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Ss BINDING LisTuuL2 1924 


Digitized by the Internet Archive 
in 2009 with funding from 
University of Toronto 


http://www.archive.org/details/publicationsstud07corn 


PUBITCA TIONS 


OF 


Cornell University 
MEDICAL COLLEGE 


oa Ui 


mel 6) el nf S: 


FROM THE 


Department of Anatomy 


© 137 ae 
VOLUME Xt 
1918-19 


NEW YORK .CAL£.T Y. 


iw] 


CONTENTS 


Seing reprints of studies published in 1918-1919. 


FURTHER STUDIES ON THE MODIFICATION OF THE GERM- 
CELLS IN MAMMALS: THE EFFECT OF ALCOHOL ON 
TREATED GUINEA-PIGS AND THEIR DESCENDANTS. 

By Charles R. Stockard and George N. Papanicolaou. 
Jour. of Experimental Zoology, Vol. XXVI, 119-226. 


THE DEVELOPMENT OF THE IDIOSOME IN THE GERM- 
CELLS OF THE MALE GUINEA-PIG. 
By George N. Papanicolaou and Charles R. Stockard. 
Am. Jour. of Anatomy, Vol. XXIV, 37-70. 


THE VAGINAL CLOSURE MEMBRANE, COPULATION, AND 
THE VAGINAL PLUG IN THE GUINEA-PIG, WITH 
FURTHER CONSIDERATIONS OF THE OESTROUS 
RHYTHM. 

By Charles R. Stockard and George N. Papanicolaou. 
Biological Bulletin, Vol. XXXVII, 222-245. 


DEVELOPMENTAL RATE AND THE FORMATION OF EM- 


BRYONIC STRUCTURES. 
By Charles R. Stockard. 


Proc. Soc. of Exp. Biology and Medicine, Vol. XVI, 93-95. 


SYMMETRY REVERSAL AND MIRROR IMAGING IN MON- 
STROUS TROUT AND A COMPARISON WITH SIMILAR 
CONDITIONS IN HUMAN DOUBLE MONSTERS. 

By Charles V. Morrill. 
Anat. Record, Vol. XVI, 265-292. 


CHANGES IN PROTOPLASMIC CONSISTENCY AND THEIR 


RELATION TO CELL DIVISION. 
By Robert Chambers. 


Jour. of General Physiology, Vol. II, 49-69. 


A REPORT OF THE RESULTS OBTAINED FROM THE MICRO- 


DISSECTION OF CERTAIN CELLS. 
By Robert Chambers. 


Trans. Royal Soc. of Canada, Series III, Vol. XII, 41-47. 


A REPORT ON CROSS-FERTILIZATION EXPERIMENTS, 
ASTERIAS BY SOLASTER. 
By Robert Chambers and Bessie Mossop. 


Trans. Royal Soc. of Canada, Section IV, 145-148—1918. 
REGENERATION OF BONE. 
By Albert A. Berg and William Thalhimer. 
Annals of Surgery, March. 1918, 331-347. 


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AUTHOR'S ABSTRACT OF THIS PAPER ISSUED Reprinted from Tue JouRNAL OF EXPERIMENTAL 
BY THE BIBLIOGRAPHIC SERVICE MARCH 2 Zob.oGy, Vol. 26, No. 1, May 1918 


FURTHER STUDIES ON THE MODIFICATION OF THE 
GERM-CELLS IN MAMMALS: THE EFFECT OF 
ALCOHOL ON TREATED GUINEA-PIGS 
AND THEIR DESCENDANTS 


CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 
From the Department of Anatomy, Cornell Medical School, New York City 


NINE FIGURES 


CONTENTS 

Pee nGrodUCbLONNK ¥..dec.e eres esicier ior eet eine ash aster Sera oa aoe se 120 
2. Quality of the experimented and control animals...................... 123 
qecelec tion Olanim als rc em erence eects sect acres sien Peni eae ges 123 
Hoop in DTCSAEN Bietocteyac eset ale ciate ayer le oi eeeeptc a area Mea othhs, Segceiete os stale a o-seci leer 124 
Cyebhe mumberotanimalsrakeoholizedacniss sores eeriecene eee 125 
3. Experimental method and the care of animals......................... 127 
4. The influence of alcohol inhalation on the individual.................. 130 

a. Contrast between the immediate effects of alcohol taken by inha- 
Jationvaridibyestomachs Weer scree cemielan a very nciee eet hae 132 

b. The vigorous condition of the animal after daily inhalation of alco- 
holifior lon gipertOds rf... rh seweravs 2) als fois sede eee ete 133 

5. A general comparison of the progeny from alcoholic lines with those 
FLOM OLIN AINE S He). atch islccas vie aces hocley eine Sais Pocus Sosa we ae ee 142 


6. Absorption of embryos in utero and abortions of parts of litters: methods 
of detecting these processes 
7. A comparison of the qualities in the different generations of the alco- 
holic lines as they become further removed from the generation di- 
TEGUly, treated Mees coat eel ar eee T Ieee ala data oisieio aidine zich 159 
8. A comparison of animals from directly treated fathers and fathers of 
alcoholic stock with animals from directly treated mothers and 
mothers of alcoholic stock and with others from both parents of 
alopholicistook:. oe cx emeripresccrrece serene tel eed tes dinate Mee aaees 168 
9. A comparison of lines from only male ancestors alcoholic with lines 
from only female ancestors alcoholic and with those from both male 
and femalerancestorspalconolicumem cic: sacsincce lace acatia tices 176 
10. Treating males with alcohol for one and two generations compared with 
treating females for one and two generations..... He daHaOree tore 183 
11. The sex-ratio in relation to paternal and maternal alcoholism and to 
the treatment of male and female ancestors with alcohol........... 187 


119 


120 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 
12. The birth weights and rate of growth in the normal and the alcoholic 


13. The records of normal males and females paired successively with nor- 
mal and aleoholic mates; the crucial demonstration of the effects of 


alcoholism onthe offspring.....<.... 2c. 2 ase ela eee ieee 205 
14. The contrasted qualities in the control and the alcoholic series........ 208 
5 General CONSiGeratiONs®.« .<.0.02 x sis.cw-a-oucie here oy heme arte mie ere ee eRe EES eRe 212 
DAGET ACUTE CLEC Gs. accsee ss repecare w\o-e,0:c%k(o/ore%s ele letecorsiattrace ce ann eReneme ce RE LS srt scorn 225 


1. INTRODUCTION 


The present contribution presents the results obtained during 
the sixth and seventh years of an experiment on the modification 
of mammalian germ cells by the treatment of parental generations 
with aleohol. A number of new facts are added to our previous 
findings, and the data now permit a more thorough analysis. 
Treating the results obtained during these two years separately 
may be looked upon as taking a cross-section of the entire experi- 
ment. And when this isolated portion of the investigation is 
compared with the previous studies, it supplies a further most 
important control for the experiment as a whole. 

The earlier reports of this investigation (Stockard, 712, 713, 
and 714; Stockard and Papanicolaou, 716) were made after the 
first two years, three years, and five years of its progress. These 
reports showed, in what seems to us a definite way, that the germ 
cells in either the male or female mammal may be changed or 
affected by a chemical treatment administered to the body of the 
individual. The progeny derived from such chemically treated 
animals showed more or less marked deviations from the normal 
in many definitely measurable qualities, such as their mortality 

-records, structural appearance, nervous reactions, and ability 
to reproduce. The treatment also affected in the male, the crucial 
germ-cell test for mammals, their ability to beget offspring when 
mated with normal females. 

In general it may be stated that the offspring produced when 
treated males were paired with normal females were inferior in 
several respects as compared with other offspring from the same 
normal mothers bred to control males of exactly the same origial 
stock. Further, when the male offspring from treated fathers 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 121 


were mated with normal females, the individuals resulting from 
such matings were as a group decidedly inferior to the young 
produced by normal females when mated with control males. 
This group inferiority was not only present in the grandchildren, 
or F, generation, but also in the F; generation descended from 
aleoholized great-grandparents. 

Fortunately, since these experiments were first reported, sev- 
_eral similar studies by other investigators using the methods here 
employed have been conducted on other mammals and birds. 

Our results have been corroborated, though the response to the 
treatment has in some instances been thought to differ from that 
shown by the guinea-pigs. Useful and suggestive interpretations 
of the results have been advanced, yet certain points of view are 
presented with which we are not always able to completely agree. 
The bearing of these studies on the present results will be dis- 
cussed in a section beyond. 

In particular we are indebted to Pearl (’17) for his recent 
characteristically clear and exact analysis of the influence of 
alcohol fumes on domestic fowls and their progeny. This study 
has suggested to us the importance of a considerable amount of 
data contained in the card-catalogue records of our animals which 
had not been fully valued in the previous discussions of the ex- 
periments. In the present report we have followed several of 
Pearl’s ideas in more completely separating the qualities to be 
contrasted between the alcoholic and control lines. 

As might be expected, various objections have been advanced 
from time to time regarding the cause and explanations of the 
results which we have reported on the effects of aleohol in the 
guinea-pig. In all cases, however, the objections have been 
raised either by persons entirely unfamiliar with these animals 
and their breeding qualities or by others who have not been 
sufficiently interested or careful to read the descriptions of the 
animals and breeding methods used. “It has been suggested on 
certain occasions that the defects and degenerate conditions 
which have been reported in our alcoholic lines were probably 
present in the original stock on which the experiment was con- 
ducted. Such a remark in the face of the experimental control 


122 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


which has been fully described scarcely warrants discussion, yet 
we should like to state in the beginning for the benefit of the 
casual critic who may not wander through the following pages 
the real nature of the original control. 

A group of forty animals, eleven males and twenty-nine fe- 
males, was obtained from a reliable breeder in the early fal of 
1910. These animals were all under one year old-and strong and 
vigorous in appearance; most of the females were pregnant. 
All the females were kept until they had produced a normal 
litter of young. Their production was what would ordinarily be 
obtained from healthy guinea-pigs; all of the young were nor- 
mal in appearance and about 80 per cent of them survived under 
the by no means perfect system of care then employed. 

Three males and six females, after the test matings, were then 
taken for alcohol treatment. The choice was entirely random, 
there being no evident marks of superiority or inferiority in any 
of them as compared with the other animals retained as normal 
control. One of the three males selected for treatment lived to 
be more than seven years old, and the others were all healthy, 
strong animals that lived long and bred vigorously. These 
treated males were mated with alcoholic females and with nor- 
mal females. The same normal females were mated at different 
times with normal males and such offspring were considered 
control. From the beginning of the experiments it may be said 
that the same normal female often serves as part of the experi- 
ment, being mated to alcoholic males and again as the control. 
The same is true of normal males; they are frequently mated 
successively with alcoholic females and normal females. From 
this original stock the normal animals, both males and females, 
have invariably given rise to average normal offspring when 
paired with normal mates, while, on the other hand, the treated 
animals being part of the same breed, have in the quality of their 
offspring shown a decidedly inferior condition even when paired 
with normal mates. 

After the experiment had been in progress for eighteen months, 
in March, 1912, a new stock of animals of an entirely different 
source from the first lot was introduced. Again, after testing 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 123 


their breeding ability by one normal mating, certain of this lot 
were taken for alcohol treatment, and these animals were bred 
both separately and with the original lot. Yet the records of 
the alcoholic and normal individuals were again different 

Finally, in October, 1915, when the experiment was five years 
old, we obtained four new stocks of guinea-pigs from different 
dealers and introduced them into the experiments in various 
ways along with our now pedigreed lines from the old stock. 
The records of these new animals as well as our old lines known 
for three or more generations regarding inbreeding and other 
conditions are to be considered in the present paper. These 
experiments bring out additional facts in the study, and we 
believe they supply an unquestionable control on the previous 
results. In other words, this may be taken as a new study con- 
sidering the conditions of 1,170 guinea-pigs born from various 
alcoholic lines as well as from normal control animals. About 
600 of the animals are born of alcoholic lines with no inbreed- 
ing in any case back through their great-grandparents. About 
300 of them are from alcoholic lines and at the same time some- 
what inbred; these are for all considerations treated separately 
from the straight alcoholic lines. The control animals with 
which the alcoholics are compared are of the same blood lines 
as the alcoholics and are also not inbred. 


2. QUALITY OF THE EXPERIMENTED AND CONTROL ANIMALS 


a. Selection of animals 


As briefly mentioned above, the control and the first treated 
individuals are derived from exactly the same original stock. 
During the progress of the experiment other animals have been 
subjected to the treatment, and these in many cases are of 
known pedigree for several generations in our colony. In all 
cases only vigorous animals are used for the treatment and they 
are invariably tested by being mated at least once before the 
treatment is commenced. This precaution is undoubtedly of 
much importance, equally as important as knowing the blood 
lines, in selecting normal breeders. These test matings are 


“ CHARLES R. STOCKARD AND GEORGE N. PAPA) f 
124 cH RGE N. PAPANICOLAOU 


further strengthened by the fact that the same normal males 
are mated with alcoholic females and with normal females, and 
normal females are mated with alcoholic males and again with 
normal males as a control, ete. In this way the experimental and 
control animals are actually in some cases the same individuals 
and in all eases they are constantly being bred together. There 
is no question that the animals treated with alcohol and the 
control are equally general or random samples of the popula- 
tion. Yet there is a marked contrast between the records of 
their offspring and descendants. 


b. Inbreeding 


The alcoholic lines which we shall analyze in detail in the 
following considerations are practically devoid of inbreeding. 
Almost all of these animals are known in our colony for three or 
more parental generations, and we mean in stating that they are 
not inbred that a given individual in their ancestry never ap- 
pears more than once back through the great-grandparent gen- 
eration. In the first table to be considered the straight alcoholie 
lines may be compared with other lines that are not only alco- 
holic, but also inbred, usually to a slight degree, and it is seen 
that inbreeding in either the alcoholic or the control to a lim- 
ited degree gives no indication of any significantly injurious 
effects. 

