia (Fig. 3.9). This suggests the hypothesis that one form secretes FSH and the other LH. The central gonadotrophs are the more active- looking cells after castration and during pregnancy. There was therefore a conflict l)etween the at one time current view that after castration the secretion is predomi- nantly follicle-stimulating and the ])roba- HYPOPHYSEAL MORPHOLOGY 193 bility that the hormone secretion during pregnancy is predominantly luteinizing. Af cer a year of investigation it was realized that the solution lay in getting rid of one of the hormones. Treatment of the adult female rat with testosterone propionate had been shown to increase the gonadotrophin content of the hypophysis and to produce a gland which contains FSH without any admixture of LH (Laqueur and Fluhmann, 1942; Greep and Chester Jones, 1950). Purves and Griesbach ( 1954) found that in female rats treated with 250 fxg. of testos- terone daily for 1 week there was a large increase in the numbers of gonadotrophs of both types which could be stained by the glycoprotein reaction. After 3 weeks treat- ment, the pale central gonadotrophs were found diminished in size and showed signs of diminishing activity. After 4 weeks treat- ment, the central gonadotrophs had prac- tically disappeared whereas large numbers of the peripheral type with a strong cyto- plasmic glycoprotein storage remained. These peripheral gonadotrophs did not seem to be actively secreting because the cells themselves were small. These observations seem to indicate with certainty that the peripheral type of gona- dotroph is exclusively the site of formation and storage of FSH, whereas the central type is probably concerned with the forma- tion and storage of LH. It has not yet been possible to obtain rat hypophyses containing LH without FSH. In glands containing large amounts of LH there are always large numbers of richly granulated central gonadotrophs and there is no reason to think that the granules of these cells contain any other hormone. The cells that secrete and store within their granules FSH are called FSH cells; the cells that secrete and store within their granules LH are called LH cells. As yet, no consistent staining difference between the FSH granules and the LH gran- ules has been obtained. The Wilson and Ezrin (1954) technique differentiates be- tween thyrotrophs and gonadotrophs, the former being PAS-red whereas the latter tend to be PAS-purple. The granules of the thyrotrophs have little affinity for acid dyes and do not take up either the orange G or methyl blue applied as counterstains to sec- tions which have first been stained by the PAS reaction. Their color is therefore a ma- genta red. Cells with a strong affinity for acid dyes take up orange G and hold it firmly. Strongly acidophilic basophils (am- phophils) of this kind are found among the /3-cells of the human pars distalis; they ap- pear brick-red after the Wilson and Ezrin staining method, the color resulting from the addition of orange G to the PAS color. These cells are also called PAS-red by Wilson and Ezrin. The difference in the two shades of red is visible in Figures 1 and 2 of Wilson and Ezrin's (1954) paper. In basophil cells with an intermediate degree of acidophilia the orange G which is taken up first is dis- placed more or less rapidly by methyl blue in the next step of the procedure. These cells therefore appear purple. The gonadotrophs of the rat have an intermediate degree of acidophilia and may be stained PAS-purple, but the colors obtained are not independent of the amount of granulation present. In this technique there is a tendency for strongly granulated cells to retain the orange G longer and to appear PAS-red whereas in lightly granulated cells the orange G is more rapidly replaced by methyl blue and the cells appear PAS-purple. The color differ- entiations obtained by Rennels (1957) and by Hildebrand, Rennels and Finerty (1957) resulted from this effect. In the normal rat the FSH cells are more densely granulated than the LH cells and can be stained PAS- red while the LH cells are stained PAS-pur- ple, but when densely granulated LH cells appear in long term castrates they too are PAS-red. Ten days after castration when both FSH cells and LH cells are only lightly granulated both stain PAS-purple. I have not found any color differentiation be- tween FSH cells and LH cells containing a similar amount of granulation. The conclu- sions of Hildebrand, Rennels and Finerty concerning the distribution of FSH cells and LH cells are therefore not likely to be cor- rect. Despite the absence of specific staining, the granules of FSH cells and LH cells can be shown to be different by solubility tests. I have made a considerable number of tests of this kind. The best results have been ol)tained by perfusion of long term castrate 194 HYPOPHYSIS AND GONADOTROPHIC HORMONES rats with an ether-saturated isotonic acetate buffer at pH 4.5 or 5. Prussian blue has been added to the perfusion medium so that the completeness of the perfusion can be checked. Under these conditions the glyco- protein of the granules of FSH cells was almost entirely extracted whereas that of the granules of LH cells was to a large ex- tent retained. Assays showed that the buffer extraction removed the FSH and some of the LH; the extracted glands contained LH without FSH. Barrnett, Ladman, McAllas- ter and Siperstein (1956) obtained a similar distinction between the granules of FSH and LH cells by immersing hypophyses in 2.5 per cent trichloracetic acid. This treat- ment removed most of the glycoprotein from FSH cells and preserved the glycoprotein of LH cells intact. Assays showed a loss of FSH and a preservation of LH. I interpret these observations as indicat- ing that basophil granules, like other secre- tion granules, contain a mixture of proteins and that the glycoproteins of LH-cell gran- ules are less soluble than those of FSH- cell granules. The loss of thyrotrophin and FSH from whole rat hypophyses immersed in 2.5 per cent trichloracetic acid must be ascribed to inactivation rather than to com- plete extraction, but it may well be that the preservation of LH under these con- ditions is related to its insolubility in 2.5 per cent trichloracetic acid. It should not be in- ferred however that all glycoproteins either insoluble in, or rendered insoluble by tri- chloracetic acid are LH. The conclusion of Barrnett, Ladman, McAllaster and Siper- stein that FSH often occurs in conjunction with LH should be regarded as unproven. I. THE PARS ANTERIOR OF THE BAT WITH SPECIAL REFERENCE TO TWO TYPES OF GONADOTROPHS The pars anterior of the hypoi:)hysis of the bat {Myotis '>nyotis) has been studied by Hcrlant (1956a). Five types of cells are distinguished by staining reactions. Two are acidophils and three basophils. Aided by the favorable staining properties of the cells and by the unusual nature of the repro- ductive cycle, Hcrlant has been able to mak(> identifications of FSH cells and LH ('(■Us in the bat that carry more conviction than the id(nitifications made in tiie rat. The distinctive staining of the granules of FSH cells and LH cells in the bat makes it possible to assert that the two types are quite separate, and do not change from one type to another at different times. The two acidophil types in the bat are: (1) Orangeophils. These are relatively sta- ble throughout the reproductive cycle and are presumed to secrete somatotrophin. (2) Carminoi)hils. These are concentrated in the anteromedian zone. They are in evidence at the time of ovulation, become less prominent towards the end of pregnancy and show in- tense activity accompanied by a high con- tent of cytoplasmic ribonucleic acid in ani- mals that lactate after parturition. They are considered to secrete prolactin. The three basophil types are: (1) Irregu- larly shaped basophils scattered through the par anterior, staining blue by trichrome methods and with a marked affinity for aldehyde-fuchsin. This type which is rela- tively stable throughout the reproductive cycle is provisionally identified as a thy- rotroph. Its response to disturbances of thyroid function has not been tested. (2) Basophils concentrated in the antero- median zone, staining blue by trichrome methods and with a slight aflfinity for alde- hyde-fuchsin. These elements are large and well granulated at the time of estrus in the autumn and continue to show an active ap- pearance throughout the period of hiberna- tion when ripe follicles are in the ovaries. They involute after ovulation in the spring and remain involuted during pregnancy. These cells are identified by Herlant as FSH cells. (3) Basophils in the i)ostcrior two-thirds of the pars anterior which show a marked tendency to retain the red component of tri- chrome staining methods and are therefore violet or puri:)le but not blue after such pro- cedures. They are brick-red in sections stained by PAS and counterstained with orange G, whereas the other two basophil types which do not stain with orange G are magenta. These cells are greatly hyper- trophied through i)regnancy but undergo |)rompt involution after parturition regard- less of whether lactation follows. Herlant identifies these cells as LH cells and this identification is in accord with the marked activity of LH cells during pregnancy in HYPOPHYSEAL MORPHOLOGY 195 the rat. By immersing bat hypophyses in 10 per cent trichloracetic acid for 12 hours before fixation Herlant found that the gran- ules of FSH cells were soluble, those of the LH cells insoluble in this medium. IX. Corticotrophin : the Problem of Its Origin A. BASOPHIL CELLS AND CORTICOTROPHIN Many attempts have been made to re- late corticotrophin secretion to one or other of the specific cell types demonstrable by staining reactions, but the results have generally been inconclusive. The distribu- tion within the anterior lobe of species which show distinct zoning is instructive. Smelser (1944) showed that corticotrophin was more concentrated in the basophil zone of the bovine pars anterior than in the acidophil zone. In the pig pars anterior, in which the zones are much more distinct, I have found that corticotrophin is present in portions of the anteromedial basophil zone that are apparently free from acidophil cells, the concentration there being 2 to 4 times that in the posterolateral acido})hil zone. The assays were made by the ascorbic acid depletion method. Giroud and Mar- tinet (1948) found that the adrenal weight- increasing action is predominantly situated in the basophil zone. Rochefort and Saffran (1957) found 4 to 13 times as much corti- cotrophin in the anteromedial basophil zone of pig and beef hypophyses as in the posterolateral parts of the acidophil zone. Certainly in both cow and pig the corti- cotrophin is not in acidophil cells. In the cow it may be in either basophils or chromo- phobes, but in the pig it seems certain that it must be in some type of basophil cell, because there are few chromophobes in the pig pars anterior and a large part of the basophil zone is free of them ancl consists of basophil cells only. Halmi and Bogdanove (1951) and Hess, Slade, Amnions and Hendrix (1955) found that the loss of acidophil granules in the thyroxine-deficient rat was not accom- panied by any change in the content of corticotrophin in the hypophysis, nor was there any change in corticotrophin output. This indicates that the hormone is not in the acidophil granules in this species and is probably not formed by acidophil cells. One indication of the possible nature of the granules to be expected in corticotro- phin-secreting cells may be made from the the chemical nature of their hormonal prod- uct. This polypeptide has some relationship to intermedin, and the amino-acid sequence — methionine, glutamic acid, histidine, phenylalanine, arginine, tryptophan, gly- cine— is common to both (Landgrebe and Mitchell, 1958) . The presence of this struc- ture in corticotrophin may account for its having some intrinsic melanin-dispersing properties. The intermedin-secreting cells of the pars intermedia may in some species have enough specific granulation to dis- tinguish them from chromophobes, but when granules are present they are basophil gran- ules containing glycoprotein and are stain- :ible with aldehyde-fuchsin. We might expect, therefore, that if corticotrophin- secreting cells contain granules they will be basophil granules, although it is not necessary that they should contain such granules. The origin of corticotrophin from baso- phil cells has gained a certain degree of acceptance based on the association of baso- phil cell adenomas with the Gushing syn- drome in man. The evidence now is that these basophil cell changes are the result of the action of excess adrenal steroids and are not the cause of the syndrome even in those cases which seem to be due to ex- cessive secretion of corticotrophin. This aspect of the subject is dealt with more fully in the later section on Gushing's syndrome. The attempt by Marshall (1951) to dem- onstrate the presence of corticotrophin in basophil cells by the use of labeled antibody must be discussed, because it has been re- garded by some as a conclusive demonstra- tion of the relation of this hormone to baso- phil cells. The antibody labeled with a fluorescent grouping demonstrated the pres- ence in basophil cells of an antigen which had been contained in the crude corti- cotrophin preparation used. There is, how- ever, nothing to associate the antigen so demonstrated with the corticotrophin. Such protein-containing preparations of corti- cotrophin seem to contain many more molecules of protein than of corticotrophin, 196 HYPOPHYSIS AND GONADOTROPHIC HORMONES and the antigen could therefore have been one of the glycoprotein hormones known to be antigenic, or some nonhormonal con- stituent. The absence of evidence of anti- genicity in corticotrophin would make the method inapplicable for this hormone unless it could be shown that the hormone w^as associated in the gland with a specific pro- tein. There is no evidence so far that this is the case, because under certain circum- stances the hormone is dialyzable from crude pituitary extracts (Tyslowitz, 1943; Geschwind, Hess, Condliffe, Evans and Simpson, 1950j , and in the extract it seems to be distributed indiscriminately among most of the proteins present (Astwood, Raben and Payne, 1952). In the rat three types of basophil cells have been identified, and the question arises whether one of these secretes corticotrophin in addition to its specific glycoprotein hor- mone. The hypothesis that thyrotrophs may secrete corticotrophin has attracted some attention and has been subjected to specific investigation. Halmi and Bogdanove (1951) concluded that corticotrophin was not pro- duced by thyrotrophs in the rat. In the pig hypophysis I have found, as Smelser (1944) found in the bovine hypophysis, that the distribution of the hormones between the basophil and acidophil zones is quite differ- ent for corticotrophin and thyrotrophin, the latter being almost exclusively in the baso- phil zone. This seems to make it certain that the same cell does not produce these two hormones. The problem of the origin of corti- cotrophin thus seems to resolve into a choice between two alternatives: either corti- cotrophin is made by gonadotrophs, or it is made by an aditional specific cell type, the "corticotroph." Inasmuch as five spe- cific cell types have been identified in the pars anterior of the rat and the bat, the search for an additional cell type which could be the corticotroph may be referred to as the search for a sixth cell type. In- vestigations aimed at the solution of this problem usually take the form of examin- ing hypophyses in which corticotrophin se- cretion is proceeding at an abnormally rapid rate, and looking for cellular responses either in sections stained by methods which reveal acidophils and basophils or in sec- tions stained by unconventional methods which might stain the granules of a sixth cell type not revealed by conventional methods. B. HYPOPHYSEAL RESPONSES TO ADRENAL ABLATION Many reports of the cytologic changes observed after bilateral adrenalectomy have been published. Some observers have not re- marked on any extensive alteration. Thus, Nicholson (1936) did not observe any change in the pars anterior of dogs after bilateral adrenalectomy, and Koneff, Holmes and Reese (1941) found that in rats to which sodium chloride was administered after adrenalectomy, cytologic changes were minimal. Houssay (1952) also remarked that in strains of rats which survive in good condition after adrenalectomy there is little hypophyseal disturbance compared with those which have severe insufficiency symp- toms. It therefore seems that the increased secretion rate or the depletion of the adreno- corticotrophin content of the hypophysis which follows adrenalectomy need not be accompanied by any marked change in the appearance after staining by the customary methods. Certain changes in the acidophil and basophil cells which are observed in certain strains of rats after adrenalectomy, if ad- ditional salt is not administered, must be related to the extensive metabolic disturb- ances produced by the adrenal insufficiency. These changes have been described by Reese, Koneff and Akimoto (1939) . Changes in the acidophil cells consist of degranula- tion with a diminution in the size and number of cells. The Golgi body in these regressing acidophils is transformed from the usual acidophil form which envelops half the circumference of the nucleus into a concentrated spherical or oval body. These changes indicate decreased activity. Cells of the basophil class also show exten- sive degranulativc changes with reduction in size and number, but some basophil cells with enlarged Golgi bodies are seen, espe- cially at short intervals after adrenalec- tomy. Tuchmann-Duplessis (1953) also found that, although most of the basophil cells undergo degenerative changes after HYPOPHYSEAL MORPHOLOGY 197 adrenalectomy, there remain always a few active normal-looking cells. Colombo (1948, 1949) reported in rats, after bilateral or unilateral adrenalectomy, a change in baso- phil cells similar to that occurring after castration and not accompanied by any change in acidophil cells. Griesbach (personal communication) ob- served that the LH cells were enlarged after adrenalectomy in the rat. There were also changes in thyrotrophs similar to those produced by exposure to cold. The changes in the thyrotrophs were prevented by thy- roxine administration. Knigge (1957) found the thyrotrophs diminished in numbers after adrenalectomy. The gonadotrophs (8-cells of Halmi) were unchanged in numbers but were hypertrophied, and some of them were hyalinized 8 weeks after adrenalectomy. Knigge suggested that the 8-cells were the source of corticotrophin. Rokhlina ( 1940) tested the effects of com- bining adrenalectomy with castration in rabbits and rats. In some rats thus treated no castration changes appeared in the baso- phil cells, whereas in others the transforma- tion was delayed. Adrenalectomy performed on the 30th day after castration had some ef- fect on the further develojimcnt of castration changes. Brokaw, Briseno-Castrcjon and Finerty (1950) performed unilateral adrenalectomy on rats. By this means a relative adrenal insufficiency should be produced without the extensive metabolic disturbances accom- panying complete adrenal deficiency. They observed a temporary increase in the pitu- itary acidophil cell percentage; the normal pituitary cytology was regained in approxi- mately 50 days. Herrick and Finerty (1940) found in adrenalectomized fowls an alteration in basophils in the pars distalis, which they correlated with the regression of the testes. The basophils showed progressive vesicula- tion, the vesicles being filled with hyaline material. They considered these basophils to be in a degenerating state. IMikami (1957) observed a degeneration of both thyrotrophs and gonadotrophs in the fowl after adrenalectomy. In addition there was a degranulation and remarkable enlarge- ment of a third basophil type in the rostral zone of the pars distalis. These cells, desig- nated by Mikami "V cells," were considered to secrete corticotrophin. In the dog, Mikami (1956) found that the ^cells of Goldberg and Chaikoff (1952a) increased in number and enlarged in size after adrenalectomy. These were the only cells showing signs of increased activity after adrenalectomy. The ^-cells of Gold- berg and Chaikoff are the cells termed "pale" cells by Purves and Griesbach (1957a) and are basophil cells with a low content of granules. C. HYPOPHYSEAL RESPONSES TO STRESS An increased secretion of corticotrophin is produced in response to many different kinds of stress. Inasmuch as adaptation to various kinds of stress wall involve changes in a number of hormones, the hypophyseal responses to stress will be less suitable for the study of corticotrophin production than the specific disturbances produced by adre- nal insufficiency. Thus, the response to cold exposure involves increased secretion of thyrotrophin as well as corticotrophin, and the increased activity in the basophil cells of the rat exposed to cold have been related thyrotro{)hin production rather than corti- cotrophin (Brolin, 1945; Allara, 1953; ]McXary, 1957). In rats exposed to cold, Griesbach, Hornabrook and Purves (1956) observed partial degranulation of thyro- trophs and early hyalinization giving rise to cells with a resemblance to Crooke's cells. This appearance is related to alteration in thyrotrophin secretion and can be pre- vented by thyroxine injections, but not by replacement with cortisone. An increased granule content in basophil cells in the rat during inanition has been re- lated to an increased storage of gonadotro- phin (Pearse and Rinaldini, 1950). The in- crease in stainable basophils observed in the hypophyses of animals subjected to various forms of stress may, therefore, be related to changes in the content of the gly- coprotein hormones which are the specific secretory products of these cells. Herlant (1936a, b) observed that the increase in stainable basophils in the rat hypophysis after ligation of the ureters or after the in- jection of hydrochloric acid, was accom- panied by an increase in the gonadotrophic potency of the pituitary tissue. 198 HYPOPHYSIS AND GONADOTROPHIC HORMONES Finerty and Binhammer (1952» and Finerty, Hess and Binhammer (1952) stud- ied the early responses of the hypophysis to the acute stress of severe burns in the rat. No changes were observed in the differential cell counts nor in the degree of specific gran- ulation of the cells stained by the azan method. There was, however, an increase in the number of acidophil cells per field in sections stained by the acid hematein method of Rennels (1951). Timmer and Finerty (1956) supplied the explanation of this discrepancy. The results of azan stain- ing were expressed by differential cell counts and did not show any change. The results of the acid hematein staining were expressed as cells per field, and the increase in this number was the result of a shrinkage in the gland which occurred after scalding. Thus, although there were no more acid hematein stained cells in the hypophysis, they were closer together after scalding. Knigge (1955) found, in thyroidecto- mized animals in which acidophil granules were absent, that there was no acid hema- tein staining before or after scalding. D. THE SIXTH CELL TYPE The pars anterior of the rat is particu- larly reactive to changes in the rate of hor- mone secretion, and the absence of any striking change in the stainable cells after adrenal ablation suggests that corticotrophin may be secreted by chromophobes in this species, or that corticotrophin is secreted by a different mechanism from that respon- sible for the secretion of other anterior lobe hormones. The discovery by Farquhar (1957) of a sixth cell type in the pars an- terior of the rat may provide an explanation of these peculiarities. These cells, which can be clearly seen only by electron micros- copy, are very different from any of the other five types. The cytoplasm is rela- tively empty and contains few formed ele- ments (mitochondria, endoplasmic reticu- lum). Secretory granules are absent. A distinctive feature of this additional cell type is its location throughout tiie an- terior lobe in groups around follicles or ductules which contain colloid of low den- sity. Some follicles are large and undoubt- edly are analogous to colloid cysts, but small follicles which jn-obably eould not be distinguished by light microscopy are much more numerous. The cells lining the follicles have angular contours, and the nucleus is eccentrically located. The amount of colloid seems to vary with the amount of corticotrophin in the gland, being increased after cortisone injection and de- creased after partial adrenalectomy. No marked response of the cells to the stimula- tion of partial adrenalectomy was observed. The identification of this sixth cell type as a corticotroph can only be regarded as ten- tative. Farquhar suggested that corticotro- phin may be stored in the form of colloid and released by some mechanism similar to that by which thyroid hormone is mobilized from the colloid of the thyroid follicles. This would explain the difficulties in relat- ing corticotrophin secretion or storage to changes in the granulated cells observed by the light microscope. My own observations show that this cell often has an extremely irregular shape with long and tenuous cytoplasmic projections extending between adjacent granulated cells or deeply indenting their cytoplasm. The cross section of the enclosed space is often elongated, suggesting that this cavity often takes the form of a cleft rather than a fol- licle or tubular ductule. X. The Pars Intermedia and Intermedin Secretion Intermedin is an adenohypophyseal hor- mone, distinct from the other adenohypo- physeal hormones. This is evident from the fact that it is formed in a different site. In addition to causing dispersion of pigment in mclanophores, intermedin acting over a long time also stimulates the formation or deposition of melanin. Intermedin is se- creted by a specific cell type in the adeno- hyjiophysis. The recognition of this specific tyjie is simple in most vertebrates because the cells are concentrated into a zone, the pars intermedia. Evidence of the specific hormone content of the pars intermedia was first obtained by Smith and Smith (1923b) in tests of the different regions of the beef hypophysis, and has been confirmed by Lewis, Lee and Astwood (1937), and r.iroiid and :\Iartinet (1948). Intermedin seems to be located in a small HYPOPHYSEAL MORPHOLOGY 199 granule fraction in sucrose solution homog- enates of pars intermedia tissue, because it sediments with the microsomal fraction on centrifugation (Jeener and Brachet, cited by Herlant, 1952a). In this respect it be- haves like the hormones secreted by baso- phil cells in the pars anterior. Evidence of specific secretion from pars intermedia tissue is provided by the inter- esting studies of Allen (1930) and Etkin (1941) in transplantation experiments in tadpoles. Their results indicate that sever- ance of the stalk connection with the hypo- thalamus removes an inhibiting influence from the pars intermedia cells, and that the resultant hypertrophy and hyperplasia is accompanied by oversecretion of intermedin which produces a permanent blackening of the animals. Etkin (1958) found that the capacity of the pars intermedia to differ- entiate and secrete was not prevented by transplantation of the epithelial primor- dium of the pituitary to the tail bud of the wood frog where it developed away from any neural tissue. It was, however, in con- tact with the neural epithelium before trans- plantation and before Rathke's pouch had begun to form. Copeland (1943) observed in Triturus viridescens that the differentiation of the pars intermedia occurs immediately before the first metamorphosis, and at this time the characteristic pigmentation of the red eft stage begins to appear. The pars intermedia, unlike the pars ner- vosa, does not undergo degeneration after stalk section in mammals (Rasmussen and Gardner, 1940; Brooks, 1938; Stutinsky, Bonvallet and Dell, 1950; Barrnett and Greep, 1951). Indeed, a hypertrophy of the pars intermedia is usually observed with signs of cellular activation. Fisher (1937) demonstrated that intermedin is still present in the cat hypophysis after the pars nervosa has degenerated after stalk section. The intensity of specific staining of pars intermedia cells varies with the species. This variation is the result of variation in the cjuantity of specific granulation. The granulation when present appears to contain glycoprotein, because it gives a positive PAS reaction and is stainable by aldehyde-fuch- sin without prior oxidation. The glycopro- tein character of the intermedia cell granu- lation has been demonstrated in the bat by Herlant (1956a) and in the frog by Ortman (1954, 1956c). In both species the granules are stained by aldehyde-fuchsin. I have ob- served the same staining reactions in the granules of intermedia cells in the cat, dog, sheep, deer, and rat (Fig. 3.16). In the rat the glycoprotein reaction and the aldehyde- fuchsin staining are negative after extrac- tion by perfusion with saline saturated with ether or by immersion in a neutral buffer after acetone treatment. Intermedia cells are, therefore, typical basophil cells contain- PI HC PA cells by aldehyde-fuchsiu (AF). The colloid of the hypophyseal cleft is not stained. PX, pars nervosa; PI, pars intermedia; HC, hypophyseal cleft; PA, pars anterior. Formol sublimate fixation, AF X 43. Fig. 3.17 (light). Section of the rat hypophysis showing the alteration of the staining prop- erties of the pars intermedia by fixation in Helly's fluid. Except for a few coarse granules of unknown nature, the pars intermedia cells are almost unstained in comparison with Figure 3.16. Key to lettering as in Figure 3.16. Helly, AF, X 150. 200 HYPOPHYSIS AND GONADOTROPHIC HORMONES ing granules with a content of soluble glyco- proteins. In the rat the granules are much more strongly stained by aldehyde-fuchsin than by PAS, and show an additional differ- ence of behavior from the basophil granules of pars anterior cells in that their staining by aldehyde-fuchsin is not enhanced by prior oxidation with acid permanganate (Halmi and Davies, 1953). The granules of intermedia cells stain a blue or purple color by trichrome staining methods (Romeis, 1940). In the cat, in which the staining reactions of intermedia cells are strong due to a high content of granulation the appearance of intermedia cells in stained sections is similar to that of typical pars anterior basophils. The relation between the specific granules and the hormone secretion of the pars inter- media cells is still obscure. The hormone can be obtained as a peptide, but it may be that a combination of this peptide with the gly- coprotein is the form in which the hormone is first produced and stored. Ortman (1954, 1956c) found in the frog that the glycopro- tein granules of the pars intermedia cells were depleted during dark adaptation, but did not find any accompanying reduction in hormone content. Not all hypophyses have a pars inter- media, although so far as is known all con- tain intermedin. In birds as a class, and in certain mammals, porpoise, whale, arma- dillo, manatee, elephant, pangolin, beaver, and man, the pars intermedia is absent. The problem of the cellular origin of the inter- medin in hypophyses of this type is of great interest because of indications that one of the basophil cell types of the human pars distalis is the intermedin secretor of this species. In the porpoise and whale inter- medin is present in the tissue of the adeno- lobe (Oldham, Last and Gelling, 1940), and although the specific cells which produce the hormone have not been identified, it must be assumed that they are present, scattered throughout the pars distalis. A similar dis- tribution of intermedin was shown in the beaver by Kelsey, Sorenson, Hagen and Clausen (1957). In birds the intermedin is found only in the rostral portion of the adcnolol)c (De Lawder, Tarr and Gelling, 1934; Mialhe-Voloss and Benoit, 1954). In hen and duck hypophyses I have found aldehyde-fuchsin positive basophil cells in the rostral zone of the pars distalis, but the correlation with the distribution of the hor- mone is obscured by the presence of other aldehyde-fuchsin positive cells in the caudal zone. In the white-crowned sparrow [Zono- trichia leucophrys gambellii) aldehyde- fuchsin positive basophils are found only in the rostral zone. Traces of thyrotrophin are present; the concentration is the same in both zones. It may be that the aldehyde- fuchsin positive cells of the rostral zone are the intermedin secretors. Much more definite information about the human hypophysis is available from the studies of ]\Iorris, Russell, Lanclgrebe and Mitchell (1956). These investigators cor- related the hormone content of small por- tions of tissue from the anterior lobe, neural lobe, and areas of basophil cell invasion in the neural lobe, with the numbers of baso- phil cells present. The selection of appro- priate parts was made by the use of an elegant method in which the presence of basophil cells could be ascertained by ex- amining a pinhead-sized fragment. The re- sults showed that intermedin was present in high concentration in the anterior lobe, whereas it was almost entirely absent from neural lobe tissue. Neural lobe tissue show- ing invasion by basophil cells was, however, equivalent in hormone content to samples from the anterior lobe. The invasion of the neural lobe by basophilic cells, which is a phenomenon peculiar to the human hy- pophysis, has permitted here the demonstra- tion that cells containing glycoprotein se- crete intermedin. For the identification of the specific type of cell responsible for inter- medin secretion in the human pituitary, we can take advantage of the fact that although a number of basophil cell types can be seen in the pars distalis, the cells that invade the n(>ural lobe are of a single tyj^e. Thej^ are composed entirely of a variety of Romeis' (1940) /?-cells which stain with aldehyde- fuchsin and take a red oi' pui|)l(' shade from the trichrome counter stain. These cells will be designated as "purple ^-cells." Morris, Russell, Landgrebe, and Mitchell (1956) referred to these invading cells as 8-cells; they are, however, quite distinctly HYPOPHYSEAL MORPHOLOGY 201 characterized as /^-cells by Romeis. The S-cells of Romeis do not invade the neural lobe. The assays of Morris and his associates were concerned with intermedin and cor- ticortrophin, and show that the invading basophil cells were not responsible for corti- cotrophin production. Although no informa- tion is available concerning the other hor- mones, it is virtually certain that the cells which secrete intermedin in the human will, like the cells secreting intermedin in other vertebrates, be responsible for this secretion only. In man this intermedin secretor is heavily granulated and stains strongly by PAS or by trichrome methods, as does the pars intermedia cell of the cat. It is par- ticularly liable to retain the red stain of the trichrome method, a tendency which is greatly strengthened after fixation in Helly's fluid. Herlant (personal communication) has found that the staining of ^-cells in the human pars distalis by aldehyde-fuchsin is greatly diminished or even fails entirely after fixation in Helly's fluid. This be- havior of the intermedin secretors of the liuman pars distalis is also found in the cells of the pars intermedia of the rat, which stain intensely with aldehyde-fuchsin after formol-sublimate fixation but stain very weakly after Helly fixation (Fig. 3.17). It has not been determined whether this be- havior is a general one for intermedin- secreting cells. In the human pars distalis the inter- medin-secreting basophils are the most con- spicuous of the basophil cells. Their preva- lence is consistent with the high intermedin content of the human hypophysis (Land- grebe and Mitchell, 1958). Their failure to respond in sympathy with disturbances of gonadotrophin and thyrotrophin secretion, which has apparently set the human pitui- tary apart from those of experimental ani- mals, need no longer be a stumbling block now that the nature of these conspicuous basophilic cells is recognized. Obviously the source of thyrotrophin and gonadotrophin must be sought in glycoprotein-containing cells other than the intermedin secretors. It is possible that some species which have a discrete pars intermedia may also have some dispersed pars intermedia cells in the pars anterior. Landgrebe, Ketterer and Waring (1955) have referred to such an invasion of the pars anterior in some breeds of pigs. XI. The Pars Tuberalis The pars tuberalis consists of a layer of adenohypophyseal cells which surrounds the neural stalk and covers the surface of the median eminence of the tuber cinereum. It is composed almost entirely of cells of a single type which, from the absence of specific granules of the kind seen in anterior lobe cells, are classed as chromophobes. There are, in addition, small numbers of typical basophil cells which, according to Romeis (1940), do not differ in any respect from the basophil cells of the anterior lobe. Acidophil cells apparently identical with the cells in the anterior lobe are extremely rare. In the larger animals, the pars tuberalis cells are arranged in cords and follicular structures ; the latter enclose small amounts of colloid. No specific hormone has been demonstrated in the pars tuberalis other than small amounts of activities probably due to contamination with adjacent anterior lobe or pars intermedia tissue. The function of the pars tuberalis is at present unknown. It may be that it is the source of some trophic influence which is carried to the anterior lobe by the hypophyseal portal system. XII. Cytologic Changes Accompanying Secretory Responses Concerned with Reproductive Function A. SEXUAL MATURATION IN THE RAT Sexual maturation in the male rat is ac- companied by a gradually increasing con- tent of glycoprotein in the gonadotrophs. In the female, however, the reaction is en- tirely different. Maturation is accompanied by a considerable degranulation of gonado- trophs. This degranulation occurred be- tween the ages of 35 and 42 days in the observations of Siperstein, Nichols, Gries- bach and Chaikoff (1954), and, in a num- ber of their rats degranulation of the pe- ripheral gonadotrophs which are thought to be follicle stimulating in activity, was found to have preceded the degranulation of the central gonadotrophs. This observa- 202 HYPOPHYSIS AND GOXADOTROPHIC HORMONES tion is significant in view of the fact that the peripheral gonadotrophs ordinarily are more strongly granulated than the central ones. The observation suggests at least that a discharge of stored FSH precedes the dis- charge of LH on the occasion of the first ovulation. B. SEXUAL MATURATION IX OTHER AXIMALS Characteristic globular basophil cells first appear in the red eft stage of Triturus viridescens simultaneously with the differ- entiation of the male and female gonads and are presumed to be gonadotrophic (Cope- land, 1943). In the opossum (Didelphys virginiana) some interesting observations have been made by Wheeler (1943). A vesiculation of the basophil cells and an ac- cumulation of hyaline material occurs in adult animals after castration, producing cells similar to the castration cells of the rat. Wheeler observed that at 100 days of age there occurs a vesiculation of basophils similar to that produced in adult animals by castration. The assumption is that during sexual maturation the hypophysis is called on to secrete gonadotrophic hormones in large amounts and that before full differ- entiation of the gonads, a temporary endo- crine situation exists similar to that pro- duced by castration. C. SEASONAL BREEDIXG In seasonally breeding animals an initia- tion of the breeding phase has been observed to be accompanied by basophil changes. In the wild cotton-tail rabbit Elder and Finerty (1943) found an increase in gonado- trophic potency in the male hypophysis in the spring, the maximal level being six times that of the level during the winter. This change was accompanied by an increase in the percentage of basophil cells from 4.4 to 13.8. In Necturus, Aplington (1942) related the seasonal activity of the testes to an increased activity in the number of granular basophil cells. A similar increased basophil granule content in Anolis rarolinensis during the spring was observed by Poris (1941). D. INDUCED SEXUAL MATURATION IN THE FEMALE RAT An important series of observations luis l)ecii made concerning the eft'ect of estrogen injections on immature female rats ap- proaching the age of sexual maturation. In the immature female rat gonadotrophin secretion is inhibited by minute amounts of estrogen and as long as this relationship ex- ists, no high estrogen levels can be naturally produced in the animal. With the approach of maturation this inhibiting action of estro- gen on gonadotrophin must diminish. Indeed it is found that a condition is reached as the time of the first ovulation approaches when estrogen, instead of inhibiting gonado- trophin secretion, triggers the sudden release of these substances. The physiologic ob- servations of Hohlweg and Chamorro (1937) indicate that a release of gonado- trophic hormones occurred between the sec- ond and fourth day after the injection of estrogen into the immature female rat ap- proaching the expected time of first ovula- tion. The effect of the release of gonado- trophic hormones may be shown by the l)roduction of ovulation and corpora lutea or by follicular enlargement only, the actual response being different in different strains of rats. The liberation of the gonado- trophins after estrogen administration re- sults in a marked drop in the hypophyseal gonadotrophin content which occurrs ap- jn'oximately between 3 and 4 days after estrogen administration (Bradbury, 1947). Purves and Griesbach (unpublished) have studied the responses of the gonadotrophs of the immature female rat after single estrogen injections and found that regard- less of dose in the range from 1 to 100 jug. of estradiol benzoate, there was no change in the hypophysis in the first 2 days. As previously mentioned, in these immature fe- male rats both FSH and LH cells are very numerous and the content of glycoprotein is high. Four days after estrogen admin- istration, the gonadotrophs of the pars an- tei'ioi- ai'e almost free of glyco]irotein and can he recogniz(>d only with difficulty as large pale cells. It is important to note that at this adolescent stage there is no differ- ential effect of estrogen on peripheral and central gonadotroi)hs or on the secretion of FSH 01' LH. The effect is rather that of the triggered discharge of the total gonado- troi)hin content of the hypophysis. At the time of glycoprotein discharge from tlic liypophysis of innnature female HYPOPHYSEAL MORPHOLOGY 203 rats after estrogen administration, large numbers of mitoses are observed in the chromophobes and granulated acidophils, an effect which is probably due to the ac- tion of the injected estrogen on these cells. The number of mitotic figures induced by this single injection of estrogen in the ado- lescent female rat is much greater than that observed after similar doses in adult animals and indicates that a proliferation of cells of the acidophil class occurs in the fe- male rat hypophysis at the time of matura- tion. Baker and Everett ( 1947) found by ac- curate measurements of mitotic index that stimulation of mitoses in acidophil cells after estrogen administration is greater in innnature female rats than in the mature animal. E. THE ACTIVE BREEDING PHASE IN THE FEMALE The relation between hypophysis and gonad during the breeding phase is quite different from that before maturation. In the rat, with its short estrous cycle, there is a re- current discharge of gonadotrophins at each ovulation and this maintains the gondaotro- phic hormone content at a low level. In ac- cordance with this the gonadotrophs whether studied by the glycoprotein staining reac- tion or by the Mallory staining reaction are inconspicuous because of the low content of specific granulation. There is during the diestrum an accumulation of specific granu- lation (Catchpole, 1949; Purves and Gries- bach, 1951a). Before the thyrotrophs and gonadotrophs were distinguished, there were observations showing a cyclic change in basophil cells during the estrous cycle. Those by Wolfe and Cleveland (1933a) and Wolfe (1935) revealed a variation of baso- phil cells in their rats which is in exact agreement with the variations of the spe- cific gonadotrophs (Table 3.2). The basophil cells observed by Wolfe, which are described as being large, oval, finely granulated, and containing a negative image of the Golgi body in most of the cells, correspond in all details with the LH cells (central gonadotrophs) observed by Purves and Griesbach (1952). Cyclic changes in the hypophysis of the dog were described by Wolfe, Cleveland and Campbell (1933) who distinguished in tlie pars anterior 4 types of cells, 3 of which contained specific granules. Goldberg and Chaikoff ( 1952a » distinguished 6 cell types of which 4 had specific granules. Type I of Wolfe, Cleveland and Campbell corresponds to the a-cell; their type II, which is selec- tively stained by azocarmine (Hartmann, Fain and Wolfe, 1946) corresponds to the e-cell of Goldberg and Chaikoff. Type III corresponds in the main to the 8-cell, but includes small numbers of cells with a pe- ripheral accumulation of granules which are probably /3-cells. The 8-cells (type III) in- creased in numbers up to 10 per cent at the proestrum and w^ere then well filled with fine purplish stained granules. At the time of estrus the number of 8-cells was at a maximum (12.6 per cent) but at the same time they showed extensive degranulation. The number of 8-cells recognized during the lutein phase of the estrous cycle and during pseudopregnancy was low (2 to 4 per cent), and during the anestrum it was 5 per cent. The variation in number of 8-cells and of the granules in these cells corresponds to what would be expected from a cell type producing FSH, if it is right to assume that this hormone is not secreted during pseudo- pregnancy. Wolfe, Cleveland and Campbell also observed changes in e-cells (type II) which, from a level of 5.8 per cent during the anestrum, rose to 7 per cent at estrus and fell during the later lutein phase of the cycle to 2.5 per cent. At this time they also showed considerable degranulation. Present interpretation of these changes would asso- ciate the changes in the €-cells with the secretion of lactogenic hormone. Cyclic changes in cells of acidophil and TABLE 3.2 (After J. M. Wolfe, Anat. Rec, 61, 321-330, 1935.) Basophil Cell Percentages Sexual State Granu- lated Nongran- ulated Total Immature (17 days) Immature (27 to 35 days) . Mature (pre-estrus) Mature (estrus) 7.2 7.4 2.8 0.9 0.6 1.3 0.7 0.9 1.9 4.4 3.5 2.7 7.8 8.6 4.8 5 2 Mature (metestrum) Mature (diestrum) 4.0 3.9 204 HYPOPHYSIS AND GONADOTROPHIC HORMONES basophil classes have also been described in the sow (Cleveland and Wolfe, 1933), rabbit (Wolfe, Phelps and Cleveland, 1934) and guinea pig (Chadwick, 1936). These ob- servations are, however, not easily cor- related with hormonal functions because the differentiation of specific cell types was not achieved. F. PSEUDOPREGNANCY AND PREGNANCY During pseudopregnancy and pregnancy five distinctive cell types are recognizable in the rat hypophysis as follows. (1) Acido- phil cells, carminophil by Dawson's (1954a) method, in close relation to blood vessels and connective tissue septa and strongly granulated. (2) Acidophil cells, orangeophil by Dawson's (1954a) method, in the interior of the cell cords and large, active, and only lightly granulated. (3) Thyrotrophs which are more prominent and active in this phase than during the estrous cycle. (4) FSH cells which are strongly granulated and at least as prominent as those in the male hypophy- sis and in the immature female. (5) LH cells which are large, round, lightly granulated cells with large and prominent negative im- ages of the Golgi body. In animals treated with thyroxine the thyrotrophs are inhibited and no longer seen. Their activity in the untreated animal is probably an indication of an increased demand for thyroxine during this phase of the reproductive cycle. The strong secretory activity which is indicated in the second type of acidophil cell is considered to be related to the secretion of lactogenic hor- mone, whereas the activity of the LH cells is considered to be related to a high level of secretion of LH. The prominence of the FSH cells indicates a retention of FSH. The increased prominence of the FSH cells does not necessarily indicate an increased se- cretion of FSH inasmuch as other condi- tions which interrupt the estrous cycle per- mit the accumulation of glycoprotein in these cells. The increased glycoprotein stor- age correlates with the finding of elevated gonadotrophin levels in the rat hypophysis during pregnancy (Evans and Simpson, 1929; Zeiner, 1952). The carminoi)hil coll in the rabbit, cat and monkey, shows during the reproductive cycle marked fluctuations in activity which can be correlated with the secretion of pro- lactin at times when its luteotrophic or lac- togenic action is manifest (Dawson and Friedgood, 1937, 1938b; Dawson, 1939, 1948; Friedgood and Dawson, 1940). G. PREGNANCY CHANGES IN THE HUMAN HYPOPHYSIS In the human hypophysis the predomi- nant change in pregnancy is the activation of large numbers of cells which are chromo- phobic in the nonpregnant state. These pregnancy cells were first described by Erdheim and Stumme (1909). They lie in the lateral regions of the pars distalis and from the seventh month contain fine acid- ophil granules. These cells have been the source of some confusion and Rasmussen (1933) , finding normal proportions of acido- phils, basophils, and chromophobes in the early months of pregnancy, denied the ex- istence of a specific pregnancy cell although he recognized the enlargement of the chro- mophobes during this condition. Romeis (1940) confirmed the observations reported by Erdheim and Stumme and described the pregnancy cells as large cells with large nuclei. In the second half of pregnancy they show an accumulation of granules which Romeis called 7^-granules. Romeis con- sidered the r;-granules to be a specific type of granule not present in the nonpregnant hypophysis. The granulated pregnancy cell closely resembles the e-cell as seen in the nonpregnant human hypophysis although there are some points of difference. The dis- tribution of the granules is similar and the granules, like those in e-cells, are orangeo- phil. a-Cells, whose granules are carmino- phil, retain their normal appearance in the pregnancy hypophysis. It is probable that pregnancy cells are e-cells in an altered functional state. Floderus (1949) examined the distribution of pregnancy cells in the lunnan hypophysis and found that they are infi'cHinent in the upper posterior part of the pars distalis and are sometimes entirely lacking in this region. They are most abun- dant in the lower lateral parts of the gland. The pregnancy cells are often centrally located in the cell cords with other types, usually ordinary acidophils, located periph- HYPOPHYSEAL MORPHOLOGY 205 erally. Cell cords composed mainly of preg- nancy cells occur and isolated cords of this nature may be conspicuous in certain re- gions. The chromophobes of the vascular zone of the anterior lobe near its attach- ment to the stalk ("zona tuberalis") are not affected by pregnancy changes. The preg- nancy cells are, therefore, not derived from chromophobes in general but from chromo- phobic cells having a specific distribution. There are two important differences be- tween human pregnancy cells and the carminophil cells of the rabbit, cat, and monkey. First, the human pregnancy cell granules are orangeophil rather than car- minophil. The distinctive color difference between a-cells and pregnancy cells is, therefore, in the human hypophysis the reverse of that seen in the other species. Second, the distribution is different from that seen in the monkey where carminophil cells are especially concentrated in the zona tuberalis. Despite these differences, the pregnancy cells appear to be functionally analogous to the carminophil cells and are presumably the source of prolactin secre- tion. H. LACTATION The characteristic feature of the pars anterior at and shortly after parturition is the increased amount of acidophil gran- ulation present. Kirkraan (1937) found in the guinea pig that the acidophils increase in numbers towards the end of pregnancy and attain a maximum soon after parturi- tion. Wolfe and Cleveland (1933b) reported a similar increase in granulation of acido- phils in the rat hypophysis towards the end of pregnancy. Everett and Baker (1945) found that after parturition in the rat, the acidophil cells increased by almost 100 per cent in the first three days of the lactation period. This increase in the num- ber of acidophil cells visualized was ac- complished without an increased number of mitoses or any increase in the size of the gland and was, therefore, due to the regranulation of acidophil cells which had been degranulated during pregnancy. Hunt (1949), however, found that mitoses were present in some but not all rat hypophyses after parturition. Even allowing for this latter observation it seems that the in- creased number of acidophil cells at the time of parturition can be accounted for by the accumulation of secretory granules in the cells of the acidophil class. Purves and Griesbach (unpublished) have found that this increase in granulation occurs in the acidophil cells which are remote from the blood vessels and connective tissue septa and which are considered to be related to prolactin secretion. It has already been noted that in the cat a granulation of carminophil cells occurs which is at its maximum at the time of parturition (Daw- son, 1946) , these cells also being considered the specific secretors of prolactin. In ac- cordance with this view, Hurst and Turner (1942) reported that in the rat, rabbit, mouse, guinea pig, and cat the prolactin content of the hypophysis was at its highest level during the first few days after parturi- tion. In connection with the question whether there is a separate lactogenic factor secreted during lactation, different from the luteo- trophic and mammogenic factor secreted during pregnancy as Turner (1939) pos- tulated, cytologic observations indicate an activity in a single specific cell type as- sociated with both mammary growth during pregnancy and lactogenesis after parturi- tion. There is, during early lactation, a phase of secretory activity in these special- ized acidophil cells which is at its maximum about the 16th day of lactation in the cat (Dawson, 1946). Thereafter the reaction wanes in a manner which suggests that a continuation of lactation is not dependent on the continued secretion of this factor. It is, therefore, probable that the continua- tion of an established lactation in those animals in which lactation is of consider- able duration is not dependent on the con- tinued secretion of prolactin. In conform- ity with this view, prolactin has been found to stimulate metabolic changes in slices of mammary gland tissue from rats on days 1 to 4 of lactation (Folley, 1952). On the other hand, purified prolactin prepa- rations have not shown any galactopoietic effect in the cow during the declining phase of lactation (Folley and Young, 1938). 206 HYPOPHYSIS AND GONADOTROPHIC HORMONES XIII. The Human Hypophysis A. STRUCTURE In the human hypophysis (Fig. 3.18j the adenolobe is closely adherent to the neural lobe. The hypophyseal cleft persists in the adult only in its distal portion. The re- mainder of the cleft is obliterated by fusion or is broken up into a number of scattered colloid-filled cysts. No pars intermedia is present, conse- quently the pars distalis adheres to the neural lobe. This feature, which is present also in the higher apes, is not found in lower mammals. In the latter there is either a pars intermedia which is adherent to the neural lobe and separates it from the pars anterior, or there is a pars distalis which is not ad- herent to the neural lobe. A full description of the structural features present in the Fig. 3.18. Diagram of a .sagittal section through the liuman hyi)oi)hysis. The anterior direction is to the left of the diagram. The adenolobe is occupied by pars distalis tissue (P.d.) containing a mixed cell population throughout and there is no pars in- termedia. Colloid cysts (C.c.) occurring in the re- gion adjacent to the neural lobe are probably rem- nants of the hypophyseal cleft. The adenolobe is adherent to the neural lobe. The neural eminence, the neural stalk, and the prolongation of the neural stalk within the neural lobe are here collectively called the pars eminens (P.e.). The pars nervosa (P.n.) shows an invasion by basophil cells (B) which migrate into the pars nervosa from the pars distalis. The extent of this invasion increases with increasing age and shows great variation in differ- ent individuals. The pars tuberalis is in(lic,il( d In- P.t. zone of contact between adenolobe and neu- I'al lobe in the human hypophysis is given Ijy Romeis (1940), who reviews the earlier literature on the subject. An invasion of the pars nervosa by baso- phil cells derived from the pars distalis is a unique feature of the human hypophysis. In lower mammals possessing a pars distalis such invasion is not possible because of lack of contact between the two parts. The cells of the pars intermedia in the more usual form of mammalian hypophysis do not show this invasion of the pars nervosa, al- though an irregularity of the plane of con- tact indicates a tendency for mutual in- terpenetration of the two tissues. As stated earlier, the cells invading the pars nervosa of the human hypophysis are intermedin- secreting cells. B. SPECIFIC BASOPHIL CELL TYPES IX THE HUMAN HYPOPHYSIS The treatise of Romeis (1940) consti- tutes a landmark in the study of the human hypophysis, as indeed for the mammalian liyi)ophysis in general. Romeis' findings in the human hypophysis have, however, de- spite their completeness and their excellent presentation, not had the influence on the development of this subject that might rea- sonably have been expected. The availabil- ity of fresh pituitary tissue from surgical hypophysectomy has changed this situation. U'hen appropriate fixing and staining techniques are used, results equivalent to those described by Romeis can be consist- ently obtained, and even those workers who, through the use of inadequate techniques, have deprived themselves of the oppor- tunity of observing the cell types described by Romeis, must accept the conclusion that such cell types are present and can be re- vealed by appropriate techniques. It is therefore advisable that those who would study the human hypophysis should iden- tify the cells they see with those so clearly delineated by Romeis rather than to em- bark on schemes of classification which Romeis' results show to be inadequate, or to use the terminology of Romeis in applica- tions other than those which he adopted. The f3-, 8-, and y-celLs of Romeis are basopiiil cells because their granules give a HYPOPHYSEAL MORPHOLOGY 201 positive PAS reaction for glycoprotein (Herlant, 1958). Romeis noticed that some of the /?-cells retained azocarraine during the azan staining of the sections previously stained with resorcin-fuchsin, whereas others retained only a blue counterstain from the aniline blue. The researches of Griesbach (unpublished) have convinced me that these staining reactions indicate two distinct types of cell whose granules are stained by resorcin-fuchsin, i.e., two types of ^- cells. After Helly fixation the difference between the two types is enhanced. Also after Helly fixation the staining of the gran- ules of one type of ^-cell by aldehyde- fuchsin is greatly weakened, an effect which allows the red counterstain by azan in this type to be more clearly seen. The y8-cells are therefore seen to comprise purple AF cells and blue AF cells. These we may call in the human pars distalis "purple /3" and ''blue ^." The 8-cells are blue non-AF after Helly fixation, and the y-cclls are usually also blue non-AF. The blue /?-cells are more variable in size and shape than the purple /?-cells and are more often found in multinucleated form than the latter. Their distribution in the gland is quite different from that of the purple /?-cells, so that they cannot be vari- ants of a single cell type. Moreover, the staining reactions of the granules, except to resorcin-fuchsin or aldehyde-fuchsin after certain fixatives, are quite distinctive. There are indeed more dissimilarities be- tween purple /3-granules and blue /3-gran- ules (Fig. 3.19) than there are between the latter and the 8-granules. Reasons have been given in the section on the pars intermedia and intermedin secre- tion for regarding the purple /?-cells as in- termedin-secreting cells corresponding to the pars intermedia cells of the usual mam- malian hypophysis. The blue /?-cells, the 8- cells, and the y-cells should therefore be homologous with the three basophil cell types in the pars anterior of such mammals as the rat, bat, and dog. C. DIFFERENTIAL STAINING OF BASOPHIL CELLS IN THE HUMAN PARS DISTALIS The y-cells of Romeis are distinctive in appearance, containing fine glycoprotein granules which are stained only feebly by acid dyes (Figs. 3.20 and 3.21). In addition they often contain droplets of glycoprotein, intensely stained by the PAS reaction. These cells have been termed "vesiculate chromo- phobes" (Pearse, 1952) . They are not likely to be confused with normally granulated jS- cells or 8-cells. The partition of the basophil cells by differential staining of the specific granules is therefore a partition of the purple ^-cells, blue ^-cells, and 8-cells (Fig. 3.22). Some of the methods used produce a differ- entiation between the purple ^-cells and the two types of blue cells. Because it was as- sumed that only two types of cell were in- volved, the distinction between purple and blue cells has been assumed to be a dis- tinction between /S- and 8-cells. Purves and Griesbach (1957b) observing purple and blue cells in Crossmon (1937) stained sections of human pars distalis wrongly assumed this to be a distinction between 13- and 8-cells. Herlant (1953b; 1954b) dif- ferentiated the purple /?-cells from the other types by counterstaining PAS stained sec- tions with orange G. The orange G stained the strongly acidophilic granules of the purple /3-cells and combined with the PAS color to produce a brick-red shade whereas the other basophil granules were magenta. Wilson and Ezrin (1954) used a method substantially the same as Herlant's PAS orange method with the addition of methyl blue. The methyl blue stained only the magenta granules of Herlant leaving the granules of the purple ^-cells brick-red. The purple cells of Purves and Griesbach, the brick-red cells of Herlant, and the PAS- red cells of Wilson and Ezrin are obviously the same cells and belong to the group of ^-cells. It is only when these staining meth- ods are applied as counterstains to sections in which /?-cells have been electively stained by aldehyde-fuchsin, as in the method of Griesbach, that the dual nature of the /3- cells is revealed. Adams and Swettenham (1958) applied aldehj^de-fuchsin or Alcian blue to sections oxidized with performic acid and followed this with PAS staining. Their R cells which were red and not stained by aldehyde- fuchsin are the purple ^-cells, their S cells which were stained by aldehyde-fuchsin or 208 HYPOPHYSIS AND GONADOTROPHIC HORMONES f^'^ Fig. 3.19 (upper left). Section of the human pars distalis showing weak staining of the granules of purple ;3-cells by aldehyde-fuchsin after fixation in Helly's fluid. Much of the density of the purple /3-cells in the photomicrograph is due to the reddish tint given to them by the azan counterstain. A single blue jS-cell (B) is strongly stained by the aldehyde-fuchsin (AF). In the section it appeared an indigo color from superimposition of a blue color from the aniline blue of the counterstain. AF, azan, X 900. Fig. 3.20 (upper right). Section of the human pars distalis showing 7-cells. The cells here show elongated forms. The granulation was stained a weak purplish shade. AF, azan, X 900. Fig. 3.21 (lower left). Section of the human pars distalis showing 7-cells stained by periodic acid-Schiff (PAS). The conspicuous PAS +ve droplets which are unusuallj^ numerous in this field are not peculiar to the 7-cell since they may be found in other cell types, both basophil and acidophil. The faint grey granulation distributed throughout the cytoplasm is considered to be the specific 7-granulation. PAS, X 900. Fig. 3.22 (lower right). Section of the human pars distalis fixed in Helly's fluid and stained with aldehyde-fuchsin (AF), counterstained with azan. Acidophils, blue /3-cells and purple /3-cells which were deeply stained in different colors in the section, appear black and cannot be clearly distinguished from one another in the photomicrograph. The numerous large rounded pale cells with grey granulation are 5-cells. The granules were stained a clear blue in the original. AF, azan, X 900. Alei;iii blue comprise the blue /i- and 8-cclls. Herlant (1956b) obtained the same distri- bution of the aldehyde-fuchsin stain in sec- tions oxidized with permanganate. It is to be noted that the effect of oxidation is to inhibit the staining of purple /?-cells by aldehyde-fuchsin and to render the 8-cells stainal)lc; the hhic /i-cells are stained with or without oxidation. The effects of jier- manganate oxidation on the aldehyde-fuch- sin staining of the human pars distalis are the same as those observed in the pars an- terior and pars intermedia of the rat (Halmi and Davies, 1953). The staining of the pars HYPOPHYSEAL MORPHOLOGY 209 intermedia cells is weakened and the S-cells of the pars anterior become stainable whereas the ^-cells remain stainable. The difference between the species is that the intermedin-secreting cells in the human hypophysis are scattered throughout the pars distalis and not segregated in the pars intermedia. Hellweg (1951) obtained by silver im- pregnation a specific staining of the 8-cells in the human pars distalis. This staining is similar to that observed by Knigge (1957) in the rat, in which a specific staining of Halmi's S-cells (gonadotrophs) was ob- tained by silver impregnation. Ezrin, Swan- son, Humphrey, Dawson and Wilson (1958) obtained specific staining of 8-cells by their iron-PAS method, the 8-cell granules being stained by dialyzed iron which is subse- quently converted to Prussian blue. A valu- able feature of the iron-PAS method is that it is applicable to postmortem material fixed in formol-saline. D. FUNCTIONS OF THE BASOPHIL CELLS OF THE HUMAN PARS DISTALIS 1. The S-Cell Neither Hellweg (1951) nor Ezrin, Swanson, Humphrey, Dawson and Wilson (1958) found 8-cells in children before the age of puberty, and the latter investigators did not find them in the hypophyses of pregnant women. This finding can be corre- lated with the reduction in gonadotrophin content during pregnancy (Herlant, 1943). It is therefore certain that the 8-cells are the source of one or more of the hypophys- eal gonadotrophins. Ezrin and his associ- ates found that the number of 8-cells de- creases with the duration of the terminal illness, being 8.5 per cent in patients dying in 24 hours and 1.8 per cent in those dying after 2 weeks or longer. This is in accord- ance with other evidence suggesting a re- duction in gonadotrojihin during chronic illness. 2. The Blue /3-Cell There is a considerable increase in the number of hypertrojihied, lightly granu- lated basophil cells in myxedema and cre- tinism (Herlant, 1954b, 1958). The staining reaction of these cells to Herlant's (1953b) PAS-orange method indicated that they are blue basophils, and inasmuch as Russell (1957j found them stainable by aldehyde- fuchsin it is probable that they are blue /?-cells and not 8-cells as Herlant supposed. The blue /3-cells are therefore probably thy- rotrophs. It should be noted that the stain- ing reactions of the blue /?- cells are the same as those of the thyrotrophs of the pars anterior of the rat, dog, bat, cat, and guinea pig. 3. The y-Cell y-Cells are well developed and fully functional in appearance in children and maintain much the same appearance in women up to the menopause. They do not seem to be altered in appearance during pregnancy (Herlant, 1958). They are quite distinct from pregnancy cells which in the latter half of pregnancy contain acidophil granules. The number of y-cells does not show any correlation with the duration of the terminal illness (Ezrin, Swanson, Hum- phrey, Dawson and Wilson, 1958). Her- lant's observation that the y-cells are in- active in aged subjects does not correlate with any known change in hormone secre- tion. It is possible that these cells secrete corticotrophin but more investigation is necessary. Crooke and Russell (1935) no- ticed in Addison's disease a variable pro- portion of very large "chromophobes." Large chromophobes ("hypertrophic am- phophils") have also been observed by Mellgren (1945, 1948) in this disease. The large chromophobes or hypertrophic am- phophils of the above authors are presum- ably the y-cells of Romeis (1940), or more precisely the remains of the y-cells, inas- much as these cells are susceptible to post- mortem autolysis and are not well preserved in autopsy material. Griesbach (private communication) has observed a large num- ber of large y-cells in the pars distalis of a patient who had been adrenalectomized for the treatment of a carcinoma some weeks before death. E. THE AMPHOPHIL CELLS OF RUSSFIELD Russfield (1957) divides the cells of the humaii pars distalis into acidophils, baso- phils, amphophils, hypertrophic ampho- 210 HYPOPHYSIS AND GONADOTROPHIC HORMONES phils, and chromophobes. The acidophils of Russfield are the a-cells of Romeis, the basophils are the conspicuous and strongly granulated ^-cells of Romeis. Russfield er- roneously considers these "normal" baso- phils to be the 8-cells of Romeis. The am- phophils consist of lightly granulated cells — 8-, y-, and c-cells — but may include «- and ^-cells if these are lightly granulated. Hypertrophic amphophils are in the main y-cells whose cytoplasm and granules have been lost by postmortem autolysis, to which these cells are especially sensitive. From the study of hypophyses of pa- tients with endocrine disturbances (Burt .■Cv Fig. 3.23 {upper). Sod ion of tlio pars distalis of a patient who suffered from the Gushing syndrome. Many typical Grooke's cells are present. The hy- ahnized zones appear homogeneous at this magnifi- cation. The remaining granules are stained in llic manner typical of puri)le /i-cell graTuilation. AF, azan, X 900. Fig. 3.24 (lower). ^wVww of a liuiuaii liypojihysis showing a basophil adenoma composed of cells with the staining leactions of purple ^-cells. In material fixed in formalin the purple ^-cells are strongly stained by aldehydo-fuchsui (.\1''). From a speci- men sui)iilicd 1)V Professor Dorothv Ru.ssell. AF, X5. and Velardo, 1954) and of hypophyseal tumors (Russfield, Reiner and Klaus, 1956) , Russfield concludes that amphophils are capable of producing all the anterior lobe hormones, but there is no implication that they produce all these hormones simultane- ously. This seems to mean no more than that large amounts of hormone may be secreted by lightly granulated cells. Russ- field's results are of importance in directing attention to the fact that more information of endocrinologic significance can be ob- tained from the study of lightly granulated cells than by the enumeration of ''typical" acidophils and basophils; they do not con- flict with the view that the cells producing different hormones are characterized by dif- ferent types of granules, whose specific character can be distinguished by appropri- ate staining methods. F. THE PUKPLE /3-CELL IN THE GUSHING SYNDROME The purple /?- or intermedin-secreting cells are only lightly granulated in infants. The amount of granulation in these cells as measured by the intensity of the PAS re- action increases with age in a smooth con- tinuous fashion which is neither accelerated nor retarded by puberty or the menopause (Herlant and Lison, 1951). The cells are not affected by pregnancy and the prog- ressive increase in granule content follows the same course in both sexes. The functional state of the purple /3-cells a])pears to be determined by the level of circulating corticosteroids and has not been shown to be related to any other factor. Al- though the physiologic significance of the re- sponses of the purple j8-cells to variations in tlie corticosteroid level is concealed in the mystery which envelops the whole subject of the function of intermedin secretion in mammals, the responses themselves are definite, striking, and consistent. High levels of corticosteroids stimulate, low levels de- press the cytologic activity of these cells. The characteristic changes in the Gushing syndrome are a degranulation and hyalini- zation of the purpl(> /3-cells. The changes were first dcscnhcd by Crooke (1935), after whom the liyaHnized cells were named (Fig. 3.23). In my own observations I have found that it is only the jiurple ^-cells HYPOPHYSEAL MORPHOLOGY 211 which are affected by hyalinization. More- over, the basophil cells which invade the neural lobe and are all purple y8-cells are sometimes hyalinized in the same way as the cells in the pars distalis. Hyalinization does not alter the staining reactions of the granules that remain in the hyalinized cells. It is notable, however, that the purple /3-cells which have invaded the neural lobe are normally less active and smaller in size than those that remained in the pars dis- talis. This difference in activity persists in the Gushing syndrome and, in consequence, the purple /3-cells in the neural lobe show a lesser degree of hyalinization than those in the pars distalis ; they may indeed not show any hyalinization in hypophyses in which many of the cells in the pars distalis are af- fected. Degranulation of Crooke's cells may be complete and the cytoplasm may be com- l)letely hyalinized. More characteristic is a partial degranulation having an annular distribution. The periphery of the cell may be granulated and the granules be pre- served around the nucleus and Golgi body, or degranulation may occur around the nucleus and Golgi body with preserva- tion of granules at the periphery. Some- times a degranulated zone occurs with pres- ervation of granules both at the periphery and in proximity to the nucleus and Golgi body. Hyalinization occurs in the degranu- lated zone and differs from the mature hy- alinization of thyrotrophs and most gonado- trophs in the rat, in that the hyaline area is not sharply limited. The surface of the hyalinized zone shows a fine vesicular struc- ture which gives to the hyalinized zone a faintly ground-glass appearance. Probably the hyalinization is similar to that of the filigree cells described by Farquhar and Rinehart (1954) in the rat. The hyaline substance is not colored by the PAS reagent and has little affinity for basic dyes. Crooke's cells are in general large cells with vesicular nonpyknotic nuclei and eas- ily visible Golgi bodies. Both McLetchie (1942a, b) and Mellgren (1945) agree with Crooke that these appearances indicate in- creased secretory activity. In the original description of the Gushing syndrome the presence of a basophil ade- noma in the hypophysis was noticed in three cases. Gushing emphasized the role of these adenomas as a primary factor in the causa- tion of the syndrome, but considered it pos- sible that the symptoms were due to stimu- lation of adrenocortical secretion by the basophil adenoma (Hubble, 1949) . However, such basophil adenomas are not found in all cases of the syndrome and are, moreover, not infrequently found in hypophyses of persons in the older age groups who have not exhibited any specific endocrine disturb- ances during life. The cells of basophil adenomas found in association with Grooko's cells contain baso- phil granules with the same staining re- actions as those of purple ^-cells (Fig. 3.24). The cells of the adenomas are usu- ally not hyalinized. In this respect they are similar to basophil adenomas induced in the rat hypophysis by thyroxine deficiency or by castration. The association of basophil adenomas with hyalinization indicates a long continued strong stimulation of the af- fected cells (Purves, 1956). G. crooke's cell changes produced by CORTICOSTEROID OR CORTICOTROPHIN ADMINISTRATION Laqueur (1950, 1951) and Thornton (1956) have reported Grooke's cell changes in patients treated with cortisone. Golden, Bondy and Sheldon (1950), Dreyfus and Zara ( 1951 ), and Thornton (1956) have also reported similar changes after treatment with corticotrophin. The corticotrophin pre- sumably acts by stimulating the adrenal cortex, the secretion from which is the ef- fective agent in producing the hyaliniza- tion. From Thornton's observations it ap- pears that hyalinization is produced in a few days by effective doses of corticos- teroids and regresses equally rapidly after cessation of treatment. Grooke's cells there- fore indicate a high level of corticosteroids in the circulation in the last few days before death. The erroneous assumption that the basophil cells which were hyalinized in the Gushing syndrome were pars anterior cells caused some investigators to postulate that the hyalinization was the expression of an increased secretion of thyrotrojihin or gonad- 212 HYPOPHYSIS AND GONADOTROPHIC HORMONES otrophin resulting from alterations of thy- roid or gonadal function produced by high levels of costicosteroids. The fact that these cells are not hyalinized in myxedema or after castration shows that this is not so. In the present state of knowledge it seems probable that Crooke's cells are actively secreting intermedin, the effects of which are antagonized by corticosteroids. Only occasionally is the Gushing syndrome as- sociated with hyperpigmentation (Ed- munds, McKeown and Coleman, 1958). It can be affirmed that Crooke's cell changes have nothing to do with an increased secre- tion of corticotrophin because they are pro- duced by conditions which cause suppres- sion of corticotrophin secretion. h. changes in the purple /3-cells in Addison's disease Reports on the human pars distalis in Addison's disease indicate a diminution in the number of basophil cells or at least a scarcity of well granulated basophils (Kraus, 1923, 1926, 1927; Berblinger, 1932; Crooke and Russell, 1935). It is the purple /?-cells which disappear, apparently passing into an inactive state in which they lose their granules. Blue basophils remain ap- parently in a normal state and it is these cells which have been referred to as Crooke- Russell cells. This interpretation is in con- formity with the staining reactions of Crooke-Russell cells reported by Russell (1956) and by Wilson and Ezrin (1954). Presumably both blue /3- and 8-cells are present ; the material at my disposal has not been suitable for differential staining of these cell types. In view of the inactivity of the purple /?-cells in Addison's disease, the hyperpig- mentation in this condition must be as- cribed to the intrinsic melanocyte stimu- lating activity of the corticotrophin which is being secreted in excessive amounts. Per- haps it is the effects of this side reaction of corticotrophin which cause suppression of int(n'mcdin secretion in this condition. I. THE PHARYNGEAL HYPOPHYSIS The pharyngeal hypophysis is a collection of cells found in the submucosa of the pos- terior pharyngeal wall and is a remnant of the epithelial stalk of the hypophysis which connects the buccal ectoderm to Rathke's pouch at an early stage of embryonic de- velopment. The pharyngeal hypophysis is found only in man and seems to be con- stantly present (Romeis, 1940) . It is a mass of cells 3 to 4 mm. in length. The cells are mainly small with scanty cytoplasm, but larger chromophobes and occasional acido- phils are present. Basophil cells are ex- tremely rare. It has been suggested that the pharyngeal hypophysis can take over some of the func- tion of the sellar hypophysis when the latter is destroyed by disease processes (Tonnis, Miiller, Ostwald and Brilmayer, 1954; Miiller, 1958). This cannot be regarded as established for the residual function may be traceable to pars distalis cells that have escaped destruction. The pharyngeal hypophysis is sometimes the site of adenoma formation (Miiller, 1958). Erdheim (1926) described a case of acromegaly in which was found an un- altered sellar hypophysis and an acidophil adenoma derived from the pharyngeal hy- pophysis. XIV. Electron Microscopy of the Adenohypophysis Many cytologic structures are so small that the details of their structure cannot be made out by means of the light microscope, the resolving power of which is limited to about 300 nifi. The limit of resolution of the electron microscope is about 1/1000 that of the light microscope. Because of the low electron density of organic materials and the consequent lack of contrast in thin sections of tissues, the available resolution of the electron microscope for cytologic detail is limited to about 1/50 of that of the light microscope, i.e., about 6 ni/x. This is a big advance, comparable to that produced by the change from the simple hand lens to the compound microscope. The most fertile application of electron microscopy in the field of cytology has been the examination of ultrathin sections of tissues fixed in solutions containing osmic acid. Osmic acid has been for many years considered the best fixative for the pres- ervation of fine structure in material exam- HYPOPHYSEAL MORPHOLOGY 2L3 ined by light microscopy. Its use is un- fortunately incompatible with most of the staining techniques on which light micro- scopists have come to rely. For the fixation of tissues for light microscopy, use has been made of a number of fixatives, which permit subsequent staining by various methods, but which do not satisfactorily preserve fine structure even at the level visible by light microscopy. These fixatives cause an extensive redistribution of the proteins of cells during fixation as is shown by an in- crease in opacity and light scattering power during fixation. No exact correspondence can be expected between the appearances of structures seen in electron micrographs of osmic acid fixed tissues and of structures rendered visible by staining in tissues fixed by other meth- ods. Cytoplasmic structures visible in electron micrographs of adenohypophyseal cells (Figs. 3.25-3.32)1 include the endoplasmic reticulum, the components of the Golgi re- gion, the Palade granules, mitochondria, secretion granules, and lipid droplets. Thus far electron microscopic observations of the pituitary have revealed nothing new with respect to the finer structures of these parts (Palade, 1953), and nothing that is sug- gestive of specific hypophyseal function. On the other hand, only a first step has been taken, but it is expected that further studies will lead to clarification of the many prob- lems to which allusion has been made. A. ENDOPLASMIC RETICULUM The endoplasmic reticulum is a cavitary system consisting of tubes, vesicles, and flattened sacs interconnected by narrower ^The electron micrographs of the cells found in the pars anterior and pars intermedia of the rat were contributed by Dr. Marilyn G. Farquhar who also supplied the descriptions. The electron micrographs, which were specially prepared for this publication are, as a result of recent technical advances, of higher quality than those appearing in the original publications. All were prepared from tissues fixed in osmium tetroxide buffered with acetate-veronal buffer to pH 7.4 and em- bedded in n-butyl methacrylate. Sections of 20 to 50 m/x were prepared with a Porter-Blum micro- tome (Servall) and examined and photographed in an RCA EMU-2 electron microscope. Further technical methods are detailed elsewhere (Farqu- har, 1956). channels to form a complicated network (Palade, 1956). The system is enclosed by a continuous membrane which is probably similar to and derived from the cell mem- brane (Howatson and Ham, 1957). The endoplasmic reticulum varies considerably in extent and form in different cells. When it is extensive the cross-sections of its mem- brane-enclosed cavities may occupy the greater part of the cytoplasm. The mem- branes are not revealed by any staining methods used in light microscopy and the cavity usually contains no stainable con- tent. In consequence light microscopy gives little indication of the existence of the endo- plasmic reticulum. The larger vesicles which form part of the endoplasmic reticulum of the basophil cells of the rat pars anterior are, however, visible in paraffin sections ex- amined by light microscopy and confer on the cytoplasm a foamy appearance which was noted by Reese, Koneff and Wainman in 1943. Under conditions of rapid secretion, hyaline substance accumulates in these vesicles which become much more easily visible by reason of their enlargement and the presence of a stainable content. That hyalinization is an accumulation of a stain- able material in cytoplasmic vesicles which are always present but normally empty was first stated by Reese, Koneff and Wain- man and confirmed by the electron micro- scope studies of Farquhar and Rinehart (1954a, b). Electron microscopy did not therefore provide the first evidence for the existence of the endoplasmic reticulum, but it showed for the first time its full extent, its continuity, and its existence in all types of cells. B. THE GOLGI REGION OR ZONE The Golgi region or zone has long been a focal point of discussion by investigators of pituitary morphology and function (Sev- eringhaus, 1932, 1933, 1939). In suitable preparations studied by< light microscopy it is often seen as a conspicuous feature of granulated cells in the pars anterior, this specialized region of the cytoplasm being made evident by the absence from it of the granulation which is distributed throughout the remainder of the cytoplasm. In sections which are sufficiently thin, an unstained zone is seen producing an appearance which 214 Tn poPHYSIS AND rxONADOTROPHIC HORMONES ' A / lri"«X cm Fi(i.3.25. l^lcriron microjir.'il'ii sliowin^ a jionioii ot an aci(loi>iul irom rhc a(lonohy]loplly^ of a rat. Part of the nucleus (AO is present above and a segment of the cell membrane (cw) crosses the field below. A few dense ovoid secretory granules {gr) are scattereti throughout the field. Several mitochorndia (w) are also seen. They show double-layered limiting membranes and internal crests {cr) or "cristae mitochondriales" of Palade (1952) which are characteristic features of all mitochondria. The endoplasmic reticulum (er) is seen in the lower portion of the cell. This cytoplasmic component occurs here in the form of parallel rows of long, membrane-limited sacs. When reconstructed in three dimensions the sacs are continuous with one another at the ends, and the membranes thus enclose a broad flat cavity. These appearances represent just one or- ganizational variant of this highly complicated system (Palade and Porter, 1954). A number of tiny dense particles {hji) (ca. lOOA) are distributed throughout the cytoplasm, but are porticularly concentrated along the membrane surfaces of the endoplasmic reticulum. These particles are generally considered to be the site of localization of cytoplasmic ribonu- cleoprotein (Palade, 1955). Components of the Golgi apparatus can also ])<> identified in tlic cytoplasm. In electron micrographs the Golgi "complex" may be resohed into 3 coniiionents: relatively empty- ajipearing vacuoles (t'oc) of varying sizes (Dalton and Felix. 1954; Sjiistrand and Hanzon, 1954; Farquhar, 1956); paired membranes (Gm) (ca. 7 m^ each) (Palade, 1952) ; small gran- ules or vesicles (Gu) (ca. 40 m^) (Palade and Porter, 1954). X 31,500. HYPOPHYSEAL MORPHOLOGY 215 / m N N cm N / <-.£»■ Fig. 3.26. Electron micrograph showing cells from the anterior pituitary of a young adult male rat. Three acidophils of the type which are thought to be responsible for the production of growth hormone (Hedinger and Farquhar, 1957; Farciuhar and Rinehart, 1954a) occupy most of the field. Their nuclei (AO are indicated. This type of acidophil is characteristically rounded or ovoid in shape, and the cells typically are arranged in groups, as shown here. In electron micrographs the most distinctive feature is their content of variable numbers of dense, ovoid secretory granules (grr) of a characteristic size (ca. 350 ni/u maximal diameter). Large numbers of secretory granules are present in this field. The cell membranes {cm) can be clearly seen separating the cytoplasm of one cell from that of another. Mitochondria (m), endoplasmic reticulum (er), and Golgi material (G) may also be distinguished. X 10,300. is termed "the negative image of the Golgi body." The negative image of the Golgi body or zone is especially conspicuous in the basophil cells of the rat pars anterior. In acidophil cells the Golgi body is usually smaller and the negative image is not seen unless the sections are thin, i.e., 2 to 3|U.. When acidophil cells are stimulated their Golgi zones become enlarged and the nega- tive image may be seen more easily. This happens in the rat hypophysis after estrogen treatment or at times when rapid secretion of the lactogenic hormone is occurring phys- iologically. The Golgi region has the ap- pearance of a spheroidal shell enclosing an area of cytoplasm. In the basophil cells of the rat pituitary, the cytoplasm enclosed by the Golgi region is more deeply stained than 21G HYPOPHYSIS AND GONADOTROPHIC HORMONES cm •m N 1* - 7-^l»,*!»^» « . cm -@r / fjdUms. ~^^. nti Fig. 3.27. Electron micrograph (if , I .-((imn iinn, il,, nm i lor ])ituitary of :i imini.-il adult female rat showing an acidojihil iti ihe i\ pu uJneh j> ihuught to be re.^pun.^iMc for the production of mammotrophic hormone (Hedinger and Farquhar, 1957; Farquhar and Rine- hart, 1954a). The nucleus (A^^) and the cell membrane (cm) of the mammotroph are shown. These cells are typically found alone, rather than in groups, in the normal, nonlactating animal. Their most distinctive feature in electron micrographs is their cytoplasmic content of very large, dense secretory granules (gr) with a maximal diameter of 600 to 900 m/i. In this cell they are predominantly found grouped to the left of the nucleus (N). The granules ap- pear very dense and do not show evidence of internal structure. Their appearance is in con- trast to that of the mitochondria (m) which are usually more elongated, much less dense, and show clear internal structure which is difficult to see in detail at this relatively low magnifica- tion. Tubular and cisternal (elongated) profiles of the endoplasmic reticulum (er) as well as vacuoles of the Golgi complex (G) may also be identified in the cytoplasm. The two areas marked A represent segments of the cytoplasm of two adjacent acidophils of the type which are presumed to be responsible for the production of growth hormone. The smaller size of the secretory granules distinguishes these cells from the mammotrophic acidophil. A portion of the nucleus of a thyrotroph (T) is seen to the right. Cytoplasmic processes of this cell extend out from the nucleus to encircle partially the mammotroph. The cell may be identified as a thyrotroph on the basis of its angular shape and content of very small secretory granules. In the lactating animal acidophils of this type with large secretory granules are very numerous and can be seen in virtually every field (Hedinger and Farquhar, 1957). X 11,700. HYPOPHYSEAL MORPHOLOGY 217 sKS.^;:*1 Fig. 3.28. Electron micrograph showing a th.yrotroph from the anterior pituitary of a young adult male rat. The nucleus (A') and cell membranes (cm) are indicated. The irregular contour characteristic of thyrotrophs is illustrated in the angular shape of this cell. In electron micrographs thyrotrophs can be distinguished by virtue of the size of their secretory granules which are smaller (maximal diameter ca. 100 iu/m) than those in any other type of anterior lobe cell (Farquhar and Rinehart, 1954b). In this cell the secretory granules are found, for the most part, lined up along the cell membrane. As seen in this cell, the endoplasmic reticulum (er) of thyrotrophs is generally present in the form of small vesicular profiles with occasional elongated (cisternal) profiles. The mitochondria (?n) usually occur in the form of short rods and show a background matrix which is much less dense than the internal matrix of gonadotroph mitochondria (see Figs. 3.29 and 3.30). A group of vacuoles of the Golgi complex (G) can also be distinguished in the cytoplasm of the thyrotroph. The thyrotroph is virtually surrounded by acidophils of the growth hormone type. The cytoplasm of these cells is labeled A. The size of their secretory granules (maximal diameter ca. 350 m/i) can be contrasted with the smaller thyrotrophic granules. X 10.000. the rest of the cytoplasm. The PAS reaction shows that this is due to a greater concen- tration of glycoprotein granules in the en- closed cytoplasm. The appearance of the darkly stained cytoplasm within the nega- tive image of the Goki body has not always been interpreted correctly and some investi- gators have mistaken it for an early stage of hyalinization. Severinghaus (1932, 1933) reported that in the rat the Golgi apparatus of the cells of the acidophil class has a different form 218 HYPOPHYSIS AND GONADOTROPHIC HORMONES / cm N nc '.?i' « " ■ ,*/ »•,■ •'^f •i M> *# i 1 '♦• * ' • ' 1 ¥■ ** ^ « . '•"•T -^ymCsi^'* «..' *•*' v* *#.f.. v*v /;•*••.-, .'/.€. "/:- *• ,••.'■ * - -^.'K- .. ^ l^li -."*#'i§fc'^^5#ff. Fig. 3.29. Electron microgmph allowing a \li>- Lugr guu:uloii(ii>li iium iln ,1111(1101 pituitary of a young adult male rat. The nucleus (iV), a nucleolus {nc), and the cell membrane {cm) are shown. This cell can be identified as a gonadotroph by virtue of its rounded contours and content of secretory granules {gr) with a maximal diameter of approximately 150 m^. The secretory granules of gonadotrophs are intermediate in size between the large secretory granules of acidophils and the small secretorj^ granules of thyrotrophs. A spherical chain of small vacuoles {vac) circumscribes the Golgi apparatus which is located above the nucleus. Elements of the Golgi complex outline a cytoplasmic area nearlv as large as the nucleus. Mitochondria (m) which have been sectioned in various planes are also visil)l(> in the cytoplasm. The mitochondria of gonadotrophs are generally more elongated and show a denser internal background matrix than other types of adenohypophyseal cells. The endoplasmic reticulum {er) is seen here in the form of numerous vesicles which vaiy greatly in size. Some are relatively small and are of a size approaching that of the secretory granules. Others are rather large, for they measure several microns across at their greatest width. It can be seen that the intermmi of the vesicles appears homogeneously grey, and has a background density greater than that of the siuTOunding cytoplasmic matrix. Gonadotrophs with this appearance have been associated with the secretion of follicle- stimulating hormone (Farquhar and Rinehart, 1954a: Farduhar and Rinehart, 1955). They differ from the luteinizing hormone-gonadotroph (see Fig. 3.30) in possessing somewhat paler nuclei, more evenly distributed granules, and prominent vesicles of the endoplasmic reticulum with the homogeneous grey internum. X 6500. HYPOPHYSEAL MORPHOLOGY 219 \' A %-. /' cm *^y^ ^ \A VQC •'.cm V* 0 * ^t ^ •' * -u .«"«' Fk;. 3,30. Election micioamphs illustiatiug another gonadotroph I'lom tlie anterior pitui- tary of a young adult male rat. Like the cell in Figure 3.29, this cell can be identified as a gonadotroph on the basis of its rounded contours, the size of its secretory granules (maximal diameter ca. 150 m^), and elongated mitochondria {m) with a dense internal matrix. The area occupied by the Golgi complex is seen directly to the right of the nucleus. It is out- lined by a number of relatively empty-appearing vacuoles {vac). This gonadotroph differs from the follicle-stimulating hormone gonadotroph illustrated in Figure 3.29 in several respects: the nucleus (iV) is more dense and shows a deep infolding, the secretory granules {gr) are aggregated into clumps, and no large vesicles of the endo- plasmic reticulum are present. In addition, there are a number of relatively open areas visible in the cytoplasm (arrows) which are occupied only by a sparse, flocculent precipitate. The endoplasmic reticulum {ex) is seen here in the form of tiny tubular profiles. Gonado- trophs with these features have been associated with the secretion of luteinizing hormone or interstitial cell-stimulating hormone (Farquhar and Rinehart, 1954a; Farquhar and Rine- hart, 1955). A portion of an acidophil (^-1) with larger secretory granules is present above the gonado- troph. X 11,700. 220 HYPOPHYSIS AND GONADOTROPHIC HORMONES cm / N N t \ ' ^ cm mv : m bp cm •• --mv % : * 5 ■ Fig. 3.31. Electron iiiici(j im.hious of sc\cial ( ( IN wlml; uii. 1,,, mix- suggested to be concerned with the formation of adrenocorticotrophic liormone (1^'arquhar, 1957). Two large nuclei (iV) and a segment of a third nucleus are shown. Such cells are typically found in groups and are arranged around large follicles or smaller ductiles. Some of the follicles are quite large, measuring several microns across and are undoubtedly analogous to so-called "colloid cysts" sometimes seen in the anterior lobe by light microscopy. Other follicles, such as the one illustrated here, are quite small and would probably escape detection by light microscopy. The cells which line the follicles or ductiles typically show tiny cytoplasmic projections or microvilli {mv) which pro.ject into the follicular lumina. In this field portions of three cells abut on the follicle and form microvilli which pro,)ect into the lumen. The follicular cells characteristically do not contain secretory granules. Furthermore, in the normal animal their cytoplasm appears relatively empty, for organized cytoplasmic structures are sparse. Only a few mitochondria {m), occasionally tubular profiles of the endoplasmic reticulum (er), and basophilic particles {hp) are encountered. In terms of their somewhat monotonous regularity, these cells resemble more closely the cells from the intermediate lobe than they do any other type of anterior lobe cell. Because of the response of these cells to alterations in adrenal activity and their lack of response to other experimental procedures, it has been tentatively suggested (Farquhar, 1957) that these cells are responsible for the formation and/or transport of corticotronhin. X 10,800. HYPOPHYSEAL MORPHOLOGY ■^c \ ^f f?- 8* 221 'i '"" *" 54r .- ^' ^" * ."■■-^ N, N cm N er H Fig. 3.32. Election micrograph showmg a field of cells from the pars intermedia of the rat pituitary. The plane of section passes through the nuclei (A^) of four of the cells, whereas only the cytoplasm of several other cells is transected. The cell membranes (cm) stand out prominently. These cells are seen to fit together like a mosaic, and they all show a similar appearance. Their cytoplasm contains relatively few secretory granules (gr) and mitochondria (m). The endoplasmic reticulum (er) is present in the form of tiny vesicular profiles with occasional tubules. Elements of the Golgi complex (G) are visible in several areas. In these cells the dense, paired Golgi membranes and granules predominate over Golgi vacuoles, which are not seen at all in this field. X 9100. from that of the cells of the basophil class. In the acidophil cells the Golgi apparatus forms a net-like cap extending over one pole of the nucleus, whereas in the basophil cells it has the appearance of a spheroidal body lying in the cytoplasm at some distance from the nucleus. These characteristic dif- ferences were confirmed by Foster (1947) using the Sudan black staining method. Among the chromophobes some cells have Golgi bodies similar to those of the granu- lated acidophils, whereas in others they are similar to those of the granulated basophils. This suggests that the chromophobic cells are not undifferentiated, rather that they consist in part at least of temporarily non- functioning but specifically differentiated cells. HYPOPHYSIS AND GONADOTROPHIC HORMONES The characteristic appearance of the Golgi body of the "normal" acidophil cell of the rat hypophysis is not retained through all the phases of activation which may be experienced by this cell. Wolfe and Brown (1942j found that in the hypophysis of the rat treated with estrogen the Golgi body of the acidophil cells enlarged and be- came more or less spheroidal in shape so that acidophil cells could no longer be dis- tinguished from basophil cells on the basis of the Golgi body appearance alone. The change of the acidophil type of Golgi body from the paranuclear net to a rounded body is not confined to conditions of strong acti- vation but is also seen after castration when the acidophil cells are undergoing a reduction in number and a shrinkage in cell and nuclei size. In mammals other than the rat distinct differences in the form of the Golgi body in different cell types have not been described, although there are distinct differences in the relative size of the Golgi bodies in different cell types. In the lizard, Anolis carolinensis, (Poris, 1941) the Golgi bodies in the acidophil and basophil cell classes are different, but in this species, unlike the rat, the type of Golgi in the acidophil cells is the more compact one. Electron microscopic studies of thin sec- tions reveal that the Golgi zone is a system of smooth membranes enclosing spaces which appear to communicate with the endo- plasmic reticulum. In Palade's (1955) ac- count of the fine structure of nerve cells, the components of the Golgi region were termed "agranular reticulum." There is an association of three distinct structures: (1) A system of double membranes formed by the apposition of flattened vesicular struc- tures. (2) A system of microvesicles with a dense content. (3) Large empty mem- brane-enclosed spaces ("vacuoles") re- sulting from the section of a hollow struc- ture with a watery content. The relationship of the three comi)onents to one another and to the classical Golgi apparatus or network is not entirely clear but it is likely that the "vacuoles" aw cross- sections of an anastomosing canalicular sys- tem (Dalton and Felix, 1953; Lacy, 1954) which is responsible for the apiiearance of the negative image of the Golgi zone. The demonstration of a network by osmium im- pregnation (Nassonov, 1923) is in the main the result of precipitation of osmium outside the canalicular space particularly at the site of the system of double membranes (Dalton and Felix, 1956; Gatenby and Lufty, 1956). Electron micrographic studies have clari- fied to some degree the relationship between the Golgi zone and secretory activity. The Golgi zone is not a region in which syn- thesis of proteins or peptides occurs. The Golgi complex appears to be concerned rather with the segregation or removal of water from maturing secretory products (Dalton and Felix, 1956) and the enclosure of the mature product in membranes to form secretory granules (Farquhar and Wellings, 1957) . The establishment of other relationships awaits other efforts. Partic- ularly promising are the techniques devel- oped by Peterson (1957) for identifying in stained sections the same fields seen in elec- tron micrographs of the adjacent section. C. PALADE GRANULES Palade granules are dense bodies 10 to 15 m/x in diameter. They occur to some extent either singly or in clusters in the cytoplasm but are for the most part attached to the membrane of the endoplasmic reticulum forming in those regions where tliey occur a "rough-surfaced" membrane (Palade, 1955, 1956). Although much too small to be visible as granules under the light micro- scope, their existence, although not their form, can be made evident by specific stain- ing. The granules contain ribonucleic acid and are therefore responsible for cyto- plasmic basophilia. They figure in the lit- erature of light microscopy as a substance, cytoplasmic ribonucleic acid or Nissl sub- stance, and not as a structure. In certain secreting cells in which rough-surfaced membranes occur as closely i)acked, flat- tened sacs they are responsible for the ap- pearance termed "ergastojihism" by Gar- nier (Pahide. 1955) . n. SECKKTORV (iRAXT'LES Secretory granules arc splici-oidal niem- bi-ane-encloscd Ixxlics with a dense homo- licncDUs coiitciit. The granules in dillVrent HYPOPHYSEAL MORPHOLOGY 223 cell types differ in size and in the density of their content. The specific differences in the chemical nature of the contents of secretory granules in different cells cannot yet be made apparent by electron microscopy. The electron microscope emphasizes the essen- tial similarity of the specific granules of acidophil and basophil cells and indeed of secretory granules generally whether in endocrine or exocrine cells. Lipid droplets are seen in electron micro- graphs as dense bodies, their density being no doubt the result of precipitation of os- mium by the reducing action of the lipid. They are irregular in outline, probably as a result of solution of the lipid and distortion during dehydration and embedding. The lipid droplets do not have an enclosing membrane. Farquhar and Rinehart have identified in electron micrographs the five granulated cell types which can be distinguished by light microscopy in the pars anterior of the rat. All five contain the same cytoplasmic structures; there is no distinctive structural element peculiar to any of the types. There are differences in the shape of the cells, in the extent and form of the endoplasmic reticulum, and in the size, density, and dis- tribution of the secretory granules which enable the different types to be distin- guished from one another. The secretory granules of the basophil cells are less than 200 mfx in size. Although such granules can be seen individually under good optical conditions with the light microscope, the coarsely granular or floc- culent appearance of the cytoplasm of baso- phil cells in stained sections is produced by the uneven distribution of the fine granules which are not individually resolved. The "basophil granules" of the basophil cells of the rat pars anterior as seen under the light microscope therefore bear the same relationship to the secretory granules as the Nissl granules of nerve cells bear to the Palade granules ; they are in fact clusters of granules made visible by the staining of their specific content. E. MICROVILLI In addition to the granulated cells the pars anterior of the rat contains cells with a distinctive structural feature, namely, microvilli projecting from the free surface of the cell into a space which is enclosed by contiguous cells of the same type. These cells are not accessible to study by light microscopy and their appearance is so dif- ferent from that of known endocrine cells that their function must be admitted to be problematical. XV. The Neurohypophysis and Neuro- hypophyseal Secretion A. NEUROSECRETORY PHENOMENA IN THE HYPOTHALAMUS AND NEUROHYPOPHYSIS Neurohypophyseal tissue contains con- nective tissue, nonmedullated axons, and interspersed neuroglial cells. In the neural eminence and neural stalk the axons run more or less parallel to one another and, although some of the axons terminate in these regions, most of them pass into the neural lobe where they end in the pars ner- vosa. The terminal enlargement which forms the neural lobe results in part from the branching of the axons in the terminal part of their course, in part from the increased number of glial cells in this region of the neurohypophysis. In fish and amphibia the axons in the neurohypophysis arise from cells in the pre- optic nucleus in the hypothalamus and form the preopticohypophyseal tract in the intra- hypothalamic part of their course. In reptiles, birds, and mammals the homologue of the preoptic nucleus is separated into two parts, the supraoptic nucleus and the paraventricular nucleus. The axons of the neurons in the paraventricular nucleus pass towards the supraoptic nucleus and after running through or closely alongside this structure join the supraopticohypophyseal tract (Laqueur in discussion, Scharrer and Scharrer, 1954) . Lesions which are produced experimentally to destroy the supraoptic nuclei or interrupt the supraopticohypo- physeal tract also interrupt the paraven- triculohypophyseal fibers and produce a total neurohypophyseal deficiency. It there- fore appeared at one time that neurohy- pophyseal activity in mammals was en- tirely dependent on the supraoptic nuclei alone, but it is now clear that the paraven- 224 HYPOPHYSIS AND GOXADOTROPHIC HORMONES tricular nuclei have the same function as the supraoptic nuclei, being spatially sepa- rated parts of the same functional unit. The organ responsible for neurohypo- physeal secretion is thus more extensive than the neurohypophysis. It includes in addition the supraoptic and paraventricular nuclei and their tracts. Functionally we may distinguish four separate regions in this hypothalamo-neurohypophyseal sys- tem. The first region contains the supra- optic and paraventricular nuclei in which the neurohypophyseal secretion is elabo- rated. The second contains the paraven- triculo- and supraoptico-hypophyseal tracts which serve two functions, the conduction of nerve impulses and the physical trans- port of neurohypophyseal secretion into the neurohypophysis. The third region is the tissue of the neural eminence, neural stalk, and in some species a portion of the neural lobe, which are in this account referred to collectively as the pars eminens. In this tissue some of the axons terminate, usually in relation to blood vessels (Scharrer and Scharrer, 1954; Scharrer, 1954) and medi- ate a secretory activity which could by vir- tue of the vascular link between this region and the pars anterior or pars distalis, mod- ify the function of the adenohypophysis. In some amphibia a very considerable pro- portion of the preopticoneurohypophyseal fibers end in this region (Dawson, 1957). In this region there are also nerve fibers from the general area of the medial fore- brain bundle and fibers from the lateral tuberal area (Green, 1951, 1956; Bargmann, 1954). The fourth division of the hypo- thalamo-neurohy])ophyseal system is the pars nervosa which in mammals contains most of the nerve fiber terminations of the neuroiiypophyseal tract and most of the stored secretion. It is also the major site of the release of the characteristic hormones which pass from it by way of systemic veins directly into the systemic circulation with- out contact with the pars anterior or pars distalis. The early investigators assumed not un- naturally that the pars nervosa was the site of elal)oration of the hormones which were secreted from it. It is obvious that there must be something unique about the nerve fibers or the glial cells, or else some unique structural element must be present to ac- count for the secretory function. Herring (1908) considered that the cells present were ordinary glial cells and did not note any peculiarity of the axons other than that they were of larger caliber than non- medullated fibers elsewhere. He, however, did discover some peculiar masses of protein without any definite structure. These bodies were only found in well fixed material and although they did not look to be likely sites for the formation of hormones attention was focused on them by the fact that they were peculiar to this gland. Bucy (1930) found that these Herring bodies were in fact large end bulbs terminat- ing some of the fibers of the neurohy- pophyseal tract. In these end bulbs the fibers were wound about like a ball of twine. Bodian (1951) confirmed this explanation of Herring bodies and considered that they are terminal malformations, negligible in number compared with normal endings. Bucy (1930) and Weaver and Bucy (1940) claimed that the neurohypophysis contained cells which were peculiar in their morphology and staining reactions and were found nowhere else in the nervous system. For these cells the term "pituicyte" was proposed. For a time the pituicytes con- tended with the Herring bodies and the nerve fibers for the responsibility for the production of the neurohypophyseal hor- mones. As objects to be studied they were certainly more promising in appearance than the nerve fibers, but it cannot be said that the studies of pituicytes have con- tributed to the elucidation of either neuro- hypophyseal or pituicyte function. The sta- tus of the pituicyte as a cell peculiar to the neurohypophysis is at the present time doubtful. Some investigators (Romeis, 1940; Slopcr. 19571 have stressed the great diver- sity of c(^ll types which can be found in the neurohypophysis, but this does not disi)ose of the i)ituicyte of Bucy. Leveque and Scharrer (1953) considered the existence of a cell specific to the neurohypophysis un- |)i'o\-cn. ilild ( I95(). in discussion I considcri'd tliat there are no consistent morphologic difTer- cnces between the glia of the neurohy- HYPOPHYSEAL MORPHOLOGY pophysis and astrocytic glia in any otlier parts of the central nervous system. Cer- tainly the pituicytes do not show any of the features of secreting cells. Recent de- velopments have made it clear that pitui- cytes are in no way responsible for the production or storage of neurohypophyseal secretion and any role that may be ascribed to them in facilitating the release of the hormones into the circulation is purely speculative. It seems unnecessary to assume that their function is different from that of similar glial cells elsewhere. The hormone activity of the neurohy- pophysis is accounted for by two octapep- tides, oxytocin and vasopressin. Both hor- mones contain cystine. There is evidence that in the glands they are combined with a specific protein referred to as the van Dyke protein (van Dyke, Chow, Greep and Rothen, 1942) . This protein has a molecular weight of approximately 30,000 and a strik- ingly high content of cystine (approxi- mately 16.0 per cent; the cystine content of insulin is 12.0 per cent). The morphologic studies of the pars ner- vosa which followed Bucy's designation of l)ituicytes as specific cells peculiar to the neurohypophysis did not contribute to the elucidation of neurohypophyseal function. The true explanation of the function of the neurohypophysis resulted from studies on neurosecretion which were proceeding con- temporaneously with the pituicyte investi- gations but were not, before 1949, considered to be related directly to neurohypophyseal secretion. "Neurosecretion" is a general term refer- ring to the presence within certain neurons of accumulations of products, usually pro- teins, which are not common to the major- ity of neurones. The first description of 8 secretion-containing nerve cells was that by Speidel (1919) who described such cells in the spinal cord of the skate. The further development of the subject is amply re- viewed by Scharrer and Scharrer (1940, 1945, 1954). Neurosecretory accumulations in tb.e hypothalamus were first described in the bony fish (Phoxiims laevis L.), and further papers by the Scharrers extended their occurrence to bony fishes generally, amphibians, reptiles, mammals, and man. The term "neurosecretion" was first applied by Scharrer in vertebrates, but it is a gen- eral term including a wide variety of secre- tory phenomena in vertebrate and inverte- brate neurones. With the investigations on neurosecretory material in invertebrates we are not here concerned, and further dis- cussion will be limited to the neurosecretory material of certain neurones of the verte- brate hypothalamus. Neurosecretion was first investigated by nonspecific methods of staining, and the evidence for the presence of a secretory product in neurones was the accumulation of protein material in large droplets and vesicles often so large as to cause distortion of the cell within which they were enclosed. Investigations by Scharrer and Scharrer (1940), disclosed a wide occurrence of such gross accumulations of an unusual material in neurones of the preoptic or supraoptic and paraventricular nuclei of many verte- brates, but the absence of any known func- tion for this material was a retarding factor in the development of the subject. More- over, man}'- species did not appear to have tliese neurosecretory droplets and in species where they were found they were often not observed in young animals but only in those of older ages. These observations seemed to make it unlikely that any es- sential function was mediated by this ma- terial. Palay (1943) was able to trace a transport of neurosecretory material along the axons of the preopticohypophyseal tract in fishes, but with the staining methods then in use it could not be shown that this was a general phenomenon in all vertebrates. This position has been entirely changed by the development of more specific methods for staining neurosecretion which permit its identification, even in traces, by staining methods which demonstrate the specific quality of the protein instead of the earlier method of recognition which depended on the physical form assumed by gross col- lections of the material. Bargmann (1949) found that Gomori's (1941) method for staining the insulin-con- taining granules of the ^-cells of the pan- creatic islets also stained intensely the accumulations of neurosecretory protein in the vertebrate hypothalamus. The intro- 226 HYPOPHYSIS AND GOXADOTROPHIC HORMONES duction of this clirome-alum hematoxylin staining method revolutionized both the technical side of investigations of neuro- secretion and the interpretation of its sig- nificance. With this method it became clear that the grosser collections of neurosecretion detected by earlier methods, which occur only in an erratic fashion, constituted but a small part of the hypothalamic neuro- secretion which is found in neurones of every vertebrate hypothalamus (Bargmann and Hild 1949; Hild, 1950; 1951a, b; Barg- mann, Hild, Ortman and Schiebler, 1950). The neurosecretory material could be seen throughout the course of the supraoptico- and paraventriculo-hypophyseal tracts and their continuations in the neural eminence and stalk into the pars nervosa (Fig. 3.33). The greater part of the stainable neuro- FlG.3.33(w/^/n, ). (■(.rnii;,! ,M.-n.,n ..I il.c I,n|m,|,|,- ysis of a normal rat stained with dilute uldehydc- fuchsin (AF) after permanganate oxidation. The pans nervosa contains a large amount of darkly stained neurosecretion. P.N. pars nervosa; P.I., pars intermedia; PA., pars anterior. KMnOi , AF, X50. Fig. 3.34 (loirer). Coronal section of tlie liypojili- ysis of a rat which was deprived of drinking water for 5 daj^s. An extreme reduction in the content of neurosecretion is apparent in comparison with Fig- ure 3.33. Key to lettering as in Figure 3.33. 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B., AND LiviNGooD, S. C. 1935. The pituitary in experimental cretinism: structural changes in the pituitaries of thyroidectomized rats. Am. J. Med. Sc, 190, 145-157. Zeiner, F. M. 1952. Pituitary gonadotrophic fluc- tuation during pregnancv in the rat. Anat. Rec, 113,255-267. PHYSIOLOGY OF THE ANTERIOR HYPOPHYSIS IN RELATION TO REPRODUCTION Roy 0. Greep, Ph.D. PROFESSOR OF ANATOMY, HARVARD SCHOOL OF DENTAL MEDICINE, BOSTON I. Introduction 241 II. The Hypophyseal CioNADOxROPHiNS. . . 241 A. Follicle stiiniilatiiig Hormone 242 1. Ch(Mnic;il f(.;i lures 242 2. Physiologic criccts in females 243 3. Physiologic etfects in males 244 4. FSH in relation to compensatory gonadal responses 244 5. Assay 245 B. Luteinizing Hormone (Interstitial Cell-stimulating Hormone) 245 1. Chemical features 245 2. Pln'siologic effects in females 246 3. Physiologic action in males 246 4. An extragonadal activity 247 5. FSH and LH in relation to estrogen secretion 247 6. FSH and LH, interactions 247 7. Assay 248 C. Luteotrophin (Prolactin) 249 1. Chemical features 249 2. Physiology of luteotrophic hor- mone (prolactin) 249 3. Detection and assay of prolactin activity " 250 III. Pituitary Gonadotrophic Hormone Content and Related Evidence of Secretory Activity 250 A. Phylogenetic Considerations 251 1. Ascidians 251 2. Fish 252 3. Amphibians 252 4. Reptiles 253 5. Birds 253 6. Mammals 254 7. (Jeneral considerations 255 li. Age, Sex, (Jonadectomy, and Repro- ductive Rhythms in Relation to Pituitary C.onadotrophins 256 1. Fetal gonadotrophins 256 2. Age and sex 256 3. (Jonadectomy 258 4. Reproductive rhythms 259 C. Effect of Dietary Restrictions on Pi- tuitary Gonadotropliic Potency and Function 261 1. Underfeeding 261 2. Vitamin deficiencies 262 3. Deficiency in intake of protein or of specific amino acids 262 IV. Gonadal-hypophyseal Interrelation- ships 263 A. Immaturity 263 B. Puberty and Maturity 264 C. Effects of Estrogens on Follicle-stimu- lating Hormone Secretion 265 D. Effect of Estrogen on Luteinizing Hormone and Luteotrophin Hor- mone Secretion 267 E. Differential Effects of Gonadal Steroids on Follicle-stimulating Hormone and Luteinizing Hormone Secretion 268 F. Effects of Androgens on Pituitary Gonadotrophins 268 G. either Steroids and Oxidation Prod- ucts 271 H. Effect of Progesterone on Pituitary Gonadotrophic Functions 271 I. Neurohypophyseal Influences on Gonadotrophin Secretion 272 V. Anatomic Features Important to Modern Concepts of Pituitary Gonadrotrophic Function 272 A. Innervation of the Hypophysis . . 273 B. The Median Eminence and the In- fundibular Stem 274 C. The Hypophyseal Portal Circulatory System 275 i). The Hypothalamic and Hypophyseal Mediated Sexual Functions 276 1. Environmental stimuli 276 2. Electrical stimulation of the hyi)o- thalamus 278 3. Lesions in the hypothalamus 279 4. Transection of the hypophyseal stalk 280 VI. References 284 240 PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 241 I. Introduction The elucidation of the major functions of the anterior lobe of the pituitary gland will likely stand as an epic in the scientific achievements of the past half century. The series of discoveries which revealed that this part of the hypophysis exercises direct or indirect control over a wide spectrum of biologic processes opened a new physiologic frontier. In no part of this new territory was understanding to be encompassed more spectacularly than by the revelation that the reproductive processes of the Vertebrata as a whole are mediated by secretions of the pituitary gland. It is intriguing to dwell on the evolutionary emergence of this gland which has so wide a control over the growth and development of both somatic and geni- tal structures of the vertebrate body. That this regulator of so many organs and proc- esses arises embryologically from the lining of the oral cavity and that it bears an an- cient, and perchance functional, relation- ship to the primitive diencephalon is in- triguing. The significance of these matters belongs to the future, but of immediate con- cern are the relationships of the pituitary to sex functions and it is to these that at- tention is addressed in this chai)ter. The coverage of literature has mainly been restricted to the period since the last edition of this work. The number of papers published in these years is large in propor- tion to the concomitant advancement of the subject. Owing to interruptions of work in reproductive physiology during World War II tmd-the postwar emphasis on those aspects of pituitary physiology which relate to the adrenal cortex and the thyroid, in- terest in the study of gonadotrophic hor- mones waned. The papers cited, by no means a complete list, are important in one or more of the following respects: a pertinent con- tribution to knowledge, an intelligent cor- relation of significant physiologic data, use- ful theorizing, or a guide to the literature in this and related fields. In several in- stances reference is made to earlier works. These help to bring into perspective current information and thought.^ ' The following review articles, books and mono- graphs are especially pertinent and valuable : Hi- saw, 1947 ; Evans and Simpson, 1950 : Ershoff, 1952 : II. The Hypophyseal Gonadotrophins During fetal development and in infancy the gonads normally come under little or no important pituitary hormonal influences; a possible exception has been noted in the rabbit (.lost, 1951). Beyond infancy, how- ever, it is clear that the arousal of gonadal functions, including the slow prepubertal, as well as the more spectacular spurt of pubertal development, is entirely dependent on gonad-stimulating hormones secreted by the anterior lobe of the hypophysis. There has been extensive exploration of the ques- tion as to whether the hypophysis secretes one or more than one gonadotrophin. Al- though there is yet very little, if any, knowl- edge of what the hypophyseal cells actually secrete, there is substantial evidence for the present widely held belief that in the mam- mals, at least, the anterior hypophysis se- cretes three trophic substances which stimu- late and govern gonadal activity. These are: the follicle-stimulating hormone (FSH) ; the luteinizing hormone (LH) or interstitial cell-stimulating hormone (IC8H) ; and pro- lactin or lactogenic hormone, which has luteotrophic properties and has been termed luteotrophin (LTH). The chemical evidence for the existence of separate gonadotrophins was reviewed by Li and Evans (1948) and Li (1949). Ex- haustive expositions of the chemistry of the adenoliypophyseal luteotrophin, the first of the gonadotroi:)hins to be isolated, have been provided by White (1949) and by Li (19571. The work on FSH and LH was brought up- to-date in 1955 by Hays and Steelman. The results of 25 years of work in these areas leave no doubt that the various biologic ac- tivities which have been demonstrated in whole anterior lobe tissue can be segregated by chemical fractionation procedures. The fact that these activities are identifiable with separate single protein fractions does not provide evidence that they are in fact secreted in the form of proteins with which the biologic activities have been identified. Furthermore, it is important to realize that Nalbandov, 1953a ; Sayers and Brown, 1954 ; Benoit and Assenmacher, 1955 ; Cowie and FoUey, 1955 ; Benson and Cowie, 1957; Burrows, 1949; Parkes, 1952, 1956 : Harris, 1955 ; Chester Jones and Eck- stein, 1955: and Pickford and Atz, 1957. 242 HYPOPHYSIS AND GONADOTROPHIC HORMONES the pituitary fractions equated with separate gonadotrophins have been regarded as chemical entities on criteria of purity which have been undergoing revision as advances are made in protein chemistry. Several attempts have been made to find extractive procedures b}^ which it would be possible to obtain all or nearly all the pi- tuitary trophic hormones, including the gon- adotrophins, from a common batch of start- ing material (Fevold, 1943; Schwenk, Fleischer and Tolksdorf, 1943; Koenig and King, 1950). Although success in this under- taking would, of course, greatly enhance the potential supply of anterior hypophyseal hormones, such procedures have thus far not proved feasible primarily because of the great differences in the solubility character- istics of the several hormones. Ellis (1958), however, has been able to obtain follicle- stimulating, luteinizing, and thyroid-stimu- lating hormone (TSH) in good yield from common batches of frozen whole sheep pi- tuitaries. Studies have been made of the biologic activity of cell-particulate fractions ob- tained by ultracentrifugation of homoge- nized anterior hypophyseal tissue, but it has not been possible to relate gonad-stimu- lating activity to any specific cellular or- ganelle (McShan and Meyer, 1952; Brown and Hess, 1957). By the use of histochemical procedures, not in themselves definitive, the secretion of FSH and LH has been ascribed, respec- tively, to specific cell types in the anterior pituitary (see chapter by Purves). A. FOLLICLE-STIMULATING HORMONE 1. Chemical Features Chow, van Dyke, Clreep, Rothen and Shedlovsky (1942), working with swine pi- tuitary, obtained an FSH i)rei)aration that was free of other contaminating activities but was physiochemically heterogeneous. In 1949 Li, Simpson and Evans ol)tained an FSH fraction (ovine) that was free of othei- trophic factors and that behaved as a single protein by electrophoretic procedures and ultracentrifugation; it was not tested for constant solubility. The hormone FSH be- haves as a protein and there is both chemi- cal and histochemical evidence indicating that it is a glycoprotein. FSH from swine and sheep has been variously estimated on the basis of incomplete data to have an iso- electric point of 4.5 and a molecular weight of 70,000 (Li, 1949). That the carbohydrate residue in FSH may play an important role in physiologic activity is suggested by the finding that the FSH activity of anterior pituitary extracts is destroyed by salivary amylase and Taka- Diastase (McShan and Meyer, 1938; Abramovitz and Hisaw, 1939). The resist- ance of FSH to proteolysis, however, is nota- ble. In fact, McShan and Meyer (1938), Chen and van Dyke (1939), Chow, Greep and van Dyke (1939), and McShan and Meyer (1940) obtained considerable puri- fication of FSH by means of a selective in- activation or destruction of LH with crude trypsin. More recently, Steelman, Lamont, Dittman and Hawrylewicz (1953) and Steelman, Lamont and Baltes (1955, 1956) have used pancreatin digestion to prepare swine FSH having an activity of 2.5 to 9 times the Armour Standard (264-151X). In 1950 van Dyke, P'an and Shedlovsky advanced the purification of swine FSH to about 80 to 85 per cent "pure." Since then, Li and Pederson (1952), Steelman and his associates (1953, 1955, 1956), and Leonora, McShan and ]\Ieyer (1956) have made addi- tional improvements in the extraction, re- covery, and purification of FSH with in- creased specific activity. Steelman, Kelly, Segaloff and Weber (1956) described the preparation of an "ap- l)arently homogenous follicle-stimulating hormone" with 30 to 50 times the activity of Armour Standard. By placing highly purified FSH on diethylaminoethyl cellulose and utilizing gradient elution, they obtained a 4- to 5-fold concentration of activity as dctcrniined by the augmentation test of Steelman and Pohley (1953). Preliminary study of this FSH in the ultracentrifuge and l)y ])aper electrophoresis revealed no evi- dence of heterogeneity. The molecular weight was calculated to be 29,000 as op- ])Osed to previous estimates. Total carbo- liydrate content was 7 to 8 per cent and was comprised of 50 per cent hexosamine and detectable quantities of mannose, galactose, and fucosc. Tests for LH, TSH, adrenocorti- cotropliic hormone (ACTH), and somato- PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 243 trophic hormone (STH) were negative at the dosage level of 0.5 mg. of the FSH prep- aration. Latest among studies of purifica- tion of FSH is that of Ellis (1958). Using metaphosphoric acid and ethanol precipita- tion, he obtained a satisfactory concentrate of FSH which he was able to purify further by chromatography on diethylaminoethyl cellulose and starch electrophoresis at pH 4. This FSH preparation was electrophoreti- cally monodisperse, free of LH and TSH, and had 30 to 40 times the potency of Ar- mour Standard. The development of satisfactory proce- dures for the isolation of FSH has been an exceedingly difficult in-ol)lcm and most workers are agreed that the methods are not finalized and that no product thus far obtained has satisfied all the modern cri- teria of purity. Certainly, also, there are too few studies with the highly imrified prepara- tions to establish the biologic characteristics of this hormone. The difficulty in ascribing to FSH the status of a homogeneous chemi- cal compound is emphasized l)y the work of Steelman, Lamont and Baltcs (1955, 1956) and by Steelman (1958). Using jiancreatin digestion followed by chromatography on hydroxyl apatite, they obtained 3 FSH frac- tions, all with considerable relative activity, and are in doubt as to whether the ])re- chromatographed material can be regarded as homogeneous. There is no doubt, as we survey the verte- brates as a whole, that there is a pituitary factor immediately concerned in follicular maturatibiTancl to this extent follicle-stim- ulating hormone is a proper term. Caution should be exercised lest terminologic con- venience be confused with the fact. Never- theless, with modern methods of protein research it is very probable that a follicle- stimulating hormone will be isolated soon, and perhaps characterized as a polypei^tide with known amino acid composition and sequence. Whatever the precise nature of FSH, it is carried to its target organs by the blood. A preliminary study (Mc Arthur, Pennell, An- toniades, Ingersoll, Oncley and Ulfelder, 1956) of postmenopausal plasma fraction- ated by the cold ethanol method of Cohn has revealed that the gonadotrophic ac- tivity, pituitary in origin and presumed to comprise mainly FSH, is contained in Frac- tions II and III. This means that FSH as such may well be in combination with pro- teins not only in the pituitary but also in the blood stream. 2. Physiologic Effects in Females One activity of the anterior lobe of the pituitary brings about the maturation of egg-bearing ovarian follicles. In mammals this property resides in the follicle-stimu- lating hormone as we know it and, in lower vertebrates, in a follicle-stimulating compo- nent of the hypophyseal complex. In mam- mals FSH acts on the ovary to promote the development of primary follicles into large fluid-filled vesicular structures of the type that engaged the attention of de Graaf in his search for the ovum. Primary follicles* consist of a large oocyte surrounded by a single layer of flattened granulosal cells. As development proceeds, additional layers (6 to 9) of granulosa cells are proliferated to form a spherical mass with the ovum at the center. It is at this stage that the follicle • becomes overtly sensitive to the action of FSH. Its further development, including the secretion of the follicular liquor, the mitotic proliferation of granulosa cells, and the molding of surrounding stroma into an in- vesting layer of thecal cells, seems to be largely controlled by FSH. In all mammals studied, the growth and maturation of the ovum itself seems independent of the action of this hormone. In the monkey (Green and Zuckerman, 1947), rat (Mandl and Zuck- erman, 1952), and hamster (Knigge and Leathem, 1956) the ovum reaches its full size well before the appearance of the fol- licular antrum. FSH, by promoting follicular enlarge- ment, controls to a large degree the growth of the ovary. In immature animals injected with different amounts of FSH, the weight of the ovary can be taken as a measure of FSH activity, providing the hormone in- jected is pure. The size to which the ovary can be forced to develop in either immature or adult mammals seems limited only by the number of responsive follicles available. With excessive dosage over periods of 5 to 10 or more days, ovaries of massive size have been developed in many species of birds (Witschi, 1955) and mammals (Ca- 244 HYPOPHYSIS AND GONADOTROPHIC HORMONES sida, ]\Ieyer, ^NlcShan and Wisnicky, 1943; Dowling, 1949; ^Vlarden, 1952), including man (Davis and Hellbaum, 1944; Maddock, 1954). In the absence of luteinizing hor- mone, a situation insured by removal of the recipient animal's own hypophysis, FSH- stimulated ovaries present the appearance of being a mass of translucent cystic fol- licles. During a normal estrous cycle the number of follicles developing to maturity differs among species, but for any given spe- cies the number is quite constant (Brambell, 1956). Inasmuch as the size of the ovaries in the continuous (nonseasonal) breeder re- mains reasonably constant, it follows that the level of FSH stimulation must also be maintained at a similar norm. Follicular en- largement, which is brought about in large measure by the accumulation of antral liquor, occurs at a constant rate for any given species (Hisaw, 1947; Brambell, 1956). Accordingly, when ovarian enlarge- ment is induced with increasingly greater dosages of exogenous FSH, the growth of individual follicles is not accelerated but more and more follicles are brought to ma- turity; the number, however, is not pre- cisely proportional to the dose. The simul- taneous development of an excessive num- ber of Graafian follicles is the basis of the many demonstrations of artificially induced superovulation and superfetation in rats (Evans and Simpson, 1940), rabbits (Pincus, 1940; Parkes, 1943), sheep (Zawa- dowsky, 1941; Hammond, Jr., Hammond and Parkes, 1942; Casida, Warwick and Meyer, 1944), and cattle (Dowling, 1949; Hammond, Jr., 1949). The administration of FSH to immature animals hastens the maturation of the ovaries and leads to marked sexual precocity in all mammals tested, including the monkey (Simpson and van Wagenen, 1953) and cattle (Casida, Meyer, McShan and Wisnicky, 1943). In- asmuch as FSH activity may be restricted to the growth of follicles, accelerated pu- berty in the instances noted must have oc- curred in part through the concomitant se- cretion of endogenous gonadotrophins, among which LH would be pi'imarily neces- sary. That FSH secretion fluctuates rhythmi- cally during reproductive cycles in sexually mature female mammals is suggested bv the evidence of Simpson, van Wagenen and Carter (1956), showing an increase in the level of pituitary FSH content during the follicular phase of the menstrual cycle in monkeys. This is in general agreement with the older literature on other mammals (van Dyke, 1939; Smith, 1939). On the other hand, the possibility that rhythmic fluctua- tion in the secretion of gonadotrophin may not be confined to FSH is discussed below. 3. Physiologic Effects in Males In males FSH acts on the spermiogenic cells in the testes with the same selectivity that it exhibits for germinal tissues in the female gonad. The growth of the testes is predominantly due to FSH and is a re- flection of an increase in the size of the seminiferous tubules and of spermatogenic activity (Greep and Fevold, 1937; Creep, van Dyke and Chow, 1942; Simpson, Li and Evans, 1951). When FSH is adminis- tered to immature male rodents the size of the testes is greatly increased, but an ac- celeration of the appearance of fully formed spermatozoa has not been observed. True sexual precocity in males has not, therefore, been obtained, even when every other as- pect of the reproductive system is fully de- veloped. Nelson (1952) cited evidence for the view that FSH may be responsible only for the proliferation of spermatogonia and primary spermatocytes. He speculates that an androgenic influence may participate in the final stages of spermatogenesis. Testes developed under the stimulation of exogenous FSH exhibited no change in the number, appearance, or secretory ac- tivity of the intertubular cells of Leydig (Greep and Fevold, 1937; Greep, van Dyke and Chow, 1942; Simi)son, Li and Evans, 1951 ) . The unchanged male accessory sexual structures register clearly the fact that no androgen was produced as a result of the treatment. 4- I'SII in lielation to Conipeusatorij Gon- adal Responses A difl'erence in the responses of male and female gonads to FSH is that under ex- ogenous FSH stimulation the testes do not develop beyond their maximal normal adult size as do the ovaries. It has been noted that tlie sexes also differ in the degree of com- PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 245 pensatory hypertrophy which is elicited by unilateral gonadectomy, a response that is l)riniarily elicited by FSH. The observation by Carmichael and Marshall (1908) that unilateral ovariectomy leads to compensa- tory hypertrophy of the remaining ovary is now commonplace. Similar experiments on tiie male were not reported until 1940 (Addis and Lew). Compensatory gonadal hyper- t rophy is much less pronounced in the male ; the 50 per cent of testis mass remaining after unilateral gonadectomy increases only to 56 per cent of the weight of paired gonads of comparable controls, whereas the ovary increases up to 70 per cent. Clearly, in no instance is all the lost tissue regained through compensatory hypertrophy. It is of incidental interest in this connection that Edwards (1940) showed that unilateral cas- tration in adult male animals leads to a halving of the sperm count. In the uni- laterally ovariectomized female it is well established that the opposite ovary sheds at each ovulation the full number of eggs characteristic of the species (Asdell, 1924; Brambell, 1956). It is also of interest that in the unilaterally spayed rat the number of primary oocytes remains at the level nor- mal for one ovary (Mandl and Zuckerman, 1950), indicating that FSH, as suggested by most older studies, does not influence ovogenesis. 5. Assay In recent years the ability of FSH to synergize with human chorionic gonado- trophin {HCCPr has been utilized in the development of methods for the bioassay of FSH. The end point is weight of ovaries from immature rats (Evans and Simpson, 1950; Steelman and Pohley, 1953) or mice (Brown, 1955) which have also been in- jected with a constant and substantial amount of HCG. These methods are more sensitive than other, older assay procedures involving ovarian weight increase following injection of FSH alone into immature, in- tact rats or mice, but they are less specific than the assay based on ovarian weight in- crease (Evans, Simpson, Tolksdorf and Jen- sen, 1939) or re-establishment of minimal follicular growth (histologic) in hypophy- sectomized immature rats (Evans and Simpson, 1950). The methods of Steelman and Pohley and of Brown have clear ad- vantages over the indirect assay based on the weight of the uteri in FSH-injected, intact, immature mice as introduced by Klinefelter, Albright and Griswold (1943). Testis weight increments in hypophy- sectomized male rodents serve as a con- venient measure of FSH activity, provided, however, that the FSH is biologically pure (Greep, van Dyke and Chow, 1940; Simp- son, Evans and Li, 1950) . The activity of FSH in an aqueous me- dium is known to be augmented by a variety of substances. The explanation of the ef- fectiveness of these cofactors is unknown, but several of them such as heme (McShan and Meyer, 1941) and zinc and copper salts, probably merely delay absorption and pro- vide a more sustained effective blood level of the hormone. B. LUTEINIZING HORMONE (INTERSTITIAL CELL-STIMULATING HORMONE ) 1. Chemical Features Working independently, Li, Simpson and Evans (1940a, b) and Shedlovsky, Rothen, Greep, van Dyke and Chow (1940) isolated LH from sheep and swine pituitaries, re- spectively. The two preparations have simi- lar physiologic properties (Greep, van Dyke and Chow, 1942; Simpson, Li and Evans, 1942a, b ) , but they differ chemically (Chow, van Dyke, Greep, Rothen and Shedlovsky, 1942) and are distinguishable by immuno- logic methods (Chow, 1942). The sheep hor- mone had an isoelectric point of 4.6 and a molecular weight of 40,000, whereas that of swine had an isoelectric point of 7.45 and a molecular weight of 100,000. Ellis (1958) has recently achieved a considerable con- centration of LH activity, using cation ex- change resins. Squire and Li (1959), using a modified extractive procedure, chromato- graphic separation, and zone electrophoresis on starch, obtained tw^o equally active "ICH" proteins; one of these, "^-ICH," has been partially characterized: it has an isoelectric point near 7.3 and a molecu- lar weight (calculated) of approximately 30,000. This fraction had "a high degree of homogeneity," showed no evidence of con- tamination with other pituitary hormones, and was active by the ventral prostate test at a total dose of 0.5 /xg. 246 HYPOPHYSIS AND GOXADOTROPHIC HORMONES LH, like FSH, is a glycoprotein. The bio- logic activity of LH is far more resistant to glycolytic enzymes than is that of FSH ; its resistance to proteolytic enzymes, how- ever, is much less. The available preparations of LH seem to be more satisfactory than those of FSH in point of comprising a hormone as a chem- ical entity. However, LH, as indicated by the recent work of Squire and Li (1959), may in turn reveal its biologic activity in some small part of a protein complex. 2. Physiologic Effects in Females The many early studies based on the ac- tion of LH extracts injected into intact im- mature rats are uninformative, since the participation of endogenous gonadotrophins cannot be excluded nor safely discounted. Histologically, the action of LH has been detected with certainty in long-term hy- pophysectomized immature female animals by repair of the atrophic ovarian inter- stitial cells (Simpson, Li and Evans, 1942a, b». The ovarian manifestations of the action of LH administered to intact immature fe- male animals are notably inconspicuous. There is no increase in ovarian weight and no evidence that LH promotes the secretion of gonadal hormones. The interstitial cells appear a little more swollen than in un- treated animals, and in guinea pigs in par- ticular so-called pseudolutein bodies may appear. Conversely, the effect of LH on ovaries of adult rats are evident macro- scopically. There occur an excessive number of ovulated follicles, many hemorrhagic fol- lich's, and widespread luteinization of me- dium to large unruptured follicles, the ovaries being generally enlarged due to tlie synergism of the administered LH with en- dogenous FSH. In hypophysectomized immature female rats under treatment with FSH, the notable effect of introducing LH is to impose an intensification of the enlargement of the ovaries and to promote a sudden growth of the reproductive tract (Greep, van Dyke and Chow, 1942». That this latter effect is due to tlie initiation of estrogen secretion is uneciuivocal. The cells of the theca in- ktcrna which remain atrophic under FSH stimulation swell and ar(|uire the cytologic and histochemical criteria of actively se- creting cells only if LH, among the pitui- tary gonadotrophins, is administered. The importance of LH in bringing about the secretion of the ovarian hormone estra- diol-17/3 has been mentioned. The facts that the thecal cells are maintained in a stimu- lated condition and that continuous uterine growth occurs during the follicular phase of a reproductive cycle suggest that LH is secreted during this time. Other lines of evi- dence likewise indicate that LH not only may be secreted throughout each estrous or menstrual cycle, but that it may play a dominant role in sexual periodicity. As early as 1937 Dempsey expressed the view that, as a step in accounting for the estrous cycle, it is necessary to assume that fluctuations occur in the secretion of LH, thus bringing about ovulation and corpus luteum forma- tion. Indeed, McArthur, Ingersoll and Worcester (1958) reported a marked eleva- tion in the urinary excretion of LH by women at the midpoint of the normal ovula- tory menstrual cycle. This view is further supported by the fact of persistent estrus in rats and guinea pigs (for reviews of litera- ture on spontaneously occurring or induced persistent estrus, see Noumura, 1958 and chapter by Everett) in which the causative factor appears to involve an inability to elicit a discharge of LH by reflex neuro- genic mechanisms. Although meager study has been made of the ovulating property of pure LH, it is known to have this function (Greep, Chow and van Dyke, 1942; Hisaw, 1947). There seems little reason to suppose that the pure hormone would behave differently in this trespect from preparations having some FSH contamination. From information availa- ble, ovulation may, with reasonable assur- ance, be ascribed to a sudden elevation in the secretion of LH. Once ovulation has taken place, the conversion of the follicle into a luteinized body — a corpus luteum — is unquestionably attributable to LH action. 3. Phi/sioloiiic Action in Males The pi'imary cITcct of LH on the male gonad is to promote the maturation, main- tenance or i-epair of the interstitial tissue leading to the elaboration of androgenic hoi'mone iC.i'eep. Fevold and Hisaw, 1936; PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 247 Fraenkel-Conrat, Li, Simpson and Evans, 1940). This function is most clearly demon- strated by the injection of LH into hy- pophysectomized, immature male rats after liyi)ophyseal deficiency has resulted in marked testicular atrophy. In these animals the shrunken, spindle-shaped, and cyto- chemically empty cells of Leydig are quickly restored to the swollen, epitheloid, lipid-rich type. The lipoid vacuoles exhibit histo- chemical reactions that are characteristic of, but not necessarily specific for, steroidal substances. More specifically, the seminal vesicles, prostate and Cowper's glands un- dergo enlargement and each elaborates its special exocrine secretion in proof of in- duced testicular endocrine function. The testes also enlarge somewhat, due to swelling of the tubules and some minimal stimulation of their spermatogenic epithelium. The lat- ter response is believed to be brought about by the direct spermatogenic action of testos- terone per se and can, in fact, be dupli- cated by the injection of this hormone alone. In hypophysectomized adult male rats given LH from the time of operation, the entire reproductive system, including all components of the testes, is maintained in a normal condition (Greep and Fevold, 1937). Here it is presumed that the action of LH is confined to stimulation of the inter- stitial cells, because all other aspects of the response can be dujilicated by exogenous androgens. 4. An Extragonadal Activity The possibility that gonadotrophins have an effect on the adrenal cortex has always been attractive. However, only in the mouse has any clear-cut action been demonstrated. LH has a trophic influence on the so-called X-zone in the adrenal glands of mice (Ches- ter Jones, 1949) . This zone of highly eosin- ophilic cells is present at birth and disap- pears at puberty in the males and during the first gestation in females. Chester Jones (1950) found that although the zone per- sists after hypophysectomy in prepubertal mice, the cells therein revert to an atrophic state. It seemed likely, then, that the zone might be responsive to a factor from the pituitary. A check of available preparations revealed that extracts rich in LH were markedly trophic to the X-zone. Neither FSH nor ACTH had any beneficial action in this regard. These findings fit nicely with the fact that the X-zone reappears after castration of postpubertal male mice (How- ard, 1939) , i.e., at a time when the secretion of LH is greatly increased (for a review see Chester Jones, 1955, 1957). 5. FSH and LH in Relation to Estrogen Secretion The relation of FSH to estrogen secretion by the ovary has not been fully elucidated. Present knowledge in this area is deficient mainly because there has been little oppor- tunity to study the activity of FSH as an entity free of LH with which it exhibits synergism. Acting with but minute amounts of LH, the quantitative responses to FSH are considerably enhanced as determined by the growth response of the female gonad (reviewed by Fevold, 1939, and by Evans and Simpson, 1950), yet the qualitative nature of the response may not be altered (Fraenkel-Conrat, Li, Simpson and Evans, 1941). To the extent of present knowledge, biologically pure FSH does not elicit the secretion of estrogen by the ovary of the hypophysectomized rat, and there is con- vincing evidence that it also does not evoke the secretion of androgen by the testes of similarly operated animals (Fevold, 1939; Greep, van Dyke and Chow, 1942; Evans and Simpson, 1950; Simpson, Evans and Li, 1950; Simpson, Li and Evans, 1951). How- ever, FSH does play an essential and pos- sibly even a primary role in the promotion of estrogen secretion by the ovary, inasmuch as it prepares the follicular apparatus on which LH can act to promote estrogen pro- duction. That the injection of highly puri- fied FSH preparations into intact, immature rats and mice (Moon and Li, 1952; Thomo- poulou and Li, 1954) has evoked the se- cretion of estrogen is explicable, perhaps, on the basis of endogenous LH release. 6. FSH and LH, Interactions LH is believed to interact in some man- ner with FSH at the ovarian level to pro- duce greater stimulation than could be expected on the basis of the sum of the separate actions of these hormones. This ef- fect has also been termed synergism or ]'»o- tentiation. It is a real and striking phenor.u'- 248 HYPOPHYSIS AND GOXADOTROPHIC HORMONES non, for which no satisfactory explanation is available. The principle of potentiation has been employed extensively to intensify artificially induced gonadal stimulation in sheep and cattle (Hammond, Jr. and Parkes, 1942; Casida, Meyer, McShan and Wis- nicky, 1943). An interplay between FSH and LH seems to operate in the accomplishment of ovula- tion. The ovulatory response to LH is well known to be intensified when acting in conjunction with FSH (Foster, Foster and Hisaw, 1937; Pincus, 1940; Chang, 1947a, b; Hisaw, 1947; ISlarden, 1952). The reader is referred to Hisaw (1947) for a thoughtful analysis of the literature. FSH acting alone in hypophysectomized animals will bring follicles to maturity, but seldom does it lead to their rupture (Knobil, Kostyo and Greep, 1959). LH, on the contrary, given as a quick acting stimulus in the presence of ripe fol- licles, is an efficient ovulator. Inasmuch as the exact nature of the ovulatory stimulus has not been elucidated, the term "ovula- tory hormone" has been widely used. It has the advantage of being less committal as to the nature of the factors operating. In reality, it would be a very difficult matter to test the ovulating capacity of LH in the complete absence of FSH, since only near- mature follicles can be ovulated, and their existence is dependent on the action of FSH. Rapid involution and disappearance of persisting corpora lutea in hypophysecto- mized adult rats (Bunde and Greep, 1936; Greep, 1938) was induced by injecting im- pure LH extracts and combinations of FSH and LH. Although pure LH did not produce the histolytic response, it is worth noting that it also did not elicit any maintenance of hiteal function (Greep, van Dyke and Chow, 1942 ». When FSH and LI I were adniinistered concurrently to intact or hypophysecto- mized, immature male rats a synergism was noted in respect to the increase in weight of the testes (Greep, Fevold and Hisaw, 193(31. 7. Assay Several means are available for detecting the activity of LH, but no one has proved fully satisfactory as a means of quantifying this hormone. Oldest of these methods is the augmentation and luteinization test, as developed in the laboratory of Fevold and Hisaw. It involved a comparison of the qualitative ovarian response of intact, im- mature female rats to FSH alone as opposed to that of animals receiving FSH and LH simultaneously. A positive test for LH was manifest by augmentation of the ovarian weight and induction of extensive luteiniza- tion, resulting in what has often been termed "mulberry ovaries." When hypophy- sectomized test animals were employed, the results were less striking both quantitatively and qualitatively, but interference by en- dogenous LH was precluded. The possibility of using the weight in- crease of the accessory sexual structures in immature male rats as a test for LH sug- gested itself from studies of the effects of the separate gonadotrophins in male ani- mals. The ventral lobe of the prostate was the most sensitive. Employing hypophy- sectomized animals and pure LH, Greep, van Dyke and Chow (1941) demonstrated a satisfactory dose-response relationship. The test is simple, ofi'ers an objective measure- ment, and is moderately sensitive. It has the disadvantage of being indirect — the prostate response is androgen-mediated and reflects the action of LH on the testicular Leydig cells. The specificity of the prostate test for LH has been questioned by Segaloff, Steelman and Flores (1956). Pursuant to a report by Grayhack, Bunce, Kearns and Scott (1955) that jirolactin enhanced the ventral j^rostate response to testosterone in castrated rats, Segaloft" and his colleagues reported that prolactin sensitized the ven- tral prostate to the action of androgen se- creted in response to LH. Lostroh and Li (1956) were not able to confirm that pro- lactin synergizes with testosterone, and Lostroh, Squire and Li (1958) found a strain variation with respect to the specific- ity of the ventral prostate as a test for ICSH. Wlien the Long-Evans strain of rats was used, neither prolactin nor growth hor- iiionc. or any combination of these, altered the prostatic response to exogenous ICSH; with the Sj)raguc-Dawley strain both growth hormone and lactogenic hormone effected significant enhancement of the re- sponse to ICSH. It seems that hypophysec- PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 249 tomized animals of any strain can be used for the assay of purified ICSH, but only those of the Long-Evans strain are suitable of the assay of impure preparations. Loraine and Diczfalusy (1958) concluded that the response of the ventral prostate of the hy- pophysectomized immature male rat to hu- man menopausal gonadotrophin and HCG is not affected by the simultaneous admini- stration of prolactin. Most important, per- haps, is the fact that FSH does not affect the prostatic assay for LH (Greep, van Dyke and Chow, 1941 ; Lostroh, Squire and Li^, 1958 j. According to Lostroh, Squire and Li (1958) , "the hypophysectomized male rat of the Long-Evans strain has been a satisfac- tory animal for assaying preparations of ICSH when the weight of the ventral pros- tate is used as a measure of the activity and the response is represented as a loga- rithmic function of the dose. . . . The sensi- tivity of the assay is such that a total dose level of from 0.010 to 0.060 mg. is sufficient for assay purposes when the subcutaneous route is employed." The Weaver finch test for LH, developed by Witschi (1940, 1955), is gaining accept- ance as a valuable means of identifying this gonadotrophin (Segal, 1957). The test is carried out on African finches of the genera Euplectes and Steganura. Regenerat- ing white feathers of the ventral pterylae in females or in males in eclipse plumage react to a single injection of LH by the formation of a black bar formed as a result of melanm-dep6sition. The test is held to be specific for pituitary LH, but the evi- dence for this view is not yet conclusive. The reaction is independent of the sex glands. The observation that the Weaver finch test does not detect LH in menopausal urine (Witschi, 1955) as does the ventral prostate method (Greep and Chester Jones, 1950b; Loraine and Diczfalusy, 1958) needs further exploration. Evidence submitted within the past year (Parlow, 1958) suggests that LH produces a fall in ascorbic acid content of the ovaries of pseudopregnant rats and promises to form the basis of a further and much more sensi- tive means of assaying the luteinizing hor- mone. C. LUTEOTROPHIN (PROLACTIN) 1. Chemical Features Prolactin has been isolated in a form satisfying all the available physicochemical criteria of protein homogeneity and ex- hibiting no biologic activity other than that believed to be attributable to the hormone (for latest evidence see Li, 1957). Prolactin was the first in this category of protein hormones to be obtained in crystalline form (White, Catchpole and Long, 1937; White, 1943) — a notable achievement even though crystallization is no necessary indication of purity. Prolactin has mainly been ob- tained from the hypophyses of beef and sheep and although there may be minor spe- cies differences in the composition of the hormone as indicated by a slight difference in solubility, the preparations otherwise be- have identically. They have an isoelectric point of 5.7 and a molecular weight of about 3200 (White. 1949). 