In our former report (’16) there were shown to be more in- 
jurious effects in the alcoholic inbred lines than in the non- 
inbred. This difference has now disappeared on account of the 
fact that the animals in the former table were more closely in- 
bred and were earlier generations than the bulk of those in the 
present consideration. The degree of inbreeding in the inbred 
lines is now much reduced as compared with the earlier table, 
and the records have improved. This difference between the 
earlier and the present results indicates that inbreeding in these 
alcoholic lines may be easily carried to a degree which will make 
the injurious effects more marked.. We have avoided even the 
slightest approach to such a degree of inbreeding in the straight 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 125 


aleoholic lines. The pedigrees of a great majority of these 
alcoholic animals could readily be given to cover several genera- 
tions, but it does not seem advisable to enter into this detail, 
since there is no possible chance that any differences which might 
exist between the normal and alcoholic lines are due to different 
degrees of inbreeding among the individuals of the two groups. 
And further, in all the groups it is entirely out of the question 
that any difference between the records of the control and the 
records of the alcoholic may be due to the control having been 
by chance originally good breeders and the alcoholics originally 
bad. The control animals are in almost all cases either sisters, 
brothers, parents or other blood relations of the treated animals. 


c. The number of animals alcoholized 


Recognizing the great variability in the breeding results from 
the different individuals in a group of higher animals, such as 
mammals, it has been deemed entirely essential to make our 
experiment on a considerable number of males and females. 
The mating records of two normal male guinea-pigs are fre- 
quently quite different even though paired with the same fe- 
males. It is also highly probable that different individuals will 
differ in their susceptibilities and responses to the treatment, 
so that the records of two or three males might easily prove con- 
fusing even though all might exhibit some effects of the experi- 
mental treatment. Thus the following twenty-eight males 
have been treated with aleohol, and a number of matings from 
each of these and their descendants have supplied the breeding 
records. The first three are from the original 1910 stock. Nos. 
4 #, 5%, and 6¢, and the remaining twenty-five are animals 
bred and reared in the colony or from the newly introduced 
stocks: Nos. 4307, 45%, 700%, 720, 8007, 81607, 67867, 88727, 


AN 
913.7, 5— 1290, 157A, 168%, 1834, 30207, 3530, 36507, 


AN 
AA Ni —=N 
57491 Sg ey N 889 #7, 1091 7, 11340, 


< ve ——S 


A 
1153 7, 13826 and 1327. 


126 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


In the case of the females an attempt has also been made to 
lessen the error caused by indiv-dual differences in breeding ca- 
pacity and in responses to the treatment by using a number of 
animals. Thirty-four individuals have been treated in all. 
Many of these females were bred for a number of times as con- 
trol before being subjected to the fume treat; ent, after wh ch 
they are placed of course among the alcohol,cs.. Their earlier 
breeding records are therefore part of the control data and their 
subsequent records part of the data included for the alcoho ic 
lines. The same thing is true of a number of the males men- 
tioned above. In none of these cases can it be objected that the 
animals had become too old for normal vigorous breeding while 
being used in the alcoholic lines. We have constantly guar ed 
against breeding the alcoholic animals after there is any question 
as to age affecting their breeding capacities when compared w th 
the normal breeding cycle of these guinea-pigs. The treat- 
ment of the large majority of the animals is begun when they are 
less than one year old, and they have a vigorous breeding span 
of at least four years. The individua! females wh ch have been 
subjected to the aleohol fumes are the following: The first six 
are from the original 1910 stock, Nos.89,92,109,119,129, 
and 34°; the following twenty-eight are animals reared in the 
colony or from the newly introduced stocks: Nos. 559, 57°, 
599, 609, 619, 629 649, 659 669, 889 909, 1179, 


1582, 1619, 65492, 84792, 8659, 9469, 1229, 2009 


ee 


NA na 

2289, 3079, 11399, A7969, 10029, 11059, N——— 
A a: 

nN 


1468 © and 1469 ¢. 

There are no contrasts between the histories and capacities 
of the experimented and control animals that can be fairly ac- 
counted for as due to differences in either their origins, blood 
lines, or relationships. As far as experiment and control with 
biological material may be practically useful, any differences 
which may exist between the records of the alcoholic guinea- 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 127 


pigs and the normal control lines are due to the treatment ad- 
ministered to the alcoholic lines. We further believe that if 
the differences which do exist between the alcoholics and control 
are so slight that the crudest mathematical calculations are ‘n- 
sufficient to indieate their presence, the experiment has then 
produced no d: of biological interest or importance since 
conducted on anu ial material of such complexity as a group of 
mammals. This statement is made with no intention or pre- 
sumption to question the real importance and value of modern 
biometrical methods, but is only what we believe should apply 
to this particular experiment. 


3. EXPERIMENTAL METHOD AND THE CARE OF ANIMALS 


Throughout these experiments alcohol has been administered 
to the guinea-pigs by a method of inhalation which was devised 
in the beginning. The animals to be treated are placed in fume 
tanks fully described and illustrated in an earlier communica- 
tion (Stockard, 712) and absorbent cotton soaked with com- 
mercial 95 per cent ethyl alcohol is placed on the floor of the tank 
beneath a wire screen on which the animals stand. The fumes 
of evaporating alcohol very soon saturate the atmosphere of 
the tanks and the guinea-pigs introduced into this saturated at- 
mosphere are allowed to remain until they show distinct signs 
of intoxication. During the earlier years of the experiment they 
remained for one hour each day in such tanks, but during the 
past twelve months we have increased the treatment to two 
hours per day for the males and three hours for the females. 

This longer treatment is much better in that the animal, of 
course, gets a larger dose and its tissues may become more quickly 
influenced by the treatment. The animals may remain until 
they are completely intoxicated, in which case they are unable 
to walk, and therefore lie in a typical drunken stupor, or they 
may be affected to such an extent that they attempt to walk 
and in so doing stagger and fall in a manner characteristic of 
the drunken state. The amount of treatment here employed, 
however, does not produce complete intoxication. 

It would be perfectly possible with an elaborate system of 
measurements to determine exactly the quantity of alcohol {umes 


128 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


ach individual receives per day, per month, or per year, but, 
as we have pointed out before, such knowledge would be of no 
advantage either to us or to others in estimating the results of 
these experiments. No two individuals would be affected to 
exactly the same degree by the same dose, and as is the case 
with man the later influences of the treatment no doubt differ 
in different individuals. There is also no particular interest 
here in the amount of aleohol used, since our primary problem is 
whether or not an active chemical substance may be given in 
sufficient amounts to the parent mammal to produce effects upon 
its offspring or descendants by modifying its germ-cells, or in 
the case of the pregnant female by acting through the mother on 
the developing embryo. 

We have thus employed, as stated in our previous reports, a 
simple physiological index of the amount of treatment, giving 
enough each day to perceptibly influence or intoxicate the ani- 
mals, but not enough to produce a complete drunken stupor. 

Animals may remain for very long times in these treatment 
tanks when alcohol fumes are not present without in any way 
suffering for want of breathing space. This method has many 
advantages so far as the general health of the individual animal 
is concerned over drinking alcohol into the stomach, as will be 
discussed in the following section. 

The only object in choosing aleohol as the treating agent is on 
account of the fact that considerable knowledge exists as to its 
physiological actions on certain animal tissues and it is known to 
be an active organic substance that might produce effects. It 
had further been used by one of us (Stockard, ’10) in producing 
various developmental abnormalities in fish embryos which could 
be treated directly with diluted alcohol, and the general nature 
of the effects on these embryos had been studied. A final advan- 
tage in using alcohol in such experiments is the ease with which 
it may be administered to the animals by the inhalation method 
which we have described. 

Caging and care of animals. All of the guinea-pigs,:both the 
experimental and the control animals, are kept in the same type 
wooden cagés. These are group cages, each containing twenty 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 129 


compartments one foot high by one foot wide by two feet long. 
Each compartment is sufficient to fully accommodate one ‘emale 
with her litter of young or three adult animals. In all of the 
cages some of the compartments are occupied by the alcoholic 
animals and others by the control so that the cage accommoda- 
tions for the two classes are identical. The cages are thoroughly 
cleaned, the floor sprinkled with sawdust and fresh hay put in 
daily. In addition to the hay, which is eaten with relish, the 
animals are fed every day with fresh carrots and several times 
per week oats are given with occasional cabbage or kale. It is 
also important for their perfect health, though not necessary for 
their existence, that guinea-pigs be given fresh water every day 
during the warmer months and several times per week during 
the winter. This is frequently neglected in keeping these ani- 
mals since it is commonly thought that they get a sufficient 
amount of water from the green foods. At the present stage of 
this experiment, along with several other problems now being 
studied, a stock of over 500 animals is constantly kept on hand. 
One reliable keeper devotes his entire time to cleaning the cages 
and feeding. He in no ease discriminates in his treatment of 
different animals and from the cage numbers is unable to know 
all of the alcoholic line animals or the controls. 

From the beginning of this experiment, in making the matings 
a male is placed in a compartment with one female during her 
heat period (Stockard and Papanicolaou, 717); in this way there 
is no opportunity for preferential or choice matings. A male 
might discriminate in his behavior between an alcoholic and a 
normal female if in a compartment with the two, as Pearl be- 
lieves his roosters have done when placed in a pen with both 
normal and alcoholic hens. After the male has remained in the 
pen for one month, the female is carefully examined and at this 
time with some practice the investigator may feel the small 
embryos in the horns of the uterus. The male is removed and 
the female remains alone in the compartment. A list of all preg- 
nant animals, both alcoholic and control, is kept and their com- 
partments are examined both morning and late afternoon of each 
day in order to detect an abortion should it occur, since the 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY. VOL. 26, No. 1 


130 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


female may devour the early aborted young. In addition to 
this, each pregnant female is reéxamined once or twice and the 
number of fetuses in the right and left horns of the uterus re- 
corded each time on her catalogue record. 

By this method it has been found that a number of females 
may often absorb their embryos, either one or all, and so give 
birth to a smaller litter than originally began development or to 
none at all. The absorption of individual embryos seems so far 
as we have detected not to interfere with the, development of 
the remaining ones. These examinations of pregnant females 
have been repeatedly controlled by opening the animals and ob- 
serving the contents of the uterus, and the examinations in all 
cases have been very accurate. This thorough watch over the 
females has furnished us much more exact data as to prenatal 
deaths, early absorptions, etc., than were contained in our former 
reports. 

The entire care of the animals has been much improved during 
the past two years. Our records for monsters and other weak- 
ened conditions are, therefore, somewhat reduced; yet the 
same marked contrast between alcoholic and control is present 
even though the weakened alcoholic lines have no doubt profited 
more by the improved methods of care and feeding than have the 
healthier controls. The defects are also the same in type as 
those formerly observed, though not so marked in degree. 


4. THE INFLUENCE OF ALCOHOL INHALATION ON THE INDIVIDUAL 


The immediate effects on guinea-pigs of inhaling alcohol are 
somewhat similar to those observed after drinking it. As stated 
above, the animals after some time become unable to walk with- 
out staggering as a result of loss of muscular coérdination and 
finally reach, with a long treatment, a state of complete alco- 
holie stupor. 

The presence of alcohol in the blood of the guinea-pig after 
the inhalation treatment is readily detected by even simple 
chemical tests, as we have frequently pointed out. Other in- 
vestigators also find that alcohol is easily introduced into the 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 131 


general system of birds and several mammals by this method. 
Pearl (’16 b) definitely recognizes the fact that alcohol is readily 
taken into the system by the inhalation method, but makes the 
following statement regarding the effect: ‘It is true that it is prac- 
tically impossible to induce by the inhalation method in animals 
habituated to alcohol that state of muscular incoérdination which 
is usually, but by no means always, the most striking objective 
symptom of the condition of being drunk.” Our observations 
on guinea-pigs show them to respond very differently in this re- 
spect from the fowls used by Pearl. In the case of guinea-pigs 
habituated to alcohol, it is very easy by the inhalation method 
to induce a state of muscular incoérdination due to the drunken 
state and finally a complete anaesthesia, the muscles being en- 
tirely relaxed and the animal unable to move. It may be that 
fowls are peculiar in their reaction to alcohol and it may also be 
extremely difficult to administer to them a highly effective dose 
without fatal results. Such an idea is suggested by the fact that 
Pearl does not get the gross symptoms of intoxication by leav- 
ing his fowls in the tanks for one hour, yet they ‘ accumulate a 
fatally toxic dose of aleohol by staying in the same tank under 
the same conditions for from twenty minutes to half an hour 
longer.”’ Guinea-pigs do not at all react in this manner after 
an hour or two in the tank they may show signs of intoxication 
by becoming groggy, with their muscles generally relaxed so that 
when lifted their bodies are almost entirely limp. Yet they 
have not consumed anything,near the fatal dose, since they may 
remain in the same tank under the same conditions for even two 
or three hours longer before becoming completely intoxicated 
so as to be unable to move; and in order to inhale a fatal dose 
they must remain still longer, at least six or seven hours 

We have treated only one fowl, a white leghorn cock, in our 
tanks. This bird responded much as the guinea-pigs do show- 
ing decided muscular incoérdination, staggering and frequently 
almost falling as it walked. He was also able to withstand a 
long treatment and never, though treated several times, did he 
show any tendency to suddenly accumulate a fatally toxic dose 
as Pearl found his fowls to do. 


132 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


a. Contrast between the immediate effects of alcohol taken by 
inhalation and by stomach 


An important point to keep in mind when considering these 
animals intoxicated by the inhalation method is that on being 
removed from the tanks they use up the alcohol in their systems 
very rapidly and also begin to throw off alcohol by respiration. 
The intoxication is, therefore, of short duration so that the 
animal may be fairly well recovered within half an hour or perhaps 
only afew minutes, depending upon the amount of the treatment. 
In other words, this is an acute short intoxication closely com- 
parable to an ether anaesthesia from which the animal readily 
recovers when the fumes are no longer inhaled, but which during 
the inhalation may give a complete intoxication. On the other 
hand, a drunken condition resulting from taking alcohol into the 
stomach is of much greater duration since the gradual absorption 
of the alcohol continues for a longer time before the system 
begins to burn it up or throw it off to such a degree that the 
amount present begins to be continuously reduced, permitting 
the animal to slowly recover from the drunken state. A guinea- 
pig receiving a dose of about 25 ee. of 15 per cent alcohol into 
its stomach will be decidedly intoxicated within fifteen or twenty 
minutes, and the extent of intoxication will increase until the 
animal becomes unable to walk or stand and lies in a drunken 
stupor. Such a condition may persist for six or seven hours or 
longer, and the bedy temperature may be lowered from one to 
even four degrees Fahrenheit. 