2. Physiology of Luteotrophic Hormone [Prolactin ) Riddle, Bates and Dykshorn (1932) pro- posed the term prolactin to describe a pitui- tary activity which stimulated the crop sac of pigeons. In mammals prolactin was shown to possess also lactogenic and luteotrophic activity. It has not been unequivocally dem- onstrated that prolactin, lactogenic hor- mone, and luteotrophin are identical nor that luteotrophic manifestations are essen- tial for luteal function except in the rat. For the purposes of this chapter, however, it is regarded as semantically legitimate to discuss LTH as a gonadotrophin, although scientifically neither its activity nor its identity with prolactin has been rigorously proved. As noted, the rat is the only animal in which a luteotrophic action of prolactin has been conclusively demonstrated (Astwood, 1941 ; Cutuly, 1941 ; Evans, Simpson and Lyons, 1941 ; Evans, Simpson, Lyons and Turpeinen, 1941 ) . Confirmatory observa- tions have been reported by Lyons (1942), Tobin (1942), Fluhmann and Laqueur (1943), Everett (1944), Sydnor (1945). Greep and Chester Jones (1950a). Tests of the luteotrophic action of prolactin in other 248 HYPOPHYSIS AND GONADOTROPHIC HORMONES non, for which no satisfactory explanation is available. The principle of potentiation has been employed extensively to intensify artificially induced gonadal stimulation in sheep and cattle (Hammond, Jr. and Parkes, 1942; Casida, Meyer, McShan and Wis- nicky, 1943). An interplay between FSH and LH seems to operate in the accomplishment of ovula- tion. The ovulatory response to LH is well known to be intensified when acting in conjunction with FSH (Foster, Foster and Hisaw, 1937; Pincus, 1940; Chang, 1947a, b; Hisaw, 1947; Marden, 1952 ». The reader is referred to Hisaw (1947) for a thoughtful analysis of the literature. FSH acting alone in hypophysectomized animals will bring follicles to maturity, but seldom does it lead to their rupture (Knobil, Kostyo and Greep, 1959 ) . LH, on the contrary, given as a cjuick acting stimulus in the presence of ripe fol- licles, is an efficient ovulator. Inasmuch as the exact nature of the ovulatory stimulus has not been elucidated, the term "ovula- tory hormone" has been widely used. It has the advantage of being less committal as to the nature of the factors operating. In reality, it would be a very difficult matter to test the ovulating capacity of LH in the complete absence of FSH, since only near- mature follicles can be ovulated, and their existence is dependent on the action of FSH. Rapid involution and disappearance of persisting corpora lutea in hypophysecto- mized adult rats (Bunde and Greep, 1936; Greej), 1938) was induced by injecting im- j)ure LH extracts and combinations of FSH and LH. Although pure LH did not produce the histolytic response, it is worth noting that it also did not elicit any maintenance of luteal function (Greep, van Dyke and Chow, 1942). When FSH and LH were adniiiristercd concurrently to intact or hypophysecto- mized, immature male rats a synergism was noted in respect to the increase in weight of the testes (Greep, Fevold and Hisaw, 1936). 7. Assay Several means are available for detecting the activity of LH, but no one has proved fully satisfactory as a means of quantifying this hormone. Oldest of these methods is the augmentation and luteinization test, as developed in the laboratory of Fevold and Hisaw. It involved a comparison of the ciualitative ovarian response of intact, im- mature female rats to FSH alone as opposed to that of animals receiving FSH and LH simultaneously. A positive test for LH was manifest by augmentation of the ovarian weight and induction of extensive luteiniza- tion, resulting in what has often been termed "mulberry ovaries." When hypophy- sectomized test animals were employed, the results were less striking both quantitatively and cjualitatively, but interference by en- dogenous LH was precluded. The possibility of using the weight in- crease of the accessory sexual structures in immature male rats as a test for LH sug- gested itself from studies of the effects of the separate gonadotrophins in male ani- mals. The ventral lobe of the prostate was the most sensitive. Employing hypophy- sectomized animals and pure LH, Greep, van Dyke and Chow (1941) demonstrated a satisfactory dose-response relationship. The test is simple, offers an objective measure- ment, and is moderately sensitive. It has the disadvantage of being indirect — the prostate response is androgen-mediated and reflects the action of LH on the testicular Leydig cells. The specificity of the prostate test for LH has been questioned by Segaloff, Steelman and Flores (1956). Pursuant to a report by Grayhack, Bunce, Kearns and Scott (1955) that prolactin enhanced the ventral prostate response to testosterone in castrated rats, Segaloff and his colleagues reported that prolactin sensitized the ven- tral prostate to the action of androgen se- creted in response to LH. Lostroh and Li (1956) were not able to confirm that i)ro- lactin synergizcs with t(>stosterone, and Lostroh, Squire and Li (1958) found a strain variation with respect to tlie specific- ity of the ventral prostate as a test for ICSH. When the Long-Evans strain of rats was used, neither i^rolactin nor growth hor- iiioiic, or any combination of these, altered the pi'ostatic response to exogenous ICSH; with the Sprague-Dawley strain both growth hormone and lactogenic hormone eftected significant enhancement of the re- sponse to ICSH. It seems that hypophysec- PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 251 sentation of the aiiiount of gonadotrophins present at the moment of death, is a result- ant of the rate of synthesis and the rate of liberation. Whether such potency measure- ments provide any reasonable clue to the actual rate of secretion has been debated. The significance of such measurements is enhanced by the preponderance of cases in which the potency has correlated well with function, as revealed by the gonads and reproductive tracts of the donors (Meyer, Biddulph and Finerty, 1946; Robinson and Nalbandov, 1951; Kammlade, Welch, Nal- bandov and Norton, 1952; Bahn, Lorenz, Bennett and Albert, 1953a; Greeley and Meyer, 1953; Nalbandov, 1953b, a review; Simpson, van Wagenen and Carter, 1956 j. These correlations, however, were not al- ways close and in a few instances, as noted elsewhere (page 261) , pituitary potency and function diverged sharply. As long ago as 1939 Smith surmised that the usefulness of whole gland assays in the study of pituitary physiology had been nearly fulfilled. The prediction seemed reasonable but the tech- nique, with some refinements, is still widely used and is one of the important means of gaining insight concerning the probable level of gonadotrophic function. It would be exceedingly helpful to be able to determine the blood level of the gonadotrophins, but even this would not reflect the rate of entry of the gonadotrophins into the blood stream unless the rate of destruction and excretion were also evaluated. For the most part the levels of circulating gonadotrophins in normal animals have been found/to be very low and are generally below the /sensitivity of the available cri- teria for defecting them. Significant gonado- trophic activity has not been found in the blood or urine of ruminants (reviewed by Benson and Cowie, 1957) and the fact has been re-emphasized by the circumstance that no trace of such activity was found in volumes of sheep blood as large as 500 ml. (Bassett, Sew^ell and White, 1955). Normal rat plasma has also yielded consistently negative results except for one unconfirmed report of a trace of LH activity (Hellbaum and Greep, 1943) . The plasma of gonadecto- mized rats, however, like the plasmas of menopausal women or gonadectomized men and women, was early found to contain demonstrable gonadotrophic activity (Em- ery, 1932). Later, Hellbaum and Greep (1943) reported finding FSH, but no LH, in 10 ml. plasma from castrated male rats. Recently this result has been confirmed in substance by Cozens and Nelson (1958). Using closely spaced injections, they ad- ministered to hypophysectomized, immature female rats up to 24 or 32 ml. plasma from spayed adult female rats over a 4-day pe- riod ; although they found a clear indication of FSH activity, ICSH was not apparent. A. PHYLOGENETIC CONSIDERATIONS The end points of cross-species testing of pituitary glands for gonadotrophic po- tency have comprised mainly gonad stimu- lation in immature male and female rats and mice and in 1-day-old male chicks, ovula- tion in estrous rabbits or pregnant mice (Ladman and Runner, 1951), ovulation or spermiation in frogs, and coloration of spar- row bill or Weaver finch feathers. The con- ditions of the assay have varied greatly; the test animals have been of different ages at the start of treatment; they have been hypophysectomized in one study and intact in the next; the dosage has been expressed in integers or fractions of whole glands or as milligrams, wet weight or dry weight, of pituitary tissue. The data often do not per- mit comparisons of assays of pituitaries from a given species, leaving aside the diffi- culties of making interspecies comparisons. The extensive literature on this topic has been reviewed repeatedly (Smith, 1939; van Dyke, 1939; Burrows, 1949; Chester Jones and Eckstein, 1955). ). Ascidians On the basis of tests in three intact 26- day-old mice, Carlisle (1950) claimed to have detected gonadotrophic activity in the neural gland of ascidians. Dodd's (1955) recent evidence of more substantial nature casts doubt on the validity of this claim — yet leaves the question open, since 1 of Dodd's 10 intact 19-day-old mice receiving as much as 230 neural glands each gave a positive response. The further statement by Carlisle (1954) that mammalian pituitary and urinary gonadotrophins promote ovula- tion and sperm discharge in the ascidians has likewise been challenged (Dodd, 19")oi. 252 HYPOPHYSIS AND GOXADOTROPHIC HORMONES 2. Fish Fish have been reported to respond to gonadotrophins of piscine origin (Hasler, Meyer and Field, 1939; Rasquin, 1951; Hisaw and Albert, 1955; Hoar, 1955; Ram- aswami and Simdararaj, 1956, 1957) by exhibiting out-of-season oocyte growth and spawning. It is to be noted, however, that satisfactory control experiments are often lacking. Fish respond less readily to am- i:)liibian pituitary. Induced spawning in rainbow trout (Migita, Matsomoto, Kino- chita, Sasaki and Ashikawa, 1952) and cat- fish (Ramaswami and Sundararaj, 1957) has been claimed to follow the injection of frog pituitary. Fish appear to be almost totally insensitive to gonadotrophins of avian and mammalian origin (de Azevedo and Canale, 1938; Hasler, Meyer and Field, 1939; Hoar, 1955); however, Dodd (1955) calls attention to some instances of doubt- ful or limited effectiveness of mammalian gonadotrophins, especially human urinary gonadotrophins, in fish. Pickford and Atz (1957) brought together the varied and con- flicting results of the multitudinous studies of reproductive physiology in fish. 3. Amphibians Amphibians are notably responsive to gonadotrophins from widely differing phy- logenetic sources, yet even among some of the anura the gonadotrophins show a high degree of species specificity (Greaser and Gorbman, 1939; Houssay, 1954). The ef- fectiveness of homoplastic anuran pitui- taries in inducing spermiation, ovulation, and oviposition has become a part of classi- cal biology. By contrast, the induction of ovulation in Rana temporaria requires 150 times as much beef as Rana pituitary (Gal- lien, 1955a), and Houssay has shown that the South American toad, Bufo arenarum, is less sensitive to rat and human hypophy- sis than to pituitaries of anuran origin. Strangely, the anura, although generally responsive to mammalian gonadotrophins of pituitary or placental origin, have reacted poorly— if at all — to injected brei of elasmo- branch pituitaries (Greaser and Gorbman, 1939) or to those of telcosts (Greaser and Gorbman, 1939; Atz and Pickford, 1954). Pursuing the matter of phyletic considera- tions further, Atz and Pickford (1954) re- port that the Russian workers, Stroganov and Alpatov (1951) found frogs more sensi- tive than fish to sturgeon pituitary; and interestingly, although their data are mea- ger, Wills, 'Riley and Stubbs (1933) ob- tained ovulation in Bujo americanus and Rana pipiens with gar pike hypophyses. The sturgeon and gar pike, as Greaser and Gorb- man (1939) point out, are closer in the phyletic scale to the amphibia than they are to the fish whose pituitaries were ineffective in amphibia. Some exploratory studies have been made of the action of mammalian FSH and LH in amphibia. The male toad, Bnfo melanos- tictus, responded to mammalian FSH and LH (Bhaduri, 1951), whereas Bujo bufo responded negatively to FSH (Thorborg and Hansen, 1950). Greze (1949) reported that LH produced a positive spermiation test in Rana esculanta, and Atz and Pick- ford (1954) , after testing a number of mam- malian pituitary preparations, concluded that LH was 40 to 100 times more active than FSH in causing sperm release in Rana pipiens; the positive responses obtained with FSH were easily attributable to an LH contaminant. Wright and Hisaw (1946) studied the response of frog ovaries to puri- fied mammalian gonadotrophins and found that, although FSH ovulated the intact frog, it was not effective in hypophysecto- mized females. Using the technique of Heil- brunn, Dougherty and Wilbur (1939), Wright and Hisaw applied sheep FSH to frog ovaries in vitro and failed to induce ovulation; but by combining FSH and LH in an appropriate ratio, ovulation was ob- tained. They believe that the principal function of FSH in the frog is to render the ovary sensitive to an ovulating stimulus. OA'iposition was induced in the salamander, Triturus viridescens, by LH but not by FSH (Mayo, 1937) and Witschi (1937) elicited ovulation in the newt, Taricha torosa, with beef and turkey hypophyses but not with pregnant mare's serum (PMS) gonado- trophin. The sensitivity of ami)hibians to human pituitary and chorionic gonadotrophins has rendered tliem extremely useful in clinical PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 253 assays for diagnostic purposes, especially for the early diagnosis of pregnancy. A number of species of frogs and toads are in use, mainly: (1) the male leopard frog, Ra7m pi-piens (Wiltberger and Miller, 1948; Robbins and Parker, 1949; Haskins and Sherman, 1952) ; positive tests are also ob- tained with hypophysectomized frogs (Kis- sen, 1954), (2) the female Xenopus laevis (Thorborg and Hansen, 1951 ; Hobson, 1952) , and (3) the male toad, Bufo arenanim (Hansel) (Galli Mainini, 1948; Allison, 1954; for review, see Houssay, 1954). The Galli Mainini test is held to be relatively more specific for the chorionic gonadotrophin in that positive responses are not obtained with urine from normal men or nonpregnant women, regardless of the presence or ab- sence of functioning gonads. This is in line with Atz and Pickford's finding that sper- miation is mainly a function of LH, the urinary output of which in normal men is very low. 4. Reptiles Few observations have been made on the interrelations of the pituitary and gonads in reptiles (Kehl and Combescot, 1955) and fewer still on the responsiveness of reptiles to heterozoic gonadotrophins. In viviparous snakes hypophysectomy led to regression of gonads and death of the embryos and pre- mature parturition (Clausen, 1940; Brag- don, 1951). Testis impairment following hy- l)ophysectomy in male garter snakes was corrected by daily implantation of pituitary glands froni the same species (Schaefer, 1933). Crude extracts of sheep pituitary promoted marked gonad stimulation in the lizard, Uromastix (Kehl, 1944; Kehl and Combescot, 1955), immature alligators, Alligator jnississippiensis (Forbes, 1937), and terrapins, Malaclemmys centrata (Ris- ley, 1941 1 . In extended studies of the cha- meleon, Anolis carolinensis, Evans (1948) oljtained positive results with mammalian gonadotrophins unspecified. 5. Birds Among birds males of all ages and mature females are responsive to gonadotrophins of both avian and mammalian sources (Breneman, 1936; Byerly and Burrows, 1938; Nalbandov and Card, 1946; Das and Nalbandov, 1955). These observations are based mainly on the domestic breeds, such as chickens, ducks, and turkeys (reviewed by Nalbandov, 1953a). Evidence suggesting qualitative differences between avian and mammalian gonadotrophins has been ob- tained by Nalbandov, Meyer and McShan (1951), Das and Nalbandov (1955), and Taber, Clay tor, Knight, Gambrell, Flowers and Ayers (1958). Thus, the ovarian cortex of the sexually immature female chicken, although notably insensitive to mammalian gonadotrophins, has responded to avian pituitary extract. Taber and her associates noted significant follicle stimulation but no precocious ovulation in immature female chickens receiving long-term treatment with acetone-dried avian anterior lobe substance. In contradistinction, the medullary portion of the immature ovary, like the testes of the male bird, responded with increase in weight and androgen production at any age and to hormones of either mammalian or avian source. Following hypophysectomy of prepubertal female birds, the medullary area remained responsive to avian, but not to mammalian, gonadotrophin. Moreover, the effectiveness of mammalian gonadotro- phins in maintaining the ovaries and comb of hypophysectomized laying hens was of short duration (Biswal and Nalbandov, 1952; Nalbandov, 1953a); egg-laying con- tinued 5 to 7 days postoperatively, and comb size was sustained for 10 to 15 days. Avian hormone, alone or in combination with mam- malian gonadotrophin, was effective over treatment periods lasting up to 35 days. The possibility that these results might have been influenced by the development of im- mune bodies was not eliminated. Observations of interest have been made on the hypophyseal control of the rudi- mentary right gonad in hens (Kornfeld and Nalbandov, 1954). Compensatory hyper- trophy of this structure, seen regularly in poulards, has been completely prevented by injections of estrogen, and to lesser extent by androgen. Hypophysectomy, moreover, causes regression of the enlarged right rudi- ment which cannot be forestalled by injec- tions of hog or sheep gonadotrophins, nor by PMS. Whether chicken gonadotrophins 254 HYPOPHYSIS AND GONADOTROPHIC HORMONES would have tlie sustaining action they could be presumed to have in this situation, has apparently not been investigated. Inasmuch as the pituitary-gonad inter- relationshii)s are complex and little under- stood, it is not to be expected that the administration of either isogeneric or heter- ozoic gonadotrophic complexes would elicit the normal pattern of sexual functions. As a demonstration in point, Witschi (1955) injected sparrows with the suspended pow- ders of human, beef, or turkey hypophyses and found that human hypophyses produced good follicle growth but the oviducts were only incompletely developed; turkey and beef glands, on the contrary, promoted intense stimulation of the reproductive tract and only minor ovarian enlargement. None of these preparations produced a balanced development of the gonads and accessory sexual organs, nor did ovulation and egg- laying occur. Prolactin is present in extracts of the hypophyses of fish, amphibians, and reptiles (Leblond and Noble, 1937) and its presence in avian pituitary was amply demonstrated by Riddle's group in the middle 'thirties. Their classic studies showed that prolactin exerts full control over the proliferative development of the crop sac in pigeons, but it also appeared to have a suppressive action on the gonads in these as well as other birds. Prolactin of mammalian origin administered to laying hens resulted in regression of ovaries, cessation of laying, and appearance of broodiness (Breneman, 1942; Nalbandov, Hochhauser and Dugas, 1945; Nalbandov, 1953a). Nalbandov expressed the opinion that broodiness is secondary to ovarian failure, with attendant reduction in estrogen secretion — a view nicely supported by the earlier finding of Godfrey and Jaap (1950) that broodiness can be quickly terminated by estrogen injections (also see chapter by Lehrman). Prolactin has also been noted to induce broodiness in cocks (Nalbandov and Card, 1946) and atrophy of the de- veloping right gonad in poulards (Kornfeld and Nalbandov, 1954). The mechanism for the suppressive influence of prolactin on the gonads of birds is unknown, but it can be presumed to involve central nervous sys- tem centers concerned in some manner with regulation of the anterior pituitary. Prolac- tin has also been noted to have a calori- genic action (Riddle, Smith, Bates, Moran and Lahr, 1936) , but it has not been possible to correlate this with the induction of broodiness. Likewise, its ability to increase the body temperature of roosters 2 to 4°F. is not contributory, since broody fowl have subnormal temperatures. 6. Menu ma Is Information on the effectiveness in mam- mals of pituitary gonadotrophins from lower orders is scanty. As would be antici- pated, avian gonadotrophins are by far the most potent. Unfractionated gonadotrophic extracts of avian pituitaries exhibited both follicle-stimulating and luteinizing activity (Leonard, 1937; Witschi, 1937; Gorbman, 1941; Traps, Fevold and Neher, 1947; Nal- bandov, Meyer and McShan, 1951). The nature of the gonadal reactions induced in mammals by avian gonadotrojjhins are ciualitatively similar to those produced by mammalian gonadotrophins (Riley and Fraps, 1942a, b; Breneman, 1945; Nalban- dov and Card, 1946; Breneman and Mason, 1951). Moderate folheuhir development and lu- teinization in rats and mice has been in- duced by chicken and turkey pituitaries, but full estrous development of the reproductive tracts is generally not attained. Riley and Fraps (1942a, b), using the mouse ovary assay, estimated the ratio of FSH to LH potency in bird hypophyses, and they along with Chance, Rowlands and Young (1939), Witschi (1940), and others, in fact, at- tempted quantitative estimations of the F8H-LH ratio in hypophyses of a variety of birds and mammals. Such precise numeri- cal values are unfortunately of little signifi- cance, owing to the limitations of the bio- assays. Nonetheless, two indications seem to emerge from these studies: the FSH-LH ratio varies greatly from one species to another, and within a given species rhythmic fluctuations have been noted. Pituitaries from such cold-blooded forms as fish, frogs, and tui'tles have at l)cst shown liarely posi- ti\-e ti'aces of gonadotrophic activity in rats and mice (for litei'aturc, see Witschi, 1955). In tact, A(hinis and Granger (1941) had to PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 255 inject 100 times as much frog as mouse hy- pophysis to obtain minimal stimulation of the mouse ovary. 7. General Considerations There are probably no generalizations that can safely be drawn from the variety of taxonomic relationships noted in regard to the gonadotrophins ; but, as Zuckerman aptly contends, "knowledge can advance little if we do not generalize." Wide dis- tribution throughout the Vertebrata is sug- gested for each of the gonadotrophins. The information concerning their ciualitative variation that is revealed by cross-species testing is of fundamental importance to both biology and medicine. Peculiarly, there is little and perhaps no qualitative variation in the gonadotrophins from various species of teleosts (Ihering, 1937; Kazanskii, 1940; Dodd, 1955), but the number of species studied is not a fair sample of the number existent. The situation is different in the amphibia. Here qualitative variation in the gonadotrophins from different s])ecies is much in evidence. Seemingly, some anurans are capable of responding to gonadotrophins from any class of vertebrate, whereas others fail to respond even to gonadotrophins from closely related amphibian species ; albeit the amphibia appear to be far more sensitive to amphibian gonadotrophins than to those from any other class of vertebrates. The reptiles, although little studied, appear to respond sluggishly to nonreptilian gonado- trophins. Birds, too, respond best to bird gonadotrophins, yet in some respects are exceedingly! sensitive to those from a wide variety of mammals. Among the mammals great variations in responsiveness are re- vealed by every exchange of gonadotrophins between their species. The variations, how- ever, are mainly of quantitative rather than of qualitative nature so far as is known. The differences in species responsiveness pose difficult problems in the bioassay of the gonadotrophins. It is obvious that the potency units assigned to a gonadotrophin preparation assayed in a foreign species have no significance as to equivalent po- tency when the same preparation is tested in other species, even when adjustment is made so that the dose per unit of body weight is the same. Moreover, an apparent ineffectiveness in one species may not nec- essarily mean that the gonadotrophin is biologically inactive; Creaser and Gorb- man (1939) state that ''the effectiveness of a gonadotrophic hormone in a foreign spe- cies tends to vary directly with the phylo- genetic proximity of the donor and recipient species." Recently, a marked qualitative species variation was demonstrated in connection with another pituitary hormone, somato- trophin. Preparations of swine, ovine, or bovine somatotrophin were markedly effec- tive in rats and dogs but not in monkeys or man (for literature, see Smith, Gaebler and Long, 1955). IVIonkey somatotrophin, how- ever, was effective in monkeys and also in rats (Knobil and Greep, 1956). Similarly, fish somatotrophin, which was active in fish, was inactive in mammals, whereas ox prepa- rations were active in fish as well as in some mammals (Pickford, 1954; Wilhelmi, 1955). We have seen that the physiologic prop- erties of a gonadotrophin are as much an expression of the sensitivity of the substrate on which the hormone acts as of the ex- citations of the hormone itself. This being the situation, the difficulties to be faced in retracing the evolutionary history of the gonadotropins are indeed formidable. How- ever, by extending lines of study that have been followed over the past 20 years, pieces of this fascinating problem may in time be fitted together. At this early departure in the quest, the dictum credited to Medawar to the effect that, "It is not the hormones which evolve but the uses to which they are put" seems grossly premature; but its value in addressing thoughtful inquiry to the matter may be considerable. Another lab- oratory aphorism to the effect that hormones can be tested down the ])hyletic scale but not in the reverse order undoubtedly contains an element of truth, but as a generalization it would seem to be fraught with much haz- ard. Along these same lines Witschi (1955), noting the spread in effectiveness of mam- malian LH — at least to the amphibia — re- marked, "One definite conclusion derives clearly ^ from the now available evidence; namely, that induction of ovulation is a much more general and more ancient func- 256 HYPOPHYSIS AND GOXADOTROPHIC HORMONES tion of LH than is its role in the formation of corpora lutea." B. AGE, SEX, GONADECTOMY, AND REPRODUC- TIVE RHYTHMS IN RELATION TO PITUITARY tiONADOTROPHINS 1. Fetal Gonadotrophins Experiments cited by Smith (1939j re- vealed no demonstrable gonadotrophic ac- tivity in the pituitary of fetuses except in the hog and horse. The amount of pooled tissue available for such tests with other species was often so small as to make the negative findings inconclusive. The original work of Smith and Dortzbach (1929) on the l)ig fetus is still the most complete. Gonado- trophin was detected in the late fetal stages. Much experimental evidence bearing on the relation of the hypophysis to the devel- opment of the fetal gonads has been ob- tained over the past several years, princi- })ally from the laboratories of Wells in Minnesota and Jost in Paris. These studies are admirably summarized by Jost (1953, 1955, 1956a) and Wells (1956). Each group developed techniques for intra-uterine ab- lation of fetal endocrine glands, including the hypophysis; the latter being accom- ])lished, in effect, by decapitation. Decapita- tion of rat fetuses on day 18 did not lead to abnormalities in the differentiation and de- velopment of either the gonads or the ac- cessory sexual structures. By contrast, re- moval of the testes on day 18 led to deficiencies in the development of the pros- tate and coagulation glands, and a failure of the Mullerian ducts to regress normally. This, according to Wells, is a deficit in maleness correctable by testosterone and is not to be construed as a feminizing in- fluence. Substitution of testosterone from the time of fetal castration prevented all the impairments of development except the regression of the Miillerian ducts. It would seem that the fetal testis exerts an influence over development of the male accessory structures and that this action is inde- pendent of any stiiiuihis fi-oni the hy])()phy- sis. The effects of fetal decapitation in I'abbits as studied by Jost differ from those in the lat in one important respect. Male fetuses decapitated on the 19th. 20th, or 21st dav of gestation were born at term in a semi- feminized state as regards the accessory ducts and external genitalia. These changes did not occur when decapitation was per- formed after the 23rd day nor when the headless fetuses were injected with gonado- trophin. By way of explanation, days 22 and 23 were held to be a determinative phase during which the prospective develop- ment of the sexual ducts is established by the fetal testis acting in response to the fetal pituitary. Cytologic examination of the fetal hypophyses during this period revealed the transient presence of McManus-positive cells on the 22nd and 23rd days, which Jost believes may signal a transitory pro- duction of gonadotrophins. That the fetal rabbit pituitary actually secretes gonado- trophin on days 22 to 24 would require more positive documentation than is pres- ently available. There are considerations that urge caution. The female rabbit ovary is known to be refractory, at least to exog- enous gonadotrophin, throughout the entire infantile period (Hertz and Hisaw, 1934), a finding common to many infant mammals. The fact that McManus-positive material appeared in the pituitary cells is suggestive, but by no means conclusive, evidence of secretion of gonadotrophins. There is also the possibility that maternal gonadotroph- ins are available to the fetus, but the evi- dence is controversial (Knobil and Briggs, 1955; Jost, 1956b). It seems clear that any function served the fetus by maternal gon- adotrophins may be of minor significance, inasmuch as the maternal pituitary gland has been removed quite early in gestation in several mammals, including the rhesus mon- key (Smith, 1954, 1955), without impair- ment to the development of the fetal male or female reproductive systems. Moreover, in man and horse the available evidence sug- gests that the pituitary gland is extremely weak in gonadotrophic activity throughout most of the gestation pciiod, including the l)uerperiuin. i. Age and Sex Brencman and ISIason (1951 ) and Brene- inan (1945, 1955) made detailed studies of the gonadotrophic i)otency of cockerel and pullet ]5ituitaries (using the chick testis as- sav) (lnrin^ the initial 3 to 4 montiis of life PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 257 and collated these data with growth of the gonads. For 3 weeks postnatally the total glandular potency remained very low in both sexes. Thereafter the cockerel pituitary gained steadily in both total and unit potency, reaching a plateau at about 90 days. The unit potency increased approxi- mately five times and was accompanied by an approximate parallel increase in the weight of the testes of the donor birds. Pul- lets over 20 days of age showed a similar rise in potency plus a sharp upturn in both unit and total potency coincident with the burst of ovarian enlargement between the 115th and 125th day of life. It is precisely at this point that female chickens were found to become overtly responsive to mam- malian gonadotrophins (Das and Nalban- dov, 1955; Breneman, 1955). Among adult fowl the pituitaries of nonlaying hens, as tested by the mouse uterine weight method, are about twice as potent as those of laying hens (Riley and Fraps, 1942b). The pitui- taries of mature male chickens are mark- edly more potent than those of females of comparable age; although the male glands are heavier, the difference in potency can- not be accounted for on this basis alone (Riley and Fraps, 1942a; Phillips, 1943; Breneman and Mason, 1951). The pituitary of the male pheasant is likewise more potent than that of the hen pheasant (Greeley and Meyer, 1953). Among the most systematic studies of gonadotrophic potency in relation to post- natal age and sex are the early ones in rats by Clark /(1935a), McQueen-Williams (1935), and Stein (1935). Gonadotrophin appears in assayable quantity around the end of the 2nd week of life, when the gonads are first becoming responsive to gonado- trophic stimulation. They found a sjjike in the age-potency curve for the female gland at the 3rd postnatal week, which was not exceeded even during sexual maturity. Pituitaries of adult male rats are mark- edly more potent in gonadotrophic complex than those of females of com})arable age. Clark's data show that the relative superior potency of the male gland is not attained until some months after puberty. Hoogstra and Paesi (1955) examined the FSH and LH content of the pituitaries of intact im- mature and adult male and female rats and. in agreement with Clark, found the total FSH content of the immature female pitui- tary greater than that of the adult female or immature male, but not as great as that of the adult male. In terms of FSH per unit of glandular tissue, adult male pituitary appeared to be about five times as potent as the pituitary from adult females of com- parable age. The LH content of the pitui- taries from immature males and females was low and approximately eciual; in adults, the amount had increased considerably and was greater in the males. Hollandbeck, Baker, Norton and Nalban- dov (1956) estimated the potency of sow pituitaries from birth through 1330 days of age, using the chick testis assay. The results were expressed in terms of the weight of the anterior lobe and correlated with age and body weight. The unit potency decreased linearly with age, whereas total potency showed a linear increase, because, of course, the weight of the pituitary increased with age. The amount of hormone available per unit of body weight was very high at birth and declined through the prepubertal period to a low level which remained nearly con- stant from puberty onward. This steady drop in available hormone per unit of body weight through the jjrepubertal period is not in accord with the general assumption that an increase in titer of circulating gonado- trophin precedes puberty. Such data do not rule out a likely increase in secretion of gonadotrophin nor, as the authors have sug- gested, the possibility that an imbalance may have occurred in the ratio at which the gonadotrophic factors, FSH and LH, were secreted. The high potency during infancy was thought to be due to the presence of mainly FSH. The initiation of cyclic ovar- ian activity at puberty was thought to signify augmented secretion of LH, yielding a more functionally balanced FSH-LH ratio. Similarly, from evidence based on the urinary excretion of FSH and LH in the human female, Brown (1958) showed that the prepubertal period may be characterized by a rising level of LH. Among the primates informative data re- lating to the ratio and content of FSH and LH in the hypophysis in relation to sex and age have been obtained only for man. Bahn and associates (1953a-d) studied the FSH 258 HYPOPHYSIS AND GOXADOTROPHIC HORMONES and LH content of the human hypophysis in infancy, childhood, maturity, and old age. The glands were obtained from persons dy- ing without prolonged terminal illness and were obtained within 1 to 4 hours after death. The anterior lobes were dissected free, weighed, and kept frozen until time of use. The glands from a given age group were homogenized, pooled, and administered to hypophysectomized immature male and female rats in total doses of homogenate corresponding to 1, 3, 10, and 30 mg. wet weight of anterior lobe tissue; FSH was indicated by follicle growth or stimulation of seminiferous tubules, LH by repair of the Leydig cells. In essential confirmation of earlier studies, they found that the hypophy- sis of the human infant has no detectable FSH or LH activity. By childhood (4 years) both hormones were present in minimal ciuantity. The hypophyses from women of reproductive age and mature men were notably potent in both FSH and LH: they were essentially equipotent per unit of tis- sue for each hormone, but the female gland being heavier had a total content greater than that of the male. Using different end points and methods of collection, Witschi (1940), Witschi and Riley (1940), and Witschi (1952) reported a sex difference — they list in round figures the total FSH unitage in hypophyses of humans dying after prolonged illness as 50 R.U. for mature males and 10 R.U. for nonpregnant mature females. In keeping with the observations of Henderson and Rowlands (1938), Witschi (1940) and Witschi and Riley (1940) found the human hypophysis to contain not more than traces of LH as determined by the Weaver finch or rat test. However, the valid- ity of this claim of the Iowa group has been challenged by the more meaningful positive findings of Bahn, Lorenz, Bennett and Al- bert (1953a, c) that the adult human hy- pophysis is moderately rich in LH. Burt and Velardo (1954) studied individ- ual hypophyses from 18 i)atients, 9 males and 9 females, ranging in age from 19 to 82 years. Most of these patients had died after prolonged illness and some had re- ceived hormone therapy. The glands were assayed by injecting 7.5 mg. of fresh gland homogenate into hypophysectomized male and female rats. Of these human liypoi)hy- ses, 6 produced reasonably good follicle growth and formation of corpora lutea, 6 produced only minimal follicle stimulation, and 6 exhibited no gonadotrophic activity. In this small series the authors were unable to detect any correlation of pituitary po- tency with sex or age. It is interesting, too, that they were unable to predict the gonado- trophic potency in individual glands on the basis of a sampling of tlie relative percent- age of cell types present. There is general agreement that the con- centration of gonadotrophin in the hypophy- sis tends to increase in old age in many animals, but it is only in women that in- creased release is thoroughly documented, this being demonstrated by the increase in the blood and urinary titer of gonadotrophin at the time of the menopause. Witschi and Riley (1940) and Witschi (1956) reported that the total FSH content of the hypophy- ses of postmenopausal women is 4- to 5-fold greater than that of women of reproductive age. The hypophyses of old men show great variability, with some glands reaching the high i)otency of those of postmenopausal women and castrated men. Older men show also a fairly consistent small increase in urinary gonadotrophin beginning at about 60 years of age (Pedersen-Bjergaard and T0nnesen, 1948). 3. Gonadectomy An increase in the gonad-stimulating po- tency of the anterior hypophysis following gonadectomy or total loss of gonadal secre- tory functions through any means other than primary pituitary failure has been found to be an almost invariable occurrence especially in the warm-blooded vertebrates. An early report of a negative finding in castrated pigeons (Schooley, 1937) needs confirmation, inasmuch as Breneman (1945) found that the capon pituitary is distinctly more potent than that of cockerels of the same age. Although the gonadotrophin con- tent in sheep pituitary is high, it is at its l)eak during the time of reproductive qui- escence (Kammlade, Welsh, Nalbandov and Norton, 1952) and increases in the ewe fol- lowing sjiaying (Warwick, 1946). An objective of recent studies has been to elucidate the effects of gonadectomy on PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 259 the separate follicle-stimulating and lutein- izing activities of the pars clistalis. Bahn, Lorenz, Bennett and Albert (1953b) found a sharp increase in the FSH content of the human hypophysis at the time of the menopause, with no coincident increase in LH. The ratio of FSH to LH was found to be 1:1 in the pituitaries of women of reproductive age and 3:1 in those from women past the menopause. The failure of LH to increase under these con- ditions in man is at variance with the ob- servation by jVIcArthur, Ingersoll and Wor- cester (1958) that appreciably more LH is excreted in the urine after the menopause than during the reproductive years. In recent years the Dutch workers, Paesi, de Jongh, Gaarenstroom, and Hoogstra in- troduced modifications in their procedure for studying the gonadotrophic activity of rat pituitaries, which provide in their views an estimate of the relative amounts of FSH and LH (for methods see Hoogstra and Paesi, 1955, 1957). Paesi, de Jongh, Hoogs- tra and Engelbregt (1955) reported that gonadectomy produced within 3 months a 5-fold increase in the FSH content of the pituitaries of females, and no conspicuous increase in males; LH increased following gonadectomy in each sex, but more so in males than in females. Their finding that gonadectomy tended to diminish the sex dif- ference in pituitary gonadotrophic content in rats is in keeping with the experience of others. 4- Reproductive Rhythms a. Cijclic Vhanges. In those species in which the reproductive activity is closely tied-in with seasonal alterations in the en- vironment, the expectation is that the peaks in i)ituitary potency would correspond with periods of heightened breeding activity. The few observations available suggest that with certain exceptions this is true. Valuable preliminary data based on a year-round collection of adult pheasant pituitary has been summarized by Greeley and Meyer (1953). The dried glands were pooled by months or pairs of months and tested in day-old chicks for testis-stimulat- ing potency. The potency of the glands was at a minimum in July when the testes of the donor birds were undergoing rapid regres- sion. Before settling to winter level pitui- tary potency showed a small increase, while the testes were in what several workers have termed a "refractory state" (see Mar- shall, 1955). The major upsurge in pitui- tary gonadotrophin, correlating well wdth weight of donor testes, occurred in January, February, and March, and reached its maxi- mum in April. The close parallel of hy- pophyseal potency and testis development throughout the annual cycle is noteworthy. It indicates that pituitary gonadotrophin content reflected accurately the rate of gonadotrophin release. Benoit's early studies showed that the duck pituitary is weakest in the off-breeding season and gathers gon- adotrophic potency as added illumination brings the animals into sexual competence; also Riley and Fraps (1942b) found a lesser potency in laying than in nonlaying hens. Some information has been obtained from mammals. The pituitary of the ground sciuirrel has a much reduced gonadotrophic potency during hibernation (Wells, 1935). A peculiar situation was found to exist with respect to the cottontail rabbit at the time of the breeding season. The pituitary of the male showed an increase in gonadotrophic content, whereas that of the female did not (Elder and Finerty, 1943). Assay of mule deer pituitaries (Grieser and Browman, 1956) has indicated that potency in yearling does is lowest in winter months, gradually increases as spring progresses, and reaches a peak by late fall. In recent years pituitary gonadotrophic potency has been studied throughout the estrous cycle in the cow, sow, and ewe. The gonadotrophic potency of the cow pitui- tary is at its lowest point during estrus (Paredis, 1950). In like manner, Robinson and Nalbandov (1951) reported that the gonadotrophin content of sow pituitaries collected during estrus was approximately half that of pituitaries taken at midcycle. The potency remained low in glands taken through the first 8 days of the cycle and then climbed sharply. Reports on cyclic variations in the gon- adotrophic potency of the ewe pituitary are at variance. According to Robinson (1951), potency is at its peak during estrus; War- wick (1946), however, found no difference between glands collected during the breed- 2G0 HYPOPHYSIS AND GONADOTROPHIC HORMONES ing season and those obtained in the non- breeding season. Kammhide, Welsh, Nal- bandov and Norton ( 1952 ) , using the same assay as Warwick- — the chick testis — found the potency highest during anestrum and lowest during estrus. Although the reproduc- tive tract of the anestrous ewe was found to be atrophic, the ovaries contained a few sizeable follicles (Hammond, 1945; Nalban- dov, 1953a, b) and ovulation was induced by exogenous gonadotroi)hins. Granting that LH is a requisite for the secretion of estro- gen as well as for ovulation, Kammlade, Welsh, Nalbandov and Norton (1952) and Nalbandov ( 1953b j speculated that anes- trum in the ewe may perhaps be attributed to an imbalance of the gonadotrophins characterized by increased pituitary FSH potency and a deficiency in circulating LH. They surmised that the follicular develop- ment was brought about by the action of virtually "pure" endogenous FSH. In accord with this view Dutt ( 1953 1 and Robinson (1952, 1954) reported that ovulation, fol- lowed in some instances by pregnancy (Robinson, 1950), can be induced in the anestrous ewe by the administration of progesterone, thereby suggesting that LH is "stored." Progesterone, as noted else- where, can be used to induce or hasten ovulation in hens, estrous rabbits, the per- sistent-estrous rat, and in monkeys during the anovulatory summer months. According to current opinion, these effects are attribut- able to excitation of neurohumoral mecha- nisms which promote the release of LH, Missing for the purpose of the present con- sideration is knowledge of whether or not the pituitary of the anestrous ewe contains LH. Unfortunately the methods used in studying the ewe pituitary have not been informative in regard to the separate gon- udotrophic activities. Especially noteworthy are the data of Simpson, van Wagenen and Carter (1956) concerning the fluctuation in pituitary gon- adotrophic potency in adult female monkeys killed at different stages in the menstrual cycle. The low titer of FSH (in unit and to- tal potency) at the beginning of tlie cycle quadrupled near the end of the follicular phase and decreased during the luteal phase. The unit and total potency of LH was high- est on (lavs 9 to 11. TheFSH to LH ratio was roughly 1:3 at the beginning and end of the cycle and 1 : 10 through the preovula- tory and ovulatory stages. This is mainly because the LH increased relatively much more during that time than did FSH. At the time of greatest concentration, the mini- mal effective dose for both FSH and LH effects was 1/10 that of pooled sheep pitui- tary powder. The two hormones were, how- ever, present in the same relative propor- tions as in sheep pituitaries. b. Pregnancy. Marked differences exist in the gonadotrophic functions of the hypophy- sis during pregnancy. In a number of ani- mals, of which the monkey, guinea pig, rat, and mouse are examples, the gonadotrophic functions of the pituitary are assumed by the placenta to the extent that the hypophy- sis can be ablated without interrupting pregnancy (for review see Smith, 1954). There are authenticated instances in which the ovaries involute especially during the later stages of gestation. Contrariwise, in many animals growth of ovarian follicles continues throughout gestation and, in some, heat ensues immediately following parturition (for review see Williams, Gar- rigus, Norton and Nalbandov, 1956, and chapter by Young on the mammalian ovary). There are many reports of mating and of spontaneous or artificially in- duced ovulation during pregnancy. It is, therefore, not unexi)ected that marked variation between species has been noted with respect to the gonadotrophic ac- tivity of the hypophysis during preg- nancy; it has been reported to increase, decrease, or to show little or no change (reviewed by Cowie and Folley, 1955). Within species, the results have not al- ways been consistent; such discrepancies are probably related to the different pro- cedures that have been used for collecting, storing, and assaying the glands. In a nota- ble example, the pituitaries of pregnant cat- tle were observed to show a steady increase in gonadotrophic content over those of non- l)rcgnant animals (Bates, Riddle and Lahr, 1935), but in a later study (Nalbandov and Casida, 1940) the potency was found to decline steadily throughout ])regnancy. Sim- ihiily. Robinson and Nalbandov (1951) found that the gonadotrojihic potency of the pregnant sow decreases throughout gesta- PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 261 tion. There is agreement that in man the pituitary gonadotrophic jiotency falls dur- ing the second month of gestation and re- mains barely detectable until a few days postpartum (Bruner, 1951). It has been assumed that in man placental gonado- trophin (HCG) is an important sustaining factor. The pregnant mare, however, also has a rich extrapituitary supply of gonado- trophin, yet shows no decline in pituitary gonadotrophic potency (Hellbaum, 1935). Some have tried to relate the instances of reduced pituitary potency during gestation to high titers of circulating estrogens, but the inconsistencies are again extreme. (For reviews of this controversial literature see Burrows, 1949; Ladman and Runner, 1953; Cowie and Folley, 1955). Recent reports have been few. Ladman and Runner (1953) in a detailed study of the pituitaries of l)regnant mice found changes suggestive of a cyclic fluctuation in potency during gesta- tion. In breeding mule deer the peak potency occurred in the fall and was followed by a sharp decline which persisted during most of gestation with a slow rise occurring near term (May or June). C. EFFECT OF DIETARY RESTRICTIONS ON PITUITARY GONADOTROPHIC POTENCY AND FUNCTION 1. Underfeeding There is a large bodj' of experimental data showing that poor nutritional status whether caused by reduced food intake or impaired assii^iilation has a deleterious in- fluence on reproductive functions (for com- IH-ehengive review of nutrition-endocrine relationships, see Ershoff, 1952 and chapter l)y Leathem). It has long been known that in the common laboratory animals con- trolled inanition and starvation produce atrophy of the gonads and accessory sex structures and varying degrees of infertility. Similarly, loss of reproductive functions have been observed in human populations during periods of restricted food intake. That the level of circulating gonadotrophins is reduced under these conditions is indi- cated by involution of the gonads and also, as shown in rats, by the fact that the gonads remain sensitive to administered gonadotrophins (Werner, 1939), the excep- tion being that stimulation of the testis requires an adequate replacement of vita- min E. Since it has been found that the pituitary gonadotrophin content remains at normal (Maddock and Heller, 1947) or somewhat greater than normal levels (^lei- tes and Reed, 1949; Rinaldini, 1949) during chronic caloric restriction, it would seem that the primary defect in pituitary func- tion is failure, not so much in the synthesis, as the release of hypophyseal gonadotroph- ins. Aladdock and Heller (1947) also em- phasized the dichotomy that is evoked through inanition between pituitary gonado- trophic content (normal) and gonado- trophic function ( low ) . This involves the important assumption that the gonadal tis- sues have not lost their sensitivity to gon- adotrophic stimuli as a result of undernour- ishment. That exogenous gonadotrophins are effective under these circumstances does not exclude the possibility of a relative reduc- tion in substrate sensitivity. It is of interest that castration type cells do not develop in the pars distalis during inanition despite the severe reduction in gonadal functions. That pituitary potency does not increase pari passu with inhibition of release of gonado- trophin is an indication that production of gonadotrophins is also impaired during these periods of restricted food intake. a. Related observations on inanition and sex function in man. Many times in the his- tory of man he has been exposed to malnu- trition of considerable duration. Currently, two-thirds of the world's population is undernourished and many authors incline to the view that actual starvation will become more widespread in the future. Although the associated endocrine disturbances have been given little attention in these conditions, there is convincing evidence that they con- stitute an extremely important part of the syndrome. Among the clinical manifesta- tions of chronic underfeeding, there are gen- erally symptoms attributable to dysfunction of the thyroid, adrenals, gonads, and pitui- tary. The few laboratory experiments in human starvation have not made full use of the various indices of endocrine function. Moreover, there is much doubt that studies of the nature of the Minnesota Experiment (Keys, Brozec, Henschel, Michelson and Tavlor, 1950) conducted under situations 262 HYPOPHYSIS AND GONADOTROPHIC HORMONES of personal security and absence of other than hunger-induced anxieties are to be compared with either mass starvation or the exigencies of war deprivations. That many of the findings in experimental animals are applicable to man is suggested by the high incidence of impaired repro- ductive functions in women during states of chronic malnutrition (Keys, 1946; Keys, Brozcc, Henschel, Michelson and Taylor, 1950; Samuels, 1948; Gillman and Gillman, 1951; Zubiran and Gomez-Mont, 1953). One of the better documented studies of the endocrine aspects of chronic malnutri- tion in man is being made in Mexico by Zubiran and Gomez-Mont. In 1953 they re- ported on 529 adult subjects, all with a long history of undernourishment, and 195 au- topsies of subjects suffering the effects of starvation. Estrogenic activity as measured by estrogen excretion and by vaginal smears was found to be absent in a high percentage of cases in women of menstrual age. The number in which menstruation had ceased was ecjually high. These evidences of the severity of the disturbances of ovarian func- tion were fully borne out by examination of ovaries at autopsy after prolonged starva- tion. The ovaries were extremely small and atrophic and not infrequently absent. It is pertinent also that Zubiran and Gomez-Mont, contrary to the work of Bis- kind (1946) and of Lloyd and Williams ( 1948) , found no evidence of hyperestrogen- ism in patients with impaired liver function. Their data show clearly that the excretion of urinary estrogen is low in a high percent- age of cases during chronic malnutrition, irrespective of the presence of cirrhosis or the extent of liver impairment. During re- covery, however, these workers often found a transitory increase in estrogen excretion sometimes of great magnitude. In males, such increases usually preceded the appear- ance of gynecomastia which generally out- lasted the period of heightened estrogen titers. Zubiran and Gomez-Mont believe that unawareness of the effect of refeeding on estrogen production may furnish an ex- planation for most of the reported cases of hyperestrogenism which have been attrib- uted to liver damage and tlie failure to in- activate estrogen. 2. Vitamin Deficiencies Gonadal dysfunctions of varying severity are also noted in animals fed diets deficient in one or another of the various B vitamins (thiamine, Drill and Burrill, 1944; imn- tothenic acid, Figge and Allen, 1942; ribo- flavin, Warkany and Schraffenberger, 1944; pyridoxine, Emerson and Evans, 1940, Nel- son and Evans, 1951; biotin, Okey, Pen- charz and Lepkovsky, 1950; and B12 , Hart- man, Dryden and Gary, 1949; the references cited are inter alia). In each instance the data suggest an impairment of the secretion of gonadotrophins and there is considerable agreement that this, in turn, is attributable to the accompanying inanition rather than to any specific vitamin deficiency per se. With respect to pituitary hormone content, Wooten, Nelson, Simpson and Evans, (1955) reported finding a striking increase in gonadotrophic potency in vitamin Be- deficient rats. In terms of the separate gon- adotrophins they found a 3- to 4-fold in- crease in FSH and little or no change in LH or LTH. With respect to vitamin E, it has been established that in rats and guinea pigs a deficiency of this factor in- jures the seminiferous tubules and causes resorption of embryos. Inconsistent findings have been reported with respect to the pitui- tary gonadotrophic potency in vitamin E- deficient rats: an increase was noted by P'an, van Dyke, Kaunitz and Slanetz (1949), and no change by Biddulph and Meyer (1941). The defects produced by ab- sence of vitamin E are in the gonads and are not correctable by administration of gonadotrophin (Mason, 1933; Drummond, Noble and Wright, 1939; Ershoff, 1943). Despite extensive study, the effect of vita- min A deficiency on pituitary gonadotrophin content has not yet been clearly defined (for a review of literature see Ershoff, 1952). 3. Deficiencji in Intake of Protein or of Spe- cific Amino Acids The effect of protein deprivation on hy- poi)hyseal functions has been lately re-ex- amined by Leathern (1958) and is dis- cussed in his chapter. He has empha- sized the importance of a lal>ile body proteni i-eser\-e and the biologic value of PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 263 dietary protein for the maintenance of normal reproductive performance and for recovery from the impairments induced by severe protein restriction. Female rats fed a diet containing less than 6 per cent pro- tein showed marked atrophy of the repro- ductive organs, whereas male rats fed pro- tein-free diets showed little impairment of spermatogenesis or loss of testis weight although the accessory sex organs were reduced by 50 per cent. Considerable data are available indicating that protein restric- tion impairs the secretion of gonadotrophin in female rats (Samuels, 1950). Less atten- tion has been paid to pituitary hormone content. Leathem (1958) noted a reduction in total gonadotrophic potency in male rats fed a protein-free diet, whereas Srebnik and Nelson (1957) obtained an approximate 2- fold increase in FSH content with no change in LH. They found also that protein restric- tion did not interfere with increases in pro- duction and release of FSH and LH follow- ing ovariotomy. Rabbits are surprisingly resistant to pro- tein depletion. Friedman and Friedman (1940) found that female rabbits main- tained on protein- free diets continued to manifest estrus and their capacity for resti- tution of the gonadotrophic hormone po- tency of the anterior pituitary following ovulation was unimpaired. The relationship of deficiencies in specific amino acids to pituitary gonadotrophic functions have not Ijeen satisfactority elucidated. The conflict- ing eviderite4s^ reviewed by Ershoff (1952). IV. Gonadal-Hypophyseal Interrelationships The dependence of the gonads on the se- cretions of the anterior lobe of the pituitary has been clearly demonstrated in the mam- mals and, although less well documented, exists for all vertebrates, to a greater or lesser degree. This dependence has brought about the consideration of the adenohy- pophysis as a "master gland," but this des- ignation connotes a degree of independent control over the endocrine function of the gonads and the other so-called target or- gans which is not strictly in accord with the facts. There is strong evidence that each of the target organs is an important factor in the determination of the functional level of the hypophysis. The gonads and the pi- tuitary play upon each other toward the achievement of a balance of function, har- monious with the fulfillment of the pro- creative functions of the organism. This reciprocal relationship, long known to endo- crinologists, has been referred to as "nega- tive feed-back" or "push-pull." Variations in these interrelationships coincide with the changing epochs of the life of the individual animal. In the mammals the main phases are immaturity, puberty, maturity, and senescence. During each there are subtleties of interplay between hypophyseal and gon- adal influences. Mention should be made of the fact that these interactions of the hy- pophysis and gonads are in large measure dependent on the permissive conditioning of the internal environment by other glands, but especially b}^ the thyroid and adrenals (see chapters by Albert and by Young on the ovary). A. IMMATURITY Following birth there is a period during which the ovaries are quite unresponsive to administered gonadotrophins. During this period, extending to about 15 days of age in rats, follicles are present which are in- distinguishable morphologically from others which respond readily at an older age. Although the reason for this ovarian insensi- tiveness to extraneous gonadotrophins dur- ing infancy is unknown, Hisaw and Ast- wood (1942) suggested that there must be ]ihysiologic differences between follicles that are morphologically similar. Zuckerman (1952), on the contrary, believes that the follicles become responsive only after a theca interna has been fully differentiated. Along this same line Hisaw (1947) specu- lated that the secretion of estrogen by the theca is a necessary preliminary to the at- tainment of follicular sensitivity to gonado- trophins. He suggested that estrogen acts on the immature follicle in the manner of an organizer, thus rendering the granulosa competent to respond to FSH. Although there is considerable evidence for a direct action of estrogen on ovarian follicles (dis- cussed more fully on page 268 and in the chapter by Young on the ovary) the assump- 264 HYPOPHYSIS AND GONADOTROPHIC HORMONES tion that initiation of ovarian secretion pre- cedes the capacity to respond morphologi- cally to gonadotrophins has not yet been substantiated by experimental evidence. Under normal circmiistances, it is pos- sible that gonadal maturation is brought about by the elaboration of gradually in- creasing amounts of pituitary gonadotro- phins, primarily FSH, or by a gradual in- crease in competence of the gonads to respond to gonadotrophins already present even in immaturity. It is tempting to sur- mise that the gonads of the immature ani- mal elaborate sufficient steroid to suppress the development of gonadotrophic func- tions. There is some evidence that this may be true in females but the situation is less clear in males. Gonadectomy at birth, as Clark (1935b I has shown, leads quickly to an increase in pituitary gonadotrophic po- tency in females but not in males. The mechanism which sustains immaturity may therefore not be the same in the two sexes. Despite the apparent refractoriness of infantile ovaries to gonadotrophins of ex- ogenous origin, they respond readily to ex- cessive endogenous gonadotrophins. Thus, ovaries of newborn rats transplanted to the anterior ocular chambers of spayed adult rats respond quickly as shown by an increase in size, follicle maturation, and ovulation, and by the re-establishment of cyclic estrus in the host (Dunham, Watts and Adair, 1941). Ovaries which would not normally mature until 45 to 70 days of age were func- tional as grafts at 10 to 20 days of age. This hastening of maturity was not influenced by the period between spaying and grafting, i.e., grafting at the time of spaying was just as effective as grafting at a later time. It is pertinent that considerable advance- ment of sexual maturation was seen by Greep and Chester Jones (1950b) in rats in which the ovaries were removed on the 26th day of life and grafted into the neck. These young animals exhibited opening of the vaginal membrane and vaginal estrus within 5 to 10 days. In this strain of rats vaginal patency normally appears at about 55 days of age. It was assumed that in the interval before circulation was re-established in the graft the i)ituitary had increased its secre- tion of gonadotrophins as it is known to do when "lonadal secretions are (>liniinate(l or rendered ineffective. The revascularized ovaries responded precociously to this heightened pituitary stimulation. Although ]Mandl and Zuckerman (1951 1 were not able to confirm these findings, it is to be noted that the vaginas in their control rats opened at a mean age of 37 days, which allows small latitude for demonstrating a hastening of vaginal opening by this procedure. Many workers have seen precocious vaginal estrus in immature female parabionts following gonadectomy of their partners (for review see Finerty, 1952» and an increase in blood level of FSH has been detected in rats 7 days after spaying (Cozens and Nelson, 1958). It is evident, therefore, that the pi- tiiitaries and gonads are in delicate hor- monal balance through the period of imma- turity, any disruption of which leads quickly to alteration of the endocrine state. B. PUBERTY AND MATURITY Puberty is characterized by comj^lex in- teractions between the gonadotrophins and the sex steroids. These become particularly evident in the cyclical episodes in the life of the mature female (Young and Yerkes, 1943). Experiments which have attempted to unravel these basic pituitary-gonadal in- terrelationships and the mechanisms re- sponsible for cyclic gonadal functions have involved, as one method of attack, the in- jection of steroid hormones into intact ])ubertal or adult female mammals. The influence of the steroid hormones, es- pecially that of the gonadal hormones and their congeners, on the pituitary has been extensively studied. The great variety of experimental procedures employed in these studies has not negated their importance, but it has made comparison of results for a given steroid or between different steroids more difficult and of less validity than might otherwise have been the case. Van Dyke (1939) and Burrows (1949) have provided comprehensive reviews of these data. It will serve the purpose here to examine recent studies wherein a more discriminatory methodology has been employed. These will illustrate tlie major ai^proaches and re- capitulate earlier findings. When any steroid, natural or synthetic, endogenous or exogenous, acts on the pitui- tarv. the alternatives ai'c that its function PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 265 will be increased, decreased, or left unaf- fected. For practical reasons alone it has been disappointing that the results achieved have been predominantly those of inhibi- tion. Consequently the physician is today fairly well armed with ways of slowing, but not of arousing, pituitary gonadotrophic ac- tivities. C. EFFECTS OF ESTROGENS ON FOLLICLE- STIMULATING HORMONE SECRETION The result of the administration of ster- oids to animals depends on not only the state of the animal, but also the dose of the hormone and duration of the treatment. Among the ^nown inhibitors of pituitary gonadotrophin secretion, the estrogens are the most effective. There is complete agree- ment that estrogen in moderate to high dosage inhibits FSH synthesis and libera- tion. No clear qualitative differences have been encountered in the effect of a variety of natural and synthetic estrogenic steroids in this regard. Quantitative differences in the capacity of the many available estro- gens to suppress FSH secretion, however, are readily demonstrable and are directh" referable to the estrogenicity of the steroid. Present evidence suggests that, although l)hysiologically equivalent dosages of a num- ber of estrogenic substances do not produce precisely comparable effects on the pitui- tary, a close correspondence exists between the ability of an estrogen to induce vaginal estrus and its inhibitory action on the pi- tuitary. "^^^-- The influence of relatively large amounts of estrogens on the secretion of gonadotro- ])hins is of interest, but particularly helpful in resolving the problems of normal physiol- ogy are those experiments in w^iich low dosages of estrogens, at or near the naturally occurring levels, have been given. Here, however, there is not agreement with respect to the meaning of the results. It has often l)een claimed that maturity may be* achieved by pituitary function independent of estrogen secretion. In the development of a broader concept. Heller and his co-workers were the first to take the position that amounts of estrogen falling within physio- logic limits have no suppressive action on pi- tuitary potency in rats nor on the secretion of gonadotrophins as measured by urinary excretion (of F8H> in women. Thus Lauson, Heller and Sevringhaus (1938) reported that ovarian development and sexual maturation in rats were not altered by chronic treatment with what they considered to be physiologic quantities of estrogen, nor did the pitui- taries exhibit any significant decrease in gonadotrophin content. Heller and Heller (1939) and Heller, Heller and Sevringhaus (1942) found that amounts of estrogen which inhibited ovarian compensatory hy- pertrophy in rats following unilateral cas- tration did not decrease pituitary potency; and in women doses of estrogen, adequate to control clinical symptoms at the meno- pause, did not diminish the excretion of gon- adotrophin. Heller, Chandler and jMyers (1944) also reported that whereas physio- logic doses of estradiol failed to prevent the typical rise in the titer of urinary gonadotro- phin following ovariectomy in women, larger doses were completely effective. This series of papers by Heller and his associates has long been puzzling in the interpretation of pituitary-gonadal relationships. It is a weak point in their evidence that they used uter- ine stimulation in recipient immature intact rats as the end point for the assay of pitui- tary gonadotrophin potency. It is doubtful if this procedure is sufficiently reliable for the establishment of this important concept. It is to be noted that in the domestic fowl Breneman (1955) likewise has observed no suppressive effects on gonadal maturation using doses of estrogen that are wathin the upper limits of normal blood estrogen levels. Heller's observations have been contra- dicted by later observations based on more critical assay procedures. Biddulph, Meyer and Gumbreck (1940) suggested that gon- adotrophic functions in rats are inhibited by doses of estrogen well below the threshold dose for the estrous reaction of the vagina. Of greatest interest and pertinence, Byrnes and Meyer (1951a) found that the minimal amount of estradiol or estrone re- quired to stimulate the uterus in the spayed member of spayed-intact parabionts was 3 to 4 times that needed to inhibit the pi- tuitary of the same animal as judged by the ovaries of the adjoined twin. In an extension of this study Byrnes and Meyer (1951b) administered estradiol in closely graded doses to single immature (30-dav-old) and 2G6 HYPOPHYSIS AND GOXADOTROPHIC HORMONES adolescent (55-day-old) rats, intact and spayed, for 10 days. The amounts given were estimated to be within physiologic limits. In the immature animals more estro- gen was required to produce positive stimu- lation of the uterus than to inhibit the pitui- tary; the evidence for a similar differential in the adolescent group was equivocal. Greep and Chester Jones (1950b), using adolescent rats, observed that a dose of estradiol benzoate which would inhibit ovar- ian development in intact rats was adecjuate for uterine maintenance in coetaneous cas- trates. Byrnes and Meyer (1951b) observed further that the quantity of estrogen re- quired to inhibit the pituitary increased from the immature to the adolescent rats by a factor of 2.77. Although dilution of hormone is involved because of the greater size of the older animals, this did not seem to invalidate the conclusion that the an- terior pituitary becomes increasingly less sensitive to the suppressing action of estro- gen as sexual maturation progresses. These data indicate that FSH secretion and estrogen bear a reciprocal relationship and emphasize that FSH secretion is not a pituitary property sui generis but depends *on the concomitant secretion of steroids. This idea has an important bearing on how a state of sexual maturity is attained. If we consider that the evidence is in favor of a prepubertal gonad-pituitary interaction, we must assume that the immature gonad has some capacity to secrete estrogen. In an un- confirmed report, Zephiroff, Drosdovsky and Dobrovolskaya-Zavadskaya (1940) have , claimed that immature rat ovaries have a significant estrogen content. Byrnes and Meyer (1951b) have postulated that re- fractoriness of the adolescent rat pituitary to estrogen permits higher levels of FSH secretion and thus allows the attainment of l)uberty. It seems clear that future progress in this area will depend on the development of satisfactory methods for measuring the blood level of pituitary and ovarian hor- mones. Until such information is available it would be helpful merely to have some of the present evidence confirmed. If, for in- stance, it were firmly established that the pituitary of the immature animal is more sensitive to estrogen than is the uterus, this would form an excellent starting point for future investigations. Exceptionally valuable information con- cerning the effect of administered steroids on pituitary function has been gained from studies of parabiotic rats. The preparation which has been most commonly employed has consisted of immature rats, one of which is castrated at the time of or following the surgical union. Given no treatment, as in control pairs, the castrated animal's pitui- tary steps up the output of gonadotrophins, thereby stimulating the gonads of the ad- joined intact member. The resulting ovarian secretions do not reach the pituitary of the castrated partner and hence exert no in- hibitory influence on it. Using substitute steroids administered from the time of spay- ing, it has thus been possible to ascertain their ability to inhibit the castration-in- duced hypersecretion of gonadotrophins. The uterus of the castrate provides an index of the estrogenicity of the administered steroid. Meyer and Hertz (1937) demonstrated convincingly that larger doses of estrogen or androgen are required to inhibit the pi- tuitary of a male than of a female rat. In each the degree of gonadal inhibition in the intact member was roughly propor- tional to the dose of sex steroid administered to the conjoined castrate. Biddulph, Meyer and Gumbreck (1940) observed that the minimal amounts of sex hormones required for complete inhibition of the postcastration rise in secretion of pituitary gonadotrophins were in females: 0.025 fxg. estradiol, 1.5 /xg. estriol, 1000 /xg. progesterone; and in males: 0.15 fxg. estradiol, 10 ^g. estriol, and 1000 /xg. progesterone. These workers also noted that the order of effectiveness of estrogens in suppressing gonadotrophin secretion paral- leled the order of their capacity to i)roduce vaginal cornification in rats, viz., estradiol, estrone, and estriol. In male animals estrogen damages the spermatogenic epithelium of the testes to the point that spermatogenesis is completely interrupted and the testes shrink to infantile size. At the same time the basophils of the pituitary are severely degranulated and re- duced in numbers relative to other cell types, and the gonadotrophic potency of PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 267 the pituitary nearly vanishes. In the human male the excretion of urinary gonadotro- phins is sharply reduced. D. EFFECT OF ESTROGEN ON LUTEINIZING HORMONE AND LUTEOTROPHIN HORMONE SECRETION An important consideration in assessing the action of steroids on pituitary function is the effect these have on the output of the luteinizing hormone. In most of the prior discussion reference to inhibition of gonado- trophin secretion has been to that of FSH. The assumption that estrogen acts in some manner to elicit a more effective luteinizing stimulus from the pituitary appears in nearly all papers dealing with pituitary- gonadal relationships. It constitutes a key point in all theories so far advanced to ex- plain sexual periodicity in the female. The idea that estrogen may act to release LH originated with the "Hohlweg effect." This author (1934) found corpora lutea appear- ing in the ovaries of immature rats as the result of a single injection of estrogen. This response has not been obtained by other workers (Bradbury, 1947; Greep and Ches- ter Jones, 1950aj using 21-day-old imma- ture rats. It seems that the response is ob- tained only if the rats are nearing sexual maturity (Burrows, 1949). In adult rats massive daily doses of estrogen induce a type of pseudopregnancy and have a de- cided effect on the corpora lutea; they in- crease inxsize to resemble corpora lutea gravidari, and-~ai'e functional as shown by their ability to maintain diestrous vaginal smears for 3 weeks in face of heavy estrogen treatment (Merckel and Nelson, 1940). To what extent such changes are due to LH or LTH, or both, is not clear. It might be ex- pected that the large amount of estrogen would suppress LH secretion. If this were the case, continued LTH secretion would be independent of estrogen titers and, fur- thermore, would be capable of both main- taining and evoking secretion from the corpus luteum. In the rat, at least, this seems to be consonant with the capacity of pituitary homografts to secrete LTH se- lectively (Everett, 1956). Hellbaum and Greep (1940, 1943) noted that the pitui- taries of castrated rats treated with estro- gen for more than 20 days gradually lost their LH potency. They presumed that the LH component was released, but they were not able to detect it in the bloodstream with certainty. Greep and Chester Jones (19501)1 tried to demonstrate by several means an LH-releasing action of estrogen in intact and gonadectomized rats of both sexes, but were forced to the conclusion that the funda- mental effect of estrogen on the pituitary appeared to be to reduce the synthesis and storage of LH. A further point against the concept that estrogen triggers off an out l^ouring of LH from the pituitary is the fact that no sudden upsurge in LH activity has been observed in the male by an injec- tion of estrogen, as judged by the condition of the testicular Leydig cells or by the size and histologic appearance of the ventral prostate. There is evidence, however, favoring the concept that estrogen promotes release of LH. Funnell, Keaty and Hellbaum (1951) administered estrogen to a selected group of patients manifesting classical menoj^ausal symptoms and were able to demonstrate LH activity in the urine where previously only FSH activity had been detectable. Con- firmatory results ha\-e been reported by Brown (1956, 1959) using patients with secondary amenorrhea. The relation of estrogen to ovulation bears on the purported LH-releasing action of this substance. Everett, Sawyer and Alarkee (1949) succeeded in hastening ovu- lation in normal cycling rats by a properly timed injection of estrogen, but the re- sponse is not specific inasmuch as other substances, including progesterone, can pro- duce the same effect. The work of Everett (1948) and co-workers (for review see his chapter in this book) suggests that estrogen may act on the anterior hypophysis in- directly through some neural, conceivably hypothalamic, mechanism. Estrogens have been used with variable, but generally poor, success in promoting ovulation. The fact that the estrous rabbit does not ovulate in response to injected estrone (Bachman, 1935) is not considered critical evidence, since ovulation in this species normally in- volves neural excitations associated with mating. More decisively the persistent- 268 HYPOPHYSIS AND GONADOTROPHIC HORMONES estrous rat, which ovulates with such regu- larity after the administration of pro- gesterone (Everett, 1940; Hillarp, 1949; Greer, 1953) or testosterone (INIarvin, 1948), does not thus respond to estrogen unless pretreated for several days with pro- gesterone (Everett, 1950). The latter result indicates that progesterone may synergize with or otherwise facilitate the LH-releasing action of estrogen. Hammond, Jr. (1945), on the other hand, succeeded in obtaining ovulation in anestrous sheep with low, but not with high, doses of estrogen. It would seem that the effect of estrogen on LH re- lease varies between species and within spe- cies and is greatly influenced by the age of the animal, dosage used, and the time in the sex cycle at which treatment is instituted. E. DIFFERENTIAL EFFECTS OF GONADAL STEROIDS ON FOLLICLE-STIMULATING HORMONE AND LUTEINIZING HORMONE SECRETION The root of the matter in the regulation of gonadal function is the interplay of FSH and LH with the sex steroids. With respect to estrogens, the results do not lead to a clear-cut conclusion. Paesi (1952) provided data on a series of immature rats treated for 7 days with doses of estradiol benzoate, ranging from 0.0002 to 100 /xg. daily. The dose-response curve representing the effect of estrogen on ovarian weight was diphasic. Doses of 0.01 to 0.05 /xg. daily decreased the ovarian weight, whereas greater amounts up to 10 fxg. increased the ovarian weight slightly; however, only with 100 /xg. did the gain attain significance. The decrease in ovarian weight with low doses represents, he believes, an impairment of LH release, inasmuch as the ovarian interstitium seemed deficient. The enhanced ovarian weight with excessive dosage was presumed to be due either to increased FSH release or to FSH enhancement by released LH, as sug- gested by the stimulated interstitium. It is to be noted, however, that even in hypophy- sectomized immature female rats, excessive doses of estrogen produced significant ovar- ian enlargement and stimulate the develop- ment of an unusual number of medium sized "solid" follicles (Williams, 1940; Pcn- charz, 1940; Gaarenstroom and de Jongh, 1946; Payne and Ilellbaum, 1955). Bradbury (1947) proposed that the effects of estrogen on pituitary gonadotrophic functions are made more accountable when the results are considered in terms of acute and chronic estrogen treatment. The latter is well known to suppress gonadotrophic function and to deplete the gonadotrophin content. His acute experiments lasted only 48 to 120 hours and revealed a significant increase in ovarian weight, which confirms the results reported by Price and Ortiz (1944) and others. Greep and Chester Jones (1950b) also obtained a confirming result and showed further than the reaction was dependent on the presence of the pituitary. In this connection it is pertinent to note that Bradbury found a concurrent reduction in pituitary gonadotrophic potency at 72 to 120 hours after the injection of estrogen and concluded that the initial action of estrogen is to release gonadotrophin (unspecified, but the effect is mainly that of FSH) . The stim- ulated ovaries showed in addition to ac- celerated follicular growth a swollen inter- stitium, suggesting that both FSH and LH had been released. The interstitial cell change was not confirmed in a later study (Greep and Chester Jones, 1950b). Thus, although it seems clear that under a given circumstance the immediate reaction to es- trogen may be a slight upsurge in either FSH release or enhancement of FSH action, it is unlikely that FSH production or re- lease is normally dependent on estrogenic action, since both its synthesis and release are greatest when no estrogen is present. Meyer, Biddulph and Finerty (1946), Gaarenstroom and de Jongh (1948), Greep and Chester Jones (1950a, b), and Byrnes and Meyer (1951b) attempted involved analyses of the effect of gonadal steroids on pituitary function in terms of the separate gonadotrophins, FSH and LH. The wide di- vergence in their accounts emphasizes how poorly these matters are understood. F. EFFECTS OF ANDROGENS ON PITUITARY GOXADOTROPHINS Considerations similar to those enumer- ated in our discussion of the estrogens apply to the influence of androgens on pituitary gonadotrophins, yet with well defined dif- ferences. The effects of androgens have been studied in females quite as much as in males. PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 269 The early idea that the gonadal hormones were sex specific has been abandoned and the possibility of abnormal functioning is hardly considered when androgens are stud- ied in the female. Perhaps it should be, be- cause in many females among the Mam- malia androgens are not known to have a physiologic role. There is much doubt also as to how importantly androgens contribute to the regulation of the pituitary in the fe- male. For the most part, injection of andro- gen into female mammals produces a dele- terious effect on ovarian development, structure, and cyclic functions. Two excep- tions may be noted: van Wagenen (1949) injected sexually immature female monkeys with androgen for long periods and obtained a remarkable hastening of the appearance of puberty. The age at first menses was, in fact, reduced by nearly one-half. Androgen injections have also been used successfully to promote ovulation in persistent-estrous rats (Marvin, 1948). Very soon after the pure androgenic ster- oids became available, it was established that these substances in adequate dosage (which varied according to the androgenic- ity of the compound) (1) caused a partial reduction of the pituitary gonadotrophic potency in castrated rats (Heller, Segaloff and Nelson, 1943), and (2) prevented the appearance of castration cells in the pitui- tary (Nelson, 1935; Nelson and Merckel, 1937a; Wolfe and Hamilton, 1937). The work of Hertz and Meyer (1937) and Nel- son (1937i^ placed androgen-pituitary rela- tionships onlTijuantitative basis. The rela- tive efficiency of testosterone propionate and dehydroandrosterone in restricting the secretion of gonadotrophins by gonadec- tomized parabionts was determined by Hertz and Meyer (1937). Each compound in adequate dosage completely suppressed secretion of pituitary gonadotrophins as shown by absence of ovarian stimulation in the intact partner. Using these compounds in dosages less than that required to pro- duce complete ovarian suppression, the de- gree of inhibition was found to be roughly proportional to the dose. The androgens evoke clear-cut alterations in pituitary FSH and LH potency and no doubt are in part responsible for the quan- titative sex differences in the pituitary content and level of secretion of these gon- adotrophins. On a comparative basis the pi- tuitaries of adult males are distinctly richer in FSH than those of adult females (Hell- baum and Greep, 1938; Greep and Chester Jones, 1950b; Hoogstra and Paesi, 1955; Paesi, de Jongh, Hoogstra and Engelbregt, 1955), yet in the absence of androgen, as after castration, FSH increases as though an inhibiting influence had been removed. Long-term castrates given large doses of testosterone showed only a partial (Heller, Segaloff and Nelson, 1943) or no reduction in gonadotrophic potency. Examining the effect of androgens on the pituitary gonado- trophins of adult intact female rats, Greep and Chester Jones (1950b) found unex- pectedly that within a specified range of dosage (0.1 to 0.5 mg. of testosterone pro- pionate per day) , FSH potency was elevated over that of untreated controls. Similar findings have been reported by Pincus (1950), Hoogstra and Paesi (1957), and Paesi, de Jongh and Willemse (1958) . Hoog- stra and Paesi made the additional observa- tion that the increase of pituitary FSH with androgen treatment (2 mg. of testos- terone propionate daily) is not limited to intact females, but occurs in intact males and gonadectomized males and females as well. Furthermore, the response was not modified by simultaneous administration of 2 fxg. of estradiol benzoate. Interest in the androgen-LH relationship is stimulated by the consideration that LH acting independently evokes androgen se- cretion by the testis, whereas its ability to elicit the secretion of estrogen by the ovary necessarily involves other factors, notably FSH. It would be expected then that the LH-testicular androgen relationship might be subject to somewhat more precise analy- sis on the basis of experimental data, but this has not been fully realized. There is some evidence that a push-pull mechanism is operative. The injection of testosterone for more than 30 days results in the elimina- tion of LH potency from the pituitary glands of long-term gonadectomized rats (Hellbaum and Greep, 1943; Paesi, de Jongh and Willemse, 1958). The latter au- thors concluded that testosterone depressed the LH content to lower levels in intact rats 270 HYPOPHYSIS AND GONADOTROPHIC HORMONES than in gonadectomized animals of the same sex. Intact adult female rats treated with tes- tosterone show cessation of cycles (Nelson and Merckel, 1937b), involution of the corpora lutea, and a marked reduction in ovarian weight (Greep and Chester Jones, 1950b j. The minimal effective inhibitory dose is 10 /tg. daily. A dosage 50 times greater (0.5 mg.j, however, does not pro- duce the same severe degree of ovarian atrophy that is seen with long-term estrogen treatment. The ovaries, although small, have a translucent blistery appearance. This is due to the presence of a small number of semi-atrophic vesicular follicles and to the relatively great reduction in amount of ovarian lipids (Greep and Chester Jones, 1950). From the foregoing evidence it would appear that androgen treatment favors FSH storage over FSH release and that androgen can suppress both the elaboration and I'elease of LH. The effect of exogenous androgens on the gonads of the intact male rat varies widely with the dose administered. Low to mod- erate doses of testosterone produced severe testicular injury (Selye and Friedman, 1941 ; Shay, Gershon-Cohen, Paschkis and Fels, 1941; Rubinstein and Kurland, 1941; Ludwig, 1950; Greep and Chester Jones, 1950b) ; maximal reduction in testes weight and complete inhibition of spermatogenesis were obtained with 0.1 mg. of testosterone propionate daily for 30 to 45 days. Large doses of androgen (2 to 20 mg. daily) main- tained testis size and sperm production even in the absence of the hypophysis (Walsh, Cuyler and McCullagh, 1934; Cutuly, Mc- Cullagh and Cutuly, 1937; Nelson, 1937; Leathem, 1944) . The response is produced by direct action on the tubular epithelium of the testes. A similar effect was produced in hypophyscctomized mice (Nelson and Merckel, 1938), ral)bits (Greep, 1939), and monkeys (P. E. Smith, 1944). Local mainte- nance of spermatogenesis has also been ob- served in the tubules in close proximity to intratesticular implants of testosterone pel- lets in hypopliysectomized rats (Dvoskin. 1947) and monkeys (P. E. Smith, 1944 ». Several androgenic steroids and their deriva- tives have been tested in regard to their ability to maintain the testes in hypopliy- sectomized rats (Nelson, 1937). It is of in- terest that the spermatogenic properties of the various androgens were not found to be related to their androgenicity as measured by ability of the compounds to stimulate the rat prostate or the capon's comb. Preg- nenolone, a nonandrogenic steroid, did not reinitiate spermatogenesis, but it did partly maintain spermatogenesis, when given im- mediately after hypophysectomy (Dvoskin, 1949). In man the testes apparently are not bene- fited by exogenous androgens at any dose level but are, on the contrary, affected ad- versely. In 1940 Heckel noted a drop in sperm count during testosterone therapy, a finding that is now a common clinical ex- perience. In the past few years much interest has centered on the recovery phase. Heller, Nelson, Hill, Henderson, Maddock, Jungck, Paulsen and Mortimore ( 1950 », Heller, Nel- son, Maddock, Jungck, Paulsen and Morti- more (1951 ), and Ewell, Munson and Salter (1950) reported finding a rebound in sper- matogenesis and in sperm counts following the cessation of androgen therapy. They ob- served that for a year or more following cessation of treatment the sperm counts may be well above the pretreatment levels and that scleroses and hyalinization of the tubular walls may be lessened. The number of cases studied has been extended (Heckel, Rosso and Kestel, 1951; Heckel and ]Mc- Donald, 1952; Swyer, 1956) with poor agreement as to improvement in sperm count. Because the already subnormal testis is further damaged by androgen, there is need for additional study of this interesting phenomenon. Although rats and man have been ex- tensively investigated, little information is available about other mammals. Wells (1943) injected male ground squirrels with testosterone in daily doses from 0.05 to 20 mg. beginning just before the peak of the annual sexual cycle. He found that the in- terstitial cells were severely damaged and the testes were somewhat reduced in size, but the spermatogenic capacity of tubular einthelium was unimpaired. His assump- tion that the release of ICSH was being inhibited has been amply substantiated by other studies. His failure to find tubular ati'()i)hv with the low doses is I'atlu'i' sur- PHYSIOLOGY OF ANTERIOR HYPOPHYSIS !71 prising and one can only conjecture that the release of FSH was not being interfered with. The higher doses were obviously adequate for direct tubular maintenance ir- respective of ICSH or FSH inhibition. G. OTHER STEROIDS AND OXIDATION PRODUCTS Several synthetic steroids related to the sex hormones but which exhibit no estro- genic activity have proved to be without ef- fect on pituitary gonadotrophin content (]\Iortimore, Paulsen and Heller, 1951). Lipoadrenal extract and desoxycorticoster- one acetate produced some uterine develop- ment and some inhibition of hypophyseal secretion of gonadotrophins, whereas corti- sone was neither estrogenic nor inhibitory of gonadotrophin secretion (Byrnes and Ship- ley, 1950). Although exogenous estrogens and androgens inhibited the development of intrasplenic ovarian grafts in guinea pigs (Lipschiitz, Iglesias, Bruzzone, Humerez and Penaranda, 1948), and rats (Takewaki and Maekawa, 1952), progesterone and desoxycorticosterone failed to do so. O. W. Smith (1944, 1945) maintained on the basis of her investigations that it is not the circulating gonadal hormones per se which influence pituitary function and po- tency but the oxidation products of these hormones. She used mainly the lactone of estrone prepared by W. W. Westerfeld and by Alan Mather. In her hands the lactone both increased the size of the pituitary and decreased its^gonadotrophic potency. The studies of Bradbury (1947) and of Morti- more, Paulsen and Heller (1951) suggested that any pituitary responses induced by es- trololactone may be attributable to its es- trogenicity, which is approximately 1/100 that of estrone in regard to both estrogenic activity and pituitary gonadotroiihic in- hii)ition. H. EFFECT OF PROGESTERONE ON PITUITARY GONADOTROPHIC FUNCTIONS Species vary in the extent to which the secretion of gonadotrophins is influenced by progesterone. In the guinea pig which has a 16- to 17-day cycle, progesterone inhibits the preovulatory swelling (generally at- tributed to LH) , but growth up to this point is unaffected (Dempsey, 1937). In rodents with short estrous cycles the influence of progesterone on the secretion of gonado- trophins appears to be minor, in that mod- erate doses do not alter ovarian maturation or cyclic functions (Greep and Chester Jones, 1950b). Although some inhibition of gonadal functions can be demonstrated after massive doses of progesterone, interpreta- tion is always complicated by the weak es- trogenicity and androgenicity of the com- l)ound. Similarly, total gonadotrophin content of the pituitary is little altered by progesterone. Although progesterone is primarily a product of the corpus luteum and exerts a major function during the luteal (and gravid ) phase of the cycle, there are strong indications that in some species the secre- tion of progesterone is initiated in follicles before ovulation (guinea pig, Dempsey, Hertz, and Young, 1936; rat, Astwood, 1939; Boling and Blandau, 1939; mouse, Ring, 1944). Consistent with this conclu- sion is the finding that the peak level of progesterone in the blood of cyclic rats coincides with the proestrum as determined by the Hooker-Forbes assay (Constan- tinides, 1947). By the same means proges- terone activity has been found in the blood of laying hens but not in nonlaying hens or roosters (Fraps, Hooker and Forbes, 1948. 1949). The preovulatory appearance of pro- gesterone is believed to be important in the elicitation of behavioral estrus (see chapter by Young ) and as a component of the hypo- thalamo-pituitary triggering mechanism for ovulation (see chapter by Everett). Current interest in the progesterone regu- lation of gonadotrophic functions has been focused on the latter mechanism. Proges- terone induces or at least hastens ovulation in cyclic-estrous rats (Everett and Sawyer, 1949a), persistent-estrous rats (Everett, 1940), rabbits (Sawyer, Everett and Mar- kee, 1950), hens (Fraps and Dury, 1943; Rothchild and Fraps, 1949), cattle (Hansel and Trimberger, 1952), anestrous ewes (T. J. Robinson, 1954), anovulatory monkeys (Pfeiffer, 1950) , and women (Rothchild and Koh, 1951 ) . Notably too, both ovulation and luteinization have been observed in ovar- ian grafts in castrated male rats following administration of progesterone (Kempf, 1949, 1950) . Such grafts normally show only HYPOPHYSIS AND GONADOTROPHIC HORMONES follicular development. Since ovulation de- pends on an acute discharge of LH (dis- cussed elsewhere), the question is raised as to the role of progesterone in this regard. Firstly, it is important to note that pro- gesterone probably is not in itself a re- leasing agent; rather, it appears to synergize with estrogen to facilitate the triggering mechanism. Moreover, although the pos- sibility exists that progesterone acts di- rectly on the hypophyseal cells to release LH, an impressive body of evidence sug- gests that progesterone promotes LH release (indirectly) by acting on the central nerv- ous system — probably the hypothalamus (for review see Markee, Everett and Saw- yer, 1952 and the chapter by Everett) . Un- der other conditions, quite the reverse of being a stimulus for ovulation, progesterone is a potent inhibitor of ovulation. There can be little doubt that the suppression of ovu- lation during the luteal and gravid phases of the reproductive cycle is due to pro- gesterone. Single doses of progesterone pre- vent ovulation in the mated rabbit, and continued high dosage to cyclic rats (Phil- lips, 1937), guinea pigs (M0ller-Christensen and Fonss-Bech, 1940), sheep (Dutt and Casida, 1948) , and cattle (Ulberg, Christian and Casida, 1951) postpones ovulation in- definitely. I. NEUROHYPOPHYSEAL INFLUENCES ON GONADOTROPHIN SECRETION Recent evidence reviewed by Benson and Cowie (1957) has tended to link the pos- terior lobe of the hypophysis with the mech- anism for release of prolactin. Many years ago Selye (1934) demonstrated that the nervous excitation of suckling served to maintain lactation. A reflex arc was believed to be involved and this has been partially identified by later studies. The stimulus of suckling has been shown to act through the central nervous system (Eayrs and Badde- ley, 1956) and the hypothalamo-neurohypo- phy.seal axis (Andcrsson, 1951a, b) and to effect the release of oxytocin (Cross and Harris, 1950, 1951). The latter causes con- traction of the myoejiithelial components of the alveoli, thereby effecting milk ejection (let-down). A number of workers (see Donker, Koshi and Petersen, 1954) have obtained evidence that tlic l)cneficial effect of oxytocin on lactation is more pro- nounced than can be accounted for on the basis of change in intra-alveolar pressure. The question then arose as to wdiether oxy- tocin has an indirect as well as a direct ac- tion on the mammary structure. Benson and Folley (1956, 1957) noted that continued administration of synthetic or commercial oxytocin retards mammary involution in lactating rats following the interruption of suckling. Inasmuch as a similar effect was obtained by injecting prolactin, Benson and Folley have speculated that oxytocin may provide the stimulus for release of prolactin, ergo LTH, from the anterior pituitary. Other lines of evidence have also sug- gested a relationship between the posterior l)ituitary and gonadotrophic functions of the anterior lobe. Desclin (1956a, b) and Stutinsky (1957) have showai in rats that injections of oxytocin at the time of estrus produce a pseudopregnancy reaction. The results suggested enhancement of LTH re- lease, but the significance of these findings has been questioned because the response is not specific for oxytocin. Pseudopregnancy has been induced by the administration of a variety of nonspecific substances, such as plant juice extract (Dury and Bradbury, 1942) and ovalbumin, etc. (Swingle, Seay, Perlmutt, Collins, Barlow and Fedor, 195i) to female rats at the time of estrus. V. Anatomic Features Important to Modern Concepts of Pituitary Gonadotrophic Function In terms of general morphology there is little to add to the classical knowledge concerning the hypophysis of mammals. Present considerations turn largely on the details of the vascular supply and the in- nervation of tlie gland. In this regard the literature has been notably enriched by Green's (1951a, 1952) comprehensive study of 76 species ranging from amphioxus to man and l)y Wingstrand's monograph (1951 ) on the structure and development of the a\ian jiituitary. The notable advances which have been made in the cytologic and eh'ctron niici'oscopic identification of cell tyi)es in the pars distalis are reviewed in the chapter by Purves. A distinct regional lobulation of the pars distalis. fii'st described in the eliicken and PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 273 duck, was subsequently shown to be widely characteristic of birds (Rahn and Painter, 1941, 18 species; Wingstrand, 1951a, 50 gen- era). According to these authors the pars distalis in birds comprises two histolog- ically distinct parts, which they designate "cephalic" and "caudal" lobes. The zonation develops during embryonic life and, in stained preparations of the adult gland, is evident to the naked eye. A conspicuous re- gional distribution of specific cell types has been noted also in the anuran hypophysis (Dawson, 1957) . These observations in lower forms provide an evolutionary background for the numerous instances of regional dis- tribution of cell types described in mam- malian pituitaries (Dawson, 1939, 1948; Halmi, 1950, 1952; Purves and Griesbach, 1951a, b, 1955; Ferrer and Danni, 1954). Ferrer (1957) noted a correlation between the arrangement of the vascular supply to the adenohypophysis in rats and the pattern of distribution of basophils. Three zones were recognized, each of which has a par- ticular basophilic picture. Dawson (1957) likewise noted a rather specific relationship of cell types to the vascular pattern in the frog. The observation that a pars intermedia is absent in chickens (Kleinholz and Rahn, 1939) has been confirmed in all species of birds thus far examined (Rahn and Painter, 1941; Wingstrand, 1951; Marshall, 1955). A. INNERVATION OF THE HYPOPHYSIS The qu^tion of the innervation of the hypophysislia^HDecome a matter of critical importance in the elucidation of mechanisms regulating the various functions of this organ, especially those of the anterior lobe. Fibers of hypothalamic origin sweep down the infundibulum in great numbers to end mainly in the processus infundibularis and, to a minor and perhajis questionable extent, along the infundibular stem. Early workers generally found some of these neurohy- pophyseal fibers entering the pars inter- media and terminating in the pars distalis, but never in numbers to inspire confidence in their significance. Vazquez-Lopez (1949, 1953) advocated the view that there is an adequate anatomic basis for neural control of the adenohy- pophysis. Using modifications of the classi- cal silver impregnation techniques of Cajal and Rio del Hortega, he observed presump- tive nerve fibers coursing through the pars distalis of the rabbit. These terminated with typical nerve endings in connection with the glandular cells. The fibers were claimed to originate from the tractus hypophysius, to cross over to the pars tuberalis in abun- dance, and in fewer numbers to follow the general course of the vascular elements to the pars distalis. In 1952 Vazquez-Lopez and Williams, reporting on their examina- tion of these relationships in the rat, de- scribed the presence of rather sizeable nerve bundles in the marginal zone of the median eminence, and in the pars tuberalis. They were uncertain of the origin and course of these fibers. In a study of the horse Metuzals (1954), using the Bielchowsky and the Gomori staining methods, traced presumptive nerve fibers from the hypo- thalamus to their endings in the pars distalis. Of the fibers seen in the pars tu- beralis, most are held by Stutinsky (1948), Christ (1951), Nowakowski (1951), and Benoit and Assenmacher (1951a) to be de- rived from similar fibers which have been observed in the marginal zone of the median eminence and along the inferior aspect of the neural stalk. A dense "secretomotor ground plexus" specifically innervating the gland cells has been described by Metuzals (1956). With respect to Metuzals' prepara- tions, A. J. Marshall (1955) states that they "undoubtedly reveal an extensive and spe- cific innervation of glandular cells in the pars distalis." In the ferret, also, sparse nonmyelinated nerve fibers and end organs of characteristic appearance have been ob- served (R. N. Smith, 1956) by a modifica- tion of Ranson's pyridine-silver method. In all these instances the nuclei of origin of the fibers remain obscure. Truscott (1944) and Wingstrand (1951a) among others described autonomic fibers entering with and terminating along the sinusoids of the anterior lobe. In the pars distalis of man Hagen (1951) reported the finding of extremely fine fibrillar networks, which were believed to enter the pars dis- talis from the capsule of the gland; and Westman, Jacobsohn and Hillarp (1943) re- ported the persistence of an autonomic fiber system in the anterior pituitaries of rabbits after cervical sympathectomy. Metuzals 274 HYPOPHYSIS AND GONADOTROPHIC HORMONES (1956), applying the Bielchowsky-Gros technique to the adenohypophysis of the duck, demonstrated in the capsule and throughout the gland the presence of an autonomic neural formation composed of large strands of nerve fibers replete with ganglion cells and end formations adjacent to parenchymal cells. The presence of even these minor neural contributions to the pars distalis has been vigorously denied by Green (1951b). He was not able to identify such perivascular fibers in the pars distalis, although they were found in the pars tu- beralis. It is significant that all attempts at in- fluencing pituitary secretory function by sympathectomy or by proximal stimulation of the cervical sympathetic trunk have yielded either negative or eciuivocal findings (Friedgood and Pincus, 1935; Friedgood and Cannon, 1940). Although bilateral re- moval of the superior cervical or stellate ganglia of the sympathetic system in fer- rets has been shown to abolish (Abrams, Marshall and Tliomson, 1954) or delay (Donovan and Van der Werff ten Bosch, 1956) the estrous response to added illumi- nation, the latter authors believe this may be accounted for on the basis of an indirect effect ; namely, a diminished amount of light impinging on the retina. All the operated animals showed a marked Horner's syn- drome, i.e., globe recession, ptosis, and nar- rowing of palpebral fissure. From the foregoing evidence, the conclu- sion seems clear that the regulation of the pars distalis by secretomotor nerves must be slight, if indeed there is such regulation. All the instances of identification of nerve fibers are open to doubt for the reason that the staining methods do not adequately differentiate nerves from reticular connec- tive tissue fibers. It is to be noted also that by use of phase contrast microscopy in combination with staining i)roccdures. Green (1951b) found no fibers in the rabbit or human pars distalis that could with cer- tainty be identified as nerves. B. TIIK MKOIAN EMINENCE AND THE I.VFUNDIBULAR STEM The eminentia has become a focus of interest in attemi)ts to understand neuro- endoci'iiic I'clationsliips, because this area is contiguous with both the hypothalamus and the hypophysis. It is present in vertebrates from amphibians to mammals, and its structure is quite uniform throughout the birds and mammals (Green, 1951a; Wing- strand, 1951b; Nowakowski, 1951). The median eminence is comprised of an inner ependymal zone, a middle coarse-fiber zone made up of the axons of the supraoptic- hypophyseal tract, and a so-called glandular zone at the surface. The name of the latter was derived from the density of the capil- lary skein on its surface — it has also been termed the peripheral or marginal zone. The glandular zone contains the capillary loops of the primary portal plexus (de- scribed below) , perpendicularly arranged fi- bers extending from cells in the ependymal zone and some fibers of nerve cell origin. The latter lie along the base of the glandular zone from which recurrent loops are said to extend toward the surface (Wingstrand, 1951b; Nowakowski, 1952; Assenmacher and Benoit, 1953a, b). Stutinsky (1951) and Vazquez-Lopez and Williams (1952) believed that they have demonstrated fibers from the tractus hypophysius crossing the marginal zone to enter the pars tuberalis, thence to follow the course of the portal veins toward the pars distalis. The exis- tence of such fibers is denied by Rumbaur (1950), Wingstrand (1951a), and Palay (1953a). The innervation of the glandular layer is, as Wingstrand (1951a) states, the key to the postulated control of the pars distalis by a neurovascular mechanism. For birds he states: "No nerve fibers have been seen leaving the eminentia but invariably turn back when they reach the surface. ..." The glandular zone contains a fiocculent colloid that is demonstrable with the azan trichrome stain, but there is no agreement as to whether this material stains selectively with chronic alum liematoxylin (Gomori- l)ositive) like tlic neurosecretory substance in tlie tractus hypophysius. Benoit finds ahmidant Gomori-positive material in the glandular zone in the duck, whereas Wing- strand's examination of a great variety of birds i'e\-eal('d faintly positive reacting colloid only in a restricted area in the rostral portion of the median eminence. He doubted that it is similar to the heavily staining neuroscci'ctoi'V substance in the supraoptic- PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 275 hypophyseal tract. The central zone ex- hibits, of course, abundant Goraori-positive material. In 1957 Dawson presented morphologic evidence of a closer neurovascular relation- ship in the median eminence of frogs than has yet been observed in mammals. Study- ing the hypothalamo-hypophyseal relation- ships at the level of the median eminence in sections stained by modifications of the chrome alum hemotoxylin-phloxine and the aldehyde fuchsin methods, he found that of the fibers from the preoptic tract a sur- prisingly large number enter and end in the median eminence and have a specific asso- ciation with the vessels of the portal system. The final simple nerve terminals (dilated with secretion) and the selectively stained secretory substance were arranged in radial patterns about the capillaries. It has not been determined whether the amount of neu- rosecretory material accumulated about the vessels in the primary plexus is of func- tional significance, but Dawson noted a specific cell type scattered along the portal vessels as they enter the anterior lobe, and felt that the "rather specific morphologic pattern displayed" suggests a functional interaction of some kind between nervous components of the hypothalamus and glan- dular units of the anterior pituitary gland, mediated by way of the portal circulation. C. THE HYPOPHYSEAL PORT.\L CIRCULATORY SYSTEM A systetH-~o^ hypophyseal portal vessels which drain the tuber cinereum above and supply the adenohypophysis below is pres- ent throughout the reptiles, birds, and mam- mals (Green, 1951). It is indicative of sig- nificance that in all these forms the portal vessels provide either the entire or the major vascular supply to the adenohypophy- sis. Wingstrand (1951a) found no afferent vessels to the pars distalis other than portal vessels in over 50 genera of birds. However, Benoit and Assenmacher (1951b) state that in ducks the pars distalis is inconstantly supplied by a few twigs passing directly from the superior hypophyseal arteries. Crooke (1952) expressed the opinion that the blood supply to the pars distalis in man must be almost entirely by way of the stalk vessels. In examination of over 300 human jntuitaries, he found minute vessels entering from the capsule in only 6 in- stances. McConnell (1953) reached the con- clusion that the pars distalis in man is supplied entirely by the hypophyseal portal vessels, a view which is substantiated by the results from a recent critical study by Xuereb, Prichard and Daniel (1954a). The latter workers (1954b), however, made a further observation wdiich is of cardinal sig- nificance. They discovered a vascular link between the neural lobe and the adenohy- pophysis. Thus, in addition to the "long" portal veins which drain the upper stalk region and supply the sinusoidal network of the anterior and lateral regions of the pars distalis, they found a variety of "short" portal vessels, which arise from an anasto- motic link between the superior and inferior hy])oi)hyseal arteries on the lower margin of the infundibular stem. These "short" ves- sels supply the jjosterior portion of the pars distalis. It will be apparent, therefore, that even though the pars distalis derives all of its blood from the portal vessels, the source of this blood is not limited to that from the superior hypophyseal arteries as was pre- viously thought, but can be supplied by the inferior hypophyseal arteries as well. The matter is of significance in that channels are now known to exist whereby some blood from the systemic circulation can reach the pars distalis without having passed through the primary plexus on the median eminence. Consequently, when the long portal vessels are severed, as in the transection of the pituitary stalk, the possibility remains that in some species collateral channels in the form of these short portal veins continue to sup])ly a portion of the pars distalis. From a study of the arrangement and structure of the hypophyseal vasculature, Wislocki and King (1936) inferred that the flow of blood in the hypophyseal portal veins was toward the anterior lobe and this has now been confirmed by decisive evi- dence from work on frogs (Green, 1947), birds (Wingstrand, 1951a), rats (Green and Harris, 1949; Barrnett and Greep, 1951), and man (McConnell, 1953; Xuereb, Prich- ard and Daniel, 1954a, b). In 1952 Nowa- kowski, on the basis of his studies of the cat, once more advanced the view that the flow of blood in the portal system is toward 276 HYPOPHYSIS AND GONADOTROPHIC HORMONES the eminentia. It was his thought that the eminentia has a sensory innervation which registers the content of adenohypophyseal hormones emanating from the pars distalis. The view seems to be completely untenable on anatomic grounds alone. The capillaries in the primary plexus of the portal vessels show an unusual arrange- ment in most mammals. From the net which spreads thickly over the surface of the me- dian eminence and the neural stalk, capil- lary loops and tufts protrude into the wall of the median eminence (Green, 1948; Xuereb, Prichard and Daniel, 1954a). They are not in close proximity with the large nerve tracts of the hypothalamo-hypophys- eal neurosecretory system. In birds and mammals they are approached but not closely surrounded by sparse fibrous ele- ments, the nature and origin of which are obscure. There is a mounting body of circum- stantial evidence suggesting that the hypo- thalamus exerts a large measure of control over the secretory functions of the anterior pituitary. Such control might be effected either through direct nervous connections, the evidence for which (as we have just seen) is scant, or through a relay chain made up of a neural and a vascular link. Anatomically, at least, the latter exists in the form of the hypophyseal portal system. The neurohumoral concept as outlined by Harris (1947, 1948a) and Green and Harris (1947) holds that pertinent exteroceptive stimuli impinge upon the hypothalamus as the first way-station in the neurovascular reflex arc. Here the sex-related impulses are integrated with existing blood levels of circulating hormones, thence effector im- pulses travel by nerve conduction to the median eminence and effect the liberation of neurohumors (chemotransmitters). The latter are held to enter the primary plexus and to be distributed to and activate the cells of the pars distalis. A weakness in this theory is at the point of transfer of chemo- transmitters from nerve to vessel in the median eminence. The anatomic relation- ship between the loops or endings of nerves of obscure origin and the capillaries, which lie either on the surface of the median eminence or extend tuft-like into its sub- stance, has not Ix'cn clearlv defined. D. THE HYPOTHALAMIC AND HYPOPHYSEAL MEDIATED SEXUAL FUNCTIONS 1. Environmental Stimuli A great number of instances are known in which reproductive processes are condi- tioned by or are dependent on environ- mental factors. Although animals breed at a season of the year that is propitious for the survival of their young — warmth and the availability of food being the major factors — this is not through choice on their part. In many birds and mammals the readying of the reproductive system for seasonal breeding and rearing of young is anchored to alterations in the physical en- vironment, especially to changes in length of daylight — a subject which will not again bear expatiation (for a recent review see Amoroso and Matthews, 1955). Darkness, too, may not be an entirely passive stimulus (Burger, Bissonnette and Doolittle, 1942; Jenner and Engels, 1952; Kirkpatrick and Leopold, 1952) , but this concept has not gone unchallenged (Hammond, Jr., 1953). Experiments involving total darkness, more- over, are often subject to the complication of reduced food intake. The ground squirrel (Wells and Zalesky, 1940) and the white- crowned sparrow (Farner, Mewaldt and Irving, 1953) respond only to changes in the ambient temperature. Propagation in some tropical birds and amphibia is in like man- ner dependent on the beginning of a rainy season. Food becoming more plentiful at this time seems to be the mediating factor for many tropical birds. In England, certain frogs (Savage, 1935) are lured to the ponds by cheraotactic stimuli arising from ripen- ing algae, but spawning is dependent on another sequence of changes induced in the ionic composition of the water by the algal cycle and perceived through the frog's skin. Animals transported to the opposite hemi- sphere, generally adapt their breeding ac- tivities rather quickly to the same charac- teristic season irrespective of when it occurs in the calendar year (for review see F. H. A. Maishall, 1942; Burrows, 1949). In many birds, even with gonads in readiness for the mating season, the actual nesting often requires another complex set of extra-organ- ismal stimuli, such as presence of food, pres- ence of mate, density of colony, suitability PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 277 of breeding site, and avian display and courtship performances (A. J. Marshall, 1955, and chapter by Lehrman). Thus, for most but not all of the seasonal breeding vertebrates, the primary excita- tions to breeding activity originate in one way or another in the external environment. Those structures most exposed to such stim- uli are the skin and the specialized sense organs, particularly those for seeing, hear- ing, and smelling. It will be recalled that the sight of his mate incubating is sufficient to initiate in the male pigeon the secretion of crop milk (Patel, 1936). It is also not infrequent, especially among birds, that the presence of a mate is a prerequisite for ovulation. Here again, interesting observa- tions have been made on the pigeon (Mat- thews, 1939). The female separately caged and denied sight of other birds does not ovulate; but if she is permitted to view, by means of a glass partition in the cage or a mirror, a separately caged male or even a female pigeon, or is allowed to see the mir- ror image of herself, ovulation and oviposi- tion ensue. The stimulus which causes ovu- lation is clearly visual and not tactile, olfac- tory, or auditory. In sheep the presence of the ram is believed to hasten ovarian activ- ity and estrus in ewes (Riches and Watson, 1954; Schinckel, 1954), and it is likely that here both sight and odors play a part. It is apparent from observation of the be- havior of domestic and laboratory mammals that strong nasogenital relationships exist, but the effeet-of'sexual odors and olfactory stimulation on the sexual functions has received only cursory study. Pseudopreg- nancy has been induced in rats by simply applying a local anesthetic to the nasal mucosa (Shelesnyak and Rosen, 1938) and similar prolongations of luteal function were induced by bilateral excision of the spheno- palatine ganglion, but not by removal of the olfactory bulbs (Rosen, Shelesnyak and Zacharias, 1940). Local irritants applied to the nasal mucosa likewise did not affect the estrous cycle. These authors felt that the nervous factor involved was limited to the nonol factory innervation of the nasal mucosa. Whittcn (1956a) showed that mice rendered anosmic by removal of the olfac- tory bulbs have significantly smaller ovaries and uteri and fewer corpora lutea. No effect was observed in anosmic males. In both male and female mice the body weights were reduced. In a recent study Whitten (1956b) found that external stimuli associated with the male mouse modify the estrous cycle in the female. The incidence of mating after pair- ing was greatest on the 3rd night and was low on the 1st, 2nd, and 4th nights. The finding suggests that in some females the cycle was delayed, and in others it was advanced, by the presence of the male. In another experiment in which males were caged in a small basket within the females' cage for 2 days before pairing, the incidence of mating on the 1st night was greatly increased. Since presence of the males' excreta had a similar effect on the recep- tivity of the females, odors were thought to be an important factor in mediating this behavioral response. The determinate-laying wild birds, i.e., those which stop laying when the number of eggs characteristic of the species has been laid, probably perceive the stimulus for cessation of egg-laying through tactile sen- sations. Tactile stimuli are also known to come into play strongly during coitus. It is well known that in several mammals (rab- bit, cat, mink, ferret, ground squirrel, short- tailed shrew) ovulation is dependent on the coital excitation of receptor areas not all of which are confined to the genitalia. The neural pathways involved are poorly under- stood. Knowledge of the effect of sound on re- productive phenomena is exceedingly lim- ited. It would be interesting to ascertain what sequential role, if any, various songs and mating calls play in releasing the mani- festations of procreative processes. It is probably of no significance that Benoit (1955) failed to alter the sexual maturation in young drakes with continuous noise, and only indicative that Vaugien (1951) found a hastening of egg-laying in parakeets placed in small containers within hearing distance of an aviary where others of the species were mating. Control females simi- larly caged beyond hearing range did not lay. Sound, however, was not the only vari- able in these experiments. Thus, many extrinsic factors act in some manner to influence the secretion of gonado- HYPOPHYSIS AND GONADOTROPHIC HORMONES trophins by the anterior hypophysis. Ob- viously, central nervous pathways are in- volved, but the anterior pituitary has so little, if any, innervation that it is almost certain that none of the impulses are carried directly to the pituitary by this means. An alternative connecting pathway between brain and hypophysis exists in the form of the portal vessels. One of the most pressing problems in endocrinology is to define the pathways and elucidate the mechanisms by which such exteroceptive stimuli as those mentioned above effect the secretory func- tions of the anterior hypophysis. The hypothalamus serves in other re- spects to receive impulses from higher affer- ent centers and to instrument these into responses that relate to vegetative func- tions, for example, breathing, sleeping, and body temperature. It is also known to be the seat of neural mechanisms concerned with sex functions. By location and neural con- nections the hypothalamus could well be attuned to the reception and integration of sex-related impulses of variable sources. It has often been observed clinically that tu- mors of the hypothalamus are associated v/ith disturbed sexual functions. Moreover, anxiety states may lead to a cessation of menstrual cycles or impotence without evi- dence of organic disease. The supposition is that chemotransmitters of some sort, arising in the diencephalon, reach the pituitary by way of the portal veins. Alternatively, no other explanation of the existing evidence is available. Such a chemotransmitter has not yet been identified (Zuckerman, 1954). Extracts of the hypothalamus have thus far ])rovided only inconclusive evidence with respect to release of another trophic factor, corticotrophin, from the adenohypophysis (Guillemin, Hearn, Cheek and Housiiolder, 1957). 2. Electrical Stimulation of the Iljipothala- mus A hopeful means of increasing the secre- tory activity of the anterior pituitary was suggested by the observation that ovulation and pscudoi)regnancy could be produced by application of a strong current to the head or spinal cord in estrous rabbits (Marshall and Verney, 1936) and rats (Harris, 1936). Witli refinement in the technique it has l)c- come possible to apply such stimulation to precise areas in the pituitary and supra- sellar region. Moreover, by use of implanted electrodes and the remote induction of stim- ulation it is possible to apply electric ex- citation of controlled intensity to precise areas for almost any length of time in un- anesthetized animals (de Groot and Harris, 1950, reviewed by Harris, 1955). The method has been used mainly in connection with the study of ovulation in rabbits and pseudopregnancy in rats, but the method is suitable for much wider application in the study of mechanisms which regulate the functions of the pars distalis. By successive steps an area wherein stimulation leads to ovulation in estrous rabbits has been fairly well delimited (Harris, 1937; Haterius and Derbyshire, 1937; Markee, Sawyer and Hollinshead, 1946; Harris, 1948b ).^ Stimulation of the various lobes of the hypophysis and of the neurohypophyseal stalk in the unanesthetized rabbit does not promote ovulation, whereas ovulation is quite regularly induced when the stimulat- ing electrodes are in the tuber cinereum just anterior to the median eminence or in areas of the anterior hypothalamus. No part of the supraoptic-hypophyseal tract yielded a positive result. These observations provide strong support for the belief that the hypo- thalamus exerts a profound effect on the reproductive functions of the pars distalis and show finite conclusively that the con- necting link to the pituitary is not neural in character. They otherwise prove nothing with respect to the essentiality of the portal circulation in tlie mediation of these re- sponses. Stimulation of the cervical sympathetic fibers leading to the hypophysis neither in- duces nor interferes with ovulation in rab- l)its, according to Haterius (1934) and Markee, Sawyer and Hollinshead (1946). The few successful instances noted by Fried- good and Pincus (1935) might have been due to spread of the stimulus to the hypo- thalamus, or a response to handling. It is })ertinent to note in this connection that neither sympathetic nor parasympathetic denervation of the hypophysis has led to any detectable change in pituitary func- tions. PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 279 3. Lesions in the H ypothahnnus The puzzling clinical association of le- sions in or trauma to the base of the brain in the region of the diencephalon and dis- orders of the reproductive system set work- ers to probing this area of the brain in experimental animals as long ago as the turn of the century (for review of the early literature and critical analysis of the evi- dence, see Anderson and Haymaker, 1948a, b). Clinical or experimental lesions, de- stroying much of the hypothalamus but leaving the hypophysis intact, ordinarily resulted in genital atrophy accompanied by obesity (Frohlich type syndomej. Diabetes insipidus did or did not ensue, depending on whether the lesion disrupted hypotha- lamic neural connections with the neuro- hypophysis. A significant advance was made in 1927 when Smith discovered quite by accident that the chromic acid he was injecting into the sella of rats to destroy pituitary tissues led to extreme obesity through damage to adjacent areas of the brain. It remained for Cahane and Cahane (1935, 1936) to dissociate in rats the se- quelae of adiposity and deficiencies in the reproductive system by means of carefully placed lesions. Rats with lesions in the in- fundibulum showed severe testicular or ovarian atrophy and cessation of estrous cycles with no obesity. Dey (1941, 1943a- c), Dey, Fisher, Berry and Ranson (1940), and Dey, Leininger and Ranson (1942) were among the first to use stereotaxic in- strumentsJBr a major study of the effect of bilaterally placed hypothalamic lesions on the reproductive organs and sexual behav- ior. They were able by this means to pro- duce two distinct categories of sexual re- sponse in female guinea pigs. (1) Animals with lesions in the median eminence ex- hibited ovarian atrophy and loss of all re- productive functions. (2) Animals with le- sions in the anterior hypothalamus remained in continuous estrus and the ovaries were filled with large Graafian follicles. The le- sions in the anterior hypothalamus seemed to interfere with the release of LH. By present thinking it is likely that the secre- tion of LH continued at a low level but that the reflex arc for acute release at the ap- propriate time in the cycle was interrupted. In guinea pigs lesions elsewhere in the hy- pothalamus produced no impairment of gonadal functions, yet some of the animals did not mate. Essentially similar findings and some- what more precise localization of the lesions were reported in rats (Hillarp, 1949). Con- stant estrus was "consistently" produced by bilateral, relatively large lesions placed im- mediately anterior and ventral to the para- ventricular nucleus, or by smaller, perfectly bilateral, symmetrical lesions within the area between the paraventricular nucleus and the stalk. Lesions in the dorsal and lateral hypothalamus did not affect the re- productive functions. Eighteen other rats with small lesions variously located in the anterior hypothalamus showed cyclic irreg- ularities and impaired reproductive capac- ity with essentially normal appearing ova- ries. Other more recent studies uniformly emphasize the harmful effect of lesions in the ventral hypothalamus and especially in the median eminence on the reproductive functions of the rat (IVIcCann, 1953; Bog- danove and Halmi, 1953) ; man (Anderson, Haymaker and Rappaport, 1950) ; cat (Laqueur, McCann, Schreiner, Rosemberg, Riocii and Anderson, 1955); sheep (Clegg, Santolucito, Smith and Ganong, 1958). Although lesions in the median eminence often produce some damage to the portal circulation, the infrequency of infarction in the anterior lobe and the usual retention of normal morjihologic aspects of this gland make it unlikely that the observed defi- ciencies in gonadotrophic functions are as- cribable to an inadequate vascular supply. It is becoming increasingly evident that the separate trophic functions of the anterior lobe can be selectively altered by appropri- ately placed lesions in the hypothalamus. The relationship of hypothalamic lesions to the thyrotrophic and adrenotrophic activi- ties of the pituitary need not be considered here, because these seem to be independent mechanisms and exhibit no significant over- lap with gonadal regulation. Evidence for the selective effect of localized hypotha- lamic lesions on pituitary trophic secretions has been presented by Bogdanove and Halmi (1953), Bogdanove, Spirtos and Halmi (1955), Ganong, Fredrickson and Hume (1955), and Bogdanove (1957). It 280 HYPOPHYSIS AND GONADOTROPHIC HORMONES seems moreover, that such lesions may also selectively influence the individual gonado- trophins, and recently evidence has been provided that lesions in the ventral hypo- thalamus of sheep may abolish the behav- ioral manifestations of estrus without alter- ing the ovarian cycle (Clegg, Santolucito, Smith and Ganong, 1958). The study by Bogdanove and Halmi (1953) also confirms an observation noted by others (Desclin, 1942; May and Stutin- sky, 1947; Stutinsky, Bonvallet and Dell, 1950) that lesions in the hypothalamus may lead to a considerable hypertrophy of the pars intermedia. Furthermore, Stutinsky, Bonvallet and Dell (1950) observed great enlargement of the sinusoids in the pars distalis following suitably placed hypotha- lamic lesions, and speculated that one means of hypothalamic mediation of hy- pophyseal function might be by way of vasomotor control of vessels in the pars dis- talis. As a modus operandi this has the drawbacks of nonspecificity and sluggish- ness. The view has not been given credence, but the observation merits study. 4. Transection of the Hypophyseal Stalk An obvious experimental procedure in studying the extent of any control that the hypothalamus may impose on the anterior hypophysis is to interrupt all anatomic con- nections between them. This has been ac- complished by either surgically transecting the stalk or occluding it with a silver clip. In either event the neural and vascular connections between the hypophysis and the brain are disrupted. Such procedures do, in fact, isolate the hypophysis from eveiy pos- sible anatomical connection with the brain, save that of the systemic blood stream. Roundabout as the latter would be, even this avenue is available only in animals having a collateral blood supply to the an- terior lobe. Present evidence indicates that this would include man and, with less cer- tainty, monkey, dog, sheep, and rabbit. A consideration of primary importance in all surgical transections of the stalk concerns the demonstrated capacity of the disrupted vessels to regenerate and reestablish vas- cular connections. Harris (1936-1955) , Har- ris and .lacobsohn (1952) and Harris and Johnson (1950) have taken the in'ccaution of placing a plate of impervious material between the severed ends of the stalk as a barrier to the regrowth of vessels. The severed neurons with nuclei in the hypo- thalamic ganglia undergo Wallerian degen- eration. Variations exist between species in the length of the pituitary stalk and in the anatomic relationship of the hypophysis to the base of the brain; consequently, it is often possible to divide the stalk at differ- ent levels, i.e., near the base of the brain (high) or near the pituitary (low). In those species, especially the rabbit, monkey, and man, which have a deep sella turcica and a relatively long infundibular stem, there is the likelihood that with high transection, portions of the portal circulation may re- main intact. Man in particular is held to have a collateral blood supply to the pars distalis (Xuereb, Prichard and Daniel, 1954a, b) by way of the trabecular and the inferior hypophyseal arteries. The level of stalk transection is also known to be important in terms of the di- abetes insipidus which follows this opera- tion. Severance in a plane near the hy- pophysis in rats (as many have already demonstrated) may result in only a mod- erate and transient polyuria, whereas after transection in a more proximal plane the polyuria is more often severe and perma- nent. A plausible explanation of these results is that after low transection, there occurs a compensatory development of neural lobe tissue at the proximal end of the severed stalk. Suprasellar reorganization of neuro- hypojihyseal tissue and restitution of neu- rohyiioi)hyscal functions have been de- scribed by Stutinsky (1951, 1953) and confirmed by Billenstien and Leveque (1955) and Benson and Cowie (1956) after hypophysectomy in the rat. (Jaupp and Spatz (1955) described comi)cnsatory de- velopment of a "suprasellar hyi:)ophysis" on the tuber cinereum following low tran- section of the stalk in rabbits. They found these growths to be composed of fibers rich in Gomori-positive granules. Some of the rabbits bearing such nodules came into late sexual maturity, which led the authors to surmise that these compensatory neural ele- ments were providing gonadotrophic stim- ulation. Tlic nodules thev described are of PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 281 great interest, but the imputation with re- spect to gonadotrophic functions is weak. No glandular parenchyma was found in the nodules. Moreover the rabbit's own pitui- tary, although isolated, remained in situ and, according to the authors, was undam- aged in cytoarchitecture. Stalk transection has led to extremely discordant findings with regard to the post- operative structure and function of the pars distalis, particularly in the earlier studies. In Dandy's (1940) often cited case of stalk transection in a young adult woman, the reproductive functions were not im- paired and no symptoms of hypophyseal deficiency appeared, except a severe and permanent diabetes insipidus. Recently Russell (1956j and Eckles, Ehni and Kir- schbaum (1958) observed varying but gen- erally severe necrosis of the anterior lobe following stalk severance in man. The men- strual cycles ceased, and there was a nota- ble regression of the gonads, adrenals, and thyroids; an increase in insulin sensitivity suggested a low output of somatotrophin as well. Quite unexpectedly either si)onta- neous lactation ensued or established milk flow persisted, suggesting output of LTH {vide infra). Russell and Eckles, and Ehni and Kirschbaum agreed that the extent of damage to the anterior lobe is associated with the level of stalk transection, this be- ing most severe wdth low transection. Since in Dandy's patient the stalk was sectioned near the midpoint, it seems likely that the maintenance of the anterior pituitary func- tions he d£Si:iribes is explicable on the basis that a portion of the circulation to the pars distalis escaped damage. The fact that Rus- sell's patients did not exhibit diabetes in- sipidus is in all probability attributable to extreme infarction of the anterior lobe and the poor state of the surviving fragments of this tissue. Brooks (1938) found that stalk-tran- sected rabbits, after a period of transitory gonadal regression, had normal appearing pituitaries and ovaries. These animals came into estrus and mated but failed to ovulate. He presumed that the operation had inter- fered with the LH-releasing mechanism. Others, too, in acute experiments have fully substantiated the fact that the postcoital ovulation in rabbits requires the presence of an intact stalk (Westman and Jacob- sohn, 1940; Brooks, Beadenkopf and Bojar, 1940). In stalk-transected rabbits which have been observed over protracted periods, extensive gonadal atrophy has usually su- pervened (Harris, 1937; Westman and Jacobsohn, 1940; Gaupp and Spatz, 1955). Nine of the 12 rabbits operated upon by Gaupp and Spatz showed extreme atrophy of the gonads and genitalia. The remaining 3 reached sexual maturity only after a delay of up to II/2 years. Much of the pioneering work on stalk transection or occlusion in relation to ad- enohypophyseal function was done in dogs (Paulesco, 1908; Crowe, Gushing and Ho- mans, 1910; Gushing and Goetsch, 1910) and monkeys (Karplus and Kriedl, 1910; ]\Iorawski, 1911). Gushing and his co-work- ers observed marked anterior lobe degen- eration, and few of their dogs escaped "ca- chexia hypophysioprivia." In their words, "the resultant condition is almost the same as if this portion of the gland had actually been remo^'ed and then reimplanted like a graft, for the circulation is almost entirely cut off." These observations were thor- oughly confirmed by Mahoney and Sheehan ( 1936) in 20 dogs with stalks occluded. Ex- tensive infarction and central necrosis of the pars distalis with concomitant impair- ment of function was seen in all. Later workers, however, have observed very different sequelae: Keller and Hamilton (1937) and Breckenridge and Keller (1948) found no deviations from the normal pat- terns of sexual functions in the majority of their stalk-sectioned dogs. The results in dogs must yet be regarded as inconclusive, inasmuch as regeneration of the portal ves- sels was not adecjuately controlled and the operation is a difficult one, which, at best, involves an indeterminate amount of trauma to the hypophyseal and median em- inence areas. Results have been more consistent in monkeys. Karplus and Kriedl (1910) and Morawski (1911) observed no untoward symptoms in stalk-transected monkeys and no obvious alteration in the histologic struc- ture of the pars distalis; and Mahoney and Sheehan (1936) completely occluded the stalk in 20 monkeys with the same results, including no polyuria or polydipsia. At 282 HYPOPHYSIS AND GONADOTROPHIC HORMONES autopsy 3 days to 10 months later the hy- pophyses appeared normal and the vas- cularity of the anterior lobes was unim- paired, as shown by perfusion with carmine gelatin. These early findings have been sub- stantiated by Harris and Johnson (1950), who made a suprasellar transection of the stalk in a large male monkey. This animal continued to ejaculate, and at the time of autopsy 89 days later the reproductive or- gans were normal in size and appearance. At autopsy the pars distalis appeared well vasculated and showed no abnormality in size or color. It was demonstrated that vascular continuity between the median eminence and the adenohypophysis had been re-established. From these particular observations one can deduce only that the severance of the neural connections to the hypothalamus are not important for the continuance of adenohypophyseal activity. Disturbance of posterior lobe functions was not observed ; this probably is accounted for by the fact that sufficient terminations of neurosecretory fibers exist proximal to the cut to sustain neurohypophyseal functions. As far as the guinea pig and rat are con- cerned, the widest possible divergence in results has been reported. In female stalk- sectioned guinea pigs Dempsey (1939) and Leininger and Ranson (1943) observed nor- mal, lengthened, or absent estrous cycles. Tang and Patton (1951) severed the stalk in male guinea pigs and observed no gonadal atrophy. Their animals were maintained for 27 to 75 days, and although the portal ves- sels could have regenerated postoperatively, no verification of this was found on histo- logic examination of the region. Equally variable responses have been described in stalk-sectioned rats by Dempsey and Uotila (1940) and Dempsey and Searles (1943). They ascribed the abnormal cycles in such operated animals to operative trauma to the anterior pituitary. Using a parapharyngeal approach for sectioning the stalk in male and female rats, Barrnett and Greep (1951) and (Ireep and Barrnett (1951) found a very high incidence of severe gonadal atro- phy along with generalized evidence of i)an- hypopituitarism. These symptoms of an- terior lobe dysfunction were consonant with the extensive infarction and necrosis of the pars distalis observed regularly following the operation. They felt that the degree of gonadal dysfunction was relatable to the extent of damage to the blood supply of the pars distalis. The pituitaries showed no capacity for recovery, and there was no re- generation of the portal vessels as deter- mined in specimens injected with India ink and studied in serial sections of the appro- priate areas. Years before, Houssay and Giusti (1930) and Lascano-Gonzalez (1935) had reported on similar infarctions in toads following severance of the vessels to the pars distalis. Much recent evidence has revealed that vascular infarction, necrosis, and dysfunc- tion of the pars distalis are consequences of procedures which destroy the portal vessels. Daniel and Prichard (1956) studied the de- gree and localization of vascular lesions in the pars distalis of rats following the appli- cation of a fine cautery to all or a portion of the long hypophyseal portal vessels which pass down the ventral aspect of the stalk. Regions of the anterior lobe supplied by short portal vessels emanating from the lower portion of the neural stalk were un- damaged. In an extension of their study of the vascular supply to the pituitary, Daniel and Prichard (1957a, b) observed in sheep that severing the stalk produced extensive necrosis of the anterior pituitary; the con- dition of the pituitary-dependent endocrine organs was not reported. The extent of damage to the pars distalis w^as remarkably similar to that seen in human cases of postpartum pituitary infarction. Harris (1949) suggested that the discrep- ancies in findings following sectioning of the stalk might be reconciled by a study of the extent of regeneration of the portal vessels. Using a transtemporal approach, he sec- tioned the stalk in a series of rats, killed them at close intervals, and injected the vessels. He demonstrated that the por- tal vessels unquestionably regenerated in ''many cases" and that the regeneration was underway by 24 to 48 hours. In a follow-up study Harris (1950) inserted between the severed ends of the stalk an impervious l)late of varying composition. Results were judged by the effect on the estrous cycle. He concluded that the regeneration of por- tal vessels correlated with the ability of the pai's distalis to sustain cyclic gonadal func- PHYSIOLOGY OF ANTERIOR HYPOPHYSIS 283 tions. The fact that the pars distalis in rats receives its blood from the portal veins and that regeneration of these vessels means restoration of nutrient supply to this organ, would seem, on the face of it, to be a plaus- ible explanation for resumption of secre- tory activity following stalk transection. However, there is strong evidence against this possibility. Grafts of the pituitary gland revascularized by vessels from other than the median eminence lose their ability to sustain ovarian functions other than progestational (Harris and Jacobsohn, 1952; Everett, 1954, 1956; Nikitovitch- Winer and Everett, 1957), and regain these functions when vascular continuity with the median eminence is restored (Nikitovitch- Winer and Everett, 1957, 1958a, b). A series of recent reports has dealt with the capacity of stalk-sectioned ferrets to respond to the stimulus of added illu- mination. Thomson and Zuckerman (1953, 1954) and Zuckerman (1955» claimed that estrus supervened in 10 of 16 operated fer- rets exposed to added illumination. In two of the animals which responded, careful study of serial sections of the operative site after India ink perfusion revealed no vas- cular connections between the pituitary and the median eminence. Thus, the essential- ity of the portal vessels and whatever hu- mors they might convey for the regulation of adenohypophyseal activities was called into question. Donovan and Harris (1956), on the contrary, found that stalk-transected ferrets in which regeneration of the portal vessels was-^recluded by a film barrier between the severed ends of the stalk did not exhibit light-induced estrus; 3 animals lacking the barrier responded with early estrus, but in each of them regeneration of a vascular link with median eminence was demonstrated. The question left unanswered by the latter experiments is whether the pituitaries in the animals which did not respond actually had the capacity to re- spond. Collateral evidence, such as reduced adrenal weight and decreased thyroid ac- tivity, suggests that the pituitaries may have been functionally incapacitated for rea- sons other than lack of a specific excitatory agent of hypothalamic origin. Campbell and Harris (1957) addressed themselves to this problem by studying the volume change of the rabbit pituitary after dividing the stalk. They found a reduction to 62-74 per cent of the normal volume for the whole gland, 68-83 per cent for the pars distalis, and 26- 27 per cent for the neural lobe. Because the extent of atrophy in operated animals was the same with or without plate insertion, they questioned the importance of ischemic damage. The rabbit, however, seems an un- fortunate choice for study of this problem because Harris (1947) had already noted that the anterior lobe in this species has an arterial as well as a portal venous blood supply. Moreover, volume changes are not a crucial index of glandular competence. Breckenridge and Keller (1948) found no correlation between retention of sex func- tions and the size of the anterior lobe remnant in dogs which had been subjected to complete removal of the stalk and partial (graded) hypophysectomy. There was, how- ever, a close correlation between mainte- nance of the genital structures and retention of normal cytoarchitecture of the remnant. The arrangement of nervous and vascular connections between the pituitary and the brain in birds is such that it is possible to section the infundibular stalk, leaving the portal vessels intact, or contrariwise, divide the vessels leaving the stalk undamaged, or by appropriate incision segregate the median eminence from the hypothalamus without disturbing either the portal vessels or the stalk. These experimental procedures have been carried out on the duck (Benoit and Assenmacher, 1953; Assenmacher and Benoit, 1953a, b). Stalk transection alone did not alter gonadal maturation in ani- mals exposed to added illumination, but the response was completely blocked by section- ing either the median eminence or the anterior portal vessels ; in birds these vessels supply the cephalic lobe of the pars dis- talis. In spite of the impaired secretion of gonadotrophins, the functional capacity of the anterior lobe tissue was otherwise ade- quate as evidenced by the fact that the thyroids and adrenals were fully main- tained. After severing both stalk and portal vessels in laying hens, Shirley and Nalban- dov (1956a, b) observed gonadal atrophy, but no change in either the thyroids or adrenals. Benoit and his associates and Shirley and Nalbandov strongly favor the 284 HYPOPHYSIS AND GONADOTROPHIC HORMONES interpretation that hypothalamic agents are involved in the excitation of the hypophysis to secrete gonadotrophic hormones. Mention has been made of the interesting preliminary observation by Eckles, Ehni and Kirschbaum (1958) of persistent lacta- tion as a sequel to stalk transection in wo- men with breast cancer. (Polyethylene plat- lets were inserted between the cut ends of the stalk.) In some women lactation was observed to continue for a year or longer. The menses ceased, hence it can be assumed that the secretion of FSH and LH was in- terrupted. Why then, was the secretion of LTH not also abated? It seems pertinent also to note here that galactorrhea has fre- quently been seen in female mammals and patients receiving tranquilizing drugs (Kehl, Audebert, Gage and Amarger, 1956; Polishuk and Kulcsar, 1956; Whitelaw, 1956; Meites, 1957; Sawyer, 1957). It would seem, in fact, that an inhibiting influence on LTH secretion had been removed by these procedures. These findings in man parallel the observations by Desclin (1950) and Everett (1954, 1956) that autografts of the pars distalis to the renal capsule secrete LTH selectively, and perhaps in increased quantities for several months (see also Niki- tovitch-Winer and Everett, 1957, 1958a, b for further variants of this study of pitui- tary autografts). Perhaps the fact that pseudopregnancy often supervenes in rats under continued treatment with certain tranquilizing agents (Barraclough, 1957; Velardo, 1958; Barraclough and Sawyer, 1959j can be explained by an abnormal re- lease of LTH from the pars distalis. In- herent in this evidence is the suggestion that the hypothalamus exercises either no in- fluence or a "tonic" suppressive influence on the secretion of LTH by the pars distalis. Other recent evidence suggests, however, that the hypothalamus may also supply a stimulus for the release and possibly the j)roduction of prolactin (LTH). Oxytocin, a neurohypoi^hyseal hormone that is almost certainly elaborated in the hypothalamus (Scharrer and Scharrer, 1945, 1954a, b; Bargmann, 1949; Olivecrona, 1957), has been shown to lead to structural mainte- nance of the mammary gland (Benson and Folley, 1956, 1957). 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Postnatal Development of the Testis 307 III. Descent of the Testis 309 IV. Breeding Patterns 315 V. Architecture of the Testis 317 VI. The Circulatory System of the Testis 318 VII. The Nervous System and the Testis 321 VIII. The Excretory Duct System 323 IX. The Seminiferous Epithelium.... 323 X. The Interstitial Tissue 329 XI. Hormones of the Testis 332 XII. Effects of the Pituitary on the Testis 335 XIII. Effects OF Steroids ON THE Testis. 