It seems to us, therefore, that the chief difference between 
inhaling alcohol and drinking it into the stomach is that in the 
first case the action of the substance on the animal system is of 
shorter duration, lasting but little longer than the length of the 
sojourn in the fume tanks—a short acute effect—while alcohol 
in the stomach is gradually and continuously absorbed for a 
considerable length of time so that the animal’s tissues are acted 
upon for hours after receiving the dose. Another very serious 
phase of the stomach alcohol, aside from the typical intoxication 
effects, is its tendency to derange the animal’s powers of diges- 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 133 


tion and thus to cause very injurious results. The inhalation 
method is accompanied by no such complications. 

We have now considerable data bearing on this problem and 
are conducting an experiment to determine the quality of the 
effects on the animal body and the progeny produced when 
dilute aleohol is taken into the stomach of guinea-pigs for long 
periods of time. The results of this study are to be compared 
with the data from the fume-treated animals. 


b. The vigorous condition of the animal after daily inhalation of 
alcohol for long periods 


A number of the guinea-pigs have now been treated with alco- 
hol fumes almost to a state of intoxication six days per week for 
from five to six years. Few guinea-pigs in captivity live so long 
atime. ‘There were two males treated for over six years, one of 
which lived to be more than seven years old. So far as we know, 
this is the longest life reported for a guinea-pig. The treat- 
ment was continued with these very old animals but they were 
not used for breeding. In no case when the treatment was be- 
gun on an animal over three months old could any injurious 
effects on its general welfare or length of life be discovered. We 
have called attention to these facts in our previous publications. 

There are certain direct injuries resulting from the inhalation 
of ethyl-aleohol fumes during the early stages of the treatment. 
The mucosa of the respiratory tract is considerably irritated 
during the first few months and secretes freely while the ani- 
mals are in the tanks, causing a watery flow from the nostrils 
and mouth. The membranes become more resistant as the 
treatment goes on and later little effect can be noticed. This 
irritation has never given rise to any noticeable inconvenience 
to the animals. The surface of the eye is also greatly irritated 
during the first few months, causing an abundant secretion from 
the lachrymal glands while in the fume tanks, and finally re- 
sulting in many instances in an opacity of the cornea. In some 
cases this opacity disappears after a few weeks and the animal 
is again able to see, yet some of the animals treated for several 
years have remained entirely blind, 


134 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


A number of the treated animals have died and many others 
have been killed at various times during the progress of the ex- 
periment. Their organs and tissues have been carefully exam- 
ined at autopsy and later studied microscopically. All tissues 
have appeared practically normal and none of the various well- 
recognized pathological conditions occurring in human alcoholism 
have been discovered. ‘Tissues from animals treated as long as 
three years have been carefully studied, and the heart, stomach, 
liver, lungs, kidney, and other organs present no noticeable con- 
ditions that might not be found in normal individuals. Aleco- 
holized animals are usually fat, but no fatty accumulation has 
been noted in the parenchyma of any organ. 

Several males and females have been semicastrated during 
the experiment, and the ovaries and testes have been found to be 
in a generally healthy condition. It has seemed, however, that 
the ovaries of treated animals as well as all animals of the alco- 
holic lines show an unusual tendency to become cystic as com- 
pared with the ovaries of normal individuals. We have not, 
however, made sufficient comparisons to give the foregoing 
statement any greater weight than a mere supposition. 

The general condition of all animals under the fume treatment 
is particularly good, and, as stated above, they continue to grow 
if the treatment is begun on individuals before they have attained 
full size, and all become fat and vigorous, taking plenty of food, 
living long, and behaving in a typically normal way. 

The accompanying illustrations of five treated animals photo- 
graphed along with control individuals show their perfectly 
normal appearance. In figure 1 is seen two male guinea-pigs 
and from the photograph as well as in life it would be impossible 
for any one to detect signs of physical inferiority on the part of 
one or the other. Yet the animal on the right, No. 8037, was 
four days less than 5 years old when the photograph was made 
and had inhaled alcohol over one hour per day, a sufficient dose 
to give signs of intoxication, for six days per week, during four 
years, two months and five days. During the last seven months 
he had inhaled alcohol fumes two hours per day. He is per+ 
fectly well and alert, as the photograph clearly shows. His 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 135 


companion on the left is a normal animal, No. 150, being 4 
years and 3 months old when photographed. The sober exist- 
ence of this male has not given him any advantage in appear- 
ance over the old alcoholic; both are very good males, each 
weighing almost 900 grams when photographed. This is well 
above the weight of the ordinary adult guinea-pig. 

Figures 2 and 3 show again on the left the same normal animal, 
No. 1507, in order that the reader may obtain a more definite 
impression of the uniformly good condition of the three aleo- 
holic males. The alcoholic male No. 72 on the right in figure 
2 was 5 years, | month and 10 days old when photographed, 


Tig. 1 The animal on the left is a normal male, No. 150, over four years 
old. The one on the right, No. 80 o’, is almost five years old and had been 
treated with the fumes of alcohol six times per week for four years and two 
months, yet is seen to be in a vigorous condition. 


weighing over 900 grams. He had been treated with alcohol 
fumes one hour per day until the last seven months, when he 
was treated two hours per day for six days per week. The 
entire duration of his treatment when photographed was four 
years, two months and five days. 

Figure 3 shows on the right alcoholic male No. 70. This 
animal was 5 years, 1 month and 11 days old when photo- 
graphed, and weighed 885 grams when 5 years old. He had re- 
ceived the same amount of alcohol treatment as the other two. 
The three were bred and reared in our colony and are above 
the average male guinea-pig in size and vigor. They have been 
good breeders as young normal specimens, as well as during 


136 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


their alcoholic careers, but there has been a decided difference 
in the quality of their offspring during the two periods. 

Figures 4 and 5 show two female alcoholics photographed along 
with the same black male No. 116. In figure 4 the alcoholic 
is an albino, No. 659. She was introduced into the experi- 
ment with the second stock from a new source in March, 1912. 
This female had been treated with alcohol fumes for two years, 
seven months and seventeen days when photographed. During 
the first two years of the treatment she inhaled one hour per day 
for six days per week and during the remaining seven months 
was treated for three hours per day, until fairly well intoxi- 
eated each time. The normal male No. 116 was 4 years, 8} 


Fig..2 The animal on the left is the same control individual, No. 150 &. 
The one on the right is an aleoholie male, No. 72, which was more than five years 
old and had been treated with alcohol fumes for four years and two months. 


months old when photographed. The female No. 65 gave r or- 
mal young before her treatment began, but now produces off- 
spring with very poor records. 

The female No. 158 is shown on the left in figure 5. This 
animal was produced in our colony from normal parentage and 
was 4 years and 3 months old when photographed. She had 
been treated for fourteen months one hour per day and for 
three hours per day during the last seven months. She is a 
large vigorous female. These photographs illustrate to some 
extent the fact that the treated animals themselves are little 
changed or injured so far as their normal appearance goes, and 
should there be inferior qualities in their offspring these cannot 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 137 


be attributed to a condition of general depression in the parents, 
but more clearly to a peculiar action of the strange chemica ma- 
terial in the blood upon the glands of reproduction or the germ 
cells of the males and females. 

In his study of the influence of alcohol inhalation on the do- 
mestic fowl, Pearl has found the treated individuals to respond 
in a way closely similar to our treated guinea-pigs. He has 
fortunately reported his results in much more thorough detail 
than we, yet the facts contained are practically the same for 
the two groups of animals. The mortality records of treated 
fowls show an advantage over similar records from untreated 


Fig. 3 Two male guinea-pigs. One on the left the normal animal, No. 150, 
more than four years old. On the right, No. 70 o, more than five years old and 
had been treated with alcohol fumes for four years and two months. 


control. Our card catalogue contains the record of every death 
that has occurred among the guinea-pigs since the beginning of 
the experiments, and we may state in a general way that the 
mortality statistics for the treated animals is certainly as good 
and perhaps slightly better than those of the control. 

Pearl has very naturally considered these findings in connection 
with the ‘‘widespread popular opinion that life-insurance statis- 
tics have ‘proved’ that even the most moderate use of alcohol 
definitely and measurably shortens human life.’ On careful 
investigation of the statistics Pearl finds them to be entirely un- 
convincing and to be based on biological evidence insufficient to 
prove anything. This is exactly in line with our own experi- 


138 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


ence in studying the literature of any phase of human alcohol- 
ism. We have studied very thoroughly the literature relating 
to the influence of alcoholism in men and women on their prog- 
eny and, including the study of Elderton and Pearson, find it 
to suffer from the defects which Pearl points out in the longevity 
studies. Some of these contributions we shall discuss beyond, 
but none give any exact statement as to the amount of alcohol 
consumed or the length of time during which it had been con- 
sumed or any definite information as to other conditions or the 
general behavior of the individuals considered. The data are 
usually collected by persons entirely untrained and incapable of 


5 


Fig. 4 On the left normal male No. 116, almost five years old, and on the 
right an aleoholic female, No. 65, more than five years old that had been 
treated with alcohol fumes for about two and one-half years. 


accumulating biological evidence. These extremely inexact 
records are often subjected to very careful and exact mathe- 
matical analysis which tends to give a scientific aspect to the 
consideration, but in no way improves the quality of the incor- 
rect data used. Unfortunately, this renders it difficult to make 
comparisons between the responses of human alcoholics and 
those of selected animals used in well-regulated experiments. 
Yet aside from the above, even should the data relating to the 
influence of aleohol on human longevity justify a comparison 
with experimental results, we feel that such a comparison could 
not properly be made with either Pearl’s observations on the 
effect of alcohol on the mortality record of fowls or ours on the 
life record of alcoholized guinea-pigs, since in both experiments 


« 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 139 


the animals have been treated by inhalation of aleohol fumes, while 
human alcoholics have taken the substance into the stomach. 
The difference between the effects on the treated individual 
of the two methods of administering alcohol cannot be too 
strongly urged. By the inhalation method the individual ex- 
periences only the stimulating or with further dosage the intoxi- 
cating and anaesthetizing effects of alcohol. As far as we have 
detected there are no injurious secondary effects on the indi- 
vidual’s welfare resulting from habitual inhalation of ethyl- 
aleohol fumes. The results are very different, however, when 
the guinea-pigs drink daily doses of 15 per cent ethyl alcohol. 


Fig.5 Thesame male, No. 116, is shown on the right and an alcoholic female, 
No. 158, is on the left. She was more than four years old and had been treated 
with alcohol for over two and one-half years, yet she is in no way injured in 
appearance. 


Only a few animals and a short time are sufficient to demon- 
strate the fact. A number of animals were given alcohol into the 
stomach at the beginning of these experiments and their diges- 
tion and metabolism were so deranged by the treatment that 
we were forced to devise and adopt the inhalation method as a 
more likely means of conducting an experiment of long duration. 

It has been shown by us for guinea-pigs, and Pearl has dem- 
onstrated with fowls, the prosperous manner in which animals 
withstand the inhalation of aleohol vapor. 

We may now give briefly the effects on three guinea-pigs of 
drinking daily doses of alcohol for only three weeks. The ani- 
mals, Nos. 173 2, 10987, and 1184.7, at the beginning of the 


140 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


experiment weighed respectively, 822, 635, and 527 grams, the 
female being old and the two males young growing specimens. 

They were each given about 20 ee. of 15 per cent ethyl aleo- 
hol in tap water daily, except that once each week they were 
given almost 30 ce., which was a completely intoxicating dose. 
The 20-ce. dose causes all of them to be groggy for a few hours 
after drinking it; the effect increases for an hour or so and then 
eradually wears off. There is only slight if any change in rectal 
temperature. The animals seem fully recovered on the follow- 
ing day and have a normal appetite, but do not eat so ravenously 
as do untreated individuals. 

When 30 ce. of 15 per cent alcohol is given in three 10-ce. 
doses at fifteen-minute intervals the animal is badly intoxicated 
and unable to walk within fifteen or twenty minutes after the 
last dose. The hind legs are particularly uncertain, the animal 
often tumbling over almost on its back, kicking frantically and 
having great difficulty in righting itself. Should its mouth come 
in contact with food the guinea-pig will chew in a peculiar man- 
ner, seeming in all reactions to be typically drunk. After one 
and a half or two hours the animal lies on its side with its trunk 
muscles often undergoing spasmodic contractions several times 
per minute, if taken up or made to move it struggles and falls 
panting in the drunken condition. By this time the body tem- 
perature may have fallen as much as 2 degrees below the pre- 
treatment record. After three hours it is still unable to stand 
or walk and is breathing heavily with a temperature as much as 
25 degrees Fahrenheit below normal. After four hours the con- 
dition is about the same and so for several hours longer until it 
gradually begins to recover and by the following morning it is 
fully recovered, but shows in its appearance the effects of the 
experience of the previous day. 

When animals are given five partial and one complete intoxi- 
cation by stomach alcohol per week they begin after a few days 
to regurgitate some of the stomach contents on receiving the 
first swallow or so of alcohol, but after this they take the dose 
without further disturbance, though they resist taking it more 
and more each time. Their desire for food is somewhat reduced 
as the treatment is continued. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 141 


After the first week No. 173, the old female that should have 
weighed the same or gained in weight under normal conditions, 
had lost 50 grams, or 6 per cent of her total weight. The two 
young males should have gained, No. 1098 gained 17 grams, only 
2.6 per cent of his weight, while No. 1184 lost 4 grams or prac- 
tically stood still. Their weight records for the indicated inter- 
vals are as follows: 


17392 | 10989 11847 

grams Prarie grams 
Nt re 822 635 527 
lh eee 772 652 523 
meter ee 740 656 477 
5 eee eee 759 659 469 
tone 2D. . cee ee 735 637 483 
inne 1 oe re 775 621 475 


The alcohol was taken from May 7 to 28, and during that 
time the first guinea-pig lost 63 grams, or 7.6 per cent of its 
original weight. Of the two young males one gained 24 grams, 
or 3.7 per cent of his weight, while the other lost 58 grams, or 
11 per cent of his original weight. During the next two weeks 
after the treatment stopped the male that had gained 24 grams 
lost 38 grams, so that at this time each animal weighed less than 
when it began to take alcohol. 

This may have been a rather strong dose, but allowing for that, 
it was readily recognized that these animals were suffering from 
the treatment, while other guinea-pigs inhaling alcohol for three 
hours per day until groggy showed no injured appearance. Ani- 
mals taking alcohol into the stomach suffer mainly on account of 
the injurious effects on their digestion. Alcohol acts on the 
gastric mucosa in such a way that the individual is placed at a 
disadvantage in handling its food and the ill effects observed are 
more largely due to this derangement of digestion than to the 
toxic action of alcohol on the animal system. Alcohol in the 
stomach makes the case complex, while we believe that inhaling 
alcohol gives effects simply due to the chemical action of alcohol 
itself on the tissues. For these reasons we do not believe that 
comparisons are easily made between the conditions of animals 


142 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


that have inhaled aleohol fumes and the condrtions of other ani- 
mals that have taken alcohol into the stomach, since the latter 
individuals may be reacting more to a deranged digestion than 
to aleoholic intoxication. Therefore, there is objection to mak- 
ing comparisons between the mortality records of animals 
treated with aleohol by the inhalation method and the reports 
on the effect of aleoholism in man. 

Yet, on the other hand, it may be possible that the influence 
of aleohol on the germ cells of an anima’ is the same whether 
the aleohol reaches the reproductive glands by being inhaled 
into the lungs or swallowed into the stomach. Such a position 
is not inconsistent with the discussion above if we take into ac- 
count the possible, though unknown, effects of the deranged 
metabolism of the parent on the germ cells. 


5. A GENERAL COMPARISON OF THE PROGENY FROM ALCOHOLIC 
LINES WITH THOSE FROM NORMAL LINES 


The consideration above has brought out the fact that the in- 
halation of aleohol fumes sufficient to produce partial intoxica- 
tion six times per week for long periods does not cause any easily 
recognized disadvantages in the general bodily condition or 
powers of existence of guinea-pigs.  Pearl’s experiments demon- 
strate the same fact in connection with the domestic fowl. This 
is, 0 course, leaving out of account the irritating effects of the 
fumes on the surface of the eye which may result in bl ndness, 
although even this is no handicap to either feeding or epro- 
duction under cage conditions. I’, then, the genera! body 
tissues are not sufficiently injured to cause an easily noticeable 
change in their powers of function, why should the germ cells 
be particularly susceptible to the treatment? The germ cells 
within the body of a mammal are undifferentiated generalized 
cells with no known function except to exist and await their 
time to develop. The soma or body, in respect to the germ 
cells, is simply a culture medium in which they live. The nour- 
ishment necessary for their existence is delivered to them by 
the body fluids. Any strange chemical substance which may find 
its way into the body fluids will reach the germ cells, and should 
this substance be sufficiently active and injurious in its effects 


€ 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 143 


the germ cells may be so modified as to render them incapable 
of normal development. This might easily occur without 
differentiated somatic tissues being sufficiently damaged to 
greatly impair their usual functions. In other cases, and prob- 
ably as a rule, the somatic tissues are also injured by any offen- 
sive substance present in sufficient quantities to modify the 
germ cells, and there are many reasons for believing this to be 
the result in several chronic human infections. 

One must not infer from these statements that the germ cells 
are readily injured by poisons taken into the system; indeed, 
they seem on the contrary to be protected to a remarkable degree 
against such effects, and for this reason it is difficult to obtain a 
substance which may be used in experimental studies on the 
modification of the germ cells. 

Should the germ cells be modified through the action of any 
substance, the point of particular importance is that all cells 
arising from such a modified germ will be similarly modified, 
since they are merely products of its division, and thus the 
soma and germ cells of the resulting individual will deviate 
from the normal in proportion to the degree of the primary modi- 
fication of the cells from which it arose. Provided the change 
is one of such a nature that the cell or its parts are unable to 
recuperate, for example, if their specific chemical or physical 
make up be altered, then not only will the generation resulting 
from the originally modified germ cells be affected, but all future 
generations arising from this modified germ plasm will likewise 
be affected. 

It seems also highly probable that should such results occur, 
the modifications to be observed in the somatic generations will 
be of a generalized nature affecting the organism in various ways 
so as to render its development less vigorous, its chance of sur- 
vival less certain, and its ability to behave in a normal fashion 
more or less hampered. In certain cases the animal might 
really show no evident signs of its altered character. It seems to 
us, on the other hand, that only through the very rarest chance, 
one in possibly thousands, would any of the small number of 
definite characters under observation happen to be modified by 
their response to the treatment. The inheritance of coat-color, 


144 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


for instance, may not be affected, although the germ plasm might 
be so seriously altered as to give rise to the most extremely ab- 
normal individuals. ‘The same would apply to the very few other 
characters in mammals the inheritance of which have been 
studied from the Mendelian standpoint. 

Finally, then, the fact that the soma seems little injured by 
the alcoholic inhalations is in no way an index of what may be 
expected from the development of the germ cells of guinea-pigs 
which have been under habitual treatment. 

Arlitt and Wells have very recently reported that the admin- 
istration of aleohol in the food of male white rats for two or 
more months results almost constantly in the appearance of 
marked degenerative alterations in the testicles although other 
organs were apparently uninjured. They find that these changes 
affect the steps of spermatogenesis in inverse order to their 
occurrence, so that for some time before sterility and complete 
aspermia result, the animal is producing spermatozoa with all 
possible degrees of abnormality. The probable relation of such 
phenomena to the production of defective offspring is obvious. 

A general survey of the progeny from the normal and alcoholic 
lines as a whole will first be undertaken and is based on the data 
presented in table 1. In this table the animals are arranged in 
four groups, the first column containing the records of those 
produced by normal control matings without inbreeding, the 
third column records of normal animals somewhat inbred, while 
the second column gives similar records for animals produced in 
the alcoholic lines without inbreeding, and the fourth-column 
animals are not only alcoholic, but also somewhat inbred. The 
table contains in all records of 1170 animals, from our catalogue 
numbers 613 to 1909 except 126 animals that could not properly 
be included such as 39 new stock adults, 22 killed for different 
purposes during early embryonic life, 31 derived from mothers 
with only one ovary, and others too heterogeneous in origin, as 
those from ancestors treated during pregnancy, ete., to be cer- 
tainly placed. They represent, as stated above, the animals 
produced during the sixth and seventh years of the experi- 
ment and none from the earlier years. 

The figures of the first horizontal space may be used to indicate 


145 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 


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THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, NO. 1 


146 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 

the productivity of the different lines. The numbers | to 5 in- 
dicate the number of young, one, two, three, four, or five 
produced by a female in a single litter. Litters of five indi- 
viduals are the largest that have occurred from this strain of 
guinea-pigs. The average number of young in a litter from the 
normal lines is 2.77, and of the 233 animals included in this 
column 24.03 per cent of them were born in litters of one or 
two young. About 39 per cent were born in litters of three, 
while 37.33 per cent of the animals were members of large litters 
of four and five individuals. There were only a few normal in- 
bred animals, as shown in the third column, but their general 
occurrence in the different-size litters was about as in the straight 
normal lines, half of the animals were born in litters of three, 
and almost 30 per cent in larger litters, and only about 20 per 
cent in litters smaller than three. The average litter happens 
to be in the small number of inbred animals a little higher than 
in non-inbred_ stock. 

The arrangement of the young in large and small litters in the 
aleoholie and alcoholic inbred lines is almost exactly the re- 
verse of what we have just seen for the normal. Again, a little 
less than half of the animals occur in litters of three. But over 
30 per cent of the individuals are from litters of only one or two, 
while about 20 per cent are born in litters of four or five. Stated 
in other words, in the normal lines one and one-half times as 
many individuals are born in litters of four or five as in litters of 
one or two, while in the alcoholic lines one and one-half times as 
many are born in litters of one or two as in litters of four or five. 

The explanation of this, we believe, is as follows: About half 
of the pregnancies in this stock of guinea-pigs should result in 
litters of three, as is found to be the ease in all of the lines of 
table 1. All litters of less than three young are due in the first 
place to a low productivity on the part of the female as is prob- 
ably indicated by the production of more than one-fifth of the 
normal young in such litters. In the seeond place, small lt- 
ters are frequently due, particularly in the alcoholic lines, to the 
death and absorption in utero or early abortion of one or more 
members of an originally large litter. The absorption in utero 
of such embryos, often of rather large size, may occur in a nor- 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 147 


mal guinea-pig, yet such a phenomenon is not very common, 
although in the alcoholic lines it is frequently observed. We 
shall consider this process below, the only point of interest here 
being its effect on the size of the litter. 

The exactly reversed percentages of individuals born in large 
and small litters in the normal and alcoholic lines, as shown by 
the table, may indicate that one-third of the animals in alcoholic 
lines that are born in litters of one or two were originally in litters 
of three, four or five. For example, the normal lines have in all 
well over 12 per cent more animals born in litters of four or five 
than in litters of one or two, and the alcoholic lines have over 
12 per cent more in litters of one or two than in litters of four 
or five, and this 12 per cent_probably has been thrown from the 
larger into the smaller litters on account of early abortions and 
absorptions which occur in the former. The too frequent oc- 
currence of small litters is undoubtedly indicative of not alone 
an actually low productivity, but a very early prenatal mortality. 

Another occurrence also partly due to an early, prenatal mor- 
tality is the failure of a mating to produce a result. No doubt 
in rare cases fertile guinea-pigs may be mated during the heat 
period of the female, as these have been, without a following 
conception. In the normal lines four out of eighty-eight matings, 
or 4.54 per cent, failed, giving negative results, while in the 
alcoholic lines three times as many matings failed, and very 
probably this excess represents those cases in which not only a 
part of the litter is lost through an early prenatal mortality, 
but the entire litter is destroyed. Of course, some cases of 
actually infertile matings are also represented. 

By this ‘early prenatal mortality’ is meant the absorption or 
loss of an embryo before it is of sufficient size to be detected on 
carefully feeling the uterus through the body wall of the mother. 
With experience an embryo eight or ten days old may be de- 
tected by an external examination of the uterus. Through our 
routine examination of the females after being with the males 
for one month, any embryo lost after this time will have been 
discovered and is definitely recorded in the third horizontal space 
of the table. If absorption or early abortion of one or more 
embryos in a litter may actually be observed to occur after as 


148 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


much as ten days of development, there must certainly be a pre- 
natal mortality of some extent previous to this time. Experi- 
ments with the eggs of lower forms which develop outside of the | 
mother, permitting direct observation, speak for the great pre- 
ponderance of an early embryonic mortality, many such eggs 
dying during the cleavage and gastrular stages when subjected 
to even slightly unfavorable conditions. We have some direct 
evidence on ‘early prenatal mortality’ in female guinea-pigs 
which have been examined by operation after repeated ‘mating 
failures.’ The ovaries of some such animals contain corpora 
lutea of pregnancy indicating that an embryo had been present 
shortly before the examination. 

Pearl records that the eggs from aleoholized fowls are to a 
high degree infertile. This he believes is due to many of the 
germ cells as such having been killed by the treatment. By 
infertile, Pearl means, of course, that no fertilization or zygote 
formation took place, yet it is extremely difficult in all cases to 
detect whether the early stages of development may not have 
occurred and been followed by death and degeneration. The 
death may have occurred during the cleavage or gastrular stages 
while the egg was yet in the uterus of the hen and many of 
the ‘infertile eggs’ might really be classed among the early pre- 
natal mortalities. We make these suggestions merely as pos- 
sibilities which to us are somewhat tempting, since if there was 
actually an early prenatal mortality in some of these ‘infertile 
eggs’ it would bring the effects of the aleohol treatment on the 
fowls and mammals still closer together. It is only through our 
recent analysis of the size of litters and mating failures, along 
with careful examination of the pregnant females, that we have 
become aware of the sometimes frequent very early embryonic 
death. 

The second horizontal space shows the number of young from 
the several lines that reached maturity, or lived over three 
months. Here again the size of the litter is an important factor. 
It may be stated generally that the power of survival of a guinea- 
pig varies inversely with the size of the litter in which it is 
born. We shall see beyond that this is also true of their birth 
weight, growth rate, and certain other qualities so that in mak- 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 149 


ing comparisons between young guinea-pigs it is important to 
know whether the individuals concerned occurred in litters of 
equal size. 

In the normal lines all individuals born singly survived, and, 
as the seventh space shows, 30 per cent of them were unusually 
large or over size when three months old. Normal animals 
born in litters of two or three survive in about 84 per cent of the 
cases and are often of large size. The members of litters of 
four survive in only 62.5 per cent of the cases and are not gener- 
ally vigorous animals. . The records show that 80 per cent of 
the young in litters of five survived, but this is very unusual 
and is due probably to the small number involved, and possibly 
to a slight extent to the extreme care with which the pregnant 
females with the larger number of young were handled. This 
extreme care, however, only saved 13.33 per cent from the same 
number of alcoholic-line young born five in a litter. 

The second column indicates that over 81 per cent of alcoholic 
animals born in litters of one or two are capable of survival. 
Such a record is almost as good as the control, showing how very 
strong the members of small litters are and indicates again that 
an early individual selection may. have played some part, since 
no doubt there has been a prenatal mortality among the weaker 
individuals which originally existed in some of these litters. 
This is emphasized further by the fact that the members of 
litters of three survive in only 60.93 per cent of the cases. Here 
the prenatal mortality has not played so severe ardle and many 
weaker individuals are born. The power of survival of animals 
born three in a litter from the control is about 23 per cent better 
than from the alcoholic lines. Only 48 per cent of the alcoholic- 
line individuals from litters of four were able to live three months. 
Recognizing the small numbers involved, only 13.33 per cent of 
the alcoholic guinea-pigs born in litters of five were viable. It 
thus appears that when the alcoholic animals produce large 
litters the quality of the young is very poor, whereas their 
small litters contain animals with good survival records. There 
is little doubt that this apparent difference in quality is in part 
due to a prenatal selection which, in the case of the small litters, 
has eliminated most of the weaker individuals and left only the 


150 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


stronger to be born. In addition to this, it must also be recog- 
nized that the ability of the female to properly nourish the mem- 
bers of the large litters is somewhat overtaxed. Three or less 
than three embryos are very well nourished by normal mothers. 
It must be recognized here that the inferior records of the alco- 
holic lines are not alone produced by alcoholic mothers, but 
come also from alcoholic fathers as following tables will show. 