337 A. Androgens 338 B. Estrogens 343 C. Adrenal Steriods 344 I). Miscellaneous Steroids and Mix- tures of Steroids 345 XIV\ Effects of Altered Endocrine States on the Testis 346 XV. Nonneoplastic Disorders of the Testis 348 XVI. Tumors of the Testis 349 XVII. Conclusion 351 XVIII. References 353 I. Introduction The function of the testis is concerned with the preservation of the species. It ac- complishes this by producing sperm and hormones. The tubular apparatus is re- sponsible for the manufacture of sperm, and the interstitial tissue gives rise to the hor- mones. These two compartments are inti- mately associated with one another embryo- logically, anatomically, and functionally. Furthermore, they are controlled by sepa- rate gonadotrophic hormones of the anterior pituitary. In turn, the secretion and me- 305 tabolism of the pituitary gonadotrophins are controlled by the tubules and the Leydig cells. Knowledge of this reciprocal control of pituitary-testis activity was well estab- lished by 1940; in general, this reciprocity is the basic frame of reference for the in- terpretation of all aspects of testicular func- tion. JMore intimate relationships are quite complex and, as will be seen, not completely understood. It would be gratifying to interpret all as- pects of the testis within this fundamental frame of reference. This is not possible at present because the literature is too con- flicting and no one has a sufficiently broad experience with testicular endocrinology to sift all of this literature competently. The extreme scatter of literature on the testis furnishes ample evidence for the disconti- nuity and heterogeneity of effort. Perhaps the main service of this chapter is the com- pilation in broad categories of the hetero- geneous literature of the past 20 years, so that the student may have a handy, albeit incomplete, guide to the subject and to several of the major problems. A preview of the material to be discussed follows. This chapter pertains to the testis in post- natal life. Acquaintance with the principal facts of the embryology of the testis and with recent developments in fetal endo- crinology of the testis is presumed. Only a short description of the postnatal develop- ment of the testis is given because en- cyclopedic coverage is to be expected in other treatises, and because the acquisition of further details of the postnatal develop- ment of the testis in various species belongs 306 PHYSIOLOGY OF GONADS more to the domain of comparative mor- phology. The basic lessons already have been learned from a few species, and only the jDrovision of an unusual specimen for study could be expected to aid the endo- crinologist. Interest in the effects of cryptorchism has shifted in the 20 years following jNIoore's (1939) summary in the second edition of this book; at that time, the main interest in the cryptorchid testis was in its capacity for hormonal production. At present, the chief concern is with its capacity for sper- matogenic function. Despite some labor and much discourse, the treatment of crypt- orchism in the human is not satisfactory. Controlled methods of management based on a reasonable working hypothesis have not been evolved, so that a definitive evalu- ation of results in terms of fertility is im- possible. The architecture of the testis has been described in terms of structural pattern and composition adapted for the formation and transport of sperm and for hormonal pro- duction. The influences of the circulatory and the nervous systems on testicular func- tion have received uneven consideration. The former system is essential for testicular function; not only does it bring the neces- sary gonadotrophic hormones to the testis but, just as important, it provides food- stuffs and oxygen and carries away metabo- lites. The testis is extremely sensitive to derangement of its blood supply. The pe- ripheral nervous system, however, appears to be relatively unimportant to the post- natal well-being of the testis. The compartments of the testis are dis- cussed in two sections of this chapter. The germinal epithelium produces sperm, and it is with regard to this compartment that major advances have been made. Quanti- tative cytologic studies have unraveled the spermatogenic cycle and have provided de- tailed information on spermiogenesis. These studies are tedious and require painstaking techniques, but there is at present no other way to obtain quantitative information. The hormonal compartment of the testis has been further clarified by morphologic methods, but the greatest advances have been made by chemists. The biogenesis of male hormone has been worked out and is discussed in detail in the chapter hj Villee. So far, the hormones manufactured by the testis have been shown to include only steroids. A flare of interest in a water-solu- ble hormone, namely inhibin, was short- lived, and this issue has been dormant in the past decade. The next two sections of this chapter, the control of the testis by the pituitary, and the effects of male hormone and other ster- oids on the testis are representative of classic endocrinology. The dual concept of testicular control by means of follicle-stimu- lating hormone (FSH) and luteinizing hor- mone (LH) for the tubular apparatus and the Leydig cells, respectively, is less secure than it was believed to be in 1939. Interest in the fractionation of pituitary gonado- trophins waned in the 1940's, and investi- gators were unable to obtain purified FSH and LH for experimental study. Further- more, the discovery that testosterone and other steroids maintained spermatogenesis in the complete absence of gonadotrophins became an irritant to the dualist's com- posure. Intensive effort in this area has re- moved some difficulties, but it has not solved the problems. Recent studies have shown that male hormone is needed for spermiogenesis and gonadotrophins for co- pious spermatogenesis, but there the prob- lem rests. The effect of alterations in the endocrine system on the testis is discussed briefly. Ex- tremely little has been done in this area ex- cept for the influence of altered thyroidal states. As will be seen, the thyroid can exert some influence on testicular function, but this depends largely on the species studied. Further understanding will evolve as more species are studied. The last sections in the chapter deal with disorders and tumors of the testis. Dis- orders of the testis, chiefly hypogonadal states, are important in both veterinary and clinical medicine. Study of some of these disorders has greatly clarified normal jihysiology. A brief survey will be given of this aspect to emphasize the pituitary regu- lation of testicular function as shown by the effects of certain spontaneous disorders of the pituitary. Brief mention will also be made of the awareness of the increasing im- ])()rtanco of genie factors and of the fetal MAMMALIAN TESTIS 30; endocrine system in basic and clinical prob- lems of the testis. The inherited types of infertility in males seem to be an especially rewarding field of investigation. Spontane- ous tumors of the testis supply interesting and instructive material for study in both clinical and veterinary medicine. Tumors induced in the testis by experimental means have contributed nothing unique to the problem of oncogenesis. They have, how- ever, provided material for the concept of ''hormonal dependence" of certain tumors; therefore, they are of importance in the field of cancer. Not included in this review are studies on the effects of nutritional deficiency, of radia- tion, and toxic substances on the testis. The first is discussed in detail in the chapter by Leathern. The second has been purposely omitted because it belongs more to the sphere of interest of the radiation biologist than to that of the endocrinologist. It must not be forgotten, however, that knowledge of the relative sensitivity of the various cells of the testis to injury, the first quanti- tative information on the spermatogenic cycle, and the mechanism of repopulation of the germinal epithelium after severe damage were contributions of the radiation biologist. The third is a hodgepodge of material which at present defies orderly condensation. De- spite this, some of the studies in this area are of potential value in providing unique experimental preparations, i.e., animals with testes containing only Leydig cells, or only Sertoli cells. Finally, a miscellany of papers dealing with the general physiology or with the general biochemistry of the testis has also been omitted. II. Postnatal Development of the Testis In the past 20 years, voluminous descrip- tive information has been compiled on the events and consequences of the postnatal de- velopment of the testis of mammals. Only a few examples will be given. The develop- mental anatomy and postnatal changes in the testis of the laboratory rat have been described. Various monographs on the rat (Farris and Griffith, 1949, for references) and Moore's chapter in the 1939 edition are available. The testis of the guinea pig has a growth spurt about the 20th day of life. The ac- cessory sex structures, such as the vas defe- rens, epididymis, and prostate, are stimu- lated somewhat later, at 30 to 40 days. The growth of these accessory organs is an in- dication of male hormone activity. The time at which hormonal secretion occurs varies among individual animals. Varia- bility (48 to 70 days) occurs also in the ap- pearance of sperm (Sayles, 1939). Under controlled conditions of breeding, Webster and Young (1951) observed that the first intromission in guinea pigs occurs at about 54 days of age. The first ejaculate occurs some 10 days later and is sterile. Fertile ejaculates begin on the average at 82 days of age. Thus, a period of adolescent sterility exists as the result of both lack of ejacula- tion and a period when there is an in- sufficiency of spermatozoa. The hamster (Bond, 1945) copulates at 30 days of age but is not fertile until 43 days of age. Ado- lescent sterility of the male may be a more common phenomenon than is generally ap- jireciated. Tile testes of the cat are descended at bh-th. Testicular growth is slow, the com- bined weight of the two testes increasing from 20 mg. at birth to 100 mg. at weaning. During the 2 months following weaning, the testes attain a weight of 130 mg. A spurt in growth occurs between the third and the fifth month of life, when the testes may weigh 500 mg. This spurt is associated with the appearance of Leydig cells and an in- crease in the size of the epididymis. Mitotic activity of the germinal epithelium is pres- ent in testes weighing 400 to 500 mg. Sper- matids appear when the testes weigh 700 mg., and sperm are found fairly uniformly when the testes are more than 1 gm. in weight. At this stage, the tubular diameter is maximal. After maturity, the weight of the testes is generally proportional to body weight. A 5-kg. cat will have testes weighing 4 gm. (Scott and Scott, 1957). From birth to 80 days of age, the testes of goats grow at a slow but uniform rate. In the immature animal, the tubules are small, measuring 30 fjL in diameter, and are composed of a single layer of cells without any lumen. The interstitial tissue contains only mesenchymal cells. At 90 days of age, 308 PHYSIOLOGY OF GONADS a lumen appears in the tubules and sper- matogenesis begins. At 94 days, maturation of the Lej^dig cells is noted, and spermio- genesis occurs. The diameter of the tubules at maturity is about 100 jx, but the tubule continues to increase to about 160 /x when the goat is 135 days of age. Formation of sperm occurs earlier in goats than in rams, bulls, and boars (Yao and Eaton, 1954). As in common laboratory animals, the time sequence in the testicular maturation of farm animals is determined by genie fac- tors, which obviously is an important phe- nomenon economically. In different strains of the ram, for instance, there is a variation of 5 weeks in the time of appearance of primary spermatocytes, of 9 weeks in the appearance of secondary spermatocytes, and of 2 weeks for spermatids (Carmon and Green, 1952). Among the primates, postnatal develop- ment has been studied intensely in the mon- key and man. In the rhesus monkey (Fig. 5.1), the tubules attain a diameter of 70 to 80 fx during fetal life (van Wagenen and Simpson, 1954). Only spermatogonia and Stertoli cells containing basal nuclei are present. Mature Leydig cells are also identi- fiable. Shortly after birth, regression occurs in the tubules, which decrease to a diameter of 50 to 60 )u, and in the Leydig cells, which dedifferentiate into mesenchymal cells. The presence of mature Leydig cells and of dif- ferentiated Sertoli cells in the fetal testis and their involution shortly after birth may be related to the secretion of a fetal morphogenic substance (c/. chapter by Burns). During the first year after birth, the few spermatogonia increase in number and size. During the second year, they become more numerous and the tubules increase in length. However, the germinal epithelium remains quiescent until late in the third year. At this time, the Sertoli cells increase in num- ber and differentiate. The Leydig cells ma- ture again. The tubular diameter now is about 100 jx. A lumen appears when the tubules are 100 to 150 /x in diameter. Sper- matids appear, and orderly spermatogenesis occurs. The prepubertal period in the mon- key is about a fifth that of man; except for the factor of time, the sequence of de- velopment in the monkey testis resembles that in man (Figs. 5.2 and 5.3). In the pubertal monkey Leydig cells and tubules are stimulated simultaneously. Some ob- servers have reported that maturation of Leydig cells occurs after tubular maturation in humans (Sniffen, 1952; Albert, Under- dahl, Greene and Lorenz, 1953a) and in bulls (Hooker, 1948). However, this point may depend on the choice of the criteria for tubular stimulation (tubular wall versus germinal epithelium) and for function of Leydig cells (morphologic differentiation versus secretory activity) . Some interesting details on the relative Grams 8000 7000 6000 5000 4000 3000 2000 1000 Tubule growth slow Centrol Sertoli nuclei ^^^ s^^^ y^ Fertile Sperm Tubule growth ropid eo^V>^ ^^/-"^^'^ Bosol Sertoli ^ — "^ ^y^ Testis lenqih|n__c|]]_^^_____ — ,^ ^^^^^^ -^^^^ -^ ' Cm 4 3 2 I Age in Years Fig. 5.1. Graphic representation of changes in testes of the rliesus monkey during de- velopment. Coordinates are body weight and age of animals, and length of testes. (From G. van Wagenen and M. E. Simpson, Anat. Rec, 118, 231, 1954.) MAMMALIAN TESTIS 309 weight of the testis in primates have been supplied by hunting and scientific expedi- tions. Schultz (1938) studied 87 adult pri- mates. The relative testicular weights (tes- ticular weight divided by body weight X 100) varied between 0.1 and 0.4 in Ameri- can monkeys. Considerably more variation was seen in Old World monkeys. The rela- tive weight in five species of macaques varied between 0.46 and 0.92, but in langurs was only 0.06. The weight ratio in the orang- utan was 0.05, in the gibbon and man 0.08, and in the chimpanzee, 0.27. Rough estima- tions of the ratio of the volume of interstitial tissue to tubular volume showed that the macacjue has a greater relative volume of interstitial tissue than man. Testicular weights vary according to race; Japanese men have smaller testes than American men (Schonfeld, 1943) . The human testis at birth consists of small tubules measuring 50 to 75 yn in di- ameter and arranged in cords containing several rows of darkly staining nuclei. The epithelium is mostly undifferentiated, but large cells with sharp boundaries are pres- ent. These are primary germ cells. The interstitium of the testis is highly developed and contains solidly packed Leydig cells. After birth the Leydig cells disappear from the interstitium in a matter of a few weeks. From this time, the testis remains generally quiescent until puberty, when Leydig cells reappear as a result of the secretion of gonadotrophin. Only mesenchymal cells re- sembling fibroblasts characterize the in- terstitium in the period from a few weeks after birth until puberty. The germinal cords acquire a lumen at approximately 6 years of age, although this landmark shows considerable variation. The nuclei of the germinal cells at this time are arranged in two layers. At puberty, which may occur at any age from 9 to 19 years, a great increase in size and tortuosity of the tubules occurs. The lamina propria develops, the Sertoli cells become differentiated, and the seminiferous epithelium gradually matures. The Leydig cells mature somewhat later than the changes noted in the tubules and become characteristically arranged in groups in the intertubular zones. After maturity is attained, the adult his- tologic pattern may be maintained into old age without pronounced changes. Spermato- genic activity varies from tubule to tubule, but an over-all picture shows spermatogene- sis proceeding in an orderly fashion, with sperm heads closely approximating the lu- minal end of the Sertoli cells. About two- thirds of the tubule is occupied by the germinal epithelium. The lumen makes up about 15 per cent of the tubule. The pro- portionate volumes for the germinal cells are as follows: spermatogonia, 24 per cent; spermatocytes, 45 per cent; spermatids and spermatozoa, 29 per cent; various abnormal cells, 2 per cent. The Sertoli cells occupy about one third of the tubular epithelium. The Leydig cells occupy 9 per cent of the total intertubular spaces. About 66 per cent of the human adult testis is composed of tubules and about 22 per cent is made up of the intertubular spaces (Table 5.1). With age, progressive fibrosis occurs in the human testis, the width of the tubular wall in- creases, and thinning of the germinal epi- thelium occurs (SnifTen, 1952; Charny, Constin and Meranze, 1952; Albert, Under- dahl, Greene and Lorenz, 1953b; de la Baize, Bur, Scarpa-Smith and Irazu, 1954; Roosen-Runge, 1956). III. Descent of the Testis The descent of the testis from an ab- dominal position in the fetus was known to the ancients (Badenoch, 1945). This change in position is a mammalian phenomenon. In the Monotremata and most of the Edentata, the testes are abdominal. Some of the In- sectivora, Cetacea, and Sirenia also have abdominal testes. The testes in marsupials lie suprapubically in a pouch that has a closed vaginal process. The testes of Aplo- dontia rufa, the most primitive rodent ex- tant, occupy a semiscrotal position during the breeding season; otherwise, the testes are abdominal (Pfeiffer, 1956). In some ro- dents, Lnsectivora and Chiroptera, the testes are intra-abdominal in the resting stage, but during the rutting season they are pulled into the scrotum by muscles. In the Ungu- lata. Carnivora, and Primates, the testes are extra-abdominal. Exceptions are the elephant and stag, whose testes are retracted in the nonrutting season. Thus, "cryptor- chism" is normal for many mammals, and 7 », ; - "^^f; <4r , »c .at^-^' ^M?-Vtt^^ > Fig. 5.5. Architecture of rat testis, showing relationship of tubules to spermatic artery. and arrangement of the Leydig cell aggregates and capillaries. (From I. MUller, Ztschr, Zellforsch. mikroscop. Anat., 45, 522, 1957.) 318 PHYSIOLOGY OF GONADS rete and mnning such a constant zigzag course within the testis that a palisade is formed. An average tubule is 30 cm. long. The tubules do not end blindly as a rule. They rarely fork or bifurcate and they never communicate with one another (Mlil- ler, 1957; Clermont. 1958). The suitability of architecture of the tubule for the process of spermatogenesis is obvious because it provides an epithe- lium with a large surface area. The arrange- ment of the tubules in arcs and palisades allows long tubules to be packed neatly in a small, ovoidal organ. The lumen of the tubule constitutes a pathway for the trans- port of sperm to the outside. Because sperm cells are transported passively, some mobile medium is needed. The obvious medium is fluid which can be transferred along the length of the tulnile. It is not known where and how such fluid enters the tubule and how it moves through the lumen as it trans- liorts sperm to the ductuli efferentes. If fluid moves constantly along the length of the tubule to carry sperm, it must be re- absorbed from the excretory duct system. The ductuli efferentes, derived from the mesonephros, may play a role analogous to that of a nephron in reabsorbing large ciuantities of fluid from the seminiferous tubules (Ladman and Young, 1958). The cytologic organization of ciliated and non- ciliated cells in the ductuli efferentes and rete testis of the guinea pig seems compat- ible with the presumption that these cells absorb fluid from the lumen and excrete it by way of the ductular system. Physio- logic evidence of the transport of fluid within the excretory duct system will l)e given in Section VIII. The architecture of the interstitium ap- pears to be well adapted for the internal secretory function of the Leydig cells. Wedges of connective tissue are present in the interstices bounded by three tubules. The wedges contain Leydig cells, blood vessels, and connective tissue. Branches of the testicular artery feed the capillary net- work in the connective tissue wedges. The wedge capillaries are in close relationship to the Leydig cells. The topography of the capillary system of the rat testis is such that blood, after contact with the intersti- tial cells, flows by the g('nerati\'c portion of the testis before entering the general circuation through the great veins at the hilum. This architecture apparently makes it feasible for hormones of the Leydig cells to exert local action on the tubule. VI. The Circulatory System of the Testis The testicular artery of mammals con- volutes before reaching the testis. It is sur- rounded by the pampiniform plexus which is thermoregulatory, serving to preheat or to precool the blood. The convolutions of the testicular artery constitute a distinctive feature of mammals (Fig. 5.6). Lower ver- tebrates have segmental arteries, the testes do not descend, and the arteries do not lengthen or convolute. Marsupials differ in that the artery forms a rete marabile from which a short artery enters directly into the testis. The testicular artery in the dog forms 25 to 30 loops before entering the tunica albuginea. In the goat, the artery convolutes many times but finally branches into 3 or 4 convolutions that enter the testis from all sides. The testicular artery in the mouse has half-loops; except for man, it shows the fewest convolutions of all the mammalian testicular arteries thus far in- vestigated by arteriography. The situation in the monkey is similar to that in the dog, whereas the artery in the cat and the guinea pig has between 5 and 10 loops. The testicular artery of man is unique in two respects. It is the longest and thin- nest artery in all the viscera, and it is also the straightest testicular artery in the 50 mammals thus far investigated. The testic- ular artery of man after giving off branches to the cord and epididymis generally runs on the posterior border of the testis. It bifurcates, each branch penetrating the tunica over its lateral and medial aspects (Harrison and Barclay, 1948). The testicu- lar artery in man has a direct anastomosis with the vasal and cremasteric arteries (Harrison, 1948a, b; 1949a, b; 1952; 1953a, b). The gradient in temperature between the i:»eritoneal and scrotal cavities varies widely among different species. A large gradient ocelli's in the goat, rabbit, rat, mouse, and ram ; a small gradient is i)resent in the mon- key, (log, guinea pig. and man. The tcm- MAMMALIAN TESTIS 319 .1 ^frv ^^fcw^ --^-sffc: Fig. 5.6. Vascular patterns of the testi.- ni ,i i. \\ maniinals. Roentgenograms of testicular artery injected with opaque medium from (1 ) (.log, (..^) goat, (3) ram, (4) mouse, (5) rat, (6) rabbit, (?) guinea pig, (S) cat, and (9) monkey. (From R. G. Harrison and J. S. Weiner, J. Exper. Biol., 26, 304, 1949.) perature gradient depends on many factors, such as the convolutions of the artery, the length of the artery, the size of the testis, the relationship between veins and arteries, and the activity of the dartos muscle (Har- rison and Weiner, 19491. Inasmuch as temperature affects the testis directly, and also indirectly by way of the circulatory system, it is necessary to deal separately with the direct effects of tem- perature on the testis, the effects of envi- ronmental temperature, and the effects of circulatory occlusion. It is generally agreed that heat applied locally is injurious to the testis. Moore's experiments in which the testes were wrapped in insulating material already have been mentioned. In the guinea pig, sex activity and fertility are depressed for 44 to 72 days after exposure to heat (Young, 1927). Similar effects may be ob- tained (Williams and Cunningham, 1940) by heating rat testes with infrared lamps or by heating dog testes with microwaves from a radar source (Williams and Carpen- ter, 1957). In men, a single bout of fever (MacLeod and Hotchkiss, 1941) that in- creased the body temperature to 40.5°C. caused a depression in the sperm count. After return of the sperm count to normal, another episode of fever induced another depression. The production of androgen is not affected by exposure to high environ- 320 PHYSIOLOGY OF GONADS mental temperature (Stein, Bader, Eliot and Bass, 1949). The local application of heat does not markedly suppress the pro- duction of androgens, judging from the older work with rats and guinea pigs. To the contrary, some evidence exists that secretion of androgens may be enhanced. After scro- tal insulation, bulls were more ready to serve and excreted more ''androgenic" ster- oid than normal. The amount of fructose in semen (an indicator of androgen) in- creased after scrotal insulation in the ram (Glover, 1956) . Increased temperature orig- inating locally may affect spermatogene- sis in man. Davidson ( 1954) studied semen in cases of oligospermia before and after re- moval of varicoceles. Removal of the vari- cocele was followed by an increased number of sperm cells and a greater incidence of fertility. Defective fertility presumably was caused by interference with normal heat transfer because of the varicocele. Local application of cold to the testis or scrotum also results in testicular degenera- tion (Harris and Harrison, 1955) ; however, testicular tissue can be frozen and stored and still retain transplantability and sub- sequently produce hormones and sperm (Parkes, 1954; Parkes and Smith, 1954; Deanesly, 1954). The effects of temperature when the en- tire animal is subjected to thermal changes depend on numerous compensatory altera- tions in testicular circulation. The compen- satory mechanisms differ in both ciuality and degree, depending on the nature of the experimental conditions. The testicular tem- perature of rodents placed in a hot room does not increase to a higher level than that of the general body temperature. This is true also for the ram. Within fairly wide limits of environmental temperature (10 to 40°C.), the intratesticular temperature of the bull is constant. However, the tempera- ture of the scrotal surface increases slightly with increases in air temperature, but re- mains below body temperature (Riemer- schmid and Quinlan, 1941). The homeostatic mechanisms for maintaining a constant, optimal testicular temperature are several. With increasing scrotal temperature, the scrotum extends and the testes lie lower. This increases heat exchange, despite ilw absence in certain species (mouse, rat, dog. cat, and rabbit) of scrotal sweat glands (Harrison and Harris, 1956). Optimal tes- ticular temperature is also maintained by means of heat exchange between vessels of the pampiniform plexus. Exact data on the transfer of heat are not available, because determinations of the blood ffow have not been done. There remains for consideration the ef- fect on the testis of severe alterations in circulation. The histologic changes pro- duced in the rat testis by temporary or permanent occlusion of the testicular artery were studied in great detail (Oettle and Harrison, 1952). Acute temporary ischemia (10 to 20 minutes in duration) produced only hyperchromasia of the spermatogonia. Normality was restored within 2 weeks. Ischemia of increasing duration produced correspondingly increased testicular dam- age. Hyperchromatic changes in the sper- matogonia, loosening and exfoliation of the germinal epithelium, and desquamation of the mesothelium of the tunica occurred. The testis shrunk, the interstitium became edem- atous, and the Leydig cells swollen. A layer of ragged and vacuolated Sertoli cells, a few spermatogonia, and an occasional pri- mary spermatocyte may be the only sur- viving elements. When the damage was ex- treme, the tubule became markedly atrojihic, the lumen disappeared, and the Sertoli cells became embedded in a collage- nous matrix. Permanent occlusion of the testicular artery in the rat can be accomplished by removing a segment of the artery within the abdominal cavity proximal to its anastomo- sis with the vasal artery (Fig. 5.7), which results in incomplete ischemia. After 1 hour of such occlusion, hyperchromasia of the spermatogonia occurs, with exfoliation of the spermatids. After 6 hours, the sper- matocytes are exfoliated. One day later the testis enlarges considerably owing to edema. Multinucleated cells appear and many show pyknosis. The cytoplasm of the Sertoli cells disintegrates. After 3 days, all tubules are abnormal ; within 1 week, they are necrotic. The damage is restricted at first to the cen- tral jwrtion, but within a week practically all tnbulcs except some near the epididymal pole have been killed. Two weeks later, vacuolation occurs in tlie Leydig cells, with MAMMALIAN TESTIS 321 an accumulation of yellow pigment. By the end of a month, the interstitium becomes invaded by fibroblasts. The tubules, al- though not yet shrunken, show a thickened basement membrane. The necrotic contents conglomerate into a mass. After 7 months, pronounced interstitial fibrosis is present, extending from the periphery toward the center. Plasma cells are seen. Some of the Sertoli cells survive. The tubular debris is removed. Thus, it seems that the Leydig cells are most resistant to arterial occlusion. The Sertoli cells are the next most resistant, followed by the resting spermatogonia. The active differentiating cells are most sus- ceptible to arterial occlusion. A different type of lesion is produced by ligation of the superior epididymal artery. Focal necrosis of the initial segment of the caput occurs (Macmillan, 1956; Harrison and Macmillan, 1954). This disrupts the pathway between the vasa efferentia and the ductus epididymidis. The vasa distal to the ligature become choked with sperm within 3 days. The testes enlarge and then atrophy. In this manner, permanent atrophy of the testes occurs, with azoospermia due to ob- struction. Vn. The Nervous System and the Testis It is difficult to see nerve endings in the parenchyma of the testis. Van Campenhout (1947, 1949a, b) described masses of para- ganglionic cells in the midportion of the genital ridge of the testis during develop- ment. The fibers of these cells are inti- mately associated with the interstitial cells. The testes of 22-day-old pigs contain nu- merous neuro-interstitial connections be- tween nerve fibers and groups of Leydig cells in the hilar zone or near the tunica. The origin of testicular nerve fibers is not entirely clear. The general belief is that the testis receives fibers from the lumbar sympathetic chain. These nerve fibers in- nervate only the blood vessels in the rat and cat. Varying reports have been made of the nervous connections in man. Apart from vasomotor and sensory nerves, few fibers enter the human testis. These follow the course of the arteries to the septula and make contact with the Leydig cells. Three types of contact are made, namely (1) peri- FiG. 5.7. Diagram of arterial supply of rat testis. The testicular artery (a), as it nears the testis, be- comes tortuous just after giving off a branch (c) to the head of the epididymis that also supplies the fatty body (upper right). On reaching the testis, the testicular artery goes to the deep surface of the tunica albuginea. After coursing around the inferior pole, the artery winds up the anterior border of the testis, entering the parenchyma at e to break up into its terminal branches. The vasal artery (6) passes along the vas to reach the tail of the epididymis, where it anastomoses with the descending branch of the artery (d) supplying the body and tail. In the experiments, the tes- ticular artery was permanently interrupted at point X (in the abdomen) or temporarily oc- cluded at point Y. In the former case, the testis still would have some blood supply via the vasal artery, the branch of the testicular artery to the tail, and the terminal part of the testicular artery. However, the testicular artery is an end-artery at point Y. (From A. G. Oettle and R. G. Harrison, J. Path. & Bact., 64, 273, 1952.) neural, in which the Leydig cells lie along- side the nerve, (2) intraneural, in which groups of Leydig cells may be found within the perineurium, and (3) interdigitational, in which the course of the nerve breaks a cluster of Leydig cells into small groups (Okkels and Sand, 1940-1941). It is not certain that nerve fibers actually pene- trate Leydig cells (Peters, 1957; Gray, 1947). Peters noted that nerve fibrils also 322 PHYSIOLOGY OF GONADS run to the walls of the tubules and enter the membrana propria to reach the Sertoli cells. Experimental studies on the significance of the sympathetic nervous system with re- gard to testicular function have been only sporadically performed in lower animals. Coujard (1952,1954) found that the sympa- thetic ganglia along the vas deferens are most important to testicular development in the guinea pig. If these ganglia are injured, hypoplasia and aspermatogenesis of the testis follow. Unilateral removal of the pros- tatovesiculodeferential ganglion causes ipsi- lateral testicular immaturity. Defects of spermatogenesis also are noted when distant lesions in the sympathetic trunk are pro- duced. Coujard concluded that the sympa- thetic system is an obligatory intermediate between gonadotrophic hormones and the testis. Somewhat similar studies have been reported on the cat (King and Langworthy, 1940) . If 7.5 cm. of the sacral and lumbar ganglionic chain are removed unilaterally, cessation of spermatogenesis occurs on the affected side within 2 or more weeks. The Leydig cells remain normal. Bilateral ex- tirpation of strips of the ganglionic chain leads to reduction in spermatogenesis. In addition Weidenmann (1952) reported a diminution in volume of the Leydig cells after lumbar sympathectomy in cats. De- struction of the spinal cord by ultrasound in mice at levels from the eighth to the tenth thoracic segment had no effect on testicular weight, morphology, or spermatogenesis (Josimovich, 1958). Lumbar sympathectomy in man has yielded variable results. Bandmann (1950) found atrophy of the testis and loss of po- tentia after unilateral lumbar sympathec- tomy; sperm examinations before and after operation disclosed deterioration in all of his cases. However, Alnor (1951) could not observe any effects in 14 patients after unilateral lumbar sympathectomy, and Kment (1951) found a temporary increase in potency in men after procaine block of the trunk. The effect of lumbar sympathec- tomy in man clearly needs more decisive study. The poor sexual status of Iniinan para- plegics has led many authors to conclude that the nervous system controls testicular function in man. Apart from the muscular disability of male paraplegics, such symp- toms and signs as gynecomastia, loss of potency, atrophy of the testes, creatinuria, proteinuria, a decreased basal metabolic rate, loss of sex hair, and decreased excre- tion of 17-ketosteroids suggest testicular in- sufficiency (Cooper and Hoen, 1949, 1952; Cooper, Rynearson, Bailey and MacCarty, 1950; Cooi^er, Rynearson, MacCarty and Power, 1950). The extent to which these changes occur in paraplegics is debatable, and certainly not all changes are always present in any one patient. A study by Talbot (1955) of 400 paraplegic and quad- riplegic patients showed that two-thirds were capable of achieving erection, and that one-third of these had successful inter- course. One-twentieth were fertile. It is obvious, then, that potency and fertility are not invariably lost. The histologic appear- ance of the testis in paraplegics has been determined by both biopsy and necropsy. In contrast to the variability in symptoms, the histologic appearance of the testis is more uniform. Atrophy of the tubule occurs, of- ten with disai)pearance of all germinal epi- thelium except the Sertoli cells. The tubular wall is thickened. Leydig cells are present but may be found in clumps, giving the appearance of hyperplasia (Keye, 1956). Perusal of most of the illustrations showing atrophic testes in paraplegic men (Stem- mermann, Weiss, Auerbach and Friedman, 1950; Klein, Fontaine, Stoll, Dany, and Frank, 1952; Bors, Engle, Rosenquist and Holliger, 1950) indicates that all stages of degeneration may be encountered. Some testes resemble those in adult seminiferous tubular failure, and others, especially those showing severe atrophy, resemble cryptor- cliid testes. It is most difficult to determine from a testis containing only Sertoli cells and clumped Leydig cells what the nature of the pathologic process was, because all tyi:)es of atrophy end in the same general histologic picture regardless of cause. jMental disease and mental stress are said to affect the testis. Jankala and Naatanen (1955) found that severely disturbed rats, presumably under "mental strain," showed marked atrophy of the testis within 6 weeks. The severity of this atrophy is evident from the finding that only Sertoli cells remained. Caged dogs, apparently under mental strain, MAMMALIAN TESTIS 323 have transient testicular atrophy. Hormonal secretion is not impaired (Huggins, Masina, Eichelberger and Wharton, 1939). Testicu- lar atrophy has been noted in schizoid pa- tients (Hemphill, 1944; Hemphill, Reiss and Taylor, 1944) and was thought to be caused by this severe mental illness. How- ever, the histopathologic appearance of the testis in schizophrenia is not specific (Blair, Sniffen, Cranswick, Jaffe and Kline, 1952; Tourney, Nelson and Gottlieb, 1953) and it may not be stated that mental illness has any direct or specific action on the human testis. VIII. The Excretory Duct System The old concept that vasectomy is fol- lowed by hypersecretion of male hormone and rejuvenation has been disproved com- pletely. Recent studies have been concerned with the effects of occlusion of the excretory ducts on the tubular apparatus. Although some reports have indicated that the testes of rats and rabbits decrease to one-half normal size after vasoligation, the majority opinion is that no change in testicular weight occurs (see Young, 1933, for re- view). Poynter (1939) did not observe any changes in the structure of the rat testis one year after vasoligation. Atrophy of the testes w^as obtained only when vasectomy was performed scrotally; under these cir- cumstances, it resulted from adhesions sub- sequent to operation. No change was ob- served in the seminal vesicles, indicating no alteration in secretion of androgen. Also, no changes were evident in the Leydig cells. Ligation of the ductuli efi'erentes, how- ever, does produce pressure atrophy of the germinal epithelium (Young, 1933; Mason and Shaver, 1952). The testis becomes swollen and tense owing to distention of the ductuli with sperm on the testicular side of the ligature. The rete is also dilated. Peri- tubular fibrosis occurs, especially in tubules at the periphery of the testis. Degeneration of the germinal epithelium then ensues. Ten weeks after ligation, only Sertoli cells are left in the tubules. The Leydig cells re- main unscathed (Harrison, 1953a). The dift'erence in effect of ligation of the excretory path distal or proximal to the epididymis is attributable to the function of the excretory duct system of reabsorbing fluid needed to carry sperm. Obstructive necrosis of the testis does not occur after ligation of the ductus deferens, because reabsorption of fluid takes place. This would also explain the absence of testicular atrophy in clinical states of inflammatory obstruction along the excretory pathway caused by gonorrhea or of obstruction caused by congenital absence of the duc- tus deferens. IX. The Seminiferous Epithelium Clarification of the spermatogenic cycle in the germinal epithelium is probably the most important development in knowledge of the testis since the second edition of this book. The difficulties in expressing sper- matogenesis in quantitative terms were great. Clear identification of each type of cell was not possible. Certain basic informa- tion on the transformation of one type of cell into another, on the renewal of certain cells, and on degenerative phenomena was lacking. Despite these difficulties, the time of a complete spermatogenic cycle in the rat was estimated by several investigators using diff"erent methods. These methods were ( 1 ) time of recovery after irradiation of the testis, (2) morphologic studies of the changes in cellular population with ref- erence to a static cell such as the Sertoli cell, and (3) turnover time of organically bound radiophosphorus in the germinal epithelium (Howard and Pelc, 1950). The introduction of the periodic acid and fuchsin sulfurous acid (PAS) stain for glycol groups such as exist in glycogen, mucoprotein, and muco- polysaccharides solved the difficulties enu- merated above. Cytologic studies have shown that the cells of the seminiferous epithelium are or- ganized in similar associations. The de- velopment of any one generation of a cer- tain type of cell is correlated with other generations present in the same part of the tubule. The changes in a certain zone of the germinal epithelium between two successive appearances of the same cellular association constitute a cycle. Different investigators do not use the same number of phases, the same classification of cell types, or the same points of reference. Depending somewhat on the cytologic detail and somewhat on the 324 PHYSIOLOGY OF GONADS point of reference, the cycle can be divided into 6, 8, 12, or more phases. Roosen-Runge (1951-1955), Roosen- Runge and Barlow (1953j, and Roosen- Runge and Giesel (1950) used eight phases to characterize a seminiferous cycle in the rat. In phase 1, no sperm cells are present in the tubule; at the end of phase 8, the sperm cells forming over the inter- vening phases have disappeared from the lumen. Two types of spermatogonia are recognized; type A is a large cell with a large nucleus and little chromatin, and type B is a smaller cell with a smaller nucleus and masses of chromatin arranged peripherally. Type A spermatogonia divide simultaneously in phase 1 and again at phases 4, 6, and 7, leading to successive doublings. Type B spermatogonia form from type A in phase 6. In phase 8, a total of 98 per cent of all the spermatogonia are type B, leaving a 2 per cent quota of type A to start the cycle over again. When the sper- matozoa are floated off the tubular wall, type B spermatogonia rapidly change into prespermatocytes. The prespermatocytes grow rapidly and become spermatocytes. Spermatid formation occurs in the first four phases. Spermatozoa are present from the end of phase 5 through phase 8. Interestingly, the Sertoli cells show a cyclic variation in volume, being largest at phases 7 and 8 and smallest at phase 1. Retraction and expansion of the Sertoli cells, with cycles of spermatogenic activity, was noted by Rolshoven (1945, 1947, 1951). When the cells retract, part of the cyto- plasm is lost, leaving a pars basalis. In ex- panding, this part of the Sertoli cell forms a fine lattice. The Sertoli cells resorb re- gressive spermatozoa and probably also the residual bodies during the spermatogenic cycle. Because PAS-positive material can be traced back to the Golgi apparatus of young spermatids (Leblond, 1950), Leblond and Clermont (1952a, b) have been able to di- vide spermiogenesis in the rat into 19 stages. In the first 8 of these, the germinal epithe- lium has old spermatids, which are released when the new crop reaches stage 8. Hence, the new crop of spermatids is alone until they reach stage 15, when another genera- ation of spermatids appear. Therefore, stage 1 and stage 15 spermatids appear together, and the succession of cells associated with this appearance marks one cycle. These authors have divided their 19 stages of spermiogenesis into four phases (Fig. 5.8). The first phase is the Golgi phase, which includes 3 of the stages. In stage 1, the idio- some is in the Golgi zone and two centrioles are near the chromatoid body. The fine filament from one centriole eventually be- comes the tail of the sperm. In stage 2, one to four granules appear in the idiosome. In stage 3, the fusion of pro-acrosomic gran- ules into one large one is accomplished. The second phase is the cap phase, which consists of 4 stages. In stage 4, the acrosome granule flattens on the nucleus. In stage 5, a membrane arising from the granule spreads over the nucleus. In stage 6, a cap is formed over the nucleus. The idiosome separates from the acrosome granule, and the two centrioles move closer to the nu- cleus. In stage 7, the cap reaches maximal size. The proximal centriole adheres to the nucleus, and the flagellum remains attached to the distal centriole. The chromatoid body is loose in the cytoplasm. The third phase is the acrosome phase, which includes 7 stages. The caudal tube is present, and the head caps are oriented to- ward the tubular wall. In stage 8, the gran- ule and cap move toward the basement membrane, and the cytoplasm shifts to the opposite pole of the nucleus. The chroma- toid body surrounds the flagellum near its insertion to the distal centriole. In stage 9, the acrosome granule elongates. In stage 10, the head cap moves toward the caudal end of the nucleus, and the apical end is pointed. In stage 11, the nucleus and head cap elongate. In stage 12, the nucleus is at its maximal size. In stage 13, the nucleus is thinner, and the distal centriole divides into a dot and ring. In stage 14, the head cap is loose over the nucleus, the cytoplasm condenses, and the sj^crmatid begins to look like a mature spermatozoon. The fourth phase is the maturation phase, which consists of 5 stages. In stage 15, the head cap has a finlike membrane; the ring centriole separates from the centriole and forms the middle piece. In stage 16, elonga- tion of the finlike membrane occurs. In stage 17. the acrosome and head cap move for- MAMMALIAN TESTIS 325 Fig. 5.8. Spermiogenesis in the rat. 1 to 3, Golgi phase. The idiosome produces two proacrosomic granules, which fuse into the single acrosomic granule. 4 to 7, cap phase. The acrosomic granule produces the head cap, which enlarges to cover a third of the nucleus. 8 to 14, acrosome phase. The nucleus and head cap elongate, whereas the acrosomic granule transforms into the acrosome. 15 to 19^ maturation phase. Near the end of this phase, the reactivity of the head cap and acrosome decreases considerably, and the sperma- tozoon is released into the lumen (19). (From C. P. Leblond and Y. Clermont, Ann. New York Acad. Sc, 55, 548, 1952.) ward. In stage 18, the perforatorium ap- pears. In stage 19, the staining capacity of the sperm is sharply reduced. The behavior of the remaining cells of the germinal epithelium now can be corre- lated. Five peaks of mitosis occur in the spermatogonia. The first three peaks give rise to type A spermatogonia, the fourth peak to type B spermatogonia, and the fifth to spermatocytes. Spermatocytes, formed in stage 6, undergo the long meiotic division and become spermatids at stage 1 of the third cycle. This quantitative method has been ap- plied to three areas which are of importance to the experimental or clinical endocrin- ologist: renewal of stem cells, postnatal de- generation of germ cells, and the effects of hypophysectomy on the germinal epithe- lium. The renewal of spermatogonia always has been puzzling. It was postulated that they were renewed from the Sertoli cells, from unequal mitosis of a spermatogonium into a spermatocyte and another spermatogo- nium or from type A cells which did not difTerentiate into type B cells. Clermont and Leblond (1953) proposed a new theory for the renewal of stem cells. Three types of spermatogonia are present in the rat and 326 PHYSIOLOGY OF GONADS SCHEMAT I C REPRESENT AT I ON PROGENY or ONE A CELL Of STAGE VIII A In B R FINAL HYPOTHESIS |b [bIb [bIb [bIb [bIb [bIb R Ir'rIrirIrirIriR JRiR jfilR iR|RlR!R"|RiRlRiR IrJrIr 0-12 12 12 0-24 24 Fig. 5.9. Diagrammatic representation of the most probable pattern for the development of spermatogonia (or "stem cell renewal theory"). The Roman numerals on either side of the diagram indicate the stages of the cycle. A, type A spermatogonia; Ad, dormant type A spermatogonia ; In, intermediate type of spermatogonia ; B, type B spermatogonia ; R, resting spermatocyte. In this hypothesis, the two daughter cells of the stage IX mitosis do not divide simultaneously. One of the granddaughter cells becomes a new dormant type A cell (Ad), ensuring the renewal of the spermatogonial population at the subsequent cycle, whereas the three other daughter type A cells divide again to produce intermediate tvpe cells, which in turn produce type B cells, which in turn produce spermatocytes. (From Y. Clermont and C. P. Leblond, Am. J. Anat., 93, 475. 1953.) mouse (Fig. 5.9). Type A spermatogonia give rise to either intermediate spermato- gonia or to dormant type A spermatogonia. The intermediate type of spermatogonia gives rise to the type B forms, which pro- duce spermatocytes. The dormant type A spermatogonia are so designated because they do not divide for 8 stages. At the 9th stage, the dormant type A spermatogonium forms 4 large type A spermatogonia. In the next cycle, one of these 4 type A spermato- gonia becomes another dormant type A spermatogonium; the others form 6 of the intermediate types of spermatogonia and eventually 24 spermatocytes. The cytologic details and the alterations in numbers of the three types of spermatogonia are illustrated in Figure 5.10. Full information can only be obtained by consulting the original pa- pers. Considerable degeneration of the primary germ cells occurs during development of the testis in the mouse and the rat (Allen and Altland, 1952). Degeneration usually ceases on the ninth day of age in the rat. Over the next 4 days, however, considerable multipli- cation occurs, but from day 14 to day 48 degenerating cells also may be seen in many tubules. Six different types of degeneration are evident — loss of cells in layers (exfoli- ation or shedding) , necrosis, loss of individ- ual cells, pyknosis, degeneration of lepto- tene forms, and abnormal mitosis in stem cells and spermatocytes. The degeneration of the germ cells, or gonocytes, soon after birth had given the impression that the spermatogonia arise from the small supporting cells that also form the Sertoli cells in the adult. Gono- cytes have a large, light, spherical nucleus, fine chromatin, and a sharp nuclear mem- brane. The supporting cells have smaller nuclei and coarse chromatin. The fourth day of life in the rat the supporting cells in- crease in number and form a palisaded layer along the basement membrane. The gono- cytes swell and begin to degenerate; how- ever, some of them look like type A sper- matogonia. By day 6, most of the spermatogonia are tyi)e A but a few inter- mediate spermatogonia and type B forms appear. By days 9 to 12, gonocytes are no longer present. Primary spermatocytes appear for the first time in resting leptotene stages. By days 15 to 18, two generations of germ cells are present. By days 23 to 26, the spermatocytes are in meiotic prophase and some spermatids are being formed. By days 33 to 50, the Sertoli cells have matured. Because the supporting cells do not divide after day 15, type B spermatogonia can- MAMMALIAN TESTIS 327 -.J 8 »< ^"'^^^':-> u;-^ 1 ^ ^i^x;: t» ? i?.- : :St- *:-*^^*to ® ;'-- © f*i'%.;-^i#% >0 -^^l |;_^. -^^, .^^-^^^^. ^ tl'^^' "Xi^''^'^,) ^.¥-'^^- • :^%^«^ \ > 13 ^ #^ .*• *» Fig. 5.10. Diagrammatic drawings of stages I to XIV of the f\cle ul ilie seminiferous epithelium. Drawings made from PAS-hematoxylin stained preparations. Numbers 1 to 19 refer to spermatids at different steps of spermiogenesis. A, type A spermatogonia; B, type B spermatogonia; Bm, mitosis of spermatogonia; R, resting spermatocytes; L, lep- totene stage; Z, zygotene stage; P, pachytene, Di, diplotene and diakinesis; SI, primary spermatocytes; Sim, primary spermatocyte metaphase; SI I, secondary spermatocytes; Slim, secondary spermatocyte metaphase; S, Sertoh element; Rh, residual body. (From R. Daoust and Y. Clermont, Am. J. Anat., 96, 255. 1955.) not arise from supporting cells (Clermont and Perey, 1957). It was known that, after hypophysec- tomy, spermatids disappear but sper- matogonia, Sertoli cells, and primary sper- matocytes remain for long periods and spermatogonial mitosis continues. Clermont and ]\Iorgentaler (1955) noted spermatids being phagocytosed by the Sertoli cells within 3 days after hypophysectomy. Young spermatids at stages 1 through 7 are present, but at 10 days after hypophysectomy, no developing spermatid has reached stage 9. A few pachytene spermatocytes are de- generating, but primary and secondary spermatocytes are present during the first week after the operation. By the tenth day, the Sertoli cells shrink but do not disin- tegrate. Spermatogonial types A and B re- main intact. Maximal regression after hy- pophysectomy is reached within 29 days. The basement membrane is thick and there are two rows of type A, intermediate, and type B spermatogonia, a few primary and secondary spermatocytes, spermatids at stages 1 through 7, and Sertoli cells. Type B cells form spermatocytes, but the sper- matocytes degenerate before and during meiosis, and only 4 per cent of them survive to produce spermatids. The spermatids de- velop to stage 7 and then disintegrate. Therefore, spermatogenesis up to stage 7 of spermiogenesis can occur in the absence of the pituitary gland but at a greatly re- duced rate. As was surmised from early ob- servations on the maintenance of sperma- togenesis by androgen, the premeiotic phase of spermatogenesis apparently can take place without gonadotrophins ; meiosis suf- fers severely from gonadotrophic depriva- tion; and the postmeiotic phase is controlled by androgen. The observation that testos- 328 PHYSIOLOGY OF GONADS <.^ ^ ; > 1^- 6 J^ ^'^ ^ r 'S ( C V Fig. 5.11. Spermiogenesis of the mouse as seen with PAS-hematoxyhn staining of Zenker- formol fixed testis. Drawings are arranged in a spiral to demonstrate stages which overlap in a cycle of the seminiferous epithelium. Orientation of spermatids in relationship to the basement membrane also is shown. ^ to S is the Golgi phase, 4 to 7 the cap phase, 8 to 12 the acrosome phase, and 13 to 16 the maturation phase. (From E. F. Oakberg, Am. J. Anat., 99, 391, 1956.) terone can maintain spermatogenesis if it is administered within a month after hy- pophyscctomy but cannot if treatment is delayed more than a month may not be so puzzling if it is assumed that androgen protects in some way the serious depletion of spermatocytes at meiosis. The plan of spermiogenesis in many spe- cies is essentially similar to that in the rat and mouse. Clermont (1954) found that the hamster shows the same successive stages of spermatogenesis except that five cycles may be represented simultaneously. In the mouse, Oakberg (1956a, b) described 16 stages, the first 12 of which constitute a cycle (Fig. 5.11 and Table 5.2). Four cycles constitute complete spermatogenesis and re- quire 34.5 days. Generally, the same plan of spermiogenesis holds for the guinea pig, cat, bull, dog, ram, monkey (Fig. 5.12), and man (Leblond and Clermont. 1952b; Cler- mont and Leblond, 1955) , although many differences in cytologic detail exist and have been documented (Zlotnik, 1943; Gresson, 1950; Gresson and Zlotnik, 1945, 1948; Bur- gos and Fawcett. 1955; Watson, 1952; Challice, 1953). Application of quantitative studies to hu- man spermatogenesis has, to date, been dis- appointing. Spermatogenesis does not pro- ceed along a wave, nor are the various stages sharply delimited, as they are in the rat. Further, the testis of the human also differs from that of the rat in that the relative pro- })ortion of differentiated germ cells to sper- matogonia is less. No helpful findings in cases of human infertility have been ob- tained by quantitative analysis of the germinal epithelium (Roosen-Runge and MAMMALIAN TESTIS 329 TABLE 5.2 Characteristic cell associations at each stage of the cycle of the seminiferous epithelium (From E. F. Oakberg, Am. J. Anat., 99, 391, 1956.) Stage of Cycle Spermatogonia Type A Intermediate Type B Spermatocytes I First Layer Second layer. . . , Spermatocytes II Spermatids (see Fig. 1) First layer Second layer 15 10 Z Dip P Dia M-I S Mil 12 A In B MI S = Spermatogonia type A = Intermediate type sperm; = Si^erniatogonia type B = First meiotic division = Secondary spermatocyte )goni M-II = Second meiotic division R = Resting L = Leptotene Z = Zygotene P = Pachytene Dip = Diplotene Dia = Diakinesis Primary spermatocytes Barlow, 1953; Eoosen-Runge, jNIarbergcr and Nelson, 1957). X. The Interstitial Tissue Although miscellaneous general informa- tion is available on the interstitial tissue of many animals, including the gorilla, the short-tailed manis, and the vampire bat (Popoff, 1947), detailed knowledge comes from common laboratory animals, such as the rat, mouse, guinea pig, rabbit, and cat. With the exception of man, however, the life history of the interstitial tissue of the testis is probably known best for the bull (Hooker, 1944, 19481. In the 1 -month-old bull, when widely separated, lumenless tubules are present, the intertubular spaces contain only mes- enchymal cells. The number of Leydig cells gradually increases up to 2 years of age; after this time, the Leydig cells become vacuolated and increase in both number and size (Fig. 5.13). From 5 to 15 years of age, loss of vacuolation and decrease in size occiu-. After 15 years of age, degeneration ensues. ^Metamorphosis of the Leydig cell begins with nuclear changes. The nucleus acquires 1 to 3 nucleoli, increases by 25 per cent in volume, and becomes spherical. Hyper- trophy and hyperplasia of the cell occur. The cell still retains its stellate appearance, but becomes polygonal in shape after gran- ules appear in the cytoplasm. After 2 years of age, vacuolation occurs and, with age, the vacuoles become larger. At 5 years of age, vacuolation is present in all Leydig cells. Regression of the Leydig cells be- gins at 7 years of age; it is manifested by a decrease in vacuolation and mitotic activity (Hooker, 1944, 1948), and ends in cellular disintegration (Fig. 5.14). In addition to regression, Leydig cells may also dedifferentiate. This occurs in the rabbit. In autografts of testis to the ear, mature Leydig cells show fusion of granules, shrinkage of cytoplasm, loss of nuclear transparency, and finally cannot be dis- tinguished as a Levdig cell (Williams, 1950). The life history of the Leydig cell in man and monkey is in general similar to 330 PHYSIOLOGY OF GONADS V vv #1. Fig. 5.12. Spermiogenesis in the monkey, i to 3 is the Golgi phase. 4 to 7 the cap phase, 8 to 12 the acrosome phase, and 13 to l-i the maturation phase. (From Y. Clermont and C. P. Leblond, Am. J. Anat., 96, 229. 1955.) that in the bull. In the human, Leydig cells are large polyhedral cells containing a large vesicular nucleus, which is not found in other cells of the interstitial tissue. The cells contain pigment, vacuoles, crystalloids, and granules. The granules vary in density, number, and arrangement within the cyto- plasm. These granules contain lipides (Nel- son and Heller, 1945) and, like those in common laboratory animals (Pollock, 1942), give reactions of steroids. Various types of Leydig cells can be distinguished on the basis of the size and nature of the granules and vacuoles. The medium-sized granular cells are believed to be vigorous producers of androgen (Sniff en, 1952; Til- linger, Birke, Franksson and Plantin, 1955) . It is difficult to determine the absolute num- ber of Leydig cells. However, rough counts made in testes of men (necropsy material) indicate that the number declines with age (Sargent and McDonald, 1948). In general, the excretion of 17-ketosteroids and the de- velopment and condition of secondary sex characteristics parallel histologic and cyto- logic evidence of secretory activity by the Leydig cells (Fig. 5.15). It is generally held that the Leydig cell MAMMALIAN TESTIS 331 ■#\ 2!^ 22 23 24 Undifferentioted cells P'' 25 26 27 Differentioting cells 28 29 /^J) // ti^^ V / ^0 ^' . Young Leydig cells / 32 V-^ 33 Mature Leydig ceils 36 (f^ Leydig cells from oid animoi ^:\ 38 Fig. 5.13. Life history of Leydig cells of the bull testis. 21 to 2S, calf 1 month old. 2.'^, calf \Vz months old; note threadlike processes extending from angulation of mesenchymal cell. 25 to 21, cells of interstitium; 25 is a fibroblast, and 26 and 21 are pre-Leydig cells. 2S, bull 4 months old. ;^9 and SO, bull 2 years old, with young Leydig cells. SI and S2, bull 28 months old; note vacuoles. SS to 35, mature Leydig cells in a 5-year-old bull. SQ to S8, bull 15 years old. (From C. W. Hooker, Am. J. Anat", 74, 1, 1944.) - ^ - ^ — ( O U #:v;(^:;-yv:;(^ Fig. 5.14. Life history of the Leydig cell of the bull. (From C. \V. Hooker, Recent Progr. Hormone Res., 3, 173, 1948.) i^ r*3i\^sc».:i;xV\ ^:^' v^^NKr^^s «*l*k. >48>i^*t* ^»^vv^ .W,^ -v^ - -, >,>s-v,.-, ■,^.'^>>tl»tt \ I f .'^J>t«Sr ■«■ -^.'t'NtliJ- \ 1 1 ■■— - ~ \ \ ^ :\ ->c ^.;^ .^.> -D>VV>«:. i^^^awr^M. C:Sb*!*&8S»*4; :S%s*^ ^ >? -Ae T?-m'i:iimt. MAMMALIAN TESTIS 333 60- * 30 0 V'- Hormone assays in normal boys (Data of Greulich el al ) . Androgen "" Gonadotropin Estrogz .^-^ 10 is 16 20 22 24 2 4 6 8 10 12 14 16 A§e in years Fig. 5.16. Frequencj- of puberty, measurements of testis and penis, and excretion of hor- mones during puberty in man. (From A. Albert, L. O. Underdahl, L. F. Greene, and N Lorenz, Proc. Staff Meet., Mavo Clin., 28, 409. 1953.) 334 PHYSIOLOGY OF GOKADS 1 2 3 4 5 6 HAIRIINE FACIAL HAIR 0 O w e © VOICE (Loryn,) T m If ■«* BREASTS 1 ^ ► « 1 .' --. AXILLARY HAIR BODY f?' \'^ III CONFICUtATION BODY HAIR (i) PUBIC HAIR -.-,. PENIS Y ? ¥ t T '. ^ LENGTH (cm.) v^ Vl/ 3.«. 45-9, 45-12 8-15 9-15 :105K CKCUMFtlENCI (cm.) m- • - m- • "° • '° ^^-105 TESTES (cc) >.• T75^ 17^B ^■■« 6 2(^A "• OR J^ -^1 PROSTATE -"■^ -^^^ PRE. POST- PUBESCENCE PUBESCENCE PUBESCENCE Fig. 5.17. Stages of sexual development and maturation. (From W. A. Schonfeld, Ai J. Dis. Child., 65, 535, 1943.) (HCG) increases the yield of testosterone from testicular slices incubated with ace- tate. Estradiol- 17-/? also has been found in the products obtained by incubating tis- sue slices with acetate. Human testicular tumors incubated with labeled acetate form labeled testosterone, androstenedione, pro- gesterone, estradiol, and estrone (Wotiz, Davis and Lemon, 1955). Mevalonic acid, a precursor of cholesterol, yields estradiol when incubated with homogenates of hu- man testis (Rabinowitz and Ragland, 1958). The biogenesis of male hormone as worked out in the stallion, rat, and human (Savard, Dorfman and Poutasse, 1952; Sav- ard, Besch, Restivo and Goldzieher, 1958; Savard, Dorfman, Baggett and Engel, 1956) by means of radioisotopic methods shows a common pathway from 17a-hydroxyprogcs- terone -^ progesterone -> 4-androstene- 3,17-dione —^ testosterone. Testosterone has been identified in the spermatic vein blood of dogs (West, Hollander, Kritchevsky and Dobriner, 1952). Also identified were A^- androstcno(lione-3-17 and 7-keto-cholester- one. In addition to confirming the presence of several biologically active steroids in the testis, the studies made in the last two decades have clarified the biosynthesis of male hormone. The peripheral metabolism of testosterone and its biologic actions in the organism are described in chapters by Villee and by Price and Williams-Ashman, respectively. In addition to these well-known steroid hormones, the presence of a water-soluble hormone in the testis has been postulated on biologic evidence. Vidgoff, Hill, Vehrs and Kubin (1939) and Vidgoff and Vehrs (1941) induced atrophy of the testis and accessory sex organs in the rat by the administration of aqueous extracts of bull testes. Because the atrophy was similar to that occurring after hypophysectomy, it was claimed that a water-soluble principle in the testis was capable of inhibiting the gonadotrophic function of the ])ituitary. This principle was called "inhibin." The theory was then con- structed that the testis secretes two hor- mones, nnnu'ly a water-soluble hormone responsible for the integrity of the germi- nal epithelium by regulating the secretion of pituitary gonadotrophin, and a fat- MAMMALIAN TESTIS 335 soluble hormone (testosterone) responsible for maintaining the accessories. The ob- servations of Vidgoff and his associates were disputed by Rubin (1941). The inhibin con- cept was supported by McCullagh and Hruby (1949) because testosterone did not inhibit the excretion of pituitary gonado- trophin and was not effective in correcting castration changes in the pituitary of crypt- orchid rats at doses that were sufficient to stimulate the accessories. Inhibin was now identified with estrogen, and the source of estrogen was claimed to be the Sertoli cell. The new evidence for this modified con- cept will now be considered. ]\IcCullagh and Schaffenburg (1952) stated that estrogen is much more effective than androgen in suppressing gonado- trophin and that estrogen is present in saline extracts of bull and human sperm. Estrogen is found in the testes, but localization of its production to the Sertoli cells is uncertain (Teilum, 1956), and is doubted by Morii (1956) and Ballerio (1954). The almost complete absence of Sertoli cells in Kline- felter's syndrome, in which values for uri- nary gonadotrophin are high, also is considered as evidence that estrogen is man- ufactured by the Sertoli cells. The high ex- cretion of gonadotrophin in Klinefelter's syndrome can be interpreted, at least in part, by the concept of Heller, Paulsen, ^lortimore, Jungck and Nelson (1952) that the amount of urinary gonadotrophin varies inversely with the state of the germinal epi- thelium. Utilization of gonadotrophins by the germinal epithelium could explain the levels of this hormone in various syndromes as satisfactorily as the lack of a hypothetic testicular inhibitory hormone. Furthermore, if the Sertoli cells secrete an inhibitory hormone, patients who have germinal aplasia (Sertoli cells only in the tubules) should have normal values for urinary gonadotrophin, whereas it is well known that this hormone is greatly increased in these patients. The proponents of the in- hibin theory claim that aqueous extracts of testes prevent the castration changes but do not repair the accessories, whereas testos- terone corrects the accessories but does not restore the normal histologic appearance of the pituitary. However, Nelson showed that cryptorchid testes produce less androgen than normal and that the order in which the above structures are affected represents dif- ferences in the degree of their sensitivity to the amount of androgen produced. The efficacy of aqueous extracts on the cytologic appearance of the pituitary has not been confirmed. Thus, evidence deduced from cryptorchism that an inhibitory hormone is produced by the germinal epithelium is inadequate. XII. Effects of the Pituitary on the Testis Little information has been added in the past 20 years to the effects of acute hypo- physeal deprivation on the mammalian testis. Smith (1938, 1939) had shown in the rat that spermatocytes as well as sperma- togonia and Sertoli cells remain for a long time after hypophysectomy. However, in the monkey, and possibly in man, all cells of the germinal line except the spermato- gonia and the Sertoli cells disappear. Even though hypophysectomy has been employed for several years as a palliative procedure in inoperable carcinoma of the prostate, no data have been obtained concerning the effects of hypophysectomy on the testis in otherwise normal man. In the dog, the testes decrease to about one-third their normal weight after surgical removal of the pituitary. Only a single row of spermato- gonia remains (Fig. 5.18). The Leydig cells are reduced in size and contain abundant quantities of fat. The lack of complete in- volution of the Leydig cells in the dog as a result of hypophysectomy is somewhat unusual, because marked involution of these cells occurs in all other mammals thus far studied. With respect to the behavior of the germinal epithelium, the dog (Huggins and Russell, 1946) seems to be more like the monkey and man than like the rat and mouse. The total relative decrease in testic- ular weight of the dog is intermediate be- tween that observed in the cat (50 per cent) and that in the rat, guinea pig, and rabbit (75 per cent). With respect to histologic features, the guinea pig and ferret are in- termediate between the rat and the monkey, because occasional spermatocytes remain in addition to spermatogonia and Sertoli cells. In the mouse, the testicular weight decreases for 25 days after hypophysectomy. Mess 336 ^, PHYSIOLOGY OF GONADS .#• .,**^,,,i*^ * .; ;« m,gm. ^ 1 Fig. 5.18. Testis of dog 60 days after hypophysectomy. (From C. Huggins and P. S. Russell, Endocrinology, 39, 1, 1946.) (1952 ( showed that early differentiation of spermatids in the rat is affected first by hypophysectomy. Spermatids degenerate, tubuhir fluid is lost, and atrophy of the germinal epithelium finally takes place (Gothie and Moricard, 1939). Some recent studies on comi^ensatory hyi)ertrophy of the remaining testis after unilateral orchiectomy have been made. Old investigations showed that compensa- tory hypertrophy occurs in boars, rabbits, and hedgehogs. Compensatory hypertrophy does not occur in mature guinea pigs or man (Calzolari, Pulito and Pasquinelli, 1950; Pasquinelli and Calzolari, 1951; Zide, 1939). In the prepubertal guinea pig and rat, however, the remaining testis shows accelerated development. The volume of the remaining testis increases in the adult rat after unilateral orchiectomy (Grant, 1956). Since the compensatory hypertrophy is suppressed by testosterone, it appears likely that the accelerated development of the remaining testis is mediated by gonado- tropliins. The effects of gonadotrophins on testicu- lar structure and function have been studied in many species. Injection of anterior jHtui- tary extract or implantation of fragments of the anterior i)ituitary into the testis of guinea pigs has residted in pronounced stim- ulation and hypertr()])hy of the Leydig cells (Petrovitch, Weill and Deminatti, 1953; Petrovic, Deminatti and Weill, 1954; Petro- vic, Weill and Deminatti, 1954; Marescaux and Deminatti, 1955). In hypophysecto- mized mice, May (1955) found that testicu- lar grafts of anterior pituitary tissue repair the atrojihic tubules and the involuted Ley- dig cells. The effects of in(li\i(lual gonadotrophins of both pituitary and placental origin have been reviewed by Greep (1937) and Fevold (1944), and in the chapter by Greep in the present volume. The established concept, as worked out in the rat, is that follicle- stimulating hormone (FSH) maintains and repairs the tubular apparatus but does not affect the function or structure of the Ley- dig cells. Luteinizing hormone (LH) main- tains the functional activity of the Leydig cells but does not dii-ectly control tubidar activity. Urinary gonadotrophin from menopausal women stimulated the tubides (Greep, 1937) and the Leydig cells (Balm. Lorenz, Ben- nett and Albert, 1953a-d). Hmnan chorionic gonadotropiiin (HCG) has little oi' no effect on tiie tubules, hut it induces ])ro- nounced stimulation of the Leydig cells. Pr(>gnaiit marc sci'uni (PMS) stimulates spermatogenic and endocrine activities of the testis. Both LH and HCG maintain spermatogcniesis after hypophysectomy. MAMMALIAN TESTIS 337 Ncitlier FSH nor LH hastens the appear- ance of sperm in the testis of immature ani- mals. No type of gonadotrophin has induced the appearance of sperm in the rat earlier that 35 days of age. Because interest in the chemical fraction- ation of animal i^ituitary tissue waned after 1945, new studies on the effects of pituitary gonadotrophins on the testis have not been performed. Instead, HCG has received at- tention. The well known hyperemia induced in the ovary by HCG, which is used as a pregnancy test, has been reported to occur also in the testis by Hartman, Millman and Stavorski (1950). Hinglais and Hinglais (1951 ) have not confirmed this. HCG causes increased testicular weight in young rats (Rubinstein and Abarbanel, 1939). The ef- fect of HCG on the rat testis has been sum- marized by Gaarenstroom (1941), who listed the following four main actions: (1) stimulation of the Leydig cells in both nor- mal and hypophysectomized animals to produce androgen; (2) increase in growth of the testis in the normal immature ani- mal; (3) maintenance of testicular tubules in hypophysectomized animals; (4) poten- tiation of the effects of i^ituitary gonado- troi)hins in either normal or hypophysecto- mized animals. All effects are interpreted as being caused by the increased liberation of androgen. This explanation probably also holds for the increased fibrosis in and around the tubular wall in hypophysecto- mized rats after administration of HCG, for the increase in the number of primary spermatocytes (Muschke, 1953; Tonutti, 1954), and for the slight increase in tes- ticular weight (Diczfalusy, Holmgren and Westerman, 1950). The effects of HCG in normal men are similar to those in animals (Maddock, Ep- stein and Nelson, 1952; Maddock and Nel- son, 1952; Weller, 1954). The Leydig cells become hyperplastic and produce more es- trogen and androgen. This is reflected first by an increase in urinary estrogen of some 5- to 20- fold and later by an increase in 17-ketosteroids of about 2-fold. The in- creased secretion of steroids by the Leydig cells is accompanied by an increase in the frequency of erections and occasionally by gynecomastia. The increased levels of es- trogen and androgen induce tubular atro- phy. The tubular diameter becomes smaller, spermatogenesis ceases, and there is an in- crease in necrosis and sloughing of the germinal cells. The basement membranes become hyalinized, and peritubular fibrosis develops. In certain eunuchoidal persons ( hy{)ogonadotrophic hypogonadism ) , use of HCG induces differentiation of the Leydig cells and hastens maturation of the Sertoli cells. Some spermatogenesis is obtained (Heller and Nelson, 1947, 1948; Maddock, Epstein and Nelson, 1952). If FSH also is administered to such eunuchoidal men, com- plete spermatogenesis occurs (Heller and Nelson, 1947). PMS acts on the rat testis in a manner intermediate between that of HCG and FSH (Creep, 1937; Kemp, Pedersen-Bjergaard and Madsen, 1943). Tubular growth and hyperplasia of the Leydig cells result. In- terstitial cell hyperi)lasia also occurs in mice (Bishop and Leathern, 1946, 1948) , although the testicular weight does not increase after the use of PMS, as it does in rats. In the opossum, PMS does not induce secretion of androgen until the the animals are 70 days of age (Moore and Morgan, 1943). PMS is able to maintain the monkey testis after hypoi)hysectomy l)ut only for 20 days, after which involution occurs. If given to a hy- pophysectomized monkey in which testicu- lar atrophy already is present, PMS causes formation of spermatocytes, but it does not induce the formation of spermatids or sperm cells (Smith, 1942). In man, PMS causes an increase in testicular weight (Hemphill and Reiss, 1945). Unfractionated extracts of pituitaries of sheep or horses induce both tubular matura- tion and androgenic formation (Sotiriadou, 1941 ) . Preparations of FSH in mice produce slightly heavier testes but do not cause androgenic secretion (Moon and Li, 1952). Purified preparations of LH produce atro- phy of the tubules and stimulation of the Leydig cells in infantile rats, and main- tenance of germinal epithelium and Leydig cells in hypophysectomized rats (Zahler, 1950). XIII. Effects of Steroids on the Testis Between 1930 and 1940, rapid advances were made in the understanding of pituitarv and gonadal interrelationships, and the eon- 338 PHYSIOLOGY OF GONADS cept of a servomechanism controlling pitui- tary-testis activities was well established. According to this concept, male hormone was considered to have its major effect on the testis by inhibiting the secretion of pi- tuitary gonadotrophins. However, it was difficult to fit into this concept the report by Walsh, Cuyler and McCullagh (1934) that testosterone was capable of maintain- ing spermatogenesis in the rat after hypoph- ysectomy. If testosterone were the medium by which spermatogenesis was maintained normally, the dualistic concept of gonado- trophic control of the testis would be in jeopardy. As can be imagined, this finding stimulated much research. By 1940 the fact that spermatogenesis is maintained in hy- pophysectomized rats, mice, and rabbits by testosterone was amply established (Cutuly and Cutuly, 1940) . A. ANDROGENS The varied effects obtained by injecting male hormone into normal and hypophysec- tomized rats depend on the nature of the androgen, the dose, the length of the treat- ment period, and the age of the animals when injections are begun. Inasmuch as most of the experimental work has been done with the rat and rats of various ages and sizes were employed, it is obvious that the dose of hormone is an important factor. Doses of testosterone of 100 /xg. per day or less can be regarded as small doses, whereas doses of 1 mg. or more can be considered as large. These definitions pertain only to the doses employed in studying the action of androgen on the testis and do not neces- sarily have any relationship to the physio- logic levels of testosterone produced by the rat testis, which is not known, or to the effects of testosterone on the accessory sex organs (Moore, 1939). In general, testosterone has no action on the undifferentiated gonad of the mouse, rat, opossum, or guinea pig (Moore and Morgan, 1942). In the immature rat small doses of testosterone propionate depress the testicu- lar weight (Zahler, 1947; Dischreit, 1939; Greene and Burrill, 1940). However, if small doses are continued for long periods, incomplete supi:)ression results. Because the testicular inhil^ition induced by small doses of testosterone apparently results from sup- pression of gonadotrophins, it seems that greater ciuantities of gonadotrophins are formed as rats grow; hence, escape from suppression may occur (Biddulph, 1939). The work of Rubinstein and Kurland (1941) indicates that even small doses of testos- terone, as already defined, may produce dift'erent effects in the rat. These investi- gators compared the effects of administra- tion of 5 and 50 fxg. testosterone propionate per day in young animals. Young rats re- ceiving the former dose showed increased testicular weight without, however, any hastening of maturation of sperm cells. The larger dose decreased testicular weight. The effect of androgen on mature rats is also dependent on dose. Small doses cause atrophy of the mature testis because of suppression of gonadotrophins. Large doses have the same suppressing effect, but this is overridden by a direct stimulating effect of androgen on the testis, and atrophy does not occur. In both instances, the Leydig cells are atrophic (Shay, Gershon-Cohen, Paschkis and Fels, 1941). Large doses of testosterone have a direct action on the testis as indicated by the protective effect exerted on the experimentally induced cryptorchid testis (Hamilton and Leonard, 1938) and on the transplanted testis (Klein and Mayer, 1942) . The aftereffects of androgenic adminis- tration also depend on the age of the animal and the duration of therapy. Using fecun- dity, libido, potency, and the state of the reproductive tract as indices of testicular function, Wilson and Wilson (1943) ex- amined rats 3 to 5 months after a 28-day period of injection of androgen. In rats age 1 to 28 days, androgen severely affected the reproductive system. Low libido, absence of fecundity, and atrophic accessories were noted 3 to 5 months after testosterone ther- apy was discontinued. However, the later this treatment was instituted in the life of the rat, the more normal was the repro- ductive system 3 to 5 months after adminis- tration of the hormone was stopped. Nelson and Merckel (1937), in a series of extensive experiments, confirmed the earlier finding that various androgens maintain spermatogenesis in the rat after hypophy- sectomy. Furthermore, they showed that the Leydig cells are atroj^hic in the face of MAMMALIAN TESTIS 339 active spermatogenesis in the androgen- treated, hypophysectomized rat. Comparing such steroids as testosterone, androsterone, dehydroisoandrosterone, androstenedione, and various isomers of androstenediol, they concluded that the ability of androgens to maintain spermatogenesis is not related to their androgenicity. In fact, the weaker the androgen the better is the maintenance of spermatogenesis after hypophysectomy. This observation is important for it shows that maintenance of spermatogenesis is not due to the induction by androgen of a favor- able scrotal environment for the testis. In further studies, Nelson (1941) showed that spermatogenesis could be maintained for ^? f.'^ 178 days after hypophysectomy by testos- terone propionate. No difference was ob- served between spermatogenesis under these conditions and that which occurs normally. Motile sperm were formed, and the animals could copulate with and impregnate females. The only difference was that the testes in the hypophysectomized animals treated with testosterone were only one-sixth nor- mal size. As is true of other effects of androgens on the testis, the time at which rats are hy- pophysectomized seems to be a critical fac- tor in the ability of testosterone to maintain spermatogenesis. Leathern (1942, 1944) showed tliat troatmcnt witli tostosterone in Fig. 5.19A. Effect of testosterone on the testis of the rat. 4, normal rat, 30 days of age. S, normal rat, 60 days of age. 6, 30-day-old rat given 10 ^g- of testosterone propionate daily for 30 days (no inhibition of spermatogenesis). 7, 30-day-old rat given 100 ^ig. of testosterone propionate daily for 30 days (suppression of spermatogenesis). 340 PHYSIOLOGY OF GONADS rats Itypophysectomized at 27 days of age resulted in the production of spermatids, but spermatogenesis did not occur. How- ever, if the animals were operated on at 33 days of age, testosterone induced the forma- tion of sperm. Furthermore, if the atrophic testes of hypophysectomized rats were stim- ulated by a gonadotrophin (PMS), testos- terone also maintained the spermatogenesis thus induced. It is not known exactly how testosterone maintains spermatogenesis after hypophy- sectomy. It seems that the "maintenance type" of spermatogenesis is not the same as spermatogenesis resulting from gonado- trophin, because the seminiferous tubules of the androgenically maintained testes in hypophysectomized rats are small. The ef- fect of androgen is not produced simply by the maintenance of sperm cells already pres- ent in the testis at the time of hypophysec- tomy because Nelson (1941) showed that spermatogenesis can be reinstituted in the testis of a hypophysectomized rat in spite of delaying treatment with testosterone for 3 to 4 weeks after hypophysectomy. This in- terval of time exceeds the normal sojourn of sperm cells in the epididymis; thus the results in terms of siring young cannot be attributed to sperm cells already present in the accessory duct system at the time of hyjjophysectomy (Figs. 5.19, A and B, and 5.20). The dose of testosterone propionate nec- essary for maintenance of spermatogenesis in the rat seems to be around 80 fig. per day. 9^-: v;: - n II Fig. 5.19B. 8, 3U-day-old mt given 1000 /ug. of testosterone propionate daily for 30 days (no suppression of spermatogenesis). .9, 30-day -old rat given 8.4 mg. of estradiol daily for 30 days (])ronoun(ed inhibition of spermatogenesis). 10, 30-day-old rat given 8.4 ng. of estradiol and 1000 mS- of lestosterone i)ro])ic)nale for 30 days (no inhir)ition of spermatogenesis). (From D. J. Jjudwig, Endocrinology, 46, 453, 1950.) MAMMALIAN TESTIS 341 • fW-" 12 Fig. 5.20. Klicct oi Ti-siostcioiic on testis oi li\|M)pli\-,~(i lomi/i d lat //, testis of norm.al rat, 30 days of age. 12, testis of 60-day-old rat li^popli^x ( tomizcd at 30 days of age. 13, testis of 60-day-old rat hypophysectomized at 30 (la.\s of age and given 1000 /lig. testosterone propionate daily for 30 days. (From D. J. Liidwig, Endocrinology, 46, 453, 1950.) However, larger doses generally have been used in experiments on the maintenance of spermatogenesis. These doses are far greater than those necessary to maintain the acces- sory sex organs of castrated animals. Tu- bules can be maintained by much smaller doses of testosterone. Dvoskin (1944) im- planted pellets of testosterone intratesticu- larly; approximately one-tenth of the amount of testosterone needed by tlie par- enteral route was effective by this route. The concept that testosterone maintains spermatogenesis in hypophysectomized rats was challenged by Simpson, Li and Evans (1942, 1944) and by Simpson and Evans (1946a, b). These investigators found that gonadotrophins, including interstitial cell- stimulating hormone (ICSH), maintained spermatogenesis in hypophysectomized rats at doses far lower than those needed to maintain the Leydig cells and the acces- sories. The testes remained in the scrotum, and motile sperm cells were produced. Inas- much as testosterone propionate can main- tain the tubules only at doses effective in maintaining the accessories, it was doubted that maintenance of spermatogenesis oc- curred by way of the direct tubular action of androgen. In addition to casting some doubt on the accepted mechanism of the spermatogenic action of androgen, this work raised doubt concerning the dualistic con- cept of gonadotrophic control of the testis. Maintenance of the testis by ICSH after hypoj^hysectomy suggests that one gonado- trophic hormone may be sufficient to main- tain testicular function in mammals. How- ever, these findings may be interpreted conventionally; i.e., that ICSH caused the Leydig cells, even though they were not re- 342 PHYSIOLOGY OF GONADS paired morphologically, to secrete androgen which by virtue of its local action on the tubules maintained spermatogenesis (Lud- wig, 1950). Testosterone maintains spermatogenesis in other species. In hypophysectomized ground squirrels, the testes are atrophic, aspermatic, and abdominal (Wells, 1942; 1943a). Hypophysectomized animals given testosterone propionate (0.5 mg. per day for 15 to 25 days) show growth of the testes, sperm formation, and testicular descent. Leydig cells remain atrophic. Because sperm formation ceases after hypophysectomy in the ground squirrel, as it does in the mon- key, rat, guinea pig, mouse, cat, and ferret, '^J^ *' ,f?t * "• • • • . »^--r- Fic. .').21. KIT(H't of .iihlhitiMi HI ,( liypophysocto- mized inoiikoj-. 1, biop-x -iniiincn from a normal 8-kg. rhesus monkey. .'. liiii|i-\ specimen from a hypophysectomized monkey .ifici- 56 da.ys, during which 1.4 gm. of testosterone propionate was ad- ministered at a daily dose of 25 mg. 3, state of testis 20 days after use of testosterone was dis- continued. Note atrophy of tubules. The Sertoli cells and spermatogonia remain. (From P. E. Smith, Yale J. Biol. & Me.l., 17, 281, 1944.) it is obvious that androgen initiated sperma- togenesis. Testosterone propionate maintains the spermatogenic activity of the testis of the hypophysectomized monkey for 20 to 50 days (van Wagenen and Simpson, 1954). A dose of about 20 mg. per day is required. When medication is discontinued, marked involution of the testis occurs within the ensuing 3 weeks. Testosterone is effective even after a lapse of 50 days between hy- pophysectomy and the institution of ther- apy. Spermatogenesis can be restored and formation of motile sperm cells induced. As in the rat, the testes maintained by androgen are smaller than normal. Pellets of testoster- one implanted locally exert a strong local ac- tion. Thus, the essential findings in the rat are duplicated in the monkey (Fig. 5.21). In man the effects of testosterone on the testis have been studied by Hotchkiss (1944a), and by Heller, Nelson, Hill, Hen- derson, Maddock, Jungck, Paulsen and Mor- timore (1950). The main effects were dis- appearance of the Leydig cells, atrophy of the tubules, arrest of spermatogenesis, and pronounced hyalinization of the basement membrane (Fig. 5.22). Complete recovery of the testis occurred 17 months after cessa- tion of therapy. In fact, the testes were histologically more normal than before treatment. The improvement in sperm pro- duction after preliminary depression of the testis by administration of testosterone has been used widely in the treatment of male infertility. Heckel, Rosso and Kestel (1951) and Heckel and McDonald (1952a, b) ob- tained an increase in spermatogenic activ- ity, as determined by sperm counts and biopsy, after cessation of treatment. This increase was termed a "rebound phenome- non"; during it, increased fertility, as de- termined by an increased incidence of preg- nancy among infertile couples, was reported. The improved quality and quantity of sperm following therai)y with testosterone are transient. Furthermore, they occur in only a small i^-oportion of men so treated (Getzoff, 1955; Heinke and Tonutti, 1956). The suppressive effect of androgen on the human testis results from inhibition of pitui- tary gonadotrophin as evidenced by meas- urement of the amount of urinary gonado- trophin before, during, and after use of MAMMALIAN TESTIS 343 1 ■•■A \fh'. Fig. 5.22. Elluci ul iC5io.stcruiie on the testis of a man with infertiUty caused by adult tubular failure. Testicular biopsies, showing the pronounced degree of sclerosis and hyaliniza- tion that occurs when an initially very poor testis is subjected to the administration of 91 consecutive injections of testosterone propionate, 25 mg. each. A, before treatment; B, at end of 91 days of treatment; C, 17 months after cessation of treatment. Note, in C, the disappearance of hyalinization, the increase in size of the seminiferous tubules, and the appearance of fairly orderly spermatogenesis. Leydig cells, not shown here, were present 17 months after treatment was stopped. (From C. G. Heller, W. O. Nelson, I. B. Hill, E. Henderson, W. O. Maddock, E. C. Jungck, C. A. Paulsen and G. E. Mortimore, Fertil & Steril., 1, 415, 1950.) testosterone. The mechanism by which gon- adotrophin is inhibited always has been assmned to be a direct effect of androgen on the pituitary. It is interesting in this regard that Paulsen (1952) showed that the use of testosterone, w^iile reducing urinary gonado- trophin, increases the amount of urinary estrogen 20-fold. Estrogen is by far the most powerful suppressant of gonadotrophin secretion known; hence, it is possible that the atrophy of the testis observed during testosterone therapy in man may be caused by estrogen. No reports of maintenance of spermatogenesis in men with pituitary in- sufficiency or after hypophysectomy are available. B. ESTROGENS Various natural and synthetic estrogens have been given to rats, guinea pigs, ham- sters, cats, bulls, boars, and man. In all forms, estrogen induces atrophy of the male gonad. The histologic appearance of the atrophic rat testis after estrogen therapy has been described by Dischreit (1940). In young rats, estradiol prevents testicular descent, produces atrophy, and inhibits spermatogenesis (Pallos, 1941; Gardner, 1949). Two weeks following atrophy in- duced by estradiol or stilbestrol, regenera- tion of the testis begins (Bourg, Van Meen- sel and Compel, 1952) and is complete wuthin 6 weeks (Lynch, 1952). However, Snair, Jaffray, Grice and Pugsley (1954) noted that the accessory sex organs re- cover before spermatogenesis resumes. The same inhibiting effects have been obtained with methylbisdehydrodoisynolic acid (Tuchmann-Duplessis and Mercier-Parot, 1952) and hydroxypropiophenone (Lacas- sagne, Chamorro and Buu-Hoi", 1950). In general the effect of estrogen in the rat is to induce atrophy of the Leydig cells and germinal epithelium, so that only sperma- tocytes, spermatogonia, and Sertoli cells remain. Uncertaint3^ exists concerning the general effects of estrogen in guinea pigs. Lynch (1952) noted that the Leydig cells are nor- mal in animals treated with estrogen, but Marescaux (1950) and Chome (1956) noted that the Leydig cells are atrophic. Mares- caux, in studying hypophysectomized guinea pigs, concluded that estrogen has a direct stimulating effect on the Leydig cell. Mas- sive tubular damage occurs in the guinea pig after administration of estrogen. In the hamster. Bacon and Kirkman (1955) found that various estrogens induce testicular atrophy. In occasional animals, hyperplasia of interstitial and Sertoli cells occurs and is attributed to direct effects of estrogen. In general, atrophy of the germinal epithelium 344 PHYSIOLOGY OF GONADS is nearly complete; only a few spermato- cytes remain in addition to the Sertoli cells. The testis of the immature cat is un- affected by estrogen (Starkey and Leathem, 1939j . Severe tubular atrophy and involu- tion of the Leydig cells are noted in bulls (Ferrara, Rosati and Consoli, 1953) and boars (Wallace, 1949j after feeding with stilbestrol. Although Haschek and Gutter (1951) found no effect of estrogen on the testis, the consensus is that any kind of estrogen pro- duces profound involution of the human testis. Temporary sterility is induced, of course, as well as impotence and gyneco- mastia (Heckel and Steinmetz, 1941). Most of the information in man has been obtained from the therapeutic administration of es- trogen in cases of prostatic carcinoma (Chome, 1956; de la Baize, Mancini and Irazu, 1951 ; de la Baize, Bur, Irazu and Mancini, 1953; de la Baize, Mancini, Bur and Irazu, 1954; Schwartz, 1945; Schiiltz, 1952, to mention only a few) and from the administration of estrogen to hypersexual and homosexual men (Dunn, 1941). Estro- gen induces atrophy of the tubules and the Leydig cells ; the latter revert to fibroblasts. The germinal epithelium shows an increase in lipids and a decrease in glycogen. Unless other disease is present, the atrophy pro- ceeds so that only the Sertoli cells remain in the tubules; even these cells may disappear with the induction of peritubular hyaliniza- tion and sclerosis. C. ADRENAL STEROIDS Tubular diameter in the testis of the mature rat remains normal despite the pres- ence of severe hypercortisonism resulting fi'oiii administration of 3 mg. cortisone per day for 6 weeks (Winter, Silber and Stoerk, 1950) or of 5 to 10 mg. per day (Ingle, 1950) . A few reports indicate that cortisone stimulates growth of the testes of young rats (Leroy, 1951) or causes degeneration of the germinal ei)ithelium of the rat (Le- roy, 1952) and mouse (Antopol, 1950). A careful study by Hanson, Blivaiss and Rosenzweig (1957) showed that the relative growth of the testis is stimulated only slightly by cortisone. Extremely little infoi-mation is avail- able on the maintenance of spermatogenesis in hypophysectomized rats by cortisone. Le- roy and Domm (1952) reported mainte- nance at doses of 5 mg. per day. The Leydig cells involuted, and the secondary sexual apparatus was atrophic. However, these findings were not confirmed by Aterman ( 1956) , who used 5 mg. hydrocortisone per day after hypophysectomy. The scrotum be- came atrophic and the testes retracted. The histologic appearance of the testes of the cortisone-treated animals was indistinguish- able from that of the hypophysectomized controls. In rabbits Arambarri (1956) re- ported only small changes in the relative weight after prolonged use of cortisone. In man, fairly large doses of cortisone given to patients with rheumatoid arthritis do not affect the histologic appearance of the testes (Maddock, Chase and Nelson, 1953). Corti- sone does bring about rapid testicular mat- uration in boys who have congenital ad- renal hyperjjlasia, but only if the bone age is near the age of puberty (Wilkins and Cara, 1954). This must not be construed as a direct effect of cortisone on testicular maturation. The action of cortisone in this instance is to inhibit the excessive release of corticotrophin (ACTH) from the pitui- tary, thus reducing the amount of 17-keto- steroids produced by the abnormal adrenals. Removal of the inhibiting effect of the androgenic steroids allow^s the formation of gonadotrophin, with resulting maturation of the testes. The consensus is that cortisone does not cause any change in the histologic appear- ance of the testis (Cavallero, Rossi and Borasi, 1951 ; Soulairac, Soulairac and Teys- seyre, 1955; Baumann, 1955). Furthermore, it causes no change in the accessory struc- tures, or in the secretion of androgen by the testis. Cortisone has no direct effect on the prostate or seminal vesicles in castrated ani- mals (Moore, 1953). It is doubtful whether cortisone can maintain spermatogenesis after hypophysectomy. The bearing of these studies on normal testicular physiologic function is questionable. Cortisone has been the main adrenal steroid studied in the rat but the rat adrenal secretes corticosterone, not cortisone. Desoxycorticosterone has been adminis- tcicfl to rats in various doses. Arvy (1942) and Overzici' (1952) I'cported that the de- MAMMALIAN TESTIS 34; velopment of the testis of the iiinnature rat was arrested by prolonged injections of this steroid. Effects from desoxycorticosterone are not evident in adrenalectomized animals (Migeon, 1952). Adult rats show atrophy of both the tubular apparatus and the Leydig cells (Naatanen, 1955; Selye and Albert, 1942a, b). Maintenance of spermatogenesis after hy])ophysectomy was described by Overzier (1952). Because cortisone even in massive doses has little effect on the testis, it would seem unlikely that ACTH would have any dra- matic effects. Li and Evans (1947) repoi'ted that ACTH depresses testicular weight and the weight of the accessories in young rats, has no effect in old rats, and does not main- tain spermatogenesis or the accessories in hypophysectomized rats. Baker, Schairer, Ingle and Li (1950) reported a small reduc- tion in testicular weight in adult rats, but spermatogenesis j^roceeded satisfactorily. Large doses of ACTH produced atrophy of the Leydig cells. Asling, Reinhardt and Li (1951) stated that large doses depress the weight of the accessory sex organs. How- ever, Moore (1953) found that administra- tion of 5 mg. ACTH per day for 10 days has no effect on the testis of young or old rats and has no extratesticular effect on the pro- duction of androgen. D. MLSCELLAXEOU.S .STEROIDS AND MIXTURES OF STEROIDS Masson (1945, 1946) studied 16 different steroids for their ability to maintain sper- matogenesis. Androstenediol, methylandro- stenediol, methylandrostanediol, A'^-preg- neninolone, and dehydroisoandrosterone are the most active compounds in maintaining spermatogenesis after hypophysectomy. No relationship is apparent between the ability to maintain spermatogenesis and the andro- genic activity of the compound as measured by stimulation of the seminal vesicles or the progestational activity (progesterone is ef- fective in maintenance but ethinyl testos- terone is not). One compound, A^-pregneninolone, was studied in detail. It prevents testicular atro- phy after hypophysectomy or following therapy with estradiol or testosterone; it does not produce atrophy of the Leydig cells. In doses of 1 to 2 mg. a day, preg- neninolone maintains spermatogenesis in young and adult hypophysectomized rats, l)ut it does not repair the tubules or Leydig cells after a 2-week delay between hypoph- ysectomy and therapy. Pregneninolone also exerts a protective effect against the damage evoked by estradiol; however, it does not affect the regeneration that occurs after cessation of estradiol treatment. In this respect, it is different from testosterone, which hastens the recovery from the estra- diol-induced damage. In fact, the accelera- tion of regeneration by testosterone is in- hibited by pregneninolone. The chief difference between pregneninolone-proges- terone and testosterone-androstenediol is that, whereas spermatogenesis is maintained by either pair after hypophysectomy, the former pair cannot restore spermatogenesis, and the latter can. ]\Iost of these effects of pregneninolone were confirmed by Dvoskin (1949). Progesterone and some new proges- tational compounds have been studied re- cently in man (Heller, Laidlaw, Harvey and Nelson, 1958). Progesterone given to normal men produces azoospermia and slight tu- bular atrophy, abolishes libido, and reduces potentia, but has no effect on the Leydig cells and the excretion of gonadotrophin, estrogen, and 17-ketosteroids. Certain doses of desoxycorticosterone or estradiol have no effect on the testis singly, l)ut when mixed produce severe depression of testicular weight (.lost and Libman, 1952). The earlier work of Emmens and Parkes (1938), showing that testosterone inhibits the debilitating action of estrone, was confirmed by Joel (1942, 1945). The testes of animals treated with estradiol are one-sixth normal size; however, when tes- tosterone propionate is added to the estro- gen, the testicular weight is one-fourth nor- mal. Furthermore, sperm cells are present in the epididymides of the group receiving testosterone. Mixtures of small amounts of androstenediol and estradiol in a constant proportion produce more profound atrophy than large doses given in the same constant proportion (Selye and Albert, 1942a, b; Selye, 1943). Furthermore, androstenediol and pregneninolone prevent the atrophy in- duced by small doses of testosterone. Plence, this protective action is not related to tes- toid activity, because the first compound is 346 PHYSIOLOGY OF GONADS a weak androgen ; the second has no andro- genic action. The protective effect possibly is due to interference with the inhibiting action of testosterone on pituitary gonadotrophin. XIV. Eflfeets of Altered Endocrine States on the Testis Apart from the pituitary, alterations in the endocrine system do not have pro- nounced effects on the testis. The thyroid has been studied extensively with regard to testicular function (Maqsood, 1952). It is difficult to generalize with respect to the total impact of the thyroid on the testis except to state that there is great variability not only from species to species, but also in different individuals of any one species. Young, Rayner, Peterson and Brown (1952a) suggested that the range of thyroid activ- ity within which normal testicular function is possible is rather wide. This may explain why many effects on the testis of altered thyroid function are marginal and why so many reports are exceedingly conflicting. Furthermore, it seems reasonable that ani- mals having a naturally high level of thy- roid activity may be impaired with respect to reproductive performance when made hypothyroid; conversely, species or indi- viduals functioning normally at relatively low levels of thyroid activity may be ad- versely affected with regard to testicular activity when made hyperthyroid (Young, Rayner, Peterson and Brown, 1952b). In laboratory animals, hypothyroidism is induced by thyroidectomy, by feeding of antithyroid substances, by administering radioiodine, or by combination of these methods. Hyperthyroidism is induced by feeding desiccated thyroid or various arti- ficial thyroproteins, or by injecting thy- roxine or triiodothyronine. Because it does not seem to matter, as far as testicular physiology is concerned, how hypothyroid- ism and hyperthyroidism are induced, do- tails of the method of altering thyroidal status will not be given. Hypothyroid rats show decreased sper- matogenesis and have smaller accessory structures than normal rats (Smelser, 1939a). However, Jones, Delfs and Foote (1946) found that adult hypothyroid rats sire litters. Young animals, made hypothy- roid at birth or shortlv thereafter, mav show delay in sexual maturation (Scow and Marx, 1945 ; Scow and Simpson, 1945) , or may have normal reproductive tracts (Goddard, 1948). Hyperthyroid rats show testicular degenera- tion associated with a decrease in sperm pro- duction and androgen secretion. The dele- terious effects of hyperthyroidism are at- tributed to an incapacity of the testis to respond to gonadotrophin. The atrophy of the accessory structures is attributed to the decrease in androgen production and to their increased requirement for androgen in states of hyperthyroidism (Smelser, 1939b). A nonendocrine explanation offered by Cunningham, King and Kessell (1941) is that testicular degeneration occurs because of the increased body heat of the animals in the hyperthyroid state. Richter and Winter (1947), however, stated that hyper- thyroidism has a stimulating effect on the rat testis and accelerates the transfer of sperm through the genital ducts. Lenzi and Marino (1947) wrote that experimental hy- perthyroidism causes a decrease in the num- ber and volume of Leydig cells. Mixtures of thyroxine and testosterone in doses that have no effect on the rat testis when given singly, produce severe atrophy in normal rats (Masson and Romanchuck, 1945). Small doses of testosterone augment the de- bilitating effect of hyperthyroidism; large doses protect the testis (Roy, Kar and Datta, 1955). Changes in thyroidal status also appear to affect the responsiveness of the testis to gonadotrophins. Meites and Chandrashaker (1948) stated that hyper- thyroidism decreases the responsiveness of the rat testis to exogenous gonadotrophin (PMS) whereas hypothyroidism increases it. The reverse holds for mice. In growing mice, sexual development is retarded by hypothyroidism and accelerated by mild liyperthyroidism (Maqsood and R('inek(\ 1950). Moreover, the effectiveness of testosterone on the seminal vesicles of mice is increased by the concomitant ad- ministration of thyroxine (Masson, 1947). indicating an increased responsiveness of the accessory reproductive tract to male hormone in the hyperthyroid state. Hyperthyroid guinea pigs have small tes- ticular tubules and fewer sperm in the semi- niferous tubules. As in the rat, Richter (1944) found that hyperthyroidism in the MAMMALIAN TESTIS 347 guinea pig was associated with a rapid dis- charge of sperm through the genital ducts. Hypothyroidism was found to have no ef- fect on the structure of the testis, on the structure of the sperm cells in the ejaculate, or on fertility (Shettles and Jones, 1942). Young, Rayner, Peterson and Brown (1952a, b ) , however, observed that the degree of fer- tility of hypothyroid guinea pigs was slightly reduced but in general the strength of the sex drive was not altered significantly by either hypothyroidism or hyperthyroidism. Other laboratory animals studied include the rabbit and the dog. Hypothyroidism in beagle puppies has no effect on spermato- genesis (Mayer, 1947j, whereas Maqsood (1951b) found atrophy of the seminiferous tubules and signs of decreased sexual drive in hypothyroid rabbits. In male farm animals, alterations in thy- roid function are associated with variable effects on the reproductive system. Atrophy of the tubules and Leydig cells occurs in the hypothyroid ram. Reduction of libido is noted in the hypothyroid ram, goat, and bull (Maqsood and Reineke, 1950). "Sum- mer sterility" of sheep is explained as being due to depression of thyroid activity brought about by hot weather. Feeding thyroidal materials increases libido and spermatogenesis in bulls (Reineke, 1946; Petersen, Spielman, Pomeroy and Boyd, 1941). The reduction in testicular activity during hypothyroidism is attributed to an altered secretion of trophic hormones by the pituitary ; the excess secretion of thyro- trophin induced by thyroid deficiency in some way reduces the secretion of gonado- trophins (De Bastiani, Sperti and Zatti, 1956). In man, Marine (1939) reported atrophy of the Leydig cells in a case of myxedema and atrophy of the tubules in a case of ex- ophthalmic goiter; however, examination of the accompanying photomicrographs is not convincing. Many conflicting claims of the effect of thyroidal materials in infertile men have been made (c/. Dickerson, 1947) but these studies are uncontrolled and deserve no further comment. A recent study by Farris and Colton (1958), if verified, indi- cates that the nature of the thyroid sub- stance used may be important after all. Thyroxine and triiodothvronine were ad- ministered to normal and subfertile men. Thyroxine depressed the number and ac- tivity of the sperm cells in the ejaculate, whereas triiodothyronine had a beneficial effect on the quality and motility of the spermatozoa. Very little can be found on the effect of altered adrenal function on the testis. Dur- ing the alarm reaction induced by the in- jection of formalin, no changes are evident in the testis when the adrenal cortex is un- dergoing its usual response (Croxatto and Chiriboga, 1951, 1952). Chronic hyper- adrenalism produced by injections of epi- nephrine is accompanied occasionally by testicular atrophy and usually by regression of the accessories (Perry, 1941). Adrenalec- tomy in dogs, cats, and man is not followed by alteration in testicular structure (Mor- ales and Hotchkiss, 1956) . In rats rendered diabetic by removal of 95 per cent of the pancreas, a slight decrease was observed in testicular weight. In the final stages of diabetic cachexia, however, severe testicular atrophy occurs (Foglia, 1945). Horstmann (1949, 1950) concluded that the impotence of diabetic men results from the combined effects of decreased androgen production and of increased andro- gen destruction. This conclusion was, how- ever, denied by Bergqvist (1954). Im- potency and loss of libido are encountered frequently in association with uncontrolled diabetes; both may be corrected by ade- quate therapy. However, men more than 35 years of age whose diabetes is well con- trolled may have irreversible loss of libido and potentia. Histologic evidence of atro- phy in the testes of such diabetic men can be found in the literature. The atrophy described seems no greater than that which may occur spontaneously in normal men at various ages, however. The pineal body has long been thought to be involved in the regulation of the testis. The following conflicting statements have been made: (1) administration of pineal extracts inhibits testicular develop- ment, (2) pinealectomy causes testicular hypertrophy, (3) the concentration of cho- lesterol esters in the testis is lowered by administration of pineal extracts, and (4) none of the above results are obtained (Simonnet and Sternberg, 1951; Simonnet 348 PHYSIOLOGY OF GONADS and Thieblot, 1951 ; Alcozer and Costa, 1954, Alcozer and Cliordano, 1954; Bailo, 1955). The reader is referred to a recent book which summarizes the literature on the pineal body (Kitay and Altschule, 1954 ) . Extensive hepatic disease is associated with testicular atrophy. Morrione (1944) induced cirrhosis in male rats by means of carbon tetrachloride. The testes of the cirrhotic rats were not affected. However, when estrogen was administered, severe testicular atrophy occurred, much greater than that induced by the same amount of estrogen in control, noncirrhotic animals. Testicular atroi^hy is said to occur in 70 per cent of men who have cirrhosis of the liver (Bennett, Baggenstoss and Butt, 1951). There is no critical information from which one could conclude that the atrophy of the testis in cirrhotic men is caused by failure of the diseased liver to inactivate estrogen. XV. Nonneoplastic Disorders of the Testis Study of certain hypogonadal disorders of man has provided information of general interest and bearing on the physiology of the mammalian testis. For an index to the large clinical literature on pituitary-testis relationships, the reader may consult Heller and Nelson (1948) and Albert, Underdahl, Greene and Lorenz (1953-1955). A group of spontaneously occurring disorders shows clearly the control of the testis by gonado- trophin. In pituitary dwarfism, the testis remains infantile even as late as 30 or 40 years of age, and perhaps for the entire life span of tlie individual so afflicted. Leydig cells are not jirescnt, and the tubules con- tain only undifferentiated cells and occa- sional spermatogonia. Pituitary dwarfism is a form of hypopituitarism in which all hor- mones of the anterior lobe may be absent. Anotiiei' type of hyi)ogonadism in man is restricted to the loss of only the gonado- trophic function of the pituitary. In this syndrome, the testis does not contain ma- ture Leydig cells or mature tul)ules. This syndrome represents a condition that can- not be duplicated in lower animals. A few instances of a selective type of gonado- tropliir insuflficiency have been described in which tubular maturation proceeds, with (liffei'entiation of the Sei'toli cells and the formation of sperm. However, Leydig cells are not present. This syndrome ("fertile eunuchs"), if interpreted in terms of the dualistic concept of pituitary control of the testis, is explainable on the basis that for- mation and secretion of FSH have occurred but that LH is absent. If pituitary lesions occur before puberty, the testes remain im- mature. Pituitary lesions occurring after maturity cause atrophy of the seminiferous epithelium, not immaturity. The adult tu- bule of man cannot dedifferentiate as does the mature Leydig cell following hypo- l^hysial deprivation. The atrophy may vary in severity from hypospermatogenesis to complete sclerosis. Lack of gonadotrophin in the adult also results in thickening of the tubular wall and atroph}^ of the Leydig cells. The most common defect in the human testis is failure of the seminiferous tubules. In contrast to the pituitary deficiencies, which generally result in both tubular and androgenic failure, disorders of sj^ermato- genesis lead only to infertility. The Leydig cells are normal, and androgenic function is unimpaired. The disordered spermatogenesis and the presence of cellular debris in the lumen are reflected by an abnormal spermo- gram. Depression of the sperm count to the point of azoos]M'rmia, abnormal sperm cells, and poor motility are characteristic find- ings. Another type of primary testicular dis- order associated with azoospermia is germi- nal aplasia, in which the tubules contain only Sertoli cells. The Leydig cells are nor- mal; hence, androgenic function is normal. Klinefelter's syndrome also is associated with azoospermia but the function of the Leydig cells is variable, ranging from se- vere insufficiency, in which the afflicted per- sons are eunuchoidal, to mild insufficiency, in which the liabitus is normal or almost so. Testicular disorders are not restricted to man. They occur in common laboratory ani- mals and in veterinary practice. Their simi- larity to some of the clinical entities just described will be evident. A genito-urinary abnormality occurs in 20 per cent of males of the A x C rat (Vilar and H(n-tz, 1958). On one side, the testis is atroi)hic and the kidney, ureter, ductus deferens, epididymis, and seminal vesicle are absent; however, the coagulating gland is |)r('scnt. The testis is noi'mal ])i'epul)ertally MAMMALIAN TESTIS 349 u}) to 10 days of age. The lumenlcss tubules contain two types of cells; one is a small cell with one nucleolus; the other is a large round cell containing two or three nucleoli. Oval cells resembling Leydig cells are pres- ent in the interstitium. At 19 to 24 days of age, both testes are ecjual in weight. The diameter of the tubules increases, a lumen is present, and the tubular wall becomes dif- ferentiated. Sertoli cells, spermatogonia, and spermatocytes are evident, and the Leydig cells are maturing. At 30 to 38 days of age, the testis on the abnormal side is noticeably smaller. The Leydig cells remain normal, but the tubules are decreased in size. Be- tween 45 and 47 days of age, spermatogene- sis ceases and the tubules become atrophic. Thick collagenous and elastic fibers are found in the tubular wall. This disorder seems to be an inherited defect with delayed somatic manifestations. In some aspects, the pathogenesis of this testicular disorder in rats resembles that in Klinefelter's syn- drome. Congenital spermatogenic hypoplasia oc- curs in guinea pigs (Jakway and Young, 1958) . It ranges from germinal aplasia in most of the seminiferous tubules to a condi- tion in which the appearance of the tubules is almost normal and the percentage of fer- tile matings is only slightly reduced. When sterility is present, the testes are smaller than those of normal males. The hormonal production, as reflected by the size of the penis and seminal vesicles and by sexual behavior, is normal. The mule has a J-shaped chromosome which is contributed by the ass (Makino, 1955). Spermatogenesis in the mule does not proceed beyond meiotic prophase, de- generation occurring without formation of the metaphase of the first division. Hence, sperm cells will not form. The testes become atroi^hic, and only a few^ spermatogonia re- main. The Leydig cells are normal. Different types of hypogonadism, some of which are inherited, are encountered in bulls. Hypoplasia associated wuth urate crystals in the semen probably results from disintegration of the seminiferous epithelium (Barron and Haq, 1948) . Idiopathic necrosis of the tubule also may cause massive tes- ticular calcification (Barker, 1956). Seven cases of hypogonadism in Belgian bulls were reported as a form of congenital sterility (Derivaux, Bienfait and Peers, 1955) ; pho- tomicrographs of the testes in these cases are similar to those of germinal aplasia in the human. Testicular hypoplasia occurs also in goats (Rollinson, 1950). Captive wild animals become sterile. Bushman, the famous gorilla at the Chicago Zoo, died at the age of approximately 22 years. Necropsy revealed neuropathy, car- diopathy, hemosiderosis, and testicular scle- rosis (Steiner, Rasmussen and Fisher, 1955). No cells of the germinal epithelium were present except occasional Sertoli cells. The Leydig cells were normal. The testicular atrophy of Bushman was similar to that of Bobby, at the Berlin Zoo. Whether this de- generative testicular lesion is caused by nu- tritional deficiency or by the "stress" of captivity is not known. XVI. Tumors of the Testis Testicular tumors are more common among lower animals than in man (Innes, 1942). Spontaneously occurring Sertoli-cell and Leydig-cell tumors of animals have been studied more than seminomas pre- sumably because of the greater endocrino- logic interest attached to them. Huggins and Pazos (1945) found 64 testicular tumors in 41 dogs; of these, 33 were Leydig-cell tu- mors, 19 were seminomas, 9 were tubular adenomas, and 3 were undifferentiated tu- mors. Zuckerman and McKeown (1938) found tumors in 35 of 243 dogs. A few of these were Sertoli-cell tumors which were associated with metaplasia of the prostate. The life span of dogs varies from 8 to 15 years, and testicular tumors occur most fre- quently at 7 years of age or older; in fact, more than half of old dogs are found to have such tumors (Scully and Coffin, 1952). The most common tumor of the dog testis is a Leydig-cell tumor. Five per cent of testicu- lar tumors in dogs occur in undescended testes. The neoplasms in cryptorchid testes are usually Sertoli-cell tumors (Greulich and Burford, 1936; Coffin, Munson and Scully, 1952; Mulligan, 1944). The veterinary diagnosis (Blum, 1954) of Sertoli-cell tumors is easily made, because the dogs become feminized. For this reason, the chief comjilaint of the owners is tlutt normal male dogs, after a brief olfaciMi} 350 PHYSIOLOGY OF GONADS reconnaissance, attempt to mount their af- flicted pets. In addition to the feminization, evidence that Sertoli-cell tumors produce estrogen comes from the finding of estrogen in the urine of tumor-bearing animals and from the extraction of estrogen from the tumor itself (Berthrong, Goodwin and Scott, 1949). In terms of estradiol, the concentra- tion of estrogen extracted from a Sertoli-cell tumor (Huggins and Moulder, 1945) was twice that found in the ovary from an es- trous bitch. Sufficient estrogen appears to be produced to cause such changes as loss of hair, depression of libido, cystic hyperplasia of the mammary glands, and atrophy of the testis. Interstitial cell tumors in dogs are usu- ally nonfunctional, but they may produce estrogen (Laufer and Sulman, 1956; Kahan, 1955). Leydig-cell tumors have been re- ported in the mule, the Brahma bull, and the saddle horse (Smith, 1954). Signifi- cantly, in the last instance, an interstitial cell tumor occurred in the undescended tes- tis of a 7-year-old horse, the descended tes- tis having been removed early in life. In man the proportion of various types of testicular tumors is different from that in lower animals. Seminomas and embryonal carcinomas are the most frequent neo- plasms. Interstitial cell tumors have been recorded in less than two dozen instances in the world literature. Several cases of Ley- dig-cell tumor have been studied by Ven- ning (1942) , Cook, Gross, Landing and Zyg- muntowicz (1952), Hertz, Cohen, Lewis and Firminger (1953), and Jungck, Thrash, Ohl- macher, Knight and Dyrenforth (1957). This tumor causes isosexual precocity in boys. Signs of androgenic activity are evi- dent in the large penis ; scrotal maturation ; the appearance of pubic, facial, and axillary hair, and acne; increased bodily growth; maturation of the larynx; and increased excretion of 17-ketosteroids. All these find- ings occur when sufficient amounts of testosterone are injected into normal pre- pubertal boys. This tumor cannot conceiva- bly be related to the secretion of LH (see subsequent material on experimental tu- mors), because the neoplasms are usually unilateral and the contralateral normal tes- tis shows no activation of the Leydig cells. Neoplasms classified as Sertoli-cell tu- mors are rich in lipids and are thought to secrete estrogen (Teilum, 1950). However, the histogenesis of these tumors is not clear, and there is doubt that Sertoli-cell tumors actually occur in man. Testicular tumors have been induced in rats by transplantation of immature testes to the spleen of castrated adult animals (Biskind and Biskind, 1945) and by radia- tion, carcinogens, and other means (Peyron and Samsonoff', 1941). Transplantation of day-old rat testes to the spleen of castrated adult rats, normal male rats, and castrated adult female rats resulted in the formation of encapsulated and sharply circumscribed tumors. Of 29 tumors thus produced, 16 were composed entirely of interstitial cells and 13 contained other testicular elements as well. One of the tumors was transplant- able into the spleen of a castrated animal. Because hyperplasia of the interstitial cells was seen in most of the transplanted testes, it was thought that the neoplasia followed the hyperplasia induced by the excess of gonadotrophin in the castrated host (Twombly, Meisel and Stout, 1949). Such Leydig-cell tumors produce estrogen (Fels and Bur, 1956). In contrast with the rat, experimental tu- mors in the mouse are not induced by any of the methods already mentioned (Gardner, 1953). Spontaneous tumors of the testis in mice do occur, however. Slye, Holmes and Wells (1919) found 28 testicular tumors in some 9000 male mice. None formed meta- static lesions. Hummel (1954) reported a spontaneous tumor in an 18-month-old mouse of the BALBC strain; this neoplasm was transplantable for three generations in normal or gonadectomized adult males or females. This was a functioning tumor as evidenced by masculinization of the sub- maxillary glands, mucification of the vagina, hypertrophy of the clitoris, and an increase in size of the uterus of the female host and of the accessory sex organs of the male host. All these findings indicate estrogenic and androgenic secretion. In general, however, interstitial cell tumors in mice are strain- limited, occurring particularly in the AC and JK strains. Spontaneous interstitial cell tumors also occur in hybrids and are as- sociated with mammary tumors (Gardner, Pfeiffcr, Trentin and Wolstenholme, 1953). MAMMALIAN TESTIS 351 This association indicates that estrogen is involved in the formation of the tumor; in- deed, it is chiefly by the use of estrogen that experimental tumors in mice have been pro- voked. Various natural and synthetic estrogens are effective. For example, Hooker, Gardner and Pfeiffer (1940) and Hooker and Pfeiffer (1942) using estradiol and stilbestrol have been able to produce interstitial cell tu- mors in the A and C strains of mice, with an incidence of 50 and 90 per cent respec- tively. Treatment for 8 months with 16.6 to 50 fjig. of estradiol dibenzoate or 0.25 /xg. stilbestrol weekly produces tumors, some of which metastasize to the renal, lumbar, and mediastinal lymph nodes. These tumors are transplantable if the hosts are given estro- gen. They are inhibited by the simultaneous injection of testosterone. Tumors also may be induced by implantation of pellets of stilbestrol and cholesterol. The implantation of a 4- to 6-mg. pellet of 10 to 25 per cent stilbestrol in cholesterol induced tumors within 5 months (Shimkin, Grady and An- dervont, 1941). Of the various natural and synthetic estrogens the triphenylethylene derivatives appear to be the most potent. Bonser (1942) and Gardner (1943) pro- duced transplantable tumors in the JK, the A, and the C 3H strains by triphenyl- ethylene. Tumors thus induced are gen- erally composed of interstitial cells. They are transplantable only in the same strain of mice and only when the hosts are given estrogen. After several generations, how- ever, the tumor may be transplanted with- out administration of estrogen in normal and in hypophysectomized mice (Gardner, 1945; Andervont, Shimkin and Canter, 1957). The tumors arise from hyperplastic inter- stitial cells. The Leydig cells enlarge, be- come foamy, and degenerate. JMacrophages or, at least, cells containing a brown pig- ment appear and phagocytose the exhausted Leydig cells. A new crop of interstitial cells appears from the mesenchyme. These may grow faster in one zone than in another. The faster-growing Leydig cells thus constitute a nodule. The Leydig cells in the nodule also become hyperplastic and foamy. These nod- ules appear as white spots and cause pres- sure atrophy of the tubules. Leydig cells in the tumor thus result from three genera- tions, since the second crop of Leydig cells is followed by a third generation containing small primitive and hyperchromatic cells. These contain brown pigment and hence give the brown color to the tumor. At this stage, the tumor may become necrotic, may metastasize by way of lymph or blood, or may invade locally. Such tumors secrete both estrogen and androgen. The consensus is that estrogen induces interstitial cell tu- mors in mice by liberation of LH (Gardner, 1953). The assumption that LH induces inter- stitial cell hyperplasia and finally a tumor has received support from studies by Simp- son and van Wagenen (1954) on young monkeys. These investigators gave ICSH for 53 days. Hyperplasia of the Leydig cells took place and nodules resulted. These nod- ules were composed of concentric laminated peritubular cells and arose from the same type of mesenchymal cell that yields the Leydig cell under normal conditions. Under the influence of HCG, the nodules secreted androgen. XVII. Conclusion The postnatal development of the mam- malian testis follows a fairly definite pat- tern. Development is slow for the variable period of prepubertal life. The testis then undergoes rapid evolution during puberty, remains fairly constant in adult life, then regresses somewhat in old age. The rapid de- velopment of the testis during puberty is brought about by the onset of gonadotrophic function of the pituitary. This develop- mental pattern is fixed for each species, but can be modified by genie and environmental factors. Once the adult status is attained, secretory controls of androgenic and sper- matogenic functions are established. A steady state of testicular function is main- tained in continuously breeding species. In those mammals which show a seasonal breeding cycle, these secretory controls, par- ticularly those of the pituitary gland, are periodically activated and deactivated. The testes of many eutherian mammals migrate from the abdomen during fetal life to the scrotum. This migration is regulated by hormones of the fetus, presumably aris- ing from the fetal testis. It is not clear just 352 PHYSIOLOGY OF GONADS why the testes occupy the scrotum. The ex- planation that scrotal residence provides "optimal testicular temperature" is not sat- isfying because one then wishes to know why the male gonad requires the cooler en- vironment afforded by the scrotum. Failure of the testes to descend may occur as a consequence of defects in the testes, prob- ably of genie origin ; or because of anatomic obstacles, representing embryologic defects, inadvertently placed along its prescribed narrow path. In either event, the testis is damaged, mildly in its endocrine function, and seriously in its spermatogenic function. Impairment of spermatogenesis of the misplaced testis is due to the relatively high temperature of the abdomen. Temperature affects the germinal epithelium directly. It also affects the testis indirectly through the circulatory system. The effect of tempera- ture, or for that matter, of any type of in- jurious agent whether it be chemical or physical, is atrophy of the seminiferous epithelium. The response of the germinal tissue to deprivation of pituitary gonado- trophin likewise is atrophy. Quantitative variation among different species does of course exist, but qualitatively, atrophy is the universal response to injury. Obviously, a common denominator must exist for this fairly general reaction on the part of the germinal epithelium. If various chemical and physical stimuli act on the testis by means of suppression or interference with the action of gonadotrophins, atrophy of the Leydig cells would also result. However, many chemical and physical agents affect only the germinal epithelium, leaving the Leydig cells unscathed. Thus, the germinal epithelium can be damaged directly and the variable damage to the components of the spermatogenic epithelium must be due to different sensitivities of its cellular com- ponents. The Sertoli cell is much more re- sistant than the cells of the germinal line, and of the seminiferous elements, the type A spermatogonia are the most resistant. Of great importance in the interpretation of the damage induced by many substances or occurring as a result of disease is the char- acteristic of the germinal epithelium to reproduce in a fixed order and sequence. It follows that the extent of injury to sper- matogenesis as a whole would 1)C determined by the relative susceptibility of the various germinal cells as well as by the nature of the noxious agent. If only sperm cells are affected, spermatogenesis will proceed through the formation of spermatid. How- ever, if spermatogonia are injured, full dif- ferentiation of the germinal epithelium will fail, and only Sertoli cells will be found in the tubule. Thus, it is possible that all sorts of injury to the testis, if sufficiently great, may result in the same end stage of tes- ticular atrophy. In spite of this common reaction pattern to severe injury, many sub- stances induce what seem to be specific le- sions in the testis. However, these represent intermediate or partial injuries, and do not necessarily constitute exceptions to the gen- eral pattern of testicular response to injury. As more is learned about the biochemistry of the germinal epithelium, it may be pos- sible to induce specific lesions. Quantitative studies on spermatogenesis have greatly clarified the role played by the pituitary gland. Spermatogenesis does proceed in hypophysectomized animals but only at a low rate. Also it appears that an- drogen, not gonadotrophin, is responsible for the maturation of the spermatid. How- ever, it must be remembered that the forma- tion of androgen is dependent on pituitary gonadotrophic function. Thus spermato- genesis is regulated entirely by pituitary gonadotrophins, which exert direct super- vision over the rate of the mitotic and meiotic activity of germ cells and indirect supervision by way of the Leydig cell over spermatid maturation, or spermiogenesis. The effectiveness of androgen in sperm for- mation is hardly equal to that of the pitui- tary. Addition of trophic hormones (except gonadotrophin) or of hormones of the target glands (tliyi'oid, adrenal cortical hormone, etc. I will ])robably not improve the ef- fectiveness of androgen. The best evidence that this surmise may be correct is obtained from jnitients with hypogonadotroi)hic hy- l)ogonadism. These i)atients have normal function with respect to the other trophic hormones of the pituitary and, therefore, normally functioning peripheral glands, but do not have sperm. The quantitative studies on the spermato- genic cycle have important bearing on other |)i'o]»lciiis wliicli have been i)uzzling to endo- MAMMALIAN TESTIS 353 crinologists. jMany unsuccessful attempts have been made to induce precocious sperm formation in the rat by chronic or massive use of various gonadotrophins. The time of a complete spermatogenic cycle is not ac- curately known. Estimates ranging from 20 to 40 days have been given, which reflects the difficulties and errors of present meth- ods. If one adds to the time at which sperm formation normally occurs in common strains of the laboratory rat (around 35 days of age ) , about 10 days borrowed from fetal life, the time of a complete spermato- genic cycle is probably between 45 and 50 days. Hence, no amount of exogenous gonadotrophin could be expected to produce precocious spermatogenesis, because a cer- tain irreducible minimum of time may be recjuired for the series of divisions which in toto constitutes a spermatogenic cycle. However, if the interval between birth and maturity is much longer than the time of a complete spermatogenic cycle, precocious spermatogenesis could be experimentally achieved, as is again indicated by an ex- ample from clinical endocrinology, i.e., the spontaneous occurrence of isosexual pre- cocity in boys. In another clinical area, the application of quantitative techniques to the study of testes of iKitients afflicted with infertility has so far not yielded helpful information. Restoration of fertility in men with adult seminiferous tubular failure has not been accomplished. Infertility, however, is re- ceiving increasing attention, especially from the standpoint of genie factors. It is in this area that the only startling development of knowledge on the testis in the past 20 years has occurred, i.e., the discovery that men with Klinefelter's syndrome are "genetic fe- males." One may, with good reason, ques- tion the suitability of the term "genetic fe- males." It arose from the application of Barr's discovery of sex dimorphism in the heterochromatin of somatic cells (Barr, 1956; Barr and Bertram, 1949; Moore and Barr, 1955) . Normal females are "chromatin positive"; normal males are "chromatin negative." This, however, may not be ab- solute. Men with Klinefelter's syndrome are chromatin positive, and if chromatin posi- tivity reflects genie constitution, it is likely that the sterility of men with this syndrome (one of its outstanding features) represents an abnormality of chromosomal division or number during gametogenesis of one of their parents. Generally similar situations may occur in lower animals; hence, the role of genie factors in fertility can be studied experimentally. Great advances have taken place in knowledge of the biosynthesis of male hor- mone by the testis. Illumination of the chemical pathway over which simple pre- cursors (acetate) or more complex ones (cholesterol) are transformed to testoster- one represents a major contribution in bio- chemistry. The enzymatic control of the various chemical steps will undoulitedly be disclosed before long. XVIII. References Albert, A., Underdahl, L. O., Greene, L. F., and LoRENz, N. 1953a. Male hypogonadism. I. The normal testis. Proc. 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Path. & Bact., 46, 7. \y^^[' J^ 6 THE ACCESSORY REPRODUCTIVE GLANDS OF MAMMALS Dorothy Price, Ph.D. PROFESSOR OF ZOOLOGY, THE UNIVERSITY OF CHICAGO and H. Guy Williams- Ashmari, Ph.D. ASSOCIATE PROFESSOR, BEN MAY LABORATORY, THE UNIVERSITY OF CHICAGO L Gross Structure, Homologies, and I. Gross Structure, Homologies, and Occurrence in Mammalian Orders. 366 Occurrence in Mammalian A. Introduction c566 „ , B. General Characteristics 367 Ureters C. Survey of the Glands 368 j^ INTRODUCTION 1. Bulbo-urethral and bulbovestibu- ^, • , , ^ , , la,!- 368 The genital system of male mammals con- 2. Male and female prostate glands. . 369 sists of three component parts. These are: 3. Seminal vesicles 376 ( 1) paired testes, the primary sex organs in 4. Ampullary glands ■ • ■ • ■ 376 ^^^j^j^j-, spermatozoa are formed and andro- U. Evolutionarv Historv of Accessory • i j. j /ov Reproductive Glands in Mammals 376 g^nic hormones are secreted; (2) accessory II. Function ofMale Accessory Glands.. 377 reproductive organs, a continuous series of A. Introduction 377 ducts in which spermatozoa are transported B. Volumetric Studies of Secretion 378 fpo^ the testes, stored in the tail of the epi- 1. Prostatic isolation operation^. 378 .^^^^ ■ ^nd finallv carried to the exterior 2. Prostatic translocation operation. 380 , - . , ,. ' , C. Chemical Composition of the Glandu- ^^hen ejaculation occurs, and various lar Secretions 380 glands, the secretions of which provide the I). Metabolism of the Prostate and Sem- carrying medium for the spermatozoa at inal Vesicle 394 emission ; ( 3) external genitalia, the penis K. Coagulation of Semen 396 , , , . , , III. Structure and Function in Relation ^r copuhitory organ and, m most mammals, TO Hormones 398 i^ scrotum in which the testes come to lie A. Introduction 398 more or less permanently, or only periodi- B. Effects of Androgens 399 p.^Hy during the breeding season. 1. Testicular andn,gens 399 ^^ ^^jj^ -^ ^j^^ epithelial lining of the 2. Adrenal androgens 423 / . i i j r l ^ c 3. Ovarian androgens 424 ^ttcrent, epididymal, and deferent parts of 4. Progesterone 425 the duct system have secretory functions, C. Effects of Estrogens 426 but all male mammals develop discrete and I). Hormonal Control ()f Spontaneous ^^^ specialized glands which are associated with 1. Benign growtU.''""^ '.'.'.'.'.'.'.'.'.'.'.'.'.'. 429 ^Pecific regions of the reproductive tract and 2. Prostatic cancer 430 eject their secretions into it at seminal E. I'^ffects of Carcinogenic Aromatic Hy emission. The degree of development of drocarbons 430 these large, conspicuous glands is a unique F. Effects of Nonsteroid Hormones 433 .ij^r^cteristic of mammals. 1. I'rolactin (LTH) 433 _, i x- i j u 2. Growth hormone (STH) 434 The accessory reproductive glands can be IV. References 435 grouped logically into those ^vhich arise 366 ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 367 embryonically from the mesonephric or Wolffian duct (ductus deferens) i.e., the ampullary glands (glandula vasis defer- entis) and seminal vesicles or vesicular glands, and those deriving from the uro- genital sinus or urethra, namely the pros- tate and bulbo-urethral or Cowper's glands (see chapter by Burns). The anatomic re- lationships established in the fetus are re- tained to a considerable degree postnatally so that the ampullary glands and seminal vesicles are associated with the ducti defer- entes. However, in some mammals the semi- nal vesicles empty into the pelvic urethra close to the openings of the deferent ducts but separate from them; no ejaculatory ducts are present. The prostatic and bulbo- urethral glands are associated with the proximal and distal urethra, respectively. The secretion of the prostate is discharged, in most cases, through multiple ducts that join the prostatic urethra at the level of the colliculus seminalis. The ducts of the bulbo-urethral glands drain into the urethra in the region of the urethral bulb. In addition to these accessory reproduc- tive glands, there are small mucus-secreting glands (of Littre) opening into the urethra along its length, and preputial glands (which are modified sebaceous glands) emi)tying their secretion on the prepuce. In many female mammals, homologues of the male prostate and bulbo-urethral glands develop in the fetus. These glands may ret- rogress prenatally, remain vestigial, or de- velop postnatally and become functionally active. These homologues are the female prostate glands (para-urethral glands of Skene) and the bulbovestibular (major vestibular or Bartholin's glands). In addi- tion, there are urethral glands (minor ves- tibular) which are homologous with the male urethral glands of Littre, and female preputial or clitoridal glands corresponding to the male preputials. The major vestibu- lar, when present, and the minor vestibular and clitoridal glands are functional in many mature females. In a few cases, well devel- oped prostate glands which are actively se- cretory have been found in females of four mammalian orders. B. GENERAL CHARACTERISTICS The male accessory reproductive glands of higher mammals have many character- istics in common. Typically, all possess (1) a secretory epithelium which is enormously increased in effective secretory area by vil- lous infoldings, or by a compound tubulo- alveolar structure, (2) an underlying layer of connective tissue (the lamina propria) and (3) smooth muscle fibers. It is now well established that the secretory activity of the epithelial cells is normally under the control of testicular hormones. The secretions pass from the cells into the lumina of the glandu- lar alveoli where they are usually stored until ejaculation. The sensory innervation includes various types of sensory nerve endings in the con- nective tissue, and free nerve endings in the epithelium. The autonomic innervation is parasympathetic (nervi erigentes) and sympathetic (hypogastric nerve) from the pelvic plexus. If the plexus is resected or the sympathetic chain above is interrupted, there is no reflex ejection of the glandular secretions. When the hypogastric nerve is stimulated, peristaltic waves of contraction occur in the ductus deferens, and there is contraction in the seminal vesicles and pros- tate which partially empties the stored se- cretion from the lumina of these glands. Stimulation of the parasympathetic system or the administration of pilocarpine results in an increased output of prostatic secre- tion. There are marked dissimilarities in gross structure, character of the epithelia, and the chemical nature of the secretions in the various glands — ^prostates, seminal vesicles, bulbo-urethral, and ampullary (Mann, 19o4a). There are also differences in struc- ture and function between homologous glands in related forms. The nomenclature that was applied to the glands in early de- scriptive studies was often based on ana- tomic relationships and gross morphologic structure in adults. This resulted in some confusion in classification, but most of the disputed points have been clarified and some of the homologies have been estab- lished by embryologic study. The extensive studies of INIann (1954a) show clearly that 368 PHYSIOLOGY OF GONADS TABLE 6.1 Occurrence of male accessory reproductive glands and their homologues in females" Male*- Female<^ Order Bu Pr Sv Am Bv Pr Genera and species with functioning female prostates Monotremata . . . + _? _ _ + _ Marsupiala + + - - + - Insectivora + + ± ± + + Erinaceoii.s europeus (Deanesly, 193-4) Hemicentetes (Lehmann, 1938) Talpa europea (Godet, 1949) Chiropt era + + ± ± + =t Coelura afra (Mathews, 1941) Taphozous sp. (Mathews, 1941) Nycteris luteola (Mathews, 1941) Carioderma cor (Mathews, 1941) Primates + + ± =b + — Carnivora ± + — zb ± — Perissodactyla... + + + + + _ Artiodactyla .... + + + ± + — Hyracoidea + + + — Proboscidea + + + + Sirenia — + + — — Cetacea — + — Edentata ± + + — Pholidota — + + — Rodentia + + + =t ± ± Arvicanthus cinereus (Rant her, 1909) Rattus norvegicus (Marx, 1931, 1932; Korenchevsky and Dennison, 1936; Korenchevsky, 1937; Witschi, Mahoney and Riley, 1938; Price, 1939; Mahoney, 1940, 1942; Mahoney and Witschi, 1947) Mastomys erythroleucus (Brambell and Davis, 1940) Apodemus sylvaticus (Raynaud, 1942, 1945) Microtus arvalis (Delost, i953a, 1953b) Lagomorpha. . . . + + ± + + ± Sylvilagus floridanus (Elschlepp, 1952) "Compiled from Oudemans, 1892; Engle, 1926a; Retief, 1949; Eckstein and Zuckerman, 1956 and others. + indicates the presence of a well developed functioning gland. — indicates either a small ves- tigial gland or the absence of any rudiment. ^ Bulbo-urethral (Cowper's), prostate, seminal vesicle and ampuUary glands. "^ Bulbovestibular (Bartholin's) and prostate glands (para-urethral glands of Skene); genera and species refer only to those in which functioning female prostates have been reported in the listed ref- erences. homologous organs do not necessarily have the same chemical functions. Finally, there is variability among orders of mannnals and families within orders, with respect to the accessory glands which are present (Table 6.1). The prostate is the only u;land that is found almost universally. C. SURVEY OF THE GL.4NDS 1. Bulbo-urethral Glands and Bulbovestihidar Bulbo-urethral (Cow^per'.s). The bulbo- urethral glands are compound tubulo-al- veolar glands resembling mucous glands in some respects. Their secretion is a viscid lubricant which is em])tied into the bulbar region of the pelvic urethra. There may be a single pair of glands as in the monotremes, primates, and rodents, or as many as three pairs (Fig. 6.1), as in some marsupials (Chase, 1939; Rubin, 1944). Their relative size, gross structure, and complexity vary widely. For example, they are small, com- pact, bean-shaped glands in man, relatively enormous, complicated glands in squirrels, and large, cylindrical glands in the boar. Bulbo-urethral glands are notably lacking in Cetacea, Sirenia, and certain carnivores such as seals, walruses, sea lions, all rauste- lids, and the bear and dog (Oudemans, 1892; Engle, 1926a; Eckstein and Zuckerman, 1956). (Oudemans made a point of the fact that tliey are not present in aciuatic mam- ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 369 mals, but it is rather doubtful if their ab- sence is related to an aquatic environment. BuLBOVESTiBULAR (Bartholin's) . The bulbovestibular or major vestibular glands are also compound tubulo-alveolar glands which resemble their male homologues in structure and secrete a mucus-like sub- stance. Their secretory function is under control of ovarian hormones and they in- volute when the ovaries are removed. They are widely distributed in the various orders of mammals although the information is fragmentary with respect to some groups. A single pair of glands is the general rule and they are usually much smaller than the bulbo-urethral. In the female opossum, the single pair of glands is homologous with the smallest of the three pairs of Cowper's glands. In the adult, they are well developed and filled with colloid (Rubin, 1944). In monotremes the ducts open at the base of the clitoris, in opossums into the urogenital sinus canal, and in hyenas (where they are well developed) into the urogenital canal close to the base of the clitoris (Eckstein and Zuckerman, 1956). In many other fe- males the ducts open into the vestibule. In the adult human female, Bartholin's glands resem])le Cowper's glands closely in histo- logic structure. 2. Male and Female Prostate Glands Male prostate. The prostate is a com- pound tubulo-alveolar gland in which the gross structure is variable and may be (1) disseminate or diffuse, in which the glandu- lar acini remain within the lamina propria around the urethra and do not penetrate the voluntary muscle of the urethra, (2) a type in which the gland forms a '"body," sometimes lobed, outside the urethral muscle, or (3) a combination of both types. A disseminate prostate is found in some marsupials (Fig. 6.1) and edentates, and in sheep, goats, the hippopotamus, and the whale. The bull and boar prostates have a disseminate region as well as a discrete body of the gland. In mammals in which there is a glandular body, there may be a solid, compact prostate as in the dog and man, or several lobes as in rodents (Figs. 6.2 and 6.3), lagomorphs (Fig. 6.4), and insectivores. Fig. 6.L Male opossum reproductive tract. B, bladder; C, Cowper's glands; D, ductus deferens; E, epididymis; P, penis; Pr, prostate I, II, III sur- rounding the urethra; T, Testis; U, ureter. (Re- drawn from C. R. Moore, Phj'siol. ZooL, 14, 1-45, 194L) A prostate gland has been found in all mammals that have been studied except monotremes, and is the only accessory gland in carnivores such as the ferret, weasel, dog, and bear, and in cetaceans — whales, dol- phins, and porpoises. Oudemans (1892) con- sidered that monotremes and marsupials lack prostate glands but possess well de- veloped urethral glands. His classification of glands as "urethral" (glands of Littrei or "prostatic" depended on whether the glandular acini remained in the urethral stroma or penetrated the muscle to form a 370 PHYSIOLOGY OF GONADS COAGULATING GLANDS SEMINAL VESICLE SEMINAL VESICLE ■1%.^ AMPULLARY GLAND DORSAL PR0STATE VENTRAL PROSTATE Fic. 6.2. Mule hamster accessory repidiluri i aspect. body outside. It is now recognized that marsupials such as the opossum have a dis- seminate prostate, and in Didelphys Virgini- an a there are three regions which differ 5EMINAL VESICLE .- RIGHT DORSAL PROSTATE ^VENTRAL PROSTATE gl.-inds. Above, ventral aspect; below, dor.sal clearly in histologic structure (Chase, 1939). It may be considered that there are three prostatic "lobes" probably differing in func- tion as well as in structure. Although Oude- ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 371 — RIGHT SEMINAL VESICLE- COAGULATING GLAND DORSAl PROSTATE LATERAL DUCTUS -DEFERENS BLADDER URETHRA Fig. 6.3. Male guinea pig accessory reproductive glands. (From E. Ortiz, D. Price, H. G. Williams-Ashman and J. Banks, Endocrinology, 59, 479-492, 1956.) mans concluded that monotremes lack pros- tate glands, he described a concentration of urethral glands at the neck of the bladder in the duckbill platypus. Ornithorhyncus poradoxicus. The diagrams in his mono- graph suggest that this concentration of complicated glands is a disseminate type of prostate. There has been confusion in the nomen- clature of the lobes of the prostate in the rat and in the descriptions of the structure of the lobes. In early studies the application of human anatomic terminology to rodents resulted in designation of the lobes as ante- rior (ventral), middle, and posterior (dor- sal). Later terminology, more suitable for cfuadrupedal animals, led to anterior (cra- nial), middle, and posterior (caudal). Un- fortunately, combinations of these two sys- tems of nomenclature still occur in the literature, and there is uncertainty as to the number of histologically distinguishable re- gions or lobes. In view of the current inter- est in the chemical composition of the glands and their secretions the subject will be reviewed. For many years the prostate was usually described as being composed of three pairs of lobes: cranial or anterior (coagulating glands) bound to the seminal vesicles; mid- dle or dorsolateral nearly encircling the urethra dorsolaterally, and the ^-entral or 372 PHYSIOLOGY OF GONADS Fig. 6.4. Rabbit accessory reproductive glands; lateral aspect. Left, domestic male; center, cottontail male; right, cottontail female. B, bulbo-urethral gland; C, coagulating gland (vesic- ular gland); D, ductus deferens; P, paraprostate ; Pr, prostate; V , urethra; VC, urogenital canal (vestibulum) ; V, vagina. (Redrawn from J. G. Elschlepp, J. Morphol., 91, 169-198, 1952.) lio.sterior (Moore, Price and Gallagher, 1930; Callow and Deanesly, 1935; Price, 1936). Korenchevsky and Dennison (1935) noted that the histologic structure of the dorsal lobe (or region) is quite similar to that of the coagulating glands whereas the lateral lobes more nearly resemble the ven- tral. This has been confirmed in histologic and functional studies (Price, Mann and Lutwak-Mann, 1955). Gunn and Gould (1957a) reported differences in histologic structure and functional activity in the two lobes. The lateral lobes can be distinguished grossly from the dorsal by anatomic rela- tionships and color, but the glandular lo- bules form a continuous mass and can be separated into distinct lateral and dorsal lobes only by dissection. This can be accom- plished with considerable accuracy in im- mature males and young adults; in large rats it is more difficult because of distention of the alveoli and overlap of lobules in contiguous regions. The ventral tips of the lateral lobes extend down and partially underlie the ventral lobes to which they are loosely bovmd. The dorsal prostate is some- what butterfly-shaped with a single cranial region and wings extending caudally along the urethra much as in the hamster (Fig. 6.2). By dissection in the midline, it can be divided into right and left lobes. The dorsal and lateral prostates are drained by 50 or more ducts opening into the roof of the pros- tatic urethra (Witschi, Mahoney and Riley, 1938). Those from the dorsal region open more dorsally; those from the lateral lobes, laterodorsally. Some of the confusion in prostatic termi- nology arises from the general application of the word "lobe" to (1) organs that are grossly anatomically distinct, (2) regions that do not form entirely discrete structures but can be distinguished histologically, (3) ])arts of the gland which contain two histo- logically different portions, and (4) regions that differ, not in histologic structure but in response to hormones and in the tendency to ])athologic growths (human and dog). The lobation of the human prostate has been the subject of controversy for some time. It is of especial interest because one region, the posterior or dorsal lobe, is com- monly the site for prostatic carcinoma and another, the more anterior or ventral region, for benign prostatic hypertrophy. The lobes have been described as posterior, anterior, middle, and two lateral, or as posterior and ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 373 anterior, or outer and inner (medullary). The component parts of these regions have been discussed extensively (see Moore, 1936; Huggins and Webster, 1948; Retief, 1949; Franks, 1954). Lowsley (1912) studied the embryologic development of the human prostate and concluded that the gland derives from five independent groups of tubules. A cranial posterior or dorsal group (lobe) arises from the dorsal wall of the prostatic urethra or urogenital sinus; right and left lateral lobes originate from the prostatic furrows and grow back to form the main part of the base of the gland; a middle lobe derives dorsally from the ure- thra between the bladder and ejaculatory ducts; a ventral or anterior lobe forms but regresses and becomes insignificant. Although these prostatic buds or tubules form independent groups in their embryonic origin there is no clear separation into such groups in the human prostate postnatally. However, Huggins and Webster (1948) were able to distinguish clearly two different re- gions, a posterior and an anterior lobe, by differential response to estrogen adminis- tration. The extent of the anterior or ventral lobe, as delimited by them, apparently in- cludes the tubules of the middle and lateral lobes as described by Lowsley. The pioneer studies of Walker (1910a) on the coagulating function of discrete glands of the prostatic complex in rats and guinea pigs (Fig. 6.3) were followed by specific identification of coagulating glands in sev- eral rodents including mice and hamsters (Fig. 6.2) and in the rhesus monkey (van Wagenen, 1936). However, a copulation plug in the vagina of females has been re- ported in some marsupials, insectivores, chiropterans, the chimpanzee among the primates (Tinklepaugh, 1930), and several genera of rodents in which coagulating glands have not been identified. Eadie (1948a) found that in an insectivore, Con- dylura cristata, there is a peculiar prostatic secretion from paired ventral lobes. It con- tains an enormous number of amyloid bodies resembling the corpora amylacea present in the prostate gland of man and some other mammals. These prostatic con- cretions are generally considered abnormal, but Eadie suggested that this unusual se- cretion, which was found in all breeding males, might be instrumental in the forma- tion of a unique type of copulation plug. A large ''urethral" gland which lies be- tween the prostate and bulbo-urethral glands and surrounds the urethra is peculiar to certain species of bats. Mathews (1941) considered it probable that the presence of this gland is correlated with the formation of a large copulation plug, but he did not ascribe a specific coagulating function to the gland (which bears a histologic resem- blance to the bulbo-urethral glands in some bats). The difficulties of homology and classifi- cation can be illustrated by the case of the rabbit. Differences of opinion have existed concerning the nomenclature and homolo- gies of the seminal vesicles (or prostatic utricle), vesicular glands (seminal vesicle or prostate), and paraprostate glands (or superior Cowper's glands). In studies on embryologic development and histologic structure, Bern and Krichesky (1943) clari- fied the problem. They established that the domestic rabbit has true seminal vesicles, vesicular glands (which are considered as probably homologous with the coagulating glands of rats), prostates, paraprostates (usually similar to the bulbo-urethrals in histologic structure but in about one-third of the cases, one or more of the parapros- tates resembled the prostate histologically), bulbo-urethral glands, and glandular am- pullae. Elschlepp (1952) compared the ac- cessory glands of the cottontail, Sylvilagus floridamis, with those of the domestic rab- bit, and concluded that coagulating glands (avoiding the usage of "vesicular glands" which has often been used synonymously with seminal vesicles) , dorsal prostates, and bulbo-urethral glands are homologous in the two species. The adult cottontail has neither paraprostates nor seminal vesicles (Fig. 6.4) . Classification of the glands in the hedgehog and shrew has also presented problems (see discussion in Eckstein and Zuckermann, 1956; Eadie, 1947). Among the Sciuridae, many possess a bulbar gland which differs from their true Cowper's glands (Mossman, Lawlah and Bradley, 1932). It is evident that among mammals 374 PHYSIOLOGY OF GONADS there are many potentialities for forming accessory glands with varied anatomic structure, histologic characteristics, and functional activities. Female prostate. In fetuses of many fe- male mammals, small cords of cells which represent the homologues of the male pros- tate bud off from the epithelial lining of the urethra. These primordia normally retro- gress or remain vestigial and only rarely continue to develop after birth. In the hu- man female, these rudimentary structures are known as para-urethral glands of Skene. They have also been referred to as peri- urethral glands. However, it seems advis- able, as Witschi, Mahoney and Riley (1938) suggested, to restrict the usage para-ure- thral and peri-urethral to the aggregations of mucus-secreting glands that have short ducts opening into the urethra. These clearly differ from the true female prostate glands. In contrast to the rudimentary prostate glands which are retained postnatally by some female mammals, relatively large, well developed female prostates have been re- ported postnatally in some insectivores, chiropterans, rodents, and lagomorphs. The male accessory glands of many species in these orders are exceptionally well devel- oped and the prostates are usually lobed. Female prostates are tubulo-alveolar glands, as are their male homologues, and they too form lobes, but the glands are never as large as those of the male. Their secretory activity is apparently dependent mainly on ovarian androgens, but the function, if any, of the secretion is obscure. Extensive re- search has shown that the administration of androgens to rodents, either to pregnant fe- males or fetuses, to fetal lagomorphs, and to pouch-young oppossums, results in the formation and retention of prostates in fe- males which normally do not have such glands (see chapter by Burns). Deanesly (1934) described vaginal glands in the female hedgehog and suggested that one pair is homologous with the external prostates of the male. The female glands ex- tend dorsolaterally on either side of the ure- thra and a single duct from each lobe opens into the vagina. They seem to be active during the breeding season and to retrogress in the anestrum. In another insectivore, Hemicentetes, there is a pair of large "paravaginal glands" which are function- ally active in the mature female and have large acini filled with secretion. They re- semble the male prostate in histologic struc- ture and anatomic position, but have no ducts (Lehmann, 1938). In adult European moles, most females have bilobed ventral prostate glands which undergo cyclic changes in the epithelium. The prostate of the male is also bilobed and ventral in posi- tion and the homology in the two sexes is clear (Godet, 1949). Mathews (1941) studied the anatomy and histophysiology of the male and female gen- ital tracts of nine species of African bats. Female prostates are well developed in four species, less conspicuous in a fifth, and ab- sent from the remaining four. There is a marked tendency for greater development of the glands in pregnant and lactating fe- males. In three species, the female prostates surround the urethra (as do their male ho- mologues), but in Nycteris luteola the fe- male prostate appears ventrally, whereas the male prostate in this species is limited to the dorsal aspect of the urethra. Mathews considered that the female prostates repre- sent greatly enlarged female urethral glands which are homologous with the male ])yos- tate. The occurrence of female prostates and their relation to hormones have been most extensively studied in rodents. The first de- scription of a well developed female pros- tate gland seems to be that of Rauther (1909), who found such a gland ventral to the neck of the bladder in the African field rat, Arvicanthis cinereus. In Rattus nor- vegicus, Marx (1931, 1932) reported the sporadic occurrence of female prostates. Korenchevsky and Dennison (1936) and Korenchevsky (1937) found prostates in 9 of 56 females and stated that the glands wei'c atrophic, but when androgens were ad- ministered, these glands resembled the male ventral prostate. On this basis, the homology of the glands of the female with the \-entral prostate of the male was sug- gested. Further studies (Witschi, Mahoney and Riley, 1938; Mahoney, 1940, 1942; Ma- lioney and Witschi, 1947) showed that the female prostate of the rat is homologous ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 375 with only the most medioventral part of the male prostate ; the lobes are bilateral or uni- lateral, with the right the preferred side; each lobe has a single duct which opens into the urethra. The incidence of female pros- tates varies markedly in different strains, and can be increased by selective inbreed- ing, w^iich also increases the occurrence of bilateral compared with unilateral lobes. The frequency of female prostates was in- creased in the Wistar stock from 28 to 99 per cent, but when selective inbreeding was stopped, the frequency declined. In young untreated female rats, the pros- tate, when present, develops a histologic structure identical with that of the male homologue, but at about 6 weeks of age the epithelium undergoes regression (Price, 1939; Mahoney, 1940) and becomes histo- logically well developed again only during pregnancy and lactation (Burrill and Greene, 1942; Price, 1942). Thus, the fe- male prostate of the rat is not only homolo- gous with a part of the ventral prostate of the male on the basis of embryologic devel- opment, but during early postnatal devel- opment and in periods of pregnancy and lactation it resembles its male homologue histologically (Fig. 6.46). In addition, it is functionally equivalent (see Section II) to the male ventral prostate in the secretion of citric acid (Price, Mann and Lutwak- Mann, 1949). Brambell and Davis (1940) found large, well developed prostate glands in every one of 104 female African mice, Mastonujs erythroleucus Temm. These glands consist of paired lobes, each draining into the urethra by a single duct. They resemble the ventral prostate of the male in position, shape, and histologic structure. On the ba- sis of this evidence it was concluded that the female glands are the homologues of the male ventral prostate. In some cases, the female prostates are nearly as large as their male homologues and are actively secre- tory. Brambell and Davis correlated hyper- trophy and secretory activity with the lu- teal phase of the cycle and gestation. Female prostate glands have also been de- scribed in the field mouse, Apodemns syl- vaenms sylvaticus. Raynaud (1942, 1945) found bilobed prostates in 51 immature and adult females collected in the vicinity of Vabre (Tarn) and in 3 females from three other regions of France. However, the lobes were macroscopically visible in only 10 fe- males; in all others, the glands were identi- fied in histologic preparations. There w^as great variability in histologic structure, but a well developed epithelium showing secre- tory activity was found during pregnancy and lactation. Raynaud established that the female prostate is homologous with a part of the male ventral prostate. He concluded that there is a probability that bilobed fe- male prostates exist normally in all females of Apodemus sylvaticus. The prostate glands in adult female field voles, Microtiis arvalis P., are considered homologous with the ventral lobes and part of the lateral lobes of the male prostate (Delost, 1953a, b). The lobes in the female are lateral in position in part of the gland, but in other regions they completely sur- round the urethra. The structure is identical with that of the ventral prostate of the male. The epithelium appears secretory in normal adult females, and during gestation this ac- tivity is intense. Bilobed female prostates were found in the 37 adult cottontail rabbits examined by Elschlepp (1952). They lie on the dorsal wall of the vagina (Fig. 6.4) and are simi- lar histologically to the prostate of the male. The glands are larger in pregnant than in nonpregnant females and contain more se- cretion. In summary, well developed female pros- tate glands are present in immature and adult females of many species. They may occur as ventral, lateral, or dorsal lobes; the lobes may be unilateral or consistently bi- lateral; their occurrence may be sporadic or reach an incidence of 100 per cent; they are found both in laboratory strains and in wild populations. The genetic studies of Witschi and his collaborators show that the incidence in rodents can be increased by selective inbreeding. In certain populations of wild rodents the character has become established. A striking example of this is the presence of large prostates in all female Masto)7iys erythroleucus. The secretory ac- tivity of the glands seems to be controlled mainly by ovarian androgens (sec Section 376 PHYSIOLOGY OF GONADS III) . No function can be ascribed to tlie se- cretion. 3. Seminal Vesicles The seminal vesicles are paired, usually elongated glands which may appear rela- tively simple externally (Figs. 6.2 and 6.3) but are subdivided internally by compli- cated villous projections. The name refers to an old misconception that they are sperm reservoirs. The seminal vesicles are rela- tively enormous and distended with secre- tion in some mammals, for example, the rat, guinea pig, and hamster; they are large in others such as the boar, and in still others, as in man, they are small and compact. Seminal vesicles are absent from the mon- otremes, marsupials, carnivores, and ceta- ceans that have been studied, and from some insectivores, chiropterans, primates, and lagomorphs. Variability exists among the edentates ; the sloths and the armadillo have seminal vesicles which are well developed in the two-toed sloth and armadillo, but are very small and rudimentary in the three-toed sloth. Among the lagomorphs, the seminal vesicle of the domestic male rabbit is a large unpaired gland whereas the semi- nal vesicles in the adult cottontail rabbit are vestigial or absent although they de- velop for a time in the fetus (Elschlepp, 1952). 