The survival records of the normal inbred lines are about the 
same as those from the straight control, and are almost equally 
superior to the alcoholic lines. 

The alcoholic inbred animals have a survival record closely 
similar to the straight alcoholic lines, and again decidedly inferior 
to either the normal or normal inbred lines. 

The fifth horizontal space contains the mortality records 
which are the reverse of the survival records just considered. 
However, we have given here not only the actual mortality in 
litters of different sizes, but have corrected the total mortality 
record on the basis of the occurrence of large and small litters 
and their mortality in the different lines as compared with the 
control. We have also expressed the mortality in numerical 
proportion in the several lines, taking the control as 100. The 
total mortality in the normal lines is 22.31 per cent. This is a 
very good record, since it not only includes the postnatal mor- 
tality, but all exact prenatal mortality as well. We mean by 
exact prenatal mortality those cases of absorption in utero and 
premature abortion which were actually observed, and not those 
calculated on the basis of size of litter, mating failures, etc., as 
was discussed in connection with the productivity of the different 
lines., 

The total mortality of the normal inbred is 21.95 per cent, or 
almost the same actually as well as when corrected for litter 
sizes as the straight normal lines. 

The total mortality of the alcoholic lines without inbreeding 
was 35.02 per cent, or almost 1.6 times greater than the mor- 
tality of the control. But this does not fully represent the real 
difference between the two lines unless it be corrected on the 
basis of the mortality record for the different-size litters in the 
alcoholic and the normal. The mortality is much higher among 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 151 


the members of the large-size litters than among those in the 
small litters, and the large litters are 1.7 times more frequent 
in the control than in the alcoholic lines. 

The mortality is corrected on the basis of the normal records 
as follows: The rate for the normal animals born one in a litter is 
zero; two in a litter, 15.21 per cent; three in a litter, 16.66 per 
cent; four in litter, 37.5 per cent, and five in litter, 20 per cent. 
On this basis what should be the number of alcoholic animals 
dying in the several different-size litters? The numbers should 
be zero instead of 7 for one in litter animals; 24.64 instead of 30 
for two in litter animals; 46.48 instead of 109 for three in litter 
animals; 37.5 instead of 52 for individuals born four in litter, and 
3 instead of 13 for five in litter. These numbers give a total of 
111.62, which divided by the number of alcoholic animals, 594, 
shows a mortality percentage of 18.79. On the basis of the 
control mortality for the different-size litters, this is what the 
mortality should have been in the alcoholic lines, yet instead of 
18.79 per cent it was actually 35.52 per cent, or almost double 
the normal rate. Again to express the corrected mortality in 
the alcoholic lines in terms of the control as 100, we find that for 
every 100 of the control animals that die 189 from the alcoholic 
lines die. 

The last column shows the 302 alcoholic inbred animals to 
present a still worse record. The actual mortality here is 39.07 
per cent, or one and three-fourths times higher than in the con- 
trol. Here again correcting as in the preceding cases, the mor- 
tality on the basis of the control record in the different-size 
litters, it should normally be 18.59 per cent, but instead the 
mortality is 2.1 times greater than this among these alcoholic 
inbred animals. In other words, for every 100 control animals 
that die 210 aleoholic inbred individuals succumb. While the 
normal inbred animals, although their numbers are small, pre- 
sent a slightly better record than the straight control, 98 of 
these dying to 100 of the control. 

In the third and fourth horizontal spaces of the table the total - 
mortality is divided into the prenatal and postnatal deaths. 
The proportion of prenatal to postnatal death in the different lines: 
presents peculiar arrangements that will be seen to exist, not only 


152 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


in this, but in several of the tables to follow. The prenatal 
records include embryos that die and are absorbed in utero, 
never passing to the outside, other embryos and fetuses which 
die and are passed out or born prematurely, and finally full- 
term young which die shortly before birth and are, therefore, 
still born or born dead. The postnatal deaths include all ani- 
mals dying before reaching three months of age, at which time 
guinea-pigs are about mature. 

In the control lines 51.92 per cent of the total mortality oec- 
curred before birth or was prenatal, while 48.08 per cent of the 
deaths occurred after birth. Considering the numbers in- 
volved, it therefore may be said that the pre- and postnatal 
mortalities are about equal in the straight control lines. There 
is no evidence here of a particular tendency on the part of the 
young animals to succumb at any given or critical stage in their 
development. 

The numbers contained in the normal inbred column are cer- 
tainly too small to be considered. 

In both the alcoholic and the alcoholic inbred lines where the 
numbers involved are considerable (the records showing 329 
deaths among 896 animals), the prenatal mortalities are double 
the postnatal deaths. The alcoholic column shows 70.14 per 
cent of the total mortality to occur before birth, while only 29.85 
per cent of the individuals that died were lost after birth. The 
last column gives for the alcoholic inbred animals 65.25 per cent 
of the total mortality as prenatal and only 34.74 per cent as 
postnatal. This consistent arrangement in the two columns 
indicates a tendency on the part of the weak and subnormal 
individuals of the alcoholic lines to succumb during early stages 
of their development. Such an interpretation is exactly in ac- 
cord with and is substantiated by the high early prenatal mor- 
tality which exists in these lines as indicated by the size of 
their litters and frequent mating failures when compared with 
the control. 

A mortality arrangement of this kind accords with what is 
known of almost all weak or diseased stocks—there is a very high 
loss during the early stages of development, as well as during 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 1538 


later embryonic or uterine life. Furthermore, many individuals 
die very soon after birth, while those that happen to survive the 
periods shortly following birth are often capable of an almost or 
quite normal existence. 

The mortality in the control is low, but half of oi. or a high 
proportion, occurs after birth. The mortality in the alcoholic 
lines is high, but only a low proportion, about one-third of this, 
occurs after birth. It may be added further that the young 
aleoholics which die after birth in the majority of cases die 
within a few days, while the control young that die after birth 
are more likely to be scattered along over a number of days 
or weeks. 

It is thus seen that in both the alcoholic and alcoholic inbred 
lines there is a decided tendency for the developing embryos and 
young to succumb during the early periods of their development. 
This would suggest that these affected individuals were often 
incapable of passing through the early critical stages of uterine 
life. But if they were sufficiently fit to survive these periods, 
their chance for existence was good, so that their postnatal 
mortality, although actually higher than the control, was pro- 
portionally much lower. Thus we have a somewhat rigid in- 
dividual selection taking place during the stages of uterine life, 
so that the sum total of the individuals at a given stage is of 
a better average quality than during any previous stage and 
vice versa. Therefore, as is clearly shown beyond, those ani- 
mals of the alcoholic lines which live to become mature and 
prove to be fertile are a strictly selected few and in each gen- 
eration the proportion of strong to weak individuals through 
this selection constantly tends to increase. 

The sixth horizontal space shows a complete absence of de- 
fective individuals in either the normal or normal inbred groups. 
It may be stated here that during the entire seven years of this 
experiment not one grossly defective or deformed individual has 
appeared in the nonalcoholic or control lines. This is a rather 
remarkable record for any group of animals, and it speaks strongly 
for the perfection of the original stocks from which both the 
control and the alcoholic lines have been derived. 


154 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 

In the alcoholic lines about 23 per cent of the individuals were 
erossly defective. By defective is meant those specimens which 
show deformities, such as one abnormally small eye, cataract 
or opaque lenses, deformed limbs, paralysis of the limbs, gross. 
tremors which make the animal incapable of locomotion or 
proper feeding, etc. There are slightly more defectives in the 
alcoholic inbred groups, 3.31 per cent in all. 

The next line records the over-size or unusually large animals, 
those weighing more than 500 grams when three months old. 
Among the control 30 per cent of the individuals born singly or 
one in a litter grew to be unusually large specimens. More than 
10 per cent of those in litters of two were also unusually large, 
and 5.53 per cent of the three in litter animals are included in 
this class. None of those born in litters of four or five were 
able to attain such a size. Of the total control animals over 
five and one-half per cent were of this large size, while only about 
half as many from the alcoholic and alcoholic inbred lines at- 
tained such a distinction, yet in both treated groups there were 
over two and one-half per cent of large specimens. 

The last line of the table shows the occurrence of unusually 
small animals, those weighing less than 300 grams when three 
months old. Among 233 control animals only one such individual 
appears, 0.42 per cent. The alcoholic lines contain more than 
three times as many of these as the control, but still very few, 
only 1.34 per cent. The numbers in the normal inbred column 
are too small for consideration. Among the 302 alcoholic in- 
bred animals there were eleven under-size specimens, or 3.64 
per cent. This is almost three times as high a percentage as 
occurred in the alcoholic lines and over eight times as high as is 
recorded for the control animals. 

Comparing the present results with those of our earlier papers, 
particularly with the similar table 2 (16), it will be noticed that 
the numbers involved are almost twice as great and the records 
of the animals considered are decidedly better than were for- 
merly shown. This improvment in the quality of all lines is due 
to several factors. In the first place, the breeding methods have 
been decidedly improved since studying the oestrous cycle of 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 155 


the females and determining the exact time of the ‘heat periods’ 
(Stockard and Papanicolaou, *17). This has enabled us to pair 
the animals at the most favorable periods and thus to obtain far 
better and more exact mating records than was possible on the 
basis of the previous conceptions of the guinea-pig’s sexual be- 
havior. Secondly, the housing, care, and feeding of the animals 
are decidedly better during the last three years than during 
previous times, and on this account the mortality in all lines 
has been reduced, but as might be expected, the weaker alcoholic 
lines have profited more by this improved condition than have 
the control animals. For example, the mortality record of the 
control has been lowered only a little more than 3 per cent, while 
in the alcoholic lines it has been lowered a much as 18 per cent. 
This improvement in the alcoholic lines is also partly due to the 
existence of more late-generation animals with many normal 
ancestors. Thus, although the lowered mortality record of the 
aleoholic may not be entirely due to the better living condi- 
tions, yet it serves as a striking illustration of the difference in 
response to the change on the part of the control animals and 
the alcoholics. The previous somewhat unfavorable state did 
not greatly impair the powers of existence of the control ani- 
mals, but it did evidently eliminate some of the weaker alco- 
holic individuals that might have survived under more ideal 
arrangements. 

It must be recognized, in the third place, that for the alcoholic 
inbred animals the degree of inbreeding among the later gen- 
erations here included is less intense than was the case with 
earlier generations in the former reports. And for this reason 
the previous rather decided differences which were shown be- 
tween the straight alcoholic group and the alcoholic inbred ani- 
mals have almost, though not entirely disappeared. 

Lastly, the fourth point of difference to be borne in mind in 
comparing the earlier and present records is that there are now 
more late-generation alcoholic descendants with less affected 
material in their total germ-cell complex than was true of the ani- 
mals in the former tables, which as a group were composed of 
generations closer to the direct alcohol treatment. For ex- 


156 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


ample, an animal derived from a directly alcoholized father 
and a normal mother could be said to contain half affected and 
half normal germ plasm, whereas another in whose pedigree the 
only alcoholic individual was an alcoholized grandfather, would 
undoubtedly contain a smaller amount of affected stuff. 

Finally, then, in the light of the facts involved, the general 
table presents an impression closely similar to that derived 
from the previous records of these experiments, but it adds data 
of much importance for a clearer understanding of the problems 
concerned. 

The improvement in the present records over the former cones 
might suggest that should the methods of breeding and caring 
for the animals reach perfection, the differences between the 
alcoholic lines and the control might be entirely erased. This 
would be possible if the improvement was due alone to method, 
but such a suggestion ignores the fact that the improvement is 
more largely due to the presence of late-generation animals with 
only a small amount of alcoholic germ plasm in their ancestry 
and a large number of normal progenitors. The analysis of the 
following table 2, in which the several generations are treated 
separately, will fully substantiate the validity of the foregoing 
statement. 

Before considering this table, however, we may discuss briefly 
the phenomenon of absorption of embryos in utero and our 
methods of examining pregnant females in order to fully record 
the fate of all embryos that begin to develop. A knowledge of 
this prenatal mortality is involved not only in the table just 
studied, but in several of those that follow. 


6. ABSORPTION OF EMBRYOS IN UTERO AND ABORTIONS OF PARTS 
OF LETTERS: METHODS OF DETECTING THESE PROCESSES 


After having observed the course of pregnancy and the size of 
the litters produced in a large number of cases, we became con- 
vinced that many of the small litters delivered at full term were 
only partial litters. Particularly in the alcoholic lines it became 
evident that abortions of one or two members of a litter might 


MODIFICATION OF THE GERM-CELLS IN MAMMALS L57 


oecur without hindering the further development to term of the 
remaining members. It was also recognized as is known even 
for the human female that embryos might be absorbed in utero. 
In the guinea-pig we have found that the absorption of one or 
more embryos in utero, as is true of partial abortion, may not 
interfere with the further normal development and birth of the 
remaining members of such litters. 

When it was realized that these absorptions and abortions of 
parts of litters were taking place, the necessity arose of definitely 
detecting each case in order to make the prenatal mortality 
records approach correctness. A systematic examination was, 
therefore, begun of every female after being with a male for one 
month up to within a week or ten days of delivery. 

The female to be examined is allowed to stand on a flat sur- 
face and the investigator with both hands presses the ventral 
abdominal wall so as to feel with the fingers the horns of the 
uterus against the dorsal abdominal wall. With considerable 
practice the small embryos and placentae may be definitely 
counted within one or both horns of the uterus. The num- 
ber of embryos and their position in the two horns of the uterus 
are noted on the record card of the female. After this initial 
examination she is reexamined once or twice during the preg- 
nancy and each time the number and position of the embryos 
with the date of examination are recorded. The number of 
young finally born helps to show how nearly correct the exam- 
inations have been. 