4- Ampullary Glands These organs are glandular enlargements arising from the ampullae of the ducti cleferentes or the posterior region of the ductus if a distinct ampullary enlargement is not present. They may be only slight glandular enlargements of the wall, or dis- crete glands which nearly encircle the duc- tus deferens as in rats, some mice, and ham- sters (Fig. 6.2). They are vestigial in certain pure line strains of mice (Horning, 1947) and lacking in guinea pigs (Fig. 6.3). In some bats, they attain very large size. In general, they are absent from many mam- malian orders and variable in others (Table 6.1). D. EVOLUTIONARY HISTORY OF ACCESSORY REPRODUCTIVE GLANDS OF MAMMALS The well developed male accessory glands which characterize the niamnialiaii class as a whole, and form such a conspicuous part of the reproductive tract in most mammals, are not found in nonmammalian vertebrates. These glands appear as anatomically dis- tinct organs in the primitive prototherian mammals, the monotremes, which are defi- nitely mammalian but which also retain certain anatomic characteristics of their reptilian ancestors and still lay shelled eggs. However, it has been suggested that in the evolution of the three groups of living mam- mals from mammal-like reptiles, the line of descent of monotremes is entirely separate from that of marsupials and placentals. Furthermore, the last two groups are prob- ably parallel branches of the mannnalian stock. The accessory glands of modern mam- mals represent, then, the parallel evolution of discrete glands that probably began their development very early in the evolutionary history of mammals. In gross structure, size, and internal complexity they are unique accessory organs among vertebrates. Mod- ern reptiles have no such glands; the semi- nal plasma is composed mainly of secretions from the epididymis and the renal tubules of the sexual segment of the long, lobulated kidney. Both these regions become highly secretory during the breeding season (see chapter by Forbes) . Parenthetically, the se- men of birds (a later offshoot from the rep- tilian line than mammals) contains only a small amount of seminal plasma (Mann, 1954a) which is secreted in the cock almost entirely by the seminiferous tubules and vasa efferentia (Lake, 1957). In modern mammals, the epididymal epithelium is still an important accessory secretory area (see chapter by Bishop) , but the bulk of the seminal plasma comes from glandular elab- orations of quite different regions, the uro- genital sinus (a derivative of the primitive cloaca) and the posterior part of the Wolf- fian ducts, the ducti deferentes. Modern monotremes are specialized forms but in certain characteristics they are primi- tive. They show almost diagramatically some of the first steps in the evolution of accessory glands. The bulbo-urethrals are already well developed but the concentra- tion of complicated urethral glands at the neck of the bladder in tlie duckbill platypus almost certainlv illustrates the derivation ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 877 of a specialized gland (the prostate) from simpler glands which are nmnerous along the urethra. The probable evolution of pros- tates and bulbo-urethral glands from smaller, simpler urethral glands has been suggested in the past. Observations of Bruner and Witschi (1946) support this concept. In experiments on fetal hamsters, it was found that masculinized females de- veloped prostate glands but the ducts joined the collecting ducts of the urethral glands and did not open directly into the urogenital sinus. According to these work- ers, this may represent an intermediate stage in the development of specialized glands. The history of the cloaca may well be im- portant in relation to development of acces- sory glands (Retief, 1949). The cloaca is retained in modern reptiles; in monotremes it is subdivided cranially into ventral uro- deum or urogenital sinus and a dorsal copro- deum; it is represented by a pocket in mar- supials but is lost as a discrete structure in all higher mammals. The first development of a separate urogenital duct or urethra as it occurs in monotremes may be correlated with the first appearance of discrete acces- sory glands from this specific region in mammals. The marsupials illustrate a more ad- vanced type of glandular development with three histologically distinguishable regions in the disseminate prostate and three pairs of bulbo-urethral glands. Seminal vesicles and ampullary glands are found only among higher mammals. The size and structural complexity of these unique glands in mammals raises the question of the adaptive value of relatively large accessory glands associated with the mammalian reproductive tract. This is a matter only for speculation. The evolution of such glands with increased surface for secretion and enlarged storage space may, perhaps, have been correlated with a tend- ency for an increase in volume of seminal plasma in the ejaculate of mammals. Mann (1954a) pointed out the variability in the volume of ejaculated semen and in the sperm density in various species. With re- gard to the volume of seminal plasma, he stated, "In lower animals it may be so scarce that the emitted semen takes the form of a very thick lump of spermatozoa, closely packed together. There is little semi- nal plasma in bird semen and even among some of the mammals, but on the whole, the higher mammals including man, produce a relatively dilute semen with a considerable l)roportion of seminal plasma." A second suggestion, more speculative, is that the evolution of large mammalian glands may also have compensated for loss of accessory reproductive function in the kidney. The kidney of mammal-like reptiles and ances- tral mammals may have contributed to the formation of seminal plasma (as is true in modern reptiles, amphibians, and fishes), but the compact kidney of warm-blooded, metabolically active mammals may be ill adapted for such a purpose. II. Function of the Male Accessory Glands A. INTRODUCTION The only known function of the male ac- cessory glands is to secrete the seminal plasma. The proportion of this fluid which originates from the various secretory or- gans, or even from different lobes of the same gland, varies greatly from one species to another. There is also remarkable species variation in the volume and composition of the individual secretions. The functional activity of the accessory glands is governed primarily by hormones of testicular origin. The output of androgens is subject to the control of the anterior hypophysis, and many factors (e.g., age, light, season, tem- perature, and diet) affect the secretory ac- tivity of the hypophysis and testis. Thus, it is not surprising that in a given individual, there may be marked fluctuations in the cjuantity and chemistry of the secretions of the accessory glands, and hence of the semi- nal plasma. The development by Charles Huggins of ingenious surgical procedures enabled the secretory activity of the canine prostate to be measured by simple volumetric methods. Such studies of prostatic secretion in the dog established the quantitative relation- ships between the function of prostatic epi- thelium and the androgenic status of the host. In other species, serial collection of the individual secretions in the same animal 378 PHYSIOLOGY OF GONADS has not been achieved for purely technical reasons. The use of "split ejaculates" has given some insight into the glandular origin of various conii)oncnts of the seminal plasma, but this techniciue does not provide uncontaminated secretions from any one gland. However, in the last two decades ex- tensive analyses of the chemical and enzy- matic constituents of the individual secre- tions stored in the accessory glands, and of the whole seminal plasma, have been per- formed. The levels of many of these sub- stances and enzymes are dependent on an- drogenic hormones. These findings have provided a basis for sensitive chemical methods for the bioassay of androgens. Moreover, knowledge of the biosynthesis of these substances by the accessory glands may point to the primary biochemical locus of action of androgenic steroids. This chemi- cal approach to the study of the accessory glands has received great impetus from the pioneer studies of Thaddeus Mann. The secretions of the accessory glands of many species are a repository for huge Cjuantities of substances which are present only in trace amounts in other tissues and body fluids. It is obvious that the seminal plasma must provide an ionically balanced and nutritive milieu suitable for the sur- vival of sperm in the vagina and uterus. Certain substances secreted by one or more of the accessory glands, e.g., fructose, un- doubtedly serve as a source of energy for the sperm. However, there is no evidence that any component of mammalian seminal plasma, or any one of the accessory glands, is absolutely indispensable for fertility. Ar- tificial insemination is successful in some mammals if sperm from the epididymis are diluted in a suital)ly prepared medium, placed in a female in the correct stage of the estrous cycle, and deposited in a region of th(; female tract where there is maximal opiiortunity for their successful ascent. Re- moval of the coagulating glands in guinea l)igs (Engle, 1926b) , or dorsolateral prostate (Gunn and Gould, 1958) in rats does not prevent insemination and fertilization. Blan- dau (1945) extirpated the seminal vesicles and coagulating glands of rats and found that when these males were mated there was no copulation plug and, evidently as a re- sult, the spermatozoa did not penetrate the vaginal canals of the females. Thus the se- cretions of the coagulating gland and semi- nal vesicles in the rat assist the transport of sperm in the female. The following section will consider the output and composition of the secretions, and their hormonal regulation, primarily from a chemical standpoint, rather than in relation to the anatomy and embryology of the structures from which they originate. B. VOLUMETRIC (STUDIES OF SECRETION 1. Prostatic Isolation Operation Volumetric studies of the secretion of canine prostatic fluid have yielded great in- sight into the factors which determine the functional activity of male accessory glands. The dog is devoid of both seminal vesicles and bulbo-urethral glands, and if the urine is suitably deviated, practically pure pros- tatic secretion can be collected from the urethra. Eckhard (1863) ligated the neck of the bladder of dogs and obtained pros- tatic fluid by urethral catheterization. This technique was used by a number of investi- gators to study the secretory activity of the i:)rostate gland (Mislawsky and Bormann, 1899; Sergijewsky and Bachromejew, 1932; Winkler, 19311. A superior modification of the operation was introduced by Farrell (1931, 1938; Farrell and Lyman, 1937). The output of prostatic fluid was increased greatly either by electrical stimulation of the nervus erigens or by the injection of cholinergic drugs such as pilocarpine. These early prostatic isolation operations suffered from the signal disadvantage that, for technical reasons, they permitted only brief experiments. In 1939, Huggins, Masina, Eichelberger and Wharton developed a simple surgical procedure which enabled frequent collection of canine prostatic secretion over long l)eriods of time. The original technique was modified slightly by Huggins and Sommer (1953) and is depicted in Figure 6.5. The bladdei' is separated from the prostate gland, the urine voided through a supra- l)ubic canula, and the animals circumcised. Healing was complete within one week after surgery, and the animals were maintained in good health. Prostatic fluid could be col- lected at fre(iucnt intervals for as long as ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 379 two years. Normal adult dogs were found to secrete 0.1 to 0.2 ml. of prostatic fluid per hour without external stimulation. Follow- ing the administration of pilocarpine, the canine prostate secreted as much as four times its weight of fluid (60 ml.) in one hour. The amount of secretion obtained in response to a standard dose of pilocar- pine remained relatively constant for three months or more, and bore no direct relation- ship to the weight of the gland. The volume and composition of the fluid varied with the time and intensity of the cholinergic stimu- lus. Huggins (1947c) found that, after a single intravenous injection of pilocarpine, the volume and the content of total pro- tein, certain enzymes (acid phosphatase, fS- glucuronidase and fibrinogenase ) , and cit- rate were maximal in the first 15 minutes, then declined progressively in three succeed- ing quarter-hour periods. But the chloride content always rose initially from the low values of the resting secretion and reached maximal levels after the first 15-minute period. If the drug was administered intra- muscularly, maximal values for total pro- tein and citrate were found in the first pe- riod, whereas those for the volume and enzyme content were higher in the second and third periods. It was concluded from experiments involving the repeated intra- venous injection of pilocarpine that acid phosphatase and fibrinogenase were defi- nitely secreted and not simply washed out of the gland. However, a "washing out" process does occur after an initial stimulus with respect to total protein and citrate levels. It was observed by Huggins, Masina, Eichelberger and Wharton (1939) that in- fectious diseases {e.g., pyelonephritis, dis- temper) often decreased the volume of stimulated fluid. This effect seemed to be due to inhibition of the hypophysis, because it could be overcome by injection of gonado- trophin. Soon after castration (7 to 23 days) the secretion ceased and was restored by the administration of testosterone propionate. Androgens also initiated secretion in imma- ture animals. In castrate dogs maintained on testosterone, neither adrenalectomy nor re- moval of the thyroid and parathyroid glands affected the rate of prostatic secretion. Huggins (1947c) observed that in normal Fig. 6.5. The canine prostatic isolation operation. The connection of the prostatic urethra with the bhulder has been severed and the prostatic secre- tion is collected by way of the penis. (From C. Huggins and J. L. Sommer, J. Exper. Med., 97, 663- 680, 1953.) animals, secretion was unaffected by in- jection of either progesterone or desoxy- corticosterone. Cystic hyperplasia of the prostate occurs in many senile dogs. The volume of fluid secreted by such hypertrophied glands in response to pilocarpine was smaller than that obtained from young adult animals (Huggins and Clark, 1940). Injection of diethylstilbestrol into normal adult dogs abolishes prostatic secretion. Ad- ministration of gonadotrophin restores se- cretion in such estrogen treated animals, which suggests that the primary effect of estrogens under these conditions is on the hypophysis (Huggins, 1947c). Estrogens also antagonize the stimulatory effects of injected androgens. In castrate dogs re- ceiving testosterone, injection of large doses of diethylstilbestrol decreases the output of prostatic fluid to very low levels (Huggins 380 PHYSIOLOGY OF GONADS and Clark, 1940). The neutralization of androgen action by estrogens in this situa- tion is pronounced but not complete. Thus the acid phosphatase activity of prostatic fluid collected from animals treated with both testosterone propionate and diethyl- stilbestrol is of the same order of magnitude as that of normal secretion, despite the fact that the volume of the secretion is ex- tremely low (Huggins, 1947c). The ratio of diethylstilbestrol recjuired to antagonize maximally the action of testosterone was found to be about 1 : 25. In dogs with either normal or cystic prostate glands, injection of amounts of estrogen sufficient to decrease prostatic secretion leads to shrinkage of the prostate. Large doses of estrogen cause the canine prostate gland to enlarge ; the dorsal segment undergoes squamous metaplasia and the ventral lobe becomes atrophic (Huggins and Clark, 1940; Huggins, 1947c). If both estrogen and androgen are adminis- tered simultaneously, the dorsal region be- comes squamous and the ventral portion of the gland retains its columnar epithelium, although the volume of the prostatic secre- tion may be drastically reduced. Fig. 6.6. The c-anine prostatic translocation op- eration. (From C. Huggins and J. L. Sommer, J. Exper. Med., 97, 663-680, 1953.) 2. Prostatic Translocation Operation Huggins and Sommer (1953) transposed the prostate gland of the dog from its natu- ral position to the perineum, as depicted in Figure 6.6. This procedure permitted the size of the prostate to be measured in the living animal, and provided prostatic fluid quite uncontaminated with other material. Pilocarpine was used as a secretory stimu- lus. Using this technique, Huggins and Som- mer found that the effects of androgens and estrogens on prostatic size and secretion were similar to those obtained with dogs that had undergone the prostatic isolation operation. C. CHEMICAL COMPOSITION OF THE GLANDULAR SECRETIONS Electrolytes. Water is the main constit- uent of prostatic and seminal vesicle secre- tions and of seminal plasma, all of which are approximately iso-osmotic with respect to blood serum. The vesicular secretion is usually more alkaline than the prostatic se- cretion and has a higher dry weight, mainly because it contains more protein. The elec- trolyte content of the secretions varies widely between different species (Huggins, 1945; Mann, 1954a). In general, sodium is the main cation, although this is not true of boar vesicular secretion which is very rich in potassium. Chloride tends to be the main anion in those species whose accessory gland secretions do not contain large amounts of citrate. In this connection it is instructive to compare the resting prostatic fluid of man, and the pilocarpine-stimulated pros- tatic fluid of the dog (Huggins, 1945, 1947c). The human secretion has a much greater citrate and calcium content, and a much smaller chloride level than the correspond- ing canine fluid, although the total concen- tration of osmotically active substances is of the same order of magnitude in both secre- tions. Zinc. Berti'aiid and X'hidesco (1921 » found large amounts of zinc in human semen. The highest concentration of zinc is present in the first fraction of the ejaculate, which is largely prostatic secretion (Mawson and Fischer, 1953). In the rat, the zinc content of the dorsolateral prostate is especially lii (sorbitol dehydrogenase) which is similar to that of the accessory glands (King and ^Nlann, 1959). Although the sper- matozoa can affect the ratio of the levels of soi'bitol and fi-uctose in seminal plasma, ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 385 most of the seminal sorbitol is probably de- rived from the accessory glands. Inositol. During his studies on the vesicu- lar secretion of the boar, Mann (1954b) iso- lated large amounts of a crystalline, non- reducing carbohydrate which he identified rigorously as ?weso-inositol. This cyclic polyol was found only in the seminal vesi- cle, being absent from the epididymis and Cowper's gland. The concentration of inosi- tol in boar vesicular secretion was as high as 2.6 gm. per 100 ml., and constituted as much as 70 per cent of the total dialyzable material therein. Using a specific microbio- logic method of estimation, Hartree (1957) found that in the boar, the inositol content of seminal plasma was usually greater than 600 mg. per 100 ml., although much smaller quantities (less than 60 mg. per 100 ml.) were present in the bull, ram, stallion, and man. In all of the species examined, the bulk of the inositol in seminal plasma was in the free state, and in amounts much greater than those in blood or cerebrospinal fluid. In most animals, seminal inositol originates from the seminal vesicles, but it has been detected in the prostate gland of the hedge- hog, and in the ampullar secretion of the stallion. The levels of inositol in human semen, to- gether with those of fructose, are increased after the administration of testosterone ac- cording to Kimmig and Schirren (1956). The physiologic function, if any, of the inositol in seminal plasma is unknown. Since boar vesicular secretion, unlike other body fluids of the pig, contains immense amounts of inositol and very little sodium chloride, j\Iann (1954b) suggested that inositol is con- cerned with the maintenance of the osmotic ecjuilibrium of boar seminal plasma. Ascorbic acid. Deproteinized extracts of the seminal plasma of many species reduce 2,6-dichlorophenol indophenol in the cold. This property has been attributed to the presence of ascorbic acid in the semen of the bull (Phillips, Lardy, Heiser and Ruppel, 1940), guinea pig (Zimmet, 1939), and man (Nespor, 1939; Berg, Huggins and Hodges, 1941; Huggins, Scott and Heinen, 1942). However, it is now established that ascorbic acid does not always account for the total reducing power of semen. In some animals, e.g., the boar, ergothioneine is responsible in TABLE 6.3 Sorbitol and fructose content of fresh seminal plasma In some oases the .samples represented semen which had lieen pooled; the number of individuals is given in brackets. (From T. E. King and T. Mann, Proc. Roy. Soc. London, ser B, 151, 226- 2-13, 1959.) Number of Species \ Samples Sorbitol ; Fructose Analyzed Ram. . . Rabbit. Bull . . . Boar. . . Stallion Dog .. . Cock... Man . . . mg./lOO ml. 150-600 (12) 40-150 (4) 120-540 (14) 20-40 (4) <1 (4) <1 (5) <1 (14) 154 (3) large i^art for the reduction of indophenol [vide infra), and bull semen contains sulfite and another, unidentified, reducing sub- stance (Larson and Salisbury, 1953) . Never- theless, ascorbic acid is undoubtedly present in seminal plasma. Employing a specific analytical method based on the formation of its dinitrophenylhydrazone, ]\Iann (1954a) found that the seminal vesicle se- cretion of the rat, bull, guinea pig, and man contains ascorbic acid in amounts varying from 5 to 12 mg. per 100 ml. Mann's values for the ascorbic acid content of human se- men (10 to 12 mg. per 100 ml.) agree well with those reported by Berg, Huggins and Hodges (1941), which were based on indo- phenol reduction. Amino sugars. After hydrolysis with acid, boar semen contains considerable amounts of amino sugars (Mann, 1954a). The epi- didymal "semen" contains more amino sugar than the vesicular secretion. Ergothioneine. The vesicular secretion of the boar (Leone and Mann, 1951 ; Mann and Leone, 1953) is a rich source of ergothione- ine. This sulfur-containing base is also found in the accessory glands of the Euro- pean hedgehog and mole (Mann, 1956) , and in the ampullar secretion of the stallion (Mann, Leone and Polge, 1956). Little or no ergothioneine is present in the semen of the bull, ram, and man. Experiments with S^^-labeled precursors suggest strongly that seminal ergothioneine 386 PHYSIOLOGY OF GONADS is not synthesized in the animal V)ody (Alel- ville, dtken and Kovalenko, 1955; Heath, Rimington and Mann, 1957). Because orally ingested S'^'"'-labeled ergothioneine l)asses into the seminal plasma of the boar (Heath, Rimington, Glover, ]Mann and Leone, 1953) , it is possible that those acces- sory glands which secrete ergothioneine concentrate this substance from the blood. Mann and Leone (1953) are of the opinion that the function of ergothioneine in seminal plasma is to protect the spermatozoa from the poisonous action of oxidizing agents. It is remarkable that the seminal fluids of the boar and the stallion, both of which contain ergothioneine, have common characteristics which would render their spermatozoa es- pecially sensitive to oxidizing agents, viz., large volume, low sperm density, and small content of glycolyzable sugars. PoLYAMiNES. Large amounts of spermine and spermidine are present in the prostate gland of many species (Harrison, 1931 ; Rosenthal and Tabor, 1956). The chemical structure of these polyamines was elucidated by Dudley, Rosenheim and Starling (1926, 1927). Human seminal plasma contains as much as 300 mg. spermine per 100 ml., most of which is derived from the prostate gland. If human semen is allowed to stand for a few hours at room temperature, the spermine present crystallizes in the form of spermine phosphate ("Boettcher's crystals").- Both spermine and spermidine are oxidized by the diamine oxidase of human seminal plasma (Zellcr, 1941; Zeller and Joel, 1941). These polyamines via their degradation products are highly toxic to spermatozoa (Tabor and Rosenthal, 1956) , and it seems unlikely that "In a letter written to llie Royal Society of London in November 1677, Antoni van Leeuwen- hoek described for the fir.'^t time tlie presence and movement of spermatozoa in human semen. In the same letter, he also mentioned that "three- sided bodies," which were "as bright and clear as if they had been crystals," were deposited in the aged semen of man. These crystals were undoubt- edly composed of spermine phosphate. The liis- tory of the discovery of spermine in semen is ad- mirably summarized by Mann (1954a), with special reference to the contributions of Louis Vauquelin (see footnote 3), and also of Alexander von Pcihl, whose claims for the therapeutic proiinities' of spermine aroused much interest aiul controversy at the end of the 19th centurv. their presence in seminal plasma is of func- tional value. Choline DERIVATIVES. Florence (1895) de- scribed the formation of brown crystals upon the addition of a solution of iodine in potassium iodide to semen. This reaction was used as the basis of a medico-legal test for semen stains. Bocarius (1902) showed that choline was responsible for the formation of this material. In the rat, the seminal fluid is by far the richest source of choline of any tissue or body fluid (Fletscher, Best and Solandt, 1935). A series of careful stud- ies by Kahane (Kahane and Levy, 1936; Kahane, 1937) revealed that human semen contains very little free choline immediately after ejaculation, but that large amounts of the free base are formed if the semen is al- lowed to stand at room temperature. Lund- quist (1946, 1947a, b, 1949) isolated phos- phorylcholine from human seminal plasma and showed that it was converted to choline and inorganic phosphate by seminal acid phosphatase. However, the French investi- gators (Diament, Kahane and Levy, 1952. 1953; Diament, 1954) isolated a-glycero- phosphorylcholine from the vesicular secre- tion of rats, and suggested that this sub- stance, rather than phosphorylcholine, was the precursor of free choline in aged semen. Lundquist (1953) also found glycerophos- l^horylcholine in tlie vc'sicular secretion of the rabbit, rat, and guinea pig. Williams- Ashman and Banks (1956) showed that the amount of glycerophosphorylcholine in rat vesicular secretion falls rapidly after castra- tion, and can be restored to normal levels by administration of testosterone. Rezek and Sir (1956) found both ]')hosi)liorylcholinf and glyceroi)hospliofylclu)line in lunnan ejaculates. A thorough study of the wat('i'-s()lul)lf choline (lerivati\-cs in seminal plasma was made by Dawson, Maiui and White (1957). They foimd (Tabh'().4i that in most species, gh^cerophosphoryh-holine is the only deriva- ti\'e preseiu, but ill man there are consider- ■a\)\v (|uantities of phosphoiyh'lioline as well. The lattei- substance is rai)idly dephospho- lylated after ejaculation, but glycerophos- phoryh'holine is not degradeil by enzymes in seminal phisnia or \'esiculai' secretion. In ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 387 TABLE 6.4 Phosphor ylchoUne and a-glycerophosphorylcholine in semen and in secretions of accessory reproductive glands Species Concentration (mg. per 100 gm.) of: Phosphorylcholine a-Glycerophosphorylcholine Ram Ram Bull Bull Bull Bull Bull Cioat Boar Boar Boar Stallion. . . Stallion. . . Man Rat Rat Rabbit... Hedgehog. Hedgehog. Monkey. . . Cock..'.... Semen Seminal pla.sma Semen Seminal plasma Vesicular secretion Epididymal secretion Ampullar secretion Semen Seminal plasma Vesicular secretion Epididymal secretion Semen Ampidlar secretion Semen Vesicular secretion Seminal vesicle Semen Secretion of "Prostate I and 11" Secretion of "Prostate III" Vesicular secretion Semen 0 0 0 0 Present 0 0 0 0 0 0 0 0 256-380 0 0 Present Present Present Present 1185-1942 1601-2040 237-460 110-496 0 1490 94 1382-1550 108-235 190 3060 38-113 120 59-90 654; 530-765" 190-515" 215-370 Present 0 0 0 ° Results from Williams-Ashman and Banks (1956); all other values from Dawson, Mann and White lf57). the bull and boar, the epidiclymi.^ is the ])rin- cii)al source of the glyceroiihosi)horylcholine of the seminal plasma. Williams-Ashman and Banks (1956) in-o- vided evidence that the choline moiety of the glycerophosphorylcholine in vesicular secre- tion is not derived from a direct reaction between glycerol and cytidine diphosphate choline. The latter nucleotide was shown to be a precursor of lecithin in rat seminal vesicle tissue. The glycerophosphorylcholine of seminal plasma may originate from the enzymatic degradation of the choline-con- taining lipids of the seminal vesicle epi- thelium. Choline and glycerophosjihorylcholine are not metabolized by spermatozoa, and do not affect their respiration (Dawson, Mann and White, 1957). There is no evidence that the water-soluble choline derivatives of seminal plasma serve any useful function. Lipids. That lipid-containing granules are l^resent in human seminal plasma has been known for more than a century. They are found in prostatic secretion (Thompson, 1861 1, and were termed "lecithin-kornchen" by Fuerbringer (1881). However, Scott ( 1945 ) showed that lecithin is absent from both of these fluids, and that the majority of the phospholipid therein is phosphatidyl ethanolamine. Neutral fat is virtually ab- sent from human seminal plasma and pros- tatic secretion, one-third of the total lipid of which can be accounted for as cholesterol. According to Boguth (1952), about one- third of the total plasmalogen in bull semen (30 to 90 mg. per 100 ml.) is in the seminal plasma. In the ram, only 10 per cent of the seminal plasmalogen is found outside the spermatozoa (Hartree and ]Mann. 1959). Large amounts of 7-dehydrocholesterol were found in the preputial gland and epi- didymis of the rat (Ward and ]\Ioore, 1953) . One gram of the hydrocarbon heptacosane (CH3(CHo)o5CH3) was isolated from an al- coholic extract of 18 liters of human semen by Wagner-Jauregg (1941). The partition of heptacosane, and of steroidal estrogens (Diczfalusy, 1954) and androgens (Dir- scherl and Kniicliel, 1950) between the sjierm 388 PHYSIOLOGY OF GONADS and plasma of human semen remains to be determined. Citric acid. Citric acid was first detected in human semen by Schersten (1929). The distribution of citric acid in the semen and in the secretions of the accessory glands of var- ious species is summarized in Table 6.5. In some animals, {e.g., the rat and man), citric acid is produced mainly by the prostate gland, and in others {e.g., the bull, boar, and guinea pig), most of it originates from the seminal vesicles. The citric acid content of the seminal plasma and of the secretions of accessory glands depends on androgenic hormones. Citric acid disappears from these fluids after castration, and is formed again after treat- ment with testosterone. This ''citric acid test" has been used to determine the time of onset of secretory function in accessory glands (Mann, Davies and Humphrey, 1949; Ortiz, Price, Williams- Ashman and Banks, 1956), hormonal influences on secretion in subcutaneous transplants (Mann, Lutwak- Mann and Price, 1948; Lutwak-jNIann. Mann and Price, 1949 L the androgenic ac- TABLE 6.5 Citric acid in semen and in the secretions of accessory reproductive glands Species Material Citric Acid Reference (tng./lOO gm.) Man Semen 140-637 Huggins and Xeal (1942) Man Prostatic secretion 480-2()88 Huggins and Neal (1942) Man .... Seminal vesicle secretion Hypertrophic adenoma of the 15-22 201-1533 Huggins and Neal (1942) Barron and Huggins (1946a) Man prostate gland Man Carcinoma of the prostate gland 12-137 Barron and Huggins (194()a) Bull Semen 510-1100 Humphrey and Mann (1949) Bull Seminal gland secretion 670 Humphrey and Mann (1949) Bull Ampullar semen 550 Humphrey and Mann (1949) Bull Epididvmal semen 0 Humphrey and IVIann (1949) Bull Epididymis 18 Humphrey and Mann (1949) Boar Semen 130 Humphrey and Mann (1949) Boar Cowper's gland secretion Epididymal semen Seminal yesicle secretion 0 Humphrey and Mann (1949) Boar 0 Humphrey and Mann (1949) Boar 580 Humphrey and Mann (1949) Ram Semen Semen 110-260 110-550 Humphrey and Mann (1949) Himiphrey and Mann (1949) Rabbit Rabbit Epididymis 54 Humphrey and Mann (1949) Rabbit Prostate (I, II and III) 62 Himiphrey and Mann (1949) Rabbit Cowper's Gland 42 Humphrey and Mann (1949) Rabbit Ampulla 273 Himiphrey and Mann (1949) Rat Seminal vesicle Coagulating gland Ampulla Dorsolateral prostate 39 0 Humi)hrey and :\Iann (1949) Hunii)hrc\ and Mann (1949) Rat Rat 0 Humplucv and Mann (1949) Rat 20 Humphrey and Mann (1949) Rat Ventral prostate Semen 122 Humphrey and Mann (1949) Mann, Leone and Polge (1956) Stallion 8-53 Stallion Seminal vesicle 77 Mann, Leone and Polge (1956) Guinea pig. . . . Seminal vesicle 153-216 Ortiz, Price, Williams-Ashman Banks (195()) Guinea pig. . . . Semiiuil vesicle sec'retion 320-357 Ortiz, Price, Williams-Ashman Baidvs (1956) Guinea pig. . . . Coagulating gland 26-40 Ortiz, Price, Williams- Ashman Banks (1956) Guinea pig. . . . Lateral i)rostate 16-20 Ortiz, Price, Williams- Ashman Banks (1956) Guinea i)ig. . . . Doi'sal ])i(is1at(> 47-75 Ortiz, Price, Williams-Ashman Banks (195(i) Dog Prostatic secretion 0-30 Barron and Huggins (1946a) Barron and Huggins (1946a) Mann, Leone and Polge (1956) Dog Prostate gland Vesicular secretion 8 Jackass 22-82 ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 389 tivity of various hormones (Mann and Par- sons, 1950; Price, Mann and Lutwak-JMann, 1955; Ortiz, Price, Williams-Ashman and Banks, 1956), and the effect of nutrition on the onset of androgen secretion and sperm formation in bull calves (Davies, Mann and Rowson, 1957). The androgen-induced changes in the cit- rate levels in semen and various accessory glands are reminiscent of similar alterations in the fructose content of these tissues. How- ever, the concentrations of these substances do not necessarily parallel one another in re- sponse to hormonal stimulation. In the post- castrate animal, the fall in citric acid and its reappearance after androgen treatment is usually more sluggish than that of fructose. Also, the seminal fructose of some species may not be secreted by the same accessory organ (or lobes of the gland ) which produces citric acid. Thus in the rat, fructose is se- creted by the anterior and dorsolateral pros- tate, whereas citric acid is derived from the seminal vesicles and dorsolateral and ven- tral prostates, but is totally absent from the anterior prostate (Humphrey and Mann, 1949). In the guinea pig, however, the semi- nal vesicles are the principal source of both fructose and citric acid (Ortiz, Price, Wil- liams-Ashman and Banks, 1956). Using a strain of rats in which the inci- dence of the female prostate is very high, Price, ]Mann and Lutwak-JVIann (1949) showed that the growth of this gland which follows the injection of testosterone is ac- companied by a tremendous increase in its content of citric acid. In this way the female prostate resembles the ventral prostate gland of the male rat. Citric acid is synthesized in the i)rostate gland by the usual reactions of the tricar- boxylic acid cycle (Williams-Ashman, 1954; Williams-Ashman and Banks, 1954b). No other organic acids are present in more than trace amounts in the secretions of those ac- cessory glands that accumulate citrate. The enzymatic machinery for the degradation of citric acid via the tricarboxylic acid cycle is jH-esent in the rat ventral prostate gland OVilliams-Ashman, 1954; Williams-Ashman and Banks, 1954b; Williams-Ashman, 1955) and there is no evidence, despite suggestions to the contrary (Awapara, 1952a), that cit- ric acid accumulates because it cannot be oxidized. It has been suggested that a com- mon denominator affecting the androgen- dependent accumulation of citric acid and fructose in the accessory glands is the intra- cellular balance between the oxidized and reduced forms of DPN and TPN (Talalay and Williams-Ashman, 1958). ^lann (1954a) has summarized the ideas of various authors concerning the possible functional role of citric acid in seminal plasma. All of these suggestions are based more upon conjecture than experimental fact. Catecholamine^. There is evidence for the presence of both epinephrine and norepi- nephrine in seminal pla.sraa (Brochart, 1948; Beauvallet and Brochart, 1949). Extracts of human prostate and seminal vesicle contain a monoamine oxidase which oxidizes cate- cholamines (Zcller and Joel, 1941). Katsh (1959) detected serotonin and histamine in human ejaculates. Amino acids. Chromatographic studies have revealed the presence of many free amino acids in human semen (Jacobbson, 1950; Lundquist, 1952), from which crystal- line tyrosine was isolated by Wagner- Jauregg (1941). According to Barron and Huggins (1946b), human prostatic adenoma is very rich in free glutamic acid, and the nonprotein amino-nitrogen of this and dog prostatic tissue is high. Bovine seminal plasma contains free serine, alanine, glycine, and aspartic and glutamic acids (Gassner and Hopwood, 1952). A similar distribution of amino acids is found in the vesicular and ampullary secretions of the bull. The free amino acid levels of bull seminal plasma fall greatly after castration. In the rat, Marvin and Awapara (1949) found that the concen- tration of free amino acids in the whole pros- tate decreased markedly following orchidec- tomy, and could be restored to normal levels in the castrate animal by treatment with androgen. In this species, Awapara (1952a) observed that the content of free amino acids in the ventral lobe of the prostate was much higher than in the dorsal lobe. After castra- tion, there was a marked drop in the content of most amino acids with the exception of aspartic and glutamic acids, which seemed 390 PHYSIOLOGY OF GOXADS to remain at almost normal levels (Awapara, 1952b j. Seminal plasma and the secretions of the male accessory glands contain a battery of proteolytic enzymes [vide infra). For this reason, changes in the levels of free amino acids in these fluids resulting from hormonal treatments should be interpreted with cau- tion. Jacobbson (1950), for example, has shown that in human semen, the nonprotein nitrogen and amino-nitrogen content in- creases many fold within 60 minutes after ejaculation. Prostaglandin. A vasodepressor sub- stance, designated j^rostaglandin, was found by von Euler (1934, 1936) in the prostatic and vesicular secretions of man, and also in the accessory glands of sheep (von Euler, 1939). The prostaglandin of ram prostate was i^urified by Bergstrom ( 1949), who sug- gested that it was an unsaturated fatty acid devoid of nitrogen. According to Eliasson (1957), the prostaglandin of human semen and of the prostate gland of sheep are iden- tical. The pharmacologic effects which result from the injection of seminal plasma icf. Kurzrok and Lieb, 1931; von Euler, 1934, 1936, 1939; Goldblatt, 1935; Cockrill, Mil- ler and Kurzrok, 1935; Asplund, 1947) are complex, and are probably due to the com- bined action of many constituents of this fluid. The hypotensive action of protein fractions of the secretions of some acces- sory organs (Freund, Miles, Mill and Wil- helm, 19581 is discussed below. Uric acid. Bull seminal vesicles may con- tain as much as 70 mg. per cent of uric acid (Leone, 1953). The uric acid content of the semen of other animals is much lowci' (Mann, 1954a). Urea. The urea content of human and ram semen is much higlici' than that found in the bull, boar, and stallion (Mann. 1954a). Major protein constitiiknts. Human seminal plasma contains from 3.5 to 5.5 gm. of protein-like material per 100 niL (Huggins, Scott and Heinen, 1942). Less than 18 per cent of this material is coagu- lable by heat, and as much as 68 per cent of it is dialyzable. Thus the majority of the seminal proteins of man can be classi- fied as proteoses. Electrophoretic analyses of the nondialyzable proteins of human seminal plasma have been performed by Gray and Huggins ( 1942 1 and by Ross, Moore and Aliller (1942). The major com- ponents bore some correspondence to those of blood serum, although the amount of albumin was small. The proteins of bovine seminal plasma are less dialyzable, and more coagulable by heat, than those of man (Larson and Salisbury, 1954). Electropho- retic studies showed the presence of three major and eight minor constituents, which seemed to be distinct from the proteins of bovine blood serum. In this species giyco- or lipoproteins were present only in very low concentrations. Larson, Gray and Salis- bury (1954) found that the bovine seminal plasma proteins are highly antigenic. They obtained immunologic evidence that the major protein constituents of this fluid are distinct from any of the main ])rot(>ins of either blood or milk. Enzymes, (i) Acid phosphatase. Kut- scher and Wolbergs (1935) discovered that human semen and prostate contain a very active phosphatase which is optimally ac- tive at pH 5 to 6. This enzyme is resjjonsi- ble for the greater phosphatase activity of male as compared with female urine. Its secretion by the prostate accounts for the fact that male urine collected from the renal pelvis exhibits very little enzyme ac- tivity (Scott and Huggins, 1942). Human prostatic acid phosjihatase hydrolyzes a number of phosphate monoesters (Kutscher and Worner, 1936; Kutscher and Pany, 1938). The enzyme has been purified ex- tensively (London and Hudson, 1953; Bo- man, 1954; London, Sommer and Hudson, 1955). In addition to hydrolyzing phos- phate esters, human prostatic acid phos- phatase catalyzes the transfer of phosphate from various donors to alcohols such as glu- cose, fructose, and methanol (London and Hudson, 1955; Jeffree, 1957). L-Tartrate inliibits tlie enzyme competitively (Abul- Kadl and King, ^1948). 'I'hc activity of acid phosphatase in the human jjrostate is low in childhood and in- creases about 20 times at i)uberty (Gutman and Gutman, 1938a). In adult men, the acid phosphatase content of semen seems to reflect the circulating levels of andro- genic hoi-niones (Gutman and Gutman, ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 391 1940). High levels of acid })hosphatase are also present in osteoplastic metastases of prostatic carcinoma (Gutman, Sproul and Gutman, 1936). Acid phosphatase does not seem to enter the circulation from the pros- tate gland in healthy individuals unless they are subject to prostatic massage. But in about 65 per cent of men with metastatic carcinoma of the prostate, the serum levels of this enzyme are abnormally high (Gut- man and Gutman, 19381); Robinson, Gut- man and Gutman, 1939; Huggins and Hodges, 1941). The diagnosis and prognos- tic evaluation of carcinoma of the prostate in men has been aided greatly by measure- ments of the acid phosphatase levels of serum. Inhibition of the acid phosphatase activity of blood serum by L-tartrate has been used as an index for the outflow of prostatic acid phosphatase into the serum in neoplastic diseases of the prostate gland (Abul-Fadl and King, 1948; Fishman and Lerner, 1953). The prostate gland of the monkey (Gut- man and Gutman, 1938a) and dog (Hug- gins and Russell, 1946), and the seminal vesicle of the guinea i)ig (Bern and Le\y, 1952) exhibit powerful acid phosphatase activity, whereas the levels of this enzyme in the prostate of the rabbit (Bern and Levy, 1952) and rat (Huggins and Web- ster, 1948) are relatively low. The proper- ties of these enzymes from different species are strikingly similar (Novales and Bern, 1953) . In the monkey and dog, the prostatic acid jihosphatase activities are controlled by androgenic hormones. This is also true in the rat (Stafford, Rubinstein and Meyer, 1949), and guinea pig (Ortiz, Brown and Wiley, 1957). {2} Alkaline phosphatase. An enzyme, activated by magnesium ions, which hydro- lyzes a variety of phosphate monoesters at pH 9, is present in the seminal fluid and accessory glands. In some species, e.g., the l)ull (Reid, Ward and Salisbury, 1948 ) , the levels of seminal alkaline phosphatase are much greater than those of the acid phos- phatase. In the rat, alkaline phosphatase activity in the prostate and seminal vesicles decreases markedly after castration (Staf- ford, Rubinstein and Meyer, 1949). (3) 5'-Nucleotidase. Reis (1937, 1938) noticed that human seminal plasma dcphos- phorylated adenosine 5'-phosphate and ino- sine 5'-phosphate very rapidly. He proposed the term ''5'-nucleotidase" for enzymes which specifically hydrolyze the 5'-mono- phosphates of ribose and its nucleosides. Mann (1945) reported that bull seminal plasma is exceedingly rich in 5'-nucleo- tidase. The enzyme was purified from this source by Heppel and Hilmoe (1951a). It was inactive towards adenosine 2'- and 3'- phosphates, but catalyzed the hydrolysis of the 5'-monophosphate esters of adeno- sine, inosine, cytidine, uridine, and ribosyl nicotinamide. The 5'-nucleotidase of bull semen is optimally active at pH 8.5, and requires magnesium ions for maximal ac- tivity. (4) Inorganic pyrophosphatase. Heppel and Hilmoe (1951b) reported the presence of an inorganic pyrophosphatase in bull seminal plasma. The enzyme was not puri- fied extensively, and it is not clear whether it is different from other in'rophosphatases in semen. (5) Nucleotide pyrophosphatases. The enzymatic hydrolysis of adenosine triphos- phate (ATP) by seminal plasma was ob- served by Mann (1945) and by MacLeod and Summerson (1946). Three distinct ATPases were isolated from bull seminal plasma by Heppel and Hilmoe (1953). The first of these enzymes catalyzed the hy- drolysis of ATP to inorganic pyrophosphate and adenosine 5'-phosphate. The other two catalyzed the liberation of inorganic ortho- phosphate from ATP, and were active at pH 5 and pH 8.5 respectively. The possible identity of any of these proteins with other enzymes which hydrolyze the pyrophos- phate linkage of ]iyridine nucleotides (Wil- liams-Ashman, Liao and Gotterer, 1958) and cytidine diphosphate choline (Wil- liams-Ashman and Banks, 1956) remains to be established. The physiologic function of any of the phosphatases in seminal plasma is un- known. {6) Proteolytic enzymes. The proteolytic activity of human semen was first noted by Huggins and Neal (1942), and has been studied extensively by Lundquist and his collaborators. An enzyme similar to pep- sinogen, and probably secreted by the semi- nal vesicles, was discovered in human semi- 392 PHYSIOLOGY OF GONADS nal plasma by Lundquist and Seedorf (1952). Three other proteolytic enzymes were partially purified from human semen by Lundquist, Thorsteinsson and Buus (1955). The first enzyme resembled chymo- trypsin, and the second was an aminopepti- dase. The third enzyme hydrolyzed benzo- ylarginine ethyl ester, and seems to be identical with the arginine ester hydrolyz- ing enzyme described in male accessory re- productive glands by Gotterer, Banks and Williams-Ashman (1956). The relationship of these enzymes to the hydrolysis of fibrin or fibrinogen by prostatic secretion is dis- cussed below with reference to the coagula- tion and liquefaction of semen. (7) Glycosidases. Using phenolphthalein glucuronide as a substrate, Talalay, Fish- man and Huggins (1946) determined the /?-glucuronidase activity of the male ac- cessory glands of the rat. The levels of this enzyme in the epididymis fall about 50 per cent after castration, and can be restored to normal levels by the administration of testosterone (Conchie and Findlay, 1959). When the corresponding phenol- or p-nitro- phenol-glycosides were employed as sub- strates, Conchie, Findlay and Levvy (1956) showed that the epididymis of the rat is particularly rich in y3-iV-acetylglucosamini- dase. The levels of this enzyme were found by Conchie and Mann (1957) to be very much greater than those of seven other glycosidases in male accessory secretions. The levels of various glycosidases in the epididymis of rodents increases enormously at puberty. In adult animals the activity of some of these enzymes {e.g., a-manno- sidase and /?-iV-acetylglucosaminidase) fell to negligible values after castration, and were restored only partially by treatment with testosterone. (8) Miscellaneous enzipnes. The kneels of a number of oxidizing enzymes in human seminal plasma were studied by Rhodes and Williams-Asluiuiii (1960», who noted the presence of a x'cry active TPN-linked iso- citric dehydrogenase. The ability of luiinan semen to hydrolyze acetylclioline is rather fe(>!)le, and the bulk of the activity resides in the seminal plasma (Zeller and Joel, 1941). According to Sekine (1951), boar semen exhibits powerful choline esterase activity, wliicli is confined mainly to the s])ermatozoa. The activity of phosphohexo- isomerase (Wiist, 1957) and lactic dehy- drogenase (MacLeod and Wroblewski, 1958) in human seminal plasma has been documented. The levels of the following soluble en- zymes have been determined in the acces- sory glands of male rodents: phenol sulfat- ase (Huggins and Smith, 1947). nonsi)ecific esterase (Huggins and ]\Ioulton, 1948), enolase, and dehydrogenases for lactate, malate, glucose 6-phosphate, 6-phosphoglu- conate and isocitrate (Williams- Ashman, 1954; Rudolph, 1956), aldolase and a-glyc- ero])hosphate dehydrogenase (Butler and Schade, 1958). The nucleoside phosphoryl- ase and adenosine deaminase activities of bull seminal vesicle were measured by Leone and Santoianni (1957). The vesicu- lar secretion of the bull is rich in flavins, and exhibits strong xanthine oxidase ac- tivity (Leone, 1953). Leone and Bonaduce (1959) described a very active diphospho- ]5yridine nucleotidase in the vesicular secre- tion of the bull. Conclusions. The foregoing survey indi- cates that, just as the size and morphology of the accessory glands differ profoundly, so there are wide species variations in the chemistry of their secretions, which com- prise the seminal plasma. Some seminal constituents {e.g., fructose) are found in many mammals. Other substances, such as ergothioneine, are present in appreciable amounts in the seminal plasma of only a few species. The biochemistry of the acces- sory glands is still in its infancy, and it may be expected that future research will disclose other species-restricted comjionents of seminal plasma. Mann (1954a, 1956) I'ightly emphasizes that the finding of sub- stantial concentrations of certain sub- stances in the semen of only relatively few species does not necessarily detract from their physiologic value. The high levels of ci-gotliioneine in the seminal plasma of the boar and stallion is a case in point. The cjacuhitcs of these species have peculiarities which may render their spermatozoa par- ticularly susceptible to the immobilizing action of oxidizing agents, and the sugges- tion (Mann and Leone, 1953; IMann, Leone and Polge, 1956) that ergothioneine, in vir- tue of its reducing properties, serves a pro- ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 393 tective function in boar and stallion semen seems an eminently reasonable one. How- ever, the accessory glands of many animals secrete certain substances {e.g., glycero- phosphorylcholine, spermine, citric acid) that do not appear to be of any particular value for the survival of spermatozoa in the male or female genital tracts. Perhaps these substances are simply by-products of the secretory mechanisms of the glands from which they originate, or represent bio- chemical vestiges. The widespread occurrence of fructose in accessory gland secretions deserves further comment. The only other situation where large amounts of fructose are present in mammalian extracellular fluids under nor- mal physiologic conditions is in the fetal blood of ungulates (Bernard, 1855; Bacon and Bell, 1948; Alexander, Huggett, Nixon and Widdas, 1955). Mammalian spermato- zoa metabolize glucose just as well as fruc- tose as a source of energy under anaerobic and aerobic conditions. Indeed, glucose has been used widely as the sole glycolyzable sugar in artifi^l diluents employed in the storage of semen for artificial insemination (Mann, 1954a). Thus fructose does not seem to be more beneficial than glucose to the well being of spermatozoa. There is evidence that the utilization of fructose, in contrast to glucose, is not impaired in the diabetic state (Chernick, Chaikoff and Abraham, 1951 ; Renold, Hastings and Nes- bett, 1954) . It is conceivable that the pres- ence of fructose in semen w^ould render the spermatozoa relatively insensitive to insu- lin. But it would seem more probable that the physiologic value of seminal fructose is related to factors other than the matura- tion or survival of spermatozoa. Mann (1954a) has pointed out that if glucose were the only glycolyzable sugar in semen, its concentration would not be expected to exceed that of blood. The transformation of blood glucose into seminal fructose by the accessory glands permits the establishment of very high levels of fructose in semen. Furthermore, the formation of seminal fruc- tose is strictly controlled by androgenic hormones, and it would be hard to conceive of a similar hormonal dependence of glu- cose levels in semen. Although the volume and chemical com- })osition of seminal plasma are influenced by many factors, androgenic hormones are undoubtedly the principal determinants of the secretory activity of the accessory glands. Chemical and enzymatic constit- uents of accessory gland secretions such as fructose, citric acid, and acid phospha- tase have proved to be exquisitely sensitive indicators of androgenic activity. The ap- plication of such "chemical tests" for an- drogen action has provided important corroborative evidence for previous con- clusions, based on purely morphologic studies, that the initiation of mature secre- tory function of the accessory glands pre- cedes the appearance of sperm in the semi- niferous tubules, and also that the adverse effects of malnutrition on the functional activity of the prostate gland and seminal vesicle are mediated via the hypophysis. Chemical investigations have established that the major portion of certain compo- nents (glycerojihosphorylcholine, glycosid- ases ) of the seminal plasma of some species originates from the epididymis. The way to the successful treatment of metastatic carcinoma of the prostate in man by anti- androgenic measures was paved by the availability of a chemical systemic index of the hormonal dependence of many of these neoplasias, viz., the acid phosphatase of blood serum. Changes in the chemistry of some accessory organs (e.g., the fructose content of the rat coagulating glandj seem to l)e more sensitive indicators of the ac- tion of exogenous androgens in castrated animals than the weights or histologic structure of these organs. The application of such chemical methods to the bioassay of an- drogens holds much promise for the future. Finally, it may be mentioned that chemi- cal studies of the secretions of the accessory glands have given insight into the homology of these organs. The finding of high con- centrations of citric acid, but not of fruc- tose, in the rat female prostate after stim- ulation with androgens shows that the secretion of this tissue resembles that of the ventral prostate gland of the male rat. On the other hand, structures which are usu- ally considered to be anatomically and functionally homologous may secrete quite different substances. Thus in the guinea pig and bull, both citric acid and fructose 394 PHYSIOLOGY OF GONADS are secreted by the seminal vesicles, whereas in the rat, citric acid is produced by the seminal vesicles and fructose is formed only in the dorsolateral prostate and coagulating glands. D. METABOLISM OF THE PROSTATE AND SEMINAL VESICLE The metabolism of the male accessory reproductive glands, and the activity of many enzymes therein, are influenced pro- foundly by steroid hormones. In adult ani- mals, excision of the testes results in a rapid decline in the respiration, but not of the anaerobic glycolysis, of slices of the prostate gland of the dog (Barron and Huggins, 1944), and of the rat prostate (Homma, 1952; Nyden and Williams-Ash- man, 1953; Bern, 1953; Rudolph and Starnes, 1954; Butler and Schade, 1958) and seminal vesicle (Rudolph and Samuels, 1949; Porter and Melampy, 1952; Rudolph and Starnes, 1954j. The post-castrate fall in oxygen consumption by these tissues can be reversed by the administration of tes- tosterone. The respiration of the epithelium (but not of the muscle) of the guinea pig seminal vesicle responds in a similar way to androgen deprivation (Levey and Szego, 1955b). The stimulatory effect of testost- erone on the respiration of the prostate gland and seminal vesicle of castrated rats is not prevented by the simultaneous ad- ministration of hydrocortisone (Rudolph and Starnes, 1954). The activity of a number of respiratory enzymes in the rat prostate gland is de- creased by castration to about the same extent as the respiration of slices of this tissue. This is true for the succinic and cy- tochrome oxidase systems (Davis, Meyer and McShan, 1949), and for fumarase, aconitase, and malic dehydrogenase (Wil- liams-Ashman, 1954). But the succinic oxi- dase levels in two other androgen-sensitive tissues are uninfluenced by castration, viz., the epithelium of the guinea pig seminal vesicle (Levey and Szego, 1955b), and the levator ani muscle of the rat (Leonard, 1950). In the rat prostate, androgens have little influence on the activity of the glyco- lytic enzymes enolase and lactic (l(>hydro- genase, and of the TPN-specific enzymes which oxidize isocitrate, glucose 6-ph()s- i:)hate and 6-phosphogluconate (Williams- Ashman, 1954; Rudolph, 1956). The enzy- matic machinery responsible for the respira- tion of the male accessory glands seems to be similar to that of other mammalian tis- sues (Barron and Huggins, 1946a, b; Nyden and Williams-Ashman, 1953; Williams- Ashman, and Banks, 1954b; Williams-Ash- man, 1954, 1955; Levey and Szego, 1955a). Glock and McLean (1955) have shown that, as in most other mammalian tissues, the levels of DPN in rodent prostate and seminal vesicle are higher than those of DPNH, whereas the content of TPNH is much greater than that of TPN. Nyden and Williams-Ashman (1953) found that the respiration-coupled synthe- sis of long-chain fatty acids from acetate by ventral prostate slices m viti'o was de- pressed by castration to a greater extent than the respiration, and could be restored to normal levels by testosterone therapy. Certain other synthetic reactions (the in- corporation of P^--labeled inorganic phos- phate into phospholipids, total nucleic acids, and phosphoproteins) were less sen- sitive to androgens under these conditions. However, in experiments involving the in- jection of P-^--labeled inorganic phosphate into animals, the administration of andro- gen increased the turnover of various acid- insoluble phosphorus containing fractions. Thus Levin, Albert and Johnson (1955) observed that testosterone increases the turnover of various phospholipids in the lirostate gland and seminal vesicle. In the seminal vesicle, Fleischmann and Fleisch- mann (1952) found that the entry of P-'- into the desoxyribonucleic acid fraction was increased 100-fold by androgen ad- ministered to castrate rats, whereas the sjiecific radioactivity of the ribonucleic acid was increased only 2-fold. Cytoplas- mic basophilia in the rat seminal vesicle (Melampy and Cavazos, 1953), and the endoplasmic reticulum of the ventral pros- tate gland (Harkin, 1957a), which are in- timately associated with cytoi)lasmic ribo- nucleic acid, are influenced profoundly by androgenic hormones. Transamination bet^^■een glutamate and cither pyruvate or a-ketoglutarate was shown by Barron and Huggins (1946b) to proceed rapidly in canine and human pros- ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 395 tate tissues. Awapara (1952a. bl reported that the ahmine (but not aspartic) trans- aminase activities of the ventral prostate gland of the rat were decreased by castra- tion, and increased by testosterone therapy. Rudolph and Starnes (1954) studied the water distribution in the rat accessory glands. The extracellular water in normal seminal vesicles and prostates was 13.8 per cent and 8.5 per cent, respectively. The corresj^onding values in castrate animals were 37.0 per cent and 31.8 per cent. The growth of the glands which resulted from treatment with testosterone was accom- panied by a greater increase in the intra- cellular water than in extracellular water. Rudolph and Samuels (1949) provided evi- dence that changes in the water content of seminal vesicles induced by treatment of castrate rats with testosterone did not \)re- cede metabolic changes (e.g., fructose syn- thesis) in this tissue. The pronounced effects of androgen ad- ministration in vivo on the metabolism and enzymatic activity of the accessary glands cannot be mimicked by the addition of an- drogens in vitro. Dirscherl, Breuer and Scheller (1955) reported that low levels of testosterone stimulated the respiration of mouse seminal vesicles if the control respi- ration was low. But others have found that the respiration and glycolysis of male ac- cessory glands are uninfluenced by the di- rect addition of androgens /// I'itro except at high concentrations (>5 X 10~^ m), at which testosterone is inhibitory (Bern, 1953; McDonald and Latta, 1954, 1956; An- drewes and Taylor, 19551. According to Farnsworth (1958), the direct addition of testosterone to prostate tissue impedes cit- rate synthesis to a greater extent than oxy- gen consumption. Williams-Ashman (1954) found that the in vitro addition of testos- terone did not affect the activity of a num- ber of respiratory and glycolytic enzymes in the rat A-entral prostate gland. The mechanism of action of androgenic hormones at a molecular level is not known. There is no evidence that androgens are di- rectly involved in the large changes in the activity of some enzyme systems in acces- sory glands which follow the administration or deprivation of these hormones. Recent studies wliich indicate that minute concen- trations of certain steroid hormones can stimulate the transfer of hydrogen between pyridine nucleotides by isolated enzyme systems deserve further comment. A soluble enzyme in human placenta catalyzes an estradiol- 17y8-dependent exchange of hy- drogen between TPNH and DPN (Talalay and Williams-Ashman, 1958). There is evi- dence in favor of the hypothesis (Talalay, Hurlock and Williams-Ashman, 1958; Tala- lay and Williams-Ashman, 1960) that es- tradiol-17^ transports hydrogen in this re- action by undergoing reversible oxidation to estrone: Estrone + TPNH + H+ ^ Estradiol-17/3 + TPN EstradioI-17/3 + DPN ^ Estrone + DPNH + H+ TPNH + DPN ^ TPN + DPNH Hagerman and Villee (1959), however, believe that estradiol- 17/;^ and estrone me- diate transhydrogenation between TPXH and DPN by a mechanism which does not involve oxido-reduction of the steroids. Hurlock and Talalay (1958) showed that a soluble 3a-hydroxysteroid dehydrogenase isolated from rat liver catalyzes hydrogen transfer between pyridine nucleotides in the presence of catalytic levels of androsterone and some other 3a-hydroxysteroids. In this instance also, it seems that the steroids act in a coenzyme-like manner by undergoing alternate oxidation and reduction. However, biologically inactive steroids such as etio- cholan-3a-ol-17-one are even more active than androgenic substances such as an- ch'osterone in this isolated enzyme system. Hurlock and Talalay (1959) reported that the particle-bound 3a- and 11^-hydroxy- steroid dehydrogenases of rat liver react at comparable rates with both TPX and DPN, and they suggest that these dehydrogenases might function as transhydrogenases in the presence of their appropriate steroid sub- strates. The hydroxy steroid dehydrogenases for which there is direct or circumstantial evidence for their ability to function as transhydrogenases are localized either in the microsomes (endoplasmic reticulum) or in the soluble cell sap. Other enzymes that catalyze the transfer of hydrogen between pyridine nucleotides are bound to the mito- chondria of many animal tissues (Stein, 396 PHYSIOLOGY OF GONADS Kaplan and Ciotti, 1959; c/. Talalay, Hur- lock and Williams-Ashman, 1958). These mitochondrial transhydrogenases do not re- quire steroid hormones as cof actors. Accord- ing to Hmiiphrey (1957), the large cyto- plasmic particles of rat prostate gland and seminal vesicle are devoid of transhydro- genase activity. Slices of human prostate gland convert testosterone to androst-4- ene-3,17-dione (and other metabolites) (Wotiz and Lemon, 1954; Wotiz, Lemon and Voulgaropoulos, 1954). This suggests that the human prostate contains a 17/3- hydroxysteroid dehydrogenase which could conceivably function as a transhydrogenase in the presence of low levels of testosterone. Baron, Gore and Williams (1960) reported the presence of androsterone-stimulated transhydrogenase reactions in the prostate gland of rodents and man. On the contrary, Williams-Ashman, Liao and Gotterer (1958), and Samuels, Harding and Mann (1960) were unable to demonstrate any ac- tivation by testosterone of hydrogen trans- fer between TPNH and DPN in rat pro- static tissue. DPNH and TPNH serve rather different metabolic functions (c/. Talalay and Williams-Ashman, 1958), and it is possible that steroid-mediated trans- hydrogenations might exert a controlling influence over the balance between the oxi- dized and reduced forms of pyridine nucleo- tides in the extramitochondrial regions of certain cells. However, at present there is no direct evidence in support of this hypothesis (cf. Talalay and Williams-Ashman, 1960). E. COAGULATION OF SEMEN Mammalian semen is emitted from the urethra as a liquid. In some species, e.g., the bull and the dog, the semen remains permanently in the liquid state. But the seminal fluid of many other mammals may undergo remarkable changes in its physical IM^operties on standing. Rodent semen clots rapidly and, if ejaculated into the vagina, forms a solid vaginal plug. This structure assists fertilization by preventing an out- flow of semen from the vagina after copu- lation (Blandau, 1945). The subsequent dissolution of the vaginal plug, probably as the result of the action of leukocytic en- zymes, was studied by Stockard and Pajia- nicolaou (1919). A copulatory plug lias also been described in certain Insectivora, Chiroptera, and Marsupiala (Camus and Gley, 1899; Engle, 1926a; Courrier, 1925; Eaclie, 1948a, bj. It has been stated that in the opposum (Hartman, 1924) and in the bat (Courrier, 1925), the vaginal plug results from the coagulation of the female secretions by seminal plasma. However, the semen of many other species clots on its own accord. Camus and Gley (1896, 1899) were the first to recognize that in the rat and guinea pig, the clotting process involves the solidi- fication of the vesicular secretion by an enzyme of prostatic origin, which they termed vesiculase. The classical experi- ments of Walker (1910a, b) showed that this enzyme is secreted solely by the an- terior prostate or "coagulating" gland. In the rhesus monkey, the secretion of the cranial lobe (but not of the caudal lobe) of the prostate gland coagulates the vesicular secretion (van Wagenen, 1936) . The "soft calculus" frequently present in the urinary bladder of male but not female rats is l^robably formed by clotting of the seminal vesicle secretion by the action of enzymes from the coagulating gland (Vulpe, Usher and Leblond, 1956) . More recently, the mechanism of action of vesiculase has been studied in consider- able detail. A crude preparation of the pro- teins of the vesicular secretion that are clotted by this enzyme can be obtained in a stable form, and the clotting process may l)e measured quantitatively by simple spec- trophotometric procedures (Gotterer, Gins- burg, Schulman, Banks and Williams-Ash- man, 1955; Gotterer and Williams-Ashman, 1957; Zorgniotti and Brendler, 1958). The over-all coagulation process is extremely sensitive to the ionic strength of the solu- tion in which it takes place, and is abolished by the addition of metal chelating agents such as Versene (ethylcnediaminetetra- acetic acid), o-i)henanthroline, and a,a- dipyridyl, and also by heavy metals such as mercuric ions. The inhibitory action of Versene can be overcome by manganous ions, or by somewhat higher concentrations of calcium ions. Experiments involving the delayed addition of either heavy metal ions or of metal chelating agents established that till' coagulation process can be separated ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 397 into two distinct phases (Gotterer and AVilliams-Ashman, 1957). The first of these requires a metal ion such as Mn++, is in- hibited by Versene, and does not necessarily involve the precipitation of insoluble ma- terial. The second phase, which is insensi- tive to the action of metal chelating agents, is inhibited by mercuric ions and leads to the formation of a coagulum. The coagu- lated material is protein in nature. Further fractionation of the vesicular secretion by Speyer (1959) led to the iso- lation of a heat-stable protein, coagulino- gen, which is the precursor of the insoluble material of the vaginal plug, but is not clotted by vesiculase. Speyer (1959) iso- lated another, heat-labile protein from vesicular secretion which he designated procoagulase, and which is converted into a clotting enzyme coagulase by the action of vesiculase. The coagulation of the semi- nal vesicle secretion by the prostatic en- zyme vesiculase thus seems to take place by the following reactions: „ , Vesiculase „ , rrocoagulase > Coagulase Coagulinogen °^^" '^^^ — ^ Coagulated protein Only the first reaction is inhibited by Versene. Partial purification of vesiculase has l>een achieved (Gotterer, Ginsburg, Schul- man. Banks and Williams-Ashman, 1955). Vesiculase is quite distinct from another enzyme in the secretion of the coagulating gland of guinea pigs which hydrolyzes, in- ter alia, tosyl-L-arginine methyl ester (TAMe) (Gotterer, Banks and Williams- Ashman, 1956). Unlike thrombin, vesicu- lase does not hydrolyze TAMe and does not clot fibrinogen. The dissimilarity between the coagulation of blood and of semen is further borne out by the failure of thrombin to clot the proteins of the vesicular secre- tion, and by the inability of TA]\Ie (which depresses the action of thrombin) to inhibit vesiculase action. Electrical stimulation of the head of the guinea pig induces ejaculation without voiding of either urine or feces (Batelli, 1922). Ejaculates obtained in this manner from normal, sexually mature guinea pigs coagulate rapidly. After castration, the se- men is no longer coagulable, but becomes so a few days after treatment with andro- gens (Moore and Gallagher, 1930). This "electric ejaculation test" can be used as an indicator for androgenic activity (c/. Sayles, 1939, 1942). It is generally believed that human se- men is ejaculated as a fluid, and then co- agulates (Lane-Roberts, Sharman, Walker and Wiesner, 1939; Joel, 1942; Huggins and Neal, 1942; Lundquist, 1949), although some authors state that it is emitted in a gelatinous form (Pollak, 1943; Hammen, 1944; Oettle, 1954). But there is no doubt that the semen from normal men subse- quently liquifies if kept at room tempera- ture.^ Human semen possesses strong fi- brinolytic activity (Huggins and Neal, 1942; Harvey, 1949; Ying, Day, Whitmore and Tagnon, 1956). The prostate gland of men secretes a proteolytic enzyme, fibrino- lysin, which is probably responsible for the phenomenon of liquefaction. Prostatic fi- brinolysin is produced in large amounts by certain cancers of the prostate in man, and seems to enter the circulation since there is a pronounced bleeding tendency in such patients (Tagnon, Schulman, Whitmore and Leone, 1953; Scott, Matthews, Butter- worth and Frommeyer, 1954; Swan, Wood and Owen, 1957). Canine semen, which does not clot, contains little fibrinolysin, but is rich in another proteolytic enzyme, fibrinogenase, which hydrolyzes fibrinogen. Little fibrinogenase is present in human semen (Huggins and Neal, 1942). The pres- ence of related proteolytic enzymes in the secretions of the male accessory glands is described above. ^ Louis Nicolas Vauquelin published the first paper on the chemistry of seminal fluid (Vau- quelin, 1791). This remarkable study includes a detailed and accurate account of the liquefaction of human semen which is quite unexcelled by later writings. It also describes the formation, in ejacu- lates which had stood for three or four days, of "cristaux transparens, d'environ une hgne de long, tres-minces, et qui se croisent souvent de maniere a representer les rayons d'une roue. Ces cristaux isoles nous ont offer, a I'aide d'un verre grossissant, la forme d'un solide a quatre pans, termines par des pyramides tres-allongees, a quatre faces." Al- though Vauquelin believed that these crystals were composed of calcium phosphate, Mann (1954a) has pointed out that he had, in reality, obser\ed the deposition of spermine phosphate in aged semen. 398 PHYSIOLOGY OF GONADS Freund and Thompson (1957) reported that intravenous injection of crude guinea pig coagulating gland secretion into rab- bits or guinea pigs induces hypotensive shock. Edema results if the secretion is injected locally. The secretion of the co- agulating gland of the rat does not possess these properties. Further studies by Freund, Miles, Mill and Wilhelm (1958) showed that two main protein fractions can be sep- arated from the secretion of the guinea pig coagulating gland by preparative starch electrophoresis. Fraction I was hypotensive and a potent permeability factor in rabbits and guinea pigs. It hydrolyzed TAMe rap- idly and may be identical with the TAMe- hydrolyzing enzyme described in guinea pig coagulating gland l)y Gotterer, Banks and Williams-Ashman (1956). The latter enzyme is not present in the coagulating gland of the rat. Fraction II isolated by Freund and his associates is ]n"obably vesic- ulase. III. Structure and Function in Relation to Hormones A. INTRODUCTION Some of the effects of removal of the testes in males have been recognized ever since castration was first practiced on man and domestic animals. Aristotle's writings include accurate descriptions of the effects of castration on secondary sex characters in birds and in man. The classical studies of John Hunter (1792) laid the basis for an understanding of the relation between the presence of the testes and the size and functional state of the accessory reproduc- tive glands of mammals, although he did not postulate the existence of testicular hormones. Hunter demonstrated experimentally that the seminal vesicles of guinea pigs are not reservoirs for semen and concluded that this a])plies to the seminal vesicles in man and in other mammals. He not only de- scribed the gross anatomy of the seminal vesicles in many species (hedgehog, iiiole. man, boar, bull, horse, buck, mouse, rat, beaver, guinea pig) and their absence fi'om others, but he observed tliat they arc smaller in the gelding than in the stallion. In refei'ence to other glands, he generalized that "the prostate gland, Cowper's glands and the glands along the urethra . . . are in the perfect male large and pulpy, secreting a considerable quantity of slimy mucus which is salt to the taste . . . while in the castrated animal these are small, flabby, tough and ligamentous, and have little se- cretion." In addition, he made the equally important discovery that the testes of mammals (and birds as well) are very small in winter in animals ''which have their seasons of copulation" and the semi- nal vesicles and prostates are "hardly dis- cernable." He concluded that "from these observations it is reasonable to infer that the use of the vesiculae in the animal oeconomy must, in common with many other parts, be dependent upon the testi- cles." Over 100 years later, many of his obser- vations were rediscovered, extended, and interpreted in the light of the first demon- stration that the testis is an endocrine or- gan (Berthold, 1849). In the early part of the 20th century the interest in attempting to isolate and characterize androgens from testis tissue and urine led to a search for rapid and dependable bioassay methods. The cock's comb provided a sensitive and convenient test object (Pezard, 1911). In addition, some of the accessory reproduc- tive glands of mammals were found to atrophy rapidly after castration and proved also to be sensitive indicators for the pres- ence of androgenic hormones. Cytologic tests using the rat prostate, seminal vesi- cles, and Cowper's glands were developed and an electric ejaculation test in the guinea l)ig was devised (Moore, 1932, 1939). Weights, sizes, cytologic structure, and mi- totic activity in mouse seminal vesicles were suggested as bioassay methods for an- drogenic hormones (Deaneslv and Parkes. 1933). After th(> successful isolation and chemi- cal characterization of androgens and estro- gens from various sources, interest cen- tered on the fundamental relationships of androgens to normal develojjment, histo- logic structure, and secretory activity of the accessory glands in many species of inainmals. The effects of estrogens and ges- tagens and the competitive and synergistic i'elationshii)s of steroid hormones were ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 399 examined. The results of this early work contributed extensively to the fields of bio- chemistry, biology, and medicine. More recently there have been studies on the rela- tion of hormones to the ultrastructure, his- tochemistry, and metabolism of the glands, and to the chemical composition of their secretions ( Section II I . In the following section, the hormonal control of structure and function will be discussed with particular reference to the luniierous studies on the prostate glands and seminal vesicles of rats and mice. B. EFFECTS OF ANDROGENS The term androgen will be used in the collective sense for substances that are capable of stimulating accessory repro- ductive glands in castrated animals and maintaining normal histologic structure and secretory activity in the epithelium. Andro- genic substances are formed by the testes, ovaries, and adrenal cortex. All androgens which have been characterized are steroids. The urine contains many androgen metabo- lites, mainly in the form of their conjugates with either glucuronic or sulfuric acids. Testosterone is the principal androgen se- creted by the testis and this substance, or tiie longer acting testosterone propionate, is most commonly used as a replacement for testicular androgen. In the last two decades a number of unnatural androgens [e.g., 17a-methyl testosterone) have been synthesized and found to possess strong biologic activity. The relationship between chemical structure of steroids and andro- genic activity in a variety of bioassay pro- cedures is discussed by Dorfman and Ship- ley (1956). 1. Testicular Androgens The effects of endogenous and exogenous androgen on weight, histologic structure, and secretory activity of the accessory glands have been reviewed by Moore (1939), Price (1947), Burrows (1949), Dorfman (1950), Dorfman and Shipley (1956) and many others. Aspects of meta- bolic activity have been treated by Roberts and Szego (1953) and Mann (1954a). The first detailed cytologic studies of male accessory glands and the changes fol- lowing castration and hormone administra- tion were made on the prostates, coagulat- ing glands, and seminal vesicles of adult rats (Moore, Price and Gallagher, 1930; Moore, Hughes and Gallagher, 1930). Ex- tensive research on structure and function of these and other accessory glands in many species followed this early work, but the cytologic structure of prostates and seminal vesicles of rats and mice remains one of the most sensitive indicators for androgenic hormones. Rat PROSTATE AND SEMINAL VESICLES. Ven- tral prostate. In the normal adult gland, the columnar secretory epithelium has basal nuclei with conspicuous nucleoli and chro- matin particles, and a supranuclear clear zone or light area in the cytoplasm corre- sponding to the position of the Golgi zone (Figs. 6.8, 6.9, and 6.14). In osmium prep- arations, the Golgi apparatus appears as Fics. (3.8 Axn 6.9. Rat ventral prostate from a normal adult male. X 5UU and lOUU. Boinn- hematoxylin preparations. (From C. R. Moore, D. Price and T. F. Gallagher, Am. J. Anat., 45, 71-107, 1930.) 400 PHYSIOLOGY OF GONADS heavy strands or networks (Figs. 6.19 and 6.22) which do not conform precisely to the shape or area of the cytoplasmic clear zone. Mitochondria are distributed as rods or granules in all parts of the cell. The se- cretion in the lumina of the alveoli is eo- sinophilic and mainly granular. A basement membrane rests on a stroma of connective tissue containing smooth muscle strands and blood vessels. Occasional small basal cells are wedged between the tall secretory cells. These observations were made by light microscopy of tissues fixed and stained by routine methods (Moore, Price and Gal- lagher, 1930). Electron microscopy (Harkin, 1957a) shows that the epithelial cells have an en- doplasmic reticulum or ergastoplasm com- posed of membrane-lined sacs with a finely granular component in the spaces between them (Figs. 6.27 to 6.29) ; the outside of the thin membrane is studded with Palade's granules (Palade, 1955). The arrangement of the sacs tends to parallel the long axis of the cells, but in cross section the pattern ai)pears concentric or lamellar, particularly in the supranuclear region (Fig. 6.29). The ergastoplasmic sacs occupy more space than the matrix apically, but basally the two are equally prominent (Fig. 6.28). The mem- TABLE 6.6 Summary of the effects of testicular androgen, on the rat prostate and coagulating glands Normal Males Castrated Males General Characteristics All lobes alveoli witli folded inueosa; secretion in the lumina.. columnar epithelial cells: cytoplasm granular or foamy. supranuclear clear zone in cytoplasm (ventral lobe) Golgi supranuclear networks mitochondria as rods or granules nuclei basal or central stroma of connective tissue and smooth muscle Size reduced; villi lost; secretion reduced Size reduced; pseudostratified; cytoplasm less dense Clear zone lost Reduced in amount; fragmented Still numerous but reduced in relative numbers Shrunken and pyknotic Increased fibromuscular tissue Specific Characteristics Ventral lobes Histochemical observations: secretion in lumina strongly PAS- and alkaline phosphatase-positive . . . cytoplasm weak PAS, strong alkaline phosphatase activity; basophilic reaction except in clear zone Golyi accumulations of PAS-positive granules stroma some alkaline phosphatase activity Electron microscopic observations: cytoplasm moderately distended ergastoplasmic sacs Golgi supranuclear microvesicular complex mitochondria numerous, prominent apically Lateral lobes Histochemical observations: cyioplasm luminal border organelle with high concentrations of zinc and basophilic material; osmiophilic, argentophilic nucleoli high concentrations of zinc and marked basophilia stroma high concentrations of zinc; basojihilic material (jresent Dorsal lobes Histochemical observations: cytoplasm in apical region strongly basophilic; basally, some zinc nucleoli high concentrations of zinc and marked basophilia stroma basophilic material; strong alkaline i)hosphatase reaction Electron microscopic observations: cytoplasm distended ergastoplasmic cisternae; Coagulating glands (anterior prostate) Histocheinical observations: secretion strongly PAS-positive cytoplasm weak P.\S reaction stroma some alkaline phosphatase activity Electron microscopic observations: cyioplasm extremely dilated ergastoplasmic cisternae Some phosphatase activity retained Phosphatase activity low Sacs collapsed; granvilar component reduced Reduced in size Reduced in relative numbers Cisternae collapse granules reduced pears unalterc Cisternae collapsed; granules reduced ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 401 hrane is continuous with the outer nuclear membrane. The Golgi complex is conspicu- ous as microvesicles midway between nu- cleus and lumen. Mitochondria lie in the matrix between the sacs and are very prom- inent in the most apical region. Microvilli project into the lumina of the alveoli with no interruption of the cytoplasmic, or plasma, membrane. The membrane at the base of the cell is double (see Fig. 6.28) ; one component, the basement membrane, continues unbroken under adjacent cells; the second forms a part of the double plasma membrane between cells. Brandes and Groth (1961) have confirmed Har- kin's findings and added further observa- tions. Nuclei contain patches of granules which are frequently along the inner nu- clear membrane; the Golgi complex consists of vesicles, vacuoles, and parallel mem- l)ranes; vesicles and granules surrounded by smooth-surfaced membranes are dis- posed in the cytoplasmic matrix and are more numerous apically; the dilated sacs or cisternae of the supranuclear region seem to intercommunicate. Histochemical studies of basophilia, al- kaline phosphatase activity, and the locali- zation of i^eriodic acid-reactive carbohy- drates (Periodic acid-Schiff or PAS reaction) add further information (Table 6.6). Davey and Foster (1950) found baso- philia (which was abolished by ribonu- clease) distributed through the cytoplasm except in the clear area described by Moore, Price and Gallagher (1930) as correspond- ing to the position of the Golgi zone. Stroma of the ventral prostate shows some degree of alkaline phosphatase activity but lumi- nal secretion and epithelial cells are strongly positive (Bern, 1949a), especially at the luminal and basal borders (Stafford, Rubenstein and Meyer, 1949). The secre- tion also gives a fairly intense PAS reac- tion whereas the epithelial cells are only slightly reactive; occasionally the Golgi apparatus is visible as PAS-positive gran- ules (Leblond, 1950). After castration, there is reduction in cell height and loss of the cytoplasmic clear zone (Fig. 6.15) within 4 days. On subse- cjuent days, cell size continues to decrease and nuclei become small and pyknotic (Figs. 6.10. 6.16 to 6.18). The Golgi ap- paratus begins to fragment by 10 days; by 20 days it consists of granules much re- duced in amount (Fig. 6.20) and the base- ment membrane of the cells disappears (Moore, Price and Gallagher, 1930). Harkin (1957a) reported changes ob- servable by electron microscopy within 24 hours after castration; distention of apical ergastoplasmic sacs and reduction in size and number of microvilli. By 2 days, there is dilation of Golgi microvesicles, collapse of the apical ergastoplasmic sacs, and re- duction in mass of apical cytoplasm; at 4 days, massive collapse of sacs, reduction in mitochondrial number, and increase in electron-dense bodies (Fig. 6.30). The granular component is not reduced until 8 days after castration or longer. Brandes and Portela (1960a) noted, briefly, collapse in the cisternae of the ergastoplasm, loss of the ribonucleic acid- (RNA) rich granules from the membranes of the endoplasmic re- ticulum, and apparent increase in mito- chondria but with a reduction in their size (Table 6.6). The distribution of alkaline phosphatase in the stroma, epithelium, and secretion is unchanged 32 days after castration; the stroma is still reactive at 120 days but the epithelium is completely atrophic (Bern and Levy, 1952). (Quantitative determina- tions of alkaline and acid phosphatases showed, however, that activities of both enzymes are reduced markedly by 8 days (Stafford, Rubenstein and Meyer, 1949). The epithelium loses the ability to secrete citric acid (see Section IT). Changes after gonadectomy are pre- vented or reversed by administration of androgenic substances. Extracts of bull testes (:\Ioore, Price and Gallagher, 1930) prevented involution of the epithelium in castrates (Fig. 6.11 and 6.21) and androst- erone, testosterone, and testosterone pro- pionate prevented or repaired castration changes CMoore and Price, 1937, 1938). The response of the castrate to androgen is rapid; cell hypertrophy begins within 23 hours after a single injection of testost- erone propionate into males castrated for 40 days ; at 35 hours mitotic activity begins and reaches a maximum at 43 hours (Burk- hart, 1942). Ergastoplasmic sacs in the epithelial cells 402 PHYSIOLOGY OF GONADS ;;^.. Da .^.^^ I I'- <; II) 0.13. Rat ventr;il lu-o-tatc (Figs. 6 10. H.U) and coagulal iim aland ( Fm- C) 12, (i.lo). All pliotomicrographs , lOUU. Fig. 6.10. 20-day cabtiatc. Fig. 6.11. 20-day ca.^tratf in- jected with testis extract. Fig. 6.12. 20-day castrate. Fig. 6.13. 20-day castrate injected with testis extract. (From C. R. Moore, D. Price and T. F. Gallagher, Am. J. Anat., 45, 71-107, 1930.) are prevented from collapsing by treatment of castrates with testosterone and the proc- ess is reversed if the androgen is given after castration changes have developed (J. C. Harkin, personal communication). Alka- line and acid phosphatase levels are essen- tially normal in castrates injected with testost(M'one propionate (Stafford, Rubin- stein and Meyer, 1949). Lateral prostate. The epithelial cells in normal adult glands are columnar and the nuclei are basal, but cell size and nuclear position arc more variable than in the ven- tral prostate ( Korenchevsky and Dcnnison. 193.51. The Golgi apparatus appears as l^rominent supranuclear networks in os- mium stainecl preparations (Rixon and Whitfield, 1959). Histochemical studies employing a di- thizone zinc stain demonstrated high con- centrations of zinc in the apical jiart of the cells (Gunn and Gould, 19o6a). Fleisch- hauer (1957) observed (macroscopically) heavy staining that was visible in this lobe after intravenous or subcutaneous injec- tions of dithizone. In mifix(Hl frozen sections, he found in tlic basal regions of all epi- thelial cells numerous stained granules which lie interpreted as zinc-positive ma- terial. 'Hie nature of a rather wide diffusely ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 403 m m IB IB m 0'^ IE m m m m Figs. 6.14-6.26 Figs. 6.14-6.22. Rat ventral prostate. Figs. 6.14-6.21. Camera lucida drawings X 3000. Figs. 6.19-6.22. Mann-Kopsch preparations for Golgi apparatus. Fig. 6.14. Normal male. Figs. 6.15- 6.18. From males castrated for 4, 10, 20 and 90 days. Fig. 6.19. Normal male. Fig. 6.20. 20-day castrate. Fig. 6.21. 20-day castrate injected with testis extract. Fig. 6.22. Normal male ; photo- micrograph X 1000. (From C. R. Moore, D. Price and T. F. Gallaglier, Am. J. Anat., 45, 71- 107, 1930.) Figs. 6.23-6.26. Rat coagulating gland. Figs. 6.23-6.25. Camera lucida drawings X 3000. Fig. 6.23. Normal male. Fig. 6.24. 20-day castrate. Fig. 6.25. 20-day castrate injected with testis extract. Fig. 6.26. Normal male; photomicrogaph X 1000; Mann-Kopsch preparation for Golgi apparatus. (From C. R. Moore, D. Price and T. F. Gallagher, Am. J. Anat.. 45, 71-107, 1930.) 404 PHYSIOLOGY OF GONADS ■^f «# Fig. 6.27. R:it xcniinl |.in~i,ii( ikhihiI mil. I ,li . 1 1 uiiiuicrograpli X 8500; LuftV ])prman- ganate fixative. Aliciuvilli lxIlikI a^ i)iul()n^atioii.-. ol the cytoplasm into the lumen; a major part of the cytoplasm is a labyrinth of ergastoplasmic sacs with scattered mitochondria ; nu- clei are basal ; half-way between nucleus and cell apex is a zone of small vesicles and canals, the Golgi complex (From J. C. Harkin, un])u})lishe(l.) stained area in the ai)ical cytoi)lasin was not clear. Ki.xoii and Whitfield 11959) re- ported high concentrations of zinc in the apical cytoplasm, nucleoli, and stroma in fixed tissues stained with dithizone. Tn the apical cytoplasm, the zinc is conccnti atcd at the tip of the cells in a "luminal l)order organelle" which is osmiophilic (distinct from the (Jo].o;i apparatus), argent()i)hilic, and basophilic. Nucleoli and subepithelial sti'oma are basophilic. Castration results in a typical pattern of involution in the epithelial cells: size is !•(■( bleed, nuclei become small and jwknotic, and changes occur in the density of the cy- toplasm (Korenchevsky and Dennison, 1935; Price, Mann and Lutwak-Mann, 1955). The zinc content of the gland (dor- ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 405 solatcral or lateral prostate) and the rate of Zn*^^ uptake decrease after gonadectomy as does the secretion of citric acid and fruc- tose (see Section II). Dorsal prostate. The epithelium in the dorsal prostate of normal rats is columnar or cuboidal depending on distention of the alveoli; nuclei are basal and stain heavily; there is cytoplasmic vacuolization which is usually limited to the basal region (Koren- chevsky and Dennison, 1935) . Brandes and Groth (1961) described the ultrastructure of two different cell types in the dorsolateral (or dorsal) lobe. These types differ in the relation of cytoplasmic matrix to endoplasmic reticulum. In both, the matrix is moderatelv homogeneous and contains small particles, but in cell type 1, the matrix appears as separate profiles and the reticulum as membrane-bounded indi- vidual cavities. In cell type 2, the reticulum forms dilated membrane-bounded cisternae which are intercommunicating and the cy- toplasmic matrix is reduced mainly to thin strands appearing isolated within the cis- ternae. Gunn and Gould (1956a) reported a zinc-negative histochemical reaction in the epithelium of the dorsal prostate. Fleisch- hauer (1957) observed a slight dithizone- stain macroscopically, and in unfixed frozen sections, individual groups of cells contain the distinctive basal zinc-positive granules that are characteristic of all epithelial cells ...\ Fl(.i. li..'N. l;;il v,l,ii;,l |Mm-;;:h, n,uM,:,l in.ih. 1 ,1. r i ,, ,i i li i in . ,-i:i [ .1, . 2(;,()()(); 1 ),-| ll ( )ll's chrome o^niic and hxatixi . Il;i>:il pari ot epithelial cell to show the character of the granular component and ergastopla>iiiic -acs which are essentially equal in amount in this region. Double basement membrane indicated by arrow. (From J. C. Harkin, Endocrinology, 60, 185-199, 1957.) 406 PHYSIOLOGY OF GONADS ■:^lVi4 l'"i(..G.2U. Rat \(;iilial lUo.-Uilu, iiuinial iiiak;. I'^lcctionnu' acid fixative with sucrose. Supranuclear region of epithelia plasmic sacs. (From J. C. Harkin, unpublished.) .-laph Is.iiOO; Paladps osmic •ell sliowing laniellatetl ergasto- in the lateral lolje. Tiiere is no diffuse stain- ing of the apical cytoplasm. Nucleoli arc intensely zinc-positive after fixation and staining with dithizone; nucleoli, apical cy- toplasm and stroma are basophilic fRixon and Whitfield, 1959). The stroma is also strongly alkaline phosphatase - positive (Bern, 1949a). Epithelial cells respond to castration l»y reduction in cell and nuclear size, and loss of granulation in the cytoplasm (Koren- chevsky and Dennison, 1935). Brandes and Portela (1960a) observed in electron micro- graphs the V)eginning of collapse of the cisternae of the endoi)lasmic reticulum, re- duction in RNA-rich particles, and changes in mitochondria. Histochemical studies ( Iicni and Levy, 1952) indicate that distri- bution of alkaline phosphatase activity re- mains unchanged. Fructose content is re- duced in the gland after castration (Price, Mann and Lut\vak-]\Iann, 1955). CocKjulating gland {anterior prostdte) . In normal males, the ei)ithelium is columnar and rests on a well marked basement mem- brane; nuclei stain heavily and homogene- ously and ai'e situated midway between the basement meml)i';iiie and lumen. The cvto- ACCESSORY MAMMALIAN REPRODUCriVE GLANDS 407 plasm is not as granular as in the ventral prostate and appears vacuolated, particu- larly in the basal region and around the nuclei; the apical cytoplasm is condensed and granular (Fig. 6.13, a gland from a castrated male injected with testicular ex- tract, illustrates essentially the characteris- tics of the normal epithelium). Golgi bodies ( Fig. 6.26) form large networks close to the luminal end of the cells (Moore, Price and Gallagher, 1930). The striking characteristic of these cells in electron microscopy (Brandes, Belt and Bourne, 1959; Brandes and Groth, 1961) is the great dilation of the cisternae of the endoplasmic reticulum (Fig. 6.31) which fill the greatest part of the cell and are par- ticularly distended in the basal region. The Fig. 6.30. Rut \entral prostate, 4-day castrate. Electronmicrograph X 26,000; Dalton's chrome osmic acid fi.xative. Portion of nucleus and di.stal region of epithelial cell. An electron dense body lies above the nucleus and below dilatated Golgi microvesicles. Arrow points to collapsed ergastoplasmic sacs. (From J. C. Harkin, Endocrinology, 60, 185-199, 1957.) 408 PHYSIOLOGY OF GONADS Fig. 6.31. Rat coagulating gland, normal male. Electronmiciogiaplis, lefl X 7200; upper and lowei- right X 39,000. Caulfield's modification of Palade's osmic acid fixative. Left, ba.sal por- tions of two epithelial cells; right, details of basal region: bni, basement membrane; ci, dilated cisternae; cm, plasma membrane; cy, cytoplasmic matrix; G, Golgi complex; bn, limiting membrane of endoplasmic reticulum; w, mitochondria; n, nucleus. (From D. Brandes, unpublished.) cytoplasmic matrix appears as strands within the cisternae. The Golgi complex is represented by parallel rows of mem- branes, vacuoles, and smaller vesicles. Histochemically (Table 6.6), the secre- tion is intensely PAS-positive and the cyto- plasm is slightly reactive (Leblond, 19501. The stroma is strongly alkaline phospha- tase-positive (Bern, 1949a). The effects of castration arc not ai)i)arent by light microscopy as early as in the ven- tral prostate and seminal vesicles. At 10 days after castration the cells are slightly smaller and the cytoplasm less dense; by 20 days, the cells are markedly reduced in size, nuclei smaller, cytoplasm clear, base- ment membrane absent or less well defined (Figs. 6.12 and 6.24). The Golgi apparatus is reduced in amount but not fragmontcnl. It still retains the shape of strands or threads which cap around tlic nucleus at 90 days of castration but the mass is re- duced (Moore, Price and Gallagher, 1930). Brandes and Portela (1960a) state that castration produces gradual and slow col- lapse of cisternae in the endoplasmic reticu- lum, changes in mitochondria, and reduc- tion and loss of RNA-rich particles from tlie membranes. Studies of functional ac- tivity show that the ability to secrete fruc- tose and vesiculase is lost (see Section II). Depending on the length of the interval between the operation and administration of the hormone, treatment of castrates with testis extracts (Figs. 6.13 and 6.25) or testosterone prevents or repairs histologic and functional changes. Seminal vc.'iicles. The secretory epithe- lium is colunuiar in normal males; nuclei are basal and contain one or two conspicu- ous nucleoli and smaller chromatin masses (Table 6.7). Seci-etion granules, surrounded ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 409 TABLE 6.7 Summary of the effects of testicular androgen on rat and mouse seminal vesicles Normal Males Castrated Males General Characteristics Rat and mouse mucosa folded; acidophilic secretion in lumen Villous folding reduced; secretion greatly reduced columnar epithelial cells: secretion granules supranuclear Cell size reduced; granules lost Golgi supranuclear networks \ Reduced in volume; fragmented mitochondria as rods or granules ' Apparently reduced in relative numbers (rat) nuclei basal; nucleoli prominent I Nuclei shrunken and pyknotic; nucleoli disappear stroma of connective tissue and smooth muscle Amount appears increased Specific Characteristics Rat Histochemical observations : secretion in lumen PAS-positive, intensity variable; acidophilic ci//opZasm slight PAS reaction; strongly acid phosphatase-positive; strongly basophilic secretion granules in epithelium strongly acid phosphatase-positive nuclei strong acid phosphatase reaction stroma slight PAS reaction; acid phosphatase-positive; strong alkaline phosphatase reaction Mouse Histochemical observations : secretion in lumen moderately PAS-positive and acidophilic cytoplasm moderately basophilic at base and lateral margins of cells;. . . apical granules acid phosphatase-positive secretion granules in epithelium weakly PAS-positive and acidophilic... Golgi region; granules PAS-positive and acidophilic stroma intensely PAS- and alkaline phosphatase-positive Electron microscopic observations: cytoplasm complex pattern of basal and lateral ergastoplasmic mem- branes; Phosphatase activity reduced Slight basophilia Activity lost Remained weakly acid phosphatase-positive Acid and alkaline phosphatase activity reduced Secretory granules less acidopliili- Weakly basophilic abundant RNA-rich granules Golgi region; parallel arrays of smooth-surfaced membranes and vesicles Reduced in number; less acidophilii Phosphatase activity reduced Ergastoplasmic channels less distended and con- torted Relative number reduced by vesicular zones are present in the supra- nuclear region (Fig. 6.36) and resemble the secretion in the lumen in staining reactions. The Golgi complex appears in osmium prep- arations as irregular networks or a vesicu- lar structure (Fig. 6.32); the basement membrane is poorly defined or absent (Moore, Hughes and Gallagher, 1930). The extracellular secretion is only slightly PAS-positive but varies in the intensity of reaction; there is little reaction in the epi- thelial cells except in some cells with stained granules; fibers of the lamina propria, smooth muscles, and walls of arterioles are weakly reactive (Leblond, 1950; ]\Ielampy and Cavazos, 1953). Stroma and capillaries are strongly alkaline phosphatase-positive (Bern, 1949a; Dempsey, Greep and Deane, 1949; :\lelarapy and Cavazos, 1953). The cytoplasm, secretion granules, nuclei, and stroma give an intense acid phosphatase reaction; the cytojilasm is strongly baso- philic and the reaction is abolished by ribo- nuclease (]\lelampy and Cavazos, 1953). The response to castration is rapid. In 2 days the cells are reduced in height mainly by reduction in apical mass ; secretion gran- ules are few, small, and indistinct. By 10 days, cells are small, secretion granules are gone, nuclei are small with heavily staining chromatin (Fig. 6.35), and Golgi bodies have begun to fragment; at 20 days, these changes are more advanced and the rem- nant of the Golgi bodies (Fig. 6.33) occu- pies almost the entire supranuclear region (Moore, Hughes and Gallagher, 1930). In a cytometric study, Cavazos and ]\Ielampy (1954) found a statistically significant re- duction in cell height by 6 hours after cas- tration; by 48 hours many nucleoli are smaller than normal and by 60 hours most nucleoli are small; nuclear diameters are reduced but change more slowly. 410 PHYSIOLOGY OF GONADS '0 (?rCf f-\ m m r-^ Q' ''1 ^ Figs. 6.32-6.36. Rat seminal vesicle; camera lucida drawing.s ,: 3000. Figs. 6.32-6.34. Manu- Kopsch preparations for Golgi apparatus. Fig. 6.32. Normal male. Fig. 6.33. 20-day castrate. Fig. 6.34. 20-day castrate injected with testis extract. Fig. 6.35. 10-day castrate. Fig. 6.36. 20- day castrate injected with testis extract. (From C. R. Moore, W. Hughes and T. F. Gallagher, Am. J. Anat., 45, 71-107, 1930.) Gonadectomy causes gradual reduction and disappearance of alkaline phosphatase activity (Dempsey, Greep and Deane, 1949; Melampy and Cavazos, 1953) and hypophy- sectomy, with consequent diminution of testicular hormones, gives similar results (Dempsey, Greep and Deane, 1949). Bern and Levy (1952) reported some retention of alkaline phosphatase activity in the fibro- muscular tissue of castrates. Acid phospha- tase activity decreased within 10 days fol- lowing castration (Melampy and Cavazos, 1953). In early experiments (Moore, Hughes and Gallagher, 1930) , administration of bull testis extracts to castrated rats maintained normal histologic structure or repaired in- volutional changes (Figs. 6.34 and 6.36), and androsterone, testosterone, and testos- terone propionate gave similar resvdts (Moore and Price, 1937, 1938). Androgen treatment in castrates produced detectable changes within 2 days. Burkhart (1942 1 observed cell hypertrophy ahd enlargement of nuclei 23 hours after a single injection of testosterone propionate into 40-day cas- trates; mitotic activity began at 35 hours and reached a maximum at 43 hours. Cava- zos and Melampy (1954) treated castrates with testosterone propionate and found in- creases in nuclear diameter within 12 hours, cell height within 24 hours, and nucleolar size l)y 36 hours; mitotic activity was evi- dent at 48 hours. The same hormone re- stoi'cd normal alkaline and acid phospha- tase activity in castrates within 10 days ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 411 (Dempsey, Grecp and Deane, 1949; Me- lampy and Cavazos, 1953). Mouse prostate and seminal vesicles. Ventral prostate. The epithelial cells in the adult gland are low to moderately tall columnar; acini are surrounded by a thin fibromuscular layer; lumina contain finely granular, acidophilic secretion. The cyto- plasm appears somewhat foamy with a clear zone in the supranuclear Golgi region and rather dense basophilia near the lumen (Franks, 1959). In approj)riate histologic preparations, the Golgi apparatus is visible as a network in the apical cytoplasm close to the nucleus and the twisted strands are oriented parallel to the long axis of the cell (Horning, 1947). Brandes and Portela (1960c) observed by electron microscopy an endoplasmic reticu- lum of cisternae or vesicles that are usually flattened but have dilations. RNA-rich granules are attached to the outer surface of the thin membranes bounding the vesicles and occur also in the cytoplasmic matrix; the arrangement of cisternae may be paral- lel or in a random pattern. The luminal margin of cells exhibits small cytoplasmic projections covered by the cell membrane. There are also extensions of the margin which ai)pear similar to fragments of cyto- plasm that seem to lie free in the lumen, and are presumably detached from the api- cal tips of cells. The lumina also contain structures that resemble profiles of the en- doplasmic reticulum and mitochondria. The supranuclear Golgi complex consists of vacuoles of various sizes and flattened vesi- cles; endoplasmic reticulum and mitochon- dria are present in the Golgi zone. Histochemical findings (Table 6.8) indi- cate alkaline phosphatase activity in the stroma with a positive reaction in the base- ment membrane, endothelium of blood ves- sels, and sheaths of smooth muscle fibers (Brandes and Bourne, 1954). With longer incubation periods of the tissue (Bern, 1949a), the epithelium and secretion in the lumina are strongly reactive and the stroma shows some activity. Brandes and Bourne (1954) reported acid phosphatase activity in the epithelium; the Golgi region gave the strongest reaction and the nuclei were moderately positive. Luminal secretion, ag- gregations of granules in the Golgi region and apical cytoplasm, basement mem- branes, and capillary endothelium were PA8-positive. Sulfhydryl and disulfide re- actions were moderately strong in epithelial cells and basement membranes. Gonadectomy results in typical cell retro- gression with reduction in cell height and nuclear size. Brandes and Bourne (1954) summarized their results as follows: after gonad removal, the Golgi apparatus showed some fragmentation and was not so dense by 12 to 14 days; alkaline phosphatase ac- tivity was slightly less intense by 4 days; changes in acid pliosphatase activity in the Golgi region were evident by 4 days and marked by 21 to 22 days; the PAS reaction was reduced by 8 days and almost lost in the epithelium by 21 to 22 days. Subcu- taneous implantation of pellets of testos- terone propionate 13 to 32 days after cas- tration produced a rapid return to normal of Golgi apparatus and phosphatase ac- tivity and a gradual recovery of normal PAS reactions. Allen (1958) reported a sig- nificant increase in mitotic activity in the epithelium of 30-day castrates within 30 to 36 hours following a single injection of 16 /xg. of testosterone propionate; peak ac- tivity was reached in 42 to 48 hours. Dorsal prostate. The epithelial cells re- semble rather closely those of the coagulat- ing gland but the cytoplasm is more granu- lar, the centrally placed nuclei darker, and the Golgi apparatus in the apical cyto- plasm (in close contact with the nucleus) is less dense than the Golgi networks in the coagulating gland (Horning, 1947). Histo- chemically, the distribution of phosphatase activities and PAS reaction in normal males, castrates, and castrates treated with testosterone propionate are similar to the findings in the coagulating gland (Bern, 1949a, 1951; Brandes and Bourne, 1954). Coagulating gland {anterior prostate). The secretory cells are columnar and the nuclei are approximately midway between basement membrane and lumen. The cyto- plasm is granular and the condensed Golgi apparatus is a flattened network oriented transversely in the most apical region of the cytoplasm (Horning, 1947). Electron microscopic studies by Brandes 412 PHYSIOLOGY OF GONADS TABLE 6.8 Summary of the effects of testicular androgen on the mouse prostate and coagulating glands Normal Males Castrated Males General Characteristics All lobes alveoli with folded lucosa; secretion in luniina. columnar epithelial cells cytoplasm of epithelial cells granular or foamy nuclei basal . stroma of connective tissue and smooth muscle Histochemical observations: secretion in lumina PAS-positive cytoplasm acid phosphatase-positive; sulfhydryl reaction intracytoplasmic granules PAS-positive; near luminal border Golgi region PAS-positive granules; strong acid phosphatase activity. basement membrane PAS- and alkaline phosphatase-positive Stroma PAS- and alkaline phosphatase-positive Electron microscopic observations: epithelial cells with microvilli Golgi complex smooth surfaced membranes and vesicles Iveolar size; loss of villi and bulk of Reduction i secretion Reduction in cell size; pseudostratification .\ppears less dense Shrunken and pyknotic Fibromuscular increase Almost completely negative Phosphatase activity reduced Almost completely negative Almost completely negative Activity retained; less intense Activity retained in sheaths of smooth muscles Cell size reduced Specific Characteristics Ventral lobes Histochemical observations : secretion in lumina strong alkaline phosphatase activity cytoplasm alkaline pliosphatase-positive Golgi loose networks in apical cytoplasm Electron microscopic observations: cytoplasm; ergastoplasm with generally flattened cisternae.. Dorsal lobes Histochemical observations : Golgi compact networks in apical cytoplasm Coagulating glands (anterior prostate) Histochemical observations : Secretion in lumina intense protein reaction; PAS-positive;. sulfhydryl reaction cytoplasm high concentrations of RNA basally and apically protein reactions, intense apically sulfhydryl reaction, especially strong apically Golgi condensed apical networks Electron microscopic observations: cytoplasm extremely dilated ergastoplasmic cisternae Reduced in amount; fragmented Cisternae collapsed; reduced granules Retluced in amount; fragmented Some PAS reaction retained Sulfhydryl reaction lost Markedly decreased Greatly reduced Reaction lost Reduced in amount; fragmented Cisternae collapsed; granules reduced and Portela (1960b) show that these epi- thelial cells are characterized by an endo- plasmic reticulum with greatly dilated cis- ternae. This dilation is more marked in the middle of the cell and in the basal re- gion (Fig. 6.37) where the dilated cisternae appear as intercommunicating channels in which the cytoplasmic matrix forms iso- lated profiles or strands containing mito- chondria and other organelles. The matrix is more abundant in the Golgi region and protrudes from the luminal margin of the cells as microprojections covered by tlu; cell membrane. Alkaline phosphatase activity is localized in the stroma (Bern, 1949a, 1951 ; Brandes and Bourne, 1954; Bern, Alfert and Blair, 1957) ; acid phosphatase activity (Brandes and Bourne, 1954) is found in the epithe- lium and is particularly strong in the Golgi zone. Brandes and Bourne (1954) and Bern, Alfert and Blair (1957) reported PAS-posi- tive reactions in the epithelial cells in the Golgi region and apical cytoplasm, and in- tense reactions in luminal secretion, base- ment membrane, and stroma. Sulfhydryl and disulfide reactions are evident in lumi- nal secretion, epithelium (especially in the a]ucal region), basement membrane, and fibromuscular tissue. The reactions are stronger than in the ventral prostate. Bern, Alfert and Blair (1957) found high con- ACCESSORY MAMMALIAN REPRODTTCTIVE GLANDS 4i; cy /Jf-!^ pc N.pc :y . .... ^ cy \ m ..^cy ^"^^ ^m V*' ^ cy IS Fig. 6.37. Mouse coagulating gland, normal male. Electronmicrograph X 39,000. Caulfield's modification of Palade's osmic acid fixative. Basal portion of an epithelial cell; insert, de- tails of basement membrane region: bm, basement membrane: ci, dilated cisternae: cm, plasma or cell membrane; cy, cytoplasmic matrix; ?n, mitochondrion: /;, nucleus. (From D. Brandes, unpublished.) centrations of RNA basally and apically in the epithelial cells. Strong protein reactions are present in luminal secretion and apical regions of the cells. The response to gonadectomy (Brandes and Bourne, 1954) includes reduction of al- kaline and acid phosphatase activity within 4 days, PAS reactions by 8 days, and slight fragmentation and loss of density of the Golgi apparatus by 12 to 14 days. Changes are more marked after longer periods of castration (Table 6.8), although Bern (1951) observed retention of stromal alka- line phosphatase for long periods. RNA concentrations in the cell (Bern, Alfert and Blair, 1957) are greatly decreased but some 414 PHYSIOLOGY OF GONADS Fig. 6.38. Mouse seininul \esick\ norniMl male. Pliotomicrograpli preparation. (From E. Howard, Am. J. Anat., 65, 105-149, 1939.) 50. Houm-liematoxvlin accunuilation remains in the apical region. The sulfhydryl reaction is partially lost luit the cells retain apical reactivity. Testosterone propionate implanted sub- cutaneously 13 to 32 days after testis re- moval (Brandes and Bourne, 1954) rapidly restored the Golgi apparatus and enzyme activity to normal, and the PAS reaction returned gradually. Allen (1958) showed that the epithelium of 30-day castrates re- sponds to a single injection of 16 fig. of testosterone propionate by an increase in mitotic activity within 30 to 36 hours. Se7ninal vesicles. The epithelial cells in adult glands are colunniai- with basal nuclei and secretory granules surrounded by halos in the supranuclear cytoplasm (Fig. 6.35) ; the epithelium rests on a layer of smooth muscle and connective tissue stroma (How- ard, 1939). In electron micrographs (Deane and Porter, 1959), it can be seen that the sur- face membranes of secretory cells Ivdvv microvilli which extend into the lumen. The supranuclear region contains secretory granules enclosed in vesicles or cisternae, smaller membrane-bound vesicles, and par- allel arrays of smooth Golgi membranes. Moderately distended ergastoplasmic clian- nels with membranes studded with pi'c- sumed libonucleoprotein ])articles lorm comi)lex convolutions along the latci'al luai'- gins and at the base of cells. The nucleus possesses clumped chromatin along the meml)rane (Figs. 6.39, 6.42, and 6.43). Fu- jita (1959) described essentially the same type of endoplasmic reticulum and the presence of microvilli and secretion gran- ules. In addition, small granules in the Golgi region were interpreted as precursors of secretory granules. Histochemical preparations (Table 6.7) show strong alkaline phosphatase activity in stromal elements (Atkinson, 1948; Bern. 1951 ) ; acid phosphatase activity is present in the apical or Golgi region of the epithe- lial cells (Deane and Dempsey, 1945). Se- cretory material and secretory granules in the lumen and cytoplasm are acidophilic and weakly PAS-positive, whereas the re- ticulum in the lamina propria is intensely PAS reactive (Fig. 6.44). Lateral margins and basal regions of the cells (Fig. 6.45) are moderately basophilic (Deane and Por- ter, 1959). Following castration of adult mice, the secretory epithelium retrogresses with loss of secretion granules and reduction in cell heiglit and nuclear size. Effects of gonadec- tomy may be retarded and not uniform in all cells (Howard, 1939), but changes within 5 days have been rei:)orted for cell and nucleai- size (Martins and Rocha, 1 929 ) . {electron mici'osco])ic and histochemical studies (Table ().7l reveal marked changes within a week aftei' gonad removal (Deane and Porter, 1959). Cell size is reduced, there are fewer secretory granules, ergas- toplasmic channels are less distended and ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 415 Fig. 6.39. Mouse seminal vesicle, normal male. Electronmierograph X 4200; osmic acid fixation with sucrose. Epithelial cells showing basal and lateral ergastoplasmic channels and membranes, and supranuclear \-psicles containing .secretory granules. (From H. W. Deane and K. R. Porter, unpubli.shed.) coin-oliitcd (Fig. 6.40). The relative number of riboniicleoprotein particles is somewhat leduced, secretory granules are less acido- philic, and the cytoplasm is only weakly basophilic (Fig. 6.45). Secretion granules were still visible by electron microscopy 10 days after castration but they were not visible at 25 days (Fujita, 1959) . Atkinson (1948) found that alkaline phos- phatase activity disappears almost com- pletely from the stroma within 10 days, but Bern, Alfert and Blair (1957) observed re- tention in the fibromuscular tissue. Martins and Rocha (1929) reported com- plete prevention of castration effects by in- jection of extracts of bull or goat testes. The epithelium of castrates responds read- ily to androgens. A single dose of 16 /x,g. of testosterone propionate in 30-day castrates resulted in increased mitotic activity be- ginning 30 to 36 hours after treatment and reached a peak at 42 to 48 hours (Allen, 1958). Administration of testosterone to castrates completely restored the fine structure to normal (Fujita, 1959). Alka- line phosphatase activity in the stroma returned to normal within 10 days with tes- tosterone propionate administration (At- kinson, 1948). The same hormone given to normal males for one week resulted in in- creased cell height, more abundant and acidophilic secretion and secretory gran- ules, increased basophilia (Fig. 6.45), more distended and convoluted ergastoplasmic channels (Fig. 6.41), and a relative increase in ribonucleoprotein particles (Deane and Porter, 1959). Discussion. The secretory cells in the epithelia of rat and mouse prostatic lobes and seminal vesicles have many histologic characteristics in common and some marked dissimilarities. In light microscopy with 416 PHYSIOLOGY OF GONADS V\v, 6 40 Mouse seminal vesicle, 7-(lay castrate. Electronmicrograph X 4200; osmic acid fixation wjtli sucrose. Note the reduction in cell height, number of secretory granules, and contortion of the ergastoplasmic membranes. Arrow indicates microvilli. (From H. W. Deane and K. R. Porter, unpublished.) routine fixation and stains, the most ob- vious differences are in cell height, position and staining intensity of the nuclei, pres- ence or absence of secretory granules, and in such cytoplasmic characteristics as the supranuclear clear zone in the rat ventral prostate and the basal vesicular region in the coagulating gland. The Golgi apparatus varies in density, structure, and position in the apical cytoplasm. Studies on ultra- structure reveal striking differences in the degree of dilation and the disposition of the endoplasmic reticulum. In the rat ventral prostate the most dilated cisternae are in the supranuclear region; in the dorsal lobe, generally distended vesicles are disposed throughout the cytoplasm in both cell types; in the coagulating gland, there is extreme dilation of the sacs, particularly in the basal region. The flattened vesicles of the mouse ventral prostate are disposed at random; the coagulating gland, like that of the rat, shows greatly dilated cisternae, especially basally. Moderately distended ergastoplasmic channels in the basal and lateral regions of cells are characteristic of the mouse seminal vesicles. Changes following castration are detect- able by light microscopy within 2 days in the rat seminal vesicle; 4 days in the ven- tral prostate; 10 days in the coagulating gland. Harkin (1957a) suggested a correlation between distention of the sacs and secretory activity of the cells in the rat ventral pros- tate. Within 24 hours after gonadectomy, he observed dilation of the sacs, but within 2 days, collapse of the apical sacs was marked and by 4 days, there was general collapse of the vesicles in other regions of the endoplasmic reticulum. At this stage secretory activity of the cells was appar- ently reduced. Brandes and Portela (1960a, b, c) dis- cussed the relation of the cisternae to secre- tion in the mouse glands. They proposed that the extremely dilated cisternae of the coagulating glands contain secretory prod- ucts which are released into the lumina of acini by some undetermined mechanism. They found no evidence that the Golgi com- plex is involved in the elaboration of secre- tory material in the cisternae, but histo- chemical findings suggest that it might take part in formation of secretory products that are not intracisternal. The ventral prostate ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 417 Fig. 6.41. Mouse seminal vesicle, intact male treated with testosterone propionate for 7 days. Electron micrograph X 4200; osmic acid fixation with sucrose. Note increase in cell height, abundance of secretory granules and contortion of the ergastoplasmic membranes. (From H. W. Deane and K. R. Porter, unpublished.) is characterized by flattened vesicles. Bran- des and Portela doubted that there is trans- port of secretory material to these cisternae and release from the intracisternal spaces into the acinar Imnen. They suggested an apocrine type of release involving extrusion of portions of the apical cytoplasm from the free margin of cells. The possibility of im- plication of the Golgi apparatus in the pro- duction of secretory material was consid- ered. From a study of the rat, Brandes and Groth (1961) concluded that the dilated cisternae of coagulating glands, dorsolat- 418 PHYSIOLOGY OF GONADS Fig. 6.42. Moil. M - :,,:ii il , i -id, , nui n, il n, ,i , I i_ (,;(,) l-.l, ,ii,m- micrograpli X 36,000: omiik- ;ici(l tixation; section liiat((l with iii.myl cicclatc to enhance the density of nucleoi)ro1(Mn.-^. Infianuclear region of an epithehal cell; nucleus at the top ; ergasto- pla.sniic channel.s with inomhianes studded with particles; arrow indicates a mitochondrion. (From H. W. Deane and K. R. Porter, unpublished.) eral and ventral prostates contain secretory products, and that it is probable that the membranes of the endoplasmic reticulum (or the granules associated with them) play an active role in the syntheses of the proteins present in the glandular secretions. Attemi)ts have been made to correlate structure of cells as observed by light and electron microscopy with histochemical lo- calizations of mucoproteins (PAS reaction), alkaline and acid phosphatase activity, and basophilic material. Various interpretations have been offered for the functional sig- nificance of these substances, all of which ACCESSORY MAMMALIAN REPRODUCTIVK GLANDS 419 are under control of andi'ogcnic hormones of testicular origin. Leblond (1950) stated that the presence of PAS-positive granules in the Golgi region supjiorts the concept of participation of the Golgi apparatus in the secretory process. Brandes and Portela (1960b) suggested that the vesicles and vacuoles in the Golgi zone might represent presecretory or secretory material. The pos- sibility that collapse of ergastoplasmic sacs after gonadectomy might be correlated with reduction in PAS-positive secretory mate- rial was proposed by Harkin (1957a). Alkaline and acid phosphatase activity is also found in the Golgi region, but the sig- nificance of this localization is not clear. Harkin (1957a) suggested that reduction in acid phosphatase activity following castra- tion might be correlated with the decrease in numbers of mitochondria. The histochemical pattern of enzyme ac- tivity has been discussed by Brandes and Bourne (1954), and the functional signifi- cance of distribution of epithelial and stro- mal alkaline phosphatase activity has been treated by Bern (1949a). Cytoplasmic basophilia that was abol- ished by ribonuclease was demonstrated in the epithelial cells of the rat seminal vesicle (Melampy and Cavazos, 1953). In the mouse seminal vesicle, Deane and Porter (1959) found cytoplasmic basophilia (all of which was attributable to ribonucleic acid) localized in regions which corre- sponded to the distribution of ergastoplas- mic membranes with their associated par- ticles of presumed ribonucleoprotein. The relative number of particles was apparentlj^ reduced after one week of castration, and increased with testosterone propionate ad- ministration to normal males. These changes were not considered marked enough to account for the pronounced reduction in basophilia following gonadectomy, and the increase with androgenic hormone treat- ment of normal males. Rixon and Whitfield (1959) found high concentrations of zinc, lipid, and basophilic material in a luminal border organelle in the lateral prostate of rats. Silver staining demonstrated fibrils, and it was suggested that zinc may be involved in the ergasto- plasmic reticulum, possibly with lipopro- tein, and would be associated with the microsome fraction in homogenatcs. In the discussion of changes in structure and histochemical localizations of sub- stances after gonadectomy and with hor- mone administration, no specific mention was made of differences in response among the glands. There arc, however, pronounced differences in rate of regression following withdrawal of testicular hormone, and in rate and degree of response to administered androgen. These differences in hormone sensitivity or threshold have been estab- lished by such end points as changes in histologic structure, weight (which includes increase in mass of cells and accumulation and storage of secretion), and secretion of specific substances such as fructose and citric acid (Mann, 1954a). In order of sensitivity they are first, secretory function, second, histologic structure, and finally, weight, whicli is frecjuently used as an end point (Dorfman and Shipley, 1956). Responsiveness of the epithelial cells de- pends on many factors and varies with spe- cific glands, age of the animal, genetic strain, and species. A few examples will illustrate these points. Following castration of adult rats the seminal vesicles retrogress more rapidly than the ventral prostate and recjuire higher doses of testosterone propi- onate to restore normal histological struc- ture (Price, 1944a). The ability of the seminal vesicles and the ventral ])rostate in young rats to respond to testosterone propionate increases with age to a peak which is specific for the organ (Price and Ortiz, 1944; Price, 1947). This is true also for the female prostate (Price, 1944b) and the accessory glands in young male ham- sters (Ortiz,' 1947). The effect of age on responsiveness in the mouse ventral pros- tate was studied by Lasnitzki ( 1955a ) who cultured glands from mice 4 to 6 weeks of age and 6 months old in normal control me- dium and in the presence of testosterone propionate. Young prostates regressed on the control medium but retained normal histologic structure when the hormone was added. In the control medium, older glands maintained normal structure and became hyperplastic with addition of the androgen. Franks (1959) also found differences re- 420 PHYSIOLOGY OF GONADS . (i 1.; .Mnii-i -(iiiiiiit Ill III ill - iiiir ~|icciiiii'ii. Ill, muilii Ml Kill, and prepara- tion as Fig. 6.39). Suprunuclcur icgiuu of au epilht'lial c-ell ; nucleus at the hottoui; numerous membrane-bound vesicles with secretory granules in the larger vesicles; arrows indicate some of the parallel arrays of smooth-surfaced Golgi membranes. (From H. W. Deane and K. R. Porter, unjiuhlished.) lated to age in the response of cultured mouse ventral prostate to testosterone pro- pionate. The Long-Evans and Sprague- Dawlcy strains of rats differ in respon- siveness of the ventral prostate of adult hypojihysectomized castrates to testosterone propionate (Lostroh and Li, 1956). Species differences in rate of retrogression of acces- sory glands in adult castrates are marked. As reported above, changes in histologic structure occur rapidly in rats, more slowly and less uniformly in mice (Howard, 1939), ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 421 Fig. 6.44. Mouse seminal vesicle, normal male. Photomicrofirapli - 250. Carnoy's fixative, stained by the periodic acid-Schiff method, counterstained with hematoxylin. Note the in- tense PAS-reaction in the reticulum in the lamina propria and surrounding the smooth muscle fibers; secretory material in the lumen is moderately reactive. (From H. W. Deane and K. R. Porter, unpublished.) ctncl slowly and somewhat incompletely in guinea pigs (Sayles, 1939, 1942) and ham- sters (Ortiz, 1953). It should be noted that in adult castrated guinea pigs the ability to secrete fructose and citric acid is appar- ently lost in cells which show only partial retrogression histologically (Ortiz, Price, Williams-Ashman and Banks, 1956). Another type of variation in responsive- ness is demonstrated when weights of sem- inal vesicles and ventral prostrates in im- mature castrated rats are used as end points for the potency of various C19 ster- oids (Dorfman and Shipley, 1956 L When lower dosages of testosterone and 17a- niethyl-A^-androstene-3^ , 17/?-diol are given the ventral prostate is more responsive than the seminal vesicles, but at high dosage lev- els the percentage increase in seminal vesi- cle weight equals or exceeds that of the pros- tate. Three other C19 steroids are far more effective on the ventral prostate than on seminal vesicles. The female prostate in adult rats re- sponds histologically and gravimetrically to a number of C19 steroids (Korenchevsky, 1937). These findings have been confirmed and extended by Huggins and Jensen (1954) and Huggins, Parsons and Jensen (1955) in hypophysectomized female rats. These workers examined the relation of mo- lecular structure to the growth-promoting ability of the steroids. Atrophy in male accessory glands of rats and mice has been reported under condi- tions of inanition and vitamin deficiency. These results are not usually attributable to reduction in responsiveness of the glands 422 PHYSIOLOGY OF GONADS Fig. 6.45. Moil-^c .^einiiial mmcIi- l'li(iiniiiicinmn|ilis x 7UU. C'ain(>\-'- ti\ati\ c-nictliylcnie blue. Top, normal mali' ; in k Idle 7-ila\ ca-tiaN , iMJitoin, intact male ticatcil wil li ic.-tostcionp proprionate for 7 days. Hasoi)lidic inatciial ( ciga-tDplasm) occurs at the hasc and along lat- eral margins of cells; the Golgi zone appears clear; secretory granules are unstained. Baso- philic material and Golgi zone are less evident after castration and more highly developed after testosterone treatment than normal. (From H. W. Deane and K. R. Porter, unjnib- lished.) themselves Init to (liiuiniition in <2;onado- trophin titer by way of pituitary inhibi- tion. Moore and Samuels (1931) showed that gonadotrophin or androgen treatment repaired the atrophied accessory glands in vitamin B-deficient rats and in those on limited food intake. Further, Lutwak-Mann and Mann (1950) demonstrated reduction of fructose and citric acid in accessory glands of rats on a vitamin B-deficient diet, but treatment with chorionic gonadotrophin not only restored the levels of these substances to normal but produced hypersecretion. Grayhack and Scott ( 1952 ) reported that the growth response to testosterone propionate of the ventral and posterior prostate in cas- trated rats on reduced food intake, vitamin- free casein, or glucose was little different from normally fed rats at lower dosages, but at higher levels there was less resj^onse in rats on limited dietarv intake. Testosterone propionate did not produce normal stimula- tion of the accesisory glands in castrated mice on limited food intake (Goldsmith and Nigrelli, 1950) . In adult rats, a folic acid an- tagonist (Aminopterin) partially prevented the reduction in jirostatic weight produced by estradiol but did not interfere with testosterone stimulation of the prostate in castrated adults or intact immature animals (Brendler, 1949). The senile changes which occur in ad- x'anced age in the prostate glands of tlie rat, mouse and of man have been describetl by Moore (1936) and interjireted on tht. basis of decrease in testicular androgen. Presenile variations in histologic structui'e are pr()l)al)ly ivhited to changes in respon- siveness to andi'ogens. In a brief report on electron microscopy (Harkin, 1957b), in- volutional changes in the rat ventral pros- tate were described. With increasing age. ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 423 eloctron-denye material is deposited in tlie Golgi region of epithelial cells and these bodies are said to resemble structures found in hyperplastic prostates in man. 2. Adrenal Androgens A large Ixxly of evidence jioints to effects of hormones from the adrenal cortex on the accessory reproductive glands of male rats and mice and on prostate glands of female rats. The significance of this relationshi}) is unknown and the effects are slight in many cases. Reviews by Parkes (1945), Ponse (1950), Courrier, Baclesse and Ma- rois (1953), Moore (1953) and Delost (1956) deal extensively with the subject. In man, a relationship between pathologies of the adrenal cortex and virilism is well rec- ognized (Dorfman and Shipley, 1956). The marked development of the ventral prostate in young castrated rats (Price, 1936) was attributed by Howard (1938) to the action of androgen from the adrenal cortex. The same explanation was sug- gested for the extensive development of the seminal vesicles and prostate in young cas- trated mice (Howard, 1939). The ventral prostate does not develop in immature cas- trated-adrenalectoraized rats according to Burrill and Greene (1939a) and Howard (1941), but Gersh and Grollman (1939) did not confirm these findings. The impairment of prostate and seminal vesicle develop- ment in young castrated-adrenalectomized mice (Howard, 1946) was considered to be the result of poor physical condition rather than loss of adrenal androgen. Gonadec- tomy in young male mice of an inbred strain (Woolley and Little, 1945a. b) pro- duced adrenal cortical carcinoma correlated w^ith strong stimulation of the prostate and seminal vesicles. Spiegel (1939) castrated young guinea pigs and found the develop- ment of adrenal-cortical tumors and evi- dence of stimulation of prostates and sem- inal vesicles. In the field vole {Microtus arvalis P.), Delost (1956) observed extensive develop- ment of the ventral prostate in young cas- trated males. Gonadectomy of adult males during the breeding season results in atro- phy of seminal vesicles, and dorsal and lat- eral prostate, whereas the ventral prostate shows an intense secretory activity by one month after testis removal. Adrenalectomy of castrates produces complete involution of the ventral prostate. Outside the breed- ing period, there is atrophy of all accessory glands except the ventral prostate which exhibits strong activation that can be pre- vented by adrenalectomy. The prostate gland of young female rats undergoes development and differentiation, and resembles the male ventral prostate with which it is homologous (Price, 1939; Mahoney, 1940). Development still occurs following ovariectomy (Burrill and Greene, 1939b; Price, 1942) or adrenalectomy (Bur- rill and Greene, 1941), but not in ovariec- tomized-adrenalectomized females. A com- parison of the responsiveness of female and male prostates indicated that the male gland is more sensiti\'e to adrenal androgens (Price, 1942). Autotransplants of adrenals into one seminal vesicle of adult castrated rats pro- duced slight local stimulation of the gland and also androgenic effects on the other seminal vesicle and on the ventral prostate (Katsh, Gordon and Charipper, 1948). But androgenic action was local and barely discernible in somewhat similar experi- ments (.lost and Geloso, 1954). Price and Ingle (1957) autotransplanted adrenals into seminal vesicles and ventral prostates of adult castrates and observed definite but local stimulation of seminal vesicles, coag- ulating glands, and ventral prostates. Neg- ative results of adrenal transplants in sem- inal vesicles of nonadrenalectomized rats were reported by Moore (1953). Takewaki (1954) failed to detect any androgenic ef- fect of autotransplants of adrenals placed subcutaneously in contact with seminal ves- icle grafts in castrated males. The finding that treatment of young cas- trated male rats with adrenocorticotrophin caused stimulation of the ventral prostate (Davidson and Moon, 1936) has been con- firmed by Deanesly (1960) who observed, in addition, a slight stimulation of the sem- inal vesicles. Nelson (1941) also found an- drogenic effects on accessory glands fol- lowing ACTH treatment but Moore (1953), van der Laan (1953), and Takewaki (1954) obtained negative results. In hypophysec- 424 PHYSIOLOGY OF GONADS tomized-castrated rats, ACTH was reported ineffective in increasing ventral prostate weight (van der Laan, 1953; Grayhack, Bunce, Kearns and Scott, 1955) , but Lostroh and Li (1957) obtained some growth of ventral pi'ostates and seminal vesicles at certain dosage levels. They emphasized that dosage is a critical factor in demonstrating the androgen-secreting ability of the ad- renal cortex under ACTH stimulation. Ad- ministration of ACTH to hypophysecto- mized-castrated-adrenalectomized rats does not affect the accessory glands. There has been no general agreement on the androgenicity of desoxycorticosterone on the accessory glands (see reviews by Parkes, 1945 and by Courrier, Baclesse and Marois, 1953). Lostroh and Li (1957) re- ported that ll-desoxy-17-hydroxy-corticos- terone and 11-dehydro-corticosterone dis- played an androgenic activity equivalent to 4 fxg. of testosterone propionate on the ven- tral prostates and seminal vesicles of hy- pophysectomized-castrated adult rats. Cor- ticosterone, cortisone, and hydrocortisone were ineffective. Grayhack, Bunce, Kearns and Scott (1955) found cortisone ineffective on the weight of ventral prostates in hy- pophysectomized castrates. In the field vole (Microtus arvalis P.), Delost (1956) pro- duced effects on the ventral prostate by cortisone administration. 3. Ovarian Androgens It has long been known that mammalian ovaries can secrete androgenic hormones which have virilizing effects in females. Dis- cussion of the evidence for androgenic ac- tivity of the ovary has been presented by Ponse (1948) and Parkes (1950). More re- cently the subject has been extensively re- viewed (Ponse, 19541), 1955). Much of the interest in ovarian androgens in the rodent has centered on the question of their site of origin in the ovary and the effects of tem- perature and gonadotrophin administration on androgen production (see reviews, and Chapter 7 by Young) . However, the use of male accessory glands as bio-indicators for ovarian androgen has contributed to our knowledge of the responsiveness of the glands. Methods for approaching this problem in- clude transplantation of ovaries into vai'i- ous sites in castrated male rats and mice; ])lacing ovarian autotransplants into the ears of females ; transplantation of male ac- cessory glands into females; and observa- tions of prostate glands in females of the so-called female prostate strains of rats. The use of such females as hosts for grafts of male prostatic tissue has permitted a di- rect comparison of responsiveness in these homologous glands. The first observations that ovarian grafts maintain normal prostates and seminal ves- icles in castrated males w^ere made in guinea pigs (Lipschiitz, 1932) and mice (de Jongh and Korteweg, 1935). Hill (1937) trans- planted ovaries into the ears of castrated male mice and obtained stimulation of the prostate and seminal vesicles. Deanesly ( 1938) reported similar findings in rats. Local effects from ovaries grafted into sem- inal vesicles of castrated rats w^ere shown by Katsh (1950). Takewaki (1953) also found local stimulating effects on seminal vesicles when rat ovaries and seminal ves- icles were transplanted close together into the spleens of gonadectomized males and females. In experiments in which ventral prostates and seminal vesicles were transplanted sub- cutaneously into adult female rats (Price, 1941, 1942) it was sliown that the ventral prostate is well maintained in virgin fe- males (Fig. 6.46) and highly stimulated during jiregnancy and lactation in the host. It is comi)letely retrogressed in spayed fe- males. Seminal vesicle grafts, however, are stimulated only rarely. This occurs only in females that have littered repeatedly and have been lactating for long periods. This indicates that the threshold of response of the ventral prostate to ovarian androgens is lower than that of the seminal vesicles. Evidence of functional stimulation with production of fructose and/or citric acid was obtained in coagulating glands, and ventral, lateral, and dorsal prostates trans- ])lanted into female rats in which the ova- ries were stimulated by gonadotrophin treatment (Price, Mann and Lutwak- Mann, 1955). It may be assumed that the effects were attributable to ovarian andro- gens since ventral prostate grafts in spayed females (Greene and Burrill, 1939) are not stimulated histologicallv when gonado- ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 425 Fig. 6.46. Rat male and female prostates. Photomicrographs X 650. Bouin-hematoxylin preparation. Top, host prostate from an adult virgin female; middle, male prostate graft from the above female host; bottom, prostate from a pregnant female. Note the semi-regressed epithelium in the female prostate of the virgin female host compared with the columnar epithelium and light areas in the male prostate graft and the prostate from a pregnant fe- male. (From D. Price, Anat. Rec, 82, 93-113, 1942.) trophin is administered. Great stimulation of ventral prostate grafts in the ovarian bursa of females was obtained by Ponse (1954a). The female prostate gland is normally jiartially retrogressed in adult females ex- cept during pregnancy and lactation (Fig. 6.46) when it appears stimulated (Burrill and Greene, 1942; Price, 1942); in spayed females it is atrophic (Price, 1942). The striking development of the female prostate in pregnancy and lactation in a number of species of mammals is discussed in Section I. Hernandez (1942) obtained stimulation of female prostates by autotransplants of oA-aries into ears, hind legs, or tails of rats. Transplantation of rat ventral prostates into virgin females shows that the male prostate has a lower threshold to ovarian androgens than the female gland and main- tains high epithelium and cellular light areas whereas the epithelium of the host prostate (Fig. 6.46) is low and retrogressed (Price, 1942). 4- Progesterone The administration of progesterone in relatively enormous doses has stimulating effects as determined by weight, histologic structure, and function of some of the ac- cessory glands in castrated male rats, mice, and guinea pigs. The literature has been reviewed by Greene, Burrill and Thomson (1940), Parkes (1950), and Price, Mann and Lutwak-Alann (1955). Burkhart (1942) treated adult 40-day- 426 PHYSIOLOGY OF GONADS castrated rats with one or two 20-mg. doses of progesterone and observed a slight stim- ulation of mitotic activity in the ventral prostate and seminal vesicles after 55 hours but a pronounced hypertrophy of epithe- lium and connective tissue in both glands. The ventral prostate is more sensitive to progesterone than the seminal vesicles. In castrated rats (Price, IMann and Lut- wak-Mann, 1955), treatment with 25 mg. of progesterone daily stimulated the secre- tion of fructose or citric acid in seminal vesicles, coagulating glands, and ventral, lateral and dorsal prostates, and produced histologic changes in the last three glands. The effect, however, was only equivalent to that of about 5 /xg. of testosterone pro- pionate. The lowest threshold to the hor- mone is in the ventral prostate and the highest in the seminal vesicles. Lostroh and Li (1957) found no effects of 0.5 mg. of progesterone daily on the ventral prostate and seminal vesicles of hypophysectomized- castrated adult rats, but the same dose of 17a-hydroxy progesterone was the andro- genic equivalent of 4 /xg. of testosterone pro- pionate. It may be noted that the following transformations are involved in the bio- synthesis of testicular androgens: choles- terol —> pregnenolone — > progesterone — > 17a-hyclroxy progesterone — > androst-4-ene- 3,17-dione -^ testosterone (Dorfman, 1957). The slight androgenic actions of pro- gesterone and 17a-hydroxy progesterone may result from their conversion to an- drostane derivatives by extragonadal tis- sues. The findings of Katsh (1950) that progesterone crystals implanted directly into the seminal vesicles of castrated rats have no stimulating influence may be sig- nificant in this regard. C. EFFECTS OF ESTROGENS Administration of estrogenic hormones to normal males affects the accessory glands both indirectly and directly. The effects fall into three categories: inhibition as evi- denced by weight changes, involution of the epithelium, and loss of secretory activity (attributable to inhibition of pituitary gon- adotro])hin and reduction in endogenous androgen) ; direct stimulation of fibromus- cular tissue; and stimulation of hyi)erplasia and stratified squamous metaplasia of the ei)ithelium with possible keratinization. In no case does estrogen induce secretory ac- tivity of epithelial cells. The reduction in seci'etion as determined cjuantitatively (see Section II) may result from castration at- rophy of secretory cells, or from hyper- plastic and metaplastic transformations of the e])ithelium witli resultant loss of normal secretory function. The observed responses to estrogen treat- ment in glands of intact and castrated males, and in organ cultures of prostatic tis- sue, represent the dual effects of androgen withdrawal and estrogen addition. The ex- tensive literature on the effects of estrogen and the evidence for so-called antagonis- tic, cooperative and synergistic effects of simultaneous administration of androgen and estrogen have been discussed exten- sivelv (Zuckerman, 1940; Emmens and Parkes, 1947; Ponse, 1948; Bern, 1949b; Burrows, 1949). The observation that administration of estrogen to intact male rats causes atrophj- of the accessory glands which is mediated by way of reduction of pituitary gonado- trophin and failure of secretion of testicular hormones was made by Moore and Price (1932). Estrogen-induced atrophy was pre- vented by simultaneous treatment with gonadotrophin or androgen. Direct stimu- lating effects were reported by Freud (1933) and David, Freud and de Jongh (1934) who observed fibromuscular growth in seminal vesicles of estrogen-treated cas- trated rats and stratification in the duct epithelium of the lateral prostate. Simul- taneous treatment with androgen enhanced the hypertrophic effect of estrogen on the fibromuscular wall of the seminal vesicle but prevented epithelial change in the lat- eral prostate ducts. Korenchevsky and Den- nison (1935) found estrogen stimulation of the muscular layer of the rat seminal vesi- cle with no effect on the epithelium, but in coagulating glands (and to a lesser degree in the dorsal prostate ) there was not only fi- bromuscular hypertrophy but also meta- plastic transformation of the epithelium with stratification; changes in the ventral and lateral lobes were slight and the epi- tiieHum was unaffected. Androgen treatment prevented the jxithologic changes induced by estrogen. Harsh, Overholser and Wells ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 427 (1939) noted stratified, sqiuinious epithe- lium in the ducts of the seminal vesicles, and ducts and acini of coagulating glands following estrogen administration. But a slight delay in castration atrophy and a weak stimulating effect of estrogen on semi- nal vesicle epithelium were observed by Overholser and Nelson ( 1935 ) and Lacas- sagne and Raynaud (1937). Ovaries transplanted into castrated male rats (Pfeiffer, 1936) induce fibromuscular hyi^ertrophy in host seminal vesicles and coagulating glands; stratified sciuamous cornified epithelium appears also in coagu- lating glands, and hyperplasia and meta- l^lasia are present in lateral prostates. Es- trogenic stimulation of fibromuscular tissue occurs (Price, 1941) in seminal vesicle grafts in normal female hosts but no such effects are evident in ventral prostate grafts. Burkhart (1942) injected a single dose of estradiol benzoate into 40-day-castrated rats and observed no effect on the ventral prostate. But in the seminal vesicles, hyper- trophy of epithelial cells occurred by 27 liours after treatment and by 55 hours, mitotic activity was evident in the epithe- lium and to some extent in the connective tissue. In histochemical studies (Bern and Levy, 1952) , metaplastic changes were observed in the seminal vesicle epithelium after es- trogen treatment but no cornification oc- curred; the replacing epithelium was alka- line phosphatase-positive in contrast to the negative reaction in the original epithelium (Table 6.7). Fibromuscular hypertrophy was found but no definite alteration in en- zyme concentrations except an absence of activity in edema of the subepithelial stroma. No metaplastic changes appeared in the coagulating gland epithelium, but the ducts of the dorsal prostate underwent metaplasia; alkaline phosphatase activity of the stroma in both glands was retained as in the castrate. The ventral prostate ejn- thelium was atrophic but still enzyme-ac- tive after 120 days of treatment and the stroma reacted positively. The effects of estrogen on the accessory glands of mice are far more marked than in rats. Long continued and strong doses of ■estrogen cause hyperplasia, metaplasia and keratinization in the epithelium of mouse coagulating glands (Lacassagne, 1933 ) . The same effects, with fibromuscular hyper- trophy, were described in coagulating glands and prostates by Burrows and Kennaway (1934), Burrows (1935a), and de Jongh (1935) who prevented epithelial metaplasia in prostates by simultaneous treatment with androgen. Burrows (1935b) studied the lo- calization of responses to estrogenic com- pounds and found that in order of time of response, the coagulating gland is first, seminal vesicles next, and finally the pros- tatic lobes. Changes begin in the urethral ends of ducts and jirogress peripherally into the acini. Li the degree of response, the co- agulating glands and seminal vesicles show the most drastic changes with the appear- ance of stratified, squamous, keratinizing epithelium and ultimate loss of acini. The effects on the lobes of the prostate include stratified, cornifying epithelium but the changes are not so i)ronounced. Some hy- pertroj^hy of fibromuscular stroma occurs in all the glands and hyperplasia is marked in the fibromuscular wall of the seminal vesicles. Tislowitz (1939) found stimulation of mitotic activity in muscle and connective tissue of seminal vesicles and ventral pros- tate glands of immature castrated mice treated with estrogen. Stratification and cornification appear in the ventral prostate epithelium, with mitoses in the basal cell layers and also in seminal vesicle epithe- lium. Allen (1956) compared the mitogenic activity of a single dose of 16 /xg. of estradiol benzoate on seminal vesicles, coagulating glands, and ventral prostates of 30-day- castrated mice. Significant increases in mi- totic activity occur in seminal vesicles and coagulating glands about 24 hours after treatment; the ventral prostate does not re- spond significantly until 72 hours and gives a low absolute value of mitoses. Horning (1947) studied some of the ini- tial changes in prostatic epithelium of in- tact mice receiving estrogen. Slight hyper- trophy of epithelial cells and extensive fragmentation and dispersal of hypertro- phied portions of the Golgi network occur by 8 days in the coagulating gland. At the same period, hypertrophic changes are less pronounced in the ejuthelium of the dorsal 428 PHYSIOLOGY OF GONADS prostate and there is only slight fragmenta- tion of the Golgi apparatus. In the ventral prostate no epithelial hypertrophy is found but the Golgi network hypertrophies with- out fragmentation or dispersal. The ventral prostate is definitely less sensitive to estro- gen than the other two glands. After longer periods of estrogen adminis- tration, Bern (1951) observed fibromuscu- lar hypertrophy of the seminal vesicle and intense alkaline phosphatase activity as in untreated intact males (Table 6.7) ; the epithelium, which is normally negative in enzyme activity, becomes positive and the beginning of metaplastic changes is occa- sionally visible. In the coagulating gland, stratified scjuamous metaplasia with masses of keratin is found and the metaplastic epithelium is strongly alkaline phosphatase- positive; enzyme activity is retained in the stroma but is variable. Bern, Alfert and Blair (1957) reported that the metaplastic coagulating gland epithelium is strongly alkaline phosphatase reactive, virtually PAS-negative, has dense homogeneous RNA concentrations decreasing in amount from base to lumen, and a dense homogene- ous cytoplasmic protein reaction with a gradient of increasing intensity from base to lumen. The enlarged vesicular nuclei of the metaplastic epithelium have lower con- centrations of deoxyribonucleic acid (DNA) than the nuclei of normal epithelial cells. The cytoplasm of these cells is as reactive as normal cells for sulfhydryl groups, and the newly formed keratin is intensely reac- tive; the greatest concentrations of disulfide groups are in superficial keratin. In the dorsal prostate (Bern, 1951), es- trogen causes fibromuscular hypertrophy with variable retention of alkaline phosi)ha- tase activity. Metaplastic changes involving basal cell proliferation and stratification be- gin and the metaplastic epithelium is in- tensely alkaline phosphatase active, a rv- versal of the normal reaction. Brandes and Bourne (1954), using di- ethylstilbestrol, observed an increase in fibromuscular stroma in coagulating glands. dorsal and ventral prostates, and epithelial hyperplasia and stratification in varying degrees. The most pronounced changes oc- curred in the coagulating gland. The effects of estrogen on Golgi networks, and on PAS and acid and alkaline phosphatase reactions are in general similar to the results of cas- tration (Table 6.8). Ventral prostate glands have been grown in culture by the watch glass method, with estrogens added to the medium. Lasnitzki (1954, 1958) reported hyperplasia and squamous metaplasia of the epithelium in young i^rostate tissue from C3H mice. In older glands, stimulation of fibromuscular tissue occurred. Franks (1959) using the C57 strain and a different culture medium observed no epithelial hyperplasia and metaplasia, but obtained increases in stroma and muscle. He attributed atrophic changes in the epithelium, which appear more marked in estrogen-treated than in control cultures, to direct inhibition by the hor- mone. Ventral prostate tissue from young mice is more sensitive to estrogen than tis- sue from adult or old males. In the dog, Huggins and his collaborators demonstrated the effects of estrogen and combinations of estrogen and androgen on histologic structure and secretion in the prostate (see Section II). Estrogen causes decrease or increase in prostatic size de- pending on the dosage and on the levels of endogenous or exogenous androgen (Hug- gins and Clark, 1940) . Sciuamous metaplasia of the epithelium of ducts and acini occurs with estrogen treatment, but only in the posterior lobe. Discussion. The results of estrogen ad- ministration to rats and mice vary with species, age of animal, specific gland under consideration, dosage, duration of treat- ment, and presence or absence of endoge- nous or exogenous androgen. Interpretation of the findings rests on the understanding that androgen directly stimulates mitotic and secretory activity in the epithelial cells. Estrogen inhibits i)ituitary function and thus i-eduees testicular androgen in intact males. It directly increases mitotic activity in th(> epithelium of the accessory glands, and inckices (>pitlielial liy|)erplasia and iiietaphisia, and fibromuscular hyperplasia. W'lietliei' the effects of simultaneous pres- ence of androgen with exogenous estrogen are classified as protective, competitive (antagonistic), or cooperative (synergistic) on the acce.ssoiy glands depends on the ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 429 relative levels of the two hormones. Both affect mitotic activity directly. In a comparison of the effectiveness of androgen and estrogen on mitotic activity, Allen (1956, 1958) showed that a dose of 16 fjig. of testosterone propionate induces statistically significant increases in mitotic activity of the epithelium of seminal vesi- cles, coagulating glands and ventral pros- tates of castrated mice in 30 to 36 hours. The same dose of estradiol benzoate in- creases mitotic activity in 24 hours in semi- nal vesicles and coagulating glands, but not until 72 hours in ventral prostates. Differences in responsiveness to estrogen are evident between rats and mice but in both species there is a gradient of reactivity with coagulating glands showing the most marked changes, seminal vesicles next, and prostatic lobes least. In glands of both spe- cies, the duct epithelium is more sensitive than acinar epithelium and the first ob- servable effects are on urethral ends of ducts. Hyperplastic and metaplastic re- sponses to estrogen occur also to varying de- grees in accessory glands of other mammals — man, monkey, dog, cat, ground squirrel, and guinea pig. Zuckerman (1940) reviewed the effects of estrogen in male and female rodents and other mammals and suggested from the evidence that "stratified squamous proliferation or metaplasia is usually a pri- mary response of tissue in whose develop- ment oestrogen-sensitive entodermal sinus epithelium has played a part." On the basis of the pathologic effects of estrogen on the mouse coagulating glands and the protective action of androgen, sev- eral workers originally suggested that be- nign prostatic hypertrophy in man might result from a primary imbalance in the nor- mal ratio of estrogenic to androgenic hor- mones in the male organism (Zuckerman, 1936). Further study has not supported this concept. D. HORMONAL CONTROL OF SPONTANEOUS PROSTATIC NEOPLASMS Spontaneous tumors of the prostate occur in rodents rarely if at all, but benign growths are extremely common in aging dogs and men, and prostatic cancer is a major prol^lem in man. It is noteworthy that neoplasms of the seminal vesicles in man are rare (Dixon and Moore, 1952). 1. Benign Growths In the dog, prostatic enlargement which is essentially due to cystic hyperplasia of the epithelium occurs in almost all senile males with functioning testes, but is not found in castrates (Huggins, 1947b). In these prostatic growths, which characteris- tically involve the entire gland, tall colum- nar secretory epithelium is always present in some acini. Canine prostatic hyperplasia is under control of testicular androgens (Huggins and Clark, 1940) and marked in- volution of these tumors as evidenced by their size and secretory activity (see Sec- tion II) can be induced by gonadectomy or treatment with suitable dosages of estrogen. Estrogen overdosage, however, causes pros- tatic enlargement and a metaplasia of the posterior lobe which does not resemble cys- tic hyperplasia. Huggins and Moulder (1945) reported that dogs feminized by es- trogen-secreting Sertoli cell tumors of the testis do not have cystic hyperplasia. The important factors in this pathologic growth seem to be age and testicular androgens (Huggins, 1947b), but prolonged adminis- tration of testosterone propionate to aged castrate dogs results in normal-appearing prostates and not cystic hyperplasia. Benign prostatic hypertrophy in man is rarely encountered before the age of 40 (Moore, 1943; Huggins, 1947b) but it is extremely common in old men. It differs markedly from prostatic hyperplasia in dogs; the lesions are limited to the medul- lary region of the prostate and are sphe- roidal neoplastic nodules involving, usually, both epithelium and fibromuscular tissue; other nodular types occur but are less fre- quent (Huggins, 1947b; Franks, 1954). The prostatic epithelium is composed of tall secretory cells (Huggins and Stevens, 1940). Despite the fact that castration may be followed by some shrinkage of hypertro- phied human prostate tissue (White, 1893; Cabot, 1896; Huggins and Stevens, 1940), it is generally admitted that this treatment is of little value. Estrogen treatment results in changes in the acini of the inner or medul- lary (periurethral) part of the prostate, and stratification with squamous metaplasia 430 PHYSIOLOGY OF GONADS of the duct epithelium, but there is little effect on nodular stroma and acini (Hug- gins, 1947bj. Since benign prostatic hyper- trophy has not been observed in men cas- trated early in life, testicular androgen is presumably involved in its etiology (Hug- gins, 1947b). However, it is doubtful whether androgens are causative agents for this disease. Lesser, Vose and Dixey (1955) found that in men over the age of 45 who had received androgen treatment for non- cancerous conditions, the incidence of be- nign enlargement of the prostate was no greater than in untreated controls. 2. Prostatic Cancer Prostatic cancer is a common disease in elderly men. This carcinoma, which charac- teristically arises in the posterior (outer) region of the prostate, consists of an abnor- mal growth of cells resembling adult pros- tatic epithelium rather than undifferentiated tissue (Huggins, Stevens and Hodges, 1941 ). It was found that these neoplasms are hor- mone-dependent and usually are influenced by anti-androgenic therapy; those which fail to respond are not adenocarcinomas with acini present in the tumor, but are un- differentiated carcinomas with solid masses of malignant cells (Huggins, 1942). How- ever, the two types intergrade and both contain large amounts of acid phosphatase and are considered cancers of adult pros- tatic epithelium. The beneficial effects of castration or estrogenic treatment or both simultaneously, on metastatic carcinoma of the prostate in man were first demonstrated by Huggins and his collaborators (Huggins and Hodges, 1941 ; Huggins, Stevens and Hodges, 1941 ; Huggins, Scott and Hodges, 1941 ; Huggins, 1943, 1947a). This discovery was facilitated by the availability of a chemical index of the activity of the neo- plasm, namely, the acid phosphatase activ- ity of blood serum. Although testosterone increases the level of serum acid phosphatase in patients with prostatic cancer (Huggins and Hodges, 1941 ; Sullivan, Gutman and Gutman, 1942 1 , androgen treatment does not always exacer- bate the growth of the tumor (Trunnel and Duffy, 1950; Brendler, Chase and Scott, 1950; Brendler, 1956; Franks, 1958) or may even decrease it (Pearson, 1957). It is ciues- tionable if androgens induce prostatic can- cer, inasmuch as their prolonged adminis- tration neither increases the incidence of this disease in man (Lesser, Vose and Dixey, 1955) nor induces it in dogs (Hertz, 1951). Tlie response of human metastasizing prostate cancer to anti-androgenic therapy is often very dramatic, but neither castra- tion nor treatment with estrogens cures this disease. The tumor may regress for consider- able periods of time, but eventually it recurs and begins to grow again. A small propor- tion of cases do not benefit at all (Huggins, 1957; Franks, 1958). Nevertheless, castra- tion and/or estrogen therapy remain the best treatment for prostatic carcinoma in man (Nesbit and Baum, 1950; Huggins, 1956; O'Conor, Desautels, Pryor, Munson and Harrison, 1959). Huggins and Scott (1945) suggested that the failure of some patients with prostatic cancer to obtain long lasting improvement from castration or estrogen treatment, or the two combined, lay in the secretion of androgenic substances by the adrenal glands. Early attempts to study the effect of bilateral adrenalectomy on human pros- tatic cancer were thwarted by the lack of suitable adrenal cortical steroids for ade- quate substitution therapy. But with the advent of cortisone, bilateral adrenalectomy could be accomplished with ease (Huggins and Bergenstal, 1951, 1952). It seems, how- ever, that adrenalectomy is of limited value to patients with prostatic cancer in relapse after orchiectomy and/or treatment with estrogens (Whitmore, Randall, Pearson and West, 1954; Huggins, 1956; Fergusson, 1958). E. EFFECTS OF CERTAIN AROMATIC HYDROCARBONS (CARCINOGENS) Spontaneous tumors have not been found in the prostate glands of rodents, but tumors can be induced in rats and mice by treat- ment with carcinogenic chemicals such as benzpyrene and metiiylcholanthrene. There lias been considerable interest in inducing such tvnnois and studying their inception and growth, and the iH'lation of steroid hor- mones to their de^•elopment. Such investi- gations have contributed to an understand- ing of early neoi)lastic changes in the rodent prostate, but have had limited applicability ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 431 to the })rol)lcm of hormonal control of pros- tatic cancer in man. The first induction of prostatic cancer in rodents was accomplished by Moore and Melchionna (1937) who injected benzpy- rene in lard directly into the rat anterior prostate (it should be noted that Moore and ]\Ielchionna used "anterior" in the sense of ventral as indicated by their histologic de- scriptions of a characteristic clear zone in the peripheral cytoplasm of the epithelial cells; this is typical only of the ventral lobe). The treatment was followed within 210 days by the development of squamous cell carcinomas in 72 per cent, and sarcomas in 5 per cent, of intact rats. Essentially simi- lar results were obtained in an equivalent number of rats castrated at the time of car- cinogen injection. Castration after tumors had developed did not cause atrophy of tu- mor cells. No metastases were found but there was anaplasia of cells and the tumors were invasive. The squamous metaplasia occurred in columnar secretory epithelium which was close to, or in contact with, benz- pyrene cysts. The sequence of changes was reduction in cell height, loss of the clear area in the peripheral cytoplasm, pseudo- stratification, true stratification, develop- ment of intercellular bridges, and formation of keratohyaline. It was concluded that testicular androgen is not an important fac- tor in the development of these squamous cell carcinomas, but on the basis of a small series of experimental animals it was sug- gested that exogenous androgen treatment in castrates may increase the incidence of sarcomas. In 1946, Dunning, Curtis and Segaloff im- planted compressed methylcholanthrene l)ellets into rat prostates (lobe not specified) and induced metastasizing squamous cell carcinomas. The tumors were transplant- able and metastasized equally well in male and female hosts. Bern and Levy (1952) in- jected methylcholanthrene in lard into ven- tral prostates of intact Long-Evans rats and induced extensive neoplasms within 7 to 9 months. All but one were squamous cell carcinomas; the exception was a sarcoma. Quantitative determinations of enzyme ac- tivity showed a loss of alkaline phosphatase in cancerous prostates but no significant changes in acid phosphatase activity. Histo- chemically, the stroma and capillaries were alkaline phosphatase reactive, but the car- cinomas had virtually lost the strong alka- line phosphatase activity of the epithelium of origin (Table 6.2). There was some pseu- doreaction or reaction in sloughed keratin and necrotic areas. Allen (1953) injected a suspension of methylcholanthrene in distilled water into ventral prostates or coagulating glands of intact and castrated rats. All were autop- sied 180 days later. A high percentage of squamous cell carcinomas and a few sar- comas developed; metastases occurred in a few cases. There was no statistically signifi- cant difference between tumor incidence in the ventral prostate and coagulating gland. Tumors of the ventral prostate were found in 70.6 per cent of the intact rats and in 100 per cent of the castrates; in castrates in- jected with testosterone propionate there were tumors in 57.7 per cent of the animals, and in castrates treated with estradiol ben- zoate, 77.8 per cent. It was concluded that tumor incidence was highest in castrates and lowest in intact males or castrates treated with testosterone propionate, and that estrogen did not affect tumor incidence. ]\Iirand and Staubitz (1956) placed methyl- cholanthrene crystals in ventral prostates of 99 intact Wistar rats and observed the ef- fects for over 300 days. The resulting tu- mors were classified as 30 squamous cell carcinomas, 3 leiomyosarcomas, and 2 ade- nocarcinomas; squamous cell carcinomas and adenocarcinomas metastasized. Frag- ments of squamous cell carcinomas were transplanted and survived and metastasized more successfully in males than in females. Horning (1946) imjiregnated strips of tissue from mouse dorsal prostates and an- terior prostates (coagulating glands) with crystals of methylcholanthrene and in- serted them as subcutaneous homografts into intact males. By this method adeno- carcinomas were induced in grafts of both dorsal prostate and coagulating gland. In mice of the RIII and Strong A strains (Horning and Dmochowski, 1947) methyl- cholanthrene in lard was injected into dor- sal and anterior prostates (coagulating glands). Squamous cell carcinomas and sar- comas developed in Strong A mice, but only sarcomas in RIII. Squamous metaplasia of 431 PHYSIOLOGY OF GONADS the epithelium occurred in the RIII strain but no malignant proliferation of metaplas- tic cells followed. It was noted that the epithelial changes which occurred with raethylcholanthrene treatment were "almost identical" with the secjuence of changes fol- lowing prolonged estrogen administration in Strong A mice (Horning, 1947). Horning (1949, 1952) studied the effects of castration, diethylstilbestrol, and testos- terone propionate on growth rates of pros- tatic tumors transplanted as grafts. Tumors were induced in ventral prostate, dorsal prostate, and coagulating gland tissue of Strong A mice by wrapping pieces of epi- thelium around crystals of methylcholan- threne and transplanting the grafts su!)cu- taneously into 75 intact males. Of the 54 tumors which developed, 42 were adenocar- cinomas or secreting glandular carcinomas, 10 were squamous cell carcinomas, and 2, spindle cell sarcomas. Neoplastic develop- ment began, apparently, in epithelium in a nonsecretory phase, and hyperplastic changes followed the sequence of mitosis, abnormal cell division, and pyknosis ac- companied by an increase in fibromuscular tissue. Three distinct types of epithelial pro- liferation then occurred; one, with tongue- like groups of early malignant cells, gave rise to secretory glandular carcinomas; the second, from acinar ejMthelium, and the third, from duct epithelium, developed into squamous cell carcinomas with keratiniza- tion and formation of keratin pearls. Some grafts had foci of the first and third type and the evidence suggested that the tumors subsequently became squamous cell carci- nomas. Both tumor types were transplanta- ble and were cari'icd through many serial transi)lantations without losing their histo- logic characteristics. In an effort to study effects of testicular androgen on growth, transplants of an ade- nocarcinoma were made into intact and cas- trated males. The tumors grew rapidly and progressively in intact mice but regressed in castrates. Testosterone propionate ad- ministration to castrated males bearing re- gressed tumors resulted in a resumption of tumor growth in some cases. Gonadectomy of the host had little effect on the growth of transplanted s(|uamous cell carcinomas. An- drogen-dependence of secreting glandular carcinomas was suggested. When stilbestrol pellets were implanted into one flank of intact males and a glandu- lar carcinoma into the opposite flank, the effects varied from slight to pronounced re- tardation of the tumor but complete re- gression did not occur. The squamous cell carcinomas were insensitive to stilbestrol. Additional experiments (Horning, 1952) involved transplanting pieces of prostatic epithelium impregnated with methylcholan- threne alone, or with the carcinogen com- bined with stilbestrol or testosterone pro- pionate into intact males (groups of 35 for each treatment). The carcinogen alone induced 8 adenocarcinomas and 5 squamous cell carcinomas; carcinogen and stilbestrol, 23 squamous cell carcinomas and 3 sar- comas; carcinogen and testosterone pro- pionate, 2 squamous cell carcinomas and 1 sarcoma. The increased tumor incidence with estradiol was interpreted as an in- hibitory action of the estrogen on secretory epithelial cells, making them more suscepti- ble to methylcholanthrenc. Brandes and Bourne (1954) made homo- grafts of pieces of ventral and anterior pros- tate (coagulating gland) impregnated with methylcholanthrenc into intact males of the Strong A strain, and studied histochemical changes. The grafts underwent squamous metaplasia and the processes of epithelial proliferation, stratification, and keratiniza- tion were completed within 10 days in some cases. Histochemical changes from the nor- mal i)attern (Table 6.8) occurred concur- rently. Alkaline phosphatase activity dis- appeared early; acid phosphatase activity became weak in nuclei and cytoplasm but keratohyalin granules were strongly reac- tive; PAS-positive reactions were gradually lost in luminal secretion and intracyto- plasmic granules but retained in the base- ment membrane. In some grafts there was transformation into squamous cell carcino- mas and when this happened phosjihatase activity was lost but the basement mem- branes were still PAS-positive. Lasnitzki (1951, 1954, 1955a. b, 1958) grew ventral jirostate glands from C3H and Strong A mice in culture by the watch-glass t('chnif|ue and added methylcholanthrenc to the medium. Hyperplasia and squamous metaplasia resulted in glands ex]ilanted ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 433 from both young and older mice. When estrone and carcinogen were added simul- taneously to cultures of young glands, squa- mous metaplasia was increased; with older glands, hyperplasia was inhibited and stro- mal increase occurred. The relation of vita- min A to the response of prostates to meth- ylcholanthrene was studied (Lasnitzki. 1955b). Vitamin A added to the medium caused an increase in secretion and deposi- tion of PAS-positive material in the secre- tory cells but did not influence growth or development; the vitamin added simultane- ously with methylcholanthrene did not in- fluence hyperplasia, but did prevent keratin formation and degenerative changes in the secretory epithelium; excess vitamin A fol- lowing the carcinogen prevented formation of keratin and decreased hyperplasia. Summary. Treatment of rat and mouse ac- cessory glands with benzpyrene or methyl- cholanthrene has induced precancerous and cancerous changes which led to the develop- ment of adenocarcinomas, squamous cell carcinomas, and sarcomas. The first type of tumor has been induced in large numbers only in mice and by the homograft method. The evidence suggests that, in mice, growth of adenocarcinomas is androgen- dependent, but squamous cell carcinomas are little affected by androgen loss or estro- gen treatment. The incidence of tumors has been increased by simultaneous administra- tion of estrogen and carcinogen but reduced by the administration of androgen with car- cinogen. In rats, it has been affirmed and denied that incidence and growth of squa- mous cell carcinomas are reduced in intact males ; estrogen has not affected tumor inci- dence. Species and strain differences in re- sponse are marked. F. EFFECTS OF NONSTEROID HORMONES There is e-\'idence that hormones from the anterior pituitary may directly affect the weight and histologic structure of accessory glands, or act synergistically with androgen. However, the findings have been somewhat conflicting. Dosage level, age, and strain of rats have varied, and questions have been raised with respect to the purity of the hor- mone preparations. Attention was focused on the pituitary in relation to accessory glands when Huggins and Russell (1946) observed that prostatic atrophy is more marked in the hypophysec- tomized than in the castrated dog. Van der Laan (1953) found the ventral prostates of hypophysectomized-castrated immature rats less responsive to testosterone pro- pionate than the glands of castrates; a crude extract of beef pituitaries restored respon- siveness in hypophysectomized-castrates. Prostates of young adult hypophysecto- mized-castrated Sprague-Dawley rats were also less responsive (total weight of dorsal and ventral prostates) to testosterone pro- pionate than those of castrates (Grayhack, Bunce, Kearns and Scott, 1955). Paesi, de Jongh and Hoogstra (1956) administered pituitary extracts simultaneously with a low dose of testosterone propionate to hy- pophysectomized-castrated rats and re- ported a slightly greater ventral prostate weight than with the androgen alone. To identify the hormones of the anterior pituitary that are capable of affecting the accessory glands or influencing their respon- siveness to androgen, the following hormone preparations have been injected alone and in various combinations into hypophysec- tomized-castrated rats: prolactin (luteo- trophin; LTH), growth hormone (so- matotrophin; STH), adrenocorticotrophin ( ACTH ) , interstitial cell-stimulating hor- mone (luteinizing hormone; ICSH; LH), follicle stimulating hormone (FSHl. In ad- dition, chorionic gonadotrophin and thy- roxine have been administered. Of these hormones, only prolactin and growth hor- mone have been shown to act directly on accessory glands (for comprehensive data on negative and positive results of these hormones see Grayhack, Bunce, Kearns and Scott, 1955; Lostroh and Li, 1956, 1957 ». The degree to which contamination with prolactin or growth hormone might influ- ence the assay of ICSH preparations by the ventral prostate test has been examined by Lostroh, Squire and Li (1958). 1. Prolactin {LTH) When Pasqualini (1953) treated castrated adult rats with testosterone propionate fol- lowed by administration of a lower dose of androgen plus LTH, the amount of secretion in the seminal vesicles was greater than with androgen alone. Prostate weights were in- creased slightly by LTH with androgen. Van der Laan (1953) reported that in adult 434 PHYSIOLOGY OF GONADS hypophysectomized-castrated rats LTH had no effect on ventral prostate weiglit. Gray- hack, Bunce, Kearns and Scott (1955) made the same observation for prostate weights in young adult Sprague-Dawley rats, but found that LTH augmented the effect of testosterone propionate on prostate weight. A difference in response between Long- Evans and Sprague-Dawley strains of rats was observed by Lostroh and Li (1956). In immature hypophysectomized-castrated Long-Evans rats, LTH alone had no effect on ventral prostate or seminal vesicle weights, and no synergistic effect when ad- ministered with a low dose of testosterone propionate; in Sprague-Dawleys, however, the weights of ventral prostates and coagu- lating glands were increased by LTH but, again, no synergism occurred with exoge- nous androgen. Chase, Geschwind and Bern (1957) reported that in immature hypo- physectomized-castrated Sprague-Dawleys, LTH did not affect weights of ventral pros- tates or coagulating glands but it did in- crease seminal vesicle weight. When LTH was administered with testosterone propio- nate, glandular tissue in the ventral prostate was increased and weights of coagulating glands (in some cases) and seminal vesicles were significantly higher than with andro- gen alone. In the immature hypophysectomized Sprague-Dawley rats that were not cas- trated, LTH alone did not affect ventral prostate weight but when given simultane- ously with ICSH it acted synergistically (Segaloff, Steelman and Flores, 1956). These results were confirmed by Lostroh, Squire and Li (1958) for the Sprague-Dawley strain, but in Long-Evans rats, LTH neither increased prostatic weight, nor augmented prostatic response to ICSH. Antliff, Prasad and Meyer (1960) have shown that in the guinea pig, LTH had no effect on seminal vesicles of castrated or hy- pophysectomized males, but when it was administered with subminimal doses of tes- tosterone propionate, seminal vesicle weight and epithelial height were increased. 2. Growth Hormone {STH) Van der Laan (1953) found no effects of STH on ventral prostate weights in young hypophysectomized-castrated rats. Huggins, Parsons and Jensen ( 1955) observed only slight effects on weights of ventral prostates and seminal vesicles with administration of STH to young hypophysectomized-cas- trated Sprague-Dawley rats, but a syner- gistic effect on weight was evident with simultaneous treatment with STH and tes- tosterone propionate. In hypophysectomized-castrated Long- Evans rats (Lostroh and Li, 1956, 1957), STH produced slight histologic changes and significant weight increases in the ventral jirostate; when administered with testoster- one propionate, an additive effect on weight was obtained. The changes in the seminal vesicles were less evident. The effects on Sprague-Dawley rats included weight in- creases in ventral prostates and seminal vesicles and a greatly enhanced weight re- sponse when STH and testosterone propio- nate were administered simultaneously (hy- pophysectomized-castrates in this strain gave a limited response to the androgen I . Chase, Geschwind and Bern (19571 found no consistent weight increases of ventral prostates, coagulating glands or seminal vesicles in young hypophysectomized-cas- trated Sprague-Dawleys treated with STH or STH and testosterone propionate. Simul- taneous administration of STH, LTH and testosterone, however, induced significant increases in all accessories above the weights produced by the androgen alone. Lostroh, Squire and Li (19581 deter- mined that STH had no effect on the ventral prostate response to ICSH in hypophysec- tomized Long-Evans rats, but })roduced an enhanced response in Sprague-Dawleys. It was concluded that the Long-Evans strain is jjreferable for the testing of crude ICSH ex- tracts, inasmuch as neither STH, LTH, nor both simultaneously, affect the response of the ventral prostate to ICSH. With regard to the action of STH on his- tologic structure of the prostate in hypopiiy- sectomized-castrated rats, it should be noted that the effects are slight; nuclei api)ear vesicular, and the connective tissue stroma is increased (Lostroh and Li, 1957). The synergistic action of STH on prostate growth in hypoi)hysectomized-castrated rats when administered simultaneously with testosterone is more striking. In a general discussion of the many biologic effects of ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS 435 growtli hormone, Li (1956) wrote, ''Does this ability to act as a synergist mean that growth hormone plays a permissive or sup- porting role in the biological action of a hor- mone or of a biological agent? It is not un- reasonable to assume that growth hormone creates the necessary and sufficient environ- ment for other biological agents to exercise the full scope of their functions." Acknowledgments. We are greatly indebted to Drs. Thaddeiis Mann, James Harkin, Helen Deane, Keith Porter, and David Brandes for generously supplying luipublished data and electronmicro- graphs. Mrs. Eva Brown provided invaluable as- sistance in the preparation of the manuscript. We wish to thank our artist. Mr. Kenji Toda, for many of the original figures. The researches of one of us (D. P.) cited in the chapter were supported in part by grants from the Dr. Wallace C. and Clara A. Abbott Fund of the University of Chicago and by Research Grants 2912 and 5335 from the National Institutes of Health, Public Health Service. IV. References Abul-F.^dl,M. A.M., AND King, E.J. 1948. Prop- erties of the acid phosphatases of erythrocytes and of the human jn'ostate gland. Biochem. J., 45, 51-60. 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Arch, exper. Path. u. Pharma- kol., 221, 447^59... R.-vuTHER, M. 1903. tJber den Genitalapparat einiger Nager imd Insektivoren, insbesondere die akzessorischen Genitaldriisen derselben. Jena Ztschr. Naturwiss., 38, 377-472. Rauther, M. 1909. Neue Beitrage zur Kennt- nis des Urogenitalsystems der Saugetiere. Denkschr. med .-naturwiss. Ges. Jena, 15, 417- 466. R.4YNAUD, A. 1942. Existence de variations dans I'etat de developpement des tubules glandu- laires de la prostate femelle d'Apodemus sylvaticus L. Compt. rend. Acad. Sc, 215, 382-384. Raynaud, A. 1945. Existence constante de deux lobes prostatiques chez les mulots (Apodemus sylvaemus sylvaticus) de sexe femelle. Bull. Soc. Zool. France, 70, 162-172. Reid, J. T., Ward, G. M., and Salisbury, R. L. 1948. Acid and alkaline phosphatase levels in consecutive semen ejaculates from bulls. Am. J. Physiol., 153, 235-241. Reis, J. 1937. tJber die Aktivitat der 5'-Nukleo- tidase in den tierischen und menschlichen Geweben. 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Effets sur les caracteres ge- neraux de lejaculat, les spermatozoides et la reproduction. Compt. rend. Soc. l)iol., 130, 1476-1479. ZoRGNioTTi, A. W., AND Brendler, H. 1958. Stud- ies in semen coagulation. Proc. Soc. Exper. Biol! & Med., 96, 195-197. ZucKERMAN, S. 1936. The endocrine control of the prostate. Proc. Roy. Soc. Med., 29, 81-92. ZucKERMAN, S. 1940. The histogenesis of tissues sensitive to oestrogens. Biol. Rev., 15, 231-271. THE MAMMALIAN OVARY William C. Young, Ph.D. PROFESSOR OF ANATOMY, UNIVERSITY OF KANSAS, LAWRENCE, KANSAS I. Introduction 449 II. FOLLICULOGENESIS 451 A. Growth of Primary and Small Ve- sicular Follicles 451 B. Growth of Vesicular Follicles 455 C. Preovulatory Swelling 455 1). Ovulation . ." 456 E. Folliculogenesis in Pregnancy and Lactation 457 III. Corpus Luteum 459 IV. Follicular Atresia 4(11 V. Hormones of the Ovary 4(i4 A. Sites of Origin 466 B. Amounts of Hormone Produced. . . . 471 VI. Age of the Animal and Ovarian Functioning 476 VII. Other Endocrine Glands and the Ovaries 478 A. Thyroid 478 B. Adrenal Cortex 480 VIII. Concluding Remarks 483 IX. References 484 I. Introduction Despite the impetus given to the study of ovarian structure and physiology by the work of Edgar Allen and Edward A. Doisy in the 1920's, knowledge of the mammalian ovary has hardly progressed beyond a de- scriptive phase. This cannot be attributed to lack of effort, although it must be realized that the ovary as an object of investigation has not held its own with the hypophysis, the thyroid, the adrenal, and the testis. Nor lias there been any failure to apply new techniques to the many problems of ovarian structure and physiology. Histochemical and cytochemical techniques were seized ujion for what they might contribute to the prol)- lem of the site of hormone production,^ and, ' Demp.sey and Bassett, 1943; Dempsey. 1948; Claesson, 1954; Claesson and Hillarp, 1947a-c ; Claesson, Diczfalusy. Hillarp and Hogberg, 1948; Claesson, Hillarp, Hogberg, and Hokfelt. 1949: in at least one series of studies (Zachariae, 1957, 1958; Zachariae and Jensen, 1958; Jensen and Zachariae, 1958), to the mecha- nism of ovulation. Methods for obtaining blood from the ovarian vein have been de- vised (Paschkis and Rakoff, 1950; Rakoff and Cantarow, 1950; Xeher and Zarrow, 1954; Edgar and Ronaldson, 1958) and re- fined techniques for the assay of secreted estrogens and progesterone have been de- veloped (Reynolds and Ginsburg, 1942; Hooker and Forbes, 1947; Emmens, 1950a, b; Haslewood, 1950; Wolstenholme, 1952; Zander and Simmer, 1954; Brown, 1955; Loraine, 1958; Sommerville and Deshpande, 1958j . The collection of follicles and corpora lutea timed more accurately with respect to the moment of ovulation has become pos- sible, and distinction between the normal and the pathologic has become clearer (Deane, 1952). Recently, the electron mi- croscope has been found to have a place, in an investigation of the finer structure of the cells of the corpus luteum (Lever, 1956), in the unraveling of the jirocesses whereby the zona pellucida is formed around the developing oocyte (Chiquoine, 1959; Odor, 1959), and in studies of ovarian oocytes and unfertilized tubal ova (Odor, 1960; Odor and Renninger, 1960) (see Figs. 14.6 to 14.8). As Villee has indicated in his chapter, great strides have been taken to- ward an understanding of the metabolic pathways in estrogen and progesterone syn- thesis and degradation. Two factors may have contributed to the McKay and Robinson, 1947; Meyer and McShan, 1950; Barker, 1951; Rockenschaub, 1951; White, Hertig, Rock and Adams, 1951 ; Deane, 1952; Nishi- zuka, 1954; Ford and Hirschman, 1955; Noach and \an Rees, 1958. 449 450 PHYSIOLOGY OF GONADS disappointment that has been expressed. First, the purification and synthesis of the hormones in the 1930's (Allen, 1939; Doisy, 1939) and the later successful development of synthetic estrogens and gestagens ( Solms- sen, 1945; Dodds, 1955; Rock, Garcia and Pincus, 1956; St. Whitelock, 1958) provided a means whereby much of ovarian physi- ology could be studied out of context with the processes by which this organ functions. Specifically, there are many effector actions of ovarian hormones, many interrelation- ships with other hormones and with each other, many problems of tissue responsive- ness, and many questions bearing on proc- esses of ovarian hormone metabolism, all of which can be studied in ovariectomized ani- mals. Secondly, there were many practical rea- sons why chemists should have striven to synthesize estrogenic substances and gesta- gens which are suitable for replacement therapy. Once prepared, these synthetic sub- stitutes are of interest, but their develop- ment and therapeutic application may well have diverted attention from studies of the ovary. If there is disappointment with tlie prog- ress that has been recorded, we would direct attention to substantial accomplishments which should stand us in good stead in the future. Among these are the numerous care- ful descriptions of the growth and matura- tion of ovarian follicles and the meticulous accounts of corpus luteum formation, struc- ture, and involution. In a general way it has become clear that in many species estrogen and progesterone are produced while the follicles are maturing, and that, during the functional life of the corpus luteum, pro- gesterone and estrogen are secreted. Esti- mates of the amounts produced have been numerous and of more than ordinary in- terest. In addition, they probably represent steps toward the determination of additional important information : the day-to-day rate of production correlated with the growth of the follicles and the development of the corpora lutea, and, in species in which variable numbers of follicles and corpora lutea develop, steps in an effort to ascertain whether, for example, 10 follicles in an in- dividual produce more hormone than 5. This knowledge, if we possessed it, might contribute significantly to current theories of gonadal-pituitary relationships because thresholds are involved in the regulatory processes r,-ini,iizs (jl (le\f'lu]iiiieiil. C: ikm Fig. 7.O. Imuiuturu li.ypophy.sectomized rat treated with Arniuui - i>il. (iiauulo.sa ha^ proliferated and follicles have developed antra. The theca is diffeientiated but the intersti- tium is deficient. (Couitesy of Dr. R. M. Melampy.) clearly the local stimulating effect of estro- gens with the ovary, as well as the systemic effect by way of the pituitary. If estrogen administration to immature or to hypophysectomized immature rats is con- tinued 7 to 10 days, the granulosa of the stimulated follicles degenerates. This atretic process differs from natural atresia in that it seems to start peripherally rather than centrally. The oocytes do not fragment or give off polar bodies as frecjuently as do oocytes in normally atretic follicles. It seems that the stimulatory effect of estrogen on the granulosa is very temporary. Its din-a- tion, however, is long enough to be coni- ])atible with the noi'inal pi'ocess of matura- tion and ovuhitiou. Before concluding the subject, a certain amount of back-tracking may be dcsii'able. One of the first suggestions to be made by Edgar Allen (1922; see also the biogi'aphic;il sketch in this book) was tliat the ovum is the dynamic center of the follicle. If the suggestion is placed in the context that has since been developed, the sequence of events would l)e an inductive influence of the oocyte on the membrana granulosa, a con- tinued inductive influence (oocyte or mem- brana granulosa?) on the surrounding con- nective tissue cells until the theca interna is formed,- and then the secretion of estro- 'Resuhs obtained by Genther (1931), Schmidt (1936), Humphreys and Zuckerman (1954). and Wcstman (1958) suggest that a similar functional iclationship exists between the granulosa and in- terstitial cells. According to Genther, x-ray-iujured o\aries composed of interstitial cells produced es- trogen only if a growing follicle was present. The in\oluted condition of the uteri in rabbits in wliich all oocytes and f()llicl(>s iiad been destroyed by x-rays led Humphreys and Zuckerman to conclude that the ovaries of these animals were not produc- ing estrogen. The results reported by Westman suggested tliat interstitial cell function continues only lor .1 limited jieriod after x-ray-induced de- generation of the granulosa cells. The results from an ingenious investigation by Ingram (1957) ar( MAMMALIAN OVARY 455 gen wliich feeds back to stimulate further growth of the meinbrana granulosa and fol- licle. Attractive as such a hypothesis is, it has at least one weakness. If gonadotrophic extract rich in FSH is administered to im- mature rats for three days, there is a gen- eralized stimulation of granulosa tissue (Fig. 7.3). Small follicles increase in size, medium sized follicles develop an antrum, and Graafian follicles become large and vesicular (Parkes, 1943), but ovulation is uncommon. At autopsy the ovaries are pale and edematous. The ovaries are markedly increased in size from numerous follicles be- coming vesicular. Histologically there is little stimulation of the theca interna. Gaar- enstroom and de Jongh (1946) recog- nized this ovarian response when they suggested that FSH be designated as Ge (gonadotrophin e])ithelial). This tissue re- sponse offers evidence that FSH is primarily f'oncerned with growth and proliferation of the granulosa cells, but there is no explana- tion to account for the failure of these granulosa cells to stimulate the differentia- tion of the theca interna and the eventual secretion of estrogens. During all of follicular growth, the pres- ence of estrogen has come to l^e assumed and its production by the growing follicle is thought to begin with the appearance of the theca interna (see below). On the other hand, the amount produced and the rate of production are unknown. The amount must increase with the growth of the follicles. Gillman and Gilbert (1946) found during their investigation of perineal turgescence in the baboon that, once the perineum reaches maximal turgescence, additional estrogen is required to maintain it. They concluded that in normal animals, during the second part of the phase of turgescence, there must be an increased output of ovarian estrogen. Direct studies have yielded little similarly suggestive. Autografts of ovarian medulla without cortical tissue or oocytes, and autografts of cortical tissue were transplanted to various sites in .sexually mature rabbits. The grafts of cortical tis- su(> p(>rsisti'(l after the medullary grafts had disap- peared. Ingram concluded that medullary tissue containing interstitial tissue but no follicles can- not survive. information. Foi'd and Hirschman (1955) estimated alkaline phosphatase activity in the ovary of the rat, but the concentrations in the theca interna and ovarian tissue as a whole were relatively constant during the phases of the cycle. B. GROWTH OF VESICULAR FOLLICLES The growth of the follicle which is de- pendent on stimulation by hypophyseal gon- adotrophins has been described for a number of animals and, for a few (cow, sow, ewe, guinea pig, rat) plotted with respect to the time of the preceding ovulation (McKenzie, 1926; Hammond, 1927; Grant, 1934; Myers, Young and Dempsey, 1936; Boling, Blan- dau, Soderwall and Young, 1941 ; von Burkl and Kellner, 1956). Data of the latter sort are especially valuable for the baselines they provide for experimental studies of the factors affecting the pituitary-gonadal re- lationships. Deviations in the shape of the curve of follicular growth, and disparities in the size of the growing follicles and in the size and structure of the corpora lutea, are clear indicators of abnormalities in function which have been too little used. C. PREOVULATORY SWELLING Without exception in the animals listed above, and probably in the horse, goat, and bat, if we may judge from the data pre- sented by Hammond and Wodzicki (1941), Wimsatt (1944), and Harrison (1946, 1948b), a linear period of growth during most of the diestrum is followed by a posi- tive acceleration (preovulatory swelling) ; shortly before estrus and ovulation. The point at which this acceleration occurs is the point in the development of the follicle where physiologic evidence for the produc- tion of progesterone by the unruptured fol- licle was first found (Dempsey, Hertz and Young, 1936; Astwood, 1939). As we will see later, however, the ^'moment" the pre- ovulatory swelling begins is not necessarily the point in time when the first progesterone is produced. In most sjiecies in which the course of the preovulatory swelling has been followed, it is a 10- to 12-hour process (Hammond, 1927; Grant, 1934; Myers, Young and 456 PHYSIOLOGY OF GONADS Dempsey, 1936; Doling, Blandau, Soder- wall and Young, 1941 ; Rowlands and Wil- liams, 1943; Rowlands, 1944), although in the cat and ferret the process is triggered by mating and extends over 25 to 30 hours. The preovulatory swelling can be initiated by injecting gonadotrophins of the LH or ICSH type, but they are effective only on well matured follicles (Hisaw, 1947; Tal- bert, Meyer and McShan, 1951). The younger follicles are not stimulated and, on the contrary, they may show an accelerated atresia. It could be postulated that the fol- licules with well developed theca interna were "competent" and that stimulated theca interna produced estrogen which favored the development of these follicles. The smaller follicles were "incompetent" in the absence of a thecal investment and became atretic. If HCG is injected into immature rats, the theca interna around the vesicular follicles hypertro])hies within 24 hours and these follicles enlarge rapidly. The vaso- dilation of the theca blood vessels is grossly evident within a few hours (Kupperman, McShan and Meyer, 1948; Sturgis and Politou, 1951; Odcblad, Nati, Selin and Westin, 1956). Explanation has been sought for the na- ture of the changes within the follicle which lead to the accelerated enlargement culmi- nating in ovulation. Studies of the staining qualities of such follicles reveal that the metachromatic polysaccharides of the gran- ulosa (hyaluronic acid and chondroitin sulfuric acid) become progressively depoly- merized and orthochromatic. This hydroly- sis of the mucopolysaccharides gives rise to an increased osmolarity which may be the major factor in the preovulatory swelling of the follicle (Harter, 1948; Catchpole, Gersh and Pan, 1950; Odeblad, 1954; Zachariae, 1958; Zachariae and Jensen, 1958; Jensen and Zachariae, 1958). Accompanying the swelling is a dispersal of the cells of the cumulus oophorus. This may be a cons(^- quence of the breakdown of the intcrcclhihii' substance in the stinudated niciubraiia gi'aii- ulosa. The time r('(|uirc(l for follicular growth and maturation fi'oin the stage when its further development is dependent on pitui- tary gonadotrophin stimulation to ovulation is related to the length of the cycle and therefore varies greatly from species to spe- cies. Somewhat less than 4 to 5 days are re- quired in the rat (Boling, Blandau, Soder- wall and Young, 1941 ) , somewhat less than 16 days in the guinea pig (Myers, Young and Dempsey, 1936), somewhat less than 21 days in the cow (Hammond, 1927), and ])resumably comparable intervals in other species. Vermande-Van Eck (1956) esti- mated that in the rhesus monkey the aver- age time required for the growth of a mature follicle from the large follicle without an antrum is 4 to 6 weeks; 11 days are esti- mated to lie necessary for the complete de- velopment of a follicle in the rabbit (De- saive, 1948) . Ovulation occurred earlier than normal when the corpora lutea from the ])receding cycle were removed, but the rate of follicular development was not altered (Dem])sey, 1937). Presumptive evidence ex- ists, however, that the rate of growth may be slower in pubescent chimpanzees (Young and Yerkes, 1943), baboons (Gillman and Gilbert, 1946), and guinea jngs (Ford and Young, 1953). D. OVULATION Ovulation, under normal circumstances, ))robably is explosive (Hill, Allen and Kramer, 1935, in the rabbit; Blandau, 1955, in the rat). In 1 of 2 human i)atients Doyle (1951) saw a gush of follicular fluid at the time of ovulation. In 163 ovulations timed l)y Blandau the interval between the rupture of the stigma and the escape of the ovum was 72 seconds when most of the folliculai' fluid escai)ed in advance of the ovum, and 216 seconds when the cumulus oophorus pre- ceded the follicular fluid. The slower, steady, continuous flow of the liquor folliculi which has been described by Walton and Ham- mond (1928) in the cow, Markee and Hin- sey (1936) in the rabbit, and by Doyle (1951) in one human sul)ject could be an artifact of the procedures used in watching the jM'Ocess. The mechanisiu heading up to formation of the stigma and rupture of the follicle is unknown. Claesson (1947), using the sub- microscoi)ical differences which can be ol)- served in ])olarized light, distinguished smooth muscle from connective tissue cells and rep()rte(l that no bundles of smooth muscle or isolated cells were found in the MAMMALIAN OVARY 457 theca externa in ovaries from the cow, pig, rabbit, and guinea pig. The earlier con- tradictory results he reviewed were at- tributed to the nonspecificity of the older staining methods. A possible clue to the mechanism of ovulation wliich does not seem to have been explored was given by the ob- servations of Boling, Blandau, Soderwall and Young ( 1941 ) when they were studying follicular growth in the rat. Immediately before ovulation, but at no other time, a large pocket at the base of the cumulus, and described as an invagination of the granulosa, is a constant feature of follicu- lar structure (Fig. 9 in their article). No guess w^as made as to its significance. In all the spontaneously ovulating infra- human mammals that have been studied, except the dog (Evans and Cole, 1931 ) , possibly other Canidae, and the mouse (Snell, Fekete, Hummel and Law, 1940) in which it takes place early in estrus, ovu- lation occurs toward the end of heat (see reviews in Young, 1941; Dukes, 1943; and more recent articles on the chimpanzee, rhe- sus monkey, baboon, cow, and mare by Young and Ycrkes, 1943; van Wagenen, 1945, 1947; Gillman and Gilbert, 1946; Cordiez, 1949; and Trum, 1950; respec- tively ) . Only in the human female in which cyclic waxing ^nd waning of sexual desire is not easily detected does uncertainty exist. Since an early period, when emphasis was given to the opinion that ovulation occurs about midway in the intermenstrual inter- val (Knaus, 1935; Hartman, 1936; Farris, 1948), much evidence has been produced indicating that it may occur at other times as well, even during menstruation (Teacher, 1935; Rubenstein, 1939; Sevitt, 1946; Berg- man, 1949; Stieve, 1952; and many others). If we may judge from what has been found in the chimpanzee (Young and Yerkes, 1943), baboon (Gillman and Gilbert, 1946), ihcsus monkey (Rossman and Bartclmez, 1946), and man (Bergman, 1949; Buxton, 1950), irregularities in the length of the preovulatory and postovulatory phases of the cycle complicate the problem and could account for some of the confusion. In the chimjianzee, baboon, and human female, in which the irregularities can be located wdth respect to the time of ovulation, age in- fluences the length of both phases, and fol- lowing pregnancies there are similar ir- regularities. In the baboon, environmental stresses result in temporary or even pro- longed inhibition of ovarian activity. There is no reason for believing that the same fac- tors have less effect on folliculogenesis in the human female; irregularities in adoles- cence (Engle and Shelesnyak, 1934) and fol- lowing pregnancy (Sharman, 1950, 1951) are common and there are many reports of psychic effects (see reviews by Kelley, 1942; Kelley, Daniels, Poe, Easser and Monroe, 1954; Kroger and Freed, 1950; Randall and McElin, 1951; Bos and Cleghorn, 1958). In all cases follicular growth is interrupted and amenorrhea follows. But if the esti- mates are correct that the average fertile woman ovulates normally about 85 per cent of the time (Farris, 1952), or that perfectly healthy women may have 3 or 4 anovulatory cycles a year (de Allende, 1956; also see table in Bergman, 1949), there must also be cases in which much of follicular growth is normal, or at least adequate to stimulate growth changes in the uterus, but ovulation does not occur. As if the complications noted above are not enough, the reviews of the methods used in determining the time of ovulation (D'Amour, 1934; Cohen and Hankin, 1960) and the critical study of Buxton and Engle (1950) in which an at- tempt was made to correlate basal body temperature, the condition of the endome- trium, and the stage of folliculogenesis in the ovary, suggest either that a really sensi- tive indicator of the time of ovulation has not been found, or if one exists, that it has not been used in a study sufficiently sys- tematic to reveal the true situation in the human female. The problem is one of the many that is with us very much as it was 20 years ago. E. FOLLICULOCJENESIS IN PREGNANCY AND LACTATION Before leaving the subject of follicular growth, its course in pregnancy and lacta- tion should be reviewed. Information has been obtained from many species, but in most cases it is not complete and a con- siderable amount of conjecture is necessary. What is certain is that pregnancy affects the process of folliculogensis in many ways ; each must be the reflection of a different in- 458 PHYSIOLOGY OF GONADS terrelationsliip betwen pituitary, gonads, and placenta. In the mare, and presumably other species in which multiple ovulations occur early in pregnancy, the involvement of chorionic gonadotrophins, pituitary gona- dotrophins, and estrogen of placental origin has been suggested (Rowlands, 1949). When folliculogenesis is inhibited just before the stage of the preovulatory swelling, as it is in many pregnant animals (see below), the nervous system may be involved. An un- usually significant investigation in which the threshold of stimulation to ovulation in the rabbit was correlated with threshold changes in cerebral activity has recently been completed (Kawakami and Sawyer, 1959). It was demonstrated that pregnancy or prolonged treatment with progesterone maintains the electroencephalogram (EEGj after-reaction threshold to low frequency stimulation of hypothalamic or rhinen- cephalic nuclei at an elevated level. At this level, gonadotrophin release does not occur in response to coitus or other ovulatory stimuli. The discovery of this fact has pro- vided a basis for understanding the various ovarian conditions associated with preg- nancy and lactation, or at least those in which follicular development proceeds to the point of preovulatory swelling and then stops. It may be that some other mechanism of inhibition accounts for the more severe retardation of folliculogenesis in si)ecies in which tliis occurs. The European hares, Lepus tiniidus L., and L. ciiniculus L., are reported as mating during pregnancy with the occurrence of superfetation (Lienhart, 1940). Pregnancy, therefore, has little or no effect on any stage of folliculogenesis in these species. The domestic rabbit appears to he somewhat more affected and perhaps more variable. Claesson, Hillarj), Hogberg and ll()kfeh (1949) state that the ovaiics of pii'gnant rab})its are composed almost entirely of interstitial gland, except for the corpoi'a lu- tea, but, according to Hannnond and Mar- shall (1925) and Dawson (1946), mature follicles ai'e present and pregnant animals will occasionally mate. However, if we may assume that the reaction of i)regnant ani- mals is similar to that of ])seudopregnant animals (Makepeace, Weinstein and Fried- man, 1938), pituitary gonadoti'opliin is not released and ovulation does not occur. From examination of the ovaries and from the fact that fertile matings can occur within a very few hours after parturition (Dempsey, 1937; Boling, Blandau, Wilson and Young, 1939; Blandau and Soderwall, 1941), it is clear that follicular development in the pregnant guinea pig and rat proceeds to a point just short of the preovulatory swell- ing. According to Nelson (1929) and Swezy and Evans (1930), cycles of oogenesis occur in laboratory rats, and, although the folli- cles may form small corpora lutea (Swezy and Evans), ordinarily they do not rupture. The musk-rat. Ondatra zibethica, and the African bat, Xycteris luteola, must display an advanced follicular development during pregnancy because there is evidence of post- l)artum estrus (Warwick, 1940; Matthews, 1941, respectively). Brown and Luther (1951) state that postpartum estrus occurs within 3 days after farrowing in the sow, if the young pigs are removed. We assume, from this latter statement and from the re- l^ort that estrus and service may occur dur- ing pregnancy in this species (Perry and Pomeroy, 1956), that large follicles are present in tlie ovaries of the pregnant sow. Heat ])eriods in the jjregnant ewe are asso- ciated with follicular growth, but ovulation does not occur, and late in pregnancy fol- licle size decreases significantly (Williams, Garrigus, Norton and Nalbandov, 1956 ) . The first heat after {parturition was an aver- age of 23.9 days later, range 1 to 61 days. According to Harrison (1948b), widespread atretic changes can be seen in all the folli- cles in the goat, beginning the 40th day of |)regnancy. By the (iOth day. no healthy fol- licles can be found. Hammond (1927) was of the opinion that during jircgnancy in the cow, follicles de- velop to the size at which the jireovulatory swelling begins, but Dukes (1943), citing a study by Weber, wrote that cows come into heat 3 to 7 weeks after parturition. Support for tliis \-iew comes from the report l)y Hafez ( 1954) that the average interval to the post- partum esti'us in another bovine, the Egyp- tian buffalo, is 43.8 days, range 16 to 76 days. The iclatixcly long postpartum inter- \al in these two species is presumptive evi- dence that follicles are relatively small at the end of pregnancy in bovines. MAMMALIAN OVARY 459 Between the 40th and 150th day of preg- nancy in the mare the ovaries contain nu- merous actively growing follicles and several functional corpora lutea (Cole, Howell and Hart, 1931; Rowlands, 1949). However, from the 150th day until the late stages, there is a regression of all the corpora lutea and an absence of large follicles. In the late stages only minute vestiges of corpora lutea and small follicles remain. If the latter is true, follicular growth must be rapid after parturition, because the first heat following foaling was between the 7th and 10th days in 77 per cent of the many mares Trum (1950) studied. In the African elephant, Loxodonta africana, there is also a replace- ment of the corpora lutea (one plus several accessory corpora lutea j about midway through pregnancy (Perry, 1953). Some are formed following ovulation and some not. They persist until term when they involute rapidly. During the late stages of pregnancy no follicles with antra are founcl. Dawson ( 1946) wrote that the domestic cat does not possess mature follicles at the time of par- turition. In nonlactating animals the pro- estrous level is reached the 4th week after parturition. Presumptive evidence exists that the folli- cles in the parturitive chimpanzee are small (Young and Yerkes, 1943 ) . In the human fe- male the appearance of the first ovulatory cycle after pregnancy is irregular (Sharman, 1950, 1951 ; AlcKeown, Gibson and Dougray, 1954). According to Sharman, it may occur about 6 weeks after delivery in nonlactating women. This suggests that follicles are small at the end of ju-egnancy in the human fe- male. Inhibitory effects of lactation on follicular development are indicated by the substance of many of the reports cited above (Dawson; Dukes; Perry; Schwartz; Sharman; Wil- liams, Garrigus, Norton and Nalbandov) and by much other information. As would be expected, the intra- and interspecies vari- ations are great. Studies in progress at Iowa (Bradbury, personal communication) are re- vealing that some women experience an atrophy of the vaginal epithelium during the second and third month of lactation. The atrophy is indicative of a lack of ovarian es- trogen and suggests that follicular develop- ment is not normal. Observations that are similarly suggestive have been made in other species. The absence of estrogen in signifi- cant ciuantities during lactation in the mouse (Atkinson and Leathem, 1946) and guinea pig (Rowlands, 1956) is believed to be the reflection of a delay in the resumption of follicular growth and ovulation. Mother rats and mice may copulate and conceive within 24 hours after delivering a litter of young. AVhile the mother is nursing the newborn lit- ter, the fertilized eggs of the new pregnancy develop into blastocysts, but these blasto- cysts fail to implant in the uterus at the usual time (Talmadge, Buchanan, Kraintz, Lazo-Wasem and Zarrow, 1954; Whitten, 1955; Cochrane and Meyer, 1957). This de- lay in implantation is apparently due to a lack of estrogen, because an injection of es- trogen will result in implantation of the blastocysts. The suppression of estrous cy- cles during lactation in the mouse and rat is influenced in part by the size of the litter. A litter of 8 to 10 young will inhibit cycles, whereas cycles are displayed if the litter is reduced to 2 or 3 young (Parkes, 1926a; Hain, 1935). The cottontail rabbit [Sijlvila- gus floridamis) seems to be a species in which ovarian follicular development is lit- tle if any affected by lactation, for Schwartz ( 1942 ) stated that suckling does not prevent ovulation after coitus, at least in the early stages of lactation. III. Corpus Liiteuni The formation of the corpus luteum has been described for many species ( see reviews in Corner, 1945; Harrison, 1948a; Brambell, 1956). In general, after rupture of the folli- cle and discharge of the ovum, the granu- losa is invaded by blood vessels from the theca interna (Bassett, 1943). They form a rich network among the enlarging granulosa lutein cells. The extent and nature of the contribution from the theca interna varies from species to species, but, as Corner states, the origin of the major part of the epithe- lioid cells of the corpus luteum from the granulosa may now be considered a fact. Whereas there may be a fairly uniform pattern of development and control of ovar- ian follicles in mammalian forms, there are diverse mechanisms for the formation and maintenance of corpoi'a lutea; consequently 460 PHYSIOLOGY OF GONADS specific examples must be presented in order to avoid the dangers of generalization. In the rabbit copulation triggers a neuro- humoral mechanism which releases gonado- trophin from the pituitary which subse- quently induces ovulation in 10 to 12 hours. The ruptured follicles form corpora lutea which have a functional span of about 28 days if pregnancy ensues but only 14 days if the mating is infertile. Crystals of estrogen implanted into a corpus luteum of a rabbit will cause its persistence while other corj^ora lutea regress (Hammond and Robson, 1951 ). This suggests that either estrogen makes the corpus luteum more sensitive to pituitary maintenance (Hammond, 1956) or estrogen protects the corpus luteum from luteolytic action. In the cat, copulation induces ovula- tion about 25 hours after mating; the cor- pora lutea function for 36 days after an in- fertile mating, but gestation lasts 62 to 64 days. The ferret ovulates about 30 hours after copulation and the corpora lutea are functional for 42 days, w^hether the mating is fertile or infertile (Brambell, 1956 1. In the unmated rat and mouse ovulation is spontaneous, but the resulting corpora lu- tea are nonfunctional and begin to regress within 2 days. After copulation the corpora lutea persist for 18 days if impregnation has occurred, but for only 12 days after an in- fertile mating. Copulation probably results in the release of enough additional gonado- trophin (LH or LTH) to activate the cor- pora lutea. In rats and mice the pituitary hormone, prolactin, is luteotrophic (LTH) (Desclin, 1949; Everett, 1956). These spe- cies have functional corpora lutea through- out lactation— actually two sets, that of pregnancy and that of the postpartum ovu- lation. Using the rat and taking weight and levels of ovarian enzymes as measures of ac- tivity, these corpora lutea were studied by Meyer and McShan and their associates and the results summarized in a ic\'i('\v (]\Ieyer and McShan, 1950). They found that "the weight of the corpora lutea of pregnancy in- creased greatly during the latt(>r half and that the amount of enzymes per corjius lu- teum was also greater. With some caution, they concluded that these corpora lutea are more highly functional during this phase of pregnancy than during the first half. Not only copulation, but also injection of estrogen at estrus is followed by the forma- tion of functional corpora lutea. The estro- gen maintenance of corpora lutea in rabbits and mice is offset by hypophysectomy (Hohn and Robson, 1949); presumably, therefore, maintenance is mediated through the anterior })ituitary. Reece and Turner (1937) showed that estrogen stimulates the rat i^ituitary to produce prolactin so the lat- ter may be the luteotrophic agent in this s]:)ecies. Moore and Nalbandov (1955) found that prolactin is luteotrophic in sheep. To date this is the only species other than the rat and mouse in which prolactin has been shown to have luteotrophic activity. In the guinea pig, monkey, man, and many other species, ovulation and the for- mation of functional corpora lutea are spon- taneous. Copulation is not known to have any neurohumoral influence in these species. The corjiora lutea of the human female func- tion for 2 to 3 months in pregnancy and for only 12 to 14 days in. an infertile cycle. Berg- man ( 1949 ) states that the duration of the luteal i^hase is limited to a maximum of 16 days. In the rhesus monkey the functional life has been estimated to be about 13.5 days in the normal cycle, and approximately 30 days when pregnancy intervenes (Hisaw, 1944) . In the bitch, ovulation is spontaneous and the corpora lutea remain functional for 6 weeks irrespective of mating or pregnancy. In the lactating African elephant the cor- pora lutea degenerate soon after parturition (Perry, 1953) ; in the lactating domestic cat they not only persist, l)ut they become ''re- juvenated" (Dawson, 1946). As a general statement, it can be said that the functional span of the corpora lutea is cithei' adequate to permit implantation or it is prolonged by copulation (as in rats and mice) so that imjilantation can occur. But inasmuch as imj:)lantation occurs in many species, including man, about the sixth day after ovulation and fertilization, the margin of safety is not great and a delay in the se- cretion of chorionic gonadotrophin by the tro])hoblast must reduce the chances of a successful pregnancy. Ill some species, e.f/., rats, mice, rabbits, an ill felt ih' mating prolongs the life of the corpora hitea. This prolonged interval of functional luteal activity is known as pseu- doj)regnancy. As Everett has noted in his MAMMALIAN OVARY 461 chapter, in pseudopregnancy tlie hormonal aspects of pregnancy are duplicated, but no fetal tissues are present. In the pseudopreg- nant bitch, for example, the hormonal as- pects of pregnancy are so nearly duplicated that lactation begins at the time a normal gestation would have terminated. The duration of pseudopregnancy in dif- ferent species offers evidence of adaptive or evolutionary mechanisms to control the du- ration of corpus luteum function, mecha- nisms that must be endogenous to the uterus. Rats and mice have a pseudopregnancy of 12 days duration after a sterile mating, cer- vical stimulation, or injection of estrogen at estrus. There is no comparable condition in guinea pigs, monkeys, or man. However, if rabbits, rats, or guinea pigs are hysterecto- mized, any subsequent corpora lutea will function for a time equivalent to the dura- tion of gestation in each species (Chu, Lee and You, 1946; Bradbury, Brown and Gray, 1950), although Velardo, Olsen, Hisaw and Dawson (1953) stated that, in the rat, hys- terectomy has no effect on the length of pseudopregnancy. Hysterectomy in the cow and sow will prolong the life of the corpus luteum (Melampy, personal communica- tion). Experimental distention of the uterus l)y beads has resulted in alteration of the length of the estrous cycle in ewes (Nal- l)andov, Moore and Norton, 1955). The only explanation which seems to account for these results is that there is a luteolytic agent in the uterus (probably in the endo- metrium) of some polyestrous species which shortens the life of the corpora lutea in non- pregnant animals. In pregnancy, or when massive deciduomas are present, if Velardo, Olsen, Hisaw and Dawson are correct, the conversion of endometrium to decidual tis- sue may cause it to lose its luteolytic ability. In future studies on the duration of the functional span of cor]5ora lutea, the possi- bility of luteotrophic and luteolytic mecha- nisms should be considered. On the other hand, a fresh start may be advisable. Few- problems in reproductive and clinical endo- crinology (Marx, 1935) seem to have been as resistant to clarification. In unmated females of species not having a spontaneous "pseudopregnancy," the cor- lius luteum involutes shortly after its forma- tion. The rat, in which 4 to 8 corpora lutea are formed in each ovary at intervals of 4 to 5 days, has recognizable involuting cor- pora lutea from the two preceding cycles, but no remnants of older ones. The early stages of involution of the corpus luteum have been described (Brewer, 1942; Boling, 1942; Dawson, 1946; Duke, 1949; Moss, Wrenn and Sykes, 1954; Corner, Jr., 1956; Rowlands, 1956; Dickie, Atkinson and Fe- kete, 1957). The timetables of cellular changes given by Brewer and by Corner, Jr. are of interest for the comparison they per- mit with physiologic estimates of the dura- tion of secretory activity by the human cor- pus luteum. On day 7 the corpus luteum seems to have reached its peak of activity, as judged by the vacuolation of its cells in Bouin's or Zenker's fluid-fixed and hematox- ylin and eosin-stained preparations. Corpora lutea of days 9 to 12 show evidence of i)ro- gressive secretory exhaustion. The later stages of cori:)ora lutea degenera- tion have not received the same careful at- tention. In women the corpus luteum under- goes a slow hyaline degeneration and the corpora albicantia persist as old scars for months or years. They may be present in ovaries 15 to 20 years after the menopause. Whether the final stage of degeneration is a process of lysis, phagocytosis, or transfor- mation into connective tissue has not been studied. IV. Follicular Atresia It was long ago estimated that the infan- tile liuman ovary contains about 400,000 oocytes (Fig. 7.4). In the 30 years of repro- ductive life about 400 ova may mature and ovulate. On this basis about 1 oocyte in 1000 achieves ovulation; the other 999 are lost through a degenerative process known as atresia. The problem is not different in any other species. Whether it is monotocous or polytocous, there is always an enormous wastage of oocytes in each cycle of follicu- logenesis. Atresia may have its onset at any stage of follicular growth or maturation and oocytes may degenerate before they have acquired a distinct membrana granulosa (Mandl and Zuckerman. 1950; de Wit, 1953; Payne, Hellbaum and Owens, 1956; Wil- liams, 1956). In advanced stages of follicu- lar development the granulosa cells may show pycnotic changes before any degenera- 462 PHYSIOLOGY OF GONADS Fig. 7.4. Ovary from 28-moiith-old child. Many primordial follicles just beneath the tunica albuginea. (Courtesy of Dr. J. T. Bradbury.) Fill. 7.5. Iimuaturc rat h grow to medium size. Many become atretic and leave stitial tissue. (Courtesy of Dr. R. M. Melampy.) MAMMALIAN OVARY 463 Fig. 7.6. Intact immature rat given progesterone for 3 days. The various-sized follicles in tliis area are in early stages of atresia. The pycnotic granulosa cells are dispersing into the follicular fluid and the oocyte in the small follicle at the upper left is denuded. (Courtesv of Dr. J. T.Bradburv.) tive clianges are evident in the oocyte. When vesicular follicles become atretic the granu- losa disintegrates and the cells disperse into the liquor folliculi (Knigge and Leathern, 19.56 ( . If the follicle has developed a distinct theca interna, the theca regresses after the granulosa has disintegrated. In rats the atretic follicles leave no recognizable histo- logic remnants. In some species the theca re- gresses back to ovarian interstitial tissue (Dawson and AlcCabe, 1951; Williams, 1956). In the ovary of the human female and rhesus monkey the atretic follicle leaves a scar (corpus atreticum) in which the mem- l)rana propria persists for months as a folded hyaline membrane within the loose fibrous remnant of the theca. The cause of follicular atresia is not known. Immediately after hypophysectomy there is a wave of atresia in the ovary of the immature rat (Fig. 7.5) and rabbit (Foster, Foster and Hisaw, 1937). In adult rats the postovulatory wave of follicular atresia has l)een attrilnited to an action of the corjiora lutea (Atkinson and Leathem, 1946). Injec- tions of androgen or of progesterone increase the incidence of atr(>sia in rat ovaries (Fig. 7.6) (Paesi, 1949b; Barraclough, 1955; Payne, Hellbaum and Owens, 1956). Fur- ther study is necessary to determine whether the atresia is due to a direct effect of andro- gen or progesterone on the follicles, or whether the postovulatory decline in gon- adotrophins, like hypophysectomy, with- draws a supi:)orting influence and permits the follicles to degenerate. The supporting influence may be estrogenic because in- jections of estrogen at the time of hypo- physectomy will prevent, or at least delay, the expected follicular atresia (Fig. 7.7). Some months after hypophysectomy the number of remaining oocytes is greater than in the ovaries of normal litter-mate sisters (Ingram, 1953). This suggests that vegetating oocytes are less liable to undergo 464 PHYSIOLOGY OF GONADS It:- '^l&^h ■ ■;%.%, Fig. 7.7. iiniuaiKi. li.\ pupliy.sectomized rat treated with estrogen (dit'thylstilbe.Ntrol). Many follicles have developed to a size appropriate for antrum formation. The interstitium i.s atrophic but the theca is differentiated. One follicle is obviously atretic. (Courtesy of Dr. J. T. Bnidburv.) atresia than those which have entered the growth phase. Hisaw (1947) discussed the problem and marshaled a number of facts in support of the idea that atresia is due to a defective differentiation of the theca interna, with a ^resulting deficiency of estrogen which is considered necessary for growth and differ- entiation of the granulosa. Ultimately the ])ituitary is involved because the success which has been achieved in the j^roduction of sui)erovulation reveals that the number of follicles maturing and ovulating is a measure of the amount of gonadotrophic hormone. But it is eciually true that there is an optimal dosage and time beyond which defects appear in the form of cystic follicles and premature luteinization (see review by Hisaw and more recently Zarrow, Caldw(>ll, Hafez and Piiicus, 19581. The disintegration of the discus proligerus of the nuiture follicle and the dissolution of the granulosa in an atretic follicle have led several investigators to suggest that the stimulus to ovulation and/or atresia is iden- tical (Harman and Kirgis, 1938; Dawson and McCabe, 1951 ; Moricard and Gothie, 1953; Williams, 1956). Moricard and Gothic found that intrafollicular injection of HCG or P]\1S caused first polar body formation within 4 liours. Control injections of estro- gen or scrum were ineffective. Dempsey (1939) noted that maturation spindles were present in ne:iily all the oocytes in medium and large follicles which had undergone atresia shortly after ovulation had been in- duced by luteinizing hormone. Inasmuch as the tubal egg ntay give off the second polar body about the time the corona radiata is lost, it is interesting to speculate on the sig- nificance of the fact that the eggs in atretic follicles may also give off polar bodies just as they are denuded of granulosa (Fig. 7.8). V. Hormones of the Ovary The hoi'mones of the ovary are the estro- gens, pi-ogesterone. androgen, and relaxin. The first three are the o\'ai"ian steroid hor- MAMMALIAN OVARY 46.- Vu,. 7.S. Aticiic lolliric in immature rat gi\en progesterone for 3 day: and second metaphase si>indle. (Courtesy of Dr. J. T. Biadbury.) mones and are among the most important hormones participating in the regulation of reproductive physiology. In the present hook, as in editions 1 and 2, the discussions of their many actions consititute one of the central themes. Relaxin is a protein rather than a steroid hormone and has always been considered more or less apart from the latter. The steps in proving its existence were reviewed by Hisaw and Zarrow in 1950, and the present status of the subject is discussed in the chap- ter by Zarrow. Unlike the other ovarian hor- mones, its clinical value is still uncertain (Swann and Schumacher, 1958; Stone, 1959). Ovarian androgens present a perplexing problem. Evidence for their production is abundant (Guyenot and Naville-Trolliet. 1936; Hill, 1937a, b; Deanesly, 1938a; Brad- bury and Gaensbauer, 1939; Greene and Burrill, 1939; Chamorro, 1943; Price, 1944; Katsh, 1950; Desclin, 1955; Johnson, 1958). l)ut the extent to which they are produced l)y the ovaries of normal females, and the nature of their action in normal females are uncertain (Parkes, 1950). It has been pos- tulated that they are produced during the normal cycle in the rat. Payne, Hellbaum and Owens (1956) suggested that the inter- stitial androgens produced at estrus are re- sponsible for the postovulatory atresia of the partially developed follicles. The pro- duction of ovarian androgens has been dem- onstrated most effectively under abnormal conditions of stimulation. The newborn rat ovary will produce androgens when stimu- lated by human chorionic gonadotrophin (Bradbury and Gaensbauer, 1939). The treated infant rat shows a marked enlarge- ment of the clitoris and may even develop the cartilage anlage of the os penis. Female guinea pigs are masculinized by injections of HOG (Guyenot and Naville-Trolliet, 1936). Ovaries transplanted to the ear of the castrate male mouse will produce enough androgen to maintain the prostate and semi- nal vesicle (Hill, 1937a, b). Ovarian trans- l^lants into the seminal vesicle may exhibit a localized androgenic stimulation of that tissue (Katsh, 1950). Ovaries of a rat in 4(36 PHYSIOLOGY OF GONADS parabiosi;? with a castrate partner become hypertrophied and produce enougli andro- gen to stimulate prostatic tissue (Jolmson, 1958) ; associated with the condition is an unusual thecal and interstitial tissue hyper- trophy. The masculinizing features of the Stein-Leventhal syndrome ( polycystic ovary) have been attributed to the presence of androgens, having their source perhaps in the hilar cells of the ovaries (Lisser and Traut, 1954) , but the action of other steroids with masculinizing properties also has been suggested (Fischer and Riley, 1952). The latter possibility might well be checked in any case when the production of androgens by the ovary is suspected. Estrogen and progesterone are the ovarian hormones whose action in the female has been studied the most extensively. The steps which led to their extraction and chemical identification were described by Doisy and by Willard Allen in the 1932 and 1939 edi- tions and will not be repeated here. Atten- tion, however, will be directed to the iso- lation and identification of several naturally occurring gestagens in addition to progester- one (Davis and Plotz, 1957; Zander, Forbes, von Miinstermann and Neher, 1958). These are metabolic degradation products which still retain progestational activity. Now as in the period 1932 to 1939, there are many unsolved problems, but it is equally true that much of interest and value has been learned. Not the least of these con- tributions has been the clarification of the l)athways of their biosynthesis (see chapter by Villeej . In the sections which follow par- ticular attention will be given to the prob- lem of their origin, to the rate of production, the manner of transport and storage, and to their "half-life." A. CELLULAR ORTOIX As Villee points out in his chajiter, estro- gen and progesterone are apparently de- rived from cholesterol by a series of chemi- cal changes. Within the ovary and in the corpora lutea of rats, there is a definite re- duction in the concentration of ascorbic acid and cholesterol after gonadotrophin admin- istration. These changes have been consid- ered (ividence for tiie activation of hormone synthesis (Everett, 1947; Miller and Ever- ett, 1948; Levin and Jailer, 1948; Aldman, Claesson, Hillarp and Odeblad, 1949; Claes- son, Hillarp, Hogberg and Hokfelt, 1949; Noach and van Rees, 1958). Efforts to identify the cell types in which these processes take place have not been al- together successful. We have noted, for ex- ample, that several tissues such as testis,, placenta, and occasionally the adrenal cor- tex can produce estrogen. These are tissues,, then, which produce more than one hormone. Gardner emphasized this during a discus- sion of Parkes' (1950) review of androgenic activity of the ovary, when he called atten- tion to Dr. Furth's observations that some ovarian tumors possess potentialities for bi- sexual hormone production. About the same time, Shippel (1950) postulated that thecal cells may be a source of estrogen and andro- gen and that the type of hormone produced may depend on particular stresses or stim- uli. On the other hand, many investigators,, and particularly those interested in the ap- jilication of histocheraical procedures,^ have l^roceeded under the assumption that there are tissues of the ovary in which one hor- mone is iH'oduced predominantly. They found, for example, that the reactions of fol- licular granulosa and theca cells are strik- ingly different (Demi)sey and Bassett, 1943; Dempsey, 1948; Shij^pel, 1950). To be sure, the results w^iich have been obtained have not led to agreement with respect to details, but there is much evidence from the reac- tions which have been described, as well as from the older morphologic studies, that tlieca interna, interstitial, and luteal cells and, perhaps to a lesser extent, granulosa cells, are active in steroid hormone synthe- sis. What is less certain than the fact that these cells, and consequently the ovaries, produce estrogen, progesterone, and andro- gen is their relative role compared with that of other tissues. Presumably it is major; nevertheless, evidence for the extra-ovarian origin of estrogenic substances is provided by the occurrence of cyclic vaginal activity in ovariectomized animals (Kostitch and Telebakovitch, 1929; Mandl, 1951; Veziris, "Dempsey and Bassett, 1943; Dempsey, 1948; Claesson and Hillarp, 1947a-c ; Claesson, Diczfa- hisy, Hillarp and Hogl)erg, 1948; McKay and Rob- inson. 1947: Sliii)iH>l. 1950: Barker. 1951: Rocken- scliauh. 1951: Wliite, Heitis, Rock and Adams. 1951: Deane. 1«)52: Fuiulue-lm, 1954: Xisliizuka. 1954. MAMMALIAN OVARY 467 1951) and by the high titer of estrogens in the urine from ovariectomized rats on a high fat diet (Ferret, 1950). Corner, as long ago as 1938, in a consider- ation of the subject, emphasized that there is only circumstantial evidence that the ovary is the major site of estrogen produc- tion. Attempts to extract estradiol or any other estrogen from ovarian tissue had yielded very small amounts. MacCorquo- dale, Thayer and Doisy (1936) processed 4 tons of hog ovaries and recovered about 6 mg. estradiol from each ton. They estimated that the concentration in liquor folliculi was of the order of 1 part in 15,000,000 and that about 0.1 of this concentration is in the rest of the ovarian tissue. There are much better sources from which "ovarian hormone" can be extracted than from the ovary, i.e., pla- centas, pregnancy urine, the urine of the stallion or boar. The adrenal is also a source and there may be other tissues as well, for Bulbrook and Greenwood (1957) reported that urinary estrogen continued to be ex- creted after oophorectomy and adrenalec- tomy of a breast-cancer patient. Whatever the relationships are quantita- tively between the follicles and the other estrogen-secreting tissues, the evidence that the follicles are a major source of estrogenic substances remains impressive. This is also true of the corpus luteum. The human cor- pus luteum produces as much, or more, es- trogen than was produced during the follicu- lar phase. In the rat and mouse, estrogen production during the luteal phase must be low because more than 1 part of estrogen nullifies the action of 1000 parts of proges- terone in these species ( Velardo and Hisaw, 1951). By contrast, ratios as high as 100 parts of estrogen to 1000 parts of progester- one enhance the progestational reactions in women (Long and Bradbury, 1951). The different tissues of the ovary — mem- brana granulosa, theca interna, and inter- stitial cells — have been studied in efforts to ascertain whether they are sites of the pro- duction of specific hormones. Inasmuch as unruptured follicles and corpora lutea se- crete both hormones, the two structures must be considered. It has long been known that corpora lutea secrete estrogen as well as progesterone. Evidence supporting the conclusion that jirogesterone is secreted by the preovulatory follicle is more recent, but it comes from many sources. The possibility was first suggested following the discoveries that the beginning of mating behavior (Dempsey, Hertz and Young, 1936) and the decrease in tissue uterine fluid (Astwood, 1939) , which depend on the presence of small amounts of progesterone, coincide with the beginning of the preovulatory swelling. More recently, progesterone has been found in the follicular fluid from sows, cows, and the human female, and, in small quantities in blood plasma of the rabbit, human female, and rhesus monkey during the follicular phase of the cycle (Duy vene de Wit, 1942 ; Forbes, 1950, 1953; Bryans, 1951; Kauf- mann, 1952; Edgar, 1953b; Buchholz, Dib- belt and Schild, 1954; Zander, 1954). Earlier in this section it was noted that there is much evidence from both histo- chemical and the older morphologic studies that theca interna, interstitial tissue, and luteal cells, and to a lesser extent, granulosa cells secrete the ovarian steroid hormones. Many who have used histochemical methods still feel that, even though these methods demonstrate steroids and their precursors, the reactions are not sufficiently specific for identification of the individual hormones. Others, however, have been more confident, and when the evidence they have presented is combined with that reported in some of the more conventional morphologic studies the following summarization of opinion seems justified. Granulosa cells of the fol- licles and granulosa lutein cells of the cor- pora lutea contain progesterone or a pre- cursor and secrete this hormone (Nishizuka, 1954; Green, 1955). Cells of the theca in- terna, theca lutein cells, and interstitial cells are believed to secrete estrogen and possibly androgen (Corner, 1938; Deanesly, 1938a; Pfeifter and Hooker, 1942; Hernandez, 1943; Claesson and Hillarp, 1947a, b; Rocken- schaub, 1951 ; Aron and Aron, 1952; Furuh- jelm, 1954; Nishizuka, 1954; Fetzer, Hille- brecht, Muschke and Tonutti, 1955; John- son, 1958). The interstitial cells have been the object of much study and will be given especial attention. Interstitial tissue or cells in the ovary is not as clear a concept as it is in the teste.-. In the latter interstitial or Leydig cells are derivatives of connective tissue elements 468 PHYSIOLOGY OF GONADS irf^'t'H-'^ . 7.'.). Aii< ^lioii.-. ral)l)U. J^aigc lulliclf^ lUulcifioinK ati< Ma Iiilcr^l il luui i,> \i>ilil<- only as the narrow wedge of granular tissue extending from the cortex into the intrafollit-u- lar septum. (Courtesy of Dr. J. T. Bradbiuy.) and can (ledift'erentiate to form connective tissue cells (Esaki, 1928; Williams, 1950). The role of Leydig cells as secretors of testicular androgen or a precursor is not (juestioned. In the ovary it is also presumed that undifferentiated connective tissue ele- ments exist, indeed much of the stroma must be composed of such cells. It is believed rather generally, although unequivocal proof has not been given, that the theca interna is derived from connective tissue elements and that, as a component of the Graafian follicle, it secretes estrogen and possibly androgen Hoc. cit.). After ovulation and corpus lu- teura formation, and after atresia in the case of follicles not rui)turing, the cells of the theca interna may pcrhajjs I'csuinc their place as connective tissue cells or they may become interstitial cells (Mossman, 1937; Dawson and McCabe, 1955; Rennels, 1951 ; Nishizuka, 1954; Williams, 1956). The |)rominence of interstitial tissue varies from species to species and also with stages of the rei)roductive cycle. Whether it is functional in i^roducing hormones has been controver- sial, but most contemporary investigators seem to feel that internal secretory capacity has been demonstrated. In all the work that has been done, supporting evidence is var- ied; in some cases it is circumstantial, but in others it is quite substantial. Interstitial tissue is deficient in the anes- trous rabbit, and even though there may be considerable follicular development, there seems to be little or no estrogen production (Claesson and Hillarp, 1947a) (Fig. 7.9).^ The liypei'ti'opliied iiitei'stitium of the es- troiis i;il)l)it (Fig. 7.10) undergoes further de\-el()pnicnt (hii'ing prc'gnancy and seems almost as luteinized as th(^ corpora lutea ' Rogr('ssi\-e changes in tlic rciirodiictixe tract and accessory structures following ovariectomy of the anestrous opossimi were taken to indicate that these parts receive estrogenic stimidation of ovar- ian origin during th(^ anestnun (Morgan, 1946; Risman. 1946). MAMMALIAN OVARY 469 Kid, 7,10. l'(isto\nl.-,l,,iy stigma is evident. Note tli of Dr. J. T. Bradburv.) epitlielioid natiii of the hypertiopliied mterstitiuin. (Courtesy (Fig. 7.11). Grossly the ovary in the anes- trous rabbit is translucent whereas the es- trous ovaries and the ovaries during preg- nancy have a chalky white opacity due to the development of the interestitium. After hypophysectomy the interstitial cells of the rat ovary exhibit a deficiency condition and the nuclear appearance has suggested the name "wheel cells." If pituitary ICSH is administered, the deficiency cells are re- stored to normal (Fig. 7.12). Hyperplastic ovarian interstitium in older women has been considered a probable source of estro- gen in some cases and of androgen in others. The stimulation of interstitium by in- jected gonadotrophins may be associated with the formation of estrogens and/or an- drogens (Bradbury and Gaensbauer, 1939; Marx and Bradbury, 1940). Some rats dis- played a permanent estrus; others, during a period of androgenic function, were mas- culinized. During this period, the theca and interstitium were not luteinized in many cases and it was concluded from the re- sponses of accessory organs that these small immature cells had secreted male hormone and perhaps female hormone, too. In rats with fully luteinized theca and interstitium and the pronounced estrous symptoms, it was considered that the androgenic effect was no longer apparent. Information ob- tained recently, however, suggests that the permanent estrus, when it was shown, may have been a consequence of an androgenic effect. Cystic follicles which might have stimulated a permanent estrus had a vagina been present, were found in many adult guinea pigs which had received androgen prenatally (Tedford and Young, 1960). Without necessarily excluding the possi- bility that the heterotypical hormone is also jiroduced, many articles contain suggestions that interstitial tissue has specific estrogenic or androgenic activity. There is the report that an ovarian interstitial-cell tumor was producing estrogens (Plate, 1957). The ob- servation that estrogen continues to be se- creted by ovaries in which the follicles have been destroyed by x-rays was reported by Parkes (1926b, i927a,^b), Brambell and 470 PHYSIOLOGY OF GONADS V J ' * k •' 6^i;>ir/ ^ V ^, Fi(. 7 11 ()\,ii\ liMiii jir. ^uaiii i.ililni l.amc luu uiiz. d < . IK m liiu. ih.niiiii ( uipu- liiii iim. Hvi)f'itioi)lue(l intPi-titium. Pninoidial t'olhcles in cortex. (Couitc^y of Di. J. T. Bra(U)iuy.) Parkes (1927), Genther (1931), Schmidt (1936), Mandl and Zuckerman (1956a, b), and others. This conclusion w^ould seem to be .strengthened by the recent report that there is no intensification of the secretion of gon- adotrophins by x-rayed rats in which there was an apparent destruction of the ova and folhcles (AVestman, 1958). Evidence of an entirely different sort for the secretion of es- trogen by interstitial tissue has been i)re- sented by Ingram (1957). Autografts of medullary tissue containing interstitial tis- sue but no follicles were made in rabbits. Five animals from which this tissue was re- covered had uteri which were not as atroi)hic as the uteri of spayed animals. He noted, however, that in the absence of the follicu- lar apparatus the capacity to secrete estro- gen is soon lost. As we have seen,- Ingram is one of several investigators who have re- lated the functioning of interstitial tissue to granulosa elements. Histochemical staining procedures for cholesterol indicated to Dempsey (1948) that the theca interna is a possible source of estrogen. The results obtained during a more extensive utilization of histochemical reac- tions in studies of the ovaries of nonpreg- nant, psoudopregnant, and pregnant rabbits, and in the ovaries of rats and guinea pigs were consistent with the conclusion that a li])i(l i)recursor of estrogenic substances is ]:)resent in interstitial tissue (Claesson, 1954; Claesson and Hillarji, 1947a, b; Claesson, Diczfalusy, Hillarp and Hclgberg, 1948). Rennels (19511, on the basis of histo- chemical reactions in the oxaries of innna- ture rats, advanced the liypotliesis that in- terstitial tissue has a dual origin. There is a primary type present between 10 and 18 (lays aftei- bii'th wliicli is dosc^ly associated with gi'anulosa ()Ut and Antoniades, 1957; Daughaday, 1959). In the jiresent context, however, othei- considei'ations are more im- portant. Protein-liinding is not confined to the es- trogens and their metabolites, but other steroidal hormones, progesterone, testoster- one, and corticosteroids, are also present in the blood in a bound-state. In studies of the binding relationships of serum albumin, the link to the esti'ogens was found to be strongest, that to the corticosteroids rela- tively weak, and that to ])rogesterone and testosterone intermediate (Sandberg, Slaun- white and Antoniades, 1957; Slaunwhite and Sandberg, 1958; Daughaday, 1959). The i;- lationships in the case of other components of i^rotein mixtures have been sliown to be 47() PHYSIOLOGY OF GONADS different, but they are ai)parently eriually specific (Daughaclay, 1959; Slaunwhite and Sandberg, 1959). A considerable specificity of the binding sites may be involved ( Sand- berg, Slaunwhite and Antoniades, 19571. Daughaday (1959) states that separate binding sites may exist for each of the steroid hormones studied, and Szego (1957) suggested that a competition for these sites may be the basis for antagonisms which are known to exist in many steroid interactions (Courrier, 1950; Hisaw and Velardo, 1951; Roberts and Szego, 1953; Velardo, 1959; Velardo and Hisaw, 1951 ; Zarrow and Neher, 1953). The most important consideration has to do with the significance of protein bind- ing for the steroid hormones, and, in the present chapter, the significance for estro- gen and progesterone. Roberts and Szego (1946, 1947) and Szego (1957) proposed that formation of the estrogen-protein com- plex is necessary for the transport and activity of endogenous and exogenous estro- gens. Riegel and Mueller (1954), on the other hand, found that the protein-estrogen complex they used had only a slight, if any, estrogenic activity, and Daughaday (1958, 1959) expressed the opinion that the un- bound steroid hormones of the plasma are probably the biologically significant moie- ties. He suggested that the degree of pro- tein binding imposes a major restraint on the passage of hydrocortisone (and pre- sumably other steroids) through the cajiil- lary membranes, but pointed out that this view has not yet been established. He then asked, in the event that the steroid-protein complex does not function in the transi)ort of hormones from the vascular component to the cell, is it likely that the presence of a steroid-protein complex stabilizes the pliysiologically significant concentration of unbound steroid very much as buffer salts stabilize the small concentration of hydro- gen ion? In this way, he continued, the organism would l)c protected against the rapid changes in concentration which char- acterize an unbuffered system. At tiic ]ir(>sent stage in this controversial sul).iect, any hypothesis with res])ect to the significance of the protein binding of steroid hormones must be tentative. It would seem, however, that whatever emerges will have validity only if it is compatible with the cyclic waxing and waning of reproductive phenomena. If the unbound, rather than the bound fractions, are the active fractions, the functioning of the ovarian steroid hormones must dei)end on the presence of unbound fractions, in some way made available at cyclic intervals to the tissues on which these hormones act. It would seem, too, that the significance of the increased capacity for l)inding in i)rcgnancy (Rakoff, Paschkis and Cantarow, 1943; Baylis, Browne, Round and Steinbeck, 1955; Daughaday, 1959; Slaunwhite and Sandberg, 1959) should be a ])art of the picture. Tentatively, this greater binding capacity on the part of the ])regnant adult, coupled with an ina- bility of the developing fetuses to bind androgens, might account for the failure of the adult to be affected by the presence of androgen at a time when the genital tracts and neural tissues of the female fetuses she is carrying are undergoing profound modi- fications (Phoenix, Goy, Gerall and Young, 1959; Diamond, 1960). VI. Age of the Animal and Ovarian Functioninij The position of the ovary is such — at one and the same time being dependent on the pituitary, possessing its own varying ca- pacity to function, and having an effective- ness which is limited by the responsiveness of the tissues on which its hormones act — that no simple consideration of the relation- shii) between the age of the animal and ovarian functioning can be given. An in- vestigation, therefore, should be planned accordingly and we find experiments in which the amount of gonadotrophic stimu- lation was varied when age was constant, and experiments in which age was the varia- ble and the amount of gonadotrophin the constant. If hypo- or hyper-responsiveness of the tissues is suspected, the point can be checked by the use of spayed animals given variable amounts of ()\-arian hormones. When information of these sorts is brought together, a fairly accurate account of the relationship between age of the animal and ovarian activity can l)e ])repared. The results fi'oni many studies have re- vealed that the ovaries in both inunature and senescent females are potentially able MAMMALIAN OVARY 477 to secrete hormones, both estrogen and progesterone, in amounts which are in ex- cess of those secreted by untreated animals. The secretion of these hormones was ele- vated in rats, mice, and hamsters by the implantation of whole pituitaries (Smith and Engle, 1927 » or by the administration of chorionic gonadotrophin (Price and Ortiz, 1944; Ortiz, 1947; Green, 1955). The re- activation of senile ovaries was first demon- strated by Zondek and Aschheim (1927) following the insertion of hypophyseal im- jilants, and later by numerous other in- vestigators listed in the review of the subject l)y Tlmng, Boot and Miihlbock (1956). In more recent experiments an enhanced secre- tion of estrogen and progesterone followed the injection of old hamsters with chori- onic gonadotrojihin (Peczenik, 1942; Ortiz, 1955). In women fertility may be lost before the menopause (Engle, 1955). Studies in progress at Iowa (Bradbury, personal com- munication) show that urinary gonado- troi')hins may be elevated before the meno- pause and the last ovarian cycles are achieved in the presence of excessive amounts of pituitary gonadotrophin. As a rule the human ovary is devoid of oocytes and produces relatively little estrogen at the time of the menopause. Frequently, how- ever, there is enough residual ovarian ac- tivity (estrogen production) to maintain the vaginal epithelium for 10 to 15 years after tlie menopause. These observations on women suggest that as the supply of oocytes l)ecomes depleted, less estrogen is produced and more gonadotrophin is released to stimulate the aging ovary. This secjuence is in harmony with the concepts of Dubreuil (1942) and Hisaw (1947), because with fewer areas of granulosa there would be fewer centers of organizer to bring al)out the differentiation of thecal tissue competent to produce estrogen. Ovarian stromal hyperplasia has been found in association with endometrial hy- perplasia after the menopause (Morris and Scully, 1958). Sherman and Woolf (1959) suggested that the postmenopausal ovary may produce abnormal sexogens which bring about an endometrial proliferation and ultimately adenocarcinoma of the endo- mi'trium. Their urinarv l)ioassay studies indicate that the patients were excreting ICSH-type gonadotrophin. The observa- tion has been made at Iowa that a few postmenopausal women with endometrial carcinoma were maintaining an estrogenic vaginal epithelium when they were ovari- ectomized at ages varying from 65 to 70 years. Subsequently the gonadotrophin ex- cretion increased to the ciuantities usually seen after the menopause. In these unusual cases the aging ovaries produce estrogen, or possibly estrogen and androgen, in quanti- ties sufficient to suppress the usual excess production of gonadotrophins. The responsiveness or sensitivity of the ovary to gonadotrophic stimulation is not constant throughout the life of an indi- vidual. If we may judge from the studies of Corey (1928) and Selye, Collip and Thomson (1935) on newborn and 10- to 15-day-old rats, Moore and Morgan (19431 on young opossums, and Price and Ortiz (1944) and Ortiz (1947) on rats and ham- sters, the prepubertal period is characterized by very rapid and great increases in re- sponsiveness to gonadotrophic stimulation. Species differences are great. The opossum ovaries do not respond to gonadotrophic stimulation until about 100 days of age (Moore and Morgan), whereas responsive- ness was first detected in the rat ovary at 4 to 10 days (Price and Ortiz) and in the hamster ovary by the 10th day (Ortiz). Such data, coupled with the appearance of the ovaries at birth, would seem to exclude the possibility of gonadotrophic stimula- tion during the prenatal period, and per- haps the capacity for being stimulated as well. Certain other species are different and present problems. There is an extensive fol- licular development and luteinization in the fetal ovaries of the giraffe which is the basis for the suggestion that the ovaries of this species are responsive to gonadotrophin before birth (Amoroso, 1955). Such a con- clusion is predicated on the assumption ei- ther that serum gonadotrophin crosses the placental membrane or that the fetal pitui- tary secretes gonadotrophin. Neither hy- pothesis has been proved. Evidence exists that the ovaries of the horse and seal are strongly stimulated before birth (Cole, Hart, Lyons and Catchpole, 1933; Amoroso, 47! PHYSIOLOGY OF GONADS Harrison, Harrison-Matthews and Row- lands, 1951; Amoroso and Rowlands, 1951), but an unusual structural condition is found in these ovaries. No vesicular follicles are present and the ovaries, which are larger than those of the adult, are composed mostly of interstitial tissue which is enclosed by a thin cortex containing short chains of germ cells and a few oocytes surrounded by a sin- gle layer of epithelial cells. Comparable in- formation does not exist for the seal, but in the horse the development of this condition is reached during the estrogenic phase and after the gonad-stimulating hormone is no longer detectable in the blood of the preg- nant adult. As a result, and cjuite apart from the belief that serum gonadotrophin does not cross the placenta (Amoroso and Row- lands, 1955 j, the massive interstitial tissue liyperplasia is thought to have been stimu- kited by estrogenic rather than by gonado- trophic action. The immediate jiostpubertal jieriod and middle age are periods of relative stability. The period of old age has been too little studied and is in need of attention. In old mice the ovaries are reported to become unresponsive to exogenous gonadotrophin (Green, 1957). Ortiz (1955), on the other hand, stated that although a certain degree of ovarian sensitivity is lost in old ham- sters, there is a surprising degree of re- sponsiveness present until death, not only after the animal is no longer fertile, but even in animals with ovaries almost com- pletely atrophic. In young animals and in old animals there are irregularities of ovarian function and irregularities in the character of the cycles which probably can be related to imbalances in the pituitary-ovarian relationship. In polytocous sjK'cies, fewer follicles ovulate in young animals (Young, Dempsey, JMyei-s and Hagquist, 1938; Ford and Young, 1953, the guinea pig; Perry, 1954, the domestic pig; Ingram, Mandl and Zuckerman, 1958. the mouse and rat), and in old animals (Perry; Ingram, Mandl and Zuckerman). These statements of fact, iiowcx-cr. do not reveal what is presumed to be more im- portant. In young and old animals the na- ture of tlie irregularities, particularly those of ovarian function, seem to differ. Evidence collecte(l fi'om rhesus monkeys (Hartman, 1932) and chimpanzees (Young and Yerkes, 1943) suggests follicular growth without ovulation and luteinization, or in the guinea pig a sluggishness of follicular growth which is followed by ovulation and the forma- tion of functional corpora lutea (Ford and Young, 1953) . In old animals there may also be abnormalities of follicular growth, but as the numerous reports are read, the im- pression is given that abnormalities of luteinization are more prominent (Deanesly, 1938b; Wolfe, 1943; Wolfe and Wright, 1943; Loci), 1948; Thung, Boot and Miihl- bock, 1956; Dickie, Atkinson and Fekete, 1957; Green, 1957). Additional investigation will be necessary before we can be sure that the pituitary-gonadal imbalance in young animals differs from that in old ani- mals. On the whole, the possibility seems to have received little attention, but its im- portance justifies more careful study. VII. Other Endocrine Glands and the Ovaries A. THYROID The relationship of the thyroid to the functioning of the ovaries was one of the first subjects of modern endocrinologic in- vestigation. Notwithstanding, disappoint- ment must be expressed that after more than 50 years of effort, little more than cautious generalization is possible. This admission is not a confession of defeat; to be sure, there is an unfortunate number of uncertainties, but we have come to know what is necessary in the way of experi- mental design and techniques to enable us to proceed with the confidence that a grati- fying clarification can be achieved. The greatest obstacle could be, not the lack of means, but rather the failure to use the means which are alnmdantly at liand for more coordinated eft'orts than many which lia\e cliaracterized this field in the past. The general l)elief that the thyroid is in- voh-ed in reproductive function is grounded in two categories of observations. The first includes those demonstrating that ovarian lioi'inones exert an action on the thyroid. 'I'lieic lia\-e been many I't'ports that in the human female the thyi'oid enlarges at pu- berty, at menstruation, and during preg- nancy (Gamier, 1921; Marine, 1935; Neu- MAMMALIAN OVARY 479 mann, 1937; and others). Modern counterparts are the reports of the in- crease during pregnancy in the concen- tration of serum precipitable iodine (Heinemann, Johnson and Man, 1948; Dowling, Freinkel and Ingbar, 1956a; Tanaka and Starr, 1959), in serum thy- roxine (Danowski, Gow, Mateer, Everhart, Johnson and Greenman, 1950), and in the accumulation of radioiodine (Pochin, 1952). Some conflicting reports should be noted. There was said to be no consistent altera- tion in the concentration of serum pre- cipitable iodine in oophorectomized women (Stoddard, Engstrom, Hovis, Servis and Watts, 1957), and Pochin (1952) found no detectable variation in P^^ uptake during the menstrual cycle in 5 women he studied. Comparable observations have been made on laboratory mammals (Greer, 1952; Soli- man and Reineke, 1954; Soliman and Bada- wi, 1956; Feldman, 1956a) and the baboon, Papio ursinus (Van Zyl, 1957), except that Brown-Grant (1956) could not agree from his findings in the rat and rabbit that the level of gonadal function exerts any striking influence on thyroid activity in the normal experimental animal. In man (Engstrom, Markardt and Lieb- man, 1952; Engstrom and Alarkardt, 1954; Bowling, Freinkel and Ingbar, 1956b) and in laboratory mammals (chiefly the rat) (Money, Kraintz, Eager, Kirschner and Rawson, 1951; Feldman, 1956a; Feldman and Danowski, 1956) the enhancement of thyroid activity is attributed to the level of circulating estrogen, whether it be endog- enous or exogenous in origin. On the other hand, many who have worked with labo- ratory mammals have not found evidence of augmented thyroid activity, and not in- freciuently decreases were reported (see Paschkis, Cantarow and Peacock, 1948; and the numerous articles cited by Farb- man, 1944; and Feldman, 1956a). The con- flicting results may perhaps be accounted for by the circumstance that the response of the thyroid seems to be related to the dura- tion of the estrogen treatment and to the estrogen that was used. Decreases in thy- roid activity have been reported when the estrogen treatment was prolonged (Feld- man, 1956a), and Money and his associates showed clearly that estrone and some other components increased the collection of P-^^ by the thyroid of rats whereas estradiol, estriol, and diethylstilbestrol decreased the collection. ]\Iany attempts have been made to ascertain the nature of the mechanism whereby the effective estrogenic substances exert their action on the thyroid (Noach, 1955a, b; Feldman, 1956b; Dowling, Frein- kel and Ingbar, 1956a, b; Bogdanove and Horn, 1958). but they are so varied and speculative that they will not be reviewed here. The second category of observations re- lated to the thyroid and ovarian functioning includes those in which there is evidence of action of thyroid hormone on the ovary. Reviews of this work are contained in the articles by Peterson, Webster, Rayner and Young (1952), Hoar, Goy and Young (1957), and Parrott, Johnston and Durbin (1960) and most of their citations of work done on the relationship of the thyroid to the ovary will not be repeated here. As they point out, many investigators have reported that thyroidectomy is fol- lowed by ovarian degeneration, arrested folliculogenesis, and failure of ovulation. Irregularity of the reproductive cycles was common and much of this in the guinea pig could be attributed to retarded and sporadic follicular development (Hoar, Goy and Young, loc cit.) . The latter in- vestigators gave especial attention to the condition of the ovaries in their hypothyroid guinea pigs. In 10 pairs from thyroidec- tomized animals (oxygen consumption and heart rate were depressed) follicular de- velopment was good in the sense that the follicles appeared healthy, but a generation of corpora lutea was missing in four. This absence of corpora lutea, which is not seen in normal adult guinea pigs, was believed to be a consequence of the involution of the older generation during the longer than nor- mal interval between ovulations. It is con- sidered significant in terms of the functional capacity of such ovaries, that although the percentage of sterile matings was higher than in the controls, that, in the course of the two studies at Kansas, 29 of 38 matings were fertile. This experience may perhaps account for the many reports (cited in tlu papers from the Kansas laboratory) that thvroidectomv or treatment with antithv- 480 PHYSIOLOGY OF GONADS roid drugs have no, or at the most rehi- tively little, effect on the ovary. To these, several additional reports should be men- tioned. In thyroid-deficient female mice, fertility and litter frequency were affected only to the extent that the estrous cycles were prolonged (Bruce and Sloviter, 1957). In the rabbit, thyroidectomy did not inter- I'upt or alter the periodicity of follicular development, but it did eliminate the final stages (Desaive, 1948). Parrott, Johnston and Durl)in (1960) express the opinion that the long i)hysiologic life of thyroid hormone may account for many of the contradictions in the reports of the relationship between thyroid deficiency and reproduction. Except as it is speculative, an unex- plained action of thyroidectomy or the ad- ministration of goitrogenic drugs is the augmentation of the ovarian response to gonadotrophins and to anterior pituitary im])lants (citations in Peterson, Webster, Rayner and Young, 1952; and see in ad- dition Janes, 1954; Janes and Bradbury, 1952; Kar and Sur, 1953). Thyroid sub- stances, on the other hand, were inhibitory. Of alternative hyjiotheses, Janes favored the suggestion that during the period of propylthiouracil treatment, provided it was short rather than long, there was an ac- cumulation of gonadotrophin in the blood and the ovarian response varied for some unknown reason according to the concentra- tion of this latter substance in the body fluids. To Kar and Sur (1953) direct in- volvement of the hypophysis could be elimi- nated; instead a direct role of the thyroid seemed more plausible. They postulated that the absence of thyroid hormone re- duced the utilization of gonadotrophic hor- mones by the ovary. The reported effects of the hyi)erthyroid state or of administered thyroid hormone on the ovary are equally conflicting. The ovaries are described as being atrophic or exhibiting incomplete folliculogenesis, or as being essentially normal or even hyper- trophied (citations in Peterson, Webster, Rayner and Young, loc. cit., Hoar, Gov and Young, loc. n't.). Irregular cycles are said to have occuitcmI in the rat and mouse. but no irregularity was detected in guinea pigs given thyroxine. A tentative explanation can be given for the many divergent reports of the relation- ship between the thyroid and the ovary, divergencies which are found in the clinical literature as well as in laboratory studies. In doing so, we will recall that there is abundant evidence that the ovary is a locus of action of thyroid hormone. The action may not be directly trophic, as is that of the pituitary, but it is assumed to be su])- l)ortive, jiossil)ly directly so. or jiossiljly indirectly through regulation of the general metabolic level. Whatever its nature, there must be great interspecies and even intra- species variation in the need of the ovary for such action. In addition, within a species there appears to be a wide range of toler- ance, for Peterson, Webster, Rayner and Young (1952) found in their study, in which the thyroid state was estimated from measurements of oxygen consumption and heart rate, that reproduction occurred in fe- males in which oxygen consumption ranged from an average of 50.0 to 93.5 cc. per 100 gr. i)er hr. (52.9 in the controls), and heait rate from 238 to 316 beats per minute (272 in the controls). In females that failed to rejiroduce, the lowest values were lower than in the animals which did reproduce; nevertheless, there was much overlai)i)ing, for in this group oxygen consumption ranged from an average of 46.7 to 94.1 cc. per 100 gr. l)er hr., and heart rate from 202 to 330 beats j)er minute. Within sucli a framework, there are bound to be more divergent results than when normal functioning depends on nioi'e narrowly circumscribed conditions, and the failure to replicate a result does not have the same significance. As a part of the investigation of sucli a problem, more and better correlated infoi'niation is re- (|uiicd, and this could be the most pressing ne('(l in the field of oxarian (and icproduc- ti\-cl functioning and the thAM'oid. B. ADRENAL CORTEX Tlu^ adrenal cortex elaborates its steroid )nn()iu's in a biosyiU lictic scciuencc \H'ry siiiiilai' to that in the o\aiy. In fact, i)ro- gcstci'onc is an intermediate substance in the synthesis of glucocorticoids. Estrogen has been found in extracts of adrenal corti- cal tissue, but whether it represents a deg- radation pi'oduct within the adrenal or an artifact resulting from the chemical i)i'o- MAMMALIAN OVARY 481 cedurcs is not clear. Occasionally adrenal tumors produce physiologically significant amounts of estrogen, but normally the ad- renal production of estrogen, if any, is not of physiologic significance. The atrophy of the female genital tract after bilateral ovar- iectomy suggests strongly that this is so. The major hormone of the adrenal cortex, hydrocortisone or corticosterone, depending on the species, has a profound effect on pro- tein and carbohydrate metabolism. An over- production, as manifested by Cushing's dis- ease, results in a wasting of body protein and other metabolic disturbances which by nonspecific influences tend to reduce gonadal function. Similarly a loss of adrenal func- tion (Addison's disease) leads to anemia, electrolyte imbalance, and hypoglycemia. There is usually a decrease in ovarian func- tion but some Addisonian patients have conceived and carried their pregnancies with only sodium and fluid supplements. There is a hereditarv metabolic defect of the human adrenal which renders it de- fective in i)roducing hydrocortisone. This is the adrenogenital syndrome. In these jiatients the adrenals produce excessive amounts of intermediate products which are excreted in the urine. Some of these com- pounds are androgenic 17-ketosteroids and may cause virilization (Bradbury, 1958). These androgens tend to inhibit the gonado- trophic activity of the pituitary and leave the ovaries unstimulated and infantile (Fig. 7.13). Replacement therapy with corticoids reduces the adrenocorticotrophic hormone ( ACTH) activity of the pituitary and then the adrenal production of androgen ceases. This then permits the pituitary to stimulate normal cyclic activity in the ovaries (Fig. 7.14). The adrenogenital syndrome thus has a profound effect on ovarian function which is specific through its production of androgen. More complete descriptions of the condition and of the rationale of treat- ment have been prepared by Wilkins ( 1949) , CHOLESTEROL PREGNENOLONE PROGESTERONE HYDROXY- PROGESTERONE Fig. 7.13. Scliematic representation of tlie interaction of tlie adrenal and tlie ovary in the adrenogenital syndrome. The process of hormone biosynthesis is defective in the adrenal (BLOCK) and the degraded by-products (17-ketosteroids) being androgenic suppress the formation of gonadotrophins (GTH). (Courtesy of Dr. J. T. Bradbury.) 482 PHYSIOLOGY OF GONADS CHOLESTEROL CORTISOL CHOLESTEROL GONAD \ ANDROGEN ESTROGEN PREGNENOLONE ; PREGNENOLONE ADHeNAL ^ \ 17-HYDROXY- PROGESTERONE PROGESTERONE PROGESTERONE 17-HYDROXY- PR06ESTER0NE Fit. 7 14. Tieatnieiit witli (oiti>one (or other glucocorticoid) reduces ACTH production and adrenal hormone .synthesis subside-^. This permits the normal pituitaiy-gonadal inter- actions to be established. (Courtesy of Dr. J. T. Bradbury.) A\'ilkins, Crigler, Silverman, Gardner and Migeon (1952), and Bradbury (1958). Milder categories of what is believed to be adrenal cortical hyperplasia have also been described and are responsive to treatment with cortisone. They are characterized by amenorrhea or oligomenorrhea, hirsutism, slightly elevated 17-ketosteroid excretion values, and difficulty in becoming pregnant (Jones, Howard and Langford, 1953; Jef- feries, AYeir, Weir and Prouty, 1958; Jef- feries, I960). Indications arc that these abnormalities, like those typical of the adrenogenital syndrome, affect the ovary, not directly, but rather by the creation of a pituitary gonadotrophic-ovarian imbal- ance. Some evidence exists for more direct re- lationships between the adrenal cortex and the ovary. These may involve actions of ovai-iaii liormones on the adrenal, and ac- tions of adrenal cortical hormones on the ovary. In general, however, the relation- ships are tenuous or at least not sharply defined. It is evident from the review by Parkes (1945) that sexual dimorphism in adrenal cortical structure has been demon- strated in a number of species, notably the mouse and rat and possibly the guinea pig, but it has not been detected in a number of other species. The effects of gonadcctomy and the injection of hormones, particularly estrogens, into gonadectomized animals are less clear, but they are suggestive of an ac- tion on the adrenal, however ill defined and variable it seems to be. A seasonal hyper- trophy of the adrenal has been reported as occurring in the mole, Talpa europaea, (Kolmer, 1918) and the ground squirrel, Citellus tridecemlineatus (Mitchill), (Fos- ter, 1934), as has enlargement at the time of estrus in the rat (Andersen and Kennedy, MAMMALIAN OVARY 483 1932; Bourne and Ziickennan, 1941bj. More recently, a significantly higher excretion of 17-hydroxy corticosteroids has been found during the second and third weeks, and therefore during the luteal phase, of the menstrual cycle (Maengwyn-Davies and Weiner, 1955). Whether a causal relationship exists be- tween these indications of a fluctuating ac- tivity within the adrenal cortex, and sea- sonal and cyclic changes within the ovaries remains to be determined. Little informa- tion exists. Bourne and Zuckerman {loc. cit.) described the changes in the adrenals of ovariectomized rats injected with estrone and concluded that the changes are inde- |)endent of the gonads. Foster's observation that the active appearance of the adrenal can be seen during pregnancy as well as during estrus suggests, but does not prove, that there is a hormonal regulation in the ground squirrel which is dependent on re- productive processes. Data with respect to possible direct effects of adrenal cortical secretions on the ovary ai'e ambiguous. Cortisone acetate adminis- tered to rabbits 5 to 33 days in daily doses of 5 to 20 mg. did not inhibit the ovulation which occurs after mating or after the in- jection of copper acetate (De Costa and Abelman, 1953). The ability of the ovary of the rat to respond after adrenalectomy was tested by the administration of gonado- trophic extracts (Brolin and Lindl)ack, 1951). They found that the ovaries could be stimulated to increase the weight of the uterus without the cooperation of the adre- nals and considered that this result does not support the view that there is a direct re- lationship between adrenal corticoids and the biosynthesis of ovarian (also testicular) hormones. In other experiments (Payne, 1951; Smith. 1955), adrenalectomy abol- ished (Payne) or interfered significantly (Smith) with the ovarian hyperemia re- sponse to injections of HCG and pituitary extract (Antuitrin T) , the response utilized by Farris (1946) as a test for early preg- nancy. Cortisone and hydrocortisone were partially effective in restoring the response in adrenalectomized animals. According to Payne, isocortisone acetate and compound A acetate were also effective, but in larger doses. No report of the use of corticosterone (comi)ound B) is gi\'en; replacement ther- apy with this hormone would have been more physiologic because it is the natural corticoid of rats. It was concluded that the hyperemia response is more nearly normal in animals with normal adrenal function; Payne believes that the response is mediated through this gland. Despite what seem to be clear-cut results which have been con- firmed, it is felt that additional closely con- trolled exi^eriments must be done in order to show whether these adrenal hormones affect the ovary directly or whether most of the effects are nonspecific metabolic al- terations. VIII. Concluding Remarks The avenue followed by investigators in- terested in the functioning of the mam- malian ovary has long carried a two-way traffic. In addition, there has been move- ment into the out of many side streets. No understanding of the pattern of the traffic in such a situation is possible and no satis- factory regulation can be achieved unless something is known about the nature, origin, and destination of the vehicles composing the traffic. Equally important, this informa- tion cannot l)e obtained by standing on one spot. This analogy contains much that is relevant for what has been attempted in this book. The problem of the ovary has been approached from the vantage point of forces and substances originating in the pituitary and the environment which act centripetally on it (Creep, Everett), and from the vantage j^oint of many of the tissues and organs on which the hormones we associate with it exert their action (the Hisaws, Cowie and Folley, Zarrow, Young in his chapter on mating behavior). In this chapter and that prepared l)y Dr. Villce positions have been taken near the ovary and attemj^ts made to bring together much of the information gathered by investigators who were in a sense looking right at it. Whether our perspective is developed from a familiarity with all the material which has been brought together or whether it is restricted by the narrower treatment given here, it is obvious that the unsolved problems outnumber by far any that have been solved, if indeed there are such. Wc have learned much about the functioning 484 PHYSIOLOGY OF GONADS of the o\'ary, but there is little we can ex- plain. As we indicated earlier, a part of this failure can be ascribed to the lack of gonadotrophic preparations which either singly or in combination will evoke changes identical with those in untreated normal animals, but this chapter alone contains an enumeration of many other problems solu- tion of which does not depend on this par- ticular advance. The disappointment we ex- press may be a reflection of what seems to be the modus operandi in science. The ex- tent of our application to unsolved prob- lems is very unequal, but more often than not it can be traced to an investigator's suc- cess in achieving a "breakthrough"*' as Edgar Allen, Doisy, Smith and Englc, Wil- lard Allen and Corner, Hisaw and other colleagues did in the twenties. At such a time, enthusiasm is intense and there follows a period of gratifying accomplishment, but obstacles are encountered and often interest lags, until another breakthrougli occurs. In the meantime, effort may have been diverted by discoveries elsewhere and the area of investigation which attracted so many is neglected and suffers. Ovarian physiology should not remain in this state for long. There is no tissue of the body in which the changes are as conspicuous and as dra- matic as those in the ovary and there is no tissue which presents more variable aspects. Many of the stages in the cycle of ovarian structure and functioning are related to changes elsewhere in the body — changes in growth, in motility, in secretion, and in be- liavior. All these changes, including those within the ovaries, offer excellent end points for continued quantitative and qualitative studies. IX. References Aldman, B., Claesson, L., Hillarp, N.-A., and Ode- BLAD, E. 1949. 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On the mechanism of action of gonadotrophin from pregnancy urine. J. Endo- crinol., 2, 12-20. Zondek, B. 1954. Some problems related to ovar- ian function and to pregnancy. Recent Progr. Hormone Res., 10, 395-423. Zondek, B., and Ascheim, S. 1927. Hypophysen- ^•OI•del•lappen und Ovarium. Beziehungen der cndokrinen Driisen zur Ovarialfunktion. Arch. Gynak., 130, 1-45. Zondek, B., and Sklow, J. 1942. Further investi- gations on the mechanisms of oestrone pro- duction in the ovary. J. Endocrinol., 3, 1-4. Zuckerman, S. 1940-41. Periodic uterine bleed- ing in spayed rhesus monkeys injected thiily with a constant threshold dose of oestron(>. J. Endocrinol., 2, 263-267. Zuckerman, S. 1951. The number of oocytes in the mature ovary. Recent Progr. Hormone Res.. 6, 63-109. 8 THE MAMMALIAN FEMALE REPRODUCTIVE CYCLE AND ITS CONTROLLING MECHANISMS John W. Everett, Ph.D. PROFESSOR OF ANATOMY, DUKE UNIVERSITY DURHAM, NORTH CAROLINA I. Introduction 497 II. Cycles Spontaneously Interrcpted. 498 III. PiTUITARY-OVARIAN DORMANCY 499 A. The Ovary in Anestrum 500 B. The Hypophysis 500 C. Relationship of the Anestrum to the Seasons 501 IV. Attainment of Maturity. Emer- gence OF Full Ovarian Function. . 502 V. Follicular Cycles. Growth and Atresia 504 A. Correlation of Ovarian Secretion with the Follicular Cycle 507 B. Cyclic Manifestations after Ovari- ectomy or Hypophysectomy 509 C. Cyclic Manifestations in the Ab- sence of Ovarian Follicles 509 D. Hypothalamus and Gonadotrophin Secretion. General Considera- tions 510 VI. Follicle Maturation and Ovula- tion 513 A. Time of Ovulation 513 B. Ovarian Steroids and Ovulation. . . 514 1. Estrogens 514 2. Gestagens 517 C. Role of the Nervous System in Ovu- lation " 520 1. The hypophyseal portal veins and the chemotransmitter hypoth- esis ' 523 2. Central depressants and ovula- tion 526 3. The central nervous system as a timing mechanism for ovula- tion 520 D. Persistent Follicle 529 VII. The Luteal Phase 530 1. Luteotrophic substances 530 2. "Nonfunctional" corpora lutea. . 531 A. Pseudopregnancy 532 1. Duration of pseudopregnancy. .. . 533 2. Neural factors in pseudopreg- nancy 534 B. Luteolytic Mechanisms 537 C. Effect of the Uterus on Luteal Func- tion 538 VIII. Concluding Comments 540 IX. References 541 I. Introduction The chain of events that constitutes the female reproductive process is character- istically repeated from time to time with considerable regularity during the adult life of an individual, and is therefore a cycle. In the broad sense, this sequence begins with ovogenesis and terminates when the progeny require no further shelter and nurture. In mammals this has become a highly complex process, involving profound maternal adjustments synchronized with successive stages in development of the ovum, fetus, and offspring. The complete mammalian cycle comprises a sequence of stages which may be identified as follows: (II follicle growth, including growth of the ovocyte; (2) ovulation, a progressive proc- ess including preovulatory maturation of follicles and ova, and the structural change of ruptured follicles to corpora lutea; (3) progravidity; (4) gravidity; (5) parturi- tion; and (6) postpartum nurture, including lactation, protection, and training. Although it is obvious that this full sequence is often realized, it may nevertheless be retarded or frankly interrupted at almost any point. In advanced human societies economic and social factors have diminished the num- ber of complete cycles to such degree that they are rarities in the lifetime of an in- 497 498 PHYSIOLOGY OF GONADS dividual and infertile ("menstmal") cycles are the rule. Inasmuch as corresponding factors operate among domesticated ani- mals, the expression "female reproductive cycle" commonly refers to those truly abortive cycles that succeed one another in the absence of insemination. The term is used in that restricted sense in this chapter. With even that restriction, the female cy- cle is actually a multiplicity of interlocking cycles, in which the rhythmic interplay be- tween hypophysis and ovary is fundamen- tal. Attention must therefore be focused on the physiology of the ovary and on the hormonal and neural mechanisms that in- tegrate hypophysis and ovary as a func- tional system. Cyclic alterations in sex ac- cessories and other nongonadal tissues are considered mainly as indicators. The "menstrual cycle," being strictly a uterine cycle, comes in this category, together with changes in behavior. No attempt is made to present an ex- haustive description of the varied adaptive modifications of the ovarian cycle among the several mammalian orders. The reader may consult works of the late F. H. A. Marshall whose full bibliography is given by Parkes (1949 1. Asdell's Patterns of Mammalian Reproduction (1946» is an- otlu'i' A-alual)lo source. II. Cycles Spontaneously Interrupted Cycles in the natural state are only im- perfectly known, from random and often er- ratic sampling. One may safely assume that, as a rule, under optimal conditions they are complete, fertile cycles. There are, then, relatively few subhuman species in wdiich the characteristics of incomplete cycles have been studied. These species are necessarily the very ones that have been amenable to some form of human restraint. Segregation of the sexes or any other interference with insemination should be re- garded as a first experimental approach to understanding the complete cycle. Such factors unriuestionably operate in nature on occasion. Controlled changes of environ- mental conditions afford another approach in which natural factors are simulated. The statement was made earlier that the complete cycle may conceivably be inter- rupted at almost any point. It has been learned that in different species segregated females interrupt their cycles at different stages and that usually the point of inter- ruption is species-characteristic. These facts have been of great service to the study of reproduction, first, by arousing the curi- osity of the investigator and, second, by supplying a variety of ready made condi- tions individually appropriate for particular experimental studies. Examples of mammalian cycles are schematically diagrammed in Figure 8.1. It is customary to state that the usual, or standard, infertile cycle is like that in pri- mates or the guinea pig. The follicular phase culminates in spontaneous ovulation, after which corpora lutea are organized and become spontaneously functional for a period of time that is usually considerably shorter than in pregnancy. In a few animals (rat, mouse, hamster) the cycle terminates shortly after ovulation before the corpora lutea become fully func- tional. Such corpora lutea are said to be inactive, in the sense that they cannot pro- duce a decidual response to uterine trauma (Long and Evans, 1922). Sterile mating or analogous stimulation induces a luteal phase which corresponds to that of the "standard" mammal. This phenomenon is not entirely limited to the small rodents, having been described in the European hedgehog (Deanesly, 1934). To this writer's knowledge there have not been described any mammalian species in which it is the rule that in isolated females the process of ovulation begins (follicle ma- turation, prelutein changes in granulosa, secretion of secondary liquor folliculi, and so on) without proceeding to eventual rup- ture of the follicles. Many cases could l3e cited, however, in which this has occurred "abnormally." Characteristically, some de- gree of luteinization occurs in the wall of such a follicle and a lutein cyst is formed. On the other hand, there are numerous species (reflex ovulators) in which the pre- ovulatory maturation of follicles and ovu- lation nearly always fail in the absence of tlie male. The known species in which this is true are widely distributed among the mammalian orders and are often closely iclated to other species in which spontane- ous ovulation is usual. The domestic rabbit MAMIMALIAN REPRODUCTIVE CYCLE 499 GUINEA PIG PRIMATE Fig. 8.1. Diagrams of cycles of representative, familiar mammals. , the follicular phase, highly schematized and inaccurate in detail ; , atresia ; i , ovulation ; • , fully active corpora lutea; O, corpora lutea regressing or otherwise not fully active. When sterile mating or equivalent stimulation (SM) is introduced, the cycles of the rat, rabbit and cat become directly comparable with those of the other species. furnishes the classic exaini)le of reflex ovu- lation. Other reflex ovulators are the domes- tic cat (Greulich, 1934), the ferret (Ham- mond and Walton, 1934), mink (Hansson, 1947), marten (Pearson and Enders, 1944), the 13-lined ground squirrel (Foster, 1934), and the mole shrew (Pearson, 1944). To this list have been added the muskrat (Miegel, 1952) and a field mouse, Microtus cali- fornicus (Greenwald, 1956). Even among the marsupials, the female Didelphijs azarae is said not to form corpora lutea in the ab- sence of the male (Martinez-Esteve, 1937). A few of these species display nearly con- stant estrus (rabbit, ferret), competent follicles being present most of the time in the isolated female during the breeding sea- son. Among even the spontaneous ovulators the cycle may sometimes not progress be- yond the follicular phase. Thus, at the approach of puberty, waves of advanced follicle development and secretion of estro- gen may take place without, however, lead- ing to ovulation or corpus luteum forma- tion. The first cycles of primates are often anovulatory ones. In the adult macacjue, at least in some colonies, such cycles are char- acteristic during the summer months (Hart- man, 1932) . A somewhat comparable sea- sonal effect has been reported in girls soon after the menarche (Engle and Shelesnyak, 1934). Menstrual cycles without ovulation have frecjuently been recognized in adult women in recent years, bearing no evident relationship to seasonal factors (Lopez Co- lombo de Allende, 1956). Anovulatory cycles were described in the mouse by Allen (1923) and have been noted occasionally in other species, but without clear measure of their incidence. III. Pituitary-Ovarian Doriiianey Varying levels of pituitary-ovarian dor- mancy are expressed in different ways from species to species or even from habitat to habitat within a given species. A general similarity exists between the anestrum of seasonal breeders and the prepubertal state. In fact, in animals that have a distinct sea- 500 PHYSIOLOGY OF GONADS son, puberty occurs at the very time when older females are emerging from anestrum. Whereas anestrum is often correlated with season of the year, there are exceptions, notably among dogs, in which the correla- tion is ill defined (Engle, 1946). In its shortest form ovarian quiescence lasts for only a few days, probably often without being recognized, between the end of one cycle and the active follicular phase of the next. In the chimpanzee it is thought to be the chief factor in the irregularity of length of the cycle (Young and Yerkes, 1943). Rossman and Bartelmez (1946) de- scribed a comparable occurrence in mon- keys. At the other extreme, anestrum may occupy the major part of the year in mones- trous animals that have a very limited breeding season. A. THE OVARY IN ANESTRUM Generally speaking, depression of ovarian function is most extreme in greatly pro- longed periods of quiescence. In the ferret, Hammond and Marshall (1930) reported that in the anestrous ovary follicles can hardly be recognized with the naked eye, because they remain small and deeply placed. The largest follicles at the "end of the season" averaged 460 /x in diameter whereas a "long time after" the average was only 240 jx, increasing again to 720 yu, at the api^roach of a new season. By con- trast, the largest follicles of animals in full heat ranged between 1220 and 1440 /x. Fol- licle atresia abounds in the anestrous ovary of the 13-lined ground squirrel (Johnson, Foster and Coco, 1933). In sheep, however, follicles of large size may be present at any time during anestrum (Kammlade, Welch, Nalbandov and Norton, 1952). Some moderate degree of secretory activ- ity of the ovary is indicated even at the depth of i^rolonged seasonal anestrum (13- lincd ground squirrel, Moore, Simmons, Wells, Zalesky and Nelson, 1934; ferret, Hill and Parkes, 1933; opossum, Risman, 1946). Although at this time uterus, vagina, and vulva are small, ovariectomy or hypo- l^hysectomy causes a further reduction. On the other hand, these structures are readily stimulated by injection of estrogens. It may be said that low-grade follicular cycles proceed throughout the anestrous interval, but whether tliere is any syn- chronization of one follicle with another is unknown. Some insight into this problem is furnished by study of (1) the transition from anestrum to the breeding season, and (2) the closely analogous phenomena of adolescence. In the report by Hammond and Marshall, it was shown that in ferrets dur- ing anestrum and proestrum there is a pro- gressive increase in size of the vulva which directly parallels the diameter of the largest follicles. The absence of overt cyclic change is not surprising in view of the fact that estrus is continuous in this species. In polyestrous animals, on the other hand, it might be expected that during anestrum fol- licle growth and accompanying estrogen se- cretion are cyclic, at least at the approach of puberty or of "the season." Important in- formation on this question has been ob- tained from some of the primates, notably the macaque (Allen, 1927; Hartman, 1932) and the chimpanzee (Zuckerman and Ful- ton, 1934; Schultz and Snyder, 1935). Slight transitory reddening of the skin of the perineum ("sex skin") of the monkey may occur at intervals for several months preceding the onset of menses, accompanied by moderate desquamation of vaginal epi- thelium. During the long intervals of amen- orrhea that some individuals exhibit during the summer, there is a tendency toward cyclic vaginal desciuamation (Fig. 8.2). The sex skin of the chimpanzee may begin to swell more than a year before the first menstruation. During the ensuing months the swelling may be irregularly cyclic or continuous. Thus, one may judge that low- grade follicular cycles, accompanied by pe- riodic increases in estrogen secretion, may succeed one another during seasonal or ])repubertal anestrum, but that in certain cases these cycles may overlap to such de- gree that rather continuous estrogen secre- tion takes place. B. THE HYPOPHYSIS The secretory activity of the anestrous ovary is apparently adequate to prevent "castration" changes in the adenohypoph- ysis, for as shown by Moore, Simmons, \^'ells, Zalesky and Nelson (1934) removal of the ovary of anestrous ground squirrels I'esults in hypertrophy of the hypophysis, MAMMALIAN REPRODUCTIVE CYCLE 501 z - 28 days 1 28 days A 28 days < " 3 - s - UJ . Q III Menses "^ v-^^/^ A> ^ Mensesllll 21 ' 15 JUNE JULY ' 15 AUGUST 15 SEPTEMBER 15 OCTOBER Fig. 8.2. Vaginal cycles during seasonal amenorrhea in a monkey. (A portion of the record of monkey ^38 from C. G. Hartman, Contr. EmbryoL, Carnegie Inst. Washington, 13, Fig. 26, p. 121, 1932.) increased gonadotrophin content thereof, and increased numbers of basophile cells. Warwick (1946) reported a highly signifi- cant increase of pituitary potency in spayed anestrous ewes. This is closely anal- ogous to the results of ovariectomy in im- mature animals (Hohlweg, 1934). As meas- ured by ovarian activity, gonadotrophin secretion (release) may be greatly dimin- ished during profound anestrum. The actual hypophyseal content of gonadotrophin seems to be markedly reduced during anes- trum in some species (Moore, Simmons, Wells, Zalesky and Nelson, 1934), but possibly not in others. Cole and Miller (1935) and Warwick (1946) reported that there is no seasonal variation in sheep. A study by Kammlade, Welch, Nalbandov and Norton (1952) indicates that the aver- age content is somewhat higher during anes- trum than it is in cycling ewes. The major factor in this difference, however, seems to be that during the cycle the potency of the pituitary drops during estrus and the early luteal phase. Somewhat similarly the potency of the immature rat hypophysis has been stated to be as high as that of the sexually active adult (Clark, 1935). The fact that the ovaries of the immature female or of the anestrous adult can be stimulated by in- jection of gonadotrophin indicates that gon- adotrophin content of, the hypophysis in these cases is not a fair measure of libera- tion of the hormone into the blood stream. Therefore, it seems justifiable to assume, as Robinson (1951) did in the interpretation of anestrum in the ewe, that, in spite of the possible absence of seasonal assay variation. there is, nevertheless, a depression of hypo- physeal gonadotrophin release during anes- trum. We may further assume that it is not completely depressed, for the ovary remains slightly active. Ovary and hypophysis are evidently in a state of equilibrium at a relatively low level of function. It seems likely that this state of affairs is brought about by the central nervous system, inas- much as the seasonal depression in some species is closely dependent on the daily ratio of light to darkness. C. RELATIONSHIP OF THE ANESTRUM TO THE SEASONS This relationship is so varied among dif- ferent species that many interesting ques- tions are raised. In many cases the midpoint of anestrum coincides approximately with the shortest days of the year (Fig. 8.3). There are other examples, however, largely among the Artiodactyla, in which it coin- cides with the longest days. Sheep are not- able examples (Robinson, 1951). Others, like the European common hare, experience a short anestrum during the time of rapidly decreasing daylight (Asdell, 1946). The Russian yak, on the other hand, is said to experience anestrum from December to May (i.e., while day length is increasing). A general explanation of these varied adap- tive manifestations is elusive. There is rea- son to believe that although illumination, or the light/darkness ratio, (Kirkpatrick and Leopold, 1952; Hammond, Jr., 1953) has a rather direct and primary effect in some cases, its role is more or less indirect in others where such things as temperature, humidity, availability of food and water assume major importance (Marshall, 1942). 502 PHYSIOLOriY OF GONADS NDJFMAMJJASON INSECTIVORA MOLE SHREW COMMON SHREW CARNIVORA BROWN BEAR (EUR.) FERRET COYOTE WILD CAT (EUR.) BAD3ER (AMER.) LAGOMORPHA HARE (ENG.) COTTONTAIL (N . ENG.) RODENTIA 13-L. GROUND SQUIRREL WOODCHUCK GRAY SQUIRREL DORMOUSE FIELD MOUSE (EUR. ) MUSKRAT (MARYLAND) (IOWA) PORCUPINE ARTIODACTYLA LLAMA ROE DEER MULE DEER GIRAFFE SHEEP (HAMP ) BIGHORN GOAT YAK (RUSS. ) INDIAN ANTELOPE PERISSODACT YLA HORSE Fig. 8.3. Some representative seasonal breeders. Solid bars indicate breeding seasons (according to Asdell, 1946); blank intervals, periods of anestrum. Months of the year repre- sented by letters at top of chart ; winter and summer solstaces marked by wavy lines. South- ern hemisphere seasons converted to corresponding ones of the northern hemisphere. End of season for the Bighorn is vmcertain. The complexity of the i)rol)lem is well illustrated by the 13-lined ground squirrel whose breeding season, like that of a multi- tude of small rodents, comes in the spring. Moore, Simmons, Wells, Zalesky and Nel- son (1934) reported that increasing illumi- nation, elevated temperature, and feeding all failed to bring the females into estrus out of season. If, however, hibernation was first induced by low temperature and dark- ness, premature estrus would follow. The conclusion was reached that hibernation it- self is a necessary prerequisite. Ovarian de- velopment actually begins, under natural conditions, in early January in the midst of hibernation. Females exix'i-imentally maintained "continually for several months in cold and darkness, with more or less normal hibernation, [exhibit] sexual de- velopment at any time of the year, and periods of estrum have thus been . . . main- tained for many months. ..." The impres- sion is given tliat tlie conditions favoring hibernation also favor sexual development to such extent that breeding potentiality continues for a few months after emergence, in spite of elevated temperatures and long periods of illumination. In another rodent, Peromyscus leucopus, however, the length of daily illumination is of paramount im- portance. Temperature changes (4 to 25°C. ) have no effect on rejiroduction when lighting is adequate (Whitaker, 1940). Whereas a similar primaiy dependence on lighting can be shown in a number of other species from several orders, it is unwise to general- ize that this is usually true. IV. Attainment of Maturity. Emergence of Full Ovarian Function Ahhough ('merg(>nce of the ovary from the state of quiescence is gradual, there is usually some outward sign that allows the observer to say that puberty has ari'ived or the breeding season has begun. In ])ri- MAMMALIAN REPRODUCTIVE CYCLE 503 mates the accepted sign is the first menstru- ation ; in rats it is the opening of the vagina ; in many animals it is the swelling and red- dening of the genitalia heralding the initial l^roestrum. In other eases, e.g., sheep, the only clear indication may be the behavior of the female toward the male. From these facts it is readily apparent that any one sign is employed simply because it happens to be accessible to easy observation. Yet the increasing output of estrogen, whether steady or cyclic, affects many parts of the organism at the same time. Furthermore, in any one individual the threshold for ex- l)ression of a given sign may be relatively liigh with respect to that of some other manifestation. Thus, in Hartman's mon- keys (1932), some were noted in which des- quamation of vaginal epithelium occurred in wave-like manner for a long time before menstruation. In others "menstrual" bleed- ing occurred with regularity while the uterus remained very small and A'aginal desqua- mation was negligible. Hartman summarized the step-wise man- ner of maturation of ovary and accessory organs of the monkey during adolescence or following amenorrheic episodes some- what as follows. The color of the sex skin may be the first to appear. A slight men- strual flow usually takes place before des- quamation of vaginal epithelium becomes measurable. "More rarely there may be one or more low desquamation cycles before a bleeding is recorded. Whole cycles marked liy jieriodic bleeding and some vaginal des- quamation may occur before there is any noticeable increase in size of the ovaries and uterus. These organs increase also in a saltatory manner, hence the term 'staircase' phenomenon for the process. Finally, the endocrines effect the acme of the reproduc- tive process — ovulation." Individual variation in the degree of ab- ruptness with which the first ovulation is achieved is well illustrated in a study of puljertal guinea pigs by Ford and Young (1953). In most cases the first period of vaginal opening was much longer than in subsequent cycles. Whatever the duration, ovulation was more closely related to the end than to the beginning of the period, as indicated by histologic study of ovaries. Even ovulation and corpus luteum for- mation do not signify that full power of reproduction has arrived. For example, the first cycle of the adolescent rat may culmi- nate in ovulation without sexual receptivity (Blandau and Money, 1943). In the ewe, an ovulation without overt signs of heat may at times take place during the anes- trum, especially just before and just after the breeding season (McKenzie and Terrill, 1936). The phenomenon is occasional in ewes during the season and has also been described in cattle (Hammond, 1946). In fact, the full manifestation of estrus in sheep seems to require the presence of a "waning" corpus luteum (Robinson, 1951). In sheep the transition from seasonal or prepubertal anestrum to the breeding sea- son may involve relatively minor changes in hypophyseal activity. Even in the im- mature rat both the hypophysis and the ovary are capable of far greater secretory function than they normally display. In the equilibrium that prevails, the ovary ap- pears to hold the upper hand by reason of a low hypophyseal threshold at which estro- gen suppresses gonadotrophin secretion in the immature individual (Hohlweg and Dohrn, 1932; Byrnes and Meyer, 1951b) and a low ovarian threshold at which gona- dotrophin stimulates estrogen secretion. Byrnes and Meyer (1951a) reported that suppression of hypophyseal gonadotrophin content in immature rats can be accom- plished with doses of estrogen much smaller than those that affect uterine growth. It is also known that the immature ovary can be induced experimentally to secrete estrogen by injection of amounts of gonadotrophin that are too small to produce significant increase of ovarian weight or follicle de- velopment (Levin and Tyndale, 1937; Moon and Li, 1952). When a gonadectomized im- mature rat is united in parabiosis (Kallas, 1929, 1930 » with a normal or hypophysec- tomized female littermate, precocious pu- berty is induced in the latter animal because insufficient estrogen passes to the first part- ner to inhibit gonadotrophin secretion (see Finerty, 1952). The somewhat analogous experiment of transplanting ovaries to the spleen produces ovarian hypertrophy in much the same way. Here again, it is thought that the hypophysis becomes hyper- active because the amount of estrogen 504 PHYSIOLOGY OF GONADS reaching the ghmd is greatly diminished, through inactivation bj^ the liver (Biskind, 1941). Although it is true that estrogens have a suppressing action on gonadotrophin se- cretion, it has become increasingly evident that they can also stimulate hypophyseal function in certain ways, as Engle pro- {)osed in 1931. Thus short-term injection of estrogen into intact immature rats and mice will invoke precocious puberty not only by stimulating the sex accessories, but also by increasing gonadotrophin secretion and thus causing ovarian growth and even ovulation. Frank, Kingery and Gustavson (1925j re- ported that after such treatment regular cycles continued after treatment was with- drawn. Lane (1935) found that when 22- day-old female rats were injected daily with estrogen there was an early increase in number of ovarian follicles, including vesic- ular stages. After the first 10 days the non- vesicular follicles became depressed al- though vesicular follicles were retained. This was interpreted to mean that for a short time estrogen actually stimulates the follicle-stimulating hormone (FSH) l)ut eventually suppresses it, although lutein- izing hormone (LH) secretion remains elevated. Hohlweg (1934) had already dem- onstrated that when somewhat older pre- pubertal rats are given single, rather large injections of estrogen, ovulation and corpus luteum formation are induced within a few days (p. 514). Obviously LH secretion is greatly increased. Various bits of evidence implicate the nervous system in the processes leading to puberty and to the onset of estrus in sea- sonal breeders. This will be discussed in the following section with respect to the general (juestion of the relationship of the hypo- thalamus to gonadotrophin secretion. V. Follicular Cycles. Growth and Atresia Attention will be focused here on the dynamic pattern of follicle development throughout the cycle, the extent to which this i)attern depends on hyi)o[)hyseal con- ti'ol, and the functional changes in the o\aiy associated with estrus in preparation foi' the more specialized events that lead to ovuhition and corpus luteum formation. Production of primordial follicles and the early growth stages have been said to be independent of the hypophysis (Smith, 1939; Hisaw, 1947). This view derives from the fact that following hypophysectomy the ovaries retain large numbers of healthy proliferating follicles below the stage of antrum formation. There are, however, sev- eral indications that these developmental stages may be accelerated by gonadotrophic stimulation. It was briefly reported by Simpson and van Wagenen (1953) that ad- ministration of purified FSH to immature monkeys caused not only a 10- to 20-fold 'increase of ovarian weight, but also stimula- tion of granulosa in follicles of all sizes. Indirect evidence comes from the fact that follicle atresia generally becomes maximal late in estrus or metestrum, when depressed FSH might be expected on theoretical grounds. Harrison (1948) reported tliat in ovaries of goats killed on the third or fourth days of estrus healthy primary ovocytes are rare. Some few, however, presumably re- main. Myers, Young and Dempsey (1936) stated that in the estrous guinea pig there are few nonatretic follicles aside from those destined for ovulation. However, small numbers of normal ai)pearing nonvesicular follicles were found. There seems to be general agreement that, very quickly after this catastrophic elimi- nation of follicles, renewed growth promptly ensues. Whether or not the wave of atresia represents a depression of FSH secretion, no one would deny that tlie new growth reflects this type of gonadotrophic stimulation. Characteristically the population of small and medium follicles is restored early in the luteal phase of the polyestrous cycle. This is clearly indicated for the guinea pig ovary (Myers, Young and Dempsey, 1936) when the data are converted from average vol- umes to average diameters (Fig. 8.4). Be- ginning on the fourth day after estrus, when the largest follicles are approximately 300 fx in diameter and when theca interna and antra have formed, rapid growth of granu- losa, theca, and antra continues for several days. This is confirmed by counts of mitotic figures obtained by the colchicine technique (Schmidt, 1942), indicating greatest mitotic activity in theca and granulosa of follicles between 300 fi and 600 fx in diameter. By the MAMMALIAN REPRODUCTIVE CYCLE 505 4 8 12 DAYS AFTER BEGINNING OF ESTRUS Fig. 8.4. A schematic repiesentation of the folhcuhir cycle in the guinea pig. The heavj^ sohd curve represents the diameters of the largest follicles, recalculated from the data of Myers, Young and Dempsey (1936). The arrow point indicates ovulation. The other solid curves and broken lines represent impressionistically the growth and atresia, respectivelj^ of other groups of follicles that are not ordinarily destined for ovulation. 11th or 12th day the largest follicles (ca. 800 /x) are "competent," i.e., capable of being ovulated (Dempsey, Hertz and Young, 1936; Dempsey, 1937). While the largest follicles are developing to this stage, multitudes of others begin to grow, being carried on to various stages of development before regression sets in. This pattern of the follicular cycle seems to be generally true among mammals that have been carefully studied, when allowance is made for the fact that from one species to another the characteristic maxima of follicle diameter are extremely variable (shrew, 350 fjL-, rat, 900 /*; cow, 19,000 /x; mare 70,000 /x; Asdell, 1946). In ovulatory cycles of poly- estrous animals the greater part of follicle growth is accomplished while the luteal phase of the preceding cycle is in progress. In successive anovulatory cycles like those of the cat the patterns of the follicular cy- cles are probably much the same (Evans and Swezy, 1931 » . In the rabbit and ferret, where more or less constant estrus char- acterizes the isolated females in season, there is probably considerable telescoping of successive waves of follicle growth such that as one set of follicles begins to undergo atresia another set is ready to take its place (Hill and White, 1933). The difference be- tween cat cycles and rabbit cycles seems to be chiefly one of degree. The writer has seen both types represented in persistent-estrous rats, among litter mates of inbred strains (Everett, 1939, and unpublished). At the end of the luteal phase of the cycle in polyestrous animals there are al- ready present several competent follicles among an extensive population of smaller ones. For example, the guinea pig corpus luteum usually shows signs of regression on day 13 of the cycle. It has been proved that ovulation can be induced as early as day 12 by injection of LH (Dempsey, 1937), several days earlier than it would normalh^ occur (Fig. 8.5). In the human and monkey it is possible that the "preferred" follicles are recognizable by their larger size during 506 PHYSIOLOGY OF GONADS 1000 750 500 Volume (lO^cu.//) 250 Normal Cycle Cycle After Removal of Corpora Luteo Progesterone Treated or Pregnant 'Corpora Luteo Removed and Oestrin Injected Fig. 8.5. The guinea pig follieular cycle and some of its experimental modifications. (After E. W. Dempsey, Am. J. Physiol., 120, 126-132, 1937.) or soon after menstruation (Allen, Pratt, Newell and Bland, 1930; Hartman, 1932). In many mammals competent follicles may be present much earlier. Ablation of corpora lutea soon after ovulation in sheep (Mc- Kenzie and Terrill, 1936) and cattle (Ham- mond, Jr., and Bhattacharya, 1944) is fol- lowed in 2 to 4 days by another ovulation, much sooner than in the guinea pig (Fig. 8.5). Removal of the primate corpus luteum, at the other extreme, produces no such im- mediate response, judging from the details of three cases among Hartman's (1932) pro- tocols (#40, #41, and #99). Whereas the next ovulations took place earlier than ex- pectation, the intervals between unilateral ovariectomy and ovulation were 16, 14, and 22 days, respectively. From detailed investigations in the rat, only the earlier stages of follicle growth may properly be regarded as pure FSH ef- fects (Lane, 1935). Lane and Greep (1935) found that addition of Lli to FSH causes a marked increase in the proportion of vesicu- lar follicles to follicles without antra. The use of more highly purified materials ((irecp, van Dyke and Chow, 1942; Fraen- kel-Conrat, Li and Simpson, 1943) has amply confirmed the necessity for combina- tion of the two gonadotrophins to yield max- imal follicle growth and estrogen secretion ill rats. Morphologic evidence indicates that LH acts selectively on thecal tissue and, therefore, on the interstitial tissue derived therefrom. Inasmuch as thecal tissue is the presumptive major source of ovarian estro- gen (see below), it follows, perhaps, as Hi- saw (1947) suggested that "the theca in- terna through the action of LH acquires competence to respond to FSH" (by se- creting estrogen) . Convincing evidence that thecal tissue and its derivatives are the principal sources of ovarian estrogen was assembled by Cor- ner (1938). The status of this question re- mains essentially the same today. Few endo- crinologists, however, would assume that no other ovarian cells have this capacity (see discussion in the chapter on the ovary). Nevertheless, there is a direct correlation in time between the marked rise in estrogen secretion as the follicular jihase of the cycle advances, on the one hand, and the organi- zation of tlicca interna of the largest fol- licles into organs of obvious endocrine char- acter, on the other. "When especially ])rominent the theca interna is referred to as the "thecal gland" (Mossman, 1937; Stafford, Collins and ]\Iossman (1942). Thecal tissue from the multitudes of atretic follicles should not be neglected as a possible additional source of estrogen. From the standpoint of chronologic rela- tions to the cycle tiiis (iiiestioii has hardly MAMMALIAN REPRODUCTIVE CYCLE 507 been touched. Pointing up our ignorance, Sturgis (1949) in a careful study of atresia of large follicles in the monkey ovary, spec- ulated that their hypertrophied thecal tissue may serve the useful purpose of estrogen secretion during the interim between follicle rupture and organization of the corpus luteum. We are in need of ciuantitative appraisals not only of the total numbers of healthy and atretic follicles of all categories present in representative species at progressive stages of the cycle, as in the work on the rat by Mandl and Zuckerman (1952), but also of the respective volumes of theca, granulosa, interstitial tissue, and corpora lutea. Lane and Davis (1939) determined in rat ovaries at four stages of the cycle the respective total volumes of theca, granulosa, and antra in all healthy follicles, as well as the sepa- rate mitotic indices of theca and granulosa. Such differential information on multiplica- tion of cells and increase of antral volume is important. Although the latter accounts for a major part of the increase in volume of the larger follicles, it represents a func- tion quite apart from protoplasmic growth per se. There is now considerable evidence that estrogen itself exerts a growth -promoting influence on the follicle and, furthermore, sensitizes it to gonadotrophic stimulation. Details may be found in papers by Pencharz (1940), Williams (1940, 1944, 1945a, b), Simpson, Evans, Fraenkel-Conrat and Li (1941) , Gaarenstroom and de Jongh (1946) , and Desclin (1949a,) . Although it seems that these effects have not been elicited by phys- iologic doses, the possibility remains that estrogen operates within the confines of the ovary as a mediator of some of the effects of the gonadotrophins. In the neighborhood of cells that produce it the estrogen concen- tration is probably far above that which would be considered physiologic for the re- mainder of the body. A. CORRELATION OF OVARIAN SECRETION W^ITH THE FOLLICULAR CYCLE Knowledge of the secretory output of the ovary during the cycle is almost entirely indirect and derives chiefly from (1) sub- stitution experiments carried out in a vari- ety of si)ecies, and (2) assays of urine, mainly human but occasionally from other forms. Satisfactory assays of blood estrogen have been very limited and chemical analy- sis of the steroid content of ovarian venous blood is in only its preliminary stages. The early substitution experiments are chiefly of historic interest (Allen, Danforth and Doisy, 1939). In great measure these investigations constitute crucial steps in proof that the ovary secretes steroid hor- mones which are fundamentally responsible for the manifestations of estrus. Con- versely, then, these manifestations might be considered to reflect an increase of estrogen secretion and their absence a relative de- crease. It has been learned, however, that the action of estrogen in certain instances may be greatly modified by progesterone, androgens, and certain adrenocortical ster- oids (notably desoxycorticosterone). Andro- gens are known to be secreted in the female by the adrenal cortex (Dorfman and van Wagenen, 1941 ; Gassner, 1952) and by the ovaries (Hill, 1937a, b; Parkes, 1950; Deanesly, 1938; Burrill and Greene, 1941; Pfeiffer and Hooker, 1942; Alloiteau, 1952). Progesterone secretion is probably not con- fined to the luteal phase of the cycle (see p. 519j. Evidence for its secretion during fol- licle maturation is considerable and its pos- sible production even earlier cannot be excluded. These considerations make it un- wise, therefore, to regard phenomena such as vaginal cornification, turgescence of vulva and sex skin, uterine growth, as direct ciuantitative measures of estrogen output. This point may be illustrated by certain ob- servations made in chim])anzees by Fish, Young and Dorfman (1941) and illustrated in Figure 8.6. Assays of urinary estrogens during the cycle exhibited two peaks, only the first of which coincided with the swelling of sex skin. The second peak of estrogen ex- cretion was unaccompanied by swelling, pre- sumably because of the coordinate increase of progesterone secretion. Had swelling been the only guide only the first peak would have been apparent. Assays of urinary estrogen in primates have often shown double peaks such as il- lustrated for the chimpanzee. Pedersen- Bjergaard and Pederson-Bjergaard (1948i. 508 PHYSIOLOGY OF GONADS I.U. '00 I.U. 0 ( »ott Androgens Curve of genital swelling 25 27 29 Day of cycle Fig. 8.6. E.strogen and androgen excretion by a female chimpanzee, Mamo. , total estrogens; , estradiol; -•-•, estrone; , estriol. Menstruation indicated by solid areas on base line. (From W. R. Fish, W. C. Young and R. I. Dorfman, Endocrinology, 28, 588,1941.) studying one woman for 2 years, found single peaks at midinterval in 8 cycles and double peaks in 12 cycles. On the average the first peak was reached on day 12 and the second on day 21. Similar double peaks were noted in blood estrogen assays in a large group of normal young women (Markee and Berg, 1944). An additional lesser rise was observed during menstruation. None of the available assays of urinary or blood estrogen can be accepted as an abso- lute measure of the rate of hormone produc- tion. Urinary assays have certain advan- tages, in spite of the fact that probably only a variable fraction of the ovarian product is measured. Intrinsically they are measures of rate, whereas assays of blood estrogen measure concentration alone at the moment of bleeding. Attempts have been made to measure estrogens in ovarian venous blood, but with little success because of the ex- treme dilution (Rakoff and Cantarow, 1950). We may hope that development of sufficiently sensitive methods of detection will soon allow systematic evaluation of ovarian output by such direct means. Tracer techniciucs have shown (Werthessen, Schwenk and Baker, 1953) in perfused ova- ries of the sow that C^^-acetate enters into the synthesis of estrone and /^-estradiol. Several years ago Corner (1940) esti- mated, from the known amounts of injected estrone required to maintain the normal status of sex skin and endometrium in cas- trates that the ovaries of an adult rhesus monkey secrete the equivalent of about 20 fig. estrone daily. On a weight basis the es- trone equivalent secreted by the ovaries of a woman would then be on the order of 300 /Ag. per day. Actual substitution data from castrated women gave an estimate of the same order of magnitude (420 ;u,g. per day). Whatever the rate of secretion may be at different times, it would seem a 'priori that effects on extra-ovarian tissues should be more directly related to amount of estrogen in circulation. The assays of human blood- estrogen in normal women by Markee and Berg (1944) and in gynecologic patients by Fluhmann (1934), although differing in ab- solute values, agree in indicating that the variation of blood estrogen concentration from one stage of the cycle to another may be relatively small. If this is true, then it nuist be supposed that cyclic changes in the accessory organs are brought about by rela- tively moderate changes in circulating estro- gen. In support of this view Markee (1948) demonstrated in the macaque that a mere 50 per cent reduction in the daily dose of es- trogen can invoke menstruation if the change is abrupt. MAMMALIAN REPRODUCTIVE CYCLE 509 B. CYCLIC MANIFESTATIONS AFTER OVARIECTOMY OR HYPOPHYSECTOMY Residual cyclic changes in the vagina liave been reported in ovariectomized mice (Kostitch and Telebakovitch, 1929) and rats (Mandl, 1951). The periodicity is very nearly that of the normal cycles, at least in the latter species. Vaginal cycles of similar duration with more extreme estrous changes are found in ovariectomized rats receiving daily injection of threshold doses of estro- gens (del Castillo and Calatroni, 1930; Bourne and Zuckerman, 1941). The same was remarked in mice by Emmens (1939) and a report by Veziris (1951) indicates that vaginal periodicity may obtain in cas- trated or menopausal women receiving es- trogen. Although sucli events have been called ''threshold cycles," the term may simply express the fact that they are most easily recognized when estrogen is given at threshold level. Hartman (1944), employing a modified Shorr stain for vaginal smears, found that castrated rats given large amounts of estrogen daily (5 to 100 fig. es- tradiol dipropionate) displayed complete cornification at 4- to 5-day intervals. Dur- ing the time intervening there was admix- ture of Shorr cells, smaller epithelial cells, and leukocytes. Analogous phenomena have been recog- nized in the endometrium of castrated mon- keys (Zuckerman, 1937, 1941) injected daily for as long as 1 year with threshold doses of estrone (10 fig.). Larger doses prevent cyclic bleeding (see Hisaw, 1942). From the report of Veziris (1951) it may be judged that threshold endometrial cycles also occur in women and that the vaginal and endo- metrial cycles are synchronized in consider- able extent. Full explanation of these phenomena is not at hand. From the standpoint of the present discussion certain considerations are especially noteworthy. (1) Vaginal "thresh- old cycles" have been obtained in castrated rats in the absence of either hypophysis or adrenals (Bourne and Zuckerman, 1941 ; del Castillo and di Paola, 1942) . The former au- thors encountered the phenomenon in two rats from which both the hypophysis and adrenals had been removed. It is important to remember, however, that the pars tu- beralis remains in situ after the usual hy- pophysectomy procedure, that accessory adrenocortical tissue is frequent in rats, and that gonadal rests might remain unrecog- nized. (2) The reported lengths of vaginal and endometrial cycles agree favorably with the cycle lengths in intact individuals of the respective species. The degree of conformity between vaginal and uterine cycles indi- cated by Veziris {loc. cit.) suggests some sort of integrating mechanism. Much more information is required, however, before one may reject the alternative view that rhyth- mic activity is an innate characteristic of these organs. C. CYCLIC MANIFESTATIONS IN THE ABSENCE OF OVARIAN FOLLICLES Many years ago Parkes (1926a, b) and Brambell, Parkes and Fielding (1927a, b) reported vaginal and uterine cycles in mice in which the entire follicular apparatus had been destroyed by x-radiation. Schmidt (1936) described the phenomenon in the guinea pig, noting that, although most of her estrous animals had one or more large atretic or cystic follicles, as she had earlier reported (Genther, 1931), a few animals exiiil)ited periodic vaginal opening of short duration and correlated proestrous vaginal smears, in the absence of follicles. Her as- says of urinary estrogen were negative in these animals, unlike the positive assays in those in which one or more follicles were demonstrable. Attempts by several workers (Drips and Ford, 1932; Levine and Witschi, 1933; Mandel, 1935) to reproduce in rats the results that Parkes and Brambell had found in mice, were unsuccessful, a fact in- dicating no estrogenic activity in ovaries completely lacking follicles and ova. Parkes (1952) more recently returned to this prob- lem, reporting vaginal cycles and "fully functional" uteri in castrated rats bearing grafts of ovaries in which all organized folli- cles and ovocytes had been destroyed by deep freezing. These were true estrous cy- cles, in the sense that the animals would mate. Many questions are posed by these ob- servations. The fundamental one seems to be whether these cycles express periodicity of hypophyseal gonadotrophin secretion. 