The records now contain several hundred such examinations 
and show that absorption of embryos may take place not only 
during early stages, but after the fetuses have attained consider- 
able size. The difference between absorption and partial abor- 
tion may usually be recognized by the fact that the embryo 
being absorbed may exist for some time as a small lump in the 
uterus, while the aborted embryo disappears from the uterus and 
leaves no palpable remains. There are exceptional cases in 
which the uterus is unusually swollen or congested after the 
abortion and these on being felt would still seem to contain a 
partial embryo. The cages of the pregnant females are exam- 


158 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


ined every morning and afternoon and aborted embryos or pla- 
centae are generally located, yet instances do occur of early 
abortion possibly during the night in which no trace of the 
aborted material is found, since the female very quickly attempts 
to eat the aborted products. 


Fig. 6 On the right a normal 19-mm. embryo taken from the right horn of 
the uterus of an alcoholic female. The left horn of the uterus contained the 
degenerating mass shown on the left which was attached to a small placenta 
and represents an embryo in the process of being absorbed in utero. The mother 
had an alcoholized father. 


During the two years which supply the data for the present 
study the females have been very carefully and consistently 
examined throughout their pregnancies, and the records of ab- 
sorbed and premature or aborted young are very accurate for all 


MODIFICATION OF THE GERM-CELLS iN MAMMALS 159 


periods after the embryos are of sufficient size to be detected by 
this method of external examination. To convey some idea of 
how accurately one may detect a structure by palpation through 
the abdominal wall of the guinea-pig, it may be stated that a 
slightly cystic ovary has frequently been diagnosed by such an 
examination. 

A normally developed embryo 19 mm. crown rump length is 
shown in figure 6 and near it is seen an amorphous embryonic 
mass 2 mm. in longest diameter which represents the other 
member of the litter. The two were in different horns of the 
uterus. The placenta of the normal embryo was of the usual 
size, while the one associated with the arrested specimen was 
only about one-half as large. The entire mass of the smaller 
ovum in the uterus was about that of a ten-day specimen, while 
the normal individual was a typical twenty-day specimen. This 
case was detected by external examination and was merely opened 
in order to use the embryos for illustrating the phenomenon. 
In the explanation of the figure the ancestry of the embryos is 
given. 

The intrauterine absorption of embryos, as stated above and 
indicated in table 1, may occur in normal guinea-pigs. A. W. 
Meyer (’t7) has very recently described the histological con- 
ditions found in partially absorbed embryos which he had ob- 
tained during a study of the prenatal growth of the guinea-pig. 
There is considerable data from our study to indicate that this 
absorption of embryos is somewhat more frequent in the alcoholic 
than in the normal lines. 


7. A COMPARISON OF THE QUALITIES IN THE DIFFERENT GEN- 
ERATIONS OF THE ALCOHOLIC LINES AS THEY BECOME 
FURTHER REMOVED FROM THE GENERATION 
DIRECTLY TREATED 


It has been mentioned in discussing the improvement of the 
records in table 1, as compared with our previous reports, that 
this advantage is partly due to the larger number of late-gen- 
eration animals at present included. We may now analyze the 
alcoholic lines for a comparison of the qualities of the early and 


160 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


late generations, I, to Fy, on the basis of their productivity and 
mortality records. Such an analysis is of particular importance 
to test in the first place whether the effects of the alcohol treat- 
ment on the germ cells are permanent, altering their qualities in 
inheritance, and in the second place whether an increasing 
amount of normal germ plasm acquired with each generation 
may tend to offset the original alcoholic effect by dilution. 

Table 2 contains the data from the non-inbred alcoholic lines 
divided into different generations. The first vertical column 
gives the records for 233 control young as a standard of com- 
parison. These are the same records shown in the normal column 
of table 1, except that in the present table we have included in 
the first horizontal line under each group the average birth weight 
of the litters produced. This is termed the average litter weight 
and is recorded in grams. For the normal stock this average 
productivity is 197.12 grams; that is, the average weight of all 
the litters at birth was this amount. The average litter weight 
is In a way associated with the average litter size, since a litter 
containing several young though each individual may not be so 
large, will probably weight more than a litter of fewer or of one 
young. Thus a group having a higher average litter than an- 
other group will also probably have a higher average litter weight, 
though this is not necessarily the case, as will be seen on com- 
paring the several columns of the table. 

The second column contains the alcoholic line animals. This 
again is the same 594 alcoholic animals shown in the second 
column of table 1 and is given here for comparison with the four 
following groups, each of which is a certain portion of this total 
column. The average productivity for the alcoholic animals is 
170 grams, or 37 grams less than the control, and when cor-- 
rected on the basis of the average litter size, it is 5.6 grams less 
than it should be according to the normal standard. 

The third column gives the records of 186 animals with one or 
both parents treated with alcohol, the F, generation. Thirty- 
three of these animals also had a slight aleoholie history in their 
ancestry, and thus the entire group are not pure F, alcoholics. 
The proportion of large and small litters in this column is about 


161 


MAMMALS 


RM-CELLS IN 


GE 


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THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, No. 1 


162 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


the same as in the total aleoholic column, 34.6 per cent of the 
animals were born in litters of less than three and only 17.74 
per cent in litters of more than three. The normal record as 
pointed out before is just about the reverse of this. The average- 
size litter in which the F, animals occur is 2.51, which is slightly 
larger than for the total alcoholic column, but the average 
weight of these litters is less than for the entire alcoholic lines, 
being 165 against 170 grams. As compared with the control the 
average productivity of this column is 32 grams low, and when 
corrected on the basis of the average-litter size, the litters are 
then more than 13 grams less than the control standard. The 
mating failures are about the average alcoholic result, 12.94 
per cent. 

The mortality record of the F, animals is not so good as for 
the entire alcoholic group, only 56.98 per cent of them living 
longer than three months as against 64.47 per cent. The total 
mortality is 43.01 per cent, and when this is corrected on the 
basis of the normal mortality for the various-size litters in which 
the individuals occurred, we find that the F, mortality is almost 
2.3 times the control record, or 230 against 100. The corrected 
mortality here as compared with the entire alcoholic group is 
230 against 189, or 41 points higher. 

The proportion of prenatal to postnatal mortality corre- 
sponds closely to that of the entire alcoholic group and contrasts 
with the control in the same way as discussed in considering 
table 1. 

Finally, then, the F; group of animals from either one or both 
treated parents, are inferior to the alcoholic group as a whole in 
having a higher mortality record and in occurring in litters of a 
lower average weight although of equal average size. 

The fourth column contains the records of animals more than 
one generation distant from the aleohol treatment; that is, those 
having treated grandparents, great-grandparents, or great-great- 
grandparents, or combinations of these, Fs, F;, and F's generations. 
All of the alcoholic animals from column 2 are included in this 
column, except the third column of F, animals; there are thus 
408 individuals. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 163 


The distribution of the animals in large and small litters is 
closely the same as in the two preceding columns, over 30 per 
cent being in litters of less than three and 20.09 per cent in litters 
larger than three. ‘The average-size litter and the average litter 
weight are just about what is found for the total alcoholic group 
and somewhat better than for the F, group. The percentage 
of surviving animals is a‘little better than the total alcoholic 
group and considerably better than the F; group. The prenatal 
and postnatal mortality proportions follow the typical arrange- 
ment for the alcoholic lines, the prenatal being about two and 
one-third times higher than the postnatal. The total mortality 
among these animals is about 10 per cent lower than for the F; 
group and slightly below the record of the total alcoholic lines. 
When the mortality is corrected in terms of the normal mortality 
for the different-size litters and stated on the basis of 100 for the 
control stock, it becomes 172 as against 230 for the F, column 
and 189 for the all generations alcoholic column. 

The fifth column records 147 animals still further removed 
from the treated generation; these had treated great-grandpar- 
ents or great-great-grandparents or both, the F; and I; genera- 
tions. Some of these animals may have had only one or two 
alcoholic ancestors out of eight or sixteen; therefore, the pro- 
portion of modified to normal germ plasm is often very small. 

The arrangement in large and small litters differs from the 
other alcoholic groups and approaches that shown by the nor- 
mal lines very closely, there being a higher percentage born in 
large litters than in small. he average-size litter is larger 
than in the three preceding columns, although still well below 
the control. The average litter weight is low when compared 
with the normal lines and only about the same as in the three 
preceding columns when taken in connection with the average 
size of the litters. When corrected for the average size, the 
weight of the litter falls more than 10 grams below the control 
record. The mating failures still show the high percentage of 
the alcoholic lines, being over three times as many as in the 
control. 


164 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


A greater percentage of individuals survived than in any of the 
preceding groups except the control. 

The proportion of prenatal to postnatal mortality shows the 
arrangement characteristic of the alcoholic groups. As a matter 
of fact, the prenatal mortality is really unusually high, and this 
is probably due to the high percentage of large litters, as among 
these the prenatal mortality is most frequent. It is as though 
the animals of this group had produced almost as high a pro- 
portion of large litters as the control animals and still they were 
not sufficiently good quality as compared with the control to 
keep down the prenatal mortality in these high litters. 

The total mortality when corrected on the normal rate for the 
litter sizes and expressed on the basis of 100 for the control 
becomes 145. This is a decided improvement over the other 
alcoholic groups, although poor in the light of the control. 

From a survey of this column it may be concluded that ani- 
mals as far as three generations removed from the direct alcohol 
treatment are still differentiated as a group from the control in 
regard to the weight of the litters in which they are born, the 
tendency of the matings to result in failure, the high proportion 
of prenatal mortality over postnatal, and the total mortality 
which is one and one-half times higher than the normal. All 
of these differences exist in spite of the fact that more and more 
normal germ plasm has been introduced during each generation 
until some of these animals may have had as many as six or 
seven normal great-grandparents against one or two treated or 
alcoholic great-grandparents, thqugh the average of course had 
somewhat more treated ancestry than this. 

One of the F; individuals, descended from treated great- 
grandparents, is shown in figure 7. The animal on the left was 
a non-inbred female, No. 803, with six of its eight great-grand- 
parents treated with alcohol and only two, on the paternal side, 
were normal. Its great-grandparents may be written thus: A 
indicating alcoholic and N normal, the 2 on the left, in the 
formulae: [(AxA) (AxA)] [(NxA) (AxN)]. The animal on the 
right is an ordinary normal guinea-pig born on the same day 
as the small degenerate specimen which weighed only one-third 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 165 


Fig. 7 On the left a non-inbred female, No. 803, with six of its eight great- 
grandparents treated with alcohol and only two on the paternal side not treated. 
She was small and degenerate and lived only one day. On the right is shown a 
normal animal born on the same day, the two being photographed on one plate. 

Fig. 8 Two F; guinea-pigs born in the same litter from a normal father and 
a mother derived from four aleoholized grandparents. The albino female, No. 
955, on the left weighed at birth 90 grams, the small defective male on the right 
weighed only 38 grams and died within two days; the sister is still alive. 


166 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


as much and lived only one day. Although some young from 
control parents do die shortly after birth, they are not so un- 
usually small nor degenerate in appearance as the defective 
young of the alcoholic lines. 

Another even more striking example of the small defective 
animals appearing in the F; generation is shown by the photo- 
graph, figure 8. The two individuals in this picture were born 
in the same litter. Their mother was a black and red animal 
from four aleoholized grandparents and their father was a nor- 
mal albino male, [(AxA) (AxA)] [N]. The F; animal on the 
left, No. 955, is an albino female weighing at birth 90 grams. 
She is thus an unusually large animal to be a member of a litter 
of three and is of the type of the normal albino father. Her 
small degenerate brother on the right weighed only 38 grams at 
birth, had a severe tremor which rendered him incapable of nor- 
mal progressive movements, and he lived only two days. His 
degeneracy and black and red color are both qualities for which 
he was indebted to his alcoholic mother. A marked discrepaney 
in either size or condition between two members of the same 
litter at birth is entirely lacking among our control lines. It is 
rarely so decided as this case illustrates, yet very frequent in 
the alcoholic lines and particularly in the F, and F; generations. 
A number of illustrations of this type could be continued to 
show that the quality of the later generations from alcoholized 
ancestors is decidedly subnormal. 

Such conditions as the above occur not only in spite of the 
introduction of normal germ plasm which tends to overshadow 
the alcohol effect, but also in spite of a rather harsh individual 
selection which is at work tending to improve the stock with 
each generation. Almost all of the badly defective individuals 
in the alcoholic lines are lost early in their career, as is shown 
by the high prenatal mortality; other less defective ones die 
soon after birth, such as those pictured above, and only the best 
live to become fertile adults. It is thus found that even this 
selected group mated with many normal individuals still pos- 
sesses enough of the medified germ plasm which resulted from the 
early alcohol treatment to cause their offspring to be inferior to 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 167 


the control animals in a number of important qualities that ren- 
der them less capable of survival. 

These two factors, the constant introduction of more normal 
germ plasm and the elimination of all the weaker alcoholic indi- 
viduals so that only the stronger reproduce, may finally in late 
generations so purify the alcoholic lines as to cause them to 
attain a condition equally as good as the normal. 

The sixth and last column of table 2 may illustrate such a 
condition, though it contains the records from only a few ani- 
mals. These animals are descended from one or more treated 
ereat-great-grandparents, the F, generation. They are four 
generations removed from the alcoholic treatment. 

The average-size litter is almost as large as in the control, 
and on the basis of its size it is actually heavier than the control 
average. It may be said from the evidence shown that the pro- 
ductivity here is equally as high as in the control. 

A higher percentage of individuals survived than among the 
control, and even though the mortality figures are small there was 
certainly no tendency toward a high prenatal mortality. On the 
contrary, there was scarcely any prenatal mortality, so that the 
record in no way resembles that of the alcoholic lines. On the 
basis of 100 for normal stock mortality, the mortality here cor- 
rected for litter size is only 84, or 16 per cent better than the 
normal. It is actually in the table 5 per cent lower than the 
control. 

After having considered the last column, the F, animals with 
their very good record, it should be recognized that these same 
animals are included with the F. and F,; imdividuals in the 
fourth and fifth columns. Their presence in these columns, 
particularly in the fifth, has tended to incline the records toward 
the normal. One must realize, therefore, that the F; and F; 
animals if considered alone would present even stronger alcoholic 
records than are indicated in the fourth and fifth columns. 