510 PHYSIOLOGY OF GONADS The answer may be long in coming. Mean- while, one would like to know whether cas- tration changes are visible in the hypophysis and whether constant estrus may be invoked by exposure to continuous light or by post- natal treatment of the host with androgen or other steroids (see p. 529 1 . D. HYPOTHALAMUS AND GONADOTROPHIN SECRETION. GENERAL CONSIDERATIONS Experimental studies, ostensibly ad- dressed to the general problem of neural control of gonadotropin secretion, have in fact often been concerned with the special problems of reflex ovulation guinea pigs regularly provoked follicle growth and estrogen secre- tion. In gregarious birds the development of ovulable follicles requires that other indi- viduals of the species be present. In the pi- geon, even the mirror image of the female constitutes a sufficient stimulus (Matthews, 1939). Studies by Flerko and his associates (1954-1957) present consistent evidence that restricted bilateral lesions in the region of the paraventricular nuclei serve to liber- ate the hypophysis from inhibitory effects of estrogen and androgen. This work is in agreement with that of Donovan and van der Werff ten Bosch in that somewhat simi- larly located lesions brought on precocious puberty. As noted elsewhere, gonadectomy in immature rats quickly results in hyper- secretion of gonadotrophin. Transplantation of the hypophysis to sites remote from the hypothalamus has produced divergent results. At the present writing, the chief divergence seems to rest between the sexes. In male guinea pigs and rats several workers have reported main- tenance of male reproductive tracts by in- tra-ocular transplants of hypophyses (May, 1937; Schweizer, Charipper and Kleinberg, 1940; Cutuly, 1941a; Courrier, 1956; Gold- berg and Knobil, 1957). Quite to the con- trary, however, there has at best been only equivocal evidence of maintenance of fe- male tracts, a matter of sex difference which needs full investigation. JNIay's (1937) re- port of 2 fertile female rats is unacceptable because of inadequate controls. Schweizer, Charipper and Haterius (1937) found in several hypophysectomized guinea pigs that intra-ocular pituitary grafts produced con- stant estrus and significant follicle stimula- tion, accompanied by uterine and mammary gland develoi)inent. Although the search for pituitary remnants in the sella turcica was reported negative, the histologic check was limited to scrapings from the sella floor. Other authors, notably Phelps, Ellison and Burch (1939), Westman and Jacobsohn (1940), Harris and Jacobsohn (1952), and Elverett ( 1956a) obtained in female rats lit- tle or no evidence of gonadotrophin secre- tion from apparently healthy, well vascu- larized grafts. The respective sites were intraniusculai', intra-ocular, in the sub- MAMMALIAN REPRODUCTIVE CYCLE 511 arachnoid space under the temporal lobe of the brain, and beneath the renal capsule — all distant from the hypothalamus. Transplantation of the pars distalis into sites close to the hypothalamus, on the other hand, is characteristically followed by maintenance of the female reproductive tract and essentially normal sex functions. Greep (1936) found that re-implantation of hypophyses into the (presumably) emp- tied capsule was frequently followed in both male and female rats by return of virtually normal reproductive powers. Females ex- hibited cycles and even went through suc- cessful pregnancy and lactation. The result observed in male rats was confirmed by Cutuly (1941a). The obvious difficulty of establishing completeness of hypophysec- tomy has been the only criticism of these instructive experiments. This fault has been eliminated by an improved procedure de- vised by Harris and Jacobsohn (1952). Hy- pophysectomy was performed by the para- pharyngeal route, after which the tissue to l)e grafted was introduced by a transtem- poral approach to a site immediately be- neath the median eminence. This permitted later histologic search for remnants of the original gland in its capsule. In many cases, including all in which the graft comprised several hypophyses from the animal's own newborn young, entirely normal gonado- trophic function was recorded. This included resumption of regular estrous cycles, typi- cally during the 2nd or 3rd postoperative week. Several of the rats became pregnant and delivered viable litters. In marked con- trast, none of the grafts that were placed under the temporal lobe gave any indication of gonadotrophin secretion, although they were as well preserved and richly vascular- ized as the others. Explanation of the dif- ference seems to be that grafts under the median eminence acquire blood supply from regenerated hypophyseal portal veins and iience a neurovascular linkage with the hy- pothalamus. The importance of this rela- tionship has been amply confirmed by Ni- kitovitch-Winer and Everett (1957, 1958d) in studies described below. In lieu of significant numbers of nerve fi- bers entering the pars distalis (see Rasmus- sen, 1938; Harris. 1948a I, the hypophyseal portal veins afford the most likely means by which the gland is brought under hypo- thalamic control. Recently it was demon- strated in rats and monkeys that these ves- sels have the power of rapid regeneration after simple stalk-section (Harris, 1949, 1950a, b). This fact at once gives a ready explanation of many of the discordant re- sults of stalk-section experiments reported in the past. Harris (1950b) explored in rats the efficacy of various materials as barriers to regeneration, with the result that numer- ous examples of partial regeneration were produced. Degree of recovery of gonado- tropliic activity by the hypophysis was strikingly correlated with degree of ana- tomic vascular recovery. Restoration of nor- mal ovarian function after simple interrup- tion of the stalk, as reported in the guinea pig by Dempsey (1939), in rats by Demp- sey and Uotila (1940) , and in the human by Dandy (1940), is thus explained by the as- sumption that portal vein regeneration had taken place. On the other hand, Westman and Jacobsohn (1937-1938), who always inserted a barrier of metal foil between the median eminence and hypophyseal capsule, consistently found ovarian atrophy, as did Harris when portal vein regeneration was completely obstructed. Attempting to prove that the portal vessels are not essential in regulating the hypophysis, Thompson and Zuckerman (1954) stated that increased illumination induced estrus in two ferrets after stalk-section and in the absence of demonstrable regeneration of portal vessels. Donovan and Harris (1954), however, ex- amining the histologic sections prepared from 1 of the 2 animals, found many such vessels that were uninfected. In their own experimental series, an estrous response to light was always associated with regenera- tion of the portal veins. Greep and Barrnett (1951) rightly em- phasized the prime importance of a good vascular supply for recovery of function by the pars distalis after either transplantation or stalk-section. They pointed to the ex- tensive central infarction and scarring that characteristically followed stalk-section by their technique, an obvious factor contribut- ing to hypopituitarism. Harris (1950a I, however, reported good function from sev- 512 PHYSIOLOGY OF GONADS eral hypophyses in which there was pro- nounced central necrosis in company with well regenerated portal vessels. A study by Nikitovitch-Winer and Everett (1957, 1958b) established beyond doubt that quali- tative functional losses after stalk-sectron or transplantation of the pars distalis result, not from impaired blood supply per se, but from the loss of the intimate neurovascular relationship with the hypothalamus. Hy- po])hyseal autografts, after first being placed under the kidney capsule for sev- eral weeks with the usual atrophy of the ovarian follicular apparatus and interstitial tissue, were later retransplanted to a site immediately under the median eminence. In the definitive series of 14 such experiments, 13 rats resumed estrous cycles spontane- ously 8 to 68 days after retransplantation ; 7 were fertile and carried litters to term. A correlated study (Nikitovitch-Winer and Everett, 1959) demonstrated clearly that on the occasion of each of these successive transplantations there was massive necrosis of the interior of the glandular mass, leav- ing but a thin shell from which the func- tional tissue of the graft was reconstituted. In spite of this double insult some special influence of the hypothalamus brought about renewed function in a surprising num- ber of cases. Together with the restoration of gonadotrophic activity there was signifi- cant improvement in thyroid-stimulating hormone (TSH) and adrenocorticotrophic hormone (ACTH) secretion. The consider- able net loss of hypophyseal parenchyma resulting from the two operations was re- flected only quantitatively in the effects on the various target organs. Ovarian weights, numbers of follicles and corpora lutea, ad- renal weights and extent of adrenal hyper- troi)liy after unilateral adrenalectomy, and thyroid uptake of P^^ were all intermediate between those of the normal female rat and control animals in which the graft remained on the kidney or was retransplanted under the temporal lobe of the brain. Regulation of pars distalis secretion by means of the stalk vessels may conceivably be carried out either by regulation of blood How or by transmission of chemical media- toi-s from the proximal capillary plexus in the median eminence to the pars distalis. An experiment describetl by Swingle, Seay, Perlmutt, Collins, Fedor and Barlow (1951) suggested that a mediator subject to Diben- amine blockade might be involved in pre- cocious puberty. Although significant uter- ine enlargement was produced in immature rats by daily stimulation of the cervix uteri for 10 days, no such effects were observed in similar rats given Dibenamine daily by stomach tube. Unfortunately, there were no controls for the possible effect of Diben- amine in nonstimulated or gonadotrophin- injected animals. Fluhmann (1952) invoked precocious vaginal opening and ovarian stimulation in immature rats by injection of neostigmine. The locus of such cholinergic action is un- known. Parenthetically, Barbarossa and di Ferrante (1950) reported follicle stimula- tion in immature rats after injection of intermedin, an effect not found in hypophy- sectomized subjects. Benoit and Assen- macher (1955) proposed that, in the drake, gonad-stimulating activity is governed by an agent contained in neurosecretory sub- stance, which is demonstrable in abundance in the retrochiasmatic region of the median eminence. Capillaries there drain selectively into an anterior set of portal venules. Oxy- tocin has been suggested as a possible medi- ator for gonadotrophin secretion (Shibu- sawa, Saito, Fukuda, Kawai, Yamada and Tomizawa, 1955; Armstrong and Hansel, 1958). There is much interest as this is being written (1958) in the jiossibility that vasopressin, oxytocin, or other agents associated with neurosecre- tory substances of the neui-ohyjioiihysis are responsible for control of produc- tion and release of the various trophic hormones of the pars distalis. As an alternative or even a supplement to neu- rochemical regulation, a vasomotor mecha- nism cannot be denied (Green, 1951), for conceivably only a slight shift in blood flow through the jiars distalis might tip the bal- ance of hormone production one way oi- an- othei-. Thus the matter stands: whereas it is apjiarent that the hypothalamus inter- venes in follicle growth and estrogen secre- tion, how it does so is little more than spec- ulati\'e. MAMMALIAN REPRODUCTIVE CYCLE 51)5 VI. Follicle Maturation and Ovulation A variety of evidence indicates disconti- nuity between growth of large follicles, on the one hand, and their preovulatory matu- ration, on the other. Such is clearly the case among "reflex ovulators." Evidence that the same is true for spontaneous ovulators will be outlined below. Follicle maturation, ovu- lation, and structural transformation of the follicles to corpora lutea seem to represent successive stages in a distinct physiologic process, superimposed on the follicle growth cycle and brought about by a relatively abrupt increase in circulating gonadotrophin (theoretically LH). Since there is evidence (p. 519) that progesterone secretion may become detectable as this process begins, there might be justification for regarding it as merely the first portion of the luteal phase. However, the fact that luteinization ( i.e., the organization per se of luteal tissue) does not necessarily lead to functional cor- |)ora lutea warrants treatment of the ovula- tion-luteinization phase as a distinct phe- nomenon. Although it is customary to state that the hypopliysis invokes ovulation by release of LH, there is considerable question about the auxiliary roles played by other gonado- trophic hormones (Hisaw, 1947). Inasmuch as the time of release has been known in only the reflex ovulators, one might look to them for information. However, the avail- able data (Hill, 1934) pertain only to the ovulating i)otency of the total gonado- trophin content of the hypophysis at various times after coitus. Substitution experiments are unsatisfactory because the presence of competent follicles implies the presence of l)oth FSH and a small amount of LH. The substitution of even the purest hormone preparations immediately after hypophy- sectomy leads to equivocal results inasmuch as it must be assumed that some FSH and LH of intrinsic origin remain in circulation. Talbert, Meyer and McShan (1951) deter- mined that in rats, when hypophysectomy is performed at the onset of proestrum, the follicles remain capable of responding to in- jected LH for about 6 hours. Morphologic signs of follicle deterioration do not appear until nuich later. Adding to the uncertainty is the fact that relatively pure preparations of either FSH or LH will ovulate an estrous rabbit (Greep, van Dyke and Chow, 1942). On the other hand, until the recent use of species-specific gonadotrophins (van Wage- nen and Simpson, 1957), the primate ovary was notoriously difficult to ovulate thera- peutically. Until effluent blood from the hy- pophysis can be assayed, there is little likelihood that the gonadotrophin complex that is normally responsible for ovulation can be known. Thus, whereas the expression, LH-release, will be employed occasionally to refer to the release of gonadotrophin that in^•okes ovulation, the term is used purely for convenience and brevity, and should be ai^propriately qualified by the reader. A. TIME OF OVUL.\TION The time of ovulation with respect to other events of the cycle is relatively easy to determine in reflex ovulators, but in spon- taneous ovulators has proven to be more elusive. In the former, laparotomy at vari- ous intervals after the stimulus enables ex- act measure to be made of the time required to accomplish ovulation. For most of the spontaneous ovulators, save the few in which the ripening follicles can be palpated as in monkeys and cattle, it has been neces- sary to attempt to correlate ovulation with some easily detectable external sign. Inas- much as the ovulation stimulus to the hy- j)oiihysis in these animals is probably in- voked by ovarian hormones and these are equally responsible for phenomena such as vaginal cornification and behavioral estrus, a considerable degree of correlation might be expected between ovulation and a given change in the vaginal smear or onset of es- trous behavior. The predictability of the relationship, however, must depend in great measure on the degree of correlation among thresholds of response in the various tissues concerned. In the primates that have no sharply limited period of sex desire the i:)roblem is even more troublesome. When reference is made to the date of the last menstruation, prediction is erratic because of the variable occurrence of postmenstrual quiescence (Rossman and Bartelmez, 1943; Young and Yerkes, 1943). Consequently, attempts must be made to find indicato:- 514 PHYSIOLOGY OF GONADS such as basal body temperature fluctuations which may bear some intrinsically closer relationship to the event in question. (See Hartman, 1936, and Buxton and Engle, 1950, for discussion of this very practical ]iroblera. ) Among mammals generally, si)ontaneous ovulation takes place sometime during es- trus (Asdell, 1946) . It is found during early estrus in the opossum, red fox, dog, mouse and hamster. In the rat some authors have placed it early (Young, Boling and Blandau, 1941) and others late (Long and Evans, 19221 with respect to vaginal estrus. In the writer's colony both relations hold, in 4-day and 5-day cycles, respectively. Ovulation in late estrus is reported for the cotton rat, bank vole, guinea pig, pig, horse, and ass. Sheep usually ovulate shortly before the end of heat, sometimes a few hours after- ward. As stated earlier, ovulation may even occur in guinea pigs, rats, sheep, and cattle without overt estrus. The cow usually ovu- lates several hours after the end of heat. The marsupial cat is said to ovulate 5 days afterward (Hill and O'Donoghue, 1913). The extreme is represented by certain bats (Asdell, 1946) which copulate in autumn and ovulate in the spring after a prolonged state of subestrus. These variations prob- ably express several factors. Among reflex ovulators there is consider- able interspecies variation in the interval between the stimulus that invokes release of gonadotroj^hin from the hypophysis and the eventual rupture of the Graafian follicles (rabbit, ca. 10 hours; ferret, ca. 30 hours; cat, 24 to 54 hours; 13-lined ground sciuirrcl, 8 to 12 hours; mink, 36 to 50 hours) . Among spontaneous ovulators the comparable in- terval is clearly defined for only the rat, 10 to 12 hours (Everett, Sawyer and Markce, 1949) . In the cow the data obtained by Han- sel and Trimberger (1951) and Hough, Bearden and Hansel (1955) i)lace the out- side limit at about 30 hours. Here again, threshold differentials among tlic vari- ous tissues of the individual are pioba- bly of gicat importance. 'I'hus in one species the threshold foi' gonadoti'ophin release may be lower than that foi' es- trous behavior with the result tliat by the time the latter makes its ai)pearance the former has already transpired and ovulation will shortly take place. The rat, for example, releases LH during the after- noon, begins to show estrous behavior around 8 p.m., and ovulates around 2 a.m. (Everett, 1948, 1956b). In other species these time relationships may be reversed. In the cow, activation of the hypophysis ap- parently occurs several hours after the onset of estrus (Hansel and Trimberger, 1951). The cow remains in heat 10 to 18 hours and ovulates 13!/2 to 151/2 hours after going out of heat (Asdell, 1946). The early termina- tion of estrus apparently reflects a refrac- tory state which sets in after estrogen activity has continued for a time, for cas- trates receiving continued estrogen therapy remain in estrus for similarly brief periods. In the mare, ovulation is delayed until a few hours before the end of estrous periods that may extend for 5 to 10 days or longer. This suggests a relative refractoriness of the LH- release mechanism in this animal. Such a state of affairs approaches that in persistent estrus or in the anovulatory cycle. B. OVARIAN STEROmS AND OVULATION 1. Estrogens Chronic administration of estrogen to the intact animal eventually produces ovarian atrophy by suppression of gonadotrophin secretion. However, some moderate basic level of continuous estrogen secretion must be compatible with normal function of the hypophyseal-ovarian system; witness the fact that blood estrogen assays in normal women (]Markee and Berg, 1944) indicate only a 2-fold increase at midinterval above a base value of considerable magnitude. Induction of corpus luteum formation by injected estrogen was first demonstrated by Hohlweg (1934) in prepubertal rats^ and the phenomenon has been repeatedly ob- served by other woi'kers (Desclin, 1935; Mazer, Israel and Aljjcrs, 1936; Westman and Jacobsolm, 1938b; Herold and Effke- mann, 1938; Price and Ortiz, 1944; Cole, 1946). The fact that the effect was not ob- tained in rats younger than 30 to 36 days l>y Piice and Ortiz, whereas Cole observed it in the age-range of 21 to 28 days, demon- ' Tlic eH'cct was later ol)1aiii(^(l witli androgens (Holilwpg, 1937; Salmon, 1938; Xathanson, Fian- spen and Sweenev, 1938). MAMMALIAN REPRODUCTIVE CYCl.E 515 B ^, ^ > 1 2 3 4 5 e A 12 3 4 5 12 3 4 5 Fig. 8.7. Experimental modifications of the 5-day cycle in rats. Two units of the ordinate represent full vaginal estrus. Time in days on abscissa, each unit 24 hours (midnight to mid- night). X, ovulation time; -p, progesterone, usually 1 to 2 mg.; e, estradiol benzoate, standard do.se 50 /xg. (From J. W. Everett, Endocrinology, 43, 393, 1948.) strates the existence of strain differences in the age factor. This probably explains the absence of luteinization in the experience of Lane (1935) and Merckel and Nelson (1940). Hohlweg and Chamorro (1937) demonstrated the importance of the hy- pophysis in the response. When hypophysec- tomy was performed 2 days after injection of estrogen no corpora liitea developed, but hypophysectomy on the 4th day did not in- terfere with corpus luteum formation. The effect could be produced in 50-gm. rats with as little as 4 |U,g. estradiol benzoate. West- man and Jacobsohn (1938b) reported that transsection of the hypophyseal stalk less than 2Vt days after injection prevented the reaction, but after that time the operation did not interfere. Bradbury (1947) assayed the gonadotrophin content of hypophyses of normal and castrated immature rats (30 to 32 days old at autopsy) 2 to 5 days after injection of estrogen or other steroids. These rats were apparently too young to form corpora lutea in response to the treatment, l)ut marked interstitial-cell stimulation, in- dicative of LH (ICSH) activity, was ob- served as early as 96 hours. In the intact animals significant loss of potency occurred 72 to 96 hours after injection, in agreement with the hypophysectomy data of Hohlweg and Chamorro (1937). In castrated rats, however, there was no loss of potency, thus suggesting that some ovarian factor in addi- tion to estrogen is essential for stimulation of the hypophysis. It is unfortunate that the study was confined to animals too young to give the full response of luteinization. Induction of ovulation in adult animals by estrogen was first reported by Hammond, Jr., Hammond and Parkes (1942) and by Hammond, Jr. (1945) in the anestrous ewe. Whereas the s])ontaneous occurrence of oc- cult ovulation was approximately 5 per cent, injection of stilbestrol was followed by corpus luteum formation in 4 of 11 ewes, with recovery of ova in 3. Injection of stil- bestrol di-n-butyrate was followed by cor- pus luteum formation in 5 of 6 ewes and ova were recovered in 3. The finding was con- firmed by Casida (1946) who stated that in cycling ewes ovulation can be invoked by injection of diethylstilbestrol on the 4th day of the cycle, but not at other times. In 1947 Everett reported the induction of ovulation in pregnant rats within 40 hours after injec- tion of estradiol benzoate (as little as 2 or 3 /xg.) or implantation of estradiol crystals or pellets. The response was not obtained in animals autopsied 24 hours after treatment nor in other animals hypophysectomized at 24 hours and autopsied the following day. In other studies with adult rats it was demon- strated (Everett, 1948) that in 5-day cyclic rats the injection of estrogen at mid-dies- trum will regularly induce ovulation 24 hours earlier than exi:)ected (Figs. 8.7D, 8.8F|. Persistent-estrous rats were refrac- tory to estrogen in this respect.- Neverthe- less, when such animals were made pseudo- pregnant by daily injection of progesterone, "The tendency toward refractoriness of similar animals with respect to induction of estrous 1) - ha\'ior had earlier been reported by Boling, Blandau, Rundlett and Young (1941). 516 PHYSIOLOGY OF GONADS B 12 3 4 12 3 4 T% ^' 2 3 4 5 1 P 2 3 4 5 e v^^ / y r-U^'f*^ 1 tA 1 12 3 4 5 Fig. 8.8. Experimental modification of the 4-day cycle in rats. Same key as in Figure 8.7. Progesterone dosage 1.5 mg. per day. Artificial 5-day cycles in D, E, and F indicated by dotted lines and numbering. (From J. W. Everett, Endocrinology, 43, 395, 1948.) B e NO OVULATION j~~r~r~r Fig. 8.9. Experiment with persistent-estrous rats. Units of ordinate and abscissa have same meaning as in Figure 8.7. A. Secjuence of "progesterone cycles." Each dose of progesterone (p) is 1.0 mg. Ovulation (x) in about 70 per cent of the cycles. B. Progesterone cycle followed by unsuccessful attempt to induce ovulation by estrogen during the second c-ycle. C . Pseudo- pregnancy maintained by daily iiijoctinn of 1.5 mg. ]irogosteronr. Ovulation induced by estrogen in several such cases. (From .1. \V. Everett, EiKhxTinolojiy. 43, ;5i»9, 194S.) ovulation and corpus luteuni loiniatioii were induced by estrogen (Fig. 8.9 1. Early attempts to induce luteinization in the guinea pig with estrogen were unsuccess- ful ( Dempsey, 1937; see Fig. 8.5), but iiioiv recently Lipschutz, Iglesias, Bruzzone, 11 u- niercz and Penaranda (1948) have shown by the use of intrasplenic ovarian autografts that luteinization is a reguhir feature in ex- periments in which estrogen is administered systcniically. Interestingly enough the im- plantation of estrogen jiellets in or near the ox'ariaii grafts had tlic coiitrai'y effect of pi'cvcnliiig luteinization. it was early I'cportcd that rabbits fail to ovulate in response to estrogen injection MAMMALIAN REPRODUCTIVE CYCLE 517 (Bachman, 1936; Mazer, Israel and Alpers, 1936 ». Hisaw (1947j inferred that this is generally true for reflex ovulators. Never- theless, it was found by Klein and Mayer ( 1946) and Klein (1947) that when pseudo- l^regnant or pregnant rabbits were treated with estrogen and then mated, new ovula- tion resulted and new corpora lutea were formed, events that do not otherwise occur. The phenomenon was further explored by Sawyer (1949). Whereas untreated rabbits, unlike cats, do not ovulate in response to mechanical stimulation of the vagina, treat- ment with estrogen on the preceding 2 days results in a positive response to this stimu- lus. In fact, his later observations (1959) indicate that estrogen priming for a longer period (4 days) occasionally results in "spontaneous" ovulation, especially during the winter and spring. In the anestrous cat, in the response to mechanical stimulation of the vagina, estro- gen facilitates the ovulation of follicles primed with equine gonadotrophin (Sawyer and Everett, 1953). Induction of ovulation by estrogen in primates remains to be demonstrated. It is of interest in this connection that Funnell, Keaty and Hellbaum (1951) observed in menopausal women an increased excretion of LH during estrogen therapy, in contrast to FSH excretion at other times. The general experience has been that injection of estro- gen during the early part of the cycle sig- nificantly postpones the next expected ovu- lation and menstruation (monkey, Ball and Hartman, 1939; baboon, Gillman, 1942; hu- man, Sturgis and ^leigs, 1942; Brown, Brad- bury and Jennings, 1948). Gillman reported that a single injection of estrogen precipi- tates widespread atresia of vesicular fol- licles. Brown and Bradbury (1947) reported IH-eliminary data that in 4 of 6 women there was increased gonadotrophin excretion during the 24 hours following estrogen ad- ministration. They proposed that delay of ovulation by estrogen given early in the primate cycle may be the result of prema- ture discharge of gonadotrophin before the Graafian follicle is competent. Sturgis and Meigs had suggested, on the contrary, that the estrogen suppresses hypophyseal func- tion. D'Amour (1940), finding in urinary assays that tlie initial peak of estrogen ex- cretion preceded the peak excretion of uri- nary gonadotrophin, postulated that the increase of estrogen stimulates the gonado- trophin release that is responsible for ovu- lation. O. W. Smith (1944) proposed that not estrogen itself, but some metabolite re- sulting from inactivation by the liver, is responsible for LH release. This interesting hypothesis has not been substantiated. 3. Gestagens Suppression of estrus and ovulation by functional corpora lutea, suggested by Beard (1898), was experimentally demon- strated in the guinea pig by Loeb (1911). It is now well established in several species that removal of the corpora lutea results in early resumption of estrus and ovulation (see p. 506), and that administration of progesterone suppresses these events. There is considerable evidence favoring the view that the primary effect is to selectively sup- press the secretion of LH. Dempsey (1937) noted that in guinea pigs receiving daily in- jection of progesterone (50 /^g.) all stages of follicle development proceeded except the maturation enlargement that heralds LH release (Fig. 8.5). Astwood and Fevold (1939) and Cutuly (1941b) found similar results in rats. Essentially the same phe- nomenon has been noted in sheep by Dutt and Casida (1948). Bradbury (1947) re- ported that in immature rats the injection of progesterone at the time of estrogen injec- tion prevented the release of gonadotrophin (LH?) which otherwise followed estrogen injection by 72 to 96 hours. In ovariecto- mized guinea pigs containing intrasplenic autografts, preparations in which luteiniza- tion can be induced by estrogen (see above) , the simultaneous administration of gesta- gens prevented this action (Lipschutz, Ig- lesias, Bruzzone, Humerez and Pefiaranda, 1948; Iglesias, Lipschutz and Guillermo, 1950; Mardones, Bruzzone, Iglesias and Lipschutz, 1951). Mardones and co-w^orkers also made the interesting observation that among several steroids having progesta- tional activity, "antiluteinizing activity is not concomitant with, or subordinated to" the former function. Proportionately very large amounts of ethinyl testosterone and ethinyl-A-'^-androstenediol exhibited very little antiluteinizing activity. There is evi- 518 PHYSIOLOGY OF GONADS denceinmice (Solve,, 1939) that siippreijsion of FSH secretion may occur when as much as 1 mg. of progesterone is injected daily. Alloiteau ( 1954 ) believes that this also oc- curs in the rat, although Cutuly (1941b) found only slight evidence of FSH suppres- sion when as much as 6 mg. progesterone were given daily for several weeks. So much emphasis has been placed on the suppressing effect of progesterone that its facilitating actions were recognized only in recent years. The first indication that pro- gesterone can promote ovulation and corpus luteum formation in mammals was encoun- tered in a study of persistent-estrous rats (Everett, 1940a, b). Daily injection of 0.25 to 1.0 mg. caused the prompt interrujition of the state of persistent follicle and the re- sumption of outwardly normal cycles. Cor- jiora lutea were formed in approximately 70 per cent of these cycles.^ The effect was obtained not only in older rats in which persistent estrus had developed spontane- ously, but also in young rats in which the condition had been induced by continuous illumination. The dose level employed is be- low that required to suppress cycles in nor- mal rats (1.5 mg. daily; Phillips, 1937). Subsequently, it was found (Everett, 1943) that the daily injection could be avoided if a single "interrupting" dose was given, fol- lowed by a single injection during proestrum or early estrus of each recurrent cycle (Fig. 8.9.4). The histologic appearance of the ovaries reverted toward the normal after a succession of "i)rogesterone cycles" and, sig- nificantly, the interstitial-cell nuclei were "repaired." Attempts in normal rats to in- voke o\'u]ati()n earliei' than the expected time were sticcessful in the 5-day cycle (Figs. S.7B, 8.8^). Injection of from 0.5 mg. to 2 mg. on the "third day of diestrum" reg- ularly (4 mg. occasionally) invoked ovula- tion (luring the coming night (Everett, 1944a, 1948) unless the treatment was given too late in the dai/ (EA'erett and Sawyer, 1949; see discussion on ]). 526 I'egarding the diurnal rhythm and ovulation). Attempts to advance ovulation in the 4-day cycle were unsuccessful, possibly because the fol- licles were not competent or the animals' ''Marvin (1947) described a similar rosull willi desoxycorticosterone acetate. intrinsic estrogen levels were not elevated sufficiently early. Ovulation induced by progesterone has been reported in several species. A direct action on the excised ovary of the toad Xeno-pns was early demonstrated by Zwar- enstein (1937) but such action is apparently not found in higher vertebrates. In the do- mestic hen injection of progesterone can invoke ovulation several hours ahead of schedule (Fraps and Dury, 1943; Rothchild and Fraps, 1949). Pfeiffer (1950) observed new corpora lutea in 10 rhesus monkeys treated with progesterone during presump- tive anovulatory cycles of the summer months. Similar attempts have been made in women (Rothchild and Koh, 1951); al- though there were said to be definite indi- cations of induced ovulation, the evidence is equivocal. On the other hand, a rei~)ort (Hansel and Trimberger, 1952) states that in heifers the injection of small doses of pro- gesterone (5 to 10 mg. ) at the beginning of estrus significantly advances ovulation time. This is in contrast with the inhibitory effect of larger doses (50 mg.) beginning before the onset of estrus (Ulberg, Christian and Casida, 1951). Even in the rabbit (Sawyer, Everett and Markee, 1950), spontaneous ovulation was noted in 4 of 10 animals after combined estrogen and progesterone injec- tion. From certain of the foregoing statements it may be inferred that whether suppression or stimulation follows administration of ju-ogestcrone depends critically on the time of injection, on the amount given, on tlie status of the ovary, and probal)ly on a l)riming action of estrogen. A significant il- lustration of the critical nature of the time factor in rats is given by the experiments represented in Figure 8.8r and E. If after the first injection of 1.5 mg. progesterone on the first day of diesti'uni, a second injec- tion follows in. about 24 lioui's. the imjiend- ing estrus and o\-ulation are retarded an additional 24 hours. However, if the second injection is given 48 hours after the first, tlie impending estrus and ovulation are ad- vanced. Evidence^ of the synergistic action of estrogen and progesterone in evoking oA'ulation is given by tlie ex]ieriments repre- sented in Figuiv 8.9/> and (\ Sawver (1952) MAMMALIAN REPRODUCTIVE CYCLE 519 investigated the synergism in rabl^ts. Em- ploying estrogen-primed animals, he found that ovulation was facilitated when pro- gesterone was injected less than 4 hours before either mating, mechanical stimula- tion of the vagina, or intravenous injec- tion of copper acetate. Inhibition was ob- tained when progesterone was injected 24 hours before such stimulation, thus confirm- ing the often-cited observations of ]Make- peace, Weinstein and Friedman (1937 » and Friedman (1941) that progesterone inhiijits ovulation in rabbits. Preovulatory secretion of gestagens now seems likely. Morphologic luteal changes in preovulatory follicles are considered in the chapter on the ovary. A variety of evidence in primates indicates that progestational clianges in the endometrium begin before ovulation (Bartelmez, Corner and Hart- man, 1951). Several workers have reported tiie excretion of small amounts of preg- nanediol during the follicular phase of the human cycle (Wilson, Randall and Oster- berg, 1939; Smith, Smith and Schiller, 1943; Davis and Fugo, 1948). Determination of plasma progesterone in women by the Hooker-Forbes test indicates the presence of significant amounts a day or two before a major rise in basal body temperature (Forbes, 1950). In monkeys a small quan- tity (ca. 0.5 to 1.0 ixg. per ml.) was detected l)etween the 4th and 9th days, rising in the 10- to 15-day period to concentrations of 2 to 6 jxg. per ml. (Forbes, Hooker and Pfeif- fer, 1950; Bryans, 1951). In both species a transient decline seems to intervene before the marked rise to still higher concentra- tions during the luteal phase. In the rat, Constantinides (1947) studied the stromal nuclei of the endometrium at different stages of the cycle and found that by the Hooker-Forbes criteria there is evidence of progesterone secretion during proestrum. Astwood (1939) on the basis of water con- tent of rat uteri concluded that progesterone secretion begins with proestrum. In the rab- l)it, Forbes (1953) assayed peripheral blood at various intervals after mating or gonado- trophin injection. Although no progesterone was detectable in controls, significant amounts appeared an average of 97 minutes after mating and 66 minutes after gonado- trophin injection. As much as 2.5 /xg. ])er ml. was found during the first 8 to 10 hours, although marked fluctuations were noted from time to time in the blood of individual animals. Verly (1951) reported that soon after mating the urine of rabbits contains significant amounts of pregnanediol. It has become customary to state that the gestagen that appears during the follicu- lar phase of the cycle is probably formed by the maturing follicle itself. Indeed, assays of fluid from Graafian follicles and cysts have indicated the presence of the hormone (Duyvene de Wit, 1942; Hooker and Forbes, 1947; Edgar, 1952, 1953). However, if it is to take part in the release of ovulat- ing hormone gestagen must be secreted ear- lier than preovulatory maturation. For this also there is some evidence. Two reports cited above indicate that in monkeys, at least, there is a detectable amount present in the blood during the early follicular phase. The known fact that a waning cor- pus luteum favors the experimental induc- tion of estrus and/or ovulation in sheep and cattle (Hammond, Jr., 1945; Robinson, 1951 ; Alarden, 1952) is suggestive. Although Hammond, Jr., Hammond and Parkes (1942) tested this possibility by progester- one sul)stitution with negative results, the amount given may have been too small, as Robinson suggested. A waning corpus lut- eum in the rat favors ovulation, as dis- closed in persistent-estrous animals in which pseudopregnancy had been induced (Eve- rett, 1939). Each of three pseudopregnancies was followed b}' a short cycle before the animals returned to persistent estrus. In the course of studies growing out of this observation evidence was advanced (Everett, 1945) which indicated that cor- pora lutea of the normal rat are not wholly inactive during the short cycle. Transient depletion of cholesterol was observed in such corpora lutea during the proestrum that followed their formation. This implies a transient increase of luteotrophin secre- tion. Significantly this occurs before the re- lease of LH. It is this writer's opinion that gestagen from such sources is not essential for the induction of ovulation but that it does facilitate the action of estrogen. 520 PHYSIOLOGY OF GONADS C. ROLE OF THE NERVOUS SYSTEM IN OVULATION Historically, the fact of neural control of reflex ovulation has been recognized in the ral)bit for many years. The comparable role of the nervous system in spontaneous ovu- lation, on the other hand, has more recently become apparent. It now seems justifiable and useful to postulate in the hypothalamus of reflex ovulators and spontaneous ovu- lators alike the existence of a mechanism that is peculiarly concerned with release of ovulating hormone. Whether it consists in a discrete anatomic entity is immaterial for the present. The suggestion has been made that the outstanding difference between reflex and sjiontaneous ovulation may be in the kinds of afferent impulses that most readily ex- cite the hypothalamus (Sawyer, Everett and Markee, 1949). The difference is not absolute, for spontaneous ovulation has been induced in rabbits by estrogen-progesterone injection (see p. 519) and reflex ovulation has been demonstrated under special cir- cumstances in rats (Dempsey and Searles, 1943; Everett, 1952a) and cattle (Marion, Smith, Wiley and Barrett, 1950). The ran- dom distribution of reflex ovulators and spontaneous ovulators among mammalian orders becomes more understandable if one assumes that dual controls are widely rep- resented and that special adaptations favor one or the other in given species. The ovulation reflex in rabbits is appar- ently initiated by afferent impulses of mul- tiple origin, among them not only impulses from the genitalia, but also propriocejUive impulses from muscles that are utilized in coitus. Brooks (1937, 1938) found that, although the sacral segments of the spinal cord and the abdominal sympathetic chains could be eliminated without jM'eventing ovu- lation, the luml)ai' cord must remain. Only by paralysis of the lower half of the body so that the female could not take pai't in coitus was ovulation pi'cvcntfMl. The neo- cortex could be removed, together with the olfactory bulbs, labyrinths, auditory ap- paratus and eyes without impairing the ovulation response. Even after complete de- cortication, ovulation followecl coitus in 1 out of 3 j'abbits. It must l)e admitted, how- ever, that, although various parts of the nervous system may thus be eliminated without changing the end result, some of them may normally play a considerable role. With little cjuestion, direct stimuli from the genitalia play a part in the natural reflex. Under certain experimental condi- tions detectable electrical activity in the rabbit rhinencephalon is associated with the induction of ovulation (Sawyer, 1955). Electrical stimulation of the amygdala will induce ovulation in rabbits and cats (Koi- kegami, Yamada and Usei, 1954; Shealy and Peele, 1957 ) . In rats, Davis (1939) found that re- moval of the neocortex had no effect on the estrous cycle and ovulation. Removal of portions or of the entire sympathetic chains of rats likewise did not interfere with ovu- lation (Bacq, 1932). Bunn and Everett (1957) reported ovulation in constant- estrous rats after electrical stimulation of the amygdala. The importance of the dorsal thalamus is unknown. The reticular activating system has been implicated as a component of the ovulation mechanism (Sawyer, 1958), but the manner of its contribution is not clear. There is little cjuestion, on the other hand, of the indispensability of the hypothalamus and its neurovascular connection to the adenoliyi:)ophysis through the median emi- nence and the hypophyseal portal veins. Although the observation by ]\Iarshall and Verney (1936) that ovulation can be induced by passing an electric current through the heads of estrous rabbits hardl}^ limited the effect to the hypothalamus it- self, it was later shown that more localized electrical stimuli api)lied to certain hypo- thalamic regions are consistently effective (preoptic area, Haterius, 1937; Christian. 1956; posterior hypothalamus or tuber cinereum, Harris, 1937, 19481); tuber ciner- eum or adjacent hypothalamic areas, Markee, Sawyer anirHollinshead. 1946; medial hypothalamus fi'om ])i-eo])tic area to mammillai'V bodies. Kui'otsiu Kurachi and Ban, 195o"; Kuiotsu, Kurachi, Tabaya- >hi and Ban, 1952). Ahliougli liypotliahimic lesions. l)oth natural and expeiimental. hax'c frecjuently been reported to interfere with normal MAMMALIAN REPRODUCTIVE CYCLE sex function (.see Harris. 1948a, 1955, for references), the majority of these reports do not api^ly to the question at issue — con- trol of ovulation. When ovarian atrophy occurs, as it frequently did in these cases, it reflects a profound depression of gonado- troi)hin secretion and absence of competent follicles. However, Dey, Fisher, Berry and Ranson (1940) and Dey (1941, 1943) 'found in guinea pigs that gross bilateral electro- lytic lesions placed in the rostral hypo- thalamus resulted in persistent follicles with continuous estrogen secretion. Similar re- sults were obtained in rats by Hillarp (1949) when small bilateral electrolytic le- sions were placed in the anterior hypothal- amic area near the paraventricular nuclei or between this region and the median emi- nence. Greer (1953) reported continuous estrus in rats after placing certain small lesions in the ventromedial nucleus, pro- vided they were bilateral. There was no correlation with obesity. There are at least four significant points in common among these several ablation experiments. ( 1 ) The effective lesions were rost rally placed and either were limited to or included the medial group of nuclei. (2) The tuber cinereum, median eminence, and stalk connection to the hypophysis were intact. (3) Although development of competent follicles was not evidently impaired, estro- gen secretion became continuous instead of cyclic. (4) The proper impetus for release of ovulating hormone from the hypophysis was absent. It would be most instructive to learn whether ovulation can be invoked in such animals by reflex stimulation or by direct electrical stimulation of the tuber. AUoiteau and Courvoisier (1953) reported that rats in constant estrus as a result of hypothalamic lesions did not undergo pseudopregnancy after stimulation of the cervix uteri. This observation, confirmed by Greer (1953), could be construed as in- direct evidence of failure of reflex ovulation, for Greer regularly obtained pseudopreg- nancy by cervical stimulation, once corpora lutea had been formed by other means. Other findings by Greer are important because of their bearing on the location and character of a presumptive ovulation center. Althougli the onset of persistent estrus after making the lesions was sometimes almost immediate (following a brief anestrous in- terval), in other cases it was preceded by several apparently normal cycles. In any event, once the condition had become estab- lished, the daily injection of small amounts of progesterone brought about the recur- rence of cycles and corpus luteum forma- tion. In about half of the cases these cycles continued for awhile after withdrawal of treatment, whereas in the remainder there was a prompt return to persistent estrus. Essentially the same results were reported by AUoiteau (1954), and the observations suggest that the areas involved in such le- sions may be of only secondary importance. The use of radioactive phosphorus for estimating energy exchange in tissues af- fords a different approach to the problem of neural control of ovulation (Borell, West- man and Orstrom, 1947, 1948). This method has the virtue that the experimental subject remains undamaged until injection of P'*- compounds 30 minutes before the end of the experiment. In rabbits there is a marked increase in phosphorus turn-over in the tuber cinereum within 2 minutes post coitum, and continuing for about an hour thereafter (Table 8.1). The adenohypoi)h- ysis shows increasing activity during the first 10 minutes which reaches a peak at about 1 hour and then regresses somewhat, although it remains relatively high for 24 hours. Response of the ovary to gonado- troi:)hin release is marked by a rapid rise during the second half-hour and another pronounced increase near the time of ovu- lation. In rats, at various stages of the estrous cycle, phosphorus exchange in both tuber cinereum and adenohypophysis is maximal during proestrum. In the ovary high values were reported during diestrum and proestrum, somewhat lower values dur- ing estrus and metestrum. Possibly correlated with the above in- formation is the observation (Gitsch, 1952b) that in rats the acetylcholine (ACh) content of the tuberal region becomes ele- vated during proestrum and estrus. It is said that ACh synthesis requires high energy phosphate (see Bain, 1952). Further investigation by Gitsch (1952a) and Gitscl: and Reitinger (1953) revealed that ACh TABLE 8.1 Sequence of events in rabbit ovulation Time Post Coitum Central Nervous System Hypophysis Ovary Circulating Blood <30 sec. <2 mill. 10 mill. 30 mill. 60 mill. 75-90 mill. 13^-2 hrs. 3-5 hrs. 6-7 hrs. 7-8 hrs. 9-11 hrs. Barbiturate-sensi- tive and atropine- sensitive mecha- nisms^ t Phosphorylation in tuber cine- reum^ t Phosphorylation in tuber ciner- reum- t Phosphoryhition in tuber cine- reum^ t Phosphorylation in tuber cine- reum- t Phosphoryhition^ Release of LH ca. 20 per cent. 6 Hy- pophysectomy prevents ovula- tion^' 12 Release of LH now- sufficient for ovul- ation."' 12 Phos- phorylation at peak^ I Phosphor\iatioii i Animal may be bled and transfused without prevent - ! ing ovulationi2 folliculi. in egg f Liquor Tetrad.' nuclei* Cholesterol deple- tion in interstitial gland. ^ Egg nu- cleus migrates, membrane dis- solves.** • " Prom- inent corona* Liquor folliculi in- creasingly vis- cous* First polar hotly'* Marked .swelling of follicles. Thecal hypertrophy,! ■ i° hyperemia OvuL.^Tion." t Phosphoryla- tion2 Bleeding and trans- fusion now prevent ovulationi2 Progesterone detect- able ^ Increased estrogen (endometrial hj-- peremia)' ' Asdell, 1946. 2 Borell, Westman and Orstrom, 1947. 3 Claesson and Hillarp, 1947a. " Fee and Parkes, 1929. '^ Forbes, 1953. « Hill, 1934. ^ Sawyer, Markee and Hollinshcad, 1947. * Pincus and Enzmann, 1935. ^ Sawyer and associates, 1947, 1949, 1950. 1" Walton and Hammond, 192S. " Waterman, 1943. '2 Weslmaii and .lacohsohn, 1936. 522 MAMMALIAN REPRODUCTIVE CYCLE 523 in the rat hypothalamus is increased also by administration of estrogen or by castra- tion, conditions that similarly increase lihosphorus exchange (Borell and Westman, 1949). The ACh content is depressed during pregnancy or when the rat has been in- jected with progesterone. It is also lowered by Pentothal anesthesia, a matter of interest in relation to the fact that the barbiturates suppress ovulation (see p. 526). The location and measurement of activity in discrete nuclei and pathways are largely in the future, although a beginning has been made in the rabbit, cat, rat, and mouse. Sawyer (1955) found in rabbits, after the combined administration of pentobarbital intravenously and histamine by way of the 3rd ventricle, that there was associated with induction of ovulation a characteristic change in intrinsic electrical activity of the rhinencephalon, extending into the preoptic area. If the olfactory tracts were cut, how- ever, this activity could not be elicited and ovulation failed. According to Porter, Cav- anaugh and Sawyer (1954), vaginal stimu- lation of estrous cats caused altered electri- cal activity in two hypothalamic regions: (1 ) in the lateral hypothalamic area at the anterior tuberal level during stimulation and for 15 to 45 seconds afterward; and (2) in the anterior hypothalamic area near the medial forebrain bundle, where response was delayed as much as 5 n:iinutes after stimulation. According to a i)reliminary account (Critchlow and Sawyer, 1955) in curarized, proestrous rats, there were i)e- riods lasting approximately 20 minutes in the midafternoon, during which altered electrical activity appeared differentially in the preoptic area or anterior hypothalamus. Another approach to localization has been described by Hertl (1952, 1955). On the pro])Osition that increased function of par- ticular cells is reflected by increased volume of their nuclei, cell nuclear volumes were measured in hypothalamic nuclei of female mice at different stages of the estrous cycle. During proestrum and estrus there was said to be a functional edema in hypothal- amic nucleus 20 of Griinthal (possibly the pars posterior of the ventromedial nucleus of Krieg) and to lesser extent in nucleus 16 (Nucl. arcuatus). 1. The Ilypophi/seal Portal Veins and the Chemotransmitter Hypothesis As noted elsewhere, hypothalamic control of the jiars distalis is probably mediated by the hypophyseal portal circulation. Evi- dence for this has been especially con- vincing with respect to control of ovulation, although indications are that other phases of the cycle are also regulated by this means. Pertinent data from numerous trans- plantation and stalk-section experiments may be summarized by the following state- ment. Aside from a questionable grafting experiment (2 rats) reported by May (1937), in no case has ovulation or luteini- zation been reported in the absence of vas- cular linkage of the pars distalis with the median eminence; on the other hand, ovu- latory cycles have often been cjuickly re- stored when the gland has been revascular- ized by the portal vessels (see especially, Harris, 1950a; Harris and Jacobsohn, 1952; Nikitovitch-Winer and Everett, 1957, 1958b). Although the importance of local vasom- otor regulation in the stalk vessels remains to be evaluated (Green, 1951), there is ex- tensive support for the hypothesis that ovu- latory release of gonadotrophin is invoked by a chemotransmitter (Harris, 1948a, 1955). If one accei)ts the prevailing opinion that nerve fibers entering the pars distalis are too few to account for its secretomotor control and that the flow of blood in the hyi^ophyseal portal vessels is toward the gland (Wislocki and King, 1936; Green, 1947; Green and Harris, 1947, 1949; Barr- nett and Greep, 1951 ; Landsmeer, 1951 ; ]McConnell, 1953; Xuereb, Prichard and Daniel, 1954; Worthington, 1955), the plausibility of the chemotransmitter hy- pothesis becomes inescapable.^ Evidence that the transmitter may l)e ^ For a dissenting view, see Zuckerman (1952). Reference should also be made to the hypothesis formulated by Spatz (1951) and associates (see Nowakowski, 1950, 1952). They postulated that a descending pathway in the spinal cord is the connecting link between hypothalamus and ova- ries. With respect to ovulation, this is clearly denied by the fact that local stimulation of the hypotlialamus provokes ovulation in rabbits in which the thoracic spinal cord has been trans- sected (Christian, Markee and Markee, 1955). 524 PHYSIOLOGY OF GONADS adrenergic was presented by Markee, Saw- yer and Hollinshead (1948), who provoked ovulation in rabbits by instilling epineph- rine directly into the pars distalis. Detailed experiments supporting this w^ere fully re- viewed by Markee, Everett and Sawyer (1952). In discussion following that paper, Sawyer reported the induction of ovulation in rabbits by the injection into the third ventricle of either epinephrine or norepi- nephrine, and suggested that the latter is "more closely related to the natural medi- ator than is epinephrine." Donovan and Harris (1956), from studies in which the rabbit hypophysis was slowdy infused in situ with solutions of epinephrine or norepi- nephrine, concluded that neither substance is the agent in question, and that the posi- tive results of Markee, Sawyer and Hollins- head (1948) were the effects of low pH and not of the drugs per se. Proof of the negative is elusive, however, and one must note that Donovan and Harris did not meet the con- ditions of timing and drug concentration that obtained in the earlier work. Intravenous injection of Dibenamine or its congener, SKF-501,-^ will usually prevent ovulation in rabl)its when injection is com- pleted within 1 minute after coitus (Sawyer and associates, 1947-50). On the other hand, when injection is delayed until 3 min- utes or later, ovulation is unaffected. The nonadrenergic hydrolysis product of Diben- amine, 2-dibenzylaminoethanol, does not have the blocking action, although its cen- tral excitatory powers are much like those of the parent substance. The failure of blockade by Dibenamine, if injection is withheld for 3 minutes, demonstrates that the drug does not interfere with the actual discharge of ovulating hormone into the l)lood stream, for that process recjuires about an hour (Fee and Parkes, 1929; West- man and Jacobsohn, 1936). The Dibena- mine-sensitive mechanism thus serves as a trigger, the gland being adequately stimu- lated within 1 01' 2 minutes post coitum. This estin^ate is in remarkal)le agreement with the earlier mentioned obscMA'ations on •'■' Dibenamine is iV,iV-dibenzyl-/:i-cliloioetliy la- mine. SKF-501 is A'-(9-fluorenyl)-.V-ethyl-/i-chlor()- ethylamine hydrochlorifle. Banthine is /i-dictliyl- aminuc'tliyl-.\antliene-9-cai'l)Oxvlak' niclliohroiniilc l)hosphorus exchange in the tuber cinereum and hypoi)hysis (p. 521, and Table 8.1 1. A mechanism that is subject to block- ade by atropine or Banthine^ evidently pre- cedes the Dibenamine-sensitive process — temjDorally if not anatomically. To ac- complish blockade in rabbits, these anti- cholinergic drugs must be injected intra- venously within about 30 seconds after coitus (Sawyer and associates, 1949-1951). It should be recalled that Foster, Haney and Hisaw (1934) reported failure of ovu- lation in several rabbits treated with small amounts of atropine before mating. ]\Iake- peace (1938), however, was unable to con- firm the effect with somewhat larger doses and the former observation was forgotten. A seemingly crucial experiment devised by Sawyer gives conclusive evidence that the atropine-sensitive process is antecedent to the Dibenamine-sensitive one. It was based on two facts: (1) intravenous injec- tion of nearly lethal doses of epinephrine does not induce ovulation in estrous rabbits, and (2) atropine protects rabbits against fatal pulmonary edema after injection of large amounts of epinephrine. In rabbits protected by atropine in dosage that was also sufficient to block the ovulation reflex, the injection of twice-lethal doses of epi- nephrine caused ovulation or significant de- grees of follicle maturation in 5 of 7 cases. These effects were not found in rabbits pro- tected by Dibenamine. Supporting evidence was adduced by Christian (1956i who found that atropine would not prevent ovulation in response to electi'ical stimula- tion of the medial preoptic area or adjacent parts of the hypothalanuis, whereas in a sig- nificant number of such rabliits ovulation was blocked by SKF-501. Extension of the blocking experiments to the rat, as an example of a spontaneous ovu- lator, disclosed that in this species also ovu- lation can be blocked by Dibenamine, SKF- 501, atropine, and Banthine, when the in- jections ai'c appropriately timed with re- spect to the stage- of the cycle and time of day (Sawyer, Everett and Markee, 1949; Everett, Sawyer and Markee, 1949; Everett and Sawyer, 1949, 1950, 1953: see \). 526). Furthermore, blockade of ])oth estrogen- imhiccd and pi'ogcstci-oiic-induced ovuhition MAMMALIAN REPRODUCTIVE CYCLE 525 was aceomi)lished with cither Dibonamine or atropine. Neither agent, however, pre- vented ovulation after injection of sheep liypophyseal LH. A report by Hansel and Trimberger (1951) stated that in cattle a significant delay of ovulation (as great as 72 hours) followed atropine administration. In control experiments the simultaneous injection of atropine and human chorionic gonadotrophin was followed by ovulation slightly earlier than the normally expected time. Treatments were begun 1 to 5 hours after the onset of estrus. This work was con- firmed and extended by Hough, Beardon and Hansel (1955). In the hen, blockade of ovulation, normal or induced by proges- terone, has been reported after administra- tion of Dibenamine, Dibenzyline, SKF-501 or atropine (Zarrow and Bastian, 1953; van Tienhoven, 1955). According to van Tienhoven, the drugs did not interfere with the ovulating action of extrinsic gonado- trophin. It is important that the same drugs will block ovulation in lioth rabbits and rats (Table 8.2) . Of ec^ual significance is the fact that several agents that are ineffective in rabbits are also ineffective in rats (notably 2-dibenzylaminoethanol, the imidazoline adrenolytic drugs, and the ganglion blocking agents). These considerations are inter- jireted to mean that spontaneous ovulation is invoked by neurohumoral mechanisms that are very like those in the reflex ovula- tion of rabbits. The suggestion that the l)locking effects might result from nonspecific stress, causing the hypophysis to be so actively secreting ACTH that gonadotroiihin secretion is in- terfered with (Dordoni and Timiras, 1952), is clearly denied by several facts. ( 1 ) In the rabbit studies, none of the various agents prevented ovulation when injected more than a minute post coitum. (2) In one study (Sawyer, Markee and Everett, 1950b) ovulation was actually induced by the intra- venous injection of "lethal" doses of epi- nephrine when the animals WTre protected by atropine. (3) In rats ovulation is un- affected by massive intravenous doses of either the imidazoline drugs or 2-dibenzyl- amionethanol in amounts known to be stressing (Sawyer and Parkerson, 1953). TABLE 8.2 Pharmacologic Agents and Blockade of Ovulation Antiadrenergics /3-Haloalkylamines Dibenamine SKF-501 Dibenzyline ImidazoHnes Priscoline Regitine Yohimbine Anticholinergics Atropine Banthine Antihistaminics Neo-antergan Ganglion blockers Tetraethvlammonium. . SC-1950/ Barbiturates Nembutal Dial Ipral Amvtal Barbital Phenobarl)ital Prominal Others Morphine Procaine, locally near tuber Procaine, systemically . Chlorpromazine Reserpine Ether 2,4-Dimtrophenol Rabbit Rat Cow Bi Bi Bi Bi 01 01 0^ 01 ■?4. 5 Bi BI B6 Bi Bi 01 01 01 01 Bi Bi Bi Bi Bi B' Bi 8 B9 BIO Bii 01 B12 B13 B14 B16 B3 I Sawyer and associates, 1947-1951; Everett and associates, 1949-1950; Christian, 1956; and see present text. ^ van Tienhoven, 1955; van Tienhoven, Nal- bandov and Norton, 1954. 3 Zarrow and Bastian, 1953. •• Fugo and Gross, 1942. 5 Sulman and Black, 1945. ^Hansel and Triml)erger, 1951; Hough, Bear- don and Hansel, 1955. ' Fraps and Case, 1953. * Doring and Goz, 1952. » Westman, 1947. 1" Barraclough and Sawyer, 1955. II Westman and Jacol)8ohn, 1942. 1- Barraclough, 1956. 13 Barraclough, 1955. " Unpublished. Temporary, during deep anes- thesia. 1^ Unpublished. EDso : 25 mg. per kg. subcu- taneously. Key: B = Blockade. 0 = No blockade. 1 = Ovulation induced by the drug. 526 PHYSIOLOGY OF GONADS Nor is it influenced by severe trauma, heat, cold, or formalin injection coincident with the known "critical period" (Everett, un- published). 2. Central Depressants and Ovulation Reported evidence of a blocking action of barbiturates on ovulation traces back to experiments by Westman (1947) who in- jected female rats with Prominal twice daily for 3 weeks. Approximately 30 per cent of these rats experienced prolonged vaginal estrus and had ovaries containing only follicles at the end of the experiment. Almost identical results were reported by Doring and Goz (1952) when rats were treated daily with phenobarbital. The agree- ment is not unexpected in view of the fact that in the body Prominal is quickly de- methylated to phenobarbital. It was shown by Everett and Sawyer (1950) that when administration of barbiturates to rats is critically timed with respect to stage of the cycle and the time of day, blockade of ovu- lation can be accomplished at will in short- term experiments (Fig. 8.10). Chronic administration introduces considerable un- certainty for reasons that are not yet clear (Everett, 1952b). In the rabbit, the rapid intravenous injection of pentobarbital or Pentothal, within as short a time as 12 seconds after coitus, generally failed to block ovulation (Sawyer, Everett and Markee, 1950) . However, it was later shown that barbiturate anesthesia will prevent the ovulation that is otherwise caused in the estrogen-primed rabbit by mechanical stimulation of the vagina, the anesthesia t)eing induced in advance of the stimulation (unjuiblished i. Other central dcpi-es^ants reported to block ovulation in the rat are morj^hine, reserj^ine, chlorpromazine (Barraclough and Sawyer, 1955; Barraclough, 1955, 1956), and even meprobamate acting synergis- tically with an anticholinergic drug (Gitscli, 1958). Special interest attaches to the mori)hine work, in that anicnoi'i lica and sterility often a('C()in|)any moiphinc addic- tion in the human female. Related studies (Sawyer, Critchlow and Barraclough, 1955), in which recordings were made of electi'ical acti\ity in various regions in the bi-ain. demonstrated in rats that morphine acts nmch like the barbiturates in depressing activity in the reticular activating system. The effect was also shown by atropine, in doses that would block ovulation. The in- ference is that all three agents block by striking at the same central elements of the LH-release apparatus. An interesting peculiarity of domestic hens with respect to barbiturates was en- countered by Fraps and Case (1953), who noted that pentobarbital induces ovulation jirematurely, and that pentobarbital and progesterone supplement each other in this capacity. Although these developments may represent pharmacologic curiosities limited to the bird, the possibility should be seri- ously considered that similar effects may occur in other animals. In fact, pento- barbital in rabbits facilitates the release of hypophyseal gonadotrophin in response to intraventricular injection of histamine, seemingly by an effect in the rhinencephalon (Sawyer^ 1955). 3. The Central Xervous System as a Timing Mechanism for Oi'ulation In the rat and the hen and probably many other species the pro-ovulatory excitation of the hypophysis is dejiendent in large meas- ure on time of day. In the rat, the blocking agents have served to delimit a critical period on the day of proestrum, before which ovulation can be blocked and after which it will occur in sjiite of injection of the blocking agent. Under controlled illumination for 14 hours daily, this critical period extends from about 2 P.M. to 4 P.M. Administration of either atroi^ine or i^entobarbital at 2 p.m. consistently blocks ovulation (Fig. 8.10), whereas injections later in the period are progressively less eff"ecti\-e ( l']\-erett and Sawyer, 1950, 1953; Everett, 1956b). Such l)redictability of the hour of pituitary acti- vation is, in itself, evidence of a relationship between this event and diunial physiologic I'liythnis. Furthei' e\-i(lence is seen in the seciuelae of pentobarbital injection (Fig. 8.11). Rep- etition at 2 P.M. on successive days re- sults in a follicular cycle and prolonged vaginal estrus with eventual atresia of all MAMMALIAN REPRODUCTIVE CYCLE <- — 24hr.— > 527 DAY NIGHT 0 Fig. 8.10. Deinoni^lration of 24-lioiir penodu-ity in the luteinizing hormone-release appa- ratus of female rats (Vanderbilt strain, 4-day cycle, controlled lighting: 14 hours per day). Schematic representations of the normal cycle (A) and of characteristic results of different regimes of Nembutal treatment (B to F). Vaginal stages indicated by Roman numerals over each time scale; symbols above these show the corresponding follicle and corpus luteum stages. The device marked l prolonged the functional life of the coi'pus luteuni in nonpregnant women. Lyon rejjorted such prolongation using lactogen alone. The Squibl) lactogen was also used by Moore and Nalbandov (1955) in prolonging the luteal phase of the cycle in the ewe. As in the human experi- ments, howe\-er. one would like to know whether lactogen is capable of initial stimu- l.'iiioii of secretory activity of corpora lutea and of maintaining their function in the ab- MAMMALIAN REPRODUCTIVE CYCLE 531 sence of the hypophysis. There is no evi- dence for or against the lactogenic liormone in this capacity, except in rats. Estrogens have direct hiteotrophic action in the rabbit (Robson, 1937, 1938, 1947). The effect does not depend on the hypophy- sis and has been produced by impLantation of estrogen crystals within corpora liitea (Hammond, Jr., and Robson, 1951; Ham- mond, Jr., 1952). Westman (1934) had ear- lier shown that operative reduction of ovarian stroma in pseudopregnant rabbits results in corpus luteum regression and that this can be prevented by administration of estrogen. Corpora lutea induced by gonado- trophin injection or by mating, as the case may be, require the presence of the hypoph- ysis for their continued function ( Smith and White, 1931; Westman and Jacobsohn, 1936). Theoretically, then, in rabbits the hypophysis liberates FSH and LH which act on the interstitial tissue to cause estrogen secretion. This in turn stimulates the cor- pora lutea to secrete progesterone. The effect of estrogen on the corpora lutea of rats is largely indirect and requires the presence of the hypophysis. Massive dosage with estrogen beginning soon after ovula- tion results in the enlargement of the cor- pora lutea and the production of sufficient amounts of progesterone to mucify the vagi- nal mucosa (Selye, Collip and Thomson, 1935; Wolfe, 1935; Desclin, 1935; Merckel and Nelson, 1940). In fact, a single injec- tion of 50 /Ag. estradiol bcnzoate on the day after ovulation is sufficient to cause pseudo- pregnancy. These effects are now judged to be the result of induced liberation of hy- pophyseal luteotrophin. vSimilar effects have been reported after administration of an- drogens (McKeown and Zuckerman, 1937; Wolfe and Hamilton, 1937; Freed, Greenhill and Soskin, 1938; Laqueur and Fluhmann, 1942). Desclin (1949b) stated that in hypophy- sectomized rats the administration of estro- gen augments the hiteotrophic action of lactogen, producing functional corpora lu- tea in the presence of subthreshold doses of the latter hormone. A physiologic synergism of the two substances has thus been indi- cated. Mayer (1951) suggested that this may explain the stimulation of corpora lutea of lactation which follows estrogen treat- ment in this species. Greep and Chester Jones (1950) postulated that estrogen fa- vors corpus luteum function in the rat by causing the luteal cells to produce choles- terol as a precursor of progesterone. Their actual data, however, indicate that the in- crease of visible cholesterol after estrogen treatment was confined to the interstitial tissue. Factors responsible for cholesterol storage and mobilization in corpora lutea of the rat were analyzed by Everett (1947). In hy- pophysectomized rats in which corpora lutea were maintained by lactogen the injection of pituitary LH induced the storage of cholesterol, but this effect did not occur in hypophysectomized rats in the absence of lactogen. It could be induced during preg- nancy or pseudopregnancy by estrogen if the hypophysis remained in place. Addition of an excess of lactogen prevented choles- terol storage. Lactogen thus tends to deplete cholesterol content of rat luteal tissue as ACTH tends to deplete adrenocortical cho- lesterol. 2. ''XonfunctionaV' Corpora Lutea In the short cycles of the rat, mouse, hamster, and so on, the corpora lutea are commonly said to be nonfunctional. The meaning of this statement, of course, is that they are incapable of supporting a decidual reaction (Long and Evans, 1922), or of lire- venting ovulation. They need not be totally inactive, however, to fail to cause these manifestations. Whereas daily injection of 1.5 mg. or more of progesterone into intact female rats will simulate pseudopregnancy and indefinitely delay ovulation (Selye, Browne and Collii^, 1936; Phillips, 1937), smaller amounts of 1.0 rag. or less are com- patible with the short cycle (Lahr and Riddle, 1936; Phillips, 1937; Everett, 1940a, b; and unpublished). In the absence of es- trogen in castrated females, daily injection of as little as 0.25 rag. progesterone will sup- port deciduomata (Velardo and Hisaw, 1951). Very small amounts of estrogen aug- ment this action of progesterone (Rothchild, Meyer and Spielman, 1940) but somewhat larger amounts are inhibitory unless the progesterone dose is proportionately in- creased (Velardo and Hisaw, 1951). In the intact animal the progestational effects of 53^ PHYSIOLOGY OF GONADS less than 1.0 mg. progesterone would be in- hibited by the periodic rise in estrogen secre- tion. Evidence that some rats, but not all, ac- tually experience low-grade corpus luteum activity during the short cycle was furnished by Everett (1945). In the comparison of ovaries from females of two strains of rats, it was noted in the supposedly normal Van- derbilt strain that on the two days im- mediately following ovulation the corpora lutea of the next youngest generation con- tained a great quantity of cholesterol, giv- ing a strong Schultz reaction. By contrast, comparable corpora lutea of the DA strain were usually free of visible lipid in Sudan preparations or the Schultz test. Adminis- tration of small amounts of lactogen (luteo- trophin I during the cycle preceding the current one, amounts inadequate to cause l)seudopregnancy, resulted in the rich depo- sition of cholesterol in these otherwise lipid- free corpora lutea. The conclusion was reached that the corpora lutea of the Van- derbilt rat must be slightly active during the short cycle and those of the DA rat less so, if at all. This would easily explain the relative indifference of the Vanderbilt rat to contin- uous light and the ease with which persistent cstrus could be induced in the DA rat by such treatment (Everett, 1942a, b). In fact, the low dosages of lactogen mentioned sub- stituted for progesterone treatment in main- taining regular cycles in persistent-estrous rats of the DA strain (Everett, 1944b). Sig- nificantly, the treatment was effective in only those animals in which a set of corpora lutea had been induced by other means at the beginning of the experiment. To be correlated with the above indica- tions of low-grade function during tlie short cycle, is the finding that corpora lutea of the Vanderl)ilt rat retain full responsiveness to luteotrophin throughout most of the di- estrous interval (Nikitovitch-Winer and Everett, 1958a). Responsiveness diminishes near the onset of i:)roestrum. Once the rat has entered proestrum these older corpora lutea are not capable of sustained function. The loss is not a function of time per se, but of stage of the cycle. .\. I'.^KTI)OPRE(;\.\XrY The terms coi-pu,^ hitcuni of ()^•ulation and corpus luteum of pscudojji'cgnancy are com- monly u.sed to differentiate the luteal bodies occurring during the normal cycles from those found during some unusually long period of luteal activity. However, the terms deny the fundamental similarity of the luteal phase in the cycles of such ani- mals as the guinea pig and the luteal phase induced by sterile mating or its equivalent in animals like the rat. In the unmated bitch the spontaneous luteal phase of the cycle is commonly called pseudopregnancy, yet it is equally common to say that the guinea pig does not experience pseudopregnancy. The truth is that the luteal phase of the canine cycle is simply longer than the luteal phase in the guinea pig and may be marked by a period of lactation near its close. In the present discussion, the expression pseu- dopregnancy will be equivalent to saying the luteal phase of the infertile cycle. Under experimental conditions it will refer to any period of sustained luteal function similar to that of the normal progestational state. Wherever appropriate, the distinction will be made between a pseudopregnancy that is spontaneous and one that is induced. In most of the familiar animals that ovu- late spontanously corpus luteum function also begins spontaneously and continues for at least several days after ovulation. With respect to the rabbit, cat, and ferret, it is often said that pseudopregnancy is invoked by sterile copulation, whereas strictly speak- ing it is only ovulation and corpus luteum formation which are invoked. The pseudo- pregnancy then follows automatically. This interpretation seems appropriate, inasmuch as in all three species the formation of corpora lutea by l)rief treatment with hy- pophyseal or chorionic gonadotrophin is fol- lowed by long periods of progesterone secre- tion which can hardly be the direct effect of the injected substances (Hill and Parkes, 1930a, b; Foster and Hisaw, 1935; van Dyke and Li, 1938). Quite different is the pseudopregnancy of the rat, mouse, and hauistei', in \\liich progestational activity is invoked by stinudation of the cervix uteri, l^verett (1952a) described the ex])eriiuental dissociation in rats of the o^•^llati()n and luteotr()])hic mechanisms. respectively. When o\-uhition is blocked (by pentobarbi- tal) in the cycle dui'ing which controlled uiatinu occurs, pseudopi-egnancy begins MAMMALIAN REPRODUCTIVE CYCLE 533 A - A. A A, A / NB Np X X X A Q- / \ A s A A s / NB NB (X) X C /^ A Fig. 8.12. Experimental dissociation in rats of the ovulation mechanism and that causing pseudopregnancy. A. Control cycles for comparison with B and C . Points on base line represent diestrum, on ascending lines proestrum, on highest level full vaginal estrum. X, ovulation. B. Blockade with Nembutal {'NB) on day of proestrum and following day (see Fig. 8.11E'). Ovulation during third night. C. Same basic procedure as B, but with copulation during first night (M). Ovulation usually failing in this cycle (contrast with B) . Corpora lutea formed after spontaneous ovulation in second cycle regularly become functional with- out further stimulation : the wavy line represents pseudopregnancy. The early copulation has introduced some change in the animal such that this pseudopregnancy "spontaneously" follows ovulation as in the standard mammalian cycle. (From J. W. Everett, Ciba Founda- tion Collofiuia Endocrinol.. 4, 172. 1952.) "spontaneously" a]ter the next cyclic estrus (Fig. 8.12). Dissociation of the two mecha- nisms is expressed in another way by cer- tain Mustelidae, e.g., the mink and marten. Ovulation in these forms is invoked by mating, whereas corpus luteum activation awaits appropriate environmental condi- tions, i.e., temperature and length of daily illumination (Pearson and Enders, 1944; Hansson, 1947). In the mink, during the pe- riod of relative luteal inactivity that fol- lows mating early in the season, recurrent estrus continues. If reraating takes place at an interval of 6 days or more, new ovula- tions are induced (Hansson, 1947). Matings late in the season are immediately followed by luteal activity. The pseudopregnant cy- cles of a representative series of mammals are much alike when conditions appropri- ate to the respective species are applied (Fig. 8.1). 1. Duration of Psciidopregndncn The length of time that corpora lutea re- main functional in the pseudopregnant cy- cle is thought to be relatively uniform in the great majority of mammals, usually about 10 to 15 days. Rarely it is shorter, e.g., the hamster, 7 days, although usually 9 to 10 days (Asdell, 1946). At the other extreme, the corpora lutea remain functional for periods corresponding to the duration of pregnancy, as in the ferret, 5 to 6 weeks. In fact, corpus luteum function lasting over a month is usual in the other two carnivores for which information is at hand: cat, 30 to 44 days (Foster and Hisaw, 1935) ; and dog, 30 days or more (Evans and Cole, 1931). These figures are only approximations, however, as the criteria on which they are based differ. In the rat, in which pseudo- pregnancy is said to last 12 to 14 days, its termination is taken to be the onset of the next estrus, whereas the corpora lutea must have undergone a decline of activity 2 or 3 days earlier (Everett, 1948). The decline is probably not abrupt, inasmuch as the vagi- nal smear during the next estrus is very strongly mucified and, as mentioned earlier (p. 519), enough progesterone seems to be secreted by the waning corpora lutea to fa- cilitate ovulation. Morphologic criteria are often employed as indicators of corpus lu- teum regression: characteristically, fatty vacuolation of luteal cells, decrease in size of the individual cells or of the entire corpus 534 PHYSIOLOGY OF GONADS luteuni. and changes in the smusoidal pat- tern suggesting reduced circulation. Such changes are first observed in the guinea pig corpora lutea on about the 13th day of the cycle. One has the choice of taking this date as the end of the pseudopregnant phase or, alternatively, the date on which the first indications of estrus are noted. Either choice is arbitrary, but the former seems preferable as it suggests that progesterone secretion is diminishing and probably is no longer suf- ficient to maintain progestational changes in the uterus. In fact, regression of the endo- metrium sets in about a day earlier than frank degenerative changes in the corpora lutea. In the cat, according to van Dyke and l.i (1938) the corpora lutea 20 days after ovulation no longer secrete enough pro- gesterone to cause motor effects of epineph- rine in the myometrium, the so-called "epi- nephrine-reversal" effect, yet by histologic criteria corpus luteum regression is not ap- parent until 28 days or later (Liche, 1939; Foster and Hisaw, 1935). In the bitch the uterus begins regression 20 to 30 days after heat, l)ut the corpora lutea are said to re- main in good condition for a longer time (see Asdell, 1946, for references). Regres- sion is so gradual that anestrum is not reached until about 85 days. In the primates the beginning of menstruation offers a means of delimiting the luteal phase, inasmuch as menstruation in the ovulatory cycle reflects a marked reduction in corpus luteum func- tion. Nevertheless, this reduction probably occurs a few days before bleeding begins. The i^eak of pregnanediol excretion in women (Venning and Browne, 1937) and of plasma progesterone concentration in women and monkeys (Forbes, 1950; Bryans. 1951 ) is passed about midway between ovu- hition and menstruation. 2. Xciivdl Factors in Pseudoprciindiicji The importance of the nervous system in control of pseudopregnancy is well recog- nized in only the few species re])resented by the rat. .\ neural effect in the mink and similar Mustelidae is implied by the rela- tion of hiteal function to daily illumination, as mentioned earlier. Beyond that fact, how- ever, no information is available. Att(>ntion will therefore be directed largely to the rat. Not only sterile mating, but se\'eral other procedures involving neural stimulation will cause rats to become pseudopregnant. Stim- ulation of the cervix by mechanical means (Long and Evans, 1922) or electric shock (Shelesnyak, 1931) have become standard methods. In fact, Greep and Hisaw (1938) obtained pseudopregnancies after electrical stimulation during early diestrum, several days before ovulation. Pseudopregnancy is also invoked by continuous stimulation of the nipples for several days (Selye and Mc- Keown, 1934). According to Harris (1936) electric shock through the head is effective. His negative results with "spinal shock" are difficult to explain. From the description of position of the electrode it seems doubt- ful that the current passed through the cord itself, yet the sacral plexus must have been stimulated. Al)dominal sympathectomy or superior cervical ganglionectomy are said to diminish the numbers of animals responding to elec- trical or mechanical stimulation of the cer- vix (Vogt, 1931; Haterius, 1933; Friedgood and Bevin, 1941). On the other hand, there is no diminution of response to sterile copu- lation, which shows that the sympathetic chains are not essential. Ball (1934) em- phasized the quantitative aspects of the problem, noting that partial resection of the uterus or excision of the cervix diminished the response to sterile copulation, but only when "single-plug" matings were allowed. Multiple plugs gave pseudopregnancy in 100 per cent of the animals. It may be assumed that Vogt's (1933) negative results after hysterectomy resulted from single-jilug cop- ulations. Kollar (1953) re-opened the cjues- tion and found that pelvic nerve resection usually pi-evented the response to mating. It is not clear, howevei', whether multiple cop- ulations wnv the rule, although it seems that the I'outiiie procedure was to leave the male with the female overnight. His contention was that cervicectomy fails to abolish the resjionse completely because the vagina re- mains sensitive. Anesthesia with ether, nitrous oxide, or ethylene ( Mcyei', Leonard and Hisaw, 1929) diniiiiished the ficciuency of response to inecliaiiical stimulation of tlu> (•er\-ix. The statement was made, although without v\\- (len(c, that spinal anesthesia |)re\-ents pseu- dopreiinanev. Aeeoi'ding to A'ogt (1933), MAMMALIAN REPRODUCTIVP] CYCLE 535 local anesthesia of the vagina and cervix by cocaine or procaine prevented the response to sterile copulation in 23 of 35 rats. Removal of neocortex (Davis, 1939) did not interfere with the pseudopregnancy re- sponse to electrical stimulation of the cer- vix, although there was slight impairment of the response to mechanical stimulation or sterile mating (single-plug?). These results taken together have been construed to mean that induction of pseudo- pregnancy in rats involves a reflex similar to the ovulation reflex in rabbits. Certain con- siderations, however, raise the possibility that it may not be a "trigger" stimulus to the hypophysis as long believed (Everett, 1952a). In the first place, it seems doubtful that a trigger stimulus would result in con- tinuation of a new pattern of secretion (luteotrophin) for as long as 10 to 12 days. Furthermore, as noted above, cervical stim- ulation during the diestrum preceding ovu- lation may induce pseudopregnancy (Greep and Hisaw, 1938). Similarly, copulation during a cycle in which ovulation is blocked by pentobarbital results in a pseudoi)reg- nancv that begins after the next estrus (Fig. 8.12)^. We turn now to experiments concerned directly with the hypothalamo-pituitary system and pseudopregnancy. Westman and Jacobsohn (1938c) cut the pituitary stalks of estrous female rats. Barriers of metal foil were inserted to prevent regeneration of nerve fibers assumed to innervate the adeno- hypophysis. Regeneration of blood vessels must have been equally impossible. Controls were simply hypophysectomized. Two to 5 hours after the operations electrical stimu- lation of the cervix was administered to all animals. Pseudopregnancies were demon- strated by deciduomas in traumatized uteri of all the stalk-sectioned animals but not in the completely hypophysectomized rats. Desclin (1950) reported the maintenance of pseudopregnancy in estrogen-treated rats in which the only remaining hypophyseal tissue was in the form of grafts in the kid- ney. Whereas in hypophysectomized con- trols the estrogen treatment (stilbestrol pel- lets) produced cornification of the vagina and no enlargement of corpora lutea, the en- grafted-estrogenized rats developed muci- fied vaginas and enlarged corpora lutea as in intact rats similarly treated with estro- gen. Desclin concluded that the grafted hy- pophysis is able to respond to estrogen by liberating luteotrophin. It is now apparent, however, that neither cervical stimulation nor estrogen treatment is needed to invoke pseudopregnancy when the gland is isolated from the hypothalamus (Everett, 1954, 1956a; Nikitovitch-Winer and Everett, 1958a; Sanders and Rennels, 1957; Desclin, 1956a, b). When autografts of anterior hypophysis were made to the re- nal capsule or near the common carotid artery on the day after ovulation in adult cyclic rats, corpus luteum function was in- voked and maintained without any stimu- lus other than the operative procedures themselves. In short-term experiments in which the uteri were traumatized 4 days after the transplantation large deciduomas were regularly found at 8 days in the proven absence of residual hypophyseal tissue at the original site (Everett, 1954). Hypophy- sectomized controls were negative. In long- term experiments, continuing luteal function was demonstrated for as long as 3 months. Here the test for luteal function was vaginal mucification in the presence of massive amounts of estrogen administered during the final week of the experiment (Everett, 1956a). Controls in which the grafts or the ovaries were removed at the beginning of such estrogen treatment responded with full vaginal cornification. Follicular apparatus and interstitial tissue of the ovaries atro- phied promptly after the grafting opera- tions, whereas corpora lutea forming at that time were maintained for the long periods without histologic sign of deterioration. In later work, the decidual reaction was used as the test for luteal function, positive re- actions being elicited as late as 2 months after the transplantation. It was discovered that function of the graft is not influenced by stage of the cycle at which transplanta- tion is carried out and that grafts in the anterior chamber of the eye secrete luteo- trophin like those on the kidney (Nikito- vitch-Winer and Everett, 1958a). Trans- section of the pituitary stalk is sufficient in itself to provoke pseudopregnancy. If an effective barrier to vascular regeneration is inserted, the pseudopregnancy will heorve permanent, but otherwise it will l:i •■ i;!*' 536 PHYSIOLOGY OF GONADS usual length of time (Xikitovitch-Winer, 1957 j. In fact, there is reason to suspect that even a transient impairment of circu- lation in the median eminence-hypophj'seal linkage can be a sufficient impetus to pseu- dopregnancy. The experiments of Tauben- haus and Soskin ( 1941 ) in which applica- tion of an acetylcholine-prostigmine mixture to the exposed hypophysis was followed by pseudopregnancy, may well be explained in some such way. It thus seems that in the rat tlie depriva- tion of, or interference with, the normal con- nection of the pars distalis with the median eminence facilitates secretion of luteotro- phin and at the same time eliminates luteo- lytic mechanisms. It is significant that transplants of pars distalis into the pituitary capsule or to the immediate vicinity of the median eminence resume cyclic function (see page 512). The hypothalamus may have an inhibitory effect on luteotrophic ac- tivity of the pars distalis during the short cycle in this species. Greep and Chester Jones (1950) made the pertinent suggestion in attempting to explain the induction of pseudopregnancy by estrogen treatment, that the fundamental action of estrogen here is the suppression of FSH and LH, after which luteotrophic secretion may "proceed apace." There is necessarily some uncertainty concerning the amount of luteotrophin se- creted by the pars distalis when dissociated from the brain. Three sets of facts indicate that the output is larger than that in the cycling animal. (1) Sufficient gestagen is secreted by the engrafted animal to main- tain a ])regnancy (Everett, 1956c; Meyer, I'rasad and Cochrane, 1958) when the pi- tuitary is trans])lanted on the day after mating. (2) After stalk-transsection, in which there is less initial destruction of glandular parenchyma than in transplanta- tion experiments, the corpora lutea enlarge to a diameter like that usually found in late pregnancy rather than remaining like those of pseudopregnancy (Nikitovitch-Winer, 1957). (3) A single homotransplant of pars distalis placed subcutaneously in an other- wise normal female mouse will maintain a se(iueiicc of pseudopregnancies that override the short cycles expected of the animal's own hypophysis (Miihlbock and Boot, 19591. This is also true of rats (Nikitovitch- Winer, uni)ublislie(l). To avoid the conclu- sion that in such preparations the grafted gland is secreting luteotrophin at an in- creased rate, one must assume that the out- l)ut of this hormone from the intact gland is only slightly below threshold and that the graft adds just enough to make the total output effective. To explain the maintenance of pregnancy one might assume that the luteotrophin output and the resulting gesta- gen secretion are no greater than during the normal short cycle and that the formation of deciduomas takes place because of the deficiency of estrogen. The results of stalk- section, however, cannot easily be explained away. The weight of evidence, then, is in favor of increased luteotrophin secretion by hypophyses isolated from the brain by severance of the stalk or transplantation. Under certain experimental conditions it has seemed that to establish pseudopreg- nancy all that is necessary is to block out the forthcoming estrus and ovulation. Thus, in cycling rats, when the hypophysis is transplanted to the kidney as late as 60 hours after ovulation, the current diestrum transforms into a permanent pseudopreg- nancy supported by the existing set of cor- pora lutea (Nikitovitch-Winer and Everett, 1958a ». Similarly, injections of chlorproma- zine (Barraclough, 1957) or Pathilon (ditsch and Everett, 1958) begun during diestrum, may transform it into a pseudo- pregnancy by blocking out the expected estrus. The Miihlhock-Boot experiment men- tioned above furnishes an instructive model of the standard mammalian cycle, in which both ovulation and pseudopregnancy are spontaneous events. Given the extra pitui- tary tissue producing luteotrophin at a pre- sumably constant rate, with the output of the normal ghmd fluctuating (juantitatively and (jualitatively, the mouse or rat under- goes one ])seudopregnancy after another. Possibly, in animals that normally liave a spontaneou.- hiteal phase, thei'c is a con- sidei'ahle poUion ot' the pars distalis which I'unctions somewhat indepeiidentl}- of the hyputhalaiiius. with a continuous output of luteotrophin as from the grafted gland in the Mvihlb()ck-P)Oot pi-epaiation. The por- tion nioic (Hrectly under control of the me(lian eminence I zona tuberaHs'.'l would MAMMALIAN REPRODUCTIVE CYCLE 537 then act like the intact hypoiihysis of the Miihlbock-Boot mouse. Such a view, un- fortunately, continues to set apart species in which the luteal phase is not spontaneous, by suggesting that only in them are special neui'al nieclianisnis involved. B. LUTEOLYTIC MECHANISMS By luteolysis we shall refer to corpus lu- teum regression in any of its manifestations. Supposedly the initial change is functional, after which overt cytologic and histologic changes appear, leading eventually to the total loss of glandular tissue. Very likely the initial stages are occult and only gradu- ally reach recognizable proportions. Men- tion was made earlier of the fact that in women and monkeys the peak of gestagen secretion is about midway between ovula- tion and menstruation. In rats indirect evi- dence from progesterone injection experi- ments leads to the deduction that toward the close of a pseudoi)regnancy gestagen secretion must drop below the estrus-sup- pressing level several days before estrous changes appear in the vaginal smear (see Fig. 8.8). A more al)rupt drop is reported for the ewe l)etween the 16th and 17th (last) days of the cycle (Edgar and Ronald- son, 1958). Long-term experiments with pituitary autotransplants indicate that at least in the rat the life span of the corpus luteum is not limited by intrinsic factors. Some agent (s) of extra-ovarian origin must, therefore, be res]5onsible for at least the initial luteo- lytic changes. Various bits of information suggest that the agent is associated with, if not identical with, FSH and/or LH. Greep (1938) noted that after hypophysectomy in rats the daily injection of LH over a pe- riod of 10 days caused the corpora lutea to regress more rapidly than otherwise. Greep, van Dyke and Chow (1942) later were un- able to repeat this with a more highly puri- fied LH C'metakentrin") , a fact suggesting that the earlier material was effective be- cause of impurity. During the short cycle of the rat, luteolysis is interrupted by trans- lilantation of the pars distalis (Everett, 1954; Nikitovitch-Winer and Everett, 1958a). Whatever regressive changes are in progress at that moment are evidently suspended forthwith. They are first appar- ent during the third day of diestrum and be- come increasingly pronounced during pro- estrum and estrus. In this connection, it should be recalled that during late diestrum and proestrum patches of cells undergoing fatty necrosis are first recognizable histo- logically (Boling, 1942; Everett, 1945). Why is it that, in the face of a continuing supply of luteotrophin in the Miihlbock- Boot preparation, or in intact animals in- jected daily with lactogen (Lahr and Riddle, 1936; Aschheim, 1954), luteolysis sets in during the 2nd week? The question ob- viously cannot be answered from present knowledge. Nevertheless, it is clear that the pseudopregnancy that transpires when a significant i)ortion of hypophyseal tissue remains in normal relation to the hypo- thalamus is far from the steady state of that which becomes established by total re- moval of the pars distalis to an extracranial site. It is also apparent that the onset of luteolysis may be postponed by such means as hysterectomy or production of artificial deciduomas (see p. 538). Furthermore, during lactation-pseudoi)regnancies in rats, Canivenc and Mayer (1953) prolonged lu- teal function to 34 days by substituting suc- cessive new litters of suckling young. This technique should prove especially valuable in experimental analysis of both luteo- trophic and luteolytic mechanisms. Benson and Folley (1956) suggested that lactogen secretion is activated by oxytocin, inasmuch as its injection prevented the nor- mal inv'olution of the mammary glands after withdrawal of the litters from lactating rats. This observation has been confirmed by McCann, Mack and Gale (1958), who also noted the interruption of lactation by le- sions of the sui)raoi)tico-hyiiophyseal tract. Selye and ]\lcKeown (1934) long ago found that pseudopregnancy could be induced in cycling rats by the introduction of a suck- ling litter. Although all this is consistent with the above-mentioned observation by Canivenc and Alayer, other workers have observed luteolytic effects of gonadotrophin- free oxytocin administered to cycling dairy heifers (Armstrong and Hansel, 1958). Fur- thermore, Grosvenor and Turner (1958), after first noting that the administration of Dibenamine, atropine, or pentobarbital to rats prevented the expected drop in assay- 588 PHYSIOLOGY OF GONADS able pituitary lactogen at nursing, found no decrease when pentobarbitalized mothers were injected with physiologic doses of oxy- tocin intravenously. When the dosage was increased 30 to 60 times there was appar- ently some moderate discharge, but the au- thors regard it as insignificant. C. EFFECT OF THE UTERUS ON LUTEAL FUNCTION This subject has been reviewed by Brad- bury, Brown and Gray (1950). In three species (Fig. 8.13) hysterectomy results in significant prolongation of the functional life-span of corpora lutea (guinea pig, Loeb, 1927; rabbit, Asdell and Hammond, 1933; rat, Bradbury, 1937). In each case the pe- riod of luteal function approximates that of normal pregnancy. The fact that corpora lu- tea in the pseudopregnant ferret normally function as long as in the pregnant animal may be a clue to the noncffect of hysterec- tomy in that species (Deanesly and Parkes, 19331. Ahhough Burford and Diddle (1936) rej^orted that in monkeys total hysterec- tomy was followed by vaginal cycles of nor- mal length, examination of their protocols shows that during the several postoperative months just 1 corpus luteum was produced among all 5 animals. The experiment thus seems inconclusive. Impairment of pelvic circulation seems to be a common factor complicating the results of hysterectomy in women and may have been one cause of the failure of luteinization in these monkeys. An interpretation given by Loeb (1927) and Bradbury, Brown and Gray (1950) for the prolongation of luteal function by hys- terectomy is that in species in which the ef- fect is demonstrable the uterus secretes a specific substance which abbreviates the life of the corpus luteum. Hechter, Fraenkel, Lev and Soskin (1940) found in rats that grafts of estrous uteri shortened the pseudo- pregnancies of hysterectomized animals to normal length. Implantation of similar tis- M 1 I I T- GUINFA pin - Actual duration 2 none 1 ^ ^ .. ^ " 90± days (Loeb)(Brouha) 70± days ? 5 _ _ " _ _ ■^ " - ~ "" ~ ^ "" ~ ~ ~ RARRIT 1 , mil mill lilll mill mill iiiii;*' 1 14-16 days 24-29 days (Asdell S Hammond) n ^ J. ^ _ ,. _ _ ^ , == _ _ 1 ~ ~ ~ — ~ ~ ~ — — ' — 30 days RAT M\ Mil nnn nmn 12 days I8±days 22 days ^H - - - - = = = = = ^ = = _ ^ ^ ~ ~ ~ ~ ~ ■~ ~ ~ ~ FERRET 6 weeks (Parkes) nil Hill lilll Hill Hill Mlllllillillllllllliilllllllliiiilll 1 1 6 weeks 6 weeks = = = = = =1 ^ = ~ ~ L Pseudopregnancy iiiiiiiiiiii iiiiiiiiiiiii Pseudopregnancy hysterectomized m^^^g^^ Mil- . Prpnnnnrv Fi(i. 8.13. Reliilive duiation.s of p.seiulopicgnaiicy in normal and hysterectomizeil animals of four species in relation to the duration of j^ostation characteristic of each. Gestation plotted as a common unit of time. (After J. T. Bradbury, W. E. Brown and L. A. Gray, Recent Proj,n-. Hormone Res., 5, 151-194, 1950.) MAMMALIAN REPRODUCTn'E CYCLE 539 SIR' which had been killed by freezing had no such effect, nor did successful grafts of uteri from diestrous donors. Bradbury, Brown and Gray (1950) found that par- tially hysterectomized rats in which the re- maining uterine tissue was continuous with the cervix, hence properly drained, experi- enced pseudopregnancies of normal length. However, when the continuity was inter- rupted the uterine remnants became greatly distended, the endometrium was destroyed, and the animals had prolonged pseudopreg- nancies. Possibly the endometrium is the source of the hypothetical "luteolytic" sub- stance.*' Under other circumstances the endome- trium of rats has a luteotrophic rather than luteolytic effect, for when deciduomas are induced by trauma the pseudopregnancies of otherwise normal animals are lengthened to 22 days or more (Ershoff and Duell, 1943; Velardo, Dawson, Olsen and Hisaw, 1953). This is not true in mice, however, and Ka- mell and Atkinson (1948) suggest that the I'eason may lie in the shorter life-span of the decidual tissue in that species. Loeb (1927) reported that deciduomas in cyclic guinea pigs ])rolonged the luteal phase, i.e., delayed the next ovulation from 3 to 7 days, which is far less than the prolongation after hys- terectomy. As an alternative to the concept of con- I rol of the corpus luteum by humoral agents " The denial by Velardo, Olsen, Hisaw and Dawson (1953) that hysterectomy in rats prolongs l)seiidopregnancy is based on operations performed later in the luteal phase than those of Bradbury, Brown and Gray and of Hechter, Fraenkel, Lev and Soskin. Whereas the latter workers had op- erated in the range from the 4th to the 7th day.^^ of p.seudopregnancy and many of Bradbury's cases lacked uteri when they entered pseudopreg- nancy, Velardo and associates excised the uteri on the 9th day. It seems possible that this dif- ference in time may be crucial, for by the 9th day of a 12-day pseudopregnancy the corpora lutea must be on the verge of regression, if that in'ocess has not already been initiated. After maintaining pseudopregnancy in estrogenized, liypophysectomized rats b}^ means of lactogen, its withdrawal is followed about 3 days later by estrous smears (Nelson and Bichette, 1943; Nel- son, 1946). A slightly longer delay occurs in non- estrogenized, intact rats at the end of a lactogen- induced pseudopregnancy (Everett, unpublished) or after withdrawal of progesterone treatment (Fig. 8C). formed in the uterus, Loeb considered the jjossibility that neural mechanisms are in- volved. The idea was not acceptable, he felt, because in partial hysterectomies the result was not detennined by the locus of the part removed. The finding by Hechter, Fraenkel, Lev and Soskin (1940) that grafts of uterine tissue in hysterectomized rats return the duration of pseudopregnancy to normal, is significant evidence pointing in the same di- rection. A third hypothesis was advanced by Heckel (1942) who found in rabbits that the extent of prolongation of luteal func- tion by subtotal hysterectomy is roughly proportional to the amount of uterine tis- sue removed. The suggestion was offered that removal of the uterus has an estrogen- sparing effect. The greater amount of estro- gen thus available to the corpora lutea pro- longs their life, according to this view. Later investigations by Moore and Nal- bandov (1953) revive the possibility that the uterus influences the ovary by way of the nervous system. In sheep the implanta- tion of a plastic bead in utero during the early luteal phase shortened the cycle by several days. Successive cycles tended to be unusually short. When the uterine segments containing the beads were denervated, how- ever, the cycles were essentially normal. Other work from the same laboratory (Hus- ton and Nalbandov, 1953) which indirectly may bear on this problem, indicates that the presence of a mechanical irritant (a thread) in the oviduct of the domestic fowl tends to block ovulation. The blockade may extend for as long as 20 days if the thread is placed in the isthmus (van Tienhoven, 1953). The ovaries remain functional, pro- ducing large follicles which may be ovulated at will by injection of LH. The authors feel that this phenomenon, like the effect of the bead in the sheep uterus, involves a neural mechanism, Init crucial information is lack- ing. It may be significant that stimulation of the ovaries was found in some hens, in place of blockade. We may hope that as more information becomes available the assortment of facts given in these paragraphs will fit into a rational system. Not until this is realized can we hope to understand the regulation of the luteal phase. 540 PHYSIOLOGY OF COXADS VIII. Concluding Comments From what has been written here it is readily apparent that present knowledge of the mechanisms controlling the reproductive cycle is extremely spotty. The number of assumptions necessary to knit the various items of factual information into an orderly pattern is disturbing. In spite of a volumi- nous literature which has grown during the last 60 years, we are really only a few steps ahead of our predecessors at the turn of the century in terms of fundamental under- standing. A brief recounting of some of these steps may be desirable. The first three decades saw the gradual development of proof that the ovary is a gland of internal secretion as well as the producer of eggs, governing the uterus and other accessory organs by secretion of hor- mones into the blood stream. For a while it seemed that the events of the reproductive cycle could be neatly explained, with the ovary in the capacity of controlling agent. Yet there were indications that the ovary itself is not independent. As early as 1909- 1910 (Aschner; Crowe, Gushing and Ho- nuins) it was noted that destruction of the hypophysis is accompanied by atrophy of the gonads and reproductive tract. In 1927 the separate investigations of Smith and Engle and of Zondek and Aschheim demon- strated conclusively that function of the ovary depends vitally on the anterior hy- jiophysis. Promptly it was learned that the hypophyseal secretion of gonadotrophic hormone is in turn modified by estrogens. In the early 1930's the "push-pull" hypothesis of pituitary-ovarian interaction was sepa- rately stated by Brouha and Simonnet and by Moore (see Moore and Price, 1932). Modified in detail as new facts appeared, this hypothesis is held to the present day in some quarters as a simple exi)lanation of how the cycle comes about in polyestrous, continuous breeders in which ovulation takes l)lacc spontaneously. Much of the investiga- tion of pituitary-ovarian i)hysiology during the 1930's was performed within the frame- work of this hypothesis. For seasonal breeders and reflex ovulators, however, the assumption was necessary that special controlling mechanisms are super- imjiosed. It bccaiiie iccognizcd also that in some vaguely defined manner even the hu- man cycle is subject to intervention of the nervous system. The possible importance of the hypothalamus was debated at some length in the twenties. In 1932 the existence of a sex center there was proposed by Hohl- weg and Junkmann. In an attempt to ex- plain the coital excitation of the rabbit hy- pophysis which causes the liberation of gonadotrophin, Hinsey and Markee (1933) suggested diffusion of a chemical substance from the posterior lobe to the anterior lobe. Hinsey (1937) later elaborated on this pos- sibility and mentioned the hypophyseal por- tal vessels as a plausible route by which the substance might travel. We have seen the later history of these ideas. The "Sexualcentrum" of Hohlweg and Junkmann was proposed as a mediator of the effects of estrogen on the anterior hy- pophysis of rats. Westman and Jacobsohn (1936-1940), on the other hand, believed that through its stalk connection with the hypophysis the rat hypothalamus governs gonadotrophin synthesis, not release. The latter they regarded as a direct effect of es- trogen on the gland. These views did not afford a common basis for spontaneous and refiex ovulation. Schweizer, Chari])i)er and Haterius ( 1937) offered the first surmise of similarity, after finding that guinea pigs bearing intra- ocular pituitary grafts developed persistent estrus and large follicles that failed to go through maturation changes. Their feeling was that the normal connection of hypophy- sis with hypothalamus may be necessary for cyclic liberation of LH. Almost concur- rently, Dempsey (1937) expressed a similar view as one alternative explanation of his experimental results with the guinea i)ig cy- cle. Suggesting cautiously that release of luteinizer may be brought about l)y a "rhythmic discharge" from the central nerv- ous system, he went on to mention the "pos- sibility that a high level of oestrin is neces- sary but not directly responsible for the release of luteinizer" (italics added). From this it is only a short transition to certain concepts set forth in the present exposition. Accoi'ding to current views: (1) Reflex ovulation and spontaneous ovulation alike are go\-enicd by a liypothalamo-pituitary MAMMALIAN REPRODUCTIVE CYCLE 541 apparatus whose final link to the pituitaiy is neurohumoral by way of the hypophyseal portal veins and whose activity precipitates release of LH. (2) The apparatus includes a hypothalamic center or centers whose ex- citation depends on estrogen-progesterone levels and afferent impulses of various kinds. (3) The sensitivity of the center (s) is influenced not only by the sex steroids, but by other poorly understood factors that vary from species to species and from time to time in individuals, e.g., the diurnal rhythm in rats. Here in bare outline is a plausible hypothesis that may be generally applied to the events immediately relating to ovulation. Satisfactory hypotheses respecting other phases of the cycle must await future de- velopments. The extent and manner of in- tervention of the nervous system in the fol- licular and luteal phases remain unsettled. Although the hypophyseal hormones con- cerned in ovarian follicle development have been characterized, their exact chemical de- scrijition has not been accomplished. The rate of their output at different stages of the cycle is largely a matter of conjecture. Structural changes that they jiroduce in the ovary are well known, but in chemical terms only the end products of ovarian activity are well recognized, and these probably incom- pletely. The fact that the estrogens, in turn, have a regulating effect on follicle-stimulat- ing activity of the hypophysis is known, but the mechanisms by which this effect is ac- complished are uncertain. The hypophyseal hormones that maintain the luteal phase are recognized with any certainty in only three species and there is a wide difference between rabbits, on the one hand, and rats and mice, on the other. For mammals gener- ally, the luteotrophic factors have not been identified. Whether the hypothalamus is actively concerned in maintenance of the luteal i)hase in the majority of mammals is unknown. The morphologic effects of luteo- trojihic stimulation on corpora lutea are well recognized, but here again the chemical mechanisms leading to the end products are obscure. The action of the corpus luteum hormone in regulation of the cycle is par- tially known, including the well established fact that its continual jiresence in large amount will suiijiress the estrogenic and ovulatory phases. Yet, one cannot say whether this effect is accomplished by direct action on the hypophysis or by indirect ac- tion through the central nervous system. Nor can one state how the hypophysis- gonad equilibrium of the luteal phase is in- terrupted in the absence of a conceptus. With respect to the ovulation mechanism itself, the hypothesis outlined above requires verification in additional species. Assuming its validity, many details remain to be stud- ied, e.g., the neural pathways and nuclei involved, identification of neurochemical activators of the pars distalis and their sources and loci of action, the precise nature of mechanisms whereby the gonadal steroids excite or suppress, the cellular mechanisms by which ovulating hormone is released into circulation by the hypoi^hysis, and the cyto- chemical effects within the ovary. All too evidently an encompassing theory of the female reproductive cycle is far from reali- zation. IX. References Allex. E., Daxforth, C. H., .and Doisy, E. A., Eds. 1939. Sex and Internal Secretions, 2nd ed. Baltimore: The Williams & Wilkins Com- pany. Allen, E. 1923. Racial and familial cyclic in- heritance and other evidence from the mouse concerning the cause of estrous phenomena. Am. J. Anat., 32, 293-304. Allen, E. 1927. 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B, 123, 441-471. ZucKERMAN, S. 1941. Periodic uterine bleeding in spayed rhesus monkeys injected daily with a constant threshold dose of oestrone. J. Endo- crinol., 2, 263-267. ZucKERMAN, S. 1952. The influence of environ- mental changes on the anterior pituitary. Ciba Foundation CoUociuia Endocrinol., 4, 213-228. ZucKERMAN, S., AND FuLTON, J. F. 1934. The menstrual cycle of the primates. VII. The sexual skin of the chimpanzee. J. Anat., 69, 38-46. ZwARENSTEiN, H. 1937. Experimental induction of ovulation with progesterone. Nature, Lon- don, 139, 112-113. 9 ACTION OF ESTROGEN AND PROGESTERONE ON THE REPRODUCTIVE TRACT OF LOWER PRIMATES Frederick L. Hisaw, Ph.D. THE BIOLOGICAL LAB0RAT0RIF:S, HARVARD TNIVERSITY, CAMBRIDGE, MASSACHUSETTS and ^ Frederick L. Hisaw, Jr., Ph.D. DEPARTMENT OF ZOOLOGY, OREGON STATE COLLEGE, CORVALLIS, OREGON I. Introduction 556 II. Ovarian Hormones and Growth of THE Genital Tract 558 III. Effects of Progesterone on the Uterus 565 IV. Synergism between Estrogen and Progesterone 567 V. Experimentally Produced Implan- tation Reactions 571 VI. The Cervix Uteri 572 VII. The Vagina 575 VIII. Sexual Skin 576 IX. Menstruation 578 X. The Mechanism of Menstru.^tion. . 583 XI. References 586 I. Introduction Cyclic menstruation is the most charac- teristic feature of primate reproduction, and distinguishes it from the estrous cycle of lower mammals. This cardinal primate event is heralded by the bloody uterine effluent emanating from the vagina, whereas in estrus the dominant characteristic is a sudden modification in behavior featuring an intense mating drive. However, the in- ternal secretions that regulate the various events in the menstrual and estrous cycles are the same, and this similarity is funda- mentally more significant than the key de- scriptive differences just mentioned. Estrus comes at the peak of the growth phase of the cycle and is associated with ovulation. In contrast, menstruation occurs in the cycle midway between times of ovulation and is not accompanied by an increase in sexual activity. From earliest times menstruation has been recognized as degenerative: the characteristic odor, and the necrotic changes in the lining of the uterus, part of which is cast off at this time, sustain this interpre- tation. Therefore, menstruation is at the opposite phase of the cycle from estrus. It is such an obvious event that menstrual cycles are dated from the onset of bleeding. Menstruation is not analogous to the pro- estrous bleeding in the dog or cow nor to the slight bleeding of primates at midpoint be- tween menstrual periods (Hartman, 1929). The study of menstruation was at first al- most entirely the province of the clinician and the material for investigation limited to w^omen. Hitschmann and Adler (1907), Meyer (1911), Schroder (1914). Novak and Te Linde (1924), and Bartelmez (1933) are among many of the earlier investigators who contributed descriptions of the cyclic changes in the human endometrium. The physiology of the menstrual cycle and at- tendant morphologic changes have contin- ued to be an area of active research interest in science and medicine. Among the many more recent contributors are Bartelmez 556 ESTROGEN AND PROGESTERONE 557 (1937), Latz and Reiner (1942), Haman (1942), Knaus (1950), Mazer and Israel (1951), and Crossen (1953). The earlier concepts regarding the men- strual cycle were based primarily on the changes occurring in the human endome- trium and for convenience of description the cycle was divided into four stages or pe- riods. The first of these was the period of active menstruation, and the length of the cycle was dated from its onset. Most au- thors agreed that menses began by leaking of blood from superficial vessels to form lakes under the surface epithelium and that there was some sloughing of tissue after the beginning of bleeding. There was consider- able disagreement as to the amount of de- struction and loss of tissue; estimates of various authors ranged from very little to almost complete denudation of the surface. Bartelmez (1933) emphasized both the wide individual variability of the amount of tis- sue lost and differences in the stage of de- velopment of the endometria at the time of menstruation. The second period immediately following menstruation began with regeneration of the surface epithelium, which started sometimes before menstrual bleeding had ceased and was completed in a very short time. This lieriod included the 5 to 7 days after cessa- tion of menses, during which the endome- trium grew in thickness. Frequent mitoses were recognized, especially in the glands which lengthened but remained straight and tubular. The third ("interval") period, lasting 6 to 10 days, was characterized by a some- what thickened endometrium, still with straight glands and showing little evidence of secretory activity. At first this was con- sidered a quiescent period as indicated by the term "interval." However, as will be shown later, such a characterization was not justified from the physiologic viewpoint. The fourth period, called the premen- strual period, included the 10 days or 2 weeks before menstruation. During this phase the glands continued to increase in size and became distended, coiled, or even sacculated. The glandular cells increased in height, and there was evidence of glyco- gen mobilization and secretion. Next, the epithelium became "frayed out" along the outer borders, then decreased in height, in- dicating secretory depletion. Decidual cells appeared in the stroma at this time. The endometrium was much thickened and ex- tremely hyperemic. At the height of this period the endometrium was approximately 5 mm. in thickness, as compared with V2 mm. toward the end of menses. The term premenstrual was usually applied to this phase but today the term progestational would seem preferable. During and after these descriptions of the changes in the human endometrium, many attempts were made to locate the time of ovulation in the menstrual cycle. When it was found, as will be discussed later, that ovulation occurred approximately midway between two menses, and was preceded by follicular growth and followed by develop- ment of a corpus luteum, it became custo- mary to refer to the two halves of the men- strual cycle as the follicular phase and the luteal phase. One advantage of this descrip- tive terminology was the emphasis it placed on the homology of the two phases of the menstrual cycle in primates with the fol- licular and luteal phases of the estrous cy- cles of lower mammals. A theory to explain menstruation, widely adopted in 1920, was formulated from this morphologic evidence. The essentials were that menstruation occurs because the lining of the uterus, prepared for implantation of the ovum, degenerates if fertilization of the egg does not occur. This required that ovu- lation and corpus luteum formation precede the i^remenstrual changes in the endome- trium. Subsequent research disclosed that menstrual cycles frequently occur in which ovulation does not take place and bleeding results from the breakdown of an "interval" rather than a progestational endometrium. The discovery of anovulatory cycles not only brought about a revision of ideas re- garding an explanation for menstruation but also raised questions as to what consti- tuted a normal menstrual cycle. The length of the cycle and the amount and duration of bleeding are approximately the same re- gardless of whether or not ovulation has taken place. The gross features of menstru- ation under these two conditions are indis- tinguishable one from the other. However, the biologic purpose of the menstrual cycle 558 PHYSIOLOGY OF GONADS is reproduction wliicli obviously cannot l)0 fulfilled unless an ovum is made available for fertilization. Therefore, in this sense it seems quite clear that anovulatory cycles should be considered incomplete and ab- normal. The investigation of changes taking place in the uterine endometrium at various pe- riods of the menstrual cycle in women was confronted with many difficulties, the chief one being that of obtaining normal tissue representative of specific times of the cycle. The entire uterus and both ovaries are es- sential for proper evaluations and it was rarely possible to meet these requirements. The material for such studies came from autopsies and surgery and tissues usually had suffered postmortem changes or the surgical condition was one involving serious pelvic disease. There have been, however, a goodly number of instances in which these difficulties were adequately overcome (Stieve, 1926, 1942, 1943, 1944; Allen, Pratt, Newell and Bland, 1930) and the clinic will continue to make important contributions (Rock and Hertig, 1942; Hertig and Rock, 1944), but quite early the need became ob- vious for a suitable primate that could be used as an experimental animal for research on the different aspects of the physiology of reproduction. Since the initial observations by Corner (1923) on the menstrual cycles of captive rhesus monkeys iMacaca mulatta) , more has been learned about the physiology of reproduction of this animal than any other primate. Monkeys of this species thrive un- der laboratory conditions, which has made it possible to devise accurately controlled experiments on normal healthy animals and obtain reliable information on the men- strual cycle, gestation, fetal development, and the interaction of hormones concerned with regulating reproductive processes. Other features that make the rhesus mon- key such an attractive animal for these pur- poses are the many morphologic and physi- ologic attributes that are strikingly like those of the human being. Tiic modal length of their menstrual cycles is 28 days but there is wide variation (Corner, 1923; Hart- man, 1932; Zuckcrman, 1937a). From an analysis of 1000 cycles recorded for some 80 females of different ages, Zuckerman (1937a) found an average cycle length of 33.5 ± 0.6 days, and the mode 28 days with an over-all range of 9 to 200 days. Ovulation occtu's api:)roximately midway between two menstrual periods, most between tlie 11th and 14th days (Hartman, 1932, 1944; van Wagenen, 1945, 1947), and although these animals breed at all seasons of the year many cycles are anovulatory, especially during the hot summer months (Eckstein and Zuckerman, 1956). A method developed by Hartman for detecting the exact time of ovulation by palpation of the ovaries in the unanesthetized animal greatly facilitated the timing of events of the menstrual cycle. This procedure also made it possible to de- termine the age of corpora lutea with great accuracy (Corner, 1942, 1945) and corre- late their develojiment and involution with corresponding changes in the endometrium (Bartelmez, 1951 ) and, in ju-egnancy, with the exact age of developing embryos ( Wis- locki and Streeter, 1938; Heuser and Streeter, 1941). The primary purpose of the present dis- cussion is to review the results of experi- mental investigations of physiologic proc- esses occurring in the female reproductive tract of lower primates during the menstrual cycle, and particularly those processes that are under hormonal control. The brief in- troductory presentation of basic observa- tions could be greatly extended and we take up the discussion of endocrine problems knowing that we must return often to the work of these authors and that of others to be cited, as conclusions based on experi- mental data take on meaning only in terms of normal function. II. Ovarian Hormones and Growth of the Genital Tract The changes that are repeated in differ- ent parts of the reproductive tract with each menstrual cycle are produced by ovarian hormones, estrogens, and progesterone. The dominant hormone of the follicular phase is estradiol-17/y, which is secreted by the Graafian follicle, and in the tissues is read- ily transformed in i)art to estrone, an estro- genic metabolite. Progesterone, secreted by the corinis luteum, is jirimarily a hormone of the luteal phase of the cycle. However, small amounts of progesterone may appear ESTROGEX AND PROGESTERONE 559 ill the blood of monkeys as early as the 7th (lay and attain a concentration of 1 fxg. per ml. of serum at ovulation, whereas a maxi- mal concentration of 10 /xg. per ml. is reached at approximately the middle of the luteal phase (Forbes, Hooker and Pfeiffer, 1950; Bryans, 1951). Also, some estrogen is present during the luteal phase, probably secreted by the corpus luteum (estrogens can be obtained from luteal tissue) or it may be partially derived from developing follicles. It is unlikely that estrogen is ever entirely absent during a normal menstrual cycle or that the presence of progesterone is completely restricted to the luteal phase. The dependence of the reproductive tract on ovarian hormones is strikingly demon- strated by the profound atrophy that fol- lows surgical removal of the ovaries. A pro- gressive decrease in size of the Fallopian tubes, uterus, cervix, and vagina takes place, and usually, the involution of the uterine endometrium involves tissue loss and bleed- ing, commonly referred to as post castra- tional bleeding. Dramatic as these effects may seem, it is equally dramatic to find that these atrophic structures can be re- stored entirely to their original condition by the administration of ovarian hormones. Therefore, it is seen at the beginning that investigations dealing with the physiology of the female reproductive tract of primates in large measure involve a study of the in- dependent and combined actions of estro- gens and progesterone on the activities of the various structures concerned. Much can be learned about the action of ovarian hormones by observing the changes they produce in the gross appearance of the atrophied reproductive tract of castrated animals. Daily injections of an estrogen in doses equivalent to 1000 I.U. or more for 10 days or a fortnight will restore the uterus, cervix, and vagina to a condition compara- l)le with that found in a normal monkey at the close of the follicular phase of a men- strual cycle (Fig. 9.1A). If a similar cas- trated monkey is given 1 or 2 mg. of pro- gesterone daily for the same length of time, very little if any change in size of the re- productive organs results. However, if first the normal condition is restored by giving estrogen and then is followed by the pro- gesterone treatment, the size of the uterus is maintained but that of the cervix and vagina decreases to an extent approaching that in a castrated animal (Fig. 9.1B). Such experiments show that an estrogen promotes growth of the reproductive tract whereas progesterone is comparatively ineffective when given alone. Yet progesterone can maintain the size of the uterus when admin- istered following an estrogen treatment but it does not prevent involution of the cervix and vagina. An additional feature of the growth-stim- ulating action of the ovarian hormones is brought out when estrogen and progesterone are administered concurrently. If, after re- pair of the reproductive tract of a castrated animal has been accomplished by a series of injections of estrogen, both estrogen (1000 I.U.) and progesterone (1 or 2 mg.) are given daily for 20 days, it will be found that the uterus is larger than when either hor- mone is given alone for a similar length of time, whereas the cervix and vagina have involuted and are approximately the size found in animals given only progesterone. Thus it can be demonstrated that a syner- gistic effect on growth of the uterus occurs when the two hormones are given simultane- FiG. 9.1. Reproductive tracts of three adolescent monkeys which were castrated and given estrogen daily for approximately 3 weeks. A shows the con- dition at the conclusion of the estrogen treatment, B the condition following the injection of pro- gesterone for an additional three weeks, and C the effects of continuing the treatment with both es- trogen and progesterone for a like period. 558 PHYSIOLOGY OF GONADS is reproduction which obviously cannot be fulfilled unless an ovum is made available for fertilization. Therefore, in this sense it seems quite clear that anovulatory cycles should lie considered incomplete and ab- normal. The investigation of changes taking place in the uterine endometrium at various pe- riods of the menstrual cycle in women was confronted with many difficulties, the chief one being that of obtaining normal tissue representative of specific times of the cycle. The entire uterus and both ovaries are es- sential for proper evaluations and it w^as rarely possible to meet these requirements. The material for such studies came from autopsies and surgery and tissues usually had suffered postmortem changes or the surgical condition was one involving serious pelvic disease. There have been, however, a goodly number of instances in which these difficulties were adequately overcome (Stieve, 1926, 1942. 1943, 1944; Allen, Pratt, Newell and Bland, 1930) and the clinic will continue to make important contributions (Rock and Hertig, 1942; Hertig and Rock, 1944), but quite early the need became ob- vious for a suitable primate that could be used as an experimental animal for research on the different aspects of the physiology of reproduction. Since the initial observations by Corner (1923) on the menstrual cycles of captive rhesus monkeys (Macaca mulatta) , more has been learned about the physiology of reproduction of this animal than any other primate. Monkeys of this species thrive un- der laboratory conditions, which has made it possible to devise accurately controlled experiments on normal healthy animals and obtain reliable information on the men- strual cycle, gestation, fetal development, and the interaction of hormones concerned with regulating reproductive processes. Other features that make the rhesus mon- key such an attractive animal for these pur- poses are the many morphologic and physi- ologic attributes that are strikingly like those of the human being. The modal length of their menstrual cycles is 28 days but there is wide variation (Corner, 1923; Hart- man, 1932; Zuckerman, 1937a). From an analysis of 1000 cycles recorded for some 80 females of different ages, Zuckerman (1937a) found an average cycle length of 33.5 ± 0.6 days, and the mode 28 days with an over-all range of 9 to 200 days. Ovulation occurs approximately midway between two menstrual periods, most between the 11th and 14th days (Hartman, 1932, 1944; van Wagenen, 1945, 1947), and although these animals breed at all seasons of the year many cycles are anovulatory, especially during the hot summer months (Eckstein and Zuckerman, 1956). A method developed by Hartman for detecting the exact time of ovulation by palpation of the ovaries in the unanesthetized animal greatly facilitated the timing of events of the menstrual cycle. This procedure also made it possible to de- termine the age of corpora lutea with great accuracy (Corner, 1942, 1945) and corre- late their development and involution with corresponding changes in the endometriun:i (Bartelmez, 1951 ) and, in pregnancy, with the exact age of developing embryos (Wis- locki and Streeter, 1938; Heuser and Streeter, 1941). The primary purpose of the present dis- cussion is to review the results of experi- mental investigations of physiologic proc- esses occurring in the female reproductive tract of lower primates during the menstrual cycle, and particularly those processes that are under hormonal control. The brief in- troductory presentation of basic observa- tions could be greatly extended and we take up the discussion of endocrine problems knowing that we must return often to the work of these authors and that of others to be cited, as conclusions based on experi- mental data take on meaning only in terms of normal function. II. Ovarian Horinones and Growth of the Genital Tract The changes that are repeated in differ- ent parts of the reproductive tract with each menstrual cycle are jiroduced by ovarian hormones, estrogens, and progesterone. The dominant hormone of the follicular phase is estradiol-17/3, which is secreted by the Graafian follicle, and in the tissues is read- ily transformed in jiart to estrone, an estro- genic metabolite. Progesterone, secreted by the corpus luteum, is primarily a hormone of the luteal phase of the cycle. However, small amounts of i)rogesterone may appear ESTROGEN AND PROGESTERONE 561 four experimental animals the only well de- veloped fundus was found in the animal on the shortest treatment, i.e., 60 days. The mitotic activity in the epithelium of the glands and surface mucosa also indicates a limited effect of estrogen. This can be demonstrated to best advantage in the endo- metria of castrated monkeys that have been on estrogen for different lengths of time and have received an injection of colchicine 8 hours before their uteri were removed. A comparison of the number of cells in mitosis per square centimeter of surface mucosa at 10, 30, 45, and 60 days is shown in Figure 9.3. From this it can be seen that mitotic activity approaches that in a castrated ani- mal. Although the five points used in draw- ing the curve are quite inadequate for an accurate analysis of the mitotic response in the epithelial components of the endome- trium, they do show that cell division is most rapid soon after the beginning of an estrogen treatment and subseciuently de- clines. The loss of responsiveness of the endome- trium to estrogen seems related more to the length of treatment than to dosage of hor- mone. An endometrium of normal thickness can be produced in 2 or 3 weeks at a dosage level of estrogen that will not maintain the growth induced for longer than about 40 days without bleeding (Hisaw, 1935; Engle and Smith, 1935; Zuckerman, 1937b). The response to a low dosage of estrogen that will prevent bleeding during the course of treatment (about 10 /xg. estradiol-17/8 daily) is one of rapid endometrial growth at first, as has been described, followed by a thin- ning of the endometrium. The refractoriness of the endometrium to estrogen becomes so pronounced after about 100 days of treat- ment that very few cell divisions are seen in the epithelium of the glands and surface mucosa. The general morphology of the en- dometrium retains the characteristic ap- pearance of the follicular phase of the men- strual cycle except that the stroma is usually more dense and the cells of the glandular epithelium have large deposits of glycogen between the nucleus and the base- ment membrane. However, metabolically such endometria are surprisingly inactive. Although they are dependent on the pres- ence of estrogen and may bleed within about Mitotic Response of Uterine Lpithelium TO looo I. u. Estrogen PER D«y. Fig. 9.3. The number of mitoses per square centi- meter of surface epithelium of the endometrium in a castrated monkey and in four castrated animals given 10 /xg. estradiol daily for 10, 30, 45, and 60 days, respectively. One-tenth of actual number of mitoses is shown on the ordinate. (From F. L. Hisaw, in A Symposium on Steroid Hormones, Uni- versity of Wisconsin Press, 1950.) 48 hours if the treatment is stopped, the activity of their oxidative enzymes and the ratio of nucleoproteins (RNA:DNA) are about the same as in the involuted endome- tria of castrated animals. The effects that accompany moderate es- trogenic stimulation become exaggerated in several respects when large doses of estrogen are given for an extended period. The dis- parity between the area of myometrium and endometrium becomes greater as the treat- ment progresses (Fig. 9.4). Kaiser (1947) described the destruction of the spiraled arterioles of the endometrium in monkeys given large doses of estrogen and Hartman, Geschickter and Speert (1941) reported the reduction of the reproductive tract to the size of that of a juvenile animal by the end of 18 months during which injections of large doses of estrogen were supplemented by subcutaneous implantation of estrogen pellets. These observations not only show that the endometrium becomes unresponsive to estrogen when the treatment is prolonged but that large doses produce injurious ef- fects. 562 PHYSIOLOGY OF GONADS Fig. 9.4. .4. i'v'< — ' ri:,,ii of the uterus of a cas- trated monkey which had received 1.0 mg. estradiol daily for 35 days. Compare with B which shows the effects of 1/10 this dosage (100 fig. daily) when gi\en for 185 days. The limited response of the endometrium to estrogen is in some respects surprising in view of its remarkable growth potentiali- ties and regenerative capacity. These quali- ties were dramatically demonstrated by Hartman (1944) who dissected out as care- fully as possible all of the endometrium from the uterus of a monkey and wiped the uterine cavity with a rough swab and yet the undetected endometrial fragments that remained were capable of restoring the en- tire structure. Also, considering the enor- mous increase in size of the uterus during gestation, it is even more difficult to ac- count for the rather sharp limitation of growtli under the influence of estrogen. The increase in tonus of the uterine mus- culature, a known effect of estrogen, has been considered as possibly exercising a re- strictive influence on growth of the endo- metrium. An attempt has been made to re- move this containing influence the muscle may have by making an incision through the anterior wall of the uterus (Hisaw, 1950) . A castrated monkey was given 10 ixg. estradiol daily for 21 days at which time the operation was performed and the treatment continued with 30 ^g. estradiol daily for 40 days. The uterus was laid open by a sagit- tal incision from fundus to cervix and most of the endometrium was removed from the anterior wall. This caused gaping of the in- cised uterus and exposure of the endome- trium on the posterior wall. The incision was not closed and after hemorrhage was completely controlled the uterus was re- turned to the abdomen. Examination of the uterus at the conclu- sion of the experiment showed no indications that endometrial growth had been enhanced. The muscularis had reunited and only a few small bits of endometrium were found in the incision (Fig. 9.5). It seemed probable that the purpose of the experiment had been de- feated by rapid repair of the uterus. There- fore, a similar experiment was done in which the musculature of the incised uterus was held open by suturing a wire loop into the incision. Yet the incision closed and no un- usual growth of tlie endometrium was de- tected (Fig. 9.6). Observations under these conditions are necessarily limited to those made on the uterus when it is removed at the conclusion of an experiment and comparisons must be made between uteri of different animals. Obviously, it would be more desirable if the response of an individual endometrium could be followed during the course of treat- ment. It is possible to meet most of these requirements under conditions afforded by utero-abdominal fistulae, exteriorized uteri, and endometrial implants in the anterior chamber of the eye. In continuing our dis- cussion we first shall present information obtained by such techni(iues that have a bearing on the response of the endometrium to estrogen. The surgical procedure used by Hisaw ( 19501 for preparing utero-abdominal fistu- lae foi' studies of the exj^erimental induction of endometrial growth by estrogen and pro- gesterone was a modification of that used by van Wagenen and Morse (1940) for ob- ESTROGEN AND PROGESTERONE 563 Figs. 9.5 and 9.6 The uteri of these two castrated monkeys were opened from fundus to cer\'ix by an incision through the anterior wall while the animals were receiving estrogen. Part of the endometrium of the anterior wall was removed and the incision in the myometrium was not closed. Fig. 9.5. Estradiol, 10 fig., was given daily for 7 days; the uterus was opened and the animal continued on 30 /xg. estradiol daily for 40 days. (From F. L. Hisaw, in A Sy7nposiii7n on Steroid Hormones, University of Wisconsin Press, 1950.) Fig. 9.6. Estradiol, 10 ^g., was given daily for 7 days; the uterus was opened and the treat- ment continued at a dosage of 30 /ig. estradiol daily for 20 days. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.) serving changes in the endometrium (luring the normal menstrual cycle. This procedure makes frequent inspections possible either by hand lens or dissecting microscope, of most of the upper part of the endometrium on the anterior and posterior walls of the uterus. The elliptic slit formed by the endo- metrium of the two opposing walls can be located easily, the two sides pressed apart by any small smooth instrument, and the surface of the endometrium examined. Changes in thickness of the endometrium cannot be ascertained without resorting to biopsies but it is free to grow out of the opened uterus if it is so inclined. However, in such preparations the growth produced in the endometrium by daily injections of 10 fjig. estradiol for periods of 2 or 3 weeks is not sufficient to show any tendency what- ever to grow out through the fistular open- ing or obstruct examination of the walls of the uterus. The limited growth observed in these experiments is in agreement with that obtained with estrogen on intact and incised uteri. The cervix uteri of the rhesus monkey is sufficiently long to make it possible to bring the entire fundus to the exterior through a midal)dominal incision. Advantage of this was taken in an attempt to exteriorize the uterus and maintain it outside the body for long enough periods to make it possible to study the growth responses of the endome- trium (Hisaw, 1950). These preparations did not i)rove satisfactory in all respects but they did contribute a number of interesting observations. The operational i^rocedure used in these experiments involved dividing the uterus transversely from fundus to cervix so that the anterior wall was deflected downward and the posterior wall upward (Fig. 9.7). The endometrium of the exteriorized uterus is difficult to maintain but with proper care it seems to retain its normal condition for at least the first few days after the uterus is opened. Small localized areas of ischemia can be seen to come and go, probably action of the coiled arteries, and there is a periodic general blanching of the endometrium as- sociated with rhythmic contractions of the muscularis. This, however, does not seem true of the whole endometrium. A zone sur- rounding the internal os of the cervix tends to retain its blood-red color even during strong contractions of the uterus and the growth reactions of the endometrium in this area are of particular interest. 564 PHYSIOLOGY OF GONADS Fig. 9.7. Exteriorized uteii. Tlie uteri were divided transversely from fundus to cervix. The anterior half is seen deflected to the right and the posterior half to the left. A and B are of the same uterus taken 13 days after exteriorization showing the "blush" and "blanch" reaction of uterine contractions. It can be seen that during blanching the endometrium of the cervix does not become ischemic. C. Uterus 18 days after exteriorization, showing response to estrogen. Ridges formed by growth of the endometrium surrounding the in- ternal OS of the cervix can be seen at the upper edge of the photograph. D, taken 83 days after exteriorization, shows response produced by a series of injections of 10 ^ig. estradiol and 1 mg. progesterone daily. The transverse ridge is formed by the two opposed lips of endometrium derived from the area surrounding the internal os of the cervix. Growth when the two hormones are given is greater than when only estrogen is injected. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.) The endometrium on the exposed anterior and posterior halves of the uterus underwent deterioration despite the best of care that could be given, but that surrounding the in- ternal OS of the cervix survived and retained its capacity to grow, in one animal, for as long as 9 months. When estrogen was given this endometrium grew rapidly and within a few days stood out as large elliptic lips surrounding the internal os (Fig. 9.7). Within 2 to 3 weeks the lips appeared to reach their full size and further growth was slow or absent. When estrogen treatments were discontinued the endometrial lips un- derwent bleeding within a few days and were entirely lost. At no time were activities observed that could be ascribed to coiled arterioles, nor did ischemia occur during in- volution previous to bleeding. It seems that the response of this tissue to estrogen is like that found in other experiments but the ab- sence of ischemia preceding bleeding is ex- ceptional. The endometrium on the anterior and posterior walls of uterine fistulae in- variably showed ischemia for several hours before active bleeding following the with- drawal of estrogen. Markee (1940) approached the problem of endometrial growth in monkeys by study- ing the changes that occur in bits of endo- metrial tissue transplanted to the anterior chamber of the eye. Such transplants retain in large measure the normal morphology of endometrial tissue and changes in their cy- clic growth parallel those going on simul- taneously in the uterus. So much so that if the animal has an ovulatory cycle, the ocu- lar implants show conditions characteristic of both the follicular and luteal phases, but if ovulation fails to occur then the luteal phase is omitted. Also, the morphologic events taking place at menstruation can be seen and recorded, since the transplants re- gress and bleed at each menstrual period. ESTROGEN AND PROGESTERONE 565 These ingenious experiments will be referred to often in the course of our discussion but at present the response of endometrial transplants in the eye to estrogen is of pri- mary interest. Monkeys having ocular transplants were given 200 to 300 R.U. of estrone daily for about 1 to 3 months. The transplants did not grow to a certain size and then remain stationary, but instead periods of rapid growth were interrupted by periods of re- gression which usually involved a marked decrease in size, and if regression was ex- tensive and rapid, bleeding ensued. It also was found that these episodes of regression in the transplants were usually accompanied by a decrease in the size of the uterus. Comparisons between the results of these experiments and those we have discussed previously may be misleading since it seems that only 1 of the 5 animals (no. 295) used was castrated. Also, the dosage of estrogen was not sufficient to maintain the endome- trium of the uterus for an indefinite period without bleeding and this also was reflected in the transplants. It seems questionable that the growth capacity of endometrial transplants in the eye can be determined un- less sufficient estrogen is given to prevent bleeding in the uterus. Therefore, it would seem that these experiments contribute less to an analysis of the effects of estrogen on endometrial growth than they do to an un- derstanding of the events that precede and accompany menstruation. In summary, it seems clear that the out- standing effect of estrogen on the uterus of the monkey is one of growth (Allen, 1927, 1928). The involuted uterus of a castrated animal can be restored to its normal size in 2 or 3 weeks by daily injections of adequate amounts of estrogen. At this time there is an increase in vascularity, a clear-cut hyper- emia as seen in rodents. There also is secre- tion of luminal fluid (Sturgis, 1942) but this does not distend the uterus as in the mouse and rat. This is accompanied by an increase in tissue fluid, especially in epithelial tissues (surface epithelium and glands) , and in the connective tissue of the stroma. Glycogen may be present at the basal ends of epithe- lial cells beneath the nuclei (Overholser and Nelson, 1936) but it apparently is not read- ily released under the action of estrogen alone (Lendrum and Hisaw, 1936; Engle and Smith, 1938) . The glands of the endo- metrium maintain a straight tubular struc- ture with some branching near the muscle layers. The condition produced experimen- tally in the monkey's uterus by short term treatments with estrogen is equivalent to that present in the normal animal at mid- cycle, or even a few days later if ovulation does not occur. If, however, an estrogen treatment is con- tinued for several months conditions de- velop in the uterus that are not found during the follicular phase of a normal men- strual cycle. When the daily dose of estrogen is small menstruation occurs at intervals during the treatment (Zuckerman, 1937b) and probably marks periods of endometrial regression as observed by Markee (1940) in eye transplants, but if the dosage is in- creased by a sufficient amount (about 10 /xg. estradiol- 17/3 daily) injections may be continued for a year or longer without bleeding. Although the size of the uterus re- mains within the range of normal variation as the injections are continued, the myome- trium tends to increase in thickness and the endometrium becomes thinner, a condition not corrected by further increases in dosage or by prolonging the treatment. The cause responsible for the limited response of the endometrium under these conditions is not known but apparently is not a restrictive in- fluence of the myometrium as similar re- sponses are given when the endometrium is exposed by incising the uterus, in abdominal fistulae, and in exteriorized uteri. III. Effects of Progesterone on the Uterus It has been mentioned that a menstrual cycle, in which ovulation occurs, can be con- veniently divided into a follicular and a luteal phase. The follicular phase extends from menstruation to ovulation and the luteal phase from ovulation to the following menstruation. It has been shown in the previous discussion that the endometrial modifications characteristic of the follicular phase of the cycle can be duplicated in a castrated monkey by the injection of estro- gen. Likewise, the progestational condition characteristic of the luteal phase can be de- veloped by giving progesterone. In fact, all 566 PHYSIOLOGY OF GONADS the morphologic and physiologic features that are known for anovulatory and ovula- tory cycles can be reproduced in castrated monkeys by estrogen and progesterone. If one designs an experiment to simulate the normal cycle in a castrated monkey then estrogen should be given first to de- velop the conditions of the follicular phase followed by progesterone for the progesta- tional development of the luteal phase. Ex- perience has shown that this is the most ef- fective procedure for the production of a progestational endometrium. Progesterone, as compared with estrogen, is a weak growth jH'omoter and although it can produce pro- gestational changes in the atrophic endome- trium of a castrated monkey when given in large doses, its action is greatly facilitated when preceded by estrogen. The first ex- periments in which progesterone was used for this purpose were planned on this prin- ciple (Hisaw, Meyer and Fevold, 1930; Hi- saw, 1935; Engle, Smith and Shelesnyak, 1935). The first noticeable effect of progesterone is an elongation of the epithelial cells of the surface membrane and necks of the glands. When the treatment is continued, this ef- fect progresses down the gland towards the base. This change is followed closely by a rearrangement of the nuclei which is more pronounced in the glands than in the surface epithelium. The nuclei under the influence of estrogen in doses which reproduce the conditions of the follicular phase of a nor- mal cvcle, are situated niostlv in the basal Fig. 9.8. Uterus of a castratefl monkey which was given 2 mg. progesterone daily tor 113 days. The endometrium is thin but bleeding occiu\s when such treatment is stopped. The myometrimn is soft and pliable and ilir l)lood vessels are cnlarsed and have thick wails. half of the cells, some of them touching the basement membrane. The nuclei retreat from the basement membrane when proges- terone is given leaving a conspicuous clear zone. This zone is produced by intracellular deposits of glycogen. These early changes usually appear before pronounced spiraling and dilation of the glands. Secretion begins in response to estrogenic stimulation and increases greatly as pro- gestational changes are established. It ap- pears first in the necks of the glands and progresses basalward. The surface epithe- lium takes a less conspicuous part in secre- tion and is usually reduced to a thin mem- brane when injections of progesterone are continued until a fully developed progesta- tional endometrium is established. This pro- gressive action of progesterone is such that it is possible to find all conditions in a single gland from active secretion and fraying in the neck region through primary swelling to an unmodified condition at the base. When treatment is continued for 25 to 30 days at doses of about 2.0 mg. daily, the glands enter a state that has been called "secretory exhaustion" (Hisaw, 1935). This condition also is seen first in the necks of the glands and progresses toward the base. The glandular epithelium decreases in thickness, and active secretion, as judged by fraying of the cells, is absent. The glands may become narrow and straight and the endometrium may resemble that in castra- tion atrophy. These involutionary changes become even more pronounced if the treat- ment is continued for several months or a year (Fig. 9.8). The endometrium by this time is extremely thin. The glands are straight, short, and narrow, and the stroma very dense. The myometrium is thick in pro- portion to the endometrium and the uterine blood vessels are large and have greatly thickened walls. Such uteri tend to be some- what smaller than normal and are soft and pHable. Thus, it is seen that when growth is pro- duced in tlie endonietiiuni of a castrated monkey by giving estrogen and then con- tinued on injections of progesterone, there follows a sequential development of all stages of the luteal phase of a normal men- stiual cycle terminating in secretory ex- haustion. However, this condition cannot ESTROGEN AND PROGESTERONE 567 be maintained by continuing the progester- one treatment, and involutionary processes set in and the endometrium is reduced to a thin structure. Yet, such degenerate endo- metria are dependent upon progesterone and will bleed within about 48 hours if the injections are stopped. It also was found that after discontinuence of progesterone daily injections of 10 /i.g. estradiol may not prevent bleeding. IV. Synergism between Estrogen and Progesterone There is considerable evidence that in primates progesterone under normal condi- tions rarely if ever produces its effects in the absence of estrogen. Large quantities of es- trogen are present in human corpora lutea (Allen, Pratt, Newell and Bland, 1930) and during pregnancy the placenta secretes es- trogens as well as progesterone (Diczfalusy, 1953) . This apparently is a common feature of primates, as indicated by the excretion of estrogens in the urine of pregnant chimpan- zees and rhesus monkeys (Allen, Diddle, Burford and Elder, 1936; Fish, Young and Dorfman, 1941 ; Dorfman and van Wa- genen, 1941). Also, correlated with this is the observation that estrogen and progester- one when given concurrently produce a greater effect on the uterus of castrated monkeys than either alone (Hisaw, Greep and Fevold, 1937; Engle, 1937; Hisaw and Greep, 1938; Engle and Smith, 1938) and that an ineffective dose of progesterone is greatly potentiated by estrogen. This syn- ergistic effect of the two hormones on the uterus of monkeys is quite different from their action on the uteri of laboratory ro- dents and rabbits. In these animals the ef- fects of progesterone can be inhibited quite easily by a surprisingly small dose of es- trogen (see chapter 7). The synergism between estrogen and pro- gesterone in the promotion of endometrial growth can be demonstrated to best advan- tage under the conditions of some of the physiologic preparations that have been dis- cussed. For instance, it was shown (Fig. 9.5) that growth of the endometrium under the influence of estrogen was not enhanced by relieving muscle tension by a midline in- cision through the anterior wall of the uterus. Now, if a similar operation is per- FiG. 9.9. Uterus of a castrated monkey that re- ceived 10 fig. estradiol and 1 mg. progesterone daily for 18 days, at which time the uterus was opened from fundus to cervix and most of the endometrium of the anterior wall removed. The incision was not closed and the treatment was continued for an additional 20 days. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.) formed on the uterus of a monkey that is receiving 10 /tg. estradiol daily and the treatment continued with the addition of a daily dose of 1 mg. progesterone, there usu- ally follows a rapid growth of endometrial tissue out through the incision until by about 3 weeks a mass is formed which ap- proximates the size of the entire uterus (Fig. 9.9). If this experiment is repeated and the same dosage of progesterone is given with- out estrogen, there is no outgrowth of the endometrium (Fig. 9.10). A similar synergistic action can be seen in utero-abdominal fistulae. We have men- tioned that estrogen does not cause excessive growth of the endometrium under these con- ditions. However, endometria that have reached their maximal response to estrogen will show a resumption of growth if 1 or 2 mg. progesterone are added daily to the treatment. By the 4th or 5th day lobes of blood-red endometrium begin to protrude 568 PHYSIOLOGY OF GONADS Fig. 9.10. Uteru.s of a castrated monkey which was given 1 mg. progesteione (lail>' for 18 days following an estrogen treatment. The uterus was opened as described for Figure 9.9, and the injec- tions of progesterone continued for 20 days. (From F. L. Hisaw, in A Syiyiposium on Steroid Hor- mones, University of Wisconsin Press, 1950.) through the opening of the fistula. Within a few days tongue-like processes of endome- trial tissue are thrust out of the opening with each uterine contraction and are en- tirely or i^artially withdrawn at each re- laxation. Such outgrowths are difficult to protect from mechanical injury and consequent tis- sue loss so it is not possible to determine ac- curately how much endometrium is pro- duced in a given time. In one experiment an animal was kept on 10 fxg. estradiol and 2 mg. progesterone daily for 98 days and it was found that the endometrium continued to grow, but the rate seemed considerably slow^er toward the conclusion of the treat- ment than at the beginning. How long an endometrium would continue to grow under these conditions was not determined, but it is obvious that much more endometrial tis- sue was produced by the treatment than is ever found at one time in the uterus of a monkey during a normal menstrual cycle. This takes on added significance when it is compared with the endometrial response in the intact uterus of an animal given the same dosage of estrogen and progesterone for a similar length of time. The progestational development of the endometrium, when both hormones are given, passes through the same stages as those following the injection of only proges- terone; i.e., presecretory swelling of the glandular epitlielium, active secretion, and secretory exhaustion. The endometrium, however, is considerably thicker than when a comparable dose of progesterone is given alone, and secretory exhaustion may not be so pronounced by the 30th day (Fig. 9.11). The glandular epithelium in the necks of the glands may be reduced to a thin mem- brane scarcely thicker than the nuclei whereas some secretion is usually present in the dilated basal parts of the glands. Also dilation of the glands in the basalis is more pronounced following a 30-day estro- gen-])rogesterone treatment than when the same amount of progesterone is given sepa- rately. Secretory exhaustion appears to be the initial indication of an involutionary proc- ess that ensues when an estrogen-progester- one treatment is continued for a long time (Hisaw, 1950). When a combination of the two hormones, known to be capable of pro- ducing a large uterus with a thick, fully de- velojied, progestational endometrium within al)out 20 days, is given for 100 days, an astonishingly different endometrium results (Fig. 9.12). It is thin, the stroma is dense and the narrow straight glands are reduced to cords of cells in the basal area. The con- dition is one suggesting inactivity and at- rophy. When such dosages of estrogen and i)ro- gesterone are given to castrated monkeys for 200 days or a year further changes in the endometrium occur. By 200 days the epithelium of the surface mucosa and glands Fk;. 9.11, .\ late i)r()ges1ati()iial condition pro- duced in tlie endometrium of a castrated monkey by giving 10 (ig. estradiol daily for 18 days followed by 10 /xg. estradiol and 2 mg. progesterone daily for 31 davs. ESTROGEN AND PROGESTERONE 569 Fig. 9.12. The endometnuin of a castrated monkey that had received 10 /xg. estradiol and 1 mg. progesterone daily for 99 days. is lost except for small glandular vestiges along the musciilaris at the base of the en- dometrium. There are no glands, coiled ar- teries, or large blood vessels in what one might yet call the functionalis. All that re- mains is a modified stroma that resembles decidual tissue (Fig. 9.13.4 and B). It is also of interest that these endometria will men- struate if the treatment is discontinued and in most if the injections of progesterone are stopped and estrogen continued, but not if estrogen is stopped and progesterone con- tinued. Even though in such experiments the en- dometrium has been under the influence of both estrogen and progesterone for a year and has undergone extremely abnormal modification, it yet is capable of responding to estrogen in a more or less characteristic way when progesterone is stopped and in- jections of estrogen continued. Apparently within about three weeks the modified endo- metrium is replaced, under the influence of estrogen, by one that has few glands which tend to be cystic, a mesenchymatous stroma, and no coiled arteries (Fig. 9.14). Under similar circumstances, if estrogen is stopped and jjrogesterone is continued, the modified endometrium is lost without bleed- ing and there is almost no repair of the en- dometrium even after a period of 3 weeks. There seems to be an incompatability be- tween the epithelial outgrowths from the mouths of the glands and the underlying stroma of the denuded surface. Conse- quently the epithelium crumbles away and epithelization of the raw surface is not ac- complished (Fig. 9.15j. How long this con- dition could continue has not been deter- mined. 570 PHYSIOLOGY OF GONADS Fk;. 9.13. The endometrium shown m .4 is (h;i( from a castraled mdiik.N wlml, l,a,l received 10 /xg- estradiol and 2 mg. progesterone daily for 200 days. In B, jiart of ilie endo- metrium of a snndai animal given the same treatment for 312 days is shown at a higher magnification. The endometrium is almost entirely a modified stroma in which glandular epithelium and coiled arteries are absent. Only vestiges of glands are present in the basal area next to the myometrium. One of the most interesting aspects of these observations is that these effects were jiroduced by dosages of estrogen and pro- gesterone that are very probably within the range of normal physiology. From this it appears that although growth of the endo- metrium is greater when the two hormones are given together, due to their synergistic interaction, this does not prevent involu- tionary changes from setting in when the treatment is continued for a period of weeks or months. In fact, greater damage to the endometrium occurs under the simultaneous action of the two hormones than when either is given alone. Also, increasing the dose in- tensifies the damaging action of both estro- gen and progesterone, so much so that very large doses will almost completely destroy the endometrium. The myometrium, however, shows a dif- ferent response to these treatments. Estro- gen stimulates myometrial growth, which is Fir;. 9.14. Uterus of a castrated monkey which was given 10 ixg. of estradiol and 2 mg. pro- gesterone daily for 307 days at which time the injections of progesterone were stopped and estro- gen continued for 20 days. Bleeding occurred the second day following discontinuance of progester- one. The absence of coiled arteries and the pres- ence of cystic glands and a mesenchymetous stroma characterize the endometrium. ESTROGEN AND PROGESTERONE 571 Fig. 9.15. Uterus of a castrated monkey which was given 10 yug. estradiol and 2 mg. pro- gesterone daily for 275 days at which time estrogen was stopped and progesterone was con- tinued for 21 days. A shows the thin endometrium and dense stroma whereas B shows failure of formation of a surface epithelium following the loss of the modified functionalis pre- sumably present at the conclusion of treatment with both hormono.^^ (see Fig. 9.13). intensified both by cln-onic treatment and high dosage, and seems to be equally ef- fective when it is given alone or in combina- tion with progesterone. Progesterone also promotes growth of the muscularis but seems less effective than estrogen and dif- fers from it by causing pronounced thicken- ing of the walls of the arcuate blood vessels. These vascular changes extend to the coiled arteries of the endometrium, which are also affected by high dosages of estrogen. It seems remarkable that estrogen is capable of preventing the action of progesterone on the myometrial blood vessels and correcting such effects after they are produced and yet at the same time it assists in the destruc- tion of the coiled arteries in the endome- trium. V. Experimentally Produced Implantation Reactions Progestational endometria of the normal menstrual cycle or those produced in cas- trated monkeys by progesterone, if mechani- cally traumatized, will develop endometrial proliferations which seem identical with those found at normal implantation sites of fertilized ova (Figs. 9.16 and 9.171 (Hisaw, 1935; Hisaw, Creep and Fevold, 1937; Wis- locki and Streeter, 1938; Rossman, 1940). The proliferated cells originate from the surface and glandular epithelium and grow into the surrounding stroma. The reaction spreads from the point of injury and within a few days may involve the entire inner l)ortion of the endometrium bordering the lumen. The implantation plaques on the 3rd or 4th day present a fairly homogeneous ap- pearance but soon thereafter certain cells attain the proportions of giant cells and many are multinucleated. The development of the plaques is most rapid during the first week, by the end of which cell division is found only in the basal half of the proliferation and evidence of re- gression is seen in the superficial portion adjoining the uterine lumen. After 10 days degenerative and phagocytic processes are the dominant features and by 24 days the ut'prus contains few or no ]iroliferation cells. Wislocki and Streeter ( 1938,1 found that im- plantation plaques during pregnancy and those experimentally induced underwent ajj- l^roximately the same development arid subsequent degeneration except for modifi- cations produced by the invading troplio- PHYSIOLOGY OF GONADS :^a.- :^^--.^ tx: ^ . Fig. 9.16. An area of the normal implantation site of a developing ovum. (From Carnegie Institu- tion, No. C467.) Fig. 9.17. An experimentally induced implanta- tion reaction in a castrated monkey showing condi- tion 6 days after mechanical traumatization of the endometrium. blast. Rossman (1940j made an extensive morphologic study of these epithelial pro- liferations and concluded that they should be regarded as typical metaplasias \vith an embryotrophic function. VI. The Cervix Uteri The cervix uteri of the rhesus monkey is remarkable for its size and complexity. It forms a large segment that is set off from the fundus by a conspicuous constriction at the level of the internal os (Fig. 9.1). A sagittal section (Fig. 9.18) shows the cervi- cal canal not straight but thrown into sev- eral sharp turns by colliculi that extend from its walls into the lumen. The largest of these projects from the midventral wall. The functional advantage of such tortuosity of the cervical canal is not obvious but since the cervix probably serves as a barrier between the bacterial flora of the vagina and the corpus uteri, this may be a useful adap- tation. The physiology of the cervix has received much less attention than has been given the uterus. This is regrettable in view of the consideration it must receive in practical obstetrics and gynecology, as well as the possibility that physiologically the monkey cervix may be homologous with that of the human regardless of morphologic difTer- ences. Recent observations indicate that this is indeed quite probable. Fig. 9.18. Sagittal section of the cervix from a normal monkey. The vagina and the external os of the cervix are shown at the left and the entrance to the fundus is at the right. ESTROGEN AND PROGESTERONE 573 Fig. 9.19. Sagittal section of the cervix of a pregnant monkey showing conditions present just previous to parturition on the 154th day of gestation. The dominant features are dilation of the cervical canal and reduction of the cervical lips (shown at the left) and the coUiculi. (From Carnegie Institution, No. C713.) Hamilton (1949) made a detailed study of the changes in the cervix of rhesus mon- keys during the menstrual cycle, paying particular attention to alterations that took place in the cells of the surface epithelium of the endocervical canal and the cervical glands. It was found that heights of the cells showed consistent increases and de- creases during the cycle. The peaks came on the 3rd, 13th to 15th, and 22nd days, the greatest of these being the 14th day which is approximately the time of ovulation. It also was observed that, following a peak, secretion was associated with the decline. Attention was called b3^ Hamilton to the rather close correlation between the fluctua- tions in height of the cervical epithelium in monkeys and the fluctuations observed by Markee and Berg (1944) in the blood estro- gens of the human menstrual cycle. It was concluded that, if similar changes in estro- gen levels also occur in monkeys, one would be justified in concluding that the increase in cell height in the cervical mucosa was due to the action of estrogen and the sudden pe- riodic drops in blood estrogen caused secre- tion and consequent regression. However, it is not clear how this could account for the abundant secretion of the cervical glands in the presence of high levels of estrogen during late pregnancy (Fig. 9.19). Much has been learned regarding the physiology of the primate cervix from ex- periments on castrated monkeys. The cervi- cal mucosa is very responsive to estrogen and castration atrophy can be repaired and a normal condition maintained by daily in- jections of small doses. Cervical secretion may become abundant when an estrogen treatment is prolonged and especially if large doses are injected. However, the amount of secretion induced by estrogen never equals that of the last half of preg- nancy, and it usually subsides if the injec- tions are continued for several months. Under conditions of chronic treatments with estrogen metaplastic aberrations in- variably appear in the epithelium of the endocervix. This reaction was first reported in monkeys by Overholser and Allen ( 1933, 1935) and has been confirmed by many in- vestigators (Engle and Smith, 1935; Hisaw and Lendrum, 1936; Zuckerman, 1937c (. Similar lesions may be found in the cervix uteri of women (Fluhmann, 1954). They seem especially prone to occur under con- ditions characterized by excessive produc- tion of estrogen, such as hyperplasia of the 574 PHYSIOLOGY OF GONADS endometrium (Hellman, Rosenthal, Kistner and Gordon, 1954) and granulosa-cell tu- mors of the ovary. Various degrees of meta- plasia may occur in the cervix during preg- nancy both in the mother and newborn but Fluhmann (1954) did not find it as fre- quently as in nonpregnant women. This reaction to estrogen as seen in the cervix of castrated monkeys is initiated by growth of small undifferentiated cells below the columnar mucous cells of the secretory epithelium. Fluhmann (1954) suggests that these cells are really undifferentiated cells of the cervical mucosa which have the po- tentiality of becoming columnar or squa- mous or simply undergoing multiplication and remaining as indifferent or reserve cells. These cells accumulate, in response to es- trogen, to form aggregates of several cells in thickness and, although this may occur in any area of the endocervix, it is generally more pronounced below the base of the glands. As this process proceeds the colum- nar mucous cells are pushed outward and are finally desquamated thus exposing the underlying metaplastic cells to the lumen of the gland (Fig. 9.20). Fig. 9.20. Al.iaph in a castrated monkey that li.-id rci-civcd 1 nig. estriol daily for 48 days. The cells of these lesions undergo a char- acteristic differentiation. When first formed they are small, cuboidal, and have spheri- cal nuclei with dense chromatin. As they in- crease in number those in the center of the cellular mass become larger and acquire an eosinophilic cytoplasm. Such collections, as seen at the base of the cervical glands, may grow in height and form cone-shaped masses with the apexes protruding through the mu- cous epithelium into the lumen or they may remain as more or less compact structures. This difference in growth seems to have a general relation to the dosage of estrogen. Large doses cause more rapid growth and cone formation with the loss of cells from the apex either singly or in groups, whereas small doses produce slower growth and des- quamated cells are seldom seen in the lu- men. However, regardless of the rate of growth, the cells at the base of the lesion remain undifferentiated and continue as the principal area of cell proliferation. Pearl formation is occasionally seen and may be quite common in animals on low dosages of estrogen. Under strong estrogenic stimulation and consequently rapid growth, these structures apparently are desqua- mated before they are completely formed. However, very early stages are frequently seen and may even be present in small clumps of metaplastic cells, but they are more commonly found in the larger collec- tions at the base of the glands. Their ap- pearance is initiated by swelling and dis- integration of one or more adjacent cells that form a center around which epidermidi- zation takes place. Further development does not proceed under the influence of es- ti'ogen, beyond the formation of a small cen- tral cavity. The most conspicuous difference between the metaplastic growths produced by estro- gen and true cancer of the cervix in the monkey (Hisaw and Hisaw, Jr., 1958) is that the former remain noninvasive even when the treatment is continued well over a year. They also involute when the treat- iiiciit is discontinued and they do not ap- peal' when progesterone is given simultane- ously with estrogen. When the injections of progesterone are started after metaplastic growths have been formed in response to estrogen, further growth is inhibited and ESTROGEN AND PROGESTERONE the keratinized cells of the lesion become vacuolated and are lost. In contrast with the effects of estrogen on the cervix, the modifications that occur as pregnancy advances are remarkable. The cervix becomes a soft thin-walled structure, the glands increase in number, and their lumina become greatly enlarged, pressing the stroma into thin partitions between them, and the amount of mucus secreted is enormous (Fig. 9.19). Attempts at duplicat- ing these changes in castrated animals by hormone therapy have been only partially successful. Estrogen produces a solid thick- walled cervix that tends to be larger than normal, an effect that is especially notice- able in young animals. Progesterone does not promote cervical growth and repair of the glands unless large doses are given and even then there is little if any secretion. The best results were obtained when both estro- gen and progesterone were given and espe- cially so when relaxin was added to the treatment (see chapter by Zarrow). VII. The Vagina The general features of the vaginal smear of rhesus monkeys have been described by several investigators (Allen, 1927; Hart- man, 1932; Westman, 1932) and a detailed study of the cellular components at differ- ent times of the menstrual cycle has been made by Lopez Columbo de Allende, Shorr and Hartman (1945). The changes in the vagina of a monkey are in most respects like those found for the human being (Papa- nicolaou, Traut and Marchetti, 1948; Lopez Columbo de Allende and Orias, 1950). Epi- thelial growth and desquamation of corni- fied cells continue at all stages of the cycle but at various rates. The epithelium is thinnest at menstruation and gradually in- creases in thickness during the follicular phase, reaching a maximum at ovulation. At this time there is a well developed basal area in which numerous mitoses can be seen and from which many papillae or ''bulbs" extend into the underlying stroma. Above this is an intermediate zone, an interepithe- lial zone of cornification (so called Dierk's layer), and a heavily cornified outer zone (Fig. 9.21). Cellular proliferation is less rapid during the luteal phase and apparently cells are desquamated more rapidly than they are re- placed. Consequently there is a decrease in the thickness of the epithelium in the luteal phase which may include an almost com- plete loss of the cornified zone (Davis and Hartman, 1935). The effects are probably due to progesterone because similar changes are seen following the introduction of pro- gesterone into a treatment in which estrogen is being given. The vaginal epithelium of a castrated monkey is remarkably sensitive to estrogen. A small daily dose of 5 to 10 /xg. estradiol will stimulate growth of an atrophic epi- thelium of 4 to 8 cells in thickness to one of 60 or even 80 layers thick within 3 weeks. One of the first things that is noticed as the vaginal epithelium thickens is the numerous mitotic figures in the stratum germinativum followed by a marked increase in the num- ber of epithelial papillae along the base- ment membrane. This condition of rapid growth, cornification, and loss of cells into the vaginal lumen is typical of the follicular phase of the menstrual cycle and can be maintained indefinitely. l''i(.. U.21. Tlie vaginal epithelium of a castuUMJ monkej' showing growth antl cornification induced by estrogen. 576 PHYSIOLOGY OF GONADS Progesterone, in contrast with estrogen, does not produce rapid growth of the vagi- nal epithelium but at the same time it is not without an effect. The vaginal epithe- lium, weeks or months after castration, has relatively few papillae projecting from its basal border into the underlying stroma. When progesterone is given, this condition is changed but not in a spectacular way. There is very slow growth without cornifica- tion. The epithelium remains thin but the papillae become more numerous. These are mostly small epithelial buds which tend to remain solid but may show enlargement of the cells in their centers. When estrogen and progesterone are given concurrently, the effects of estrogen on the vaginal mucosa are modified. If an estrogen treatment has continued for a sufficient time to produce full cornification and then pro- gesterone is added, the first indication of an inhibition of estrogen is a decrease in mi- totic activity. This is followed by a con- tinuation of cornification and loss of cells faster than they are replaced ; consequently, most of the functionalis is lost and the epi- thelium becomes thinner. There is also a noticeable decrease in the intensity of corni- fication, which in the monkey is never as pronounced as in rodents, and under these conditions is quite incomplete, each cell re- taining a conspicuous nucleus. Partly corni- fied cells may be present for several weeks .-•^ss;^ 4* ' V ^ § Fig. 9.22. ^^•tgi^al epithelium of a pregnant mon- key showing condition on the 154th day of gesta- tion. (From Carnegie Institution, No. C713.) when both estrogen and progesterone are given, but eventually they almost entirely disappear and the epithelium attains a con- dition resembling that of late pregnancy. The inhibitory effect of progesterone on the action of estrogen is shown perhaps even better when a castrated monkey having a fully involuted reproductive tract is first given progesterone for a few days and then (>strogen is added to the treatment, or when injections of the two hormones are started at the same time. In such experiments es- trogen has little effect on the vaginal mucosa even in doses that would produce marked cornification if given alone. These observa- tions show that a fully cornified vaginal epithelium cannot be produced or main- tained by estrogen when an effective dosage of progesterone is included in the treatment (Hisaw, Greep and Fevold, 1937). Estrogens and progesterone are the domi- nant hormones of gestation and their simul- taneous action is reflected by the changes in the vaginal epithelium. The fully corni- fied vagina, present at the time of ovulation, is gradually modified as pregnancy pro- gresses into a condition strikingly like that seen in experiments when estrogen and pro- gesterone are given concurrently. In late pregnancy the most striking feature of the thin, uncornified epithelium is the presence of numerous epithelial buds extending deeply into the underlying stroma. They may branch and rebranch and along their course there is conspicuous enlargement of the more centrally situated cells among which cavities ai^pear, enlarge, and join each other (Fig. 9.22). It seems quite prob- able that this process may be of considerable importance in increasing the diameter of the vagina. VIII. Sexual Skin A so-called sexual skin is jiresent in most catarrhine monkeys, is not found in platyr- iliine monkeys, and among the anthropoids occurs regularly only in the chimpanzee I l^]ckstein and Zuckcrman, 1956). Changes in the sexual skin during the menstrual cy- cle have been observed most extensively in the monkey (Macaca), the baboon (Papio), and the chimpanzee (Pan). The sexual skin of t!ie baboon and chimpanzee undergo jiro- ESTROGEN AND PROGESTERONE 577 nounced swelling during the follicular phase of the cycle. A maximal size is attained by the middle of the cycle followed by a rapid regression and loss of edema which at least in the baboon is associated with a marked increase in the output of urine (Gillman, 1937a; Krohn and Zuckerman, 1937). The subsidence of the sexual skin begins approxi- mately at the time of ovulation and remains in the reduced condition throughout the luteal phase, followed by a subsequent ini- tiation of swelling during or soon after menstruation (Zuckerman, 1930, 1937e; Zuckerman and Parkes, 1932; Gillman and Gilbert, 1946; Young and Yerkes, 1943; Nissen and Yerkes, 1943). A w^ell developed sexual skin is present in the monkey {Macaca mulatta) only dur- ing adolescence. With the appearance of the menstrual cycles the sexual skin under- goes a process of maturation into the adult condition in which cyclic changes in edema are absent and the most noticeable feature is a vivid red color. Such coloration is due to vascular engorgement rather than pig- ment (Collings, 1926) and involves the perineum, the buttocks, and may extend for various distances down the legs and over the symphysis pubis. The development and maturation of the sexual skin have been de- scribed in considerable detail by several in- vestigators (Hartman, 1932; Zuckerman, van Wagenen and Gardiner, 1938) . The sexual skin has been of considerable interest both as to the nature of its re- sponsiveness to ovarian hormones and the manner in which its grossly visible changes during the menstrual cycle parallel events occurring in the reproductive tract. The sudden loss of edema at the conclusion of the follicular phase not only signals ovula- tion but also raises the question as to whether the loss of tissue fluid is due to a decrease in estrogen or is the direct effect of progesterone. The importance of this becomes obvious when it is considered that a similar process also goes on simultaneously in the endometrium and raises the question again as to the respective roles played by estrogen and progesterone in endometrial growth and menstruation. That the development and edema of the sexual skin of adolescent rhesus monkeys depend on the ovaries was first demon- strated by Allen ( 1927 ) . Involution and loss of color follow castration, and the normal condition can be restored by the injection of estrogen. Also, when estrogen treatment is continued for several weeks maturation of the sexual skin occurs and a condition char- acteristic of that in the adult is established (Zuckerman, van Wagenen and Gardiner, 1938). The genital area loses its edema and develops a brilliant red color which is re- tained as long as estrogen is administered. Once this mature condition is established the response of the sexual skin to subse- quent estrogen treatments is limited to a change in color. Similar experiments have been performed on the chacma baboon, Papio porcarius (Parkes and Zuckerman, 1931; Gillman, 1937b, 1938, 1940a). The large sexual skin of these animals is very responsive to es- trogen and development equal to that of the follicular phase of the menstrual cycle can be readily induced by daily injections for about 2 weeks. However, the perineal swell- ing of the baboon differs from the sexual skin of the genital area of the rhesus mon- key in that it does not "mature" under the influence of estrogen. When large doses of estrogen are given to a rhesus monkey a generalized edema of the skin occurs beyond the genital area. This first appears as deeply indented swell- ings along the sartorii from groin to knee, and next appears at the base of the tail and spreads gradually upward until it involves the entire dorsal portion of the trunk. At the same time, the skin of the face, scalp, and supraorbital ridges becomes swollen and finally the edema may extend out on the arms and down the legs to the ankles (Bach- man, Collip and Selye, 1935; Hartman, Geschickter and Speert, 1941). A daily dose of 500 /xg. or more of estriol or estradiol w^ll produce this condition within 2 to 3 weeks and, when the treatment is con- tinued for an extended period the effect tends to subside. Progesterone has a strong inhibitory ac- tion on the effects produced by estrogen on both the genital and extragenital sexual skin of the monkey. If daily injections of progesterone are added to the treatmeiu after full development of the sexual skin has been induced by estrogen, there is a 578 PHYSIOLOGY OF GONADS noticeable loss of edema by the 4th or 5th day followed by rapid involution and re- duction of the turgid folds of skin to loose, flabby wrinkles within about 10 days. When estrogen and progesterone are given con- currently to a castrated monkey from the beginning of treatment edema does not ap- pear but the sexual skin regains its normal color. In fact, progesterone alone, like es- trogen, can restore the color to the sexual skin of castrated adult monkeys (Hisaw, Greep and Fevold, 1937; Hisaw, 1942). The interaction of estrogen and i)ro- gesterone on the sexual skin of rhesus mon- keys can best be demonstrated by the reac- tion of the skin of the sexual area in adolescent animals. The most striking effect and probably the most important is the sequence of events initiated by a single dose of progesterone when given to an ani- mal on continuous estrogen treatment. Un- der such treatment a full response of the sexual skin is obtained by the end of 20 days. If at this time 1 mg. progesterone is given in a single dose and the estrogen treatment continued uninterruptedly, the first indication of an effect of the luteal hor- mone is a slight loss of edema and color of the sexual skin on the 4th or 5th day there- after. The sexual skin is markedly reduced by the 8th day, almost gone by the 9th, and at the end of about a fortnight regains its ability to respond to estrogen as shown by a return of color and swelling. However, the most remarkable eventuation of such treat- ment is menstruation which usually begins on about the 10th day (Hisaw, 1942). Involution of the sexual skin and men- struation following a single injection of progesterone also have been produced in the baboon by Gillman (1940a). He found that 5 mg. progesterone, when given on the 8th day of a normal menstrual cycle, would cause an appreciable loss of edema of the swollen perineal sexual skin by the day after injection. This was followed by a progres- sive involution of the perineum until the 13th day and swelling was re-initiated by the end of the 15th day. Reduction of the sexual skin at this dosage of progesterone was not associated with menstruation. How- ever, when tlie dose was increased to 20 mg. both deturgescence of the sexual skin and menstruation occurred. These effects pro- duced by progesterone in the presence of endogenous estrogen have much in common with those described above as occurring in castrated monkeys on continuous estrogen treatments. IX. Menstruation An experimental ai^proach to the physi- ology of menstruation dates from the ob- servations of Allen (1927) that uterine bleeding would occur in castrated monkeys following the discontinuance of an estrogen treatment. He suggested that normal men- struation is due to a fluctuation in estrogen secretion and proposed the "estrogen-with- drawal" theory to account for the observed facts. This concept led to an extensive in- vestigation of the effects of estrogens on the endometrium and of conditions that modify their action. It was soon found that in both castrated monkeys and human beings there was a quantitative relationship between the dosage of estrogen given and the mainte- nance of the endometrium. Bleeding oc- curred during treatment when the daily dose of estrogen was small, but with larger doses a point was reached at which the injections could be continued for months or even years without bleeding (Werner and Collier, 1933; Zuckerman, 1937b, d). Estrogen also will inliihit i)ostop('rative bleeding which usually follows total castra- tion, provided the ovaries are removed be- fore or soon after ovulation (Hartman. 1934). With the advent of a corpus luteum and development of a progestational endo- metrium it becomes progressively more diffi- cult, following castration, to prevent men- struation by injecting estrogen. Similar results are obtained when estrogen is given during a normal menstrual cycle. Small doses may not prevent the onset of men- struation, but if continued, subsequent men- strual periods are delayed (Corner, 1935). Large doses when given during the luteal phase of the cycle do not disturb the normal menstrual rhythm, but may do so if the treatment is started during the follicular phase (Zuckerman, 1935. 1936a). Progesterone, in contrast with estrogen, will prevent menstruation from an endome- trium representative of any stage of the nor- mal cycle. It will delay onset of the next men- ses even when the treatment is started only ESTROGEN AND PROGESTERONE 579 a few days before the expected menstrua- tion (Corner, 1935; Corner and Allen, 1936) . Also, the bleeding that invariably follows the discontinuance of a long treatment with estrogen can be inhibited indefinitely by giving progesterone (Hisaw, 1935; Engle, Smith and Shelesnvak, 1935; Zuckerman, 1936b). An impression held by many of the ear- lier investigators was that progesterone could not produce its effects on the primate endometrium unless it w^as preceded by the action of estrogen. It is true, of course, that progesterone is a comparatively weak growth promoter and its effects can be dem- onstrated to best advantage on an endo- metrium that has been developed by estro- gen. However, Hisaw, Greep and Fevold (1937) produced a progestational endome- trium in a monkey that had been castrated 242 days previously by giving synthetic progesterone. Also, the endometrium of this animal was found capable of forming a decidual plaque upon traumatization. Soon afterwards Hartman and Speert (1941) ob- served menstruation following the with- drawal of progesterone in castrated monkeys that had not been given estrogen and more recently similar results have been reported by Eckstein ( 1950) . At the same time it has l)een found that progesterone will induce menstruation in women suffering from amenorrhea and also that uterine bleeding can l)e jirecipitated l)y similar treatment (hiring tlie follicular j^hase of the cycle (Zondek and Rozin, 1938; Rakoff, 1946). These observations have been confirmed and extended by Krohn (1951; 1955) who finds that menstrual bleeding can be in- duced in monkeys wdth secondary amenor- rhea by the injection of 5 daily doses of progesterone. Progesterone (5 mg. daily for 5 days) also precipitates uterine bleeding in castrated monkeys at intervals of about 8 days provided the treatment is started innnediately a menstrual bleeding has been induced either by removel of the ovaries or withdrawal of estrogen. The most in- teresting aspect of these observations is that the number of short 8-day cycles that can be obtained in this way in a castrated animal seems to be related to the size of the initial dose of estrogen used to induce with- drawal bleeding. This also applies to pro- gesterone-withdrawal bleeding, so the ef- fect does not depend upon the particular hormone used to obtain the bleeding. It also is of interest that such conditioning of the endometrium to subsequent responses to the 5-day treatments with progesterone may last for several months on a continuous re- gime. It is surprising that such a series of responses cannot be initiated unless the first injection of progesterone is given within 6 days following the initial withdrawal bleeding. These observations have much in common with those of Phelps (1947) who also studied the influence of previous treat- ment on experimental menstruation in mon- keys. There seems to be a quantitative relation- ship between the dosage of progesterone given in combination with estrogen and the ability of estrogen to prevent bleeding after the injections of progesterone are stopped. It has been mentioned that once a fully developed i^rogestational reaction has been produc(Hl l)y progesterone, it is extremely difficult, if not impossible, to inhibit men- struation by giving estrogen following the withdrawal of progesterone. However, Hi- saw and Greep (1938) found that pro- gestational endometria produced ijy small doses of estrogen plus api^roximately 0.5 mg. progesterone daily for 18 to 21 days did not bleed following progesterone with- drawal when continued on 10 to 20 times the original dosage of estrogen. In fact, such endometria were brought back to a condi- tion typical for the action of estrogen and again transformed into a presecretory pro- gestational state without the intervention of bleeding. Similar observations were made previously by Zuckerman (1936a, 1937d). These experimental results give grounds for some doubt as to the adequacy of the estrogen-withdrawal theory to account fully for menstruation. Not only can progesterone bring about menstruation without the in- tervention of estrogen but other steroid hor- mones are capable of pi'oducing similar ef- fects. Desoxycorticosterone in large doses can inhibit estrogen-withdrawal bleeding in castrated monkeys (Zuckerman, 1939, 1951 ) and induce phases of uterine bleeding in rapid succession in normal monkeys (Krohn, 1951). So too can testosterone pre- vent estrogen-withdrawal bleeding (Hart- 580 PHYSIOLOGY OF GONADS man, 1937; Engle and Smith, 1939; Duncan, Allen and Hamilton, 1941) and inhibit pro- gesterone-withdrawal bleeding as well (En- gle and Smith, 1939). Testosterone also will precipitate bleeding during an estrogen treatment (Hisaw, 1943) and in normal monkeys if given early in the cycle (Krohn, 1951). Just what specific action these com- pounds have in common that enables them to produce these effects or whether there are different modes of action that lead to the same results is not known, but, before men- tioning certain possibilities, it may be help- ful to consider information regarding the influence of estrogen-progesterone interac- tions on menstruation. Among the most significant observations regarding primary causes of menstruation are a few indications that there may be an intrinsic difference in the ways in which es- trogen and progesterone produce their ef- fects on the endometrium. One of the first indications of this was the discovery that a short series of injections of progesterone during treatment with estrogen will precipi- tate menstruation (Corner, 1937; Zucker- man, 1937d; Hisaw and Greep, 1938). This can be demonstrated by giving a castrated monkey a maintenance dose of estrogen daily for 2 or 3 weeks, then adding a daily injection of progesterone for 5 to 10 days and continuing the estrogen treatment. As a rule bleeding appears within 2 or 3 days after stopping progesterone. The most in- teresting point brought out by such experi- ments is that bleeding can occur under these conditions in the presence of an otherwise maintenance dosage of estrogen. Perhaps the most surprising as well as most important fact brought out by subse- quent experiments was the small amount of progesterone required to bring about bleed- ing under these conditions. It was found that only a single injection of 1 mg. was required for animals on chronic treatment with a maintenance dose of estradiol (1000 LIT.) and some bled when 0.5 mg. progesterone was given (Hisaw, 1942). The sequence of events following the injection of progester- one can be seen to best advantage in an adolescent monkey whose sexual skin also respond" to the estrogen treatment. When the 1 mg. ]irogesterone is given on the 20th day of estrogen treatment the edema of th(^ sexual skin will have attained its maximal development. The first indication of an ef- fect of progesterone is a slight loss of edema and color of the sexual skin which appears on the 4th or 5th day and by the 9th or 10th day the edema is almost gone and the sexual skin is pale. Blood apjiears in the vaginal lavage between the 7th and 10th days, of about 70 per cent of the animals on this dos- age. The sexual skin may remain markedly reduced and pale until about the 15th day after which both color and edema rapidly return. These effects can also be seen when 1 mg. progesterone is given for a series of days. However, neither the time of appear- ance nor loss of edema of the sexual skin is significantly hastened, and if the injections extend over no more than 5 days the time between the first injection and bleeding re- mains approximately the same. Similar observations have been made by Gillman and Smyth (1939) on the South African baboon iPapio porcarius). They found that 3 mg. or more of progesterone when given in a single injection during the follicular phase of the cycle would cause the relatively enormous perineal swellings to pass rapidly through deturgescence and reach a flabby resting condition within 5 to 7 days, and after a delay of about 24 hours once again begin to swell. As much as 10 or 15 mg. in a single dose caused perineal de- turgescence without bleeding, whereas 20 mg. in a single dose or a total of 15 mg. if divided into 2 or 3 injections and given at 3 or 4 day intervals, produced both detur- gescence and bleeding (Gillman, 1940b). The l)aboon diff"ers from the monkey in that larger doses of progesterone are required to produce the effects and the sexual skin does not ''mature" on repeated treatments and lose its responsiveness; otherwise the basic physiology of the reaction in both animals seems to be the same. The most important fact l)rought out by these experiments is that the effects of a single injection of progesterone can continue in the presence of estrogen for as long as 10 to 15 days. It is highly imi^robable that pro- gesterone lingers in the body for so long a time (Zarrow, Shoger and Lazo-Wasem, 1954). In general it is considered the most ephemeral of the sex steroids and is prob- ablv inactivated within at least a few hours ESTROGEN AND PROGESTERONE 581 after it is administered. It seems more lilvely that progesterone modifies the sexual skin in a way that renders it unresponsive to es- trogen and that about a fortnight is required to recover the original condition. This takes on added significance when the possibility is considered that effects similar to those seen in the sexual skin might also be going on simultaneously in the uterine endometrium. An appreciable dehydration of the endometrium occurs just previous to menstruation (van Dyke and Ch'en, 1936) and a loss of interstitial fluid before bleed- ing has been observed in endometrial im- plants in the eyes of monkeys and described in detail by Markee (1940) . This was shown by periodic regression in size and compact- ness of the grafts which resulted in a de- crease in area of 25 to more than 75 per cent. Because cyclic changes in endometrial grafts in the eye are correlated with events of the menstrual cycle there is reason to be- lieve that similar reactions were going on in the endometrium of the uterus. Endometrial regression, as described by Markee, did not always lead to menstrua- tion although it invariably preceded, accom- panied, and followed menstrual bleeding. Menstruation occurred only when regression was rapid and extensive. This was seen in the endometrial grafts in the eye during a normal menstrual cycle at the time of in- volution of a corpus luteum and during an anovulatory cycle soon after the involution of a large follicle. It also begins soon after the last of a series of injections of estrogen or i^'ogesterone. A slow decrease in size of the ocular grafts, without concomitant bleeding, can be induced in castrated mon- keys by gradual withdrawal of estrogen, and when estrogen is given in amounts that are inadequate for maintaining the endome- trium for an extended period the "break through" bleeding that eventually ensues is preceded by a rapid and extensive endome- trial regression. Because this reaction also occurs w^hen menstruation is induced by such an unusual procedure as spinal transection (Markee, Davis and Hinsey, 1936), it prob- ably is a phenomenon that always precedes menstruation. It seems from these observations that the changes in the endometrium preceding men- struation are initiated by a sudden with- drawal of a stimulus on which the endome- trium at the time relies for the maintenance of a particular physiologic condition, and bleeding and tissue loss are incidents that occur during the readjustment necessary for the return to an inactive state. What this in- volves is only partly known, but an under- standing of the initial changes in the endo- metrium that usher in menstruation most certainly holds the explanation of the real cause. This has been a perennial subject for discussion and many suggestions and the- ories have been set forth in an extensive literature to account for various aspects of menstruation. Among the more recent gen- eral discussions are those by Zuckerman (1949, 1951), Corner (19511, and Zondek ( 1954 ) . The estrogen- withdrawal or estrogen-de- privation theory proposed by Edgar Allen has received more attention than any other. From what has been mentioned earlier it is clear that this theory can account for uter- ine bleeding subsequent to the discontinu- ance of a series of estrogen injections and also perhaps menstruation at the conclusion of an anovulatory cycle. However, it is not so obvious as to how this theory can explain the occurrence of menstruation at the close of the luteal phase of a normal cycle. Estro- gen in large doses will not inhibit such bleeding, but it is postponed if progesterone is given. It is equally difficult to see how this theory is helpful in accounting for the fact that a small dose of progesterone will pre- cipitate bleeding in the presence of a main- tenance dosage of estrogen. As little as 2 /xg. progesterone will induce bleeding when applied topically to the endometrial lips of an exteriorized uterus (Fig. 9.7) in a mon- key that is receiving 10 fig. estradiol daily (Hisaw, 1950). Uterine bleeding precipitated by admin- istering progesterone during an estrogen treatment has been explained on the grounds that progesterone in some way interferes with the action of estrogen on the endome- trium. Therefore, it is assumed that an ani- mal receiving both estrogen and progester- one is in a sense "deprived" of estrogen. That is, when the two hormones are given simultaneously, progesterone itself is capa- ble of maintaining the endometrium with- out bleeding; but when it is stopped, the 582 PHYSIOLOGY OF GONADS suggestion is that the animal is physiologi- cally deprived of estrogen and literally de- prived of progesterone (Corner, 1951). Al- though this view is in descriptive agreement with the observed facts the idea of the in- hibitory effect of progesterone does not take into consideration the synergistic interac- tion of the two hormones on the endome- trium. The physiologic function of progesterone is the conversion of an estrogen-endome- trium into a progestational endometrium suitable for receiving and nourishing a de- veloping blastocyst. Such an endometrium is adapted for this specific reproductive function and accordingly its physiologic na- ture must be quite different from that of the follicular phase of the cycle. Indeed, it is known that these two structures (follicular and luteal phase endometrial are morpho- logically and biochemically unlike in a num- ber of respects. This gradual transformation, following ovulation, occurs as progesterone becomes the dominant hormone, and conse- quently, as this proceeds, the endometrium progressively loses competence to respond to estrogen. However, this does not imply that estrogen is without effect in the general economy of the progestational endometrium. It has been shown in a number of ways that the action of progesterone on the primate endometrium is greatly facilitated by the presence of estrogen. In fact, it seems prob- able that rarely if ever does progesterone perform its function in the absence of estro- gen (Hisaw, 1959; chapter by Zarrow). After consideration of the endometrial specializations brought about by ]irogester- one, it seems rather jwintless to hark back to the follicular phase and inject the past recoi'd of accomjilishments and prerogatives that estrogen had at that time into tlie ex- planation of an entirely different hormonal situation. It seems more in keeping with the facts to state outright that menstruation following the involution of a corpus luteum or the discontinuance of progesterone, even though estrogen is present, is due to a de- crease or absence of progesterone. It also has become less certain that men- struation at the conclusion of an anovula- tory cycle is really an estrogen-withdrawal bleeding. This is possible, of couisc, but at the same time the exceedingly small amount of progesterone required to induce bleeding in the presence of estrogen makes it difficult to be sure what the situation might be. Even a negative test for progesterone in the blood, by our present methods, does not necessarily indicate the absence of a physiologically ef- fective amount of progesterone. Zarrow, Shoger and Lazo-Wasem (1954) found that in rabbits an intramuscular injection of 40 mg. progesterone was required to produce an appreciable concentration of the hor- mone in the blood as determined by the Hooker-Forbes method. Yet, 0.2 mg. pro- gesterone daily for 5 days will produce a progestational reaction in the uterus equiva- lent to that of the 5th day of normal pseudo- pregnancy. In monkeys 0.5 mg. daily when given with 10 fxg. estradiol is an adequate dosage of progesterone to induce unques- tionable progestational changes in the en- dometrium and much less will cause bleed- ing. These observations indicate that the minimal effective concentration of proges- terone in the blood may be less than is pos- sible to detect by our present methods. This also seems to hold for the human be- ing. Estimates of secretion and metabolism of progesterone in the human being have been based primarily on the recovery of its excretory product sodium pregnanediol glu- curonidate in the urine. It seems obvious that such determinations must be only gen- eral approximations because only about 20 per cent of the progesterone secreted or in- jected can be accounted for by the pregnane- diol in the urine. Also, it is generally known that a physiologically effective dosage of progesterone does not necessarily lead to the excretion of pregnanediol (Hamblen, Cuylcr, Powell, Ashley and Baptist, 1939; Seegar, 1940). In other words, the threshold dose of progesterone for endometrial stimulation is l)elow that at which the hormone is excreted as pregnanediol. In fact, it has been sug- gested by some investigators that there is no quantitative relationship between the l)rogesterone present in the blood and the pregnanediol excreted in the urine (Buxton, 1940; Sommerville and Marrian, 1950; Kaufmann, Westphal and Zander, 1951). These findings and the wide variation in the amount of prc'gnancdiol excreted during ESTROGEN AND PROGESTERONE 58.3 a menstrual cycle (Venning and Browne, 1937) suggest that, even in the absence of ovulation, sufficient progesterone may be present to influence menstruation. There also is the possibility of progestational hor- mone from some extra-ovarian source, such as the suprarenal cortex. This was suggested by Zuckerman (1937b, 1941 j as a possible explanation for periodic bleeding in mon- keys on a constant submaintenance dose of estrogen. This thought becomes more plausi- ble in view of the fact that progesterone is one of the precursors in the metabolic syn- thesis of androgens, estrogens, and adrenal cortical steroids (Dorfman, 1956). Also, it has been shown that desoxycorticosterone acetate is converted to progesterone in vivo (Zarrow, Hisaw and Bryans, 1950). There- fore, progesterone is not restricted to ovar- ian luteal function but instead is of rather general occurrence in the body and the likelihood is that small amounts are a con- stant constituent of the blood. Also, the amount of progesterone from extra-ovarian sources may fluctuate, as sug- gested by Zuckerman (1949), and conse- quently disturb the normal menstrual rhythm and probably cause bleeding in monkeys on a continuous submaintenance dose of estrogen. However, as to the latter, there is an alternative explanation. Cas- trated monkeys on a continuous treatment of 10 fxg. of estradiol daily do not show "break-through" bleeding, and a synergistic effect on growth of the uterus is seen when 0.5 mg. or more of progesterone daily is in- troduced into the treatment. However, the simultaneous administration of 0.25 mg. or even 0.125 mg. progesterone daily in similar exj^eriments results in bleeding between a!)out the 10th to 16th day of the combina- tion treatment. Thus, a dosage of progester- one less than that required for synergism or prevention of bleeding when given alone, modifies the endometrium so that it can no longer be maintained by 10 fxg. estradiol daily (Hisaw, Jr., unpublished). When it is considered that the endometrium becomes increasingly dependent on estrogen during a chronic treatment, even after maximal growth is attained (Hisaw, 1942), it seems plausible that the effectiveness of a dosage of estrogen only slightly alcove the thresh- old for bleeding may be decreased suffi- ciently by the endogenous progesterone from extra-ovarian sources to precipitate bleed- ing. Although it is obvious that the normal menstrual cycle is primarily under the con- trol of the ovarian estrogens and progester- one, it is also equally clear that menstrua- tion is not due to a specific hormonal action. Experimental evidence indicates that any natural or synthetic compound having the capacity for promoting growth or sustaining an existing metabolic state in the endome- trium is also capable of inducing with- drawal bleeding. However, this does not im- ply that all compounds capable of inducing menstruation do so by the same biochemical action; in fact, there is considerable evi- dence that this is not so (see chapter by Villee). Yet in each instance a series of events is set in motion that leads up to ac- tive bleeding. X. The Mechanism of Menstruation The immediate cause and mechanism of menstruation has continued to be a topic of special interest for many years and the sub- ject of frequent general discussions. A gen- eralization in keeping with our present knowledge is that no gross morphologic fea- ture of the endometrium is distinctive of menstruation. A menstruating endometrium may be representative of any stage of the follicular or luteal phase of the cycle. The most frequently discussed hypothesis re- garding the mechanism of menstruation is that proposed by Markee (1940, 1946) which is based on direct observations of vascular changes in endometrial grafts in the anterior chamber of the eye of monkeys (see p. 564). The changes observed in the endometrium shortly before bleeding are, briefly, as follows. (1) There is extensive and rapid regression of the endometrium due to loss of ground substance from the stroma (Fig. 9.23). (2) The rapid regression brings about a disproportion between the length of the coiled arteries and thickness of the en- dometrium with the formation of additional coils. (3) The increased coiling of the ar- teries retards the circulation of blood through them and their branches. This stasis begins 1 to 3 davs before the onset of the 584 PHYSIOLOGY OF GONADS Repair Fig. 9.23. A diagram indicating correlated changes in ovary and endometrium during an ovulatory cycle of rhesus monkey. Thickness of endometrium, density of stroma, gland form, and three types of arteries are indicated. There is a gradual rise in thickness up to the time of ovulation, and a brief decline followed by development of the luteal or progesta- tional phase with accumulation of secretion in the glands due to relaxation of the myometrium. This is followed by loss of ground substance from the stroma, which is the primary factor in the premenstrual regression of the ischemic phase. This is a prelude to extravasation and shedding of tissue. Incidentally, secretion is extruded and glands col- lapse. There is further regression throughout the phase of menstruation. More than the basal zone (coarse stipple) survives menstruation. During repair, thickening of the endo- metrium is associated with increase in ground substance in the stroma and growth in the glands. (From G. W. Bartelmez, 1957, Am. J. Obst. & Gynec, 74, 931-955, 1957, with some modification of description.) flow, and is associated with leukocytosis in the endometrium. (4) The portion of the coiled arteries located adjacent to the mus- cularis constricts 4 to 24 hours before the onset of the flow. This vasoconstriction per- sists throughout the menstrual period ex- cept when individual coiled arteries relax and blood circulates through them for a few minutes. Markee postulated that the im- mediate cause of menstruation under these conditions was the injurious effect of anox- emia upon the tissues of the endometrium l)rought about by mechanical compression and constriction of the coiled arteries. Therefore, the coiled arteries and their mod- ifications become the central feature upon which the theory is based. Although this offers an explanation for many of the facts, it falls short in that now it is known that menstruation can occur in the absence of coiled arteries. Kaiser (1947) showed that no spiral arteries are present in the endometrium of three species of South American monkeys known to menstruate. He also found that the coiled vessels of the endometrium could be destroyed almost completely by giving large doses of estrogen and yet bleeding followed estrogen with- drawal. Several experimental conditions under which the coiled vessels of the endometrium are destroyed have been mentioned in the present discussion and in each instance bleeding invariably followed withdrawal of the supporting stimulus. The extremely atrophic endometrium present at the conclu- sion of a prolonged treatment with proges- terone (Fig. 9.8) will bleed when the injections are stopped, and if estrogen in- jections are started immediately thereafter the endometrium that develops is normal with the exception of the absence of coiled arteries; even so, it also will bleed when the treatment is stopped. Even a more ESTROGEN AND PROGESTERONE 58c drastic destruction of endometrial struc- tures occurs when both estrogen and proges- terone are given for several months. Not only are the coiled arteries destroyed but also the glands and the luminal epithelium. All that remains is a modified stroma pene- trated by a few small blood and lymph ves- sels and scattered glandular rudiments along the myometrium (Fig. 9.13). Yet, in spite of this, bleeding follows discontinuance of the treatment. These observations prove conclusively that the spiral arteries of the endometrium do not hold the solution to the menstrual process. However, the descriptive account by Markee of the events that take place in the endometrium during the cycle remains one of the major contributions to our knowl- edge of the primate endometrium. Phelps (1946) also made a very careful study of the vascular changes in intraocular endometrial transplants in ovariectomized monkeys re- ceiving estrogen and progesterone, and con- cluded that the primary function of the coiled arteries is concerned with vasculari- zation of the implantation site of a develop- ing embryo. There also is reason for doul^ting that ischemia is a determining factor in the menstrual process. That constriction of the endometrial vessels does occur is well estab- lished, but that tissue destruction and bleed- ing are consequences of prolonged anoxemia may be questioned. The endometrium around the internal cervical os as seen in incised exteriorized uteri (Fig. 9.7) contains very few coiled arteries and does not take part in the periodic blushing and blanching of the fundus, but instead remains blood-red even during menstruation. Also, certain tongues of endometrium in a uterine fistula may become crowded by their neighbors to an extent of being partly or completely de- prived of blood, yet they do not bleed even though their unfavorable situation leads to deterioration within a few days. Emmel, Worthington and Allen (1941) attempted to induce menstruation in mon- keys by operative ischemia. Circulation to the fundus of the uterus was interrupted by means of a tourniquet for periods of 1 to 8V4 hours, and in two instances for 19 hours. This procedure did not precijiitate uterine bleeding nor did it hasten the onset of an expected bleeding following estrogen with- drawal. In fact, when the uterus was de- prived of blood for periods longer than 3 hours impairment of the bleeding response to estrogen withdrawal was observed, and 19 hours of ischemia caused atrophy of the uterus without bleeding. It also has been reported that a toxic sub- stance formed in the endometrium is re- sponsible for menstruation. This menstrual toxin is supposed to be present in the endo- metrium just previous to and during men- struation, and to be a substance resembling or identical with necrosin, a material found in pleural exudate following an inflamma- tory reaction (Smith and Smith, 1951). Zondek (1953) reports that menstrual blood, when obtained under relatively sterile con- ditions, is no more toxic to experimental animals than sterile tissue extracts. He also found that death of animals given injections of menstrual blood was due to bacteremia, an effect that could be prevented by giving antibiotics. Nor was he able to demonstrate a toxic substance in the premenstrual or menstrual endometrium. It might be men- tioned in this connection that endometrial tissue destroyed by experimental ischemia in the experiments by Emmel, Worthington and Allen (1941), obviously did not influ- ence menstruation nor did involuting endo- metrial tissue in uterine fistulae (p. 564). Therefore, the presence of a specific toxin that may induce menstruation has not been conclusively demonstrated. Regardless of the specific cause of men- struation, the evidence shows that it can occur in the absence of coiled arteries, en- dometrial glands, or surface mucosa, and is unrelated to the thickness of the endome- trium. This statement is based on condi- tions that have been experimentally induced in the monkey and they strongly indicate that menstruation, whatever the cause, is a stromal phenomenon. This view seems to be in agreement with the observations reported by Bartelmez in his elegant studies of the morphology of the endometrium of both monkeys and the human being. He empha- sizes changes taking place in the connective tissue elements of the stroma and points out that much less tissue is lost at menstruation 586 PHYSIOLOGY OF GONADS than i.< commonly thought (Bartehnez, 1957). The reduction in thickness is clue pri- mariiy to loss of ground substance from the stroma, and conversely, the outstanding feature of repair is the increase in stromal ground substance (Fig. 9.23). ^Mitoses are rarely seen in the stroma during repair and arc not abundant enough in any phase ac- cording to Bartelmez to account for the ob- served increase in thickness of the endome- trium. Our present knowledge indicates that an explanation of menstruation may be found in the metabolic effects induced in the stromal connective tissue of the endome- trium by a sudden withdrawal of a support- ing hormonal stimulus. XI. References Allen, E. 1927. The menstrual CA'cle in the mon- key, Macacus rhesus: observations on normal animals, the effects of removal of the ovaries and the effects of injections of ovarian and placental extracts into the .spayed animals. Contr. Embrvol., Carnegie Inst. Washington, 19, 1-44. Allen. E. 1928. 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XL The part played by oestrogenic hormone in the men- strual cycle. Proc. Roy. Soc, London, ser. B., 123,441-471. ZucKER.MAN, S. 1937c Effects of prolonged oes- trin-stimulation on the cervix uteri. Lancet, 1, 435-437. Zuckerman, S. 1937d. Further observations on endocrine interaction in the menstrual cvcle. J. Physiol., 89, 49-51. Zuckerman, S. 1937e. The duration and phases of the menstrual cycle in Primates. Proc. Zool. Soc London, ser. A., 1937, 315-329. Zuckerman, S. 1939. The effect of sex hormones, cortin, and vasopressin on water-retention in the reproductive organs of monkeys. J. Endo- crinol., 1, 147-155. Zuckerman, S. 1941. Periodic uterine bleeding in spayed rhesus monkeys injected daily with constant threshold dose of oestrone. J. Endo- crinol., 2, 263-267. Zuckerman, S. 1949. The menstrual cvcle. Lan- cet, 2, 176. Zuckerman, S. 1951. The hormonal basis of uter- ine bleeding. Acta endocrinol., 7, 378-388. Zuckerman, S., and P.arkes, A. S. 1932. The menstrual cycle of the primates. V. The cycle of the baboon. Proc Zool. Soc. London, 1932, 139-191. Zuckerman, S., van W.agenen, G., and Gardiner, R. H. 1938. The sexual skin of the rhesus mon- key. Proc Zool. Soc, London, ser. A., 108, 385-401. 10 THE MAMMARY GLAND AND LACTATION A. T. Cowie and S. J. FoUeij NATIONAL INSTITUTE FOR RESEARCH IN DAIRYING, SHINFIELD, READING, ENGLAND I. Introduction I. Introduction 590 II. Development of the Mammary Gland 591 A. Histogenesis 591 B. Normal Postnatal Development . 593 1. Methods of assessing mammary development 593 2. Mammary development in the nonpregnant female 594 3. Mammary growth in the male . . 595 4. Mammary development during pregnancy 596 5. Mammary involution 598 C. Experimental Analysis of Hormonal Influences 598 1. Ovarian hormones in the animal with intact pituitary 598 2. Anterior pituitary hormones. . . 601 3. Metabolic hormones (corticoids, insulin, and thyroid hormones) 604 III. Endocrine Influences in Milk Se- cretion 606 A. Anterior Pituitary Hormones 606 1. Initiation of secretion (laeto- genesis) 606 2. Maintenance of milk secretion — galactopoiesis 609 3. Suckling stimulus and the main- tenance of lactation 611 B. Hormones of the Adrenal Corte.x . . 612 C. Ovarian Hormones 613 D. Thyroid Hormones 617 E. Parathyroid Hormone 618 F. Insulin 619 IV. Removal of Milk from the Mammary Glands: Physiology of Suckling AND Milking 619 A. Milk-Ejection Reflex 619 B. Role of the Neurohypophysis 621 C. Milk-Ejection Hormone 622 D. Effector Contractile Mechanism of the Mammary Gland 623 E. Inhibition of Milk Ejection 624 F. Neural Pathways of the Milk-Ejec- tion Reflex 625 G. Mechanism of Suckling 626 V. Relation between the Reflexes Concerned in the Maintenance of Milk Secretion and Milk Ejection 627 VI. Pharmacologic Blockade of the Re- flexes Concerned in the Main- tenance OF Milk Secretion and Milk E.tection 630 VII. Conclusion 632 VIII. References 632 This account of the hormonal control of the mammary gland is in no way intended as an exhaustive treatment of mammary gland physiology, but rather an attempted synthesis of current knowledge which it is hoped will be of interest as an exposition of the authors' conception of the present status of the subject. Since the publication of the second edition of this book, the emphasis in the field under review has tended to shift towards the development of quantitative techniques for assessing the degree of mam- mary development, towards attempts at a ])enetration into the interactions of hor- mones with the biochemical mechanisms of the mammary epithelial cells, and towards an increasing preoccupation with the in- terplay of nervous and endocrine influences in certain phases of lactation. The reader's acquaintance with the classical foundations of the subject as described in the second edition of this book (Turner, 1939) and in other subsequent reviews (Follcy, 1940; Petersen, 1944, 1948; Folley and Malpress, 1948a, b; Mayer and Klein. 1948, 1949; Follev, 1952a, ]9r)6; Dabelow. 1957) will 590 MAMMARY GLAXD AND LACTATION 591 therefore be assumed and used as a point of departure for the present account which can most profitably be concerned mainly with developments which have occurred since the last edition was published. Refer- ence will freciuently be made to these re- views in which authority will be found for the many ex cathedra statements that will be made, but original sources will be cited wherever appropriate.^ As an aid to logical treatment of the sub- ject the scheme of classification proposed by Cowie, Folley, Cross, Harris, Jacobsohn and Richardson (1951) will be followed in this chapter. Besides introducing a system of terminology in respect of the physiology of suckling or milking, these writers have put forward a classification scheme which is an extension of one previously proposed by one of the present authors (Folley, 1947). This scheme considers the phenom- enon of lactation as divisible into a number of phases as follows: [ [Milk synthesis I Milk secretion ■! Passage of milk from I I the alveolar cells Lactation Fig. 10.2. Relative mammary gland growth in the female hooded Norway Cowie. J. Endocrinol.. 6, 145-157, 1949.) (From A.T. analysis were made in the rat by Cowie (1949) and Silver (1953a, b) and in the mouse by Flux (1954a, b), and their results will now be summarized. In the rat the total mammary area increased isometrically with the body surface (a = 1.1 as compared with the theoretic value of 1.0) until the 21st to 23rd day when a phase of allometry (a = 3.0) set in. The onset of the allometric phase could be prevented by ovariectomy on the 22nd day (see Fig. 10.2). Since estrous cycles do not begin until the 35th to 42nd day in this strain of rat, it is clear that the rapid extension of the mammary ducts be- gan well before puberty. In the immature male rat the increase of mammary area on body surface was slightly but significantly allometric; this was not altered by castra- tion at the 22nd day. Earlier ovariectomy, i.e., when the pups were 10 days old, was followed by a phase of slightly allometric growth of the mammary glands in the fe- males (a = 1.5). With regard to the female mouse (CHI strain) a i)hase of marked al- lometry in mammary duct growth set in about the 24th day (a = 5.2) which could also be prevented by prior ovariectomy. It is clear that the presence of the ovary is essential for the change from isometry to allometry, but the nature of the mechanisms governing the change is still uncertain (for further discussion, see Folley, 1956). 3. Mammary Growth in the Male The testes have apparently little effect on mammary duct extension in the rat inas- much as the gland in the male grows iso- metrically or nearly so and its specific growth rate is unaffected by castration. Cas- tration at 21 days, however, does prevent for a time development of the lobules of al- veoli, first described by Turner and Schultze (1931 ) , which are characteristic of the mam- mary gland in the male rat. Eventually. 596 PHY,SI(3L0GY OF GONADS however, some alveoli do develop in the mammae of immaturely castrated male rats (Cowie and Folley, 1947d; Cowie, 1949; Ahren and Etienne, 1957) and it has been ])Ostulated that these arise from the en- hanced production by the adrenal cortex of mammogenic steroids (androgens or proges- terone) due to the hormone imbalance brought about by gonadectomy (see Folley, 1956 L In a recent study, Ahren and Etienne (1957) have shown that the ducts and al- veoli in the mammary gland of the male rat are remarkable in that their epithelial lining is unusually thick, being composed of sev- eral layers of cells. It had been previously noted by van Wagenen and Folley (1939) and Folley, Guthkelch and Zuckerman (1939) that testosterone caused a thickening of the mammary duct epithelium in the monkey and sometimes papillomatous out- growths of epithelium into the lumen of the duct. It would thus seem that, although the hormone of the testis is capable of eliciting alveolar development, these alveoli and ducts differ from those occurring in the fe- male in the nature of their epithelium. It w^as further observed by Ahren and Etienne (1957) that in the castrated male rat the alveoli, which eventually developed, had a simple epithelial lining somewhat similar to that seen in the normal female rat, suggest- ing that, if the adrenals are responsible, the mammogenic steroid is more likely to be progesterone than an androgen. A study of considerable clinical interest is that of Pfaltz (1949) on the developmental changes in the mammary gland in the human male. The greatest development reached was at the 20th year; by the 40th year there occurred an atrophy first of the l)arenchyma and later of the connective tissue. In the second half of the fifth decade tliere was renewed growth of the paren- chyma and connective tissues. The hor- monal background of these changes and the possible relationship with prostatic hyjier- trophy are discussed by Pfaltz. (Further details of the microscopic anatomy of the mammary gland of the human male may be found in the studies by Graumann, 1952, 1953, and Dabclow, 1957.) 4- Mammary Development during Preg- nancy It has been customary to divide mam- mary changes during pregnancy into two phases, a phase of growth and a secretory phase. In the former there occurs hy- perplasia of the mammary parenchyma whereas, in the latter, the continued increase in gland size is due to cell hypertrophy and the distension of the alveoli with secretion (see Folley, 1952a j . Although it was realized that these two phases merged gradually, re- cent studies have confirmed earh^ reports {e.g., those of Cole, 1933; Jeffers, 1935) that a wave of cell division occurs in the mam- mary gland towards the end of parturition or at the beginning of lactation. Al'tman (1945) described a doubling in number of cells per alveolus, in the mammary gland of the cow at parturition, but the statistical significance of his findings is difficult to assess. More recently, how^ever, Greenbaum and Slater (1957a) found that the DNA content of the rat mammary gland doubled between the end of pregnancy and the 3rd day of lactation, a finding which they in- terpret as resulting in the main from hyper- plasia of the gland cells. Likewise in the mouse mammary gland, Lewin (1957) ob- served between parturition and the 4th day of lactation a great increase both in the DNA content of the mammary gland and in the total cell count. Studies on the factors controlling this wave of cell division are awaited with interest. Also associated with the onset of copious milk secretion is a con- siderable increase in cell volume and coinci- dent ally the mitochondria elongate and may increase in diameter (Howe, Richardson and Birbeck, 1956). Cross, Goodwin and Silver (1958) have followed the histologic changes in the mammary glands of the sow, by means of a biopsy technique, at the end of pregnancy, during parturition, and at wean- ing. At the end of pregnancy there was a ])i'()gr('ssi\-c' distension of the alveoli, tlie existing hyaline eosinoi)hilic secretion within the alveoli was gradually replaced by a ba- sophilic material, and fat globules appeared. At i)arturition the alveoli were contracted and their walls appeared folded (Fig. 10.3). MAMMARY GLAND AND LACTATION 597 Fig. 10.3. Sections of biopsy specimens from the mammary gland of a sow before and din-ing parturition. A. Six days before parturition: the mammary alveoh are small and con- tain a nongranular eosinophilic secretion. B. Two days before parturition: alveoli have in- creased in size and fat globules are conspicuous. C. Fifteen hours before parturition: alveoli are now distended with secretion which consists of an outer zone of eosinophilic material and fat globules, and a central zone of basophilic granular secretion. D. During parturition: alveoli contracted with folded epithelium and sparse secretion. (From B. A. Cross, R. F. W. Goodwm and L A. Silver, J. Endocrinol., 17, 63-74, 1958.) 598 PHYSIOLOGY OF GONADS 5. Mam /nary Involution The involutionary changes which occur in the mammary gland after weaning in vari- ous species were described in the previous edition of this book (Turner, 1939) and in a later review by Folley (1952a). Since that time, a few further studies have appeared. There is evidence that the course of the histologic changes in the regressing mam- mary gland may differ according to whether the young are weaned after lactation has reached its peak and is declining, or whether they are removed soon after parturition, when the effects of engorgement with milk seem to be more marked (see, for example, Williams, 1942, for the mouse). In rats whose young were weaned soon after par- turition Silver (1956) was able to re-estab- lish lactation provided suckling was resumed within 4 or 5 days; after that time irre- versible changes in the capillary blood sup- ply to the alveoli had set in. A further point arises from a study on the cow by Mosimann (1949) which indicates that the course of the regressive changes in a gland which has undergone one lactation only may differ from those seen in glands from muciparous animals. Oshima and Goto (1955) have used quantitative histometric methods in a study of the involuting rat mammary gland ; the values which they obtained for the per- centage parenchyma 7 to 10 days after re- moval of the young agree quite well with tiiose reported by Benson and Folley ( 1957b) for rats weaned at the 4th day and killed 9 days later. The biochemical changes occurring in mammary tissue during involution arc of some interest and have been studied in our laboratory by McNaught (1956, 1957). She studied mammary slices taken from rats whose young were removed at the 10th day and also slices from suckled glands, the es- cajie of milk from which was prevented by ligation of the galactophores, the other glands in the same animals remaining intact and serving as controls. Her results, some of whicli are summarized in Figure 10.4, sug- gest that functional changes which may be taken as indicative of involution (decrease in oxygen up-take, respiratory quotient (R.Q.), and glucose up-take; increase in lactic acid prcxUiction ) are seen as early as 8 to 12 hours after weaning. Continued suckling without removal of milk retards tlie onset of these changes, but only for some hours. Injections of oxytocin into the rats after weaning (see page 607) did not retard these biochemical changes. Essentially simi- hii' results were independently reported by Ota and Yokoyama (1958) and Mizuno and Chikamune (i958). C. EXPERIMENTAL ANALYSIS OF HORMONAL INFLUENCES 1. Ovarian Hortnones in the Animal with Intact Pituitary We shall see later (page 602) that the mammogenic effects of the ovarian hor- mones are largely dependent on the integrity of the a'nterior pituitary and thus to ana- lyze accurately the role of hormones in mam- mary development it is necessary to use hy- pophysectomized animals. Information of considerable academic and practical impor- tance has been obtained, however, from studies in the animal with intact pituitary and these we shall now consider. Early studies involving hormone adminis- tration pointed to the conclusion that estro- gens were in general resi)onsible for the growth of the mammary (hicts, whereas pro- gesterone was necessary for complete lobule- alveolar growth (see reviews, l)y Turner, 1939; Folley and Malpress, 1948a; Folley, 1952a). The foundation for i^liis general statement is now more sure, for as a result of experimental studies over the last 10 years, what seemed to be exceptions to this generalization have been shown to be other- wise. In some species (mouse, rat, guinea \)ig, and monkey) it is true that progester- one alone, if given in sufficiently large doses, will evoke duct and alveolar development in the ovariectomized animal, but this is prob- ably a pharmacologic rather than a physio- logic effect. There are great differences in the response of the mammary ducts to estro- gen and on this basis it has become usual to divide species into three broad categories (see FoUey, 1956). It is, however, necessary to add the warning that in the estrogen- tre.'ited spayed animal progesterone from the a(h'eiial eoiiex may synergize with the ex- ogenous estrogen (see Folley, 1940; Trentin and 1'ui'iier, 1947; Hohn, 1957) and it mav MAMMARY GLAND AND LACTATION 599 O2 Uptake G\ ucose uptake Lactic acid production. s 12 ■Hours Fig. 10.4. Oxygen uptake, respiratory quotient, glucose uptake, and lactic acid production of mammary gland slices from lactating rats killed at various times after weaning (A — A) and from rats in which svickling was maintained, but in which the galactophores of certain glands were ligatured (• •) to prevent the escape of milk, the nonligatured glands (O O) acting as controls. (Courtesy of Dr. M. L. McNaught.) be that the I'eal basis for the categories is to be found largely in differences in endoge- nous progesterone production by the adrenal cortex. The first category comprises those in which estrogens, in what are believed to be physiologic doses, evoke primarily and mainly duct growth; alveoli may appear, but only if high doses are given and the administration is prolonged. Examples of this class are the mouse, rat, rabbit, and cat. Silver (1953a), using the relative-growth technique, has obtained information on the levels of estrogen necessary for normal mammary duct growth in the nonpregnant rat. In the young ovariectomized rat, the normal mammary growth rate was best imi- tated by injecting 0.1 ;u,g. estradiol dipro- pionate every second day (from 21 days of age) and increasing the dose step- wise with body weight. In the ovariectomized mouse, Flux (1954a) found it necessary to give 0.055 /jLg. estrone daily to attain mammarv duct growth comparable with that obser\-( . i in intact mice. In the second category are those s]:»ecies (JOO PHYSIOLOGY OI-' GONADS in which estrogen in physiologic doses causes growth of the ducts and the lobule-alveoL^r system, the classical example being the guinea pig in which functional mammae can be developed after gonadectomy in either sex by estrogen alone. A recent study by Hohn (1957), however, strongly suggests that progesterone from the adrenal cortex participates in the effect. The earlier view, moreover, that complete mammary growth can be evoked in the gonadectomized guinea l)ig by estrogen alone (Turner and Gomez. 1934; Nelson, 1937.) does not find support in the recent study of Benson, Cowie, Cox and Goldzveig (1957), who, using both sub- jective and objective methods of assessing the degree of mammary development, found that over a wide dose range of estrone, fur- ther development of the mammary gland was obtained when jirogesterone was also administered; essentially similar conclu- sions have been reached by Smith and Rich- terich (1958). Also in this second category are cattle and goats in which, however, the male mammary gland is not equipotential with that of the female. The early studies on these species have been reviewed at length by FoUey and Malpress (1948a) and Fol- ley (1952a, 1956). Briefly it may be said that these studies clearly showed that estro- gen alone induced extensive growth of lob- ule-alveolar tissue of which the functional capacity was considerable although the milk yields in general were less than those ex- pected from similar animals after parturi- tion. The response to estrogen treatment was, moreover, very erratic. It was generally believed that the deficiencies of this treat- ment could be made good if progesterone were also administered, a view supported by the observations of Mixner and Turner (1943) that the mammary gland of goats treated with estrogens, when examined his- tologically, showed the i)resence of cystic alv(>oli, an abnormality which tended to disappear when jirogestcrone was also ad- ministered. When progesterone became more readily available, an extensive study of tlie role of estrogen and progesterone in mammary de- velopment in the goat was carried out (Cowie, Folley, ^lalpress and Richai'dson. 1952; Benson, Cowie, Cox, Flux and Folley, 1955). The mammary tissue was examined histologically and the procedure devised by Richardson (see page 594) used to estimate the area and "porosity" of the alveolar epi- thelium. The udders grown in immaturely ovariectomized virgin goats by combined treatment with estrogens and progesterone in various proportions and at different ab- solute dose levels were compared with ud- ders resulting from treatment with estrogen alone. As in the earlier observations of Mix- ner and Turner (1943) , histologic abnormal- ities were noted, the more widespread being a marked deficiency of total epithelial sur- face, associated with the presence of cystic alveoli, in the udders of the estrogen-treated animals. The addition of progesterone pre- vented the appearance of many of these ab- normalities and increased the surface area of the secretory epithelium. JMoreover, when estrogen and progesterone were given in a suitable ratio and absolute level the milk yields obtained were remarkably uniform as between different animals and the glan- dular tissue was virtually free from abnor- malities. Studies in the cow have been less exten- sive, but there is evidence that both estrogen and progesterone are necessary for complete normal mammary development (Sykes and Wrenn, 1950, 1951; Reineke, INIeites, Cairy and Huffman, 1952; Flux and Folley, cited by Folley, 1956; Meites, 1960). The case for the inclusion of the monkey in the present category has been strength- ened by the excellent monograph of Speert ( 1948) who has had access to more extensive material than many of the earlier workers whose results are reviewed by him (see also Folley, 1952a). The sum total of available evidence now justifies the conclusion that estrogen alone will cause virtually complete growth of the duct and lobule-alveolar sys- tems of the monkey breast. Extensive lobule- alveolar development in the monkey breast in response to estrogen is shown in Figure 10.5. The synergistic effect of estrogen and jirogesterone on the monkey breast has not yet been adequately studied, but from avail- able evidence it does not seem to be very dramatic. If it is permissible to argue from pi'iinates to man. it seems jiossible that coidd MAMMARY GLAND AND LACTATION 601 Fig. 10.5. Wliole mounts of breast of an ovariectomized immature female rhesus monkey before (left) and after (right) e.strogen treatment. (From H. Speert, Contr. Embrvol., Carnegie Inst. Washington, 32, 9-65, 1948.) the necessary experiments be done the human breast would show a considerable growth response to estrogen alone. Finally, in the third category are those species in which estrogen in physiologic doses causes little or no mammary growth. The bitch and probably the ferret seem to belong to this class (see Folley, 1956). There has been considerable discussion in the past regarding the ratio of proges- terone to estrogen optimal for mammary growth. Only recently, however, has this question been fully investigated in any spe- cies. Benson, Cowie, Cox and Goldzveig (1957) have shown that in the guinea pig the absolute quantities of progesterone and estrogen are the crucial factors in controlling mammary growth; altering the dose levels but maintaining the ratio gave entirely dif- ferent growth responses. In view of the varying ability of the different estrogens to stimulate mammary duct growth (Reece, 1950) it is essential in discussing ratios to take into consideration the nature of the estrogen used, a fact not always recognized in the past. 2. Anterior Pituitary Hormones Soon after the discovery by Strieker and Grueter (1928, 1929) of the lactogenic ef- fects of anterior iiituitarv extracts, it was shown that anterior i)ituitary extracts had a mammogenic effect in the ovariectomized animal and that the ovarian steroids had little or no mammogenic effect in hypophy- sectomized animals. C. W. Turner and his colleagues postulated that mammogenic ac- tivity of the anterior pituitary was due to specific factors which they termed "mam- mogens"; other workers, in particular W. R. Lyons, believed the mammogenic ef- fect was due to prolactin. The theory of spe- cific mammogens has been fully reviewed in the past (Trentin and Turner, 1948; Folley and Malpress, 1948a) and we do not propose to discuss it further for there is now little evidence to support it. Damm and Turner ( 1958) , while recently seeking new evidence for the existence of a specific pituitary mam- mogen, concur in the view expressed by Folley and Malpress (1948a) that final proof of the existence of a specific mam- mogen will depend on the development of l)etter assay techniques and the characteri- zation or isolation of the active principle. The mammogenic effects of prolactin were observed in the rabbit by Lyons (1942) who injected small quantities of prolactin directly into the galactophores of the suit- ably prepared mammary gland. IV'Iilk secre- tion occurred but Lyons also noted that the l)rolactin caused active growth of the alveo- 602 PHYSIOLOGY OF CIOXADS lar epithelium. Recently, Mizuno, lida and Naito (1955) and Mizuno and Naito (19561 have confirmed Lyons' observations on the mammogenic effect of intracluct injections of prolactin in the rabbit both by histologic and biochemical means (DNA estimations) and there seems little doubt that the pro- lactin is capable of exerting a direct effect on the growth of the mammary parenchyma, at least in the rabbit whose pituitary is in- tact. In the last 18 years much information on the role of the anterior pituitary in mam- mary growth has been obtained by Lyons and his colleagues in studies on hypophy- sectomized, hypophysectomized-ovariecto- mized, and hypophysectomized-ovariecto- mized-adrenalectomized (triply operated) rats of the Long-Evans strain. In 1943 Lyons showed that in the hypophysecto- mized-ovariectomized rat, estrogen + pro- gesterone + prolactin induced lobule- alveolar development, but the degree of development was less than that obtained in the ovariectomized rat with intact pitui- tary receiving estrogen and progesterone. When supplies of purified anterior-pitui- tary hormones became available the experi- ments were extended (Lyons, Li and Johnson, 1952) and it was shown that if somatotrophin (STH) was added to the hormone combination of estrogen -f pro- gesterone + prolactin, the degree of lobule- alveolar development obtained in the hy- pophysectomized-ovariectomized rat was much enhanced. The omission of prolactin from the hormonal tetrad prevented lobule- alveolar development from occurring. In the hypophysectomized-ovariectomized-ad- renalectomized rat the above hormonal tet- rad could also evoke lobule-alveolar devel- opment, provided the animals were given saline to drink (Lyons, Li, Cole and John- son, 1953). In yet more recent experiments Lyons, Li and Johnson (1958) observed that somatotrophin has a direct stimulatory ef- fect on duct growth, but in the hypophysec- tomized-ovariectomized rat, the presence of estrogen is also necessary to evoke normal duct development (Fig. 10.6a, b, c) ; Like- wise, in the triply operated rat, STH plus estrogen is mammogenic, but the presence of a corticoid is r('([ui]'ed to o])tain full duct de- velopment (Fig. 10.6r/). Lyons and his col- leagues were able to build up the mammary glands of triply operated rats from the state of bare regressed ducts to full prolactational lobule-alveolar development by giving es- trogen + STH + corticoids for a period of 10 days to obtain duct proliferation fol- lowed by a further treatment (for 10 to 20 days) with estrone + progesterone -I- STH -I- prolactin + corticoid to induce lobule- alveolar development. Alilk secretion could then be induced by a third course of treat- ment lasting about 6 days in which only prolactin and corticoids were given (Fig. 10. 6e, /). Essentially similar results have been obtained in studies with the hooded Norway rat (Cowie and Lyons, 1959). Studies on mammogenesis in the hypo- physectomized mouse have revealed some differences in the response of the mammary gland of this species in comparison with that of the rat and indications of strain differences within the species. The mam- mary gland of the hypophysectomized male weanling mouse of the Strong A2G strain shows no response to the ovarian steroids alone, to prolactin, or to STH alone, but it responds with vigorous duct proliferation to combinations of estrogen + progesterone + prolactin, or of estrogen 4- progesterone + STH (Hadfield, 1957; Hadfield and Young, 1958). In the hypophysectomized male mouse of the CHI strain slight duct growth occurs in response to estrogen + jirogesterone and this is much enhanced when STH is also given; the further addi- tion of prolactin then results in alveolar development (Flux, 1958). Extensive studies in triply operated mice of the C3H 'HeCrgl strain have been reported by Nandi (1958a, b). In this strain some duct growth was ob- served in triply operated animals in re- sponse to steroids alone (estrogen -I- pro- gesterone + corticoids), but normal duct develojmient was believed to be due to the action of estrogen + STH + corticoids, a conclusion in agreement with Lyons' ob- servations in the rat. Extensive lobule- ahcohii' development could be induced by a number of hormone coml)inations, one of the most effective being estrogen + pro- gesterone + corticoids + prolactin + STH, milk secretion occurring when the ovarian MAMMARY C5LAND AND LACTATION 603 Fig. 10.6. Typical areas of whole mounts of the abdominal mammary gland of rat.s after the following treatments: A. Untreated rat on day 31, 14 days after hypophysectomy. The gland has regressed to a bare duct system. B. Rat hypophysectomized and ovariectomized on day 30 and injected daily with 2 mg. somatotrophin (STH) for 7 days. Note the presence of end clubs, r. Rat treated as in B but which received, in addition to the STH, 1 ^g. estrone. Note profuse eiid-rhil' ] iroliferatiou. D. Rat li.\|M)]ili\s(>ctomized on day 30. ovariectomized and adri'nali^ctoinized on day 60, and injected daily from days 60 to 69 with 1 mg. STH + 0.1 mg. DCA + 1 fig. estrone. Note again the profuse number of end buds indicative of duct proliferation. E. Same treatment as in D followed by 10 days treatment with 5 mg. prolactin + 2 mg. STH + 1 /xg. estrone + 2 mg. progesterone + 0.1 mg. DCA + 0.05 mg. prednisolone acetate. Note excellent lobule-alveolar growth. F. Same treatment as in D followed by 20 days treatment with 5 mg. prolactin + 2 mg. STH + 1 fig. estrone + 2 mg. progesterone + 0.1 mg. DCA + 0.05 mg. prednisolone acetate; thereafter given 0.1 mg. pro- lactin locally over this gland and 0.1 mg. DCA + 0.1 mg. prednisolone acetate systemically for 6 days. Note fully developed lobules with ah'eoli filled with milk. (All glands at the same magnification.) (From W. R. Lyons. C. H. Li and R. E. Johnson, Recent Progr. Hormone Res., 14, 219-254, 1958.) 604 PHYSIOLOGY OF GONADS steroids were withdrawn, while the })rohic- tin, STH, and Cortisol were continued. A further interesting observation made by Nandi is that in the C3H/HeCrgl mouse STH can replace prolactin in the stimula- tion of all phases of mammary development and in the induction of milk secretion; en- hanced effects were obtained, however, when prolactin and STH were given together. Nandi also considers that progesterone plays a greater role in duct development in the mouse than in the rat. The above experiments clearly indicate that both in the triply operated rat and mouse, it is possible to build up the mam- mary gland to the full prolactational state by injecting the known ovarian, adrenal cortical, and anterior pituitary hormones. There would thus seem to be no necessity to postulate the existence of other uniden- tified pituitary mammogens. It must be recognized, however, that in normal preg- nancy the placenta may be an important source of mammogenic hormones. The pla- centa of the rat contains a substance or sub- stances possessing luteotrophic, mammo- genic, lactogenic, and crop-sac stimulating properties, but it is uncertain whether this material is identical with pituitary prolactin (Averill, Ray and Lyons, 1950; Canivenc, 1952; Canivenc and Mayer, 1953; Ray, Averill, Lyons and Johnson, 1955). There is also some evidence of the presence of a somatotrophin-like principle in rat placenta (Ray, Averill, Lyons and Johnson, 19551. 3. Metabolic Hormones {Corticoids, Insulin, and Thyroid Hormones) We have already noted that Lyons and his colleagues were able to obtain full duct development in the triply operated rat only when corticoids were given. Early studies of the role of the adrenals in mammary de- velopment have given conflicting and un- certain results (see review by Folley, 1952a). Recent studies have not entirely clarified the position. Flux (1954b) tested a number of 11 -oxygenated corticoids, and found that not only were they devoid of mammogenic activity in the ovariectomized virgin mouse, but that they inhibited the gi'owth-promoting effects of estrogen on the mammary ducts, whereas 11-desoxycorti- costerone acted synergistically with estro- gen in promoting duct growth. In subsequent studies it was shown that injections of ad- renocorticotrophin (ACTH) into intact female mice did not influence mammary growth (Flux and ]\lunford, 1957), but that Cortisol acetate in low doses (12.5 /^g. l)er day) stimulated mammary develop- ment in ovariectomized and in ovariec- tomized estrone-treated mice, whereas at higher levels (25 and 50 ftg. per day) it was without effect (Munford, 1957). In the vir- gin rat, on the other hand, glucocorticoids are said to stimulate mammary growth and to induce milk secretion (Selye, 1954; John- son and Meites, 1955). Some light on these conflicting results has been shed by the studies of Ahren and Jacobsohn (1957) who investigated the effects of cortisone on the mammary glands of ovariectomized and of ovariectomized-hypophysectomized rats, both in the presence and absence of exogenous ovarian hormones. In the hypo- physectomized animals, cortisone promoted enlargement and proliferation of the epi- thelial cells lining the duct walls, but nor- mal growth and differentiation did not oc- cur, nor did the addition of estrogen and progesterone appreciably alter these effects ; in rats with intact pituitaries, however, cortisone stimulated secretion but not mammary growth, whereas the addition of estrogen and progesterone promoted both growth and al)undant secretion. Ahren and Jacobsohn concluded that "the effect elic- ited by cortisone in the mammary gland should be analysed with due regard to the endocrine state of the animal both as to its effects on the structures of the mammary gland and to the consequences resulting from an eventual upset of the general meta- bolic equilibrium." They consider that in circumstances optimal for mammary gland growth and maintenance of homeostasis the predominant actions of cortisone are en- hancement of alveolar growth and stimula- tion of secretion, whereas under conditions ill which the metabolic actions of cortisone are not efficiently counteracted, gland growth is either inhibited or an abnormal development of certain iiianimaiy cells may be e^■()ked. That the general metabolic milieu may indeed profoundly influence the response of the iiuuiimarv gland to hormones has MAMMARY GLAND AND LACTATION 605 been emiiha.-^ized by the recent experiments of Jacobsohn and her colleagues. Following on the work of Salter and Best (1953) who showed that hypophysectomized rats could be made to resume body growth by the in- jections of long-acting insulin, Jacobsohn and her colleagues (Ahren and Jacobsohn, 1956; Ahren and Etienne, 1958; Ahren, 1959) found that treatment with estrogen and progesterone would stimulate consid- erable mammary duct growth in hypophy- sectomized-gonadectomized rats when given with suitable doses of long-acting insulin (Fig. 10.7). This growth-supporting effect of insulin could be nullified if cortisone was also administered (Ahren and Jacobsohn, 1957) but could be enhanced by giving thy- roxine (Jacobsohn, 1959). The thyroid would thus appear to be an- other endocrine gland whose hormones affect mammary growth intlirectly by altering the metabolic environment. Studies in this field, reviewed by Folley (1952a, 1956), indicate that in the rat some degree of hypothyroid- ism enhances alveolar development wdiereas in the mouse, hypothyroidism seems to inhibit mammary development. Chen, John- son, Lyons, Li and Cole (1955) have shown that mammary growth can be induced in hypophysectomized - adrenalectomized-thy- roidectomized rats by giving estrone, pro- gesterone, prolactin, STH, and Cortisol, no replacement of the thyroid hormones being necessary. These investigations on the effect of the metabolic environment on mammary devel- opment seem to ])e opening up new avenues of approach to the advancement of our understanding of the mechanisms of mam- mary growth and we would recommend. 0-5 cm. Fig. 10.7. Whole mount preparation of .second thoracic mammary gland of : ^. Ovariec- tomized rats injected with estrone and progesterone. B. Hypophysectomized-ovariectomized rat injected with estrone and progesterone. C. Hypophysectomizcd-o\ariectomized rat. D. Hypophysectomized-ovariectomized rat injected with estrone, progesterone, and insulin. (From K. Ahren and D. Jacobsohn, Acta physiol. scandinav., 37, 190-203, 1956.) GOG PHYSIOLOGY OF GONADS to those seeking further information about this important new fiekl, the recent review by Jacobsohn (19581. III. Endocrine Influences in Milk Secretion A. ANTERIOR PITUITARY HORMONES 1. Initiation of Secretion iLactogenesis) The early experiments leading to the view that the anterior pituitary was not only necessary for the initiation of milk secretion, but in fact i)rovided a positive lactogenic stimulus, are now well known and the reader is referred to the reviews by Folley (1952a, 1956) and Lyons (1958) for further particulars. That pituitary prolactin can evoke milk secretion in the suitably de\-eloped mammary gland of the rabbit with intact pituitary has been amply con- firmed, and the original experiments of Lyons (1942) involving the intraduct in- jection of prolactin have been successfully repeated by Meites and Turner (1947) and Fk;. 10.8. Liictation.'il lespon.scs in pseudoincg- nant rabbit to different doses of prolactin injeclcd intraductallv. (Fiom T. R. Bradley and P. M. Clarke, J. Endo.ninol., 14, 28-36, 1956.) Bradley and Clarke (1956) (Fig. 10.8). However, endogenous pituitary hormones may have participated in the response in such experiments and in the last 20 years there has been considerable discussion as to whether prolactin should be regarded as the lactogenic hormone or as a component of a lactogenic complex. This whole question has been fully discussed in recent years (see Folley, 1952a, 1956) and it now seems reasonably certain that lactogenesis is a response to the co-operative action of more than one anterior pituitary hormone, that is, to a lactogenic hormone complex of which prolactin is an important component, as first suggested by Folley and Young (1941 ) . The recent reports by Nandi (1958a, b) that STH -I- Cortisol can induce milk secre- tion in triply operated mice with suitably developed glands is further strong evidence against regarding prolactin as the lactogenic hormone. Secretory activity is evident in the mam- mary gland during the second half of preg- nancy, but abundant milk secretion does not set in until parturition or shortly there- after. The nature of the mechanism control- ling the initiation of abundant secretion has been the subject of speculation for many years. The earlier theories w^ere discussed l)y Turner ( 1939 ) in the second edition of this book, and included the theory put forward by Nelson with reference to the guinea pig, that the high levels of blood estrogen in late pregnancy suppressed the secretion or release of prolactin from the pituitary and had also a direct inhibitory cttcct on the mammary parenchyma, the fall in the levels of estrogen occurring at parturition then allowing the anterior pitui- tary to exert its full lactogenic effect. This concept proved inadequate to exjilain ob- servations in other species and it was later extended by Folley and Malpress (1948b) to embrace the concept of two thresholds for oi:)posing influences of estrogen upon jiituitary lactogenic function, a lower threshold for stimulation and a higher one for inhibition. Subsequent observations on the inhibitory role of progesterone, in the pix'sence of estrogen, on milk secretion, how- ever, necessitated further modification of the theorv. Before discussing these modifica- MAMMARY GLAND AND LACTATION 607 tions it is convenient to refer to the ingeni- ous tlieory put forward by Meites and Turner (1942a, b; 1948) which was based on their extensive investigation of the pro- lactin content of the pituitary in various physiologic and experimental states. Ac- cording to Meites and Turner, estrogen elicits the secretion of prolactin from the anterior pituitary thereby causing lacto- genesis, whereas progesterone is an inhibi- tory agent, operative in pregnancy, inhibit- ing or over-riding the lactogenic action of estrogen. The induction of lactation was thus ascribed to a fall in the body level of progesterone relative to that of estrogen heheved to occur at the time of parturition. Subsequent studies in the rabbit by jVIeites and Sgouris (1953, 1954) revealed that combinations of estrogen and progesterone could inhibit, at the mammary gland level, the lactogenic effects of exogenous prolactin. This effect was, however, relative and by in- creasing the prolactin or decreasing the ster- oids, lactogenesis ensued. Inasmuch as the theory of Meites and Turner did not take into account the eventuality that estrogen and progesterone act at the level of the mam- mary gland, Meites ( 1954) modified the con- ('ei)t, postulating that milk secretion was held in check during pregnancy first by the combined effect of estrogen and proges- terone which make the mammary gland re- fractory to prolactin and, secondly, by a low rate of prolactin secretion. The role of progesterone in over-riding the stimulatory effect of estrogen on the pituitary he now considered to be of only minor importance. Meites also explained the continuance of lactation in pregnant animals by postulat- ing that the initial level of prolactin was sufficiently high as a result of the suckling stimulus to overcome the inhibitory action of the ovarian hormones on the mammary gland. One of us (Folley, 1954, 1956) put forward a tentative theory, combining vari- ous features of previous hypotheses, which seemed capable of harmonizing most of the known facts regarding the initiation of milk secretion. In this it was emphasized that measurements of the prolactin content of the pituitary were not necessarily indica- tive of the rate of prolactin release (a recent study bv Grosvenor and Turner (1958c) lends further support to this contention) and were best considered as largely ir- relevant; low circulating levels of estrogen activate the lactogenic function of the an- terior pituitary whereas higher levels tend to inhibit lactation even in the absence of the ovary; lactogenic doses of estrogen may be deprived of their lactogenic action by suitable doses of progesterone, the com- bination then acting as a potent inhibitor of lactation, this being the influence oper- ating in pregnancy; at parturition the rela- tive fall in the progesterone to estrogen ratio removes the inhibition which is replaced by the positive lactogenic effect of estrogen acting unopposed. It was observed by Gaines in 1915 that although a colostral secretion accumulated in the mammary gland during pregnancy, the initiation of copious secretion was as- sociated with functioning of the contractile mechanisms in the udder responsible for milk ejection; later Petersen (1944) also suggested that the suckling or milking stim- ulus might be partly responsible for the onset of lactation. Recent studies have pro- vided evidence that this may well be so, and these will be considered later when dis- cussing the role of the suckling and milking stimulus in the maintenance of milk secre- tion (see page 611). During the past decade a fair amount of information has been obtained about the biochemical changes which occur in mam- mary tissue near the time of parturition, and which are almost certainly related to lactogenesis. The earlier work has been re- viewed in some detail by one of us (Folley, 1956) and need only be referred to briefly here. Folley and French (1949), studying rat mammary gland slices incubated in media containing glucose, showed that — QOo in- creased from a value of about 1.3 in late pregnancy to a value of about 4.4 at day 1 of lactation, and thereafter increased still further. At the same time the R.Q. which was below unity (approximately 0.83) at the end of pregnancy, increased to unity soon after parturition, and by day 8 had reached a value of 1.62 at approxi- mately which level it remained for the rest of the lactation period. In accord with the G08 PHYSIOLOGY OF GONADS increased respiratory activity of the tissue about the time of parturition in the rat mammary gland, Moore and Nelson (1952) reported increases in the content of certain respiratory enzymes, succinic oxidase and cytochrome oxidase, in the guinea pig mam- mary gland at about this time. Greenbaum and Slater (1957b) made similar observa- tions about mammary gland succinic oxi- dase in the rat. Recent work is beginning to throw light on the metabolic pathways in- volved in this increase in respiratory ac- tivity. Thus McLean (1958a) has adduced evidence indicating an increase in the ac- tivity of the pentose phosphate pathway in the rat mammary gland at about the time of parturition. Mammary gland slices taken from rats at various stages of the lactation cycle were incubated in media containing either glucose 1-C^^ or glucose 6-C^-^, and the amount of radioactivity appearing in the respiratory CO2 was determined. The results given in Figure 10.9 show that al- though the recovery of C^^'Oo from C-6 was relatively unaffected by the initiation of lac- tation, the C^^Oo originating from C-1 be- gan a striking increase at the time of parturition (see also Glock, McLean and Whitehead, 1956, and Glock and McLean, 1958, from which Figure 10.9 was taken). pregnancy in\'oliition Imc;. 1().<», The relative amounts of C'Oi; formed fioin iiiilucosc 1-C'^ and glucose 6-C" by rat niani- maiy gland slices. O O, C'^Oi formed from glucose 1-C^'. • • . C^'Oi! formed from glucose 6-C". (From G. E. Glock and P. McLean, Proc. Roy. Soc, London, ser. B, 149, 354-362, 1958.) Despite the well known pitfalls which sur- round the interpretation of C-1: C-6 quo- tients in experiments such as these, it seems clear that lactation is associated with an increase in the metabolism of glucose by the pentose phosphate cycle, whereas the proportion going by the Embden-Meyerhof jmthway would appear to be relatively un- affected. These conclusions are supported by the fact that the levels in rat mammary tissue of two enzymes concerned in this pathway of glucose breakdown, glucose 6-phosphate dehydrogenase and 6-phospho- gluconate dehydrogenase, show very strik- ing increases at the time of parturition (Glock and McLean, 1954; McLean, 1958a). Other enzymes concerned in glucose break- down whose activities in mammary tissue begin to increase at parturition are hexo- kinase and phosphoglucose isomerase (]\Ic- Lean, 1958a). In connection with the glu- cose metabolism of rat mammary tissue it may be noted that addition of insulin to the incubation medium markedly increases the — QOo and R.Q. of rat mammary slices metabolizing glucose or glucose plus acetate (see page 619), and that this tissue only becomes sensitive to insulin just after par- turition (Balmain and Folley, 1951). It is interesting to speculate which of the two above-mentioned pathways of glucose breakdown in mammary tissue resjjonds to the action of insulin. According to Abraham, Cady and Chaikoff (1957) addition of in- sulin in vitro increased the production by lactating rat mammary slices of C^'^Oo from glucose l-C^'*, but not from glucose 6-C^'*, which might indicate that insulin stimulates preferentially the pentose phosphate path- way. Against this, insulin increased the in- corporation of both these carbon atoms (and also the 3:4 carbon atoms of glucose) into fatty acids of the slices to about the same extent. McLean (1959) believes that the stimulatory effect of insulin on the pentose jihosphate pathway in the lactating rat mammary gland is secondary to its stimulating effect on lipogenesis. The latter l)rocess generates the oxidized form of tri- l)hosphopyridine nucleotide (TPN) which is needed for the first two steps of the pentose phosphate cycle. The inci-casc in the R.Q. of mammary MAMMARY GLAXD AND LACTATION 009 tissue beginning at parturition observed by Folley and French (1949) was inter- i:)reted as indicating that this tissue assumes the power of effecting net fatty acid syn- thesis from ghicose at this time. Much sub- sequent evidence confirming this idea has been reviewed by Folley (1956). It only rt'mains to add that Ringler, Becker and Nelson (1954), Lauryssens, Peelers and Donck (1956), and Read and Moore (1958) ha^-e shown that the amount of coenzyme A in mammary tissue undergoes an increase at parturition. Moreover, the recent findings of McLean (1958b), who showed that the levels of pyridine nucleotides in the mam- mary gland of the rat begin to increase at parturition, reaching a high level by the end of lactation, may be significant in this connection. McLean found that although the increase in the tissue levels of diphos- l^hopyridine nucleotide was almost entirely due to an increase in the oxidized form (DPN), in the case of TPN it was the re- duced form (TPNH) which increased. The latter might well be used for reductive syn- theses such as lipogenesis. The rate of synthesis of milk constituents other than fat must also begin to increase at parturition, and Greenbaum and Greenwood (1954) showed that an increase in the levels of glutamic aspartic transaminase and of glutamic dehydrogenase in rat mammary tissue occurs at this time. The authors be- lieve these enzymes are concerned in the provision of substrates for the synthesis of milk protein. It is significant in connection with milk protein synthesis that the mam- mary gland ribonucleic acid (RNA) in the rat undergoes a marked rise at parturition (Greenbaum and Slater, 1957a). The above - mentioned biochemical changes in mammary tissue which occur at al)out the time of parturition are almost certainly closely related to the effect on this tissue of members of the anterior pituitary lactogenic complex, and particularly pro- lactin. Attempts have been made to elicit the characteristic respiratory changes, de- scribed above, in mammary slices in vitro by addition of prolactin and adrenal gluco- corticoids to the incubation medium (see Folley, 1956). So far, however, definitive re- sults luive not been obtained and it is doubt- ful whether any biochemical changes in lactating mammary gland slices in vitro have been demonstrated which could with certainty be ascribed to the action of pro- lactin (in this connection see also Bradley and Mitchell. 1957). 2. Maintenance of Milk Secretion — Galac- topoiesis It is well known that the removal of the pituitary of a lactating animal will end milk secretion (for references see Folley, 1952a). The cessation of milk secretion has been generally ascribed to the loss of the anterior lobe, but when the importance of the neurohypophysis in milk ejection be- came established (see page 621), it was clear that in the hypophysectomized animal it was necessary to distinguish between a failure in milk secretion and a failure in milk ejection, since either would lead to failure of lactation. It has now been shown in the rat that adequate oxytocin therapy ensuring the occurrence of milk ejection after hypophysectomy will not restore lac- tation (Cowie, 1957) and it may thus be concluded that the integrity of the anterior lobe is essential for the maintenance of milk secretion. The effect of hypophysec- tomy on milk secretion is dramatic, be- cause in the rat, milk secretion virtually ceases within a day of the operation and biochemical changes in the metabolic ac- tivity of the mammary tissue can be de- tected within 4 to 8 hours (Bradley and Cowie, 1956). It is of interest to note that these metabolic changes are similar to those observed during mammary involution (see page 598). Since the second edition of this book, there have been surprisingly few studies on replacement therapy in hypophysectomized lactating animals. In such studies we would stress the need for rigorous methods of assessing the efficacy of treatment. In the past the presence of milk in the gland as revealed by macroscopic or microscopic examination has been regarded as an indi- cation of successful replacement. This, however, gives no measure of the degree of maintenance of lactation and some measure of the daily milk yield of such animals should be obtained (see also Cowie, 1957). GIO PHYSIOLOGY OF GONADS It is abo now obvious that oxytocin may have to be injected to ensure milk ejection; under certain circumstances, however, the neurohypophyseal tissue remaining after the removal of the posterior lol^e may be capable of releasing oxytocin and permit- ting milk ejection (see Benson and Cowie, 1956; Bintarningsih, Lyons, Johnson and Li. 1957, 1958). The earliest report on the maintenance of lactation after hypophysectomy is that of Gomez (1939, 1940), who found that hy- pophysectomized lactating rats could rear their litters if given anterior-pituitary ex- tract, adrenal cortical extracts, glucose, and posterior pituitary extract. These experi- ments are difficult to assess because they are reported only in abstract, but the use of pos- terior pituitary extract at a time when the role of oxytocin in milk ejection was not generally recognized is worthy of note. Re- cently, slight maintenance of milk secretion in hypophysectomized rats has been ob- tained with prolactin alone, and greater maintenance when adrenocorticotrophic hormone ( ACTH I or STH was administered with prolactin (Cowie, 1957). Similar studies were reported by Bintarningsih, Lyons, Johnson and Li (1957, 1958) (see also Lvons, Li and Johnson, 1958) in which I « c -^ 4 -0 I 1 £ Z -6 z J E :^ 2 ^^, TV 2 ^ Fig. 10.10. Effect on the luilk yield of the cow of injected hormones of the anterior pituitary. (From the results of P. M. Cotes, J. A. Crichton, S. J. Folley and F. G. Young, Nature, London. 164, 992-993, 1919.) considerable maintenance of milk secretion was obtained in hypophysectomized rats with prolactin and certain corticoids. Of related interest is the observation by Elias (1957) that Cortisol and prolactin can in- duce secretory activity in explants of mouse mammary gland growing on a synthetic medium. (Tissue culture techniques have been little exploited in mammary studies and further developments in this field may be expected.) The evidence to date suggests that, in the rat, prolactin is an essential component of the hormone complex involved in the main- tenance of lactation with ACTH and STH also participating, but further studies are recjuired to determine the most favorable balance of these factors. Preliminary studies on the maintenance of lactation in the goat after hypophysectomy suggest that both prolactin and STH are im- portant in the initiation and maintenance of milk secretioii (Cowie and Tindal, 1960). Our knowledge of the process in other spe- cies is derived from studies on the effect of exogenous anterior pituitary hormones on established lactation in intact animals— galactopoietic effects (for reference see Folley, 1952a, 1956). In the cow, consider- able increase in milk yield can be obtained by injecting STH (Cotes, Crichton, Folley and Young, 1949), whereas prolactin has a negligible galactopoietic effect (Fig. 10.10; for discussion see also Folley, 1955). Re- cently the precise relationship between the dose of STH (ox) and the lactational re- sponse in the cow was established in our lab- oratory by Hutton (1957) who observed a highly significant linear relationship be- tween log doses of STH (single injection) and the increase in milk yield obtained (Fig. 10.11 ) ; increases in fat yield relative to the yield of nonfatty solids also occurred. In the lactating rat, on the other hand, STH has no galactopoietic effect (Meites, 1957b; Cowie, Cox and Naito, 1957), whereas pro- lactin has (Johnson and Meites, 1958). Such studies must be interpreted with caution as endogenous pituitary hormones were pres- ent ; nevertheless, it seems reasonable to conclude that STH is likely to be an im- poi'tant factor in the maintenance of lacta- tion in the row. MAMMARY GLAND AND LACTATION 611 mq qro\Om hormone (onthmeTTc scale) fa-25 12-5 25-0 50-0 100-0 200-0 S-«^0 'Zoo-o Fig. 10. IL Effect of graded doses of growth hormone on milk yield of row. Upper curve, doses plotted on arithmetic scale. Lower curve, doses plotted on logarithmic scale. (From J. B. Hutton, J. Endocrinol., 16, 115-125, 1957.) C)ther hormones of the anterior pituitary in all probability influence milk secretion through their target glands and these will be dealt with later. 3. Suckling Stimulus and the Maintenance of Lactation It has been long believed that regular milking is an important factor in main- taining lactation and that if milk is allowed to accumulate in the gland, as occurs at weaning, atrophy of the alveolar epithelium and glandular involution occur. Evidence in support of this concept was obtained in studies showing that ligature or occlusion of the main ducts of some of the mammae of a lactating animal resulted in atrophy of the glands concerned although the other glands were suckled normally (Kuramitsu and Loeb, 1921; Hammond and Marshall, 1925; Fauvet, 1941a). Studies by Selye and his colleagues, however, revealed that such occluded glands did not atrophy as quickly as did glands of animals in which the suck- ling stimulus was no longer maintained (Selye, 1934; Selye, Collip and Thomson, 1934) and it was postulated that the suck- ling stimulus evoked from the anterior pituitary the secretion of prolactin which maintained the secretory activity of the gland. This theory has been widely accepted 012 PHYSIOLOGY OF GONADS although it has been suggested that a com- plex of hormones rather than prolactin alone is released (Folley, 1947). Williams (1945) showed that prolactin could in fact main- tain the integrity of the mammary gland in the unsuckled mouse thus mimicking the effects of the suckhng stimulus; other sup- porting evidence has been reviewed by Folley (1952a). Recent studies in goats, however, have shown that milk secretion may continue more or less at the normal level after complete denervation of the ud- der (Tverskoi, 1958; Denamur and Mar- tinet, 1959a, b, 1960) and it may be that in some species the suckling or milking stimu- lus is loss important in the maintenance of milk secretion. Milk secretion is essentially a continuous process whereas the suckling or milking stimulus is intermittent ; indeed the milking stimulus may be of remarkably brief dura- tion (in the cow about 10 minutes in all per 24 hours) and it is therefore likely that the stimulus triggers off the release of sufficient galactopoietic complex to maintain mam- mary function for some hours. Grosvenor and Turner (1957b) reported that suckling causes a rapid drop in the prolactin content of the pituitary in the rat, and that the prenursing level of prolactin in the pituitary is not fully regained some 9 hours later. It is difficult, however, to relate pituitary levels of prolactin to the rate of its secre- tion into the circulation and, although these observations are interesting, further ad- vances are unlikely until a method of assay for blood prolactin becomes available and the "half-life" of prolactin in circulation is known. The experiments of Gregoire (1947) on the maintenance of involution of the thymus during nursing suggests that the suckling stimulus releases ACTH which, as we have seen, is galactopoietic in the rat; thus, so far as the rat is concerned, there would appear to be good evidence that the suckling stim- ulus releases at least two known important components of the galactopoietic complex. The milking and suckling stimulus is also responsible for eliciting the milk-ejection reflex and the relation between the two re- flexes will be discussed later in this chapter (sec ])age 619 1. B. HORMONES OF THE ADRENAL CORTEX Adrenalectomy results in a marked in- hibition of milk secretion and the early ex- periments in this field were reviewed by Turner in 1939. Since then, however, puri- fied adrenal steroids have become available enabling further analysis to be made of the role of the adrenal cortex in lactation. Gaunt, Eversole and Kendall (1942) con- sidered that in the rat the defect in milk secretion after adrenalectomy could be re- paired by the administration of the adrenal steroids most closely concerned with carbo- hydrate metabolism, whereas we came to the somewhat opposing view that the defect was best remedied by those hormones primarily concerned with electrolyte metab- olism (Folley and Cowie, 1944; Cowie and Folley, 1947b, c). The reasons for these differing observations are not yet entirely clear. Virtually complete restoration of milk secretion was subsequently obtained in our strain of rat by the combined ad- ministration of desoxycorticosterone acetate (DCA) and cortisone, or with the halogen- ated steroids, 9a-chlorocortisol and 9a- fluorocortisol (Cowie, 1952; Cowie and Tindal, 1955; Cowie and Tindal, unpub- lished; see also Table 10.1). It would there- fore seem that both glucocorticoid and mineralocorticoid activity was necessary to maintain the intensity of milk secretion at its normal level. The interesting observation was made by Flux (1955» and later con- firmed by Cowie and Tindal (unpublished) that the ovaries contribute to the mainte- nance of lactation after adrenalectomy, a contribution which could be simulated in the adrenalectomized-ovariectomized rat by the administration of 3 mg. progesterone daily. The differences in the size of the ovarian contribution may partly accoimt for the ap- parent differences in various strains of rat of the relative importance of mineralo- and glucocorticoids in sustaining milk secretion after adrenalectomy. The only other species in which the maintenance of lactation after adrenalectomy has been studied is the goat in which, as in the rat, lactation can be maintained with cortisone and desoxycorti- costerone, the latter being apparently the more critical steroid (Cowie and Tindal. 1958; Figs. 10.12a, b). MAMM.\RY GLAND AND LACTATION 613 There have been several studies on the effects of corticoids and adrenocorti- eotrophin on lactation in the intact animal. ACTH and the corticoids depress lactation in the intact cow (Fig. 10.10) (Cotes, Crich- ton, Folley and Young, 1949; Flux, Folley and Rowland, 1954; Shaw, Chung and Bunding, 1955; Shaw, 1955), whereas in the rat ACTH and cortisone have been reported as exhibiting galactopoietic effects (Meites, private communication; Johnson and Meites, 1958). With larger doses of corti- sone, however, an inhibition of milk secre- tion in the rat has been reported (Mercier- Parot, 1955). The main function of the cortical steroids in lactation is still uncertain. They may act in a "supporting" or "permissive" manner (see Ingle, 1954), maintaining the alveolar cells in a state responsive to the galacto- ])oictic complex, or they may act by main- taining the necessary levels of milk precur- sors in the blood. Biochemical studies are, however, Ix'gin- ning to add to our information on the role of the corticoids in lactation. In the rat, adrenalectomy prevents the increase in liver and mammary gland arginase which occurs during normal lactation and it has been suggested that this depression of arginase activity interferes with deamination of amino acids, and thereby inhibits any in- crease in gluconeogenesis from protein and thus starves the mammary gland of non- nitrogenous milk precursors (Folley and Greenbaum, 1947, 1948). As there is little arginase in the mammary gland of other species {e.g., rabbit, cow, goat, sheep), this mechanism may not have general validity (for further discussion see Folley, 1956). Other biochemical studies have suggested that the steroids of the adrenal cortex may be concerned in mammary lipogenesis, but the results so far have been conflicting and no firm conclusions can as yet be drawn (see Folley, 1956). C. OVARIAN HORMONES There is no evidence that ovariectomy has any deleterious effect on lactation (Kura- mitsu and Loeb, 1921; de Jongh, 1932; Fol- ley and Kon, 1938; Flux, 1955); neither is there evidence for the belief, once TABLE 10.1 Replacement therapy in lactating rats adrenalectomized on the fourth day of lactation (From A. T. Cowie and S. J. Folley, J. Endocrinol., 5, 9-13, 1947.) Treatment Num- ber of Litters Number of Pups per Litter Litter-growth Index* gm. + S.E. Control Adrenalectomy Adrenalectomy + corti- sone + DC A (tablet implantsf) 8 9 7 8 8 8 15.6 + 0.5 7.5 ± 0.6 14.9 ± 0.6 (Above results from Cowie, 1952) Control 6 8 14.5 ± 0.8 Adrenalectomy 6 8 6.2 ± 0.4 Adrenalectomy + chloro- 5 8 13.1 ± 0.5 cortisol (100 Mg per day) (Above results from Cowie and Tindal, 1955) Control 8 12 17.7 ± 0.8 Adrenalectomy 8 12 7.5 ± 0.5 Adrenalectomy + ovari- 5 12 3.6 ± 0.5 ectomy Adrenalectomy + ovari- 7 12 14.5 ± 0.7 ectomy + fiuorocorti- sol (200 Mg per day) (Above results from Cowie and Tindal, unpublished) * The litter-growth index is defined as the mean daily gain in weight per litter over the 5-day pe- riod from the 6th to the 11th days. t 2 X 11 mg. tablets cortisone giving mean daily absorption of 850 ^ig., and 1 X 50 mg. tablet DCA giving mean daily absorption of 360 ng. widely held, that ovariectomy increases and prolongs lactation in the nonpregnant cow (see Richter, 1936). Although the integrity of the ovary is not essential for the maintenance of lacta- tion, there can be no doubt that ovarian hormones, in certain circumstances, pro- foundly influence milk secretion. Estrogens have long been regarded as possessing the power to inhibit lactation, a concept on which Nelson based his theory of the mech- anism of lactation initiation (see page 606 1 . Some workers, however, have expressed doubts that the effect is primarily on milk secretion, and have suggested that in ex- 614 PHYSIOLOGY OF GONADS periments on laboratory animals the ap- parent failure in milk secretion could be a secondary effect due to either a toxic action of the estrogen causing an anorexia in the mother, interference with milk ejection, or disturbance of maternal behavior or to toxic effects on the young, whose growth rate serves as a measure of lactational perform- ance, through estrogens being excreted in milk. The evidence to date shows that in the intact rat estrogens even in very low doses inhibit milk secretion, their action depending on the presence of the ovary ; the ovarian factor concerned appears to be pro- gesterone, estrogen and progesterone acting locally on the mammary gland and render- ing it refractory to the lactogenic com- plex. In the ovariectomized rat much larger doses of estrogen are necessary to inhibit lactation, and the evidence is not entirely Body Goat 478 weight ^^L :.) 45 L Plasma Na (m-equiv./l.) ^^^^ Plasma K (m-equiv./l Milk K 40 - (m-equiv./l.) 30 Milk Na , (m-equiv./l.) - Solids-not- fat (%) Yield of solids-not- fat (g) Fat (%) Milk yield (kg) Goat died-* 5 15 25 4 14 24 Mgr. Apr. Fig. 10.12i4. Effect of replaconi(>nt tlierapy with (losoxycoiticostcM-oiu c-ortisone aoetate (CA) on milk yield, milk composition, and concent (DCA) and tion of Na and K in milk and blood plasma of the goat after adrenalectomy. Duration of replacement therapy (pellet implantation) indicated by horizontal lines; the names of steroids and their mean daily absorption rates are given adjacent to the lines. Note in Figure 12.4 the considerable maintenance of milk vield with DCA alone. See also Figure 12/?. (From A. T. Cowic and J. S. Tindal. J. Endocrinol., 16, 403-414, 1958.) MAMMARY GLAND AND LACTATION 6L Goat 515 Body 5Q _ weight — (kg) 40 150 Plasma Na ^ ^. / /I \ ^40 — (m-equiv./l) — 130 Plasma K (m-equiv./l) Milk K (m-equiv./l.) Milk Na (m-equiv./l.) Solids-not- ^ H fat {%) 7 U Yield of 200 - solids-not- — fat (g) 100 - Fat (- ^ Fat yield Milk yield (kg) 13 23 2 12 22 2 12 22 Oct. Nov Dec. Fig. 12B. 11 21 31 10 20 Jan. Feb conclusive that there is a true inhibition of milk secretion (see Cowie, 1960). In the cow estrogen in sufficient doses depresses milk yield, but its mode of action has not been fully elucidated. In women, estrogens are used clinically to suppress unwanted lactation, but as the suckling stimulus is also removed about the same time, the role of the estrogen is difficult to assess (see Meites and Turner, 1942a). It has been well established that proges- terone by itself has no effect on milk secre- tion (see Folley, 1952a), save in the ad- renalectomized animal (see page 612), and so it would appear that the physiologic inhibition of lactation is effected Ijy estrogen and progesterone acting synergistically as first demonstrated by Fauvet (1941b) and confirmed by others including Masson (1948), Walker and Matthews (1949), GIG PHYSIOLOGY OF GONADS Cowie, FoUey, Malpress and Richarcl.son (1952J,, and Meites and Sgouris (1954). There is clear evidence that the estrogen- progesterone combination acts at least partly on the mammary parenchyma (Des- clin, 1952; Meites and Sgouris, 1953) but the mechanism of the action is unknown. The hormonal interplay and complex endo- crine interactions in the process of lactation inhibition with estrogen has recently been discussed at length by von Berswordt-Wall- rabe (1958). Lactogenic effects of estrogens have al- ready been mentioned; these have been demonstrated most strikingly in cows and goats, in which milk secretion has been in- duced in udders being developed by exog- enous estrogen. These experiments have been reviewed in some detail by Folley and Malpress (1948b) and Folley (1956).^ It is generally assumed that estrogens act by stimulating the production of lactogenic and galactopoietic factors by the anterior pituitary. In experiments on the ovari- ectomized goat we have shown (Cowie, Folley, Malpress and Richardson, 1952; Benson, Cowie, Cox, Flux and Folley, 1955) that it is possible to select a daily dose of estrogen which will induce mammary growth but relatively little secretion in the sense that the udder does not become tense and distended as will happen when a lower dose of estrogen is given — an observation we may quote in support of the "double-thresh- old" theory of estrogen action. The lacto- genic effect of the lower dose of estrogen could be abolished, however, by administer- ing progesterone simultaneously with the estrogen (Fig. 10.13), an observation in accord with those of other workers on the rabbit and rat (see above). In 1936 one of us (Folley, 1936) reported Fig. 10.13. Photographs of goat uddois dovelopcd by daily injections of hoxoostiol (HX) with and without progesterone (PG). The hibels indicate the daily dose in mg. of each substance. (Results from A. T. Cowie, S. J. Folley, F. H. Malpre.ss and K. C. Ricliardson, J. Endocrinol., 8, 64-88, 1952.) MAMMARY GLAND AND LACTATION GK that certain dose levels of estrogen in the lactating cow produced long-lasting changes in milk composition characterized by in- creases in the percentages of fat and non- fatty solids. This was regarded as an exam- ple of galactopoiesis and was termed the "enrichment" effect. The effect, however, w^as somewhat erratic and it has recently been re-investigated by Hiitton (1958) who con- firmed and extended the earlier observations. Hutton found that galactopoietic responses (Figs. 10.14 and 10.15) were obtained only within a restricted dose range, the limits of which were affected by the stage of preg- nancy and the breed of the cow. Hutton further concluded that in the normal cow changes in milk composition and yield as- sociated with advancing pregnancy were probably determined by the progressive rise of blood estrogen levels. D. THYROID HORMONES Studies on the effect of removal of the thyroids on milk secretion have been re- viewed by one of us (Folley, 1952a) ; the evidence strongly suggests that the thyroid glands are not essential for milk secretion, but in their absence the intensity and dura- tion of lactation is reduced. Histologic and cytologic studies of the thyroid of the lac- tating cat suggest that there is a consider- able outpouring of the thyroid secretion in the early stages of lactation (Racadot, 1957), and Grosvenor and Turner (1958b) have reported that the thyroid secretion rate is higher in lactating than in nonlactat- ing rats. Since the last edition of this l)ook, a great volume of experimental results has been published on the use of thyroid-active ma- terials for increasing the milk yield of cows. These experiments have been extensively reviewed by Blaxter (1952) and Meites (1960) and we need here only touch on the salient points. In the early studies i^reparations of dried thyroid gland were fed to cows or injections of DL-thyroxine were given, but the use on a large scale of thyroid-active materials for increasing the milk yield of cows only became feasible when it was shown that certain iodinated proteins exhibited thyroid- like activitv when given in the feed. Al- 9-9 97 o 9-3 ^ 9-1 8-9 •'' Guernsey Shorthorn 8-5 •^U^ri I L 0 20 40 60 80 100 Oestradiol monobenzoite (mg) Fig. 10.14. Effect of graded doses of estradiol benzoate on percentage of nonfatty solids in milk from cows of three breeds. (From J. B. Hutton, J. Endocrinol., 17, 121-133, 1958.) Oestradiol monobenzoate (mg) (arith. scale) 0 10 20 30 40 50 6-25 12-5 250 500 Oestradiol monobenzoate (mg) (log scale) Fig. 10.15. Effect of graded doses of estradiol benzoate on fat content of cows' milk. Upper curve, doses plotted on arithmetic scale. Lower curve, doses plotted on logarithmic scale. (From J. B. Hutton, J. Endocrinol., 17, 121-133, 1958.) though these materials were readily made and were economical for large-scale use, they possessed several disadvantages. Their ac- tivity was difficult to assay and standardize, they were frequently unpalatable, and their administration entailed a considerable in- take of iodine which could be undesirable. Nevertheless, a large number of experiments were carried out all over the world with this type of material. In 1949, however, a new and improved method for the synthesis of L-thyroxine was developed (Chalmers, Dickson, Elks and Hems, 1949) and thyrox- ine became available in large quantities. It was then shown jjy Bailey, Bartlett and Folley (1949) that this material was ealac- 618 PHYSIOLOGY OF GONADS , /" \^ Cont-rol. A<' / " "^ - ^* — • DO m§. .^''\ / V- -' v;. 100 m|. ^..^-Av / V 150mg. ^^^4?^^/ . V ,.-•*.. \ vv / .^-r \ \ • — •• tva --^-^ y \ \ \ •••\-'^\ \ x- ^ .. \ ^ \. *-^ '• •■*— . \ \ *■*•—., \ \ \ \ .... -.... "•N-:w- -o ■;;16 - i:/^ c u= «14 / J3 as^g/mi. 8 12 Z' j^p^ £) 0-Vg/ml. — 10 y rf^ ,-fP 1 3 8 si r^^ r-f^ y^ Control o ° M J A y^ ^cr ,^y^ 4J z Pr( .iif^ jy''^^ 4 ^ M^ ^r^ 2 L_l 1 1 1 1 \ 1 \ 1 1 \ 1 15 30 60 90 120 Time (min) 150 Fig. 10.17. Effect of various concentrations of insulin on the respiratory metabolism of slices of rat mammarj' glands. (From J. H. Balmain, C. P. Cox, S J. Folley and M. L. McNaught, J. Endo- crinol., 11, 269-276, 1954.) portion of the milk secreted by the alveohir cells in the intervals between suckling or milking remains within the alveoli and the fine ducts. Only a small portion passes into the larger ducts and cisterns or sinuses from which it can be immediately removed by suckling, milking, or cannulation; its re- moval requires no maternal participation and has been termed passive withdrawal (see Cowie, Folley, Cross, Harris, Jacob- sohn and Richardson, 1951, and page 612). The larger portion of the milk in the alveoli and fine ducts becomes available only with the active participation of the mother and requires the reflex contraction of special cells (see page 623) surrounding the alveoli in re- sponse to the milking or suckling stimulus to eject the milk from the alveoli and fine ducts into the cistern and sinuses of the gland. The occurrence of this reflex has long been known, although its true nature has only recently been generally recognized.^ -H. K. Waller {Clinical Slujlits un Lnrfallon, London: Heinemann, 1938), and later one of us (S. J. Folley, Physiology and Biochemistry of Lac- tation, London and Edinburgh: Oliver & Boyd, 1956) have drawn attention to the fact that the theme of the "milk-ejection reflex" was the inspira- tion of a paiming by II Tintoretto entitled "The Origin of the Milky Way" which hangs in the 111 the past it has been termed the "draught" in lactating women (see Isbister, 1954) and the "let-down" of milk in the cow. The latter term is particularly misleading since it implies the release of some restraint, whereas there is, in fact, an active and forceful expulsion of milk from the alveoli and we have, therefore, urged that this term be no longer used in scientific literature and that it be replaced by the term "milk ejec- tion" (Folley, 1947; Cowie, Folley, Cross, Harris, Jacobsohn and Richardson, 1951), a term, incidentally, which was used by Gaines in 1915 in his classical researches on the phenomenon (see below j. The true nature of the milk removal pro- cess was for many years not recognized, probably because it was assumed that the mammary gland could not contain all the milk obtainable at a milking, and this as- sumption made it necessary to postulate a very active secretion of milk during suckling or milking. Even as late as 1926 two phases of milk secretion were described in the cow ; the first phase was one of slow secretion occurring between milkings, the second phase was one of very active secretion oc- curring in response to the milking stimulus when a volume of milk about equal to that produced in the first phase was secreted in a matter of a few minutes (Zietzschmann, 1926). That some physiologic mechanism National Gallery, London. Both authors point out tliat the picture shows evidence of a considerable intuiti^■e understanding of the physiologic nature of the milk-ejection reflex. Thus, it illustrates, first, that the application of the suckling stimulus causes a considerable increase in intranianiinai >• jiressure resulting, in this instance, in a sjnni cii' milk from the nipples, and second, that ihv Muklmg stimulus applied to one nipple gives rise to a systemic rather than a localized effect, for the milk is forcibly ejected from the suckled and unsuckled breasts ahke. The same theme was also treatetl by Rubens in a picture called "The Birth of the Milky Way" which can be seen in the Prado Museum, Madrid. This picture differs from Tintoretto's in one im- portant detail, the stream of milk coming only from one breast. The forcible ejection of milk from the nipple has doubtless been the subject of many statues. An ex- ample known to the authors is the fountain in the Sfiuare at Palos Verdes, near Los Angeles, Cali- fornia. The center piece of this fountain has a nude female torso at each of its four corners from whose nipples spurt streams of water. MAMMARY GLAND AND LACTATION 621 was involved in the discharge of preformed milk from the mammary gland had, how- ever, been recognized. Schafer (1898) con- sidered that milk discharge was aided by contraction of plain muscle w^ithin the gland and pressure on the alveoli produced by vasodilation. The first full investigation of the physi- ology of milk removal was that by Gaines in 1915. Unfortunately, his remarkably ac- curate observations and perspicacious conclusions aroused little general interest and were almost wholly overlooked for more than quarter of a century. It is now of interest to recall the more important of Gaines' observations. First, he made a clear distinction between milk ejection and milk secretion — "Milk secretion, in the sense of the formation of the milk constituents, is one thing; the ejection of the milk from the gland after it is formed is quite another thing. The one is probably continuous; the other, certainly discontinuous." Secondly, he concluded that "Nursing, milking and the insertion of a cannula in the teat, excite a reflex contraction of the gland musculature and expression of milk. There is a latent period of 35 to 65 seconds. . . . Removal of milk from the gland is dependent on this reflex, and it may be completely inhibited l)y anaesthesia. The conduction in the reflex arc is dependent upon the psychic condition of the mother." He also observed that the increased flow of milk following the latent period after stimulation was associated wath a steep rise in pressure within the gland cistern and that the reflex could be condi- tioned. Thirdly, with reference to the gland capacity, he reported that "the indication is that practically the entire quantity of milk obtained at any one time is present as such in the udder at the beginning of milking." Lastl3^ he confirmed earlier ob- servations that injections of posterior pitui- tary extract caused a flow of milk in the lactating animal and he postulated that "pituitrin has a muscular action on the ac- tive mammary gland causing a constriction of the milk ducts and alveoli with a con- sequent expression of milk. This action holds, also, on the excised gland in the absence of any true secretory action." Gaines regarded the milk-ejection reflex as a l)urely neural arc although he emphasized that the effect was "very similar to that produced by pituitrin." All that is required to bring these views of milk ejection in line with present day concepts is to recognize that the reflex arc is neurohormonal in char- acter, the efferent component of which is a hormone released from the neurohypoph- ysis. When Gaines was carrying out these experiments hardly anything was known of neuro-endocrine relationships and there was no background of knowledge to lead anyone to conceive that the effects of the posterior pituitary extract might represent a physio- logic rather than a pharmacologic effect. In 1930 Turner and Slaughter hinted at a possible physiologic role of the posterior pituitary in milk ejection and, as we have noted (page 610), Gomez (1939) used pos- terior pituitary extract in replacement ther- apy given to hypophysectomized lactating rats. It was not until 1941, however, that the role of the posterior pituitary in milk ejection was seriously postulated by Ely and Petersen (1941) who, having shown in the cow that milk ejection occurred in the mammary gland to which all efferent nerve fibers had been cut, suggested that the reflex was neurohormonal, the hormonal compo- nent being derived from the posterior pitui- tary, and being, in all likelihood, oxytocin. The neurohormonal theory of Ely and Peter- sen and the subsequent work of Petersen and his colleagues (see reviews by Petersen, 1948; and Harris, 1958), unlike the earlier work of Gaines, aroused wide interest and its practical applications permitted rationaliza- tion of milking techniques in the cowshed thereby improving milk yields. Despite the attractiveness of the concept, however, a further 10 years were to elapse before un- equivocal evidence of the correctness of the theory was forthcoming and this evidence we shall now briefly review. B. ROLE OF THE NEUROHYPOPHYSIS The first reliable indication that the suckling or milking stimulus does in fact cause an outpouring of neurohypophyseal hormones were the observations that in- hibition of diuresis occurred following the application of the milking or suckling stimulus (Cross, 1950; Peeters and Cous- 622 PHYSIOLOGY OF GONADS sens, 1950; Kalliala and Karvoncn, 1951; Kalliala, Karvonen and Leppanen, 1952). It was also shown that electrical stimulation of the nerve paths to the posterior pituitary resulted in milk ejection (Cross and Harris, 1950, 1952; Andersson, 1951a, b, c; Popo- vich, 1958 », and that when lesions were placed in these tracts the milk-ejection re- flex was abolished (Cross and Harris, 1952) . Further evidence was adduced when it was found that removal of the posterior pituitary immediately abolished the milk- ejection reflex in the lactating rat, and that it was necessary to inject such animals sev- eral times a day with oxytocin if their litters were to be reared (Cowie, quoted by Folley, 1952b). Earlier workers had claimed that the posterior lolie was not essential for lac- tation (Smith, 1932; Houssay, 1935), but an explanation of these discordant conclusions was provided when it was shown that the impairment of the reflex after removal of the posterior lobe is not permanent and that the reflex re-establishes itself after some weeks, presumably because the remaining portions of the neurohypophysis take over the functions of the posterior lobe (Benson and Cowie, 1956). That the neurohypophy- sis participates in milk ejection would now appear to be beyond question. The discovery of the role of the neurohy- pophyseal hormones in milk ejection has provided an explanation of some longstand- ing clinical observations on what has been termed the natural "sympathy" between the uterus and the breasts. Thus the benefi- cial effects of the suckling stimulus and the occurrence of the "draught" {i.e., milk ejec- tion) in causing uterine contraction after parturition were emphasized over a century ago by both Smith (1844) and Patcrson (1844). 0})servations have also been made on the I'cciprocal process of stimuli arising from the reproductive organs apparently causing milk ejection. In domestic animals two such examples were mentioned by Mar- tiny (1871). According to Herodotus, the Scythians milk their mares thus: "They take l)lowpipes of bone, very like flutes, and put them into the genitals of the mares and blow with their mouths, others milk. And they say that the I'cason why thoy do so is this, that when the marc's \-cins ai'c filled with air, the udder cometh down" (transla- tion by Powell, 1949). Kolbe (1727) de- scribed a similar procedure of blowing air into the vagina used by the Hottentots when milking cows which were normally suckled by calves and in which, presumably, milk ejection did not occur in response to hand nnlking. A drawing depicting this procedure from Kolbe's book was recently published in the Ciba Zeitschrift (No. 84^ 1957) along with a photograph of African natives still using the method!-^ In 1839, Busch described the occurrence of milk ejection, the milk actually spurting from the nipple, in a lactating woman dur- ing coitus. A satisfactory explanation of these curious observations is now forth- coming. Harris (1947) suggested that coitus might cause the liberation of oxytocin from the neurohypophysis and, within the next few years it was demonstrated that stimula- tion of the reproductive organs evoked milk ejection in the cow (Hays and VanDemark. 1953) and reports confirmatory of Busch's long forgotten observations also appeared (Harris and Pickles, 1953; Campliell and Petersen, 1953).^ C. MILK-EJECTIOX HORMONE There is much circumstantial evidence to confirm the belief that the milk-ejection hormone is oxytocin (see Cowie and Policy. 1957). Attemi)ts, however, to demonstrate oxytocin in the blood after application of the milking stimulus have given rather in- conclusive results. Early claims that the hormone could be demonstrated in blood are ^ A similar drawing, also apparently from Kolbe '.•< book, has been used in the campaign for clean milk production! Heineman (1919) discussing sanitary l^recautions in the cowshed says of the picture "another picture shows a nude Hottentot milking a cow while another one is liolding the tail of the cow to prevent its dropping into the open pail. This ])icture might well serve as a model to some modern producers who do not take such precautions and calmly lift the tail out of the milk with their hands wlicn it hnjipens to switch into the pail." ' W(- h;i\(' hi'cii able to find only one painting illustrating this plienomenon. It is a picture by a contemporary French painter, Andre Masson, en- titled "Le Viol" and painted in 1939. It illustrates in Masson 's personal idiom the act of rape and it is interesting to note that a stream of milk is depicted as being I'orcibly (\iected from one breast of the MAMMARY GLAND AND LACTATION 623 of doiil)tful validity, because the milk-ejec- tion effect observed may have been due to 5-hydroxytryptamine (see Linzell, 1955), and more recent attempts to assay the level of oxytocin in the blood have not been entirely satisfactory or conclusive. There seem to be other polypeptide substances in blood which possess oxytocic activity, al- though the thiogly collate inactivation test indicates that these are different from oxy- tocin (Robertson and Hawker, 1957), and no marked changes in the blood oxytocic activity associated with suckling or milking have been detected (Hawker and Roberts, 1957; Hawker, 1958). However, it would seem doubtful whether the present assay techniques are sufficiently sensitive and spe- cific to detect changes in blood oxytocin of the magnitude likely to be associated with milking or suckling. In the lactating cow the intravenous injection of 0.05 to 2.0 I.U. oxytocin will cause milk ejection (Bilek and .Tanovsk>% 1956; Donker, 1958), in the goat 0.01 to 1 I.U. (Cowie, cited by Folley, 1952b; Denamur and Martinet, 1953), in the sow 0.2 to 1.0 I.U. (Braude, 1954; Whit- tlestone, 1954; Cross, Goodwin and Silver, 1958) in the rabbit 0.05 I.U. (Cross, 1955b) , and in the lactating woman 0.01 I.U. (Bel- ler, Krumholz and Zeininger, 1958) . If these (loses give any indication of the quantity of endogenous oxytocin released, then the concentration in the peripheral blood is likely to be very small ; indeed Cross, Good- win and Silver (1958) calculated that a threshold dose (10 mU.) of oxytocin in the sow w^ould give a plasma concentration of about 1 (U,U. per ml, and until it can be shown that the assay techniques are suf- ficiently sensitive to detect the changes in oxytocin concentration produced by in- travenous injections of "physiologic" doses of oxytocin, no great reliance can be placed on the results of assays. Attempts have been made to demonstrate alterations in the hormone content of the neural lobe following the suckling or milk- ing stimulus. In the goat and cow no de- tectable changes have been reported, but in the smaller species (dog, cat, rat, guinea pig) decreases have been described (see Cowie and Folley, 1957). It is likely that in many species the amount released is small relative to tlie total hormone content of the gland and within the limits of error of the assay. D. EFFECTOR CONTRACTILE MECHANISM OF THE MAMMARY GLAND In the last 10 years considerable research has been devoted to a study of the effector contractile tissue in the mammary gland; this work has recently been reviewed in some detail (see Folley, 1956) and only the salient features need be mentioned here. Although earlier histologists had from time to time figured myoepithelial or "bas- ket" cells in close association with the mam- mary alveoli, the morphology and distribu- tion of the cells remained vague until Richardson (1949) published a detailed and illuminating description (Fig. 10.18). His beautiful observations have since been con- firmed and supplemented by Linzell (1952) and Silver (1954). Richardson also disposed of the oft repeated view that smooth-mus- cle fibers around the alveoli played an im- l)ortant role in milk ejection. From a study of the general orientation of the myoepithe- lial cells and the precise relationship between these cells and the folds in the secretory epi- thelium from contracted glands, Richardson considered it reasonable to regard the myo- epithelium as the contractile tissue in the mammary gland which responds to oxytocin causing contraction of the alveoli and wid- ening of the ducts. The evidence adduced by Richardson, although good, was neverthe- less circumstantial, and it was desirable that attempts be made to visualize the contrac- tion of the myoepithelial cells in response to oxj^tocin. In this connection it is of interest to recall that Gaines (1915) reported that when a drop of pituitrin was placed on the cut surface of the mammary gland from a lactating guinea pig, minute white dots ap- peared within a few seconds beneath the pituitrin and slowly swelled to tiny milky rivulets streaming beautifully through the clear liquid. Much later the local effects of posterior pituitary extract on the mammary gland were studied by Zaks (1951) in the living mouse, when it was reported that it caused contraction of the alveoli and ex- pansion of the ducts. These observations were considerablv extended bv Linzell 624 PHYSIOLOGY OF GONADS Fig. 10.18. Surface view of contracted alveoli (of goat) showing myoepithelial cells. (Courtesy of K. C. Richardson.) Fig. 10.19. Recording of pressure changes witliin a galactophore of a forcibly restrained lactating rabbit. The litter was allowed to suckle the non- cannulated mammary glands but obtained only 8 gm. milk, there being only a slight rise in the milk pressure probably associated with a slight contraction of the myoepithelium in response to mechanical stimulation. When 5 mU. oxytocin were injected (5P) there was a rapid milk ejection response which could be inhibited by injecting 1 yug. adrenaline (lA) just before the oxytocin. After a few minutes 5 mU. oxytocin were again effective and the litter obtained 44 gm. milk when they were allowed to suckle. A more complete milk ejection respon.so was obtained with 50 mU. oxytocin (50P) and the young obtained a further 59 gm. milk. Anesthesia did not enhance the milk-ejection re- sponse to 50 mU. oxytocin. During emotional in- hibition of milk ejection the mammary gland thus remains responsive to oxytocin. (From B. A. Cross, J. Endocrinol., 12, 29-37, 1955.) (19ooi who studied the local effects of liighly purified oxytocin and vasopressin and a number of other drugs on the mam- mnry gland, and confirmed that oxytocin and vasopressin produced alveolar contrac- tion and widening of the ducts. Although in these experiments the myoepithelial cells themselves could not be visualized, never- theless the effects observed leave little doubt that the effector mechanism was the niyoei)ithelium. The myoepithelium is responsive to stim- uli other than those arising from the pres- ence of neurohypophyseal hormones in the blood inasmuch as partial milk ejection may occur in response to local mechanical stimulation of the mammary gland (Cross, 1954; Yokoyama, 1956; see also Fig. 10.191. These observations may explain the recent reports by Tverskoi (1958) and Denamui- and Martinet (1959a, b) that milk yields can be maintained in goats in the absence of the milk-ejection reflex. E. INHIBITION OF MILK EJECTION (laines (1915) stressed that the conduc- tion in the milk-ejection reflex pathway was dei)endent on the psychic condition of the MAMMARY GLAND AND LACTATION 625 mother. Many years later Ely and Petersen (1941) confirmed this and, having shown that injections of adrenaline blocked the milk-ejection reflex, postulated that the in- creased blood level of adrenaline in emo- tionally disturbed cows interfered with the action of oxytocin. In the last few years, the nature of the inhibitory mechanisms has been more fully investigated. Braude and Mitchell (1952) showed in the sow that adrenaline exerts at least part of its inhibi- tory effect at the level of the mammary gland and that, whereas the injection of adrenaline before the injection of oxytocin blocked milk ejection, less inhibition oc- curred if both were given together. Cross (1953, 1955a) confirmed these observations in the rabbit and demonstrated that electri- cal stimulation of the posterior hypothala- mus (sympathetic centers) inhibited the milk-ejection response to injected oxytocin, an effect which was abolished after adrenal- ectomy. Cross concluded from his experi- ments that any central stimulation causing sympathetico-adrenal activity inhibits the milk-ejection response and that the effect appears to depend on a constriction of the mammary blood vessels resulting from the release of adrenaline and excitation of the sympathetic fibers to the mammary glands. Whereas such a mechanism could account for the emotional disturbance of the reflex. Cross was careful to point out that there was no direct proof that this was so and he later demonstrated (Cross, 1955b) that in rabbits in which emotional inhibition of milk ejection was present, milk ejection could be effected by the injection of oxy- tocin (Fig. 10.19). In such cases there was clearly no peripheral inhibitory effect of milk ejection. Cross concluded that the main factor in emotional disturbance of the milk- ejection reflex is a partial or complete in- hibition of oxytocin release from the pos- terior pituitary gland. At present nothing is known of the nature of this central inhibi- tory mechanism.