The table shows that the nearer to the direct alcohol treatment 
an animal is produced, the more inferior in quality it will be as a 
result of the high amount of modified germ plasm contained in the 
germ-cell complex from which it arises. Therefore, the records 


168 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


of F, individuals are worse than the records of the sum of all 
alcoholic generations, as is seen on comparing the third column 
with the second. The later generations being further and 
further removed from the treatment and having less and less 
modified germ plasm on account of the constant introduction of 
normal stock are progressively improved until finally the Fs gen- 
eration has its modified germ plasm diluted to such a degree that 
its record is on par with the control. 

The ancestors of these late-generation animals were also suc- 
cessively selected from the least affected of the alcoholic stock, 
being those animals capable of survival and reproduction, while 
the most highly affected died or were sterile and incapable of 
reproduction. We assume the probability that the more nearly 
normal animals with stronger bodies also carry germ cells that 
are less affected than those in the more degenerate individuals. 
Most of the grossly defective individuals which reach maturity 
are sterile as evidence in this direction. Thus individual 
selection being in this case a selection of germ plasm as well 
as soma, helps materially to improve the quality of the later 
generations. 


8. ACOMPARISON OF ANIMALS FROM DIRECTLY TREATED FATHERS 
AND FATHERS OF ALCOHOLIC STOCK WITH ANIMALS FROM 
DIRECTLY TREATED MOTHERS AND MOTHERS OF 
ALCOHOLIC STOCK AND WITH OTHERS FROM 
BOTH PARENTS OF ALCOHOLIC STOCK 

Are the general conditions induced by directly treating the 
father with alcohol the same as those resulting from treating the 
mother, and are they equal in extent? Do fathers of alcoholic 
ancestry beget offspring of better or worse quality than off- 
spring produced by mothers of similar aleoholic ancestry? Or 
are the effects of the alcohol treatment on the germ cells, which 
is expressed through several generations, carried with equal 
degree by both the alcoholic father and the alcoholic mother? 
We shall attempt in this and the following section to supply 
data which may serve to partially, at least, satisfy these queries 
as well as furnish an analysis of several other more detailed 
propositions. 


9 


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IN MAMMALS 


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170 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


Table 3 is an arrangement of the records of animals on the 
basis of paternal and maternal alcoholism. The first group of 
animals are those from parents treated directly and having no 
other alcoholic history. Thus the total 1538 differs from the total 
186 animals with treated parents in the third column of table 2, 
since in thirty-three cases the former group had not only treated 
parents, but also treated ancestors. ‘The second group contains 
all the animals with parents of alcoholic descent, but not di- 
rectly treated, the total number is 408; these are the same ani- 
mals that compose the fourth column of table 2. The third or 
last group contains all of the 594 non-inbred animals of the 
alcoholic lines. 

The individuals in each of the three groups are separated into 
three classes. The classes of the first group are those with only 
father treated, those with only mother treated, and those with 
both parents treated. In the second group the young are classi- 
fied as those from only father alcoholic, which means the father 
was descended from treated ancestors which may have been 
either treated males or females. In other words, this is not the 
record of a pure alcoholic male line, but merely the alcoholic 
effects, if any, that reach the recorded individual through an 
alcoholic father regardless of the origin of his alcoholism. The 
second column of this group shows the records of animals from 
alcoholic mothers. Here again the mother’s alcoholism may be 
due to treatment of any of her ancestors, male or female. It is 
not a purely female alcoholic line, but a maternal alcoholic line. 
The third column of the second group shows records of animals 
from parents both of which were alcoholic. 

In the entire second group the alcoholism of the parents is 
ancestral, not being due to direct treatment, while in the third 
group the alcoholism is either direct, ancestral, or both. The 
third group is, therefore, an arrangement of all the animals from 
alcoholic lines for a comparison of the influences of maternal and 
paternal alcoholism. 

In the first column of table 3 it is seen that when the father 
only is treated the results contrast decidedly with the control. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 171 


There is a high percentage of small litters and a low percentage of 
large litters, thus giving next to the lowest average litter con- 
tained in all the records, only 2.30 against 2.77 for the normal. 
The average litter weight is very low on account of the small 
average litter size. When this is corrected for the proportion of 
weight to number of individuals in the control litters these 
small litters from the treated fathers weigh more for their size 
than do the control, being over 6 grams heavier. This is not 
an actual advantage since the majority of young born in small 
litters of one and two are larger than those born in high litters 
of four or five. The percentage of mating failures is unusually 
high, 23.52 per cent against only 4.54 per cent in the control. 
All of these facts would seem to indicate that the treatment of 
the fathers had evidently lowered their productivity or fertility, 
causing them to fail to sire offsprmg in almost one-quarter of 
the matings and to beget unusually small litters in the other 
three-quarters of the cases. There must have also been a high 
‘early prenatal mortality’ in view of the remarkably great per- 
centage of small litters and high percentage of mating failures. 
We must necessarily divide the mortality into prenatal and 
postnatal, and the prenatal again into ‘early prenatal,’ as in- 
dicated by the small average size litter and high number of 
mating failures, and ‘late prenatal’ based on the exact observa- 
tions of absorptions, abortions, and still births. 

Two-thirds of the offspring from treated fathers survived 
against over three-fourths from the control. The prenatal mor- 
tality is a larger proportion of the total than in the normal. The 
total mortality when corrected to the normal rate for the differ- 
ent-size litters in which the animals were born is 178 in terms of 
the control as 100. This is only slightly below the mortality 
rate of 189 for the entire non-inbred alcoholic group. 

When the mother alone was treated the records of the off- 
spring differ considerably from the above. The percentage of 
small litters is only slightly higher than the percentage of large 
litters, and the average-size litter, 2.78, is as large as the nor- 
mal. There are very few mating failures, in this regard again 


172 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


almost a normal record. The productivity of these treated 
mothers is high and the size of the litters would indicate a very 
low ‘early prenatal mortality.’ Here, however, their good 
records stop. 

Although the litters contained as many individuals as the con- 
trol litters, their average weight was 26 grams below the nor- 
mal. The large litters from treated mothers actually weighed 
only as much as the very small litters from treated fathers; there- 
fore, the individual members of the litters from treated mothers 
were unusually small animals. The ‘late prenatal mortality’ 
was proportionately very high—three times the postnatal. Thus 
many of the young died in utero or were still-born, and those 
that were born alive were small specimens. The total mor- 
tality was 51.08 per cent, corrected for the litter sizes, and ex- 
pressed in terms of the control as 100 it becomes 281—the 
highest mortality on record. 

We see from the table that treating the mother with alcohol 
does not appreciably affect her productivity, but greatly depreci- 
ates the quality of offspring to which she gives rise. While in 
the case of the alcoholic father the productivity is greatly re- 
duced, and although the quality of offspring which he begets 
does not compare favorably with the control, it is considerably 
superior to that from the treated mother. In the treated mother 
the alcohol may act not alone on the ova or germ cells, but on 
the developing embryo as well, while in the father it acts, of 
course, on the germ cells alone. Does the difference between 
the qualities of the offspring from these two cases represent the 
action of the treatment on the developing young in utero? 
Further, does the reduced productivity on the part of the 
treated male indicate that the spermatozo6n or male germ cells 
are more sensitive to the treatment than the egg? The remain- 
ing columns of this and the following table may throw some light 
on these questions. 

During the period of the experiments now under consideration 
practically no matings between treated males and females have 
been made, as the third column of this group shows. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 173 


The next group in table 3 are animals derived from parents 
of alcoholic descent which had not themselves been treated. 
These are the same 408 animals recorded in the fourth column of 
table 2. The first class in this group are animals obtained from 
fathers of aleoholie ancestry and normal mothers; the second 
class are from mothers of aleoholic ancestry and normal fathers, 
and the third class are animals produced by two alcoholic parents. 
As mentioned above, the alcoholic father or mother may owe 
their condition to either male or female or to both male and 
female ancestors. These are not purely male or female alcoholic 
lines such as will be found in the next table. 

A comparison of these three columns with the normal records 
shows clearly the alcohol effects, though not so strongly ex- 
pressed as when the father or mother is directly treated. The 
father and mother columns of this group differ very little from 
one another, which is in marked contrast to the striking differ- 
ences when the fathers and mothers are directly treated, as seen 
in columns | and 2. In the present columns all of the modified 
conditions are due to an injury of the germ cells in the treated 
ancestral generations. This is equally as true of the alcoholic- 
mother column as of the alcoholic father. For example, the 
mortality records in the alcoholic father and mother columns are 
about the same, while there is a remarkable discrepancy between 
the mortality records of young from treated fathers and treated 
mothers in the first two columns. The extremely high mortality, 
largely late prenatal, among the offspring of directly treated fe- 
males is to some extent due to the direct action of the alcohol 
upon the early developing embryo in utero. If this action 
could be eliminated the treated father and mother columns 
of the first group might become as nearly similar as the alcoholic 
father and mother columns of the second group. The individuals 
in the latter two columns are on an average about the same dis- 
tance removed from the ancestral alcohol treatment, and, there- 
fore, the records would be little affected by a correction on the 
basis of the generations treated. 

When both parents are from alcoholic ancestry the produc- 
tivity is considerably lowered as shown in the third column by 


174 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU . 


the high percentage of small litters and low percentage of large 
litters and consequently the very low average litter of 2.31. 
This is likely due to the male partner in the combination, as the 
preceding columns would suggest. The average litter weight, 
however, is high, so that the individual members of the litter 
are as heavy as the normal; this, again, may be due to the male 
influence as expressed in the high early prenatal mortality. 

The mortality records, though markedly inferior to the nor- 
mal, show an advantage over the two previous columns. ‘There 
is probably a high ‘early prenatal mortality’ as indicated by the 
low average litter, but the ‘late prenatal mortality’ is lower 
than in any of the foregoing columns éxcept that of the treated 
fathers, where again the litter was very small and the prob- 
able ‘early prenatal mortality’ high. This close association be- 
tween the small litters and the low late prenatal mortality makes 
it seem all the more probable that the litter size is associated 
with an ‘early prenatal mortality’ that occurs so near the begin- 
ning of development that it cannot be directly observed. On 
the other hand, this result could be interpreted as due to a 
lowered fertility. If this were brought about through an elimi- 
nation of the weaker germ cells we might except also the asso- 
ciated low late prenatal and postnatal mortality, and would 
have a condition in exact accord with Pearl’s interpretation of 
the results on fowls. We should be glad to accept such an ex- 
planation, but for the considerable amount of evidence in our 
records which points towards a high ‘early prenatal mortality’ 
rather more than infertility as the underlying cause of the small 
litters and low late mortality. It must also be remembered 
that the infertility among the fowls was found in the females as 
well as the males, while here it would be confined to the males 
only. 

The slight advantages which appear in favor of the records 
from both parents alcoholic as compared with records from alco- 
holic mothers or fathers are due largely to the distance from the 
treatment of the generations concerned. In the majority of 
cases the generations are more remote in the both-parent column 
than in either the father or mother column, and on the basis of 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 175 


the evidence shown in table 2 this may readily explain the 
apparent advantages. 

The last three columns show the results of the first two groups 
combined and in addition contain a few records from mixed 
eases that could not be properly included in any of the previous 
classes; for example, animals with one parent of alcoholic ancestry 
and the other parent directly treated, ete. 

Here again there is considerable contrast between the aleoholic- 
father and the alcoholic-mother columns, these differences being 
due to the influence on the totals of the F, records from the 
treated-father and treated-mother columns of the first group. 
The productivity when only the father is alcoholic is low, the 
litters being small and over 21 per cent of the matings result in 
failure. It may be inferred that there was a rather high ‘early 
prenatal mortality.’ The average litter weight, however, was 
about as good as normal. The late prenatal and postnatal 
mortality records are better than those from the alcoholic 
mothers. 

The average-size litters from the alcoholic mothers was rather 
large and the mating failures were much less frequent than from 
the alcoholic fathers, indicating a lower probable ‘early prenatal 
mortality.’ The average litter weight was lower than from alco- 
holic fathers, taking into account the size of the litters in the 
two classes. The total mortality from alcoholic mothers was 
high and the proportion of late prenatal to postnatal was ex- 
cessive. It is thus seen that a high prenatal mortality is fol- 
lowed by a low postnatal death rate, and this is in accord with 
our assumption that a high ‘early prenatal mortality’ will be 
followed by not only a low postnatal, but also a low late pre- 
natal mortality. In other words, the more thorough the elimi- 
nation of defective embryos and fetuses the greater the prob- 
ability of survival for the selected few that remains to be born. 

The last column with both parents aleoholic has a mortality 
record as good as the aleoholic-father column and better than 
the alcoholic-mother, but this is only apparent and not real. 
The column contains only one individual from directly treated 
parents, and consequently the alcoholic treatment was applied 


176 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


on the average to more remote generations than was the case in 
the two single alcoholic-parent columns. 

In spite of the generations concerned, there is a higher per 
cent of small litters and a lower per cent of large litters here than 
in any other class in the entire table. Consequently there is 
also the lowest average litter. In so extreme a case there was no 
doubt a high early prenatal mortality. The average litter weight 
is actually low, but allowing for the small-size litter the average 
birth weight of the individuals is about as much as the control, 
again indicating that an individual selection has occurred through 
an elimination of the weaker embryos during the early develop- 
mental stages. 

The extremely small-size litter and the high ‘early prenatal 
mortality’ may also in addition to the generations concerned ex- 
plain to some extent the relatively low total mortality and es- 
pecially the lower rate of late prenatal mortality as compared 
with postnatal. 

The questions involved in the present section may be still 
further analyzed by rearranging the data on the basis of only male 
ancestors treated or only female ancestors treated instead of 
only father alcoholic and only mother aleoholic. Table 4 pre- 
senting this arrangement will be reviewed in the following sec- 
tion, after which several points of interest may be better 
discussed. 