^ ^ A curious form of the suckling stimulus is il- lustrated in carvings which siumount the main door of the church of Sainte Croix in Bordeaux. The carvings illustrate penances prescribed for wrong doers who have committed one of the seven deadly sins. The penance for indulgence in the sin of luxiu y is the application to the breasts of serpents or toads. Inhibition of the milk ejection reflex may also occur when the mammary gland be- comes engorged with secretion to such an extent that the capillary circulation is so re- duced that oxytocin can no longer reach the myoepithelium (Cross and Silver, 1956; Cross, Goodwin and Silver, 1958). F. NEURAL PATHWAYS OF THE MILK-EJECTION REFLEX Interpretation of some of the earlier studies on neural pathways is difficult be- cause investigators did not realize that, al- though the milk ejection reflex normally occurs in response to the suckling stimulus, it can become conditioned and can then oc- cur in response to visual or auditory stimuli associated with the act of nursing. In such cases an apparent lack of effect on milk ejection of section of nerves or nerve tracts would not necessarily imply that the nerves normally carrying the stimuli arising from the suckling had not been cut. Studies on the effects of hemisection of the spinal cord in a few goats led Tsakhaev (1953) to the conclusion that the apparent pathway used by the milk-ejection stimulus was un- crossed. More recently pathways within the spinal cord have been investigated by Eayrs and Baddeley (1956) who found inter alia that lactation in the rat was inhibited by lesions to the lateral funiculi, and by section of the dorsal roots of nerves supplying the segments in which the suckled nipples were situated. With few exceptions hemisection of the spinal cord abolished lactation when the only nipples available for suckling were on the same side as the lesion, but not when the contralateral nipples were avail- able. It was concluded that the pathway used by the suckling stimulus enters the central nervous system by the dorsal routes and ascends the cord deep in the lateral funiculus of the same side. Inasmuch as in these experiments lactation was assessed from the growth curve of the pups, it is not always clear whether the failure of lactation was due to a cessation of milk secretion or to loss of the milk-ejection reflex. It was noted, however, that injections of oxytocin in some It may be questioned whether this unusual form of the suckling stimulus would not inhibit rather than evoke the milk-ejection reflex. 626 PHYSIOLOGY OF GONADS cases restored lactation for up to 2 days after it had ceased as a result of lesions of the cord which would suggest a primary interference with milk ejection. In the goat, Andersson (1951b) considered that stim- uli may reach the hypothalamus by way of the medial lemniscus in the medulla, but little definite information is available con- cerning the pathways used by the stimuli to reach the hypothalamus and there is here scope for further investigations. (For fur- ther discussion see review by Cross, 1960.) From the hyopthalamus there is little doubt that the route to the posterior lobe is by way of the hypothalamo-hypophyseal tract which receives nerve fibers from the cells in the hypothalamic nuclei, and in the main from the paraventricular and supra-optic nuclei. It was generally assumed that the posterior lobe hormones were secreted in the posterior lobe from the pituicytes in re- sponse to stimuli passing down the hypo- thalamo-hypophyseal tract. In the last dec- ade, however, much evidence has come to light which suggests that the so-called pos- terior lobe hormones are in fact elaborated in the cells of the hypothalamic nuclei and are then transported down the axones as a neurosecretion and stored in the posterior lobe (see Scharrer and Scharrer, 1954). Before leaving the neural pathways of the milk-ejection reflex, brief reference must be made to the recent discovery by Soviet phys- iologists that there is also a purely nervous reflex (segmental in nature) involved in the ejection of milk. It is said that within a few seconds of the application of the milking stimulus, reflex contraction of the smooth muscle in the mammary ducts occurs, caus- ing a flow of milk from the ducts into the cistern. This reflex contraction of the smooth muscle is also believed to occur in response to stimuli arising within the gland between milkings thus aiding the redistribution of milk in the udder. This purely nervous reflex is stated to occur some 30 to 60 seconds be- fore the reflex ejection of milk from the alve- oli by oxytocin (for further details sec review by Baryshnikov, 1957). The condi- tioned reflexes associated with suckling and milking have been the subject of numerous investigations l)y Grachev (see Grachev, 1953, 1958) ; these and other Russian re- searches into the motor apparatus of the ud- der have been fully reviewed by Zaks (1958). G. MECHANISM OF SUCKLING In the past, various theories have been put forward as to how the suckling obtains milk from its mother's mammary gland. In the human infant some considered that the lips formed an airtight seal around the nip- ple and areola thus allowing the child to suck, whereas others believed that compres- sion of the lacteal sinuses between the gums aided the expulsion of the milk (see Ardran, Kemp and Lind, 1958a, b for review) . In the calf the act of suckling was studied by Krzywanek and Briiggemann (1930) who described how the base of the teat was pinched off between upper and lower jaws and the teat compressed from its base to- wards its tip by a stripping action of the tongue. Smith and Petersen (1945) on the other hand, concluded that the calf wrapped its tongue round the teat and obtained milk by suction. Much misunderstanding about the nature of the act of suckling has arisen because the occurrence of milk ejection was overlooked or its significance was not appreciated. As a result, the idea became prevalent that suc- cess or failure in obtaining milk could be reckoned solely in terms of the power behind the baby's suction. This erroneous concept was vigorously attacked by Waller (1938), who pointed out that once the "draught" had occurred the milk at times flowed so freely from the breast that the baby had to break off and turn its head to avoid choking. A similar observation had been made by Sir Astley Cooper in 1840 who in describing the "draught" in nursing women wrote, "If the nipple be not immediately caught by the child, the milk escapes from it, and the child when it receives the nipple is almost choked l)y the rapid and abundant flow of the fluid; if it lets go its hold, the milk spurts into the infant's eyes." An even earlier comment was made by Soranus, a writer on paediatrics in the cai'ly half of the second century A.D., that it was unwise to allow the infant to fall asleep at the breast since the milk some- MAMMARY GLAND AND LACTATION 627 times flowed without suckling and the infant choked. It must thus be emphasized that once milk ejection has occurred the milk in the gland cisterns or sinuses is under con- siderable pressure and the suckling has merely to overcome the resistance of the sphincters in the nipple or teat to obtain the milk. Recently the use of cineradiograjihy has allowed a more accurate analysis of the mechanism of suckling. Studies by Ardran, Kemp and Lind (1958b) have shown that the human infant sucks the nipple to the back of the mouth and forms a "teat" from the mother's breast; when the jaw is raised this teat is compressed between the upper gum and the tip of the tongue resting on the lower gum, the tongue is then applied to the lower surface of the "teat" from be- fore backwards pressing it against the hard palate. Suction may assist the flow of milk so expressed from the nipple, but is only of secondary importance. Studies by Ardran, Cowie and Kemp (1957, 1958) in the goat have extended these observations, because it was possible in this species to follow the withdrawal, from the udder, of milk made radiopaque with barium sulfate. As with the infant, the neck of the teat was obliter- ated between the tongue and the palate of the kid and the contents of the teat sinus were displaced into the mouth cavity by a suitable movement of the tongue; while the first mouthful w^as being displaced into the pharynx, the jaw and tongue were low- ered to allow the refilling of the teat sinus. The normal method of obtaining milk is, therefore, for the suckling to occlude the neck of the teat and then to expel the con- tents of the teat sinus by exerting positive pressure on the teat (120 mm. Hg in the goat), so forcing the contents through the teat canal or nipple orifices into the mouth cavity, a process which may be aided by negative pressure created at the tip of the teat. Human infants, goat kids, and calves can obtain milk through rubber teats by suction alone provided the orifice is large enough (see Krzywanek and Briiggemann, 1930; Martyugin, 1944; Ardran, Kemp and Lind, 1958a) , but this procedure occurs only w^hen the structure of the rubber teat is such that the suckling is unable to ol)literate the neck of the teat and cannot, therefore, strip the contents of the teat by positive pressure. V. Relation between the Reflexes Con- cerned in the Maintenance of Milk Secretion and Milk Ejection We have seen that the suckling or milk- ing stimulus is responsible for initiating the reflex concerned wath the maintenance of milk secretion and also the milk-ejection re- flex; the question now arises as to what ex- tent their arcs share common paths. It would seem logical to assume that a common path to the hypothalamus exists and parts of this, as we have seen, have been partially elucidated. Although the hypothalamo-hy- pophyseal nerve tracts provide an obvious link between hypothalamus and the pos- terior lobe, the connections between the hy- pothalamus and anterior pituitary are still a matter of some controversy. The possible avenues of communication to the anterior lobe are neural and vascular and these may be subdivided into central and peripheral neural connections and into portal and sys- temic vascular connections. The various ex- perimental findings relating to these routes have recently been critically discussed by Sayers, Redgate and Royce (1958), and by Greep and Everett in their chapters in this book, and it is clear that at present no defi- nite conclusions can be reached concerning their relative importance. So far as the spe- cific question of maintenance of milk secre- tion is concerned, the experiments of Harris and Jacobsohn (1952), which showed that pituitary grafts maintained lactation when implanted adjacent to the median eminence in hypophysectomized rats, were consistent with the existence of a hormonal transmit- ter, passing by w^ay of the hypophyseal por- tal system. On the other hand, transplanta- tion studies by Desclin (1950, 1956) and Everett ( 1954, 1956) have revealed that in the rat the anterior lobe can spontaneously secrete prolactin in situations remote from the median eminence, and Donovan and van der Werff ten Bosch (1957) have reported that milk secretion continued in rabbits in wiiich the pituitary portal vessels had been completely destroyed, although there was, however, an inferred change in milk compo- sition. Evidence has recentlv been obtained 628 PHYSIOLOGY OF GONADS which has confirmed that pituitary tissue grafted under the kidney capsule in rats ap- parently secretes prolactin and will give slight maintenance of milk secretion in hy- pophysectomized animals, this maintenance being considerably enhanced if ACTH or STH is also administered (Cowie, Tindal and Benson, 1960). It would thus seem that the cells of the anterior lobe have the ability when isolated from the hypo- physeal portal system to secrete prolactin, but the experiments cited above allow no conclusions to be drawn regarding the route by which the galactopoietic function of the pituitary is normally controlled. Recent reports that bilateral cervical sympathectomy in the lactating goat causes a fall in the milk yield suggest that the ga- lactopoietic functions of the anterior lobe may be influenced by the sympathetic nerv- ous system (Tsakhaev, 1959; Tverskoy, 1960) . Declines in milk yield also occur after section of the pituitary stalk in the goat, but it is not clear in such cases whether the ef- fects are due to the interruption of nervous or vascular pathways within the stalk (Tsakhaev, 1959; Tverskoy, 1960). In these studies on stalk section the cut ends of the pituitary stalk were not separated by a plas- tic plate, so some restoration of the hy- l^ophyseal portal system may have occurred. Further experiments on the effects of sec- tion of the pituitary stalk on lactation in which restoration of the hypophyseal portal is prevented by the insertion of a plate are being conducted in our laboratory and also in the Soviet Union. Another possible mode of communication between hypothalamus and anterior pituitary has been investigated by Benson and Folley (1956, 1957a, b) who have suggested that the oxytocin released from the neurohypophysis in response to the suckling stimulus may directly act on the cells of the anterior lobe and stimulate the release of the galactopoietic complex. The careful anatomic researches of Landsmeer (1951), Daniel and Prichard (1956, 1957, 1958) and Jewell (1956) have demonstrated in several species the existence of direct vascular connections from the neurohy- lK)physis to the anterior lobe so that the neurohypophyseal hormones liberated into the blood stream would in fact be carried direct to the anterior pituitary cells in very high concentrations. Clearly such a concept would provide a simple explanation of how the hormonal integration, coordination, and maintenance of mammary function is achieved. It has already been noted (see page 607) that a connection between milk ejection and the onset of copious lactation has been suggested. There is considerable evidence that oxytocin is liberated during parturition in sufficient quantities to cause contraction of the alveoli and milk ejection (see Harris, 1955; Cross, 1958; Cross, Good- win and Silver, 1958) ; if, therefore, oxytocin can release the lactogenic and galatopoietic complexes from the anterior pituitary, a simple explanation of the mechanism trig- gering off the onset of copious milk secre- tion, before the application of the milking stimulus, is available. We must now consider what experimental evidence there is to support this rather at- tractive theory. First, Benson and Folley (1956, 1957a, b) demonstrated that regular injections of oxytocin can retard mammary regression after weaning in a similar fash- ion to injections of prolactin (see page 610), and they have shown that the pres- ence of the pituitary is essential for oxytocin to elicit this effect. Synthetic oxytocin proved equally effective, thus discounting the possibility of a contaminant in natural oxytocin being concerned (Fig. 10.20) . These experiments have so far only been carried out in rats, but they strongly suggest that oxytocin can elicit the secretion of prolactin. In agreement with this concept are several observations that regular injections of oxy- tocin have galactopoietic effects in lactating cows and that oxytocin has luteotrophic ef- fects in rats (see review by Benson, Cowie and Tindal, 1958) . There is, moreover, some evidence that the suckling stimulus may cause the release of vasopressin or the anti- diuretic hormone (ADH) from the neuro- hypoi)hysis (see page 621), and it has been shown that ADH or some material closely associated with it may cause the secretion of ACTH from the anterior lobe (see review by Benson, Cowie and Tindal, 1958) ; so there are some grounds for supposing that the hormones of the posterior lobe evoke the secretion of several components of the MAMMARY GLAND AND LACTATION Fig. 10.20. Sections from abdominal mammary gland of rats from wliuli Ur- pups were removed on the fourth day of lactation and which received thereafter for 9 daj^s: A. LO I.U. synthetic oxytocin three times daily. B. Saline daily. Note the maintenance of gland structure in A. (Courtesy of Dr. G. K. Benson.) galactopoietic complex from the anterior lobe. It was hoped to gain further evidence on this point by studies on hypophysecto- mized rats bearing pituitary homografts under the kidney capsule (see Benson, Cowie, Folley and Tindal, 1959) . As already noted, such grafts secrete prolactin and will give a slight maintenance of milk secretion, but these grafts will not maintain normal milk secretion even when such animals are injected with oxytocin and ADH (Cowie, Tindal and Benson, 1960). It must, there- fore, be assumed that if these posterior pituitary hormones are responsible for the release of the galactopoietic complex, some other hypothalamic factor is also necessary to maintain the anterior lobe in a responsive condition. Everett (1956) suggested that the hypothalamus by way of its neurovas- cular connections with the anterior lobe, normally exerts a partial inhibitory effect on prolactin secretion. It may thus be that when the anterior lobe is removed from hypothalamic influence, the synthetic ac- tivities of its cells are centered on prolactin production to the detriment of the other components of the galactopoietic complex, so that these are no longer available for re- lease in response to neurohypophyseal hor- mones. There is need, however, for experi- mentation in other species. The theory that the release of the galac- topoietic complex is effected by the hor- mones of the posterior lobe secreted in re- sponse to the suckling stimulus is attractive in that it appears to afford a simple explana- tion of the hormonal integration of mam- mary function, but it must be pointed out that the observations on the maintenance of mammary structure after weaning by injec- tions of oxytocin do not prove that prolactin or the galactopoietic complex is released in response to oxytocin under normal condi- tions of milking or suckling, and more re- search, particularly in species other than the rat, is necessary. Grosvenor and Turner (1958a) injected oxytocin into anesthetized lactating rats and, on the basis of assays of the pituitary content of prolactin, considered that oxytocin caused no significant release of 630 PHYSIOLOGY OF GONADS prolactin. They had previously shown that there was an immediate fall in the pituitary content of prolactin after nursing (Gros- vcnor and Turner, 1957b) and therefore concluded that their findings were contrary to the hypothesis that oxytocin is a hor- monal link in the discharge of prolactin. This, however, cannot be regarded as con- clusive because of the difficulties of relating pituitary content of a hormone to blood levels of the hormone and also the difficulty of determining the physiologic dose of oxy- tocin, for if the oxytocin is carried directly from the neurohypophysis into the anterior lobe, then the concentration in the blood reaching the anterior lobe may be relatively great (see also Cowie and Folley, 1957). Other theories of the reflex maintenance of milk secretion have been put forward. In 1953 Tverskoi, observing that repeated in- jections of oxytocin were galactopoietic in the goat, suggested that alveolar contraction stimulated sensory nerve endings in the alveolar walls which reflcxly caused the re- lease of prolactin. It is obvious that his observations could be explained on the basis of the Benson-Folley theory of direct pitui- tary stimulation by oxytocin. This possi- bility was indeed considered by Tverskoi. but rejected on the grounds that oxytocin did not affect the prolactin content of the pituitary (Meites and Turner, 1948). In 1957 Tverskoi found it necessary to revise his theory, having found that full lactation could be maintained in the goat after com- plete and repeated denervation of the udder provided oxytocin was regularly given to evoke milk ejection. He then suggested that alveolar contraction stimulates the syn- thetic activities of the mammary epithelium causing an uptake of prolactin from the blood, the fall in the blood prolactin level then stimulating the further production of prolactin by the anterior lobe. Although these latter observations of Tverskoi might again be explained on the basis of direct pituitary stimulation by exogenous oxy- tocin, more recent studies on goats have cast doubts on the validity of such an ex- planation. Tverskoi (1958) and Denannir and Martinet (1959a, b, 1960) have shown that lactating goats will continue to lactate, giving nonnal or onlv niodcratelv reduced milk yields after section of all nervous con- nections between the udder and brain (cord section, radicotomy, bilateral sympathec- tomy) and without their receiving oxytocin and in the absence of conditioned milk- ejection reflexes. It has already been noted that milk ejection in such animals may re- sult from mechanical stimulation of the myoepithelial cells by udder massage (see page 624) , but the release of the galacto- poietic complex from the anterior pituitary would seem in these goats to have been in- dependent of neurohormonal reflex activi- ties. AVhether in such animals the release is spontaneous or dependent on the level of hormones in the blood as suggested by Tverskoi (1957) is a matter for further re- search. VI. Pharmacologic Blockade of the Re- flexes Concerned in the Maintenance of Milk Secretion and Milk Ejection Various attempts have been made to investigate the mechanism controlling re- lease of anterior pituitary hormones by the use of dibenamine, atropine, and other drugs. In reviewing such experiments, Harris (1955) concluded that there was no con- vincing evidence of the participation of adrenergic, cholinergic, or histaminergic agents in the control of gonadotrophic and adrenocorticotrophic hormone release. Re- cently Grosvenor and Turner (1957a) re- ported that various ergot alkaloids, diben- amine, and atropine blocked milk ejection in the rat; the ergot alkaloids doing so within 10 minutes of administration, the atropine and dibenamine within 2 to 4 hours. Inasmuch as milk ejection occurred in re- sponse to exogenous oxytocin, it was con- cluded that these drugs acted centrally, and the presence of adrenergic and cholinergic links in the neurohormone arc was postu- lated to be responsible for the discharge of oxytocin. Later, on tlie basis of assays of jntuitary prolactin after nursing in drug- injected lactating rats, it was suggested that cholinergic and adrenergic links are iinohcd in the reflex resi)onsible for pro- lactin release (Grosvenor and Turner, 1958a). Ergot alkaloids, however, adminis- tered in our laboratory to lactating rats had no significant effect on the lactational per- MAMMARY GLAND AND LACTATION 631 fonnance as judged by the growth of the litters in comparison with the growth of litters of pair-fed control rats, showing that apparent inhibitory effects of the alkaloids on lactation were due to depressed food in- take of the mothers (Tindal, 1956a). Inas- much as growth of the litter depends on efficient milk secretion and milk ejection, Tindal's observations seem to throw doubt on the importance of the adrenergic link in these reflexes. On the other hand, IVIeites (1959) has reported that adrenaline and acetylcholine can induce or maintain mam- mary development and milk secretion in suitably prepared rats, observations which could be interpreted as supporting the pres- ence of adrenergic and cholinergic links as postulated by Grosvenor and Turner (1958a). There have been clinical reports of wo- men developing galactorrhoea after treat- ment with trancjuilizing drugs {e.g., Sulman and Winnik, 1956; Marshall and Leiber- man, 1956; Piatt and Sears, 19561 and in- teresting observations have recently ap- peared on the lactogenic effects of reserpine in animals. Milk secretion has been initiated both in virgin rabbits after suitable estrogen priming and in the pseudopregnant rabbit by reserpine (Sawyer, 1957; Meites, 1957a). On the other hand, in our laboratory Tindal (1956b, 1958) had been unable to detect any mammogenic or lactogenic effects with chlorpromazine or reserpine in rabbits (Dutch breed), rats, or goats, nor did reser- pine stimulate the crop-sac when injected into pigeons. Recently, using New Zealand White rabbits, Tindal (1960) has induced milk secretion with reserpine. The reason for these contradictory results is not entirely clear, although breed differences in the re- sponse would appear to exist in the rabbit. In our laboratory, Benson (1958) has shown that reserpine is strikingly active in re- tarding mammary involution in the lactat- ing rat after weaning, the effect being of such a magnitude as has so far only been equalled by a combination of prolactin and STH (Fig. 10.21). It has been tentatively suggested that the tranquilizing drugs may ^^:f/ mm\"^>m.-Wi ■w^- .•^^:j^-^ f4kr 1" Fig. 10.2L Sections from the abdominal mammary gland of rats from whicli the pujis were removed on the fourth day of lactation and which received thereafter for 9 days: A 100 fj.g. reserpine daily. B. Sahne dailJ^ Note the retardation of involution effected by reserpine. (Courtesy of Dr. G. K. Benson.) 632 PHYSIOLOGY OF GONADS remove .some hypothalamic restraining mechanism on the release of jn'olactin and probably of other anterior-pituitary hor- mones (Sulman and Winnik, 1956; Benson, Cowie and Tindal, 1958), an effect which, if confirmed, may throw light on the be- havior of pituitary transplants in sites re- mote from the median eminence. VII. Conclusion Any reader familiar with the chajiter on the mammary gland in the previous edition of this book cannot fail to note the main directions in which the subject has advanced in the intervening two decades. These re- flect, as they are bound to do, the road taken by the science of endocrinology itself, a road leading to greater biochemical understand- ing on the one hand and to ever closer rap- prochement with neurophysiology on the other. The mammary gland offers unique oppor- tunities of studying the biochemical mecha- nisms of hormone action because it is an organ with quite exceptional synthetic capa- bilities, an organ which is perhaps the most comprehensive hormone target in the mam- malian body. Biochemists are entering this promising field in increasing numbers and we may expect to reap the fruits of their labors in the future. VIII. References Abraham, S., Cady, P., and Chaikoff, I. L. 1957. Effect of insulin in vitro on pathways of glu- cose utilization, other than Embden-Meyerhof, in rat mammarv gland. J. Biol. Cliem., 224, 955-962. Ahren, K. 1959. 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[Tsakhabv, G. a.] II,axaeB, F. A. 1953. O npiipoAe a(j)(j)epeHTHbix ny xett pe(|).TieKca MOJiOKooTjia'm. Dokl. Akad. Nauk S.S.S.R., 93, 941-944. [Tsakhaev, G. a.] UaxaeB, F. A. 1959. CeKpe- TopHaa (j)yHKiiiiH Mo.noHHoft :xeJie3M b ycjio- BiiHx neHepBauiiii rnno(})ii3a. In Bonpocw (j[)II3HO.nOrmi CejIbCK0.\03HHCTBeHHbIX JLHBO- THbix. M. G. Zaks. Ed., pp. 3-19. Vilnius, Kau- nas: Akademiya Nauk Litovsk. S.S.R. Turner, C. W. 1939. The mammary glands. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., Ch. XL Baltimore: The Williams & Wilkins Com- pany. Turner, C. W. 1952. The Mammary Gland. Vol. 1. The Anatomy of the Udder of Cattle and Domestic Animals. Columbia, Mo.: Lucas Bros. Turner, C. W.. .-^nd Gomez, E. T. 1933. The normal development of the mammary gland of the male and female albino mouse. Res. Bull. Missouri Agric. Exper. Sta., No. 182. Turner, C. W., and Gomez, E. T. 1934. The ex- perimental development of the mammary gland. II. The male and female guinea pig. Res. Bull. Missouri Agric. Exper. Sta No 206. Turner, C. W., and Schultze, A. B. 193L A study of the causes of the normal development of the mammary gland of the albino rat. Res Bull. Missouri Agric. Exper. Sta., No. 157. Turner, C. W., and Slaughter, I. S. 1930. The physiological effect of pituitary extract (pos- terior lobe) on the lactating mammarv gland J.Dairy Sc, 13, 8-24. [TvERSKoi, G. B.] TBepcKOH, F. B. 1953. O npiipOAe HyBCTBIITeJILHblX ctumv.tiob c BbmeHH, yyacTByiouiiix b pe(J)JieKTopHoft pery.nHmin ceKpei;Hn MO.noKa. Zhur. Obshchel Biol., 14, 349-359. [TvERSKoi, G. B.] TBepcKOH, F. B. 1957. O pojiH HyBCTEHTejIbHOH IIHHepBaAHII xMOJIOMHOa JKe.nesbi b pe({).neKTopHoii pery.nnmiH ceKpeAHii MOJioKa a mo-jiohhofo H,iipa. Zhur ObshcheT Biol., 18, 169-184. [TvERSKoi, G. B.] TsepcKofi F. B. 1958 CeKpeuna MO.ioKa y K03 noc.Tie no.HHoft nepepe3KH cnnHHoro Moara. Dokl. Akad. Nauk. SSSR 123, 1137-1139. ' TvERSKOY, G. B. 1960. Influence of cervical sym- pathectomy and pituitary stalk section upon milk secretion in goats. Nature, London, 186, 782-784. VAN W.-iGENEN, G., AND FOLLEY, S. J. 1939. The effect of androgens on the mammary gland of the female rhesus monkev. J. Endocrinol 1, 367-372. Vendrely, R. 1955. The deoxyribonucleic acid content of the nucleus. In The Xucleic Acids: Chemistry and Biology, E. Chargaff and J. N. Davidson, Eds., Ch. 19, pp. 155-180. New York : Academic Press, Inc. VON Berswordt-Wallrabe, R. 1958. Versuch ei- ner theoretischen Erklarung der Hemmung der Galaktopoese der Albinomaus durch Dieno- stroldiacetat. Arch. Gynak., 190, 619-637. Walker, S. M., and M.^tthews, J. I. 1949. Ob- servations on the effects of prepartal and post- partal estrogen and progesterone treatment on lactation in the rat. Endocrinology, 44, 8-17. Waller, H. 1938. Clinical Studies\)i Lactation. London: William Heinemann, Ltd. Whittlestone, W. G. 1954. Intramammary pres- sure changes in the lactating sow. HI. 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T. 1958. administration of the substrate of the enzyme, tryptophan, would lead to an increase in the activity of the en- zyme which was maximal in 6 to 10 hours. Evidence that the increased activity of enzyme following the administration of cortisone represents the synthesis of new protein molecules is supplied by experi- STEROID SEX HORMONES 653 ments in which it was found that the in- crease in enzyme activity is inhibited by ethionine and this inhibition is reversed by methionine. The amino acid analogue ethionine is known to inhibit protein syn- thesis and this inhibition of protein syn- thesis is overcome by methionine. The injection of estrogen into the im- mature or castrate rodent produces a strik- ing uptake of water by the uterus followed by a marked increase in its dry weight (Astwood, 1938). Holden (1939) postu- lated that the imbibition of water results from vasodilatation and from changes in the permeability of the blood vessels of the uterus. There is clear evidence (Mueller, 1957) that the subsequent increase in dry weight is due to an increased rate of syn- thesis of proteins and nucleic acids. The sex hormones and other steroids could be pictured as reacting with the protein or lipoprotein membrane around the cell or around some subcellular structure like a surface-wetting agent and in this way in- ducing a change in the permeability of the membrane. This might then increase the rate of entry of substances and thus alter the rate of metabolism within the cell. This theory could hardly account for the many notable specific relationships between steroid structure and biologic activity. Spaziani and Szego (1958) postulated that estrogens induce the release of histamine in the uterus and the histamine then alters the permeability of the blood vessels and pro- duces the imbibition of water secondarily. The uterus of the ovariectomized rat is remarkably responsive to estrogens and has been widely used as a test system. After ovariectomy, the content of ribo- nucleic acid of the uterus decreases to a low level and then is rapidly restored after injection of estradiol (Telfer, 1953). A single injection of 5 to 10 yu,g. of estradiol brings about (1) the hyperemia and water imbibition described previously; (2) an increased rate of over-all metabolism as reflected in increased utilization of oxygen (David, 1931; Khayyal and Scott, 1931; Kerly, 1937; MacLeod and Reynolds, 1938; Walaas, Walaas and Loken, 1952a; Roberts and Szego, 1953a) ; (3) an increased rate of glycolysis (Kerly, 1937; Carroll, 1942; Stuermer and Stein, 1952; Walaas, Walaas and Loken, 1952b; Roberts and Szego, 1953a) ; (4) an increased rate of utilization of phosphorus (Grauer, Strickler, Wolken and Cutuly, 1950; Walaas and Walaas, 1950) ; and (5) tissue hypertrophy as re- flected in increased dry weight (Astwood, 1938), increased content of ribonucleic acid and protein (Astwood, 1938; Telfer, 1953; Mueller, 1957), and finally, after about 72 hours, an increased content of desoxy- ribonucleic acid (Mueller, 1957). An important series of experiments by Mueller and his colleagues revealed that estrogens injected in vivo affect the metab- olism of the uterus which can be detected by subsequent incubation of the uterus in vitro with labeled substrate molecules. Mueller (1953) first showed that pre- treatment with estradiol increases the rate of incorporation of glycine-2-C^'* into uter- ine protein. He then found that estrogen stimulation increases that rate of incorpo- ration into protein of all other amino acids tested: alanine, serine, lysine, and trypto- phan. The peak of stimulation occurred about 20 hours after the injection of estra- diol. In further studies (Mueller and Her- ranen, 1956) it was found that estrogen increases the rate of incorporation of gly- cine-2-C^^ and formate-2-C^'* into protein, lipid, and the purine bases, adenine and guanine, of nucleic acids. A stimulation of cholesterol synthesis in the mouse uterus 20 hours after administration of estradiol was shown by Emmelot and Bos (1954). In more detailed studies of the effects of estrogens on the metabolism of "one-carbon units" Herranen and Mueller (1956) found that the incorporation of serine-3-C^'* into adenine and guanine was stimulated by pretreatment with estradiol. The incorpora- tion was greatly decreased when unlabeled formate was added to the reaction mixture to trap the one-carbon intermediate. In contrast, the incorporation of C^^02 into uridine and thymine by the surviving uter- ine segment was not increased by pretreat- ment with estradiol in vivo (Mueller, 1957). To delineate further the site of estrogen effect on one-carbon metabohsm, Herranen and Mueller (1957) studied the effect of estrogen pretreatment on serine aldolase, the enzyme which catalyzes the equilibrium 654 PHYSIOLOGY OF GONADS between serine and glycine plus an active one-carbon unit. They found that serine aldolase activity, measured in homog- enates of rat uteri, increased 18 hours after pretreatment in vivo with estradiol. It seemed that the estrogen-induced increase in the activity of this enzyme might explain at least part of the increased rate of one- carbon metabolism following estrogen in- jection. They found, however, that incuba- tion of uterine segments in tissue culture medium (Eagle, 1955) for 18 hours pro- duced a marked increase in both the activity of serine aldolase and the incorporation of glycine-2-C^'* into protein. The addition of estradiol to Eagle's medium did not produce a greater increase than the control to which no estradiol was added. Uterine seg- ments taken from rats pretreated with estra- diol for 18 hours, with their glycine-incor- porating system activated by hormonal stimulation, showed very little further stimulation on being incubated in Eagle's medium for 18 hours. With a shorter period of i^retreatment with estradiol, greater stim- ulation occurred on subsequent incubation in tissue culture fluid. These experiments suggest that the hormone and the incuba- tion in tissue culture medium are affecting the same process, one which has a limited capacity to respond. When comparable ex- periments were performed with other labeled amino acids as substrates, similar results were obtained. Mueller's work gave evidence that a con- siderable number of enzyme systems in the uterus are accelerated by the administration of estradiol — not only the enzymes for the incorporation of serine, glycine, and formate into adenine and guanine, but also the en- zymes involved in the synthesis of fatty acids and cholesterol and indejX'ndent en- zymes for the activation of amino acids by the formation of adenosine monoiihosphate (AMP) derivatives. The initial step in protein synthesis has been shown to be the activation of the carboxyl grou]) of the amino acid with transfer of energy from ATP, the formation of AMP -"amino acid, and the release of jiyrophosphate (Hoagland, Keller and Zamecnick, 1956). This reversible step was studied with ho- mogenates of uterine tissue, P^--labeled ]n'rni)liosi)liate, and a variety of amino acids (Mueller, Herranen and Jervell, 1958). Seven of the amino acids tested, leucine, tryptophan, valine, tryosine, methi- onine, glycine, and isoleucine, stimulated the exchange of P^^ between pyrophosphate and ATP. Pretreatment of the uteri by estradiol injected in vivo increased the activity of these three enzymes. The activating effect of mixtures of these amino acids was the sum of their individual effects, from which it was inferred that a specific enzyme is involved in the activation of each amino acid. Since estrogen stimu- lated the exchange reaction with each of these seven amino acids, Mueller con- cluded that the hormone must affect the amount of each of the amino acid-activat- ing enzvmes in the soluble fraction of the cell. Mueller (1957) postulated that estrogens increase the rate of many enzyme systems both by activating preformed enzyme mole- cules and by increasing the rate of de novo synthesis of enzyme molecules, possibly by removing membranous barriers covering the templates for enzyme synthesis. To explain why estrogens affect these enzymes in the target organs, but not comparable enzymes in other tissues, one would have to assume that embryonic differentiation results in the formation of enzymes in different tissues which, although catalyzing the same re- action, have different properties such as their responsiveness to hormonal stimula- tion. As an alternative hypothesis, estrogen might affect some reaction which provides a substance required for all of these en- zyme reactions. The carboxyl group of amino acids must be activated by ATP be- fore the amino acid can be incorporated into proteins; the synthesis of both purines and pyrimidines requires ATP for the activation of the carboxyl group of certain precursors and for several other steps; the synthesis of cholesterol requires ATP for the conversion of mevalonic acid to squalene; and the synthesis of fatty acids is also an energy-requiring process. Thus if (>strogens acted in some way to increase the amount of biologically useful energy, in the form of ATP or of energy-rich thioesters such as acetyl coenzyme A, it would increase the rate of syntliesis of all of these compo- STEROID SEX HORMONES 655 nents of the cell. This would occur, of course, only if the supply of ATP, rather than the amount of enzyme, substrate, or some other cofactor, were the rate-limiting factor in the synthetic processes. When purified estrogens became avail- able, they were tested for their effects on tissues in vitro. Estrogens added in vitro in- creased the utilization of oxygen by the rat uterus (Khayyal and Scott, 1931) and the rat pituitary (Victor and Andersen, 1937). The addition of estradiol- 17^ at a level of 1 fxg. per ml. of incubation medium increased the rate of utilization of oxygen and of pyruvic acid by slices of human endome- trium and increased the rate at which la- beled glucose and pyruvate were oxidized to C^-^Os (Hagerman and Villee, 1952, 1953a, 1953b) . In experiments with slices of human placenta similar results were obtained and it was found that estradiol increased the rate of conversion of both pyruvate-2-C^'* and acetate-l-Ci4 to C^^Os (Villee and Hagerman, 1953) . From this and other evi- dence it was inferred that the estrogen acted at some point in the oxidative pathway common to pyruvate and acetate, i.e., in the tricarboxylic acid cycle. Homogenates of placenta also respond to estradiol added in vitro. With citric acid as substrate, the utilization of citric acid and oxygen and the production of a-ketoglutaric acid were increased 50 per cent by the addition of estradiol to a final concentra- lion of 1 fjig. per ml. (Villee and Hagerman, 1953). The homogenates were separated by differential ultracentrifugation into nuclear, mitochondrial, microsomal, and nonparticu- late fractions. The estrogen-stimulable sys- tem was shown to be in the nonparticulate fraction, the material which is not sedi- mented by centrifugating at 57,000 X g for 60 minutes (Villee, 1955). Experiments with citric, as-aconitic, isocitric, oxalosuc- cinic, and a-ketoglutaric acids as sub- strates and with fluorocitric and trans- aconitic acids as inhibitors localized the estrogen-sensitive system at the oxidation of isocitric to oxalosuccinic acid, which then undergoes spontaneous decarboxylation to a-ketoglutaric acid (Villee and Gordon, 1955). Further investigations using the en- zymes of the nonparticulate fraction of the human placenta revealed that, in ad- dition to isocitric acid as substrate, only DPN and a divalent cation such as Mg+ + or Mn++ were required (Villee, 1955; Gor- don and Villee, 1955; Villee and Gordon, 1956). The estrogen-sensitive reaction was formulated as a DPN-linked isocitric de- hydrogenase: Isocitrate + DPN* -^ a-ketoglutarate + CO2 + DPXH + H* It was found that the effect of the hor- mone on the enzyme can be measured by the increased rate of disappearance of citric acid, the increased rate of appearance of a-ketoglutaric acid, or by the increased rate of reduction of DPN, measured spectro- photometrically by the optical density at 340 m/x. As little as 0.001 /xg. estradiol per ml. (4 X 10~^ m) produced a measurable increase in the rate of the reaction, and there was a graded response to increasing concentrations of estrogen. The dose-re- sponse curve is typically sigmoid. This sys- tem has been used to assay the estrogen content of extracts of urine (Gordon and Villee, 1956) and of tissues (Hagerman, Wellington and Villee, 1957; Loring and Villee, 1957). Attempts to isolate and purify the estro- gen-sensitive enzyme were not very success- ful. By a combination of low temperature alcohol fractionation and elution from cal- cium phosphate gel a 20-fold purification was obtained (Hagerman and Villee, 1957). However, as the enzyme was purified it was found that an additional cofactor was re- quired. Either uridine triphosphate (UTP) or ATP added to the system greatly increased the magnitude of the estrogen ef- fect and, subsequently, adenosine diphos- phate (ADP) was recovered from the in- cubation medium and identified by paper chromatography (Villee and Hagerman, 1957). Talalay and Williams-Ashman (1958) confirmed our observations and showed that the additional cofactor was triphosphopyridine nucleotide (TPN) which was required in minute amounts. This find- ing was confirmed by Villee and Hagerman (1958) and the estrogen-sensitive enzyme system of the placenta is now believed to be a transhydrogenase which catalyzes the transfer of hydrogen ions and electrons 656 PHYSIOLOGY OF GONADS fromTPNHtoDPN: TPXH + DPN^ -> DPNH + TPN^ The transhydrogenation system can be coupled to glucose 6-phosphate dehydro- genase as well as to isocitric dehydrogenase (Talalay and Williams-Ashman, 1958; Vil- lee and Hagerman, 1958) and presumably can be coupled to any TPNH-generating system. If the estrogen-stimulable transhydrogen- ation reaction were readily reversible, an enzyme such as lactic dehydrogenase which requires DPN should be stimulated by estrogen if supplied with substrate amounts of TPN, catalytic amounts of DPN, and a preparation from the placenta containing the transhydrogenase. Experiments to test this prediction were made using lactic de- hydrogenase and alcohol dehydrogenase of both yeast and liver (Villee, 1958a). It was not possible to demonstrate an estrogen stimulation of either enzyme system in either the forward or the reverse direction. The stimulation of the lactic dehydrogen- ase-DPN oxidase system of the rat uterus by estrogens administered in vivo reported by Bever, Velardo and Hisaw (1956) might be explained by the stimulation of a transhydrogenase, but it has not yet been possible to demonstrate a coupling of this transhydrogenase and lactic dehydrogenase. The stimulating effect of a number of steroids has been tested with a system in which the transhydrogenation reaction is coupled to isocitric dehydrogenase (Villee and Gordon, 1956; Hollander, Nolan and Hollander, 1958). Estrone, equilin, equi- lenin, and 6-ketoestradiol have activities essentially the same as that of estradiol- 17 j3. Samples of 1 -methyl estrone and 2- methoxy-estrone had one-half the activitj'' of estradiol. Estriol is only weakly estro- genic in this system; 33 fig. estriol are less active than 0.1 fig. estradiol- 17/3 (Villee, 1957a). The activities of estriol and 16- epiestriol are similar, whereas 16-oxoestra- diol is more active than either, with about 10 per cent as much activitv as csti'adiol- 17/3. Certain analogues of stilbestrol have been shown to be anti-estrogens in vivo. When applied topically to the vagina of the rat, they prevent the cornification normally in- duced by the administration of estrogen (Barany, Morsing, Muller, Stallberg, and Stenhagen, 1955). One of these, 1,3-di-p- hydroxyphenylpropane, was found to be strongly anti-estrogenic in the placental system in vitro: it prevented the accelera- tion of the transhydrogenase-isocitric de- hydrogenase system normally produced by estradiol- 17/3 (Villee and Hagerman, 1957). The inhibitory power declines as the length of the carbon chain connecting the two phenolic rings is increased and 1 , 10-di-p- hydroxyphenyldecane had no inhibitory ac- tion. Similar inhibitions of the estradiol- sensitive system were observed with stil- bestrol, estradiol-17a, and a smaller anti- estrogenic effect was found with estriol (Villee, 1957a). The inhibition induced by these compounds can be overcome by adding increased amounts of estradiol-17^. When stilbestrol is added alone at low con- centration, 10~' M, it has a stimulatory ef- fect equal to that of estradiol-17^ (Glass, Loring, Spencer and Villee, 1961). The quantitative relations between the amounts of stimulator and inhibitor suggest that this inhibition is a competitive one. It was postulated that this phenomenon in- volves a competition between the steroids for specific binding sites on the estrogen- sensitive enzyme (Villee, 1957b; Hagerman and Villee, 1957). When added alone, estriol and stilbestrol are estrogenic and increase the rate of the estrogen-sensitive enzyme. In the presence of both estradiol and estriol, the total enzyme activity observed is the sum of that due to the enzyme combined with a potent activator, estradiol- 17^, and that due to the enzyme combined with a weak activator, estriol. When the concen- tration of estriol is increased, some of the estradiol is displaced from the enzyme and the total activity of the enzyme system is decreased. Two hypotheses have been proposed for the mechanism of action of estrogens on the enzyme system of the placenta. One states that the estrogen combines with an inactive form of the enzyme and converts it to an active form (Hagerman and Villee, 1957). When this theory was formulated the evi- dence indicated that the estrogen acted on a specific DPN-linked isocitric dehydrogen- ase. The theory is equally applicable if the STEROID SEX HORMONES 657 estrogen-sensitive enzyme is a transhydro- genase, as the evidence now indicates. The results of kinetic studies with the coupled isocitric dehydrogenase-transhydrogenase system are consistent with this theory (Gordon and Villee, 1955; Villee, 1957b; Hagerman and Villee, 1957). Apparent binding constants for the enzyme-hormone complex (Gordon and Villee, 1955j and for enzyme-inhibitor complexes have been cal- culated (Hagerman and Villee, 1957). The observation that estradiol and es- trone, which differ in structure only by a pair of hydrogen atoms, are equally ef- fective in stimulating the reaction suggested that the steroid might be acting in some way as a hydrogen carrier from substrate to pyridine nucleotide (Gordon and Villee, 1956). Talalay and Williams-Ashman (1958) suggested that the estrogens act as coenzymes in the transhydrogenation reac- tion and postulated that the reactions were: Estrone + TPNH + H* — Estradiol + TPN^ Estradiol + DPN+ — Estrone + DPNH + H* Sum : TPNH H* - DPN^ — TPN^ + H^ DPNH This formulation implies that the estro- gen-sensitive transhydrogenation reaction is catalyzed by the estradiol-17y3 dehydro- genase characterized by Langer and Engel (1956). This enzyme was shown by Langer (1957) to use either DPN or TPN as hy- drogen acceptor but it reacts more rapidly with DPN. Ryan and Engel (1953) showed that this enzyme is present in rat liver, and in human adrenal, ileum, and liver. How- ever, no estrogen-stimulable enzyme is demonstrable in rat or human liver (Villee, 1955). The nonparticulate fraction obtained by high speed centrifugation of homogen- ized rabbit liver rapidly converts estradiol to estrone if DPN is present as hydrogen acceptor, but does not contain any estrogen- stimulable transhydrogenation system. It will not be possible to choose between these two hypotheses until either the estro- gen-sensitive transhydrogenase and the estradiol dehydrogenase have been sepa- rated or there is conclusive proof of their identity. Talalay, Williams-Ashman and Hurlock (1958) reported a 100-fold puri- fication of the dehydrogenase without sepa- ration of the transhydrogenase activity and found that both activities were in- hibited identically by sulfhydryl inhibitors. In contrast, Hagerman and Villee (1958) obtained partial separation of the two ac- tivities by the usual techniques of protein fractionation, and reported that a 50 per cent inhibition of transhydrogenase is ob- tained with p-chloromercurisulfonic acid at a concentration of 10~^ m whereas 10"^ m p-chloromercurisulfonic acid is required for a 50 per cent inhibition of the dehydro- genase. The evidence that these two ac- tivities are mediated by separate and dis- tinct proteins has been summarized by Villee, Hagerman and Joel (1960). The transhydrogenase present in the mitochondrial membranes of heart muscle was shown by Ball and Cooper (1957) to be inhibited by 4 X 10"^ m thyroxine. The estrogen-sensitive transhydrogenase of the placenta is also inhibited by thyroxine (Vil- lee, 1958b). The degree of inhibition is a function of the concentration of the thyrox- ine and the inhibition can be overcome by increased amounts of estrogen. Suitable con- trol experiments show that thyroxine at this concentration does not inhibit the glu- cose 6-phosphate dehydrogenase or isocitric dehydrogenase used as TPNH-generating systems to couple with the transhydro- genase. Triiodothyronine also inhibits the estrogen-sensitive transhydrogenase but tyrosine, diiodotyrosine and thyronine do not. The thyroxine does not seem to be inhibiting by binding the divalent cation, Mn + + or ]Mg+ + , required for activity, for the inhibition is not overcome by increasing the concentration of the cation 10-fold. In the intact animal estrogens stimulate the growth of the tissues of certain target organs. The estrogen-sensitive enzyme has been shown to be present in many of the target organs of estrogens: in human endo- metrium, myometrium, placenta, mammary gland, and mammary carcinoma, in rat ven- tral prostate gland and uterus, and in mam- motrophic-dependent transplantable tumors of the rat and mouse pituitary. In contrast, it is not demonstrable in comparable prepa- rations from liver, heart, lung, brain, or 658 PHYSIOLOGY OF GONADS kidney. The growth of any tissue involves the utilization of energy, derived in large part from the oxidation of substrates, for the synthesis of new chemical bonds and for the reduction of substances involved in the synthesis of compounds such as fatty acids, cholesterol, purines, and pyrimidines. The physiologic responses to estrogen action, such as water imbibition and protein and nucleic acid synthesis, are processes not directly dependent on the activity of trans- hydrogenase. However, all of these processes are endergonic, and one way of increasing their rate would be to increase the supply of biologically available energy by speeding up the Krebs tricarboxylic acid cycle and the flow of electrons through the electron transmitter system. Much of the oxidation of substrates by the cell produces TPNH, whereas the major fraction of the biologi- cally useful energy of the cell comes from the oxidation of DPNH in the electron trans- mitter system of the cytochromes. Hormonal control of the rat of transfer of hydrogens from TPN to DPN could, at least in theory, influence the over-all rate of metabolism in the cell and secondarily influence the amount of energy available for synthetic processes. Direct evidence of this was shown in our early experiments in which the oxy- gen consumption of tissue slices of target organs was increased by the addition of estradiol (Hagerman and Villee, 1952; Vil- lee and Hagerman, 1953). This theory assumes that the supply of energy is rate-limiting for synthetic proc- esses in these target tissues and that the activation of the estrogen-sensitive enzyme does produce a significant increase in the supply of energy. The addition of estradiol in vitro produces a significant increase in the total amount of isocitric acid dehy- drogenated by the placenta (Villee, Loring and Sarner, 1958) . Slices of endometrium to which no estradiol was added in vitro utilized oxygen and metabolized substrates to carbon dioxide at rates which paralleled the levels of estradiol in the blood and urine of the patient from whom the endometrium was obtained (Hagerman and Villee, esses in these target tissues and that the 1953b). Estradiol increases the rate of syn- thesis of ATP by liomogenates of human placenta (Villee, Joel, Loring and Spencer, 1960). The reductive steps in the biosynthesis of steroids, fatty acids, purines, serine, and other substances generally require TPNH rather than DPNH as hydrogen donor. The cell ordinarily contains most of its TPN in the reduced state and most its DPN in the oxidized state (Glock and McLean, 1955). If the amount of TPN+ is rate- limiting, a transhydrogenase, by oxidizing TPN and reducing DPN, would permit further oxidation of substrates such as iso- citric acid and glucose 6-phosphate, which require TPN+ as hydrogen acceptor and which are key reactions in the Krebs tri- carboxylic acid cycle and the hexose mono- phosphate shunt, respectively. Furthermore, the experiments of Kaplan, Schwartz, Freeh and Ciotti (1956) indicate that less bio- logically useful energy, as ATP, is obtained when TPNH is oxidized by TPNH cyto- chrome c reductase than when DPNH is oxidized by DPNH cytochrome c reductase. Thus, a transhydrogenase, by transferring hydrogens from TPNH to DPN before oxidation in the cytochrome system, could increase the energy yield from a given amount of TPNH produced by isocitrate or glucose 6-phosphate oxidation. The in- creased amount of biologically useful energy could be used for growth, for protein and nucleic acid synthesis, for the imbibition of water, and for the other physiologic effects of estrogens. Estrogen stimulation of the transhy- drogenation reaction would tend to decrease rather than increase the amount of TPNH in the cell. Thus the estrogen-induced stimu- lation of the synthesis of steroids, fatty acids, proteins, and purines in the uterus can be explained more reasonably as due to an increased supply of energy rather than to an increased supply of TPNH. The theory that estrogens stimulate trans- hydrogenation by acting as coenzymes which are rapidly and reversibly oxidized and reduced does not explain the pro- nounced estrogenic activity in vivo of stil- bestrol, 17a-ethinyl estradiol, or bfsdehy- drodoisynolic acid, for these substances do not contain groups that could be readily oxidized or reduced. The exact mechanism STEROID SEX HORMONES (359 of action of estrogens at the biochemical level remains to be elucidated, but the data available permit the formulation of a detailed working hypothesis. The notable effects of estrogens and androgens on be- havior (see chapter by Young) are pre- sumably due to some direct or indirect ef- fect of the hormone on the central nervous system. The explanation of these phenomena in physiologic and biochemical terms re- mains for future investigations to provide. B. ANDROGENS Although there is a considerable body of literature regarding the responses at the bio- logic level to administered androgens and progesterone, much less is known about the site and mechanism of action of these hor- mones than is known about the estrogens. The review by Roberts and Szego (1953b) deals especially with the synergistic and antagonistic interactions of the several steroidal sex hormones. The rapid growth of the capon comb fol- lowing the administration of testosterone has been shown to involve a pronounced increase in the amount of mucopolysac- charide present, as measured by the content of glucosamine (Ludwig and Boas, 1950; Schiller, Benditt and Dorfman, 1952). It is not known whether the androgen acts by increasing the amount or activity of one of the enzymes involved in the synthesis of polysaccharides or whether it increases the amount or availability of some requisite cofactor. Many of the other biologic effects of androgens do not seem to involve mucopolysaccharide synthesis and the relation of these observations to the other roles of androgens remains to he de- termined. Mann and Parsons (1947) found that castration of rabbits resulted in a decreased concentration of fructose in the semen. Within 2 to 3 weeks after castration the amount of fructose in the semen dropped to zero, but rapidly returned to normal fol- lowing the subcutaneous implantation of a pellet of testosterone. Fructose reappeared in the semen of the castrate rat 10 hours after the injection of 10 mg. of testosterone (Rudolph and Samuels, 1949). The coagu- lating gland of the rat, even when trans- planted to a new site in the body, also re- sponds by producing fructose when the host is injected with testosterone. The amount of citric acid and ergothioneine in the semen is also decreased by castration and in- creased by the implantation of testosterone pellets (Mann, 1955). The experiments of Hers (1956) demonstrate that fructose is produced in the seminal vesicle by the reduction of glucose to sorbitol and the subsequent oxidation of sorbitol to fructose. The reduction of glucose re- quires TPNH as hydrogen donor and the oxidation of sorbitol requires DPN as hydrogen acceptor. The sum of these two reactions provides for the transfer of hy- drogens from TPNH to DPN. If androgens act as cofactors which are reversibly oxi- dized and reduced, and thus transfer hy- drogens from TPNH to DPN as postulated by Talalay and Williams-Ashman (1958), one would expect that an increased amount of androgen, by providing a competing sys- tem for hydrogen transfer, would decrease rather than increase the production of fruc- tose. The marked increases in the citric acid and ergothioneine content of semen are not readily explained by this postulated site of action of androgens. An increase in the activity of /3-glu- curonidase in the kidney has been reported following the administration of androgens (Fishman, 1951). This might be interpreted as an arlaptive increase in enzyme induced by the increased concentration of substrate, or by a direct effect of the steroid on the synthesis of the enzyme. The respiration of slices of prostate gland of the dog is decreased by castration or by the administration of stilbestrol (Barron and Huggins, 1944). The decrease in respi- ration occurs with either glucose or pyruvate as substrate. The seminal vesicle of the rat responds similarly to castration. Rudolph and Samuels (1949) found that respiration of slices of seminal vesicle is decreased by castration and restored to normal values within 10 hours after the injection of tes- tosterone. Experiments by Dr. Phillip Corf- man in our laboratory with slices of prostate gland from patients with benign prostatic hypertrophy showed that oxygen utilization was reduced 50 per cent by estradiol added 660 PHYSIOLOGY OF GONADS in vitro at a level of 1 /xg. per ml. Respira- tion of slices of the ventral prostate gland of the rat is decreased by castration and in- creased by administered testosterone (Ny- den and Williams-Ashman, 1953). These workers showed that lipogenesis from ace- tate-l-C^* in the prostate is also sig- nificantly diminished by castration and restored to normal by administered testos- terone. The succinic dehydrogenase of the liver has been found to be increased by castration and decreased by the administration of tes- tosterone (Kalman, 1952; Rindani, 1958), the enzyme is also inhibited by testosterone added in vitro (Kalman, 1952). In contrast, Davis, Meyer and McShan (1949) found that the succinic dehydrogenase of the prostate and seminal vesicles is decreased by castration and increased by the admin- istration of testosterone. An interesting example of an androgen effect on a specific target organ is the de- creased size of the levator ani and other perineal muscles of the rat fol- lowing castration. The administration of androgen stimulates the growth of these muscles and increases their glycogen content (Leonard, 1952). However, their succin- oxidase activity is unaffected by castration or by the administration of testosterone. Courrier and Marois (1952) reported that the growth of these muscles stimulated by androgen is inhibited by cortisone. The remarkable responsiveness of these muscles to androgens in vivo gave promise that slices or homogenates of this tissue incu- bated with androgens might yield clues as to the mode of action of the male sex hor- mones. Homogenates of perineal and mas- seter muscles of the rat responded to andro- gens administered in vivo with increased oxygen consumption and ATP production iLoring, Spencer and Villee, 1961). The ex- periments suggested that the activity of DPNH-cytochromo r reductase in these tissues is controlled by aiKh'ogeiis. C. PROGESTERONE Attempts to clarify the biochemical basis of the role of progesterone have been ham- pered by the requirement, in most instances, for a previous stimulation of the tissue by estrogen. The work of Wade and Jones (1956a, b) demonstrated an interesting ef- fect of progesterone added in vitro on sev- eral aspects of metabolism in rat liver mito- chondria. Progesterone, but not estradiol, testosterone, 17a-hydroxyprogesterone, or any of several other steroids tested, stimu- lated the adenosine triphosphatase activity of rat liver mitochondria. This stimulation is not the result of an increased permeability of the mitochondrial membrane induced by progesterone, for the stimulatory effect is also demonstrable with mitochondria that have been repeatedly frozen and thawed to break the membranes. Other experiments showed that ATP was the only substrate effective in this system ; progesterone did not activate the release of inorganic phosphate from AMP, ADP, or glycerophosphate. In other experiments with rat liver mito- chondria (Wade and Jones, 1956b), proges- terone at a higher concentration (6 X lO"'* m) was found to inhibit the utilization of oxygen with one of the tricarboxylic acids or with DPNH as substrate. This inhibition is less specific and occurred with estradiol, testosterone, pregnanediol, and 17a-hy- droxy progesterone, as well as with proges- terone. The inhibition of respiration by high concentrations of steroids in vitro has been reported many times and with several dif- ferent tissues; it seems to be relatively un- specific. Wade and Jones were able to show that progesterone inhibits the reduction of cytochrome c but accelerates the oxidation of ascorbic acid. They concluded that pro- gesterone may perhaps uncouple oxidation from phosphorylation in a manner similar to that postulated for dinitrophenol. The site of action of this uncoupling appears to be in the oxidation-reduction path between DPNH and cytochrome c. Mueller (1953) found that progesterone added in vitro de- creases the incorporation of glycine-2-C^'* into the protein of strips of rat uterus, thus counteracting the stimulatory effect of es- tradiol administered in vivo. Zander (1958) reported that A4-3-keto- pregnene-20-a-ol and A4-3-ketopregnene- 20-^-ol arc effective gestational hormones in the mouse, rabbit, and man, although somewhat less active in general than is progesterone. An enzyme in rat ovary which converts progesterone to pregnene-20-a-ol, and also catalyzes the reverse reaction, was STEROID SEX HORMONES 661 described by Wiest (1956). The conversion occurred when slices of ovary were incu- bated with DPN. Wiest postulated that the progesterone-pregnene-20-a-ol system might play a role in hydrogen transfer, in a manner analogous to that postulated by Talalay and Williams-Ashman (1958) for estrone-estradiol- 17^, but his subsequent experiments ruled out this possibility, for he was unable to demonstrate any pro- gesterone-stimulable transhydrogenation re- action. The nature of the effect of progesterone and of estrogens on myometrium has been investigated extensively by Csapo. Csapo and Corner (1952, 1953) found that ovari- ectomy decreased the maximal tension of the myometrium and decreased its content of actomyosin. The administration of estra- diol to the ovariectomized rabbit over a period of 7 days restored both the actomyo- sin content and the maximal tension of the myometrium to normal. The concentration of ATP and of creatine phosphate in the myometrium is decreased by ovariectomy but is restored by only 2 days of estrogen treatment. This suggests that the effect on intermediary metabolism occurs before the effect on protein {i.e., actomyosin) synthe- sis. Csapo (1956a) concluded that estrogen is a limiting substance in the synthesis of the contractile proteins of myometrium, but he could not differentiate between an effect of estrogen on some particular biosynthetic reaction and an effect of estrogen on some fundamental reaction which favors synthe- sis in general. He was unable to demonstrate any comparable effect of progesterone on the contractile actomyosin-ATP system of the myometrium. Other observations provide an explana- tion for the well known effect of progester- one in decreasing the contractile activity of myometrium, not by any effect on the con- tractile system itself, but in some previous step in the excitation process. Under the domination of progesterone the myometrial cells have a decreased intracellular concen- tration of potassium ions and an increased concentration of sodium ions (Horvath, 1954). The change in ionic gradient across the cell membrane is believed to be respon- sible for the altered resting potential and the partial depolarization of the cell mem- brane which results in decreased conductiv- ity and decreased pharmacologic reactivity of the myometrial cell. The means by which progesterone produces the changes in ionic gradients is as yet unknown. Csapo postu- lates that the hormone might decrease the rate of metabolism which in turn would lessen the rate of the "sodium pump" of the cell membrane. The contractile elements, the actomyosin-ATP system, are capable of full contraction but, because of the partial block in the mechanism of excitation and of propagation of impulses (Csapo, 1956b), the muscle cells cannot operate effectively; the contractile activity remains localized. Csapo (1956a) showed that the progesterone block is quickly reversible and disappears if progesterone is withdrawn for 24 hours. He concluded that the progesterone block is necessary for the continuation of pregnancy and that its withdrawal is responsible for the onset of labor. ]\Iost investigators who have speculated about the mode of action of steroids — whether they believe the effect is by acti- vating an enzyme, by altering the permea- bility of a membrane, or by serving as a coenzyme in a given reaction— have empha- sized the physical binding of the steroid to a protein as an essential part of the mecha- nism of action or a preliminary step to that action. They have in this way explained the specificities, synergisms, and antago- nisms of the several steroids in terms of the formation of specific steroid-protein com- plexes. 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J., and Engel, L. L. 1953. The inter- conversion of estrone and estradiol-17i3 by rat liver slices. Endocrinology, 52, 277-286. Schiller, S., Benditt, E., and Dorfman, A. 1952. Effect of testosterone and cortisone on the hexosamine content and metachromasia of chick combs. Endocrinology, 50, 504-510. Solomon, S., Levitan, P., and Lieberman, S. 1956. Possible intermediates between cholesterol and pregneneolone in corticosteroidogenesis. Ab- stract. Proc. Canad. Physiol. Soc, Rev. Canad. Biol., 15, 282. Spaziani, E., and Szego, CM. 1958. The influ- ence of estradiol and Cortisol on uterine hista- mine of the ovariectomized rat. Endocrinol- ogy, 63, 669-678. Stuermer, V. M., .AND Stein, R. J. 1952. Cyto- dynamic properties of the human endome- trium. V. Metabolism and the enzymatic ac- tivity of the human endometrium during the menstrual cycle. Am. J. Obst. & Gynec, 63, 359-370. Tal.alay, p., and Williams-Ashman, H. G. 1958. Activation of hydrogen transfer between pyr- idine nucleotides bv steroid hormones. Proc Nat. Acad. Sc, 44, 15-26. Tal.alay, P., Willi.ams-Ashman, H. G., and Hur- LOCK, B. 1958. Steroid hormones as coen- zymes of hydrogen transfer. Science, 127, 1060. Tchen, T. T., and Bloch, K. 1955. In vitro conversion of squalene to lanosterol and cho- lesterol. J. Am. Chem. Soc, 77, 6085-6086. Telfer, M. a. 1953. Influence of estradiol on nucleic acids, respiratory enzymes and the distribution of nitrogen in the rat uteru.*. Arch. Biochem., 44, 111-119. Victor, J., and Andersen, D. H. 1937. Stimula- tion of anterior hypophysis metabolism by theelin or dihydrotheelin. Am. J. Physiol., 120, 154-166. Villee. C. A. 1955. An estradiol-induccd stimu- lation of citrate utilization by placenta. J. Biol. Chem., 215, 171-182. Villee, C. A. 1957a. Effects of estrogens and antiestiogens in vitro. Cancer Res.. 17, 507- 511. STEROID SEX HORMONES 665 ViLLEE, C. A. 1957b. Role of estrogens in regu- lating the metabolism of the placenta and endometrium. Fertil. & Steril., 8, 156-163. ViLLEE, C. A. 1958a. Estrogens and uterine en- zymes. Ann. New York Acad. Sc, 75, 524- 534. ViLLEE, C. A. 1958b. Antagonistic effects of es- trogens and thyroxine on an enzyme system in vitro. In Proceedings IV International Con- gress Biochemistry {Vienna), p. 115. London: Pergamon Press. ViLLEE, C. A., AND GoRDON, E. E. 1955. Further studies on the action of estradiol in vitro. J. Biol. Chem., 216, 203-214. ViLLEE, C. A., AND GoRDON, E. E. 1956. The stimulation by estrogens of a DPN-linked isocitric dehydrogenase from human placenta. Bull. Soc. chim. belg., 65, 186-201. ViLLEE, C. A., AND Hagerman, D. D. 1953. Effects of estradiol on the metabolism of himian pla- centa in vitro. J. Biol. Chem., 205, 873-882. ViLLEE, C. A., and Hagerman, D. D. 1957. Studies on the estrogen-sensitive isocitric dehydro- genase of the human placenta. 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The metabo- lism of uterine muscle studied with radio- active phosphorus P^". Acta physiol. scandinav 21, 18-26. Wal-aas, O.. Wal.aas, E., .\nd Loken, F. 1952a. The effect of estradiol monobenzoate on the metaboli-sin of rat uterine muscle. Acta endo- crinol., 10,201-211. Wal.'^as, 0., Walaas, E., and Loken, F. 1952b. The effect of estradiol monobenzoate on the metabolism of the rat endometrium. Acta en- docrinol., 11, 61-66. Werbin, H. and LeRoy, G. V. 1954. Cholesterol: a precursor of tetrahydrocortisone in man. J. Am. Chem. Soc, 76, 5260-5261. West. C. D., Damast, B. L., Sarro, S. D., and Pearson, 0. H. 1956. Conversion of testos- terone to estrogens in castrated, adrenalecto- mized human females. J. Biol. Chem., 218, 409-418. WiEST, W. G. 1956. The metabolism of pro- gesterone to A4-pregnen-20a-ol-3-one in evis- cerated female rats. J. Biol. Chem., 221, 461- 467. Zander, J. 1958. Gestagens in human pregnancy. In Proceedings Conference on Endocrinology of Reproduction, C. W. Lloyd, Ed. New York: Academic Press, Inc. Zondek, B., and Hestrin, S. 1947. Phosphorylase activity in human endometrium. Am. J. Obst. ct Gvnec, 54, 173-175. 12 NUTRITIONAL EFFECTS ON ENDOCRINE SECRETIONS James H. Leathern, Ph.D. PROFESSOR OF ZOOLOGY, RUTGERS, THE STATE UNIVERSITY, NEW BRUNSWICK, NEW JERSEY I. Introduction' 666 I, Introduction II. Nature of Problems in Nutritional -j-. -, .^ ^ ,• ,• t , Studies 668 Despite the accumulation of many data A. Thyroid Cxland, Nutrition, and Re- in the field of reproductive endocrinology production ()68 during the past 20 years and the long es- B. Adreiial Gland, Nutrition, and Re- ^ _^ tablished awareness of a nutritional in- C. Diabetef^Mellitus, Nutrition, and '' ^f^f on fertility and fecundity, knowl- Reproduction ()72 edge bearing on nutrition and the endocrine D. Sterile-Obese Syndrome 673 glands subserving reproduction has ad- E. Diet and the Liver 673 vanced comparatively slowly. However, re- III. Hypophysis and Diet 674 markable advances have been made in each A. Inanition 674 speciality SO that nutritional-endocrine ^roein. .. w problems should continue to be a fruitful C. Carbohvdrate and Fat ()7() ^ „ ^i-r^x i-ii D Vitamins 676 ^^^^^ ^^^' ^tudy. Data which have yet to IV. Male Reproductive System (i77 be obtained eventually w'ill contribute to A. Testis 677 the coherence one would prefer to present 1. Inanition 677 now. 2. Protein 678 'y\^q endocrinologist appreciates the deli- 4 Vitamins (i8() ^^^^ balance which exists between the hy- B. Influence of Nutrition on the Respon- ' pophysis and the gonads. In a sense, a simi- siveness of Male Reproductive Tis- lar interdependence exists between nutrition sues to Hormones 681 and the endocrine glands, including those 1. Testis ■ . . 681 ^j^|-^ reproductive functions. Not only does 2. feeminal vesicles and i)rostate 682 x •<• • n xi • j i -• V. Female Reproductive System 683 nutrition influence synthesis and release of A. Ovaries 683 hormones, but hormones in turn, through 1. Inanition 683 their regulation of the metabolism of pro- 2. Protein 684 teins, carbohydrates, and fats, influence nu- 3. Carbohydrate 685 trition. Thus, dietary deficiencies may create 5 Vitamins 685 endocrine imbalance, and endocrine imbal- B. Influence of Nutrition on the ResiK)n- '^^ce may create demands for dietary fac- siveness of Female Pe])roxamination of the possibility sliould he made. C. DIABETES MELLITUS, NUTRITION, AND REPRODUCTION (llycosuria can be induced experimentally by starvation, overfeeding, and shifting NUTRITIONAL EFFECTS 673 diet^ from one of high fat content to one i; which is isocaloric but high in carbohydrate (Ingle, 1948). Force feeding a high carbo- hydrate diet will eventually kill a rat de- spite insulin administration aimed at con- trolling glycosuria (Ingle and Nezamis, 1947). In man excessive eating leading to obesity increases insulin demand and, in many diabetics of middle age, obesity pre- cedes the onset of diabetes. With our present knowledge we must conclude that overfeed- ing is wrong when glycosuria exists and that vitamin B supplements may be of value in diabetes (Meites, Feng and Wilwerth, 1957; Salvesen, 1957). In man urinary 17-ketosteroids and an- drogen levels are subnormal in diabetes (Horstmann, 1950), and in the diabetic rat pituitary gonadotrophins are reduced (Shipley and Danley, 1947), but testis hy- aluronidase does not change (Moore, 1948) . When hyperglycemia exists in rats, semen ■] carbohydrates increase (Mann and Lut- wak-Mann, 1951). Hypoglycemia influences the male re- productive organs. In rats tolbutamide or insulin produce lesions of the germinal epithelium which can be prevented by \' simultaneous administration of glucose. When 2 to 5 hypoglycemic comas are in- duced, such testis injuries increase pro- gressively in number and frequency, and only a partial return to normal is observed a month later (Mancini, Izquierdo, Hein- rich, Penhos and Gerschenfeld, 1959). It is well known that the incidence of infertility in the pre-insulin era was high in young diabetic women. Fertility is also reduced in diabetic experimental animals, and rat estrous cycles are prolonged (Davis, Fugo and Lawrence, 1947) . Insulin is cor- rective (Sinden and Longwell, 1949; Ferret, Lindan and Morgans, 1950). Pregnancy in women with uncontrolled diabetes may terminate in abortion or stillbirth, possibly l)ecause toxemia of pregnancy is high (Ped- ersen. 1952). In rats pancreatectomy per- formed the 8th to 12th day of pregnancy increased the incidence of stillbirths (Hult- ciuist, 1950). In another experiment almost one- fourth of 163 animals with diabetes induced by alloxan on the 10th to 12th day of pregnancy died before parturition and about 25 ])er cent of the survivors aborted (Angcrvall, 1959). D. STERILE-OBESE SYNDROME A sterile-obese syndrome in one colony of mice has been shown to be a recessive mono- genic trait (Ingalls, Dickie and Snell, 1950). Obesity was transmitted to subsequent gen- erations by way of ovaries that were trans- planted from obese donors to nonobese re- cipients (Hummel, 1957). Obesity was transmitted by obese females receiving hor- monal therapy and mated to obese males kept on restricted food intake (Smithberg and Runner, 1957). In addition to the in- vestigations of the hereditary nature of the sterile-obese syndrome, the physiologic basis for the sterility has been studied in reference to the presence of germ cells, via- bility of ova and sperm, integrity of the ovary, and response of the uterus to estro- gen (Drasher, Dickie and Lane, 1955). The data indicate that sterility in some obese males can be prevented by food restriction and that sterility in certain obese females can be corrected. E. DIET AND THE LIVER The concentration of hormones which reaches the target organs in the blood is the result of the rate of their production, me- tabolism, and excretion. How hypophyseal hormones are destroyed is not clear, but current data make it apparent that pitui- tary hormones have a short half-life in the circulatory system. Exerting a major con- trol over circulating estrogen levels is the liver, with its steroid-inactivating systems. Zondek (1934) initially demonstrated that the liver could inactivate estrogens and this finding has had repeated confirmation (Cantarow, Paschkis, Rakoff and Hansen, 1943; De:\Ieio, Rakoff, Cantarow and Paschkis, 1948; Vanderlinde and Wester- field, 1950). Other steroids are also inac- tivated by the liver with several enzyme systems being involved; the relative con- centration of these enzymes varies among species of vertebrates (Samuels, 1949). The liver is a labile organ which readib.' responds to nutritional modifications; the induced liver changes alter the steroid-in- activating systems of this organ. Thus, in- anition (Drill and Pfeiffer, 1946; Jailer, G74 PHYSIOLOGY OF GONADS 1948) , vitamin B complex deficiency (Segal- off and Segaloff, 1944; Biskind, 1946), and protein restriction (Jailer and Seaman, 1950) all influence the capacity of the liver to detoxify steroids. Reduced protein intake is a primary factor in decreasing the effec- tiveness of the steroid-inactivating system (Jailer and Seaman, 1950; Vanderlinde and Westerfield, 1950). Rats fed an 8 per cent casein diet lose their capacity to inactivate estrone within 10 days. However, ascorbic acid alone or in combination with gluta- thione restored the estrone-inactivating sys- tem (Vasington, Parker, Headley and Van- derlinde, 1958). Failure of steroids to be inactivated will influence the hypophyseal-gonadal axis. In turn the excess of estrogen will decrease gonadotrophin production by the hypoph- ysis and thus reduce steroid production by the gonad. In addition nutritional modi- fications influence hypophyseal and possibly gonadal secretory capacity directly. Con- ceivably, nutritional alterations could modify the amount of steroid secreted or interfere with complete steroid synthesis by a gland. Fatty infiltration of the liver and a general increase in fat deposition occur in fed and fasted rats after the injection of certain pituitary extracts and adrenal ster- oids and after the feeding of specific diets. Impaired estrogen inactivation has been associated with a fatty liver, but Szego and Barnes (1943) believe that the major in- fluence is inanition. In fact, estrogens, es- pecially ethinyl estradiol, interfere with fatty infiltration of the liver induced by a low protein diet (Gyorgy, Rose and Ship- ley, 1947) or by a choline-deficient diet (Emerson, Zamecnik and Nathanson, 1951). Stilbestrol, however, did not prevent the in- crease in liver fat induced by a protein-free diet (Glasser, 1957). Estrogens that are effective in preventing fatty infiltration may act by sparing methionine or choline or by inhibiting growth hormone (Flagge, Mar- asso and Zimmerman, 1958). Ethionino, the antimetabolite of nu'- thioniiK', Avill induce a fatty liver and in- hibit hepatic protein synthesis in female, but not in male rats (Farber and Segaloff, 1955; Farber and Corban, 1958). Pretreat- ment of females with testosterone prevents the ethionine effect, but this blockage of ethionine action need not be related to androgenic or progestational properties of steroids (Ranney and Drill, 1957). III. Hypophysis and Diet Studies involving acute and chronic star- vation have shown that gonadal hypofunc- tion during inanition is primarily due to diminished levels of circulating gonado- trophins. Because of the similarity to changes following hypophysectomy, the endocrine response to inanition has been referred to as "pseudohypophysectomy." A. INANITION The hypophysis has been implicated in human reproduction disturbances asso- ciated with undernutrition. Hypophyseal atrophy and a decrease in urinary gonado- trophins have been observed in chronic malnutrition (Klinefelter, Albright and Griswold, 1943; Zubiran and Gomez-]\Iont, 1953) and anorexia nervosa (Perloff, Lasche, Nodine, Schneeberg and Vieillard, 1954). Refeeding has restored urinary gonadotrophin levels in some cases, but hypoj^hyseal damage may result from severe food restriction at puberty (VoUmer, 1943; Samuels, 1948). The influence of inanition on the re- productive organs of lal)oratory rodents is well recognized but the cft'ects on the hypophysis cannot be presented conclu- sively. In support of prior investigations, Mulinos and Pomerantz (1941a, b) in rats and Giroud and Desclaux (1945) in guinea pigs observed a hypophyseal atrophy fol- lowing chronic underfeeding as well as a decrease in cell numbers and mitoses. In fact, refeeding after chronic starvation resulted in only a partial recovery of hy- pophyseal weight (Quimby, 1948). Never- theless, complete starvation did not in- fluence relative gland weight in female rats (Meites and Reed, 1949), and cytologic evidence (periodic acid-Schiff (PAS) test) of an estimated 3- fold increase in gonado- ti'ophin content was claimed following chronic starvation (Pearse and Rinaldini, 1950). Assays of hypophyseal gonado- trophin content in chronically starved rats of both sexes have l)een reported as de- creased (Mason and Wolfe, 1930; Werner, NUTRITIONAL EFFECTS 675 1939), unchanged (,]\Iarrian and Parkes, 1929; Pomerantz and Mulinos, 1939; Mad- dock and Heller, 1947; Meites and Reed, 1949; Blivaiss, Hanson, Rosenzweig and McNeil, 1954), or increased (Rinaldini, 1949; Vanderlinde and Westerfield, 1950). An increase in pituitary gonadotrophin was evident when hormone content was related to milligrams of tissue (Meites and Reed, 1949). Thus, the hormone release mecha- nism may fail in starvation, and eventually gonadotrophin production will be reduced to a minimum (]\laddock and Heller, 1947). Gonadectomy of fully fed rats is followed by an increase in hypophyseal gonado- trophin content. Chronic starvation, how- ever, prevented the anticipated changes in the pituitary gland following gonadectomy in 8 of 12 female rats (Werner, 1939). On the other hand, if adult female rats were subjected to 14 days of reduced feeding 1 month after ovariectomy, no change in the elevated gonadotrophin levels was noted (Meites and Reed, 1949). In contrast, Gomez-Mont (1959) observed above normal urinary gonadotrophins in many meno- pausal and postmenopausal women despite undernutrition. It is apparent that uniformity of opinion as to how starvation influences hypophyseal gonadotrophin content has not been at- tained. Several explanations can be given for the discrepancies. (1) There have been un- fortunate variations in experimental design. IVIaddock and Heller (1947) starved rats for 12 days, whereas Rinaldini (1949) used a low calorie diet of bread and milk for 30 days. Other variations in feeding have included feeding one-half the intake re- quired for growth (Mulinos and Pomerantz, 1941b I, regimens of full, one-half, one- ciuarter, and no feeding for 7 and 14 days (Meites and Reed, 1949), and feeding in- adequate amounts of a standard rat diet for 1 to 4 months (Werner, 1939). (2) Hypophyseal implants and anterior pitui- tary extracts should not be compared, for variable gonadotrophin production may fol- low implantation procedures, depending on whether necrosis or growth occurs (Mad- dock and Heller, 1947). (3) There has been an insufficient standardization of experi- mental materials. The assay animal has usually been the immature female rat, but occasionally the immature mouse has been used, and Rinaldini (1949) used the hypo- physectomized rat. B. PROTEIN The need for specific food elements by the hypophysis warrants consideration, for the hormones secreted by this gland are protein in nature and the amino acids for protein synthesis must be drawn from body sources. However, dietary protein levels can vary from 15 per cent to 30 per cent without influencing hypophyseal gona- dotrophin content in rats (Weatherby and Reece, 1941), but diets containing 80 per cent to 90 per cent of casein increased hypophyseal gonadotrophin (Tuchmann- Duplessis and Aschkenasy-Lelu, 1948). Re- moval of protein from the diet will decrease hypophyseal gonadotrophin content in adult male rats in comparison with pair-fed and ad libitum-ied controls, but the de- crease may or may not be significant in a 30-day period; luteinizing hormone (LH) seemed to be initially reduced. Extension of the period of protein depletion another 2 months resulted in a significant lowering of hy]iophyseal gonadotrophin levels (Table 12.4) (Leathern, 1958a). On the other hand, an increased FSH with no decrease in LH activity was observed in the hy- pophyses of adult female rats following 30 to 35 days of protein depletion (Srebnik and Nelson, 1957). The available data in- dicate that not only may a sex difference exist, but also that species may differ; resti- tution of gonadotrophin in the discharged rabbit pituitary was not influenced by in- TABLE 12.4 Influence of a protein-free diet on hypophyseal gonadotrophin content (From J. H. Leathern, Recent Progr. Hormone Res., 14, 141, 1958.) Days on PFD* Xo. of Rats Anterior Pituitary Weight Recipient Ovarv Weight nig. mg. 0 9 8.3 74 30 9 7.3 54 50 7 7.0 33 90 17 6.0 23 L^ntreated recipient ovarian weight = 15.4 mg. * PFD = Protein-free diet. 67G PHYSIOLOGY OF GONADS adequate dietary protein (Friedman and Friedman, 1940). When anterior pituitar}^ glands of 60- gm. male rats were extracted and adminis- tered to immature female recipients, ovarian weight increased from 13.0 to 37.3 mg. After feeding 20 per cent casein or fox chow ad libitum for 14 days, the hypophyses of male rats contained almost twice as much gonadotrophin per milligram of tissue as did the hypophyses of the initial controls. Removal of protein from the diet for 14 days, however, reduced hypophyseal gona- dotrophin concentration below the level of the initial controls (Leathem and Fisher, 1959). Data on the hypophyseal hormone con- tent as influenced by specific amino acid deficiencies have not come to the author's attention. Cytologically, however, Scott (1956) noted that an isoleucine-deficient diet depleted the pituitary gonadotrophic cells of their PAS-positive material and re- duced the size of acidophilic cells. Omission of threonine, histidine, or tryptophan in- voked similar effects. The changes prob- ably represent the interference of a single amino acid deficiency with protein metab- olism rather than specific effects attribut- able to the lack of amino acid itself. Exces- sive amino acid provided by injecting leucine, methionine, valine, tyrosine, or glycine caused release of gonadotrophin (Goth, Lengyel, Bencze, Saveley and Maj- say, 1955). Administration of 0.1 mg. stilbestrol for 20 days to adult male rats eliminated de- tectable hypophyseal gonadotrophins. Hor- mone levels returned during the postinjec- tion period provided the diet contained adequate protein, whereas a protein-free diet markedly hindered the recovery of hypo- physeal gonadotrophins. The gonadotrophin content of the pituitary gland correlated well with the recovery of the reproductive system, indicating that gonadotrophin pro- duction was subnormal on ]irotein-free feed- ing (Leathem, 1958a). C. CAHBOHYDR.\TE .\ND FAT Reproduction does not appear to be in- fluenced by carbohydrates per se and hy- pophyseal alterations have not been noted. Fat-deficient diets, however, do influence reproduction and the hypophysis exhibits cellular changes. Pituitary glands of fe- male rats fed a fat-free diet contain a sub- normal number of acidophiles and an in- creased number of basophiles (Panos and Finerty, 1953) . In male rats the feeding oi a fat-free diet increased hypophyseal baso- philes, followed progessively by more cas- tration changes (Finerty, Klein and Panos, 1957; Panos, Klein and Finerty, 1959). D. VITAMINS Despite the many investigations relating reproduction to vitamin requirements, rela- tively few have involved hypophyseal hor- mone estimations. Thus in 1955, Wooten, Nelson, Simpson and Evans reported the first definitive study which related pyri- doxine deficiency to hypophyseal gonado- trophin content. Using the hypophysec- tomized rat for assay, pituitary glands from Bo-deficient rats were shown to have a 10-fold increase in FSH per milligram of tissue and a slightly increased LH content. Earlier studies had revealed that vitamin Bi-free diets decreased pituitary gonado- trophins in male rats (Evans and Simpson, 1930) and a similar effect of the folic acid antagonist, aminopterin, in the monkey was found later (Salhanick, Hisaw and Zar- row, 1952). Male rats deficient in vitamin A exhibited a 43 per cent increase, and castrated vitamin A-deficient rats a 100 per cent increase in hypophyseal gonadotrophin potency over the normal controls (Mason and Wolfe, 1930). The increase of gonadotrophin was more marked in vitamin A-deficient male than in vitamin A-deficient female rats. Associated with the increase in hormone level was a significant increase in basophile cells (Sutton and Brief, 1939; Hodgson, Hall, Sweetman, Wiseman and Converse, 1946; Erb, Andrews, Hauge and King, 1947). A re\-iew of the literature up to 1944 per- mitted Mason to suggest that the anterior hypophysis was not the instigator of re- productive disturbances in vitamin E de- ficiency. Nevertheless, Griesbach, Bell and Livingston (1957), in an analysis of the pituitary gland during progessive stages of NUTRITIONAL EFFECTS 677 tocopherol deprivation, observed cytologic changes in the hypophysis which preceded testis changes. The "peripheral or FSH gonadotrophes" increased in number, size, and activity. The LH cells exhibited a hyperplasia of lesser extent, but possibly sufficient to increase LH in circulation and to cause hypertrophy of the male accessory glands. Gonadotrophic hormone content of pituitary glands from vitamin E-deficient rats may be decreased (Rowlands and Singer, 1936), unchanged (Biddulph and Meyer, 1941), or increased to a level be- tween normal and that of the castrate, when the adult male rats were examined after 22 weeks on a deficient diet (Nelson, 1933; Drummond, Noble and Wright, 1939). Using hypophysectomized male rats as as- say animals, evidence was obtained that FSH was increased in the pituitary glands of vitamin E-deficient male and female rats (P'an, Van Dyke, Kaunitz and Slanetz, 1949). IV. Male Reproductive System A. TESTIS The two basic functions of the male gonads are to produce gametes and secrete steroids. Spermatogenic activity can be estimated from testis morphology and ex- amination of semen samples. Androgen se- cretion can be estimated from urinary ster- oid levels, accessorj^ gland weight, and from analyses of accessory sex gland secre- tions, i.e., fructose and citric acid. In nor- mal maturation in the rabbit, rat, boar, and bull, androgen secretion precedes sper- matogenesis (Lutwak-Mann, 1958). On this basis it would appear that well fed young bulls may come into semen production 2 to 3 months sooner than poorly fed animals (Brat ton, 1957). 1. Inanition Complete starvation will pre^•ent matura- tion of immature animals. Furthermore, marked undernutrition in 700 boys, 7 to 16 years of age, was associated with genital infantilism in 37 per cent and crypt- orchidism in 27 per cent (Stephens, 1941). Restriction of food intake to one-half of the normal in maturing bull calves had a marked delaying effect on the onset of seminal vesicle secretion, but a lesser de- laying effect on spermatogenesis (Davies, Mann and Rowson, 1957) . Limiting the food intake to one-third of the normal did not prevent the immature rat testis from form- ing spermatozoa at the same time as their controls (Talbert and Hamilton, 1955). When testis maturation was prevented by inanition, a rapid growth and maturation occurred on refeeding (Ball, Barnes and Visscher, 1947; Quimby, 1948) but Schultze (1955) observed that full body size was not attained. The reproductive organs of the adult are more resistant to changes imposed by diet than are those of the immature animal. Thus, Mann and Walton (1953) found that 23 weeks of underfeeding produced little change in sperm density and motility in ma- ture animals although seminal vesicle func- tion was reduced. Li the male rat testis hypofunction follows partial or complete starvation (]\Iason and Wolfe, 1930; Muli- nos and Pomerantz, 1941a; Escudero, Her- raiz and Mussmano, 1948), but there is no reduction in testicular nitrogen (Addis, Poo and Lew, 1936). Loss of Leydig cell func- tion precedes cessation of spermatogenesis (Moore and Samuels, 1931) and is evident by the atrophy of the accessory sexual or- gans (^lulinos and Pomerantz, 1941a) and by an alteration in accessory gland secretion (Pazos and Huggins, 1945; Lutwak-]\Iann and Mann, 1950). Evidence of a tubular effect is provided by the lack of motile sperm (Reid, 1949). Severe dietary re- striction is associated with the absence of spermatozoa in the seminiferous tubules and epididymis (Mason, 1933; Menze, 1941). The human male suffering from chronic malnutrition exhibits hypogonadism. The testes atrophy and exhibit a decrease in size of the seminiferous tubules; basement mem- brane thickening and small Leydig cells are seen. These individuals excrete signifi- cantly subnormal amounts of 17-ketoster- oids (Zubiran and Gomez-Mont, 19531. Acute starvation may also decrease urinary 17-ketosteroid and androgen levels as much as 50 per cent, with recovery evident on re- feeding (Perloff, Lasche, Nodine, Schnee- berg and Vieillard. 1954). 6/ PHYSIOLOGY OF GONADS TABLE 12.5 Effect of diet on the testes of immature rats (From J. H. Leathern, in Re-productive Phijsiology and Protein Nutrition, Rutgers University Press, New Brunswick, N. J., 1959.) Sperm Initial control . . . 20 per cent X 30 days 6 per cent X 30 days 3 per cent X 30 days 0 per cent X 30 days 0 per cent + 5 per cent liver. , 0 per cent + 5 per cent yeast G5 per cent X 30 davs No. of Rats Testis Weight mg. mg./lOOgm. 10 329 825 10 1694 1035 16 824 890 12 380 930 10 140 346 10 112 291 10 119 296 10 1747 1040 0 100 50 0 0 0 0 100 2. Protein The minimal amount of dietary protein which will support reproduction, lactation, and growth is 16.7 per cent (Goettsch, 1949) . Thus, it is not surprising that maturation of testes and accessory sex organs was pre- vented in immature rats (Horn, 1955) and mice (Leathern and DeFeo, 1952) when they were fed a protein-free diet for 15 to 30 days after weaning. Furthermore, supple- ments of 5 per cent liver to the casein-free diet had no effect. After a month, the testes, averaging 329 mg., decreased to 140 mg. in rats fed 0 per cent casein, but the weight increased to an average of 1694 mg. and 1747 mg. in rats fed 20 per cent and 65 per cent casein, respectively (Table 12.5). Fol- lowing protein depletion, there was a de- crease in tubular ribonucleic acid and an in- crease in lipid. Accumulation of gonadal lipid in the inactive testis may be an ab- normal assimilation of a degenerative na- ture or simply nonutilization. A diet con- taining 6 per cent casein permitted the formation of spermatozoa in some animals (Guilbert and Goss, 1932). When the 6 per cent casein diet was fed to immature ani- mals for 30 days 50 per cent of the rats exhibited some spermatozoa; in addition, testis weight increased slightly and seminal vesicle weight doubled, but body weight was not improved (Horn, 1955). Thus, as we noted earlier, the reproductive system may gain special consideration for protein allot- ments when supplies are limited. Gain in testis weight in immature male rats and the biochemical composition of the immature testis are influenced by the nutri- tive value of the protein fed. The testes of normal immature rats contain 85 per cent water, 10.5 per cent protein, 4.5 per cent lipid, and detectable glycogen (Wolf and Leathern, 1955). Proteins of lower nutritive value (wheat gluten, peanut flour, gela- tin ) may permit some increase in testis weight, but testis protein concentration de- creased, percentage of water increased, and lipid and glycogen remained unchanged (Table 12.6). The enzyme ^fi-glucuronidase, which has frequently been associated with growth processes, exhibited no change in concentration as the testis matured or was jirevented from maturing by a protein-free diet (Leathem and Fisher, 1959). Not only are the weight and composition of the testis influenced by feeding proteins of varied bio- logic value, but the release of androgen is more markedly altered. When a 22-day-old male rat was fed a 20 per cent casein diet for 30 days, the seminal vesicle and ventral prostate weights increased 9- to 10-fold in comparison with initial control weight. Sub- TABLE 12.6 Niiiritioiial effects on testis-coin position in immature rats (From R. C. Wolf and J. H. Leathem, Endocrinology, 57, 286, 1955.) 20 pel' cent ca- sein 20 per cent wheat gluten 20 per cent i)ea- nut flour 20 i)er cent gela- tin 5 per cent casein Fox chow Initial control . . No. of Rats Final Body Weight gm. 7 128 8 82 8 81 5 53 5 61 8 115 7 61 72.3j30.4 04.634.0 1468 1017 1257 66.1 2101 28.8 0.11 0.18 0.11 0.10 684,62.4 29.2 0.26 1515'70.3 30.3 0.15 273 72. 7:30. 10.19 NUTRITIONAL EFFECTS 679 stitution of wheat gluten and peanut flour for casein, limited the increase in the weight of the seminal vesicles to less than 100 per cent. In fact, seminal vesicle weight as re- lated to body weight did not increase in animals fed 20 per cent wheat gluten. The withholding of dietary protein from an immature rat for 30 days, during which time maturation occurs in the fully fed ani- mal, did not impose a permanent damage. Refeeding of protein permitted the rapid re- covery of testis weight and the appearance of spermatoza, 70 per cent of all animals having recovered in 30 days when fully fed, whereas only 25 per cent recovered when 6 per cent casein was fed. Recovery of androgen secretion was somewhat slower than that of the tubules as estimated by seminal vesicle weight. Variations in protein quality are a re- flection of amino acid patterns, and amino acid deficiencies interfere with testis matu- ration (Scott, 1956; Pomeranze, Piliero, Medeci and Plachta, 1959). Alterations in food intake which follow amino acid de- ficiencies have required forced feeding or pair-fed controls, but it is clear from what w^as found in the controls that the gonadal changes were not entirely due to inanition (Ershoff, 1952). If the diet is varied so that caloric intake per gram is reduced to half while retaining the dietary casein level at 20 per cent, im- mature rat testis growth is prevented. The effect is unlike that obtained with this level of protein in the presence of adequate calo- ries. Furthermore, the caloric restriction may increase testis glycogen (Leathem, 1959c). Protein anabolic levels are higher in the tissues of young growing animals and the body is more dependent on dietary protein level and quality for maintenance of the metabolic nitrogen pool than in adult ani- mals. On the other hand, body protein re- serves in adult animals permit internal shifts of nitrogen to the metabolic pool and to tissues when dietary sources are reduced or endocrine imbalances are imposed. Thus, Cole, Guilbert and Goss (1932) fed a low protein diet to adult male rats for 60 to 90 days before the sperm disappeared, but the animals would not mate. Amount of semen and sperm produced by sheep have been re- TABLE 12.7 Arlult rat testes and seminal vesicles after protein depletion (From J. H. Leatliem, Recent Progr. Hormone Res., 14, 141, 1958.) Days on No. of Testis H2O Protein Total Seminal PFD* Rats Weight Protein Vesical nig. % %dry gm. mg. Control 9 2852 85.9 66.7 0.28 1276 30 9 2600 86.0 66.1 0.24 689 50 7 2398 85.4 64.1 0.22 320 90 25 1429 85.7 69.6 0.13 168 * PFD = Protein-free diet. lated to the dietary protein level (Popoff and Okultilschew, 1936). Removal of pro- tein from the diet for 30 days had little effect on the adult rat testis weight, sper- matogenesis, or nitrogen content (Leathem, 1954). However, seminal vesicle w^eight was reduced 50 per cent (Aschkenasy, 1954). Prolonged protein depletion was required before the testis exhibited a loss in protein and a reduction in size. A loss of sperma- tozoa was not observed consistently, al- though some testes were completely atrophic (Table 12.7). Accessory organ weight de- crease reflected the disappearance of andro- gen (Leathem, 1958a). Interstitial cell at- rophy has also been noted in rats fed a low vegetable protein (cassava) diet (Adams, Fernand and Schnieden, 1958). Sterility may or may not be induced with diets containing 65 per cent protein (Reid. 1949; Leathem, 1959c) but a 15 to 18 per cent dietary level of a poor protein such as maize or gelatin will decrease sperm mo- tility and increase the number of abnormal sperm. The influence of proteins having dif- ferent nutritional values in support of the growth of testes from the level to which they were depressed by stilbestrol indicated that casein, lactalbumin, and wheat gluten are equally competent to support testis growth whereas gelatin is deficient. Whole proteins may have several amino acid de- ficiencies, but the administration of amino acid antagonists may help to identify im- portant individual amino acids. As an ex- ample, ethionine causes severe seminiferous tubule atrophy and Leydig cell hypoplasia (Kaufman, Klavins and Kinney, 1956; Goldberg, Pfau and Ungar, 1959). Studies in man have indicated a sharp reduction in 680 PHYSIOLOGY OF GONADS spermatozoa after 9 days on an arginine- deficient diet (Holt, Albanese, Shettles, Kajdi and Wangerin, 1942). Adequate dietary protein cannot main- tain reproductive function if the diet is calorie deficient. Thus, a decrease in seminal vesicle weight could be related to a decrease in dietary calories while protein levels were constant (Rosenthal and Allison, 1956). However, the accessory gland weight loss imposed by caloric restriction could be slowed by increasing the dietary protein (Rivero-Fontan, Paschkis, West and Can- tarow, 1952). Alterations in testis function imposed by inadequate protein are corrected when pro- tein is returned to the diet at normal levels (Aschkenasy and Dray, 1953). Neverthe- less, the nutritional state of the animal as a factor influencing recovery has been dem- onstrated with stilbestrol-treated adult male rats. While being fed an 18 per cent casein diet, adult male rats were injected with 0.1 mg. stilbestrol daily for 20 days. Testis weight decreased from 2848 to 842 mg., spermatogenesis was abolished, and testis water and protein content were significantly reduced. Despite a reduction in food intake, pair-fed controls exhibited no effect on re- productive organs. When a protein-free diet was substituted for the normal diet during the administration of stilbestrol, atrophy of the reproductive system was observed. Ces- sation of hormone administration was fol- lowed by a rapid return of testicular func- tion toward normal when 18 per cent casein was fed both during the injection period and the recovery period. Within 30 days spermatogenesis and testicular composition were fully recovered. However, when 18 per cent casein was fed in the postinjection period to rats that had received a protein- free diet while being given stilbestrol, re- covery was clearly slow. After a month, spermatozoa were observed in only 30 per cent of the testes and testis weight was sub- normal. Despite the seeming similarity of response by the two nutritional groups dur- ing the injection period, the postinjection re- covery on identical dietary intake revealed marked differences in rate of recoverv (Lea- them, 1958a). 3. Fat Linoleic, linolenic, and arachidonic acids are designated as essentially fatty acids, but the physiologic role of these substances is not clearly understood. Nevertheless, the male reproductive organs are influenced by dietary essential fatty acid levels. High fat diets may enhance testicular weight (Kaunitz, Slanetz, Johnson and Guilmain, 1956) whereas removal of fat from the diet resulted in a degeneration of the seminifer- ous tubules as evidenced by intracellular vacuolation and a reduction in spermatids and spermatozoa (Panos and Finerty, 1954). After 5 months of feeding a fat- free diet, the rat testis may be devoid of sperm (Evans, Lepkovsky and Murphy, 1934). Testis degeneration occurred despite dietary supplements of vitamins A and E and in animals whose health appeared quite normal (Ferrando, Jacques, Mabboux and Prieur, 1955; Ferrando, Jacques, Mabboux and SoUogoub, 1955). Weanling rats fed 14 per cent arachis (pea- nut) oil for 15 weeks exhibited a marked impairment of spermatogenesis (Aaes-Jor- gensen, Funch and Dam, 1956) and 28 per cent arachis oil induced testicular dam- age of such an order that 15 weeks of feeding ethyl linoleate did not restore fertility (Aaes-Jorgensen, Funch and Dam, 1957). 4. Vitamins Testicular dysfunction as judged by fail- ure of sperm formation or atrophy of the secondary sex organs has been observed in deprivations of thiamine, riboflavin, pyri- doxine, calcium pantothenate, biotin, and vitamins A and E. One must distinguish, however, between effects of inanition as- sociated w^ith a vitamin deficiency and a specific vitamin effect (Skelton, 1950) ; one must also consider species differences (Bis- kind, 1946). There is no question but that vitamin E deficiency in the rat results in a specific and irreversible damage to the testis. Tubu- lar damage may proceed to the point where only Sertoli cells remain and yet the in- terstitial cells are not influenced (Mason, 1939). Similar changes followed vitamin E deficiency in the guinea pig (Pappenheimer and SchogolefT, 1944; Curto, 1954; Ingel- NUTRITIONAL EFFECTS 681 man-Siindberg, 1954) , hamster (Mason and Mauer, 1957), and bird (Herrick, Eide and Snow, 1952; Lowe, Morton, Cunningham and Vernon, 1957). However, little or no effect of an absence of vitamin E was noted in the rabbit (Mackenzie, 1942) and mouse (Bryan and Mason, 1941), or in live stock (Blaxter and Brown, 1952), or man (Lut- wak-Mann, 1958), although vitamin E is present in human testes (Dju, Mason and Filer, 1958). Treatment of low-fertility farm animals with wheat germ oil or to- copherol or the use of this vitamin clinically have provided only inconclusive results (Beckmann, 1955) . Although some positive effects have been reported in man, the re- sults may be due in part at least to the spar- ing action of tocopherol toward vitamin A. Vitamin A deficiency influences the testis but changes are closely associated with the degree of inanition. In the rat, a vitamin de- ficiency sufficient to cause ocular lesions did not prevent sperm formation, but a de- ficiency of such proportions as to cause a body weight loss did cause atrophy of the germinal epithelium (Reid, 1949). Vitamin A deficiency will induce sterility in mice (McCarthy and Cerecedo, 1952). A gross vitamin deficiency in bulls before expected breeding age prevented breeding ; adult bulls may exhibit a lower quality semen but they remain fertile (Reid, 1949). Vitamin A de- ficiency induces metaplastic keratinization of the epithelium lining the male accessory sex organs (Follis, 1948) and thus may in- fluence semen. Testis damage induced by vitamin A de- ficiency can be reversed, but vitamin A therapy in man for oligospermia not due to vitamin lack was without effect (Home and Maddock, 1952) . Age of the animal and dosage are factors which influence the results obtained in male rats with administered vitamin A. Immature male rats given 250 I.U. of vitamin A per gram of body weight daily exhibited a loss of spermatocytes, an effect which was ac- centuated by tocopherol (Maddock, Cohen and Wolbach, 1953). Little or no effect of similar treatment was observed in adult rats. The liver is the major storage depot for vitamin A and the fact that the male rat liver is more quickly depleted and less capa- ble of storage than is the liver of the female should be considered in any attempted cor- relation of the vitamin and hormone levels (Booth, 1952). Other vitamin deficiencies have been shown to influence the testis. A lack of thiamine had little effect on testis weight, but did influence the Leydig cells and pre- vented growth of the accessory sex organs (Pecora and Highman, 1953). A chronic lack of ascorbic acid will cause a degenera- tion of both Leydig cells and seminiferous tubules. The effects of vitamin deficiency on the testis has been distinguished from those due to inanition and have been related to changes in carbohydrate metabolism (Mu- kherjee and Banerjee, 1954; Kocen and Cavazos, 1958) . The importance of ascorbic acid in the testis as related to function is not evident, but concentrations of this vita- min are maximal at 1 week of age (Coste, Delbarre and Lacronique, 1953). Serious anatomic and functional impair- ments of testes were noted in pantothenic acid deficiency (Barboriak, Krehl, Cowgill and Whedon, 1957), and development of the rat testis and seminal vesicles was re- tarded by a biotin deficiency (Bishop and Kosarick, 1951; Katsh, Kosarick and Al- pern, 1955), but the animals did not exhibit marked alterations in other endocrine or- gans (Delost and Terroine, 1954). Testos- terone hastened the development of vitamin deficiency and enhanced the severity of bio- tin deficiency in both sexes, thereby sug- gesting a hormone-vitamin relationship (Okey, Pencharz and Lepkovsky, 1950). On the other hand, testosterone had no effect on the tolerance of mice for aminopterin, but castration increased the tolerance (Goldin, Greenspan, Goldberg and Schoenberg 1950). B. INFLUENCE OF NUTRITION ON THE EE- SPONSIVENESS OF MALE REPRODUCTIVE TISSUES TO HORMONES 1. Testis a. Inanition. The testes of birds on limited food intake were more responsive to hypo- physeal gonadotrophin than fully fed birds (Byerly and Burrows, 1938; Breneman, 1940). In the rat several investigators have shown that the testis will respond to gonado- trophin despite inanition (Moore and Sara- 682 PHYSIOLOGY OF GONADS TABLE 12.8 Influence of diet and pregnant mare serum (PMS) on testes and seminal vesicles of immature male mice (From V. J. DeFeo and J. H. Leathern, unpublished.) Diet (Per cent Protein X Days Fed) 0 per cent X 10 0 per cent X 10 0 per cent X 20 0 per cent X 20 l.u. 0 3 0 3 stage of Spermatogenesis 4 1 1 5 5 Sper- ma- tids Seminal Vesicles mg. 2.7 4.5 2.7 3.5 uels, 1931; Funk and Funk, 1939; Meites, 1953), with a stimulation of Leydig cells, an increase in testis size, and, in 40 days, a return of spermatozoa. Underfed males in- jected with gonadotrophin sired litters (Mu- linos and Pomerantz, 1941a, b). Improved nutrition aided by unknown liver factors enhanced the response to androgen in se- vere human oligospermia (Glass and Rus- sell, 1952). b. Protein. Feeding a protein-deficient diet to adult male rats for 60 to 90 days did not prevent stimulation of the testes and seminal vesicles after pregnant mare's serum (PMS) administration (Cole, Guilbert and Goss, 1932). As we have noted, immature animals are prevented from maturing when diets lack protein. Nevertheless, a gonadal response to injected gonadotrophin was ob- tained in immature mice fed a protein-free diet for 13 days; tubules and Leydig cells were stimulated and androgen was secreted (Table 12.8). Refeeding alone permitted a recovery of spermatogenesis which was not hastened by concomitant PMS (Leathem, 1959c). The maintenance of testis weight and spermatogenic activity with testosterone propionate in hypophysectomized adult male rats is well known, but these studies have involved adequate nutrition. If hypo- physectomized rats were fed a protein-free diet and injected with 0.25 mg. testosterone propionate daily, testis weight and sper- matogenesis were less well maintained than in rats fed protein. Testis protein concentra- tion was also reduced. These data suggest that influences of nutrition on the testis can be direct and are not entirely mediated through hypophyeal gonadotrophin changes (Leathem, 1959b). c. Fat. The rat fed for 20 weeks on a fat- free diet exhibits a degeneration of the seminiferous epithelium within the first weeks which progresses rapidly thereafter. Chorionic gonadotrophin or rat pituitary ex- tract started during the 20th week failed to counteract the tubular degeneration, but testosterone propionate proved effective (Finerty, Klein and Panos, 1957). The re- sult shows that the ineffectiveness of the gonadotrophins could not be due to the fail- ure of androgen release (Greenberg and Ers- hoff, 1951). d. Vitamins. Gonadotrophins failed to promote spermatogenesis in vitamin A- (Mason, 1939) or vitamin E- (]Mason, 1933; Geller, 1933; Drummond, Noble and Wright, 1939) deficient rats, but in another experiment the atrophic accessory sex or- gans of vitamin A-depleted rats were stimu- lated (Mayer and Goodard, 1951). Lack of vitamin A favored an enhanced response to PMS when the ratios of seminal vesicle weight to body weight were computed (Meites, 1953). The failure of gonadotro- phins to stimulate testis tubules suggests a specific effect of avitaminosis A and E (Mason, 1933) on the responsiveness of the germinal epithelium. Subnormal responses of rats to PMS, as measured by relative seminal vesicle weight, were obtained when there were individual vitamin B deficiencies, but the influence was due largely to inanition (Drill and Burrill, 1944; Meites, 1953). Nevertheless, sufficient response to chorionic gonadotrophin was ob- tained so that fructose and citric acid levels were restored to normal. Such an effect was not observed to follow dietary correction un- less an unlimited food intake was allowed (Lutwak-Mann. 1958). 2. Sc il W.siclfx and Prosfate a. Inanition. Although the accessory re- productive organs resppnd to direct stimu- lation despite an inadequate food intake (Mooi'c and Samuels. 19311, tlio increase NUTRITIONAL EFFECTS 683 in weight may be subnormal in mice and rats (Goldsmith, Nigrelli and Ross, 1950; Kline and Dorfman, 1951a, Grayhack and Scott, 1952), or above normal in chickens (Breneman, 1940). Complete deprivation of food reduced the quantity of prostatic fluid in the dog, but exogenous androgen re- stored the volume, increased acid phospha- tase, and induced tissue growth (Pazos and Huggins, 1945) . 6. Protein. The response of the seminal vesicles to androgen was investigated in im- mature rats, using weight and /5-glucuroni- dase as end points. Castration and 10 days on a protein-free diet preceded the 72-hour response to 0.25 mg. testosterone propionate. The lack of protein did not prevent a nor- mal weight increase, and enzyme concentra- tion was unchanged. If an 18 per cent diet was fed during the 3-day period that the androgen was acting, no improvement in weight response was noted, but enzyme con- centration increased 100 per cent. Thus, when protein stores are depleted, the andro- gen response may be incomplete in the ab- sence of dietary protein (Leathern, 1959c). Nevertheless, varied protein levels do not influence seminal vesicle weight-response when caloric intake is reduced (Rivero- Fontan, Paschkis, West and Cantarow, 1952). c. Vitamins. Vitamin deficiencies do not prevent the seminal vesicles from respond- ing to androgen. In fact, in vitamin B de- ficiency, testosterone restored fructose and citric acid levels to normal despite the need for thiamine in carbohydrate metabolism (Lutwak-Mann and Mann, 1950). In the male, unlike the female, the effects of folic acid deficiency in reducing responsiveness to administered androgen were largely due to inanition in both mice and rats (Gold- smith, Nigrelli and Ross, 1950; Kline and Dorfman, 1951a) , and vitamin A deficiency which leads to virtual castration does not prevent an essentially normal response of the accessory glands to testosterone pro- pionate (Mayer and Truant, 1949). Re- stricting the caloric intake of vitamin A- deficient rats retarded the curative effects of vitamin A in restoring the accessory sex glands of the A-deficient animals (Mason, 1939). V. Female Reproductive System A. OVARIES 1. Inanition Mammalian species generally exhibit a delay in sexual maturation when food in- take is subnormal before puberty, and ovarian atrophy with associated changes in cycles if inanition is imposed on adults. In human beings a decrease in fertility and a greater incidence of menstrual irregulari- ties were induced by war famine (Zimmer, Weill and Dubois, 1944). Ovarian atrophy with associated amenorrhea and sterility were invoked by chronic undernutrition (Stephens, 1941). The ovarian morpliologic changes were similar to those of aging. Urinary estrogens were subnormal in 22 of 25 patients exhibiting amenorrhea associ- ated with limited food intake (Zubiran and (_lomcz-Mont, 1953). The nutritional requirements of jM'imates other than man have been studied in female baboons. The intake of vitamins and other essential nutrients was found to be of the same order as that recommended for man. Caloric intake varied with the menstrual cycle, being least during the follicular phase and maximal during the 2 to 7 days preced- ing menstruation (Gilbert and Gillman, 1956). Various diets were also studied to assess their importance in maintaining the normal menstrual rhythm. The feeding of (a) maize alone, (b) assorted vegetables and fruit, or (c) maize, skimmed milk, and fat led to menstrual irregularities or to amenorrhea. The mechanism regulating ov- ulation was the first to be deranged. The addition of various vitamins or of animal protein did not correct the menstrual dis- orders. However, inclusion of ox liver in the diet did maintain the menstrual rhyth- micity, but the beneficial effect could not be attributed to its protein content (Gill- man and Gilbert, 1956 ) . In lower mammals that have been studied, inanition will hinder vaginal opening, and delay puberty and ovarian maturation and functioning. In adult rats and mice ostrous cycles are interrupted and the reprorUictive system becomes atrophic when body weight loss exceeds 15 per cent. The ovaries be- 684 PHYSIOLOGY OF GONADS come smaller, ovulation fails, and large vesicular follicles decrease in number with an increase in atresia, but primary follicles show a compensatory increase (Marrian and Parkes, 1929; Mulinos and Pomerantz, 1940; Stephens and Allen, 1941; Guilbert, 1942; Bratton, 1957). The ovarian intersti- tial cells mav be markedly altered or absent (Huseby and Ball, 1945; Rinaldini, 1949) and the ovary may exhibit excessive luteini- zation (Arvy, Aschkenasy, Aschkenasy- Lelu and Gabe, 1946) or regressing corpora lutea (Rinaldini, 1949) . However, the ovar- ian changes induced by inanition may be reversed by refeeding, with a return to re- productive capacity (Ball, Barnes and Vis- scher, 1947; Schultze, 1955). The effect of feed-level on the reproductive capacity of the ewe has been reported (El-Skukh, Nulet, Pope and Casida, 1955), but one must re- alize that high planes of nutrition may ad- versely influence fertility (Asdell, 1949). Nevertheless, additional protein and cal- cium added to an adequate diet extended the reproductive life span (Sherman, Pear- son, Bal, McCarthy and Lanford, 1956). 2. Protein The availability of just protein has an important influence on the female repro- ductive system. In immature rats ovarian maturation was prevented by feeding diets containing 0 per cent to 1.5 per cent protein (Ryabinina, 1952) and low protein diets de- creased the number of ova but without al- tering their ribonucleic acid (RNA) or gly- cogen content (Ishida, 1957). Refeeding 18 per cent protein for only 3 days was marked by the appearance of vesicular follicles and the release of estrogen in mice previously fed a protein-free diet (Leathem, 1958a). In experiments involving the opposite extreme, in which 90 per cent protein diets were used, a retardation of ovarian growth, and a de- lay in follicular maturation, in vaginal opening, and in the initiation of estrous cycles were noted (Aschkenasy-Lelu and Tuchmann-Duplessis, 1947; Tuchniann- Duplcssis and Aschkenasy-Lelu, 1948). Adult female rats fed a protein-free diet for 30 days exhibited ovaries weighing 22 mg. compared with ovaries weighing 56 mg. from i)air-fed controls fed 18 per cent casein. Ovarian glycogen, ascorbic acid, and cho- lesterol were all influenced by protein deprivation and anestrum accompanied the ovarian changes. Furthermore, uterine weight and gl3^cogen decreased in rats fed protein-free diets (Leathem, 1959b). In adult rats the feeding of 3.5 per cent to 5 per cent levels of protein (GuillDert and Gross, 1932) was followed by irregularity of the cycles or by their cessation. The cycles became normal when 20 to 30 per cent protein was fed (Aschkenasy-Lelu and Aschkenasy, 1947). However, abnormally high levels of casein (90 per cent) in- duced prolonged periods of constant estrus (Tuchmann-Duplessis and Aschkenasy- Lelu, 1947). Nevertheless, not all species responded to protein depletion in the same manner. For example, the rabbit exhibited estrus and ovulation despite a 25 per cent body weight loss imposed by 0 to 2 per cent protein diets (Friedman and Friedman, 1940). Despite a normal level of protein in the diet, inadequate calories will interfere with reproductive function and induce ovarian atrophy (Escudero, Herraiz and Mussmano, 1948; Rivero-Fontan, Paschkis, West and Cantarow, 1952). Furthermore, the effects of 15 per cent and 56 per cent protein levels on estrous cycles could not be distinguished when calories were reduced 50 per cent (Lee, King and Visscher, 1952). Returning mice to full feeding after months of caloric de- ficiency resulted in a sharp increase in re- productive performance well above that ex- pected for the age of the animal (Visscher, King and Lee, 1952). This type of rebound phenomenon has not been explained. Reproductive failure assigned to dietary protein may be a reflection of protein quality as well as level. Specific amino acid deficiencies lead to cessation of estrus (White and AVhite, 1942; Berg and Rohse, 1947) and thus feeding gelatin or wheat as the protein source and at an 18 per cent level was quickly followed by an anestrum (Leathem, 1959b). Supplementation of the wheat diet with lysine corrected the re- productive abnormalities (Courrier and Raynaud, 1932), but neither lysine (Pear- son, Hart and Bohstedt, 1937) nor cystine (Pearson, 1936) added to a low casein diet was beneficial. Control of food intake must be considered in studies involving amino NUTRITIONAL EFFECTS 685 acids, for a deficiency or an excess can cre- ate an imbalance and alter appetite. Oppor- tunity to study the amino acids in reproduc- tion is now possible because of the work of Greenstein, Birnbaum, Winitz and Otey (1957) and Schultze (1956) , who maintained rats for two or more generations on syn- thetic diets containing amino acids as the only source of protein. Similarly, the amino acid needs for egg-laying in hens has been reported (Fisher, 1957). Tissue culture methods also permit the study of the nu- tritional requirements of embryonic gonadal tissue, the success of avian gonadal tissue in culture being judged by survival, growth, and differentiation. In experiments in which this technique was used it was found that a medium made up of amino acids as the basic nitrogen source can maintain gonadal ex- plants very successfully, even though the choice of amino acids does not exactly cor- respond to the 10 essential amino acids rec- ommended for postnatal growth (Stenger- Haffen and Wolff, 1957). 3. Carbohydrate The absence of dietary carbohydrate does not appreciably affect the regularity of estrous cycles in rats provided the caloric need is met. However, the substitution of 20 per cent sucrose for corn starch induced precocious sexual maturity which was fol- lowed by sterility (Whitnah and Bogart, 1956). The ovaries contained corpora lutea, but the excessive luteinization of unruptured follicles suggested a hypophyseal disturb- ance. Substitution of 20 per cent lactose for corn starch had no effect. Increased amounts of lactose retarded the gain in body weight and blocked ovarian maturation, possibly because the animal could not hydrolyze ade- quate amounts of the disaccharide. Ad- dition of whole liver powder to the diet counteracted the depressing action of 45 per cent lactose on the ovary (Ershoff, 1949). 4. Fat There seems to be little doubt that dietary fat is reciuired for normal cyclic activity, successful pregnancy, and lactation, and that the requirements for essential fatty acids are lower in females than in males (Deuel, 1956). Conception, fetal development, and par- turition can take place in animals fed a diet deficient in fatty acids (Deuel, Martin and Alfin-Slater, 1954) , despite a reduction in total carcass arachidonic acid (Kum- merow. Pan and Hickman, 1952). Earlier reports indicated that a deficiency of es- sential fatty acids caused irregular ovula- tion and impaired reproduction (Burr and Burr, 1930; Maeder, 1937). The large pale ovaries lead Sherman (1941) to relate es- sential fatty acid deficiency to carotene metabolism. In this regard the removal of essential fatty acids from an adequate diet supplemented with vitamin A and E lead to anestrum and sterility while maintaining good health (Ferrando, Jacques, Mabboux and Prieur, 1955). Perhaps the differences in opinion regarding the effects of fatty acid deficiency can be related to the duration of the experimental period. Panos and Finerty (1953) found that growing rats placed on a fat-free diet exhibited a normal time for vaginal opening, normal ovarian weight, follicles, and corpora lutea, although in- terstitial cells were atrophic. However, reg- ular estrous cycles were noted for only 20 weeks, thereafter 60 per cent of the animals exhibited irregular cycles. A decrease in reproductive function may be invoked by adding 14 per cent arachis oil to the diet (Aaes-Jorgensen, Funch and Dam, 1956) . Increasing dietary fat by add- ing rape oil did not influence ovarian func- tion but did cause the accumulation of ovarian and adrenal cholesterol (Carroll and Noble, 1952). Essential fatty acid deficiency is as- sociated with underdevelopment and atrophic changes of the uterine mucosa. Adding fat to a stock diet enhanced uterine weight in young animals at a more rapid pace than body weight (Umberger and Gass, 1958) . 5. Vitamins Carotenoid pigments are present in the gonads of many vertebrates and marine in- vertebrates, and, in mammals, are par- ticularly prominent in the corpus luteum. However, no progress has been made in determining either the importance of the carotenoids in the ovary or of the factors controlling their concentrations. It is well known that vitamin A deficiency induces 686 PHYSIOLOGY OF GONADS a characteristic keratinizing metaplasia of the uterus and vagina, but estrous cycles continue despite the vaginal mucosal changes. Furthermore, ovulation occurs regularly until advanced stages of de- ficiency appear. The estrous cycle becomes irregular in cattle fed for a long period of time on fodder deficient in carotene. The corpora lutea fail to regress at the normal rate and ovarian follicles become atretic and cystic ( Jaskowski, Watkowski, Dobrowol- ska and Domanski, cited by Lutwak-Mann, 1958). The alterations in reproductive or- gans associated with a lack of vitamin A may be due in part to a vitamin E deficiency since the latter enhances the rate at which liver stores of vitamin A are depleted. Definite effects of hypervitaminosis A have been observed on reproduction. Masin (1950) noted that estrus in female rats could be prolonged by administration of 37,000 I.U. of vitamin A daily. The im- plications, however, have not been studied. The effect of hypervitaminosis A may actu- ally induce secondary hypovitaminoses. The displacement of vitamin K by excess A is almost certain and similar relationships appear to exist with vitamin D (Nieman and Klein Obbink, 1954). The failure of vitamin E-deficient fe- male rats to become pregnant is apparently due to disturbances of the implantation process rather than to the failure of ovu- lation. There is no direct proof of ovarian dysfunction (Blandau, Kaunitz and Slanetz, 1949). However, the ovary of the rat de- ficient in vitamin E may have more con- nective tissue and pigment, and Kaunitz (1955) showed by ovarian transplantation that some nonspecific ovarian dysfunction appears to exist (cited by Cheng, 1959 (. Vitamin E is essential for birds, but there is little evidence for a dependency in most mammals; sheep, cows, goats, and pigs have been studied. Treatment of low-fertility farm animals with tocopherol has not pro- vided conclusive data favoring its use (Lut- wak-Mann, 1958), nor has the treatment of human females been rewarded with any indication that vitamin E might be helpful in cases of abnormal cycles and habitual abortion (Beckmann, 1955). No specific reproductive disturbances in man, the rhesus monkey, or the guinea pig have been associated with vitamin C de- ficiency (Mason, 1939). Nevertheless, the high ascorbic acid content of ovarian and luteal tissue and of the adrenal cortex sug- gests a physiologic role in association with steroid synthesis. (3varian ascorbic acid varies with the estrous cycle, dropping sharply in the proestrum (Coste, Delbarre and Lacronique, 1953), and decreasing in resjionse to gonadotrophin (Hokfelt, 1950; Parlow, 1958). Virtually no ascorbic acid is present in bovine follicular fluid (Lutwak- Mann, 1954) or in rat ovarian cyst fluid (Blye and Leathem, 1959). Uterine ascorbic acid decreased in immature mice treated with estrogen, but remained unchanged in rats following thiouracil administration (Leathem, 1959a). Its role in the uterus awaits elucidation. Delayed sexual maturation and ovarian atrophy have been described when there are deficiencies of thiamine, riboflavin, pyri- doxine, pantothenic acid, biotin, and B12 (Ershoff, 1952; Ullrey, Becker, Terrill and Notzold, 1955). However, as we noted when deficiencies of the vitamins were being con- sidered, much of the impairment of repro- ductive function can be related to inanition rather than to a vitamin deficiency (Drill and Burrill, 1944). Pyridoxine deficiency, although not affecting structure (Morris, Dunn and Wagner, 1953) , markedly reduces the sensitivity of the ovary to administered gonadotrophin (Wooten, Nelson, Simpson and Evans, 1958) . Bird, frog, and fish eggs contain consider- able quantities of vitamins. In fact, the daily human requirements for vitamins may be contained in a hen's egg and thus it is not surprising that hatchability is decreased l)y virtually any vitamin deficiency. Lut- wak-Mann (1958) has provided an excellent survey of these data with numerous refer- ences to studies of frogs and fishes. Nearly all the B vitamins are present in fish roe and the pantothenic acid concentration in cod ovaries {Gadus morrhua) exceeds most otlicr natural sources. The amount of the latter varies with the reproductive cycle, d(>creasing to its lowest level before spawn- ing. Riboflavin and vitamin B12 , on the other h;ui(l, do not change (Braekkan, 1955). NUTRITIONAL EFFECTS 687 B. INFLUENCE OF NUTRITION ON THE RESPON- SIVENESS OF FEMALE REPRODUCTIVE TISSUES TO HORMONES 1. Ovary a. Inanition. Marrian and Parkes (1929) were the first to show that the quiescent ovary of the underfed rat can respond to injections of anterior pituitary as evidenced by ovulation and estrous smears. Subse- quently the ovaries of underfed birds, rats, and guinea pigs were found to be responsive to serum gonadotrophin (Werner, 1939; Stephens and Allen, 1941; Mulinos and Pomerantz, 1941b; Hosoda, Kaneko, Mogi and Abe, 1956). A low calorie bread-and- milk diet for 30 days did not prevent ovar- ian response to rat anterior pituitary or to chorionic gonadotrophin. In these animals an increase in ovarian weight with repair of interstitial tissue, as well as folhcle stimulation and corpus luteum formation, were observed (Rinaldini, 1949). Rats from which food had been withdrawn for 12 days could respond to castrated rat pitui- tary extract with an increase in ovarian and uterine weight (Maddock and Heller, 1947). Nevertheless, differences in the time and degree of responsiveness to adminis- tered gonadotrophin were noted in rabbits. Animals on a high plane of nutrition re- sponded to gonadotrophin at 12 weeks, whereas rabbits on a low plane of nutrition responded at 20 weeks and fewer eggs were shed (Adams, 1953). b. Protein. Protein or amino acid defi- ciencies in the rat do not prevent a response to administered gonadotrophin (Cole, Guil- bert and Goss, 1932; Courrier and Raynaud, 1932) . However, the degree and type of gonadal response is influenced by the diet. Thus, immature female mice fed 0 to 6 per cent casein for 13 days exhibited only fol- licular growth in response to pregnant mare serum, whereas the ovarian response in mice fed 18 per cent casein was suggestive of follicle-stimulating and strongly lutein- izing actions (Table 12.9). Furthermore, the ovarian response was significantly less after 20 days of nonprotein feeding than after 10 days of depletion (Leathem, 1958a). Ovarian stimulation by a gonado- trophin involves tissue protein synthesis and thus the type of whole protein fed could influence the responses. Yamamoto and Chow (1950) fed casein, lactalbumin, soybean, and wheat gluten at 20 per cent levels and noted that the response to gon- adotrophin as estimated by tissue nitrogen was related to the nutritive value of the protein. The ovarian weight response to chorionic gonadotrophin was less in rats fed 20 per cent gelatin than those fed 20 per cent casein (Leathem, 1959b). Inasmuch as the hypophysis may influence the gon- adal response to injected hormone despite the diet, hypophysectomized rats fed a pro- tein-free diet for 5 weeks and hyophysecto- mized rats on a complete diet were tested for response to gonadotrophins. The re- sponse to FSH was not influenced by diet, but the protein-depleted rats were twice as sensitive to interstitial cell-stimulating hormone (ICSH), human chorionic gonado- trophin (HCG), and PMS as the normal rats (Srebnik, Nelson and Simpson, 1958). Protein-depleted, normal mice were twice as sensitive to PMS as fully fed mice (Lea- them and Defeo, 1952 1 . c. Vitamins. In the female vitamin B deficiencies do not prevent ovarian re- sponses to gonadotrophin (Figge and Allen, 1942), but the number of studies is limited. Be deficiency in DBA mice was associated with an increased sensitivity of the ovary to gonadotrophins (Morris, Dunn and Wag- ner, 1953), whereas pyridoxine deficiency in the rat decreased ovarian sensitivity, especially to FSH (Wooten, Nelson, Simp- TABLE 12.9 Influence of dietary protein and pregnant mare serum {PMS) on the mouse ovary (From J. H. Leathem, Recent Progr. Hormone Res., 14, 141, 1958.) Diet fPer cent Protein X Days Fed) c-5, •g-s 1* k r 1 11 c 1 I.U. mg. mg. 0 per cent X 23 0 1.2 0 0 4.8 0 per cent X 2.3 3 2.8 13 0 10.8 0 per cent X 13 0 1.4 0 0 4.9 0 per cent X 13 3 4.4 16 1 15.1 6 per cent X 13 0 3.2 6 0 7.7 6 per cent X 13 3 5.6 12 1 31.9 18 per cent X 13 0 5.0 10 2 51.6 18 per cent X 13 3 8.0 7 4 51.3 088 PHYSIOLOGY OF GONADS son and Evans, 1958 j. Administration of vitamin C concomitant witli gonadotropliin has been claimed to enhance ovarian re- sponse (DiCio and Schteingart, 1942), but in another study the addition of ascorbic acid inhibited the hiteinizing and ovulating action of the gonadotrophin (Desaive, 1956). Whether induced by vitamin deficiency or by inanition, the anestrum in rats which follows 2 to 3 weeks' feeding of a vitamin B-deficient diet has been explored as a method for the assay of gonadotrophin. Pugsley (1957) has shown that there is considerable convenience of method and a satisfactory precision of response for the assay of HCG and pregnant mare serum. 2. Uterus and Vagina a. Inanition. Limited food intake does not prevent an increase in uterine weight after estrogen. Testosterone propionate will markedly increase uterine growth despite a 50 per cent reduction in food intake (Leathem, Nocenti and Granitsas, 1956). Furthermore, dietary manipulations involv- ing caloric and protein levels did not pre- vent the uteri of spayed rats from re- sponding to estrogen (Vanderlinde and Westerfield, 1950). More specific biochem- ical and physiologic responses must be measured because starvation for 4-day pe- riods clearly interferes with deciduoma for- mation (DeFeo and Rothchild, 1953). A start in the direction of studying tissue- composition changes has been made by measuring glycogen. However, no changes were noted in uterine glycogen in fasting rats (Walaas, 1952), and estrogen promoted glycogen deposition in the uteri of starved rats as well as in the uteri of fully fed rats (Bo and Atkinson, 1953). b. Fat. Interest in the hormone content of fat from the tissues of animals treated with estrogen for the purpose of increasing body weight has raised the question of tissue hor- mone content. If estrogen was to be de- tected in tissues, an increase in dietary fat was necessary. However, the increase in dietary fat decreased the uterine response to stilbcstrol (I'mberger and Gass, 1958), thus complicating the assay. c. Vitainins. Stimulation of the uterus by estrogen does not require tliianiinc, ribo- flavin, pyridoxine, or pantothenic acid. On the other hand, a deficiency of nicotinic acid appears to enhance the response to low doses of estrogen (Kline and Dorfman, 1951a, b). However, Bio appears to be needed for optimal oviduct response (Kline, 1955) and is required for methyl group syn- thesis from various one-carbon precursors including serine and glycine (Johnson, 1958). Response of the bird oviduct to stilbestrol requires folic acid (Hertz, 1945, 1948). It was shown subsequently that stilbestrol and estrone effects in frogs, rats, and the rhesus monkey also require folic acid. A folic acid deficiency can be induced by feeding ami- nopterin. In this way the estrogen effects can be prevented. Aminopterin also prevents the action of progesterone in deciduoma formation, from which it may be inferred that folic acid is necessary for deciduoma formation in the rat. Increased steroid or folic acid levels can reverse the antagonist's effect (Velardo and Hisaw, 1953). The mechanism of folic acid action is not clear. It may function in fundamental meta- bolic reactions linked with nucleic acid synthesis. Brown (1953) showed that desoxyribonucleic acid could be substituted for folic acid in the bird. In the rat ami- nopterin interferes with the increase in uterine nucleic acids, and with nitrogen and P-^- uptake by nucleic acids following estrogen. Folic acid has been implicated in the metabolism of several amino acids (Davis, Meyer and McShan, 1956). Rats ovariectomized at weaning and maintained on a vitamin E-free diet for 6 weeks to 10 months responded to estradiol in the same manner as rats supplemented with tocopherol. This finding suggests that an intimate physiologic relationship be- tween estradiol and vitamin E is not very probable (Kaunitz, Slanetz and Atkinson, 1949). Nevertheless, vitamin E has been re]:»orted to act synergistically with ovarian hormones in dc^ciduoma formation (Kehl, Douard and Lanfranchi. 1951 ) and to in- fluence nucleic acid turnover (Dinning. Simc and Day, 1956). A vitamin-hormone interrelationship is apparent when estrogen and vitamin A are considered. Vitamin A-deficient female rats present evidence of a metaplastic uterine NUTRITIONAL EFFECTS 689 epithelium in 11 to 13 weeks, but similar changes failed to develop in ovariectomized rats. Vitamin A-deficient castrated rats quickly developed symptoms of metaplasia when estrogen alone was administered, but no adverse effect followed the administra- tion of estrogen combined with vitamin A (Bo, 1955, 1956). The vagina is different. Its epithelium becomes cornified in vitamin A-deficient normal and castrated rats. The cornification is histologically indistinguish- able from that occurring in the estrous rat and can be prevented by vitamin A. In fact, vitamin A will quantitatively in- hibit the effect of estrogen on the vaginal mucosa when both are applied locally (Kahn, 1954). Conversion of ^-carotene to vitamin A is influenced by tocopherol, vita- min Bi2 , insulin, and thyroid, with evidence for and against a similar action by cortisone (Lowe and Morton, 1956; Rice and Bo, 1958). An additional vitamin-hormone re- lationship is suggested by the augmentation of progesterone action in rabbits given vita- min Do . 3. Mammary Gland Inanition prevents mammary growth, but feeding above recommended requirements for maintenance and growth from birth to the first parturition also seems to inter- fere with mammary growth. Furthermore, steroid stimulation of the mammary gland is influenced by nutritional factors. Using the male mouse, Trentin and Turner (1941) showed that as food intake decreased, the amount of estradiol required to produce a minimal duct growth w^as proportionately increased. In the immature male rat a limited food intake prevented the growth of the mammary gland exhibited by fully fed controls. Nevertheless, the gland was com- petent to respond to estrogen (Reece, 1950) . Inasmuch as the glands of force-fed hy- pophysectomized rats did not respond to es- trogen (Samuels, Reinecke and Peterson, 1941; Ahren, 1959), one can assume that, despite inanition, a hypophyseal factor was present to permit the response of the mam- mary gland to estrogen. However, inanition (IMeites and Reed, 1949) , but not vitamin deficiencies (Reece, Turner, Hathaway and Davis, 1937), did reduce the content of hypophyseal lactogen in the rat. Growth of the mammary gland duct in the male rat in response to estradiol re- quires a minimum of 6 per cent casein. Pro- tein levels of 3 per cent and 0 per cent failed to support growth of the duct (Reece, 1959) . C. PREGNANCY The human male after attaining adult- hood is confronted with the problem of maintaining the body tissues built up during the growth period. However, in the human female it has been estimated that the re- placement of menstrual losses may require the synthesis of tissue equivalent to 100 per cent of her body weight (Flodin, 19531. In the event of pregnancy and in all vivi- parous species, the female is presented with even more formidable demands and a limi- tation of nutritional needs can lead to loss of the embryo or fetus. The role of nutrition at this point in reproduction has always re- ceived considerable attention and is compli- cated by the circumstance that many food substances influence pregnancy (Jackson, 1959). However, in many instances there is no evidence that fetal loss or malformation induced by nutritional modifications has been the consequence of an endocrine im- balance and thus limitation of the immense literature is permissible. During the first 15 days of pregnancy, a rat may gain 50 gm. Since the fetuses and placentas are small, most of the gain is ma- ternal and is associated with an' increase in food intake of as much as 100 calories per kilogram of body weight (Morrison, 1956). During the first 2 weeks of preg- nancy, marked storage of fat and water oc- curs in the maternal body and the animal's positive nitrogen balance is above normal. Liver fat also increases (Shipley, Chudzik, Curtiss and Price, 1953). The increased food intake in early pregnancy may therefore provide a reserve for late fetal growth, as food intake may decline to 65 per cent of the general pregnancy level during the last 7 days (Morrison, 1956). During this last week, fetal growth is rapid. The rapid growth has been related to (1) greater de- mands of the fetus, (2) greater amounts of food in the maternal blood, and (31 greater permeability of the placenta. Certainly the anabolic potential of fetal tissues is high and the mother can lose weight while the 690 PHYSIOLOGY OF GONADS fetuses gain. But it is also important to recall that there is a shift in protein, because its distribution in organs of pregnant rats differs from that in nonpregnant animals (Poo, Lew and Addis, 1939). Other changes in the maternal organism were enumerated b}^ Newton (1952) and by Souders and Morgan (1957). A measure of nitrogen balance during pregnancy, rather than weight of young at birth, has been suggested as a means of determining a diet adequate for reproduc- tion (Pike, Suder and Ross, 1954). After the 15th day, a retention of body protein increases, blood amino nitrogen and amino acids decrease, and urea formation de- creases. These metabolic activities suggest an increase in growth hormone although the levels of this hormone have not been esti- mated (Beaton, Ryu and McHenry, 1955). Placental secretions have also been asso- ciated with the active anabolic state of the second half of pregnancy, because removal of the fetuses in the rat did not change the anabolic activity, whereas removal of the placentas was followed by a return to nor- mal (Bourdel, 1957). A sharp increase in liver ribose nucleic acid has been observed during late pregnancy in mice and rats and the effect attributed to a placental secretion or to estrogen. Species differences also in- fluence the results because only a modest change in liver RNA was observed in guinea pigs and no change occurred in cats (Camp- bell and Kostcrlitz, 1953; Campbell, Innes and Kosterlitz, 1953a, b). Clinical observations have related both TABLE 12.10 Nutrition and pregnancy in rats (From J. H. Leathern, in Recent Progress in the Endocrinology of Reproduction, Academic Press, Inc., New York, 1959.) Calories/kg. Body Weight Fetuses, Day 20 No. Average weight 18 per cent casein 18 per cent casein 6 per cent casein 0 per cent casein 18 per cent gelatin 18 per cent gelatin 200 100 250 200 200 100 8 6 0 0 0 0 gm. 6.1 3.5 toxemia of pregnancy (Pequignot, 1956) and prematurity to inadequate nutrition (Jeans, Smith and Stearns, 1955). The po- tential role of protein deprivation in the pathogenesis of the toxemia of pregnancy prompted studies in sheep and rats. In sheep nutritionally induced toxemia simulates the spontaneous toxemia (Parry and Taylor, 1956), but only certain aspects of toxemia were observed in the pregnant rat subjected to low protein diets. When rats were fed 5 per cent casein and mated, fluid retention was observed (Shipley, Chudzik, Curtiss and Price, 1953) and pregnancy was com- pleted in only 48 per cent of the animals Curtiss, 1953). Gain in body weight in the adult rat and gain in fetal weight were sub- normal as the result of a low protein feeding during pregnancy. Complete removal of protein from the diet beginning at the time of mating did not prevent implantation but did induce an 86 to 100 per cent embryonic loss. The effect was not related solely to food intake (Nel- son and Evans, 1953) , as we will see in what follows when the relationship between pro- tein deficiency and the supply of estrogen and progesterone is described. Limiting pro- tein deprivation to the first 9 to 10 days of pregnancy will also terminate a pregnancy, but when the protein was removed from the diet during only the last week of pregnancy, the maternal weight decreased without an effect on fetal or placental weight (Camp- bell and Kosterlitz, 1953). As would be an- ticipated, a successful pregnancy requires protein of good nutritional quality and the caloric intake must be adequate. Thus, an 18 per cent gelatin diet failed to maintain pregnancy when 200 calories per kilogram were fed, whereas a similar level of casein was adequate (Table 12.10). However, re- ducing caloric intake to 100 calories despite an otherwise adequate protein ration in- fluenced the number and size of fetuses (Leathern, 1959b). Additional proteins should be studied and related to biochemi- cal changes in pregnancy and to the need for specific amino acids; for example, elimina- tion of methionine or tryptophan from the diet may or may not be followed by resorp- tion (Sims, 1951; Kemeny, Handel, Kertesz and Sos, 1953; Albanese, Randall and Holt, 1943). Excretion of 10 amino acids was in- NUTRITIONAL EFFECTS 691 creased during normal human pregnancy (Miller, Ruttinger and Macey, 1954). That a relationship exists, between the dietary requirements just described to the endocrine substances which participate in the control of pregnancy, is suggested by the fact that the deleterious effects of a protein- free diet on pregnancy in rats have been counteracted by the administration of es- trone and progesterone. Pregnancy was maintained in 30 per cent, 60 to 80 per cent, and 0 per cent of protein-deficient animals by daily dosages of 0.5 /xg., 1 to 3 fig., and 6 jug. estrone, respectively. On the other hand, injection of 4 to 8 mg. progesterone alone maintained pregnancy in 70 per cent of the animals (Nelson and Evans, 1955) , and an injection of 4 mg. progesterone with 0.5 //.g. estrone provided complete replacement therapy (Nelson and Evans, 1954). Food intake did not increase. The results suggest that reproductive failure in the absence of dietary protein was due initially to lack of progesterone and secondarily to estrogen, the estrogen possibly serving as an indirect stimulation for luteotrophin secretion and release. It is well known that hypophysec- tomy or ovariectomy shortly after breeding will terminate a pregnancy and that re- placement therapy requires both ovarian hormones. Thus, the protein-deficient state differs somewhat from the state following hypophysectomy or ovariectomy, but the factors involved are not known. Pregnancy alters nutritional and meta- bolic conditions in such a way that labile protein stores of the liver and other parts of the body are influenced, but similar effects are imposed by a transplanted tumor, es- pecially when it reaches 10 per cent of the body weight. ' Thus, transplantation of a tumor into a pregnant animal would place the fetuses in competition with the tumor for the amino acids of the metabolic pool. Under these circumstances will the pregnancy be maintained? An answer to the question may not yet be given. Nevertheless, Bly, Drevets and Migliarese (1955) observed various de- grees of fetal damage in pregnant rats bear- ing the Walker 256 tumor, and 43 per cent fetal loss was obtained with a small hepa- toma (Paschkis and Cantarow, 1958). Essential fatty acid deficiency, at least in the initial stages, does not interfere with development of the fetuses or parturition in the rat, but the pups may be born dead or they do not survive more than a few days (Kummerow, Pan and Hickman, 1952). A more pronounced deficiency has induced atrophic changes in the decidua, resorption of fetuses, and prolonged gestation. Death of the fetuses appears to be secondary to placental injury. Hormonal involvement, if any, when there is fatty acid deficiency and pregnancy seems not to have been investi- gated. Pregnancy and lactation are major fac- tors influencing vitamin requirements. It is not surprising, therefore, that vitamin de- ficiencies influence the course of a preg- nancy. The subject has recently been re- viewed by Lutwak-jMann (1958). A deficiency of vitamin A does not no- ticeably affect early fetal development, but later in gestation placental degeneration oc- curs with hemorrhage and abortion. When the deficiency is moderate the pregnancy is not interrupted, but the fetuses are damaged (Warkany and Schraffenberger, 1944; Wil- son, Roth and Warkany, 1953; Giroud and Martinet, 1959). In calves and pigs the abnormalities are associated with the eyes and palate (Guilbert, 1942) ; in birds skele- tal abnormalities are seen (Asmundson and Kratzer, 1952). The use of hormones in an effort to counteract the effects seems to have been attempted only in the rabbit where 12.5 mg. progesterone improved reproduc- tion impaired by vitamin A lack (Hays and Kendall, 1956). Vitamin A excess also proves highly detrimental to pregnancy, as resorption and malformations occur. Ad- ministration of excessive vitamin A on days 11 to 13 of pregnancy induced cleft palate in 90 per cent of the embryos (Giroud and Martinet, 1955) . In another experiment the effect of excessive vitamin A was augmented by cortisone (Woollam and Millen, 1957). Vitamin E deficiency has long been known to influence pregnancy in rodents and fetal death appears to precede placental damage and involution of the corpora lutea. Gross observations of the abnormal embryos have been reported (Cheng, Chang and Bairn- son, 1957). Estrogen, progesterone, and lac- togen were not effective in attempts at cor- rective therapy (Ershoff, 1943), but estrone and progesterone markedly reduced the in- 692 PHYSIOLOGY OF GONADS cidence of congenital malformations asso- ciated with vitamin E lack (Cheng, 1959). In the test of a possible converse relation- ship, estradiol-induced abortion in guinea pigs was not prevented by vitamin E (Ingel- man-Sundberg, 1958) . Fat-soluble vitamins incorporated in the diet may be destroyed by oxidation of the unsaturated fatty acids. To stabilize the vitamins, the addition of diphenyl-p-phen- ylenediamine (DPPD) to the diet has proven successful, but recent studies show that DPPD has an adverse effect on re- production and thus its use in rat rations is contraindicated (Draper, Goodyear, Bar- bee and Johnson, 1956). Vitamin-hormone relationships in preg- nancy have been studied with regard to thiamine, pyridoxine, pantothenic acid, and folic acid. Thiamine deficiency induced still- births, subnormal birth weights, resorption of fetuses, and loss of weight in the mother. However, as in the case of protein defi- ciency, pregnancy could be maintained with 0.5 fjLg. estrone and 4 mg. progesterone (Nel- son and Evans, 1955). Estrone alone had some favorable effect on the maintenance of pregnancy in thiamine-deficient animals, but it was less effective in protein-deficient animals. Fetal death and resorptions as well as serum protein and nonprotein nitrogen (NPN) changes similar to those reported for toxemia of pregnancy (Ross and Pike, 1956; Pike and Kirksey, 1959) were induced by a diet deficient in vitamin Be . Adminis- tration of 1 fxg. estrone and 4 mg. progester- one maintained pregnancy in 90 per cent of vitamin Be-deficient rats (Nelson, Lyons and Evans, 1951). However, the pyridoxine- deficient rat required both steroids to re- main pregnant and in this regard resembled the hypophysectomized animal (Nelson, Lyoas and Evans, 1953). Nevertheless, a hypophyseal hormone combination which was adequate for the maintenance of preg- nancy in the fully fed hypophysectomized rat (Lyons, 1951) was only partially suc- cessful when there was a deficiency of pyri- doxine. An ovarian defect is suggested. The folic acid antagonist, 4-amino- pteroylglutamic acid, will rapidly induce the death of early implanted embryos in mice. rats, and man (Thiersch, 1954j . Removal of folic acid from the diet or the addition of x-methyl folic acid will induce malfor- mations when low doses are given and re- sorptions when high doses are given. Fur- thermore, this effect is obtained even when the folic acid deficiency is delayed until day 9 of a rat pregnancy or maintained for only a 36-hour period. A deficiency of pantothenic acid will also induce fetal re- sorption. The vitamin is required for hatch- ing eggs (Gillis, Heuser and Norris, 1942). In animals deficient in folic acid or in pantothenic acid, estrone and progesterone replacement therapy did not prevent fetal loss, suggesting that the hormones cannot act (Nelson and Evans, 1956). In the above mentioned deficiencies replacement of the vitamin is effective. However, vitamins other than those specifically deleted may provide replacement, thus ascorbic acid seems to have a sparing action on calcium pantothenate (Everson, Northrop, Chung and Getty, 1954) . Pregnancy can be interrupted by altering vitamins other than those discussed above, but the hormonal aspects have not been explored. Thus, the lack of choline, ribo- flavin, and Bi2 will induce fetal abnormali- ties and interrupt gestation (Giroud, Levy, Lefebvres and Dupuis, 1952; Dryden, Hart- man and Gary, 1952; Jones, Brown, Rich- ardson and Sinclair, 1955; Newberne and O'Dell, 1958) . Choline lack is detrimental to the placenta (Dubnov, 1958), riboflavin deficiency may impair carbohydrate use (Nelson, Arnrich and Morgan, 1957) and/or induce electrolyte disturbances (Diamant and Guggenheim, 1957) , and Bjo spares cho- line and may be concerned with nucleic acid synthesis (Johnson, 1958). Excessive amounts of Bio are not harmful. It is in- teresting to note that uterine secretions and rabbit blastocyst fluid are rich in vitamin B]2 (Lutwak-Mann, 1956), but its presence in such large amounts has not been ex- plained. An additional substance, lithospermin, ex- tracted from the plant, Lathijrus odoratus, is related to hormone functioning; it is anti- gonadotrophic when eaten by nonpregnant animals and man. The feeding of this sub- stance to prciiiiant rats terminated the preg- NUTRITIONAL EFFECTS 693 nancies about the 17th day. Treatment with estrogen and progesterone was preventive (Walker and Wirtschafter, 1956). It is as- sumed, therefore, that lithospermin inter- fered with the production of these hormones. A repetition of the experiment on a species in which the hypophysis and ovaries are dispensable during much of pregnancy would be of interest. In retrospect it has been found that a deficiency in protein and the vitamins thiamine, pyridoxine, pantothenic acid, and folic acid individually can interrupt a preg- nancy. Furthermore, a combination of es- trone and progesterone which is adequate to maintain pregnancy after hypophysectomy and ovariectomy, is equally effective in pro- tein or thiamine deficiency. This suggests that the basic physiologic alteration is a deprivation of ovarian hormones. However, protein- and thiamine-deficiency states dif- fer from each other as shown by the re- sponse to estrogen alone (thiamine defi- ciency is less responsive), and these states differ from hypophysectomy in which es- trone alone has no effect. A pyridoxine de- ficiency seems to involve both ovary and hypophysis, for neither steroids nor pitui- tary hormones were more than partially successful in maintaining pregnancy in rats. Lastly, pantothenic acid and folic acid deficiencies may not create a steroid defi- ciency. What is involved is not known; many possibilities exist. Pantothenic acid, for example, participates in many chemical reactions. Furthermore, it is known that thiamine is essential for carbohydrate me- tabolism but not for fat metabolism whereas pyridoxine is involved in fat metabolism and in the conversion of tryptophan to nico- tinic acid. It is clear, though, that much ground must be covered before the formula- tion of fruitful hypotheses may be antici- pated. VI. Concluding Remarks The development, composition, and nor- mal functioning of the reproductive system is dependent on adequate nutrition. How- ever, the requirements are many and only gradually are data being acquired which are pertinent to the elucidation of the nutri- tional-gonadal relationship. The demands for nutrient substances is not always the same. During pregnancy and lactation there is a need for supplemental feeding. A similar need exists in birds and in the many cold-blooded vertebrates in which reproduction is seasonal. Atypical en- docrine states create imbalances and a need for nutrient materials which vary, unpre- dictably, we must acknowledge, until the numerous interrelationships have been clari- fied. At many points where determination of cause and effect are possible, an indirect action of dietary factors on reproduction is indicated. No other conclusion seems pos- sible in view of the many instances in which the effect of dietary deficiencies can be counteracted by the administration of a hormone or combination of hormones. The direct action is not immediately apparent; it probably is on the processes by which metabolic homeostasis is maintained, and is in the nature of a lowering of the respon- siveness to the stimuli which normally trig- ger these processes into action. The processes may be those by which pituitary and gon- adal hormones are produced or they may be the mechanisms by which these hormones produce their effects on the genital tracts and on the numerous other tissues on which they are known to act. Because of the many interrelationships, some of which are antagonistic and some supportive, determination of the role of spe- cific dietary substances is not easy. For those who work with laboratory species, the problem is further complicated by the many strain differences. For everyone, the problem is complicated by the many species differences which are the result of an evolu- tion toward carnivorous, herbivorous, or omnivorous diets, to say nothing of the countless specific preferences within each group. Finally, it is something of a paradox in our culture that much of our effort has been de- voted to investigations of the effects of de- ficiencies and undernutrition rather than to the effects of excesses and overnutrition. 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