9. A COMPARISON OF LINES FROM ONLY MALE ANCESTORS ALCO- 
HOLIC WITH LINES FROM ONLY FEMALE ANCESTORS 
ALCOHOLIC AND WITH THOSE FROM BOTH MALE 
AND FEMALE ANCESTORS ALCOHOLIC 


The records tabulated on the basis of male or female ancestors 
treated supplement the arrangements in table 3, where the 
groups are classed for only father or mother alcoholic. In table 
3 the alcoholic father may owe his alcoholism to the treatment 
of any of his ancestors, either male or female or both. The 
alcoholic effects, if any, are there due to the, paternal ancestry. 
The same applies to the groups with only mother alcohotic. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 177 


In table 4, on the other hand, the groups with only male 
ancestors treated owe their modified conditions, if such exist, 
entirely to the effects of the treatment on male animals, though 
the individual being considered may have inherited this alco- 
holie effect through its mother. Thus animals in the columns 
with only male ancestors treated were not necessarily derived 
from alcoholic fathers; but may have been produced by alco- 
holic mothers which, however, owe their alcoholic condition 
to one or more treated male ancestors. The table permits a 
comparison of the action of the treatment on the male germ cells 
and the transmission of the effects with the action of the alcohol 
treatment on the female germ cells and the effects transmitted 
to the different generations. While the last table permitted a 
comparison of the animals derived from males of alcoholic stock 
with others derived from females of alcoholic stock. The two 
tables serve to analyze very completely the problem of the 
parts played by the sexes in the acquisition and transmission 
of the effects of the alcohol treatments. 

The three columns in the first group of table 4 are the same 
as those of the first group of table 3, being the records of F; 
animals derived from treated fathers, which have only one male 
ancestor treated according to the table 4 arrangement, and F; 
animals derived from treated mothers or from only the one 
female ancestor treated. This group was discussed in review- 
ing the third table. The points of chief interest in the present 
connection are the decidedly inferior conditions of the off- 
spring from the treated females as compared with those from the 
treated males, in so far as their measured mortality records and 
birth weights per litter are concerned. On the other hand, the 
records from treated males suffer as regards the ‘early prenatal 
mortality’ indicated by the small average-size litter and the 
high percentage of mating failures, while the records of the 
treated females in regard to these conditions are equally as 
good as those of the control animals. 

The next three columns of table 4 are highly important, since 
they contain the results of matings when, first, only male an- 
cestors are treated; second, when only female ancestors are 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, NO. 1 


178 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


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MODIFICATION OF THE GERM-CELLS IN MAMMALS 179 


treated, and third, when both male and female ancestors are 
treated with all first generation, F, offspring, excluded. The 
modified conditions shown by these records are due to an heredi- 
tary transmission of the defects and not in any case to the 
direct influence of the treatment on the developing animals. 

The fourth column from only male ancestors when compared 
with the normal stock in tables 1 and 2 shows a higher ‘early 
prenatal mortality’ based on the average litter size and high 
number of mating failures, a lower average litter weight, a higher 
late prenatal mortality, and a higher total mortality. The re- 
sults of these matings are, therefore, from any point of view 
worse than the results of normal matings. And they prove the 
hereditary transmission of the defects arising from the treat- 
ment of the male animals. 

The same can be said for the female column, the results shown 
here also being worse than from the normal matings. The 
‘early prenatal mortality’ is higher, the average litter weight, 
indicating the total productivity is smaller, the late prenatal 
and total mortality are higher, while the mating failures are 
about the same as in the control records. Therefore, the treat- 
ment of female individuals also induces effects that are trans- 
mitted to later generations through the germ cells. 

When, however, the records of the fourth and fifth columns 
are compared, it is found that the treatment of male ancestors 
gives in every point considered more marked effects on the 
qualities of the descendants than the treatment of female an- 
cestors. Among the descendants of treated males there is a 
higher early and late prenatal mortality, a decidedly higher 
total mortality, and more mating failures than among those 
from treated female ancestors, while the first and second columns 
show the opposite to prevail so far as litter weight and mortality 
are concerned for first-generation, F;, animals from directly 
treated males and females. These inferior results, so far as 
late prenatal and total mortality are concerned on the part of 
the offspring from the directly treated female, may be interpreted 
as due to the direct influence of the treatment upon the young 
in utero. On the other hand, the improved records from the 


180 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


treated-female line during later generations can probably be 
explained in part by the higher mortality of the offspring in the 
first generation, thus bringing about a greater elimination of the 
weaker individuals. In other words, these animals from only 
female ancestors treated have withstood a somewhat more 
severe selection during the first generation than have the off- 
spring from only treated male ancestors. 

As a final possibility it must be recognized that the superior 
records of the late generations descended from treated female 
ancestors as compared with the records of similar generations | 
descended from treated males, may be due to a smaller influence ~ 
of the alcohol treatment on the ova, or female germ cells, than 
on the spermatozoa. 

The sixth column from treated male and female ancestors 
shows, in comparison with the two preceding columns, the highest 
‘early prenatal mortality’ based on the many small-size litters. 
There is also the lowest average litter weight. The late prenatal 
mortality, total mortality, and mating failures, while higher than 
for the treated-female line, are lower than in the treated-male 
line. The complete absence of matings between directly treated 
animals as seen in the third column of this table makes com- 
parisons and explanations of the results in this sixth column 
very difficult. 

The last three columns of the table show the combined re- 
sults from all generations. The column for both male and 
female ancestors treated shows the highest early prenatal mor- 
tality, the male treated line the highest late prenatal mortality, 
and the female treated line the highest postnatal mortality. 

In general it may be stated after reviewing this and the fore- 
going table that the treatment of males produces in their de- 
scendants a high early mortality, especially early prenatal. The 
treatment of females produces in their descendants a high later 
mortality, especially late prenatal and postnatal. The treatment 
of male and female ancestors produces in their descendants 
the highest early prenatal mortality, but the lowest late pre- 
natal and postnatal mortality. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 181 


In table 4, male and female ancestors treated does not neces- 
sarily indicate that all records in the column were derived from 
matings between two alcoholic parents, since both males and 
females may have been treated among either the ancestors of 
the mother or the father, but not necessarily both. For this 
reason the sixth and ninth columns of table 3, in which both 
parents were in all cases from alcoholic ancestry, show more 
decidedly that two alcoholic parents when mated together give 
the very highest early prenatal mortality, but a low late pre- 
natal and postnatal mortality. This last conclusion is extremely 
_interesting in connection with Pearl’s results on fowls. 

Pearl found that when two alcoholic fowls were mated to- 
gether, the percentage of infertile eggs was higher than from 
any other combination, while the prenatal mortality, embryos 
dying in shell, and the postnatal mortality were the lowest. 
This is exactly what the guinea-pig records show, provided 
our ‘early prenatal mortality’ (indicated by the small litter 
size, the frequent mating failures, and the observed mortality 
occurring in utero during all later stages of development) can 
be considered the same as many of Pearl’s ‘infertile eggs.’ 
Without intending any adverse criticism of the designation 
‘infertile,’ we may again suggest the possibility that a certain 
proportion of these eggs had really begun development, but had 
died in the early cleavage or gastrular stages, and yet on ex- 
amination, other than a minute microscopic study, they ap- 
peared as infertile or unfertilized eggs. If this were true, they 
could be classed in the early prenatal mortality records. Such 
an adjustment would serve to harmonize the fowl and guinea- 
pig records in another important respect. 

Pearl has attributed the good qualities of the offspring from 
his alcoholic parents to a germinal selection which has tended to 
cause all weak germ cells to be completely put out of commis- 
sion by the alcohol treatment and only the very best have sur- 
vived to produce embryos, and these therefore show a low per- 
centage of deaths in shell and a low postnatal mortality. A 
selection is also playing its réle in the case of the guinea-pigs, 
but here it is not acting alone on the germ cells, but more evi- 


182 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


dently on the developing individuals. The selection in our case 
is a continuous selection of individuals, eliminating, no doubt, 
certain of the least resistant germ cells, but continuing to act on 
the embryonic population to eliminate the most defective of 
these during very early developmental stages, and so on until 
the individuals born are a mixture of strong specimen and others 
only sufficiently strong to have reached birth and possibly to 
survive in a subnormal fashion for a shorter or longer time. This 
continuous, both germinal and individual, selection seems to us 
more to be expected than the abruptly broken germinal selec- 
tion advocated by Pearl, which completely eliminates all weak 
germs, and therefore no weak individuals begin development. 
We must admit that the data from Pearl’s double alcoholic 
matings considered alone strongly suggest only a germinal selec- 
tion, but the results from our double alcoholic matings, while 
leaning in the same direction, still show a greater late prenatal 
and postnatal mortality than do the control matings, and in 
addition present much evidence to suggest a very high early 
embryonie elimination. This same early embryonic elimination 
may be included among the high percentage of infertile eggs re- 
sulting from the matings of two alcoholic fowls, and in the 
case of the fowls it may be so much more severe that the later 
mortality records compare favorably with the control. This 
again would lead us to an abrupt break after the high very 
early prenatal mortality and might be thought to vitiate our 
entire supposition, yet the guinea-pig records show almost all 
gradations up to the condition for the fowls. 

Our results show that in the alcoholic lines the higher the 
early prenatal mortality and consequently the smaller the aver- 
age-size litter, the lower the late prenatal and postnatal death 
rate, much as Pearl also finds for fowls. These findings will be 
still further discussed in connection with the sex ratio, table 6. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 183 


10. TREATING MALES WITH ALCOHOL FOR ONE AND TWO GENERA- 
TIONS AGAINST TREATING FEMALES FOR ONE AND 
TWO GENERATIONS 


An experiment has now been in progress for some time in 
which straight male lines have been treated with alcohol for sev- 
eral generations in order to compare the results with those from 
the treatment of straight female lines for several generations. 
That is, the original males are treated, their sons are then di- 
rectetly treated, their grandsons, great-grandsons, and so on; 
these we consider the straight male lines. The treated females, 
their directly treated daughters, granddaughters, and so on 
constitute the straight female lines. There are now a few third- 
and fourth-generation individuals, though not a sufficient num- 
ber to tabulate. We shall thus for the present confine our 
attention to the records from the originally selected and treated 
males and females and the treated sons of these males and 
daughters of the females. 

The records are arranged in table 5. In considering the 
table it must be stated that the original animals in this experi- 
ment have been carefully selected’ large strong specimens that 
were particularly good breeders. Such a choice has been made 
on account of the severity of the treatment to which the de- 
scendants are to be subjected through a number of generations. 
Only the best animals are likely to produce descendants suffi- 
ciently strong to be treated with alcohol and to continue to re- 
produce for one generation after another. The fact that such 
a selection is possible does not reflect on the general population, 
since no population is so perfect that certain individuals are not 
better than others. This selection probably accounts for the 
presence in all the groups of table 5 of some offspring of unusually 
large size. 

The pedigrees or conditions of the animals in the different 
generations are expressed by the following symbols or formulae. 
A normal animal is represented by the letter N and one treated 
with alcohol by A. The symbol for the male is placed to the 
right of that for the female. Thus the first column NN are the 
normal control animals for comparison, the second column NA 


184 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


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MODIFICATION OF THE GERM-CWLLS IN MAMMALS 185 


show records of offspring derived from a treated father A and 
a normal mother N. The third column are offspring from 
treated males which were also derived from treated fathers and 


normal mothers, mated with normal females, N. The 


A 
A”? 
next straight male generation treated and paired with normal 
NA 


females would be expressed by N A _, the offspring from such 
a combination would have had their father, a grandfather, and 
a great-grandfather treated with aleohol and their mother, 
grandmothers, and great-grandmothers all normal, and so on for 
later generations. Animals of these higher pedigrees will be 
recorded in a future communication. In the table 5 only records 
from treated fathers are given in column 2 and from treated 
father and grandfather in column 3. The fourth column shows 
records from normal fathers and treated mothers, AN, and the 
fifth column from normal fathers mated with treated females 


r 


4 ; AN 
which were derived from treated mothers, ae IN 


The numbers in all of the columns are rather small, but in 
every case the records differ from the control.- There is a re- 
markable similarity between the two treated-male groups and 
also between the two treated-female groups, but a striking 
contrast exists between the male records as a class and the 
female records. 

In the two male columns the average litter is very small and 
the mating failures high. The percentage of surviving young, 
though well under the control record, is equally above the fe- 
male records. The corrected total mortality in both columns 
is over 180 against 100 for the control. The proportion of late 
prenatal to postnatal mortality is slightly contrasted in the 
one treated male generation column, but more so in the two 
treated generations column. There are no defective animals in 
the NA column, but a small per cent of such are seen in the 


N oe group. 


186 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


The average litter size is high in both female columns and the 
mating failures lower than in the male groups. While the total 
mortality is extremely high, being in the two-generation treated 
column on the basis of litter size over twice as high as either 
treated-male column and about four times the control record. 
The proportion of late prenatal to postnatal mortality is in the 
first female column over three to one and in the last column 
over six to one. There were some defective animals in both 
female groups. The records of the females in this and the two 
preceding tables are out of accord with the records from fowls 
and do not fit an explanation based on a germinal selection or 
partial infertility. The total productivity is good and the late 
prenatal and the postnatal mortality are high. 

It is seen at once that the records from the treated-female 
generations are far worse than from the treated-male generations; 
in fact, so much worse that we are led to conclude that the alco- 
hol has not acted on exactly the same things in the two ceases. 
The increased effect of the treatment in the double female column 
is much more evident than in the two male generation column. 
The results in the male columns are due only to an action of the 
treatment on the spermatozoa or male germ cells, while the re- 
sults in the female columns are also due to the effects of the 
treatment on the germ cells or ova, but more largely to the 
effects of the alcohol on the developing embryos within the 
uterus of the treated mother. Provided the effects of alcohol 
were equal on the sperm and ova of guinea-pigs, the difference 
between these two sets of records would then represent the 
action of the treatment on the developing embryo itself. 

Although the records in table 5 involve only small numbers, 
we are led to believe that they represent the true trend of the 
effects, since they harmonize so perfectly with the data of dif- 
ferent composition yet much more complete shown in tables 
3 and 4. 

Here again, as in tables 3 and 4, the treated-male lines show 
the early prenatal mortality (based on the average litter size 
and frequent mating failures) to be unusually high while in the 
female line it is low. In the male lines the late prenatal and 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 187 


total mortality is low while in the female lines the late pre- 
natal mortality is extremely high and the total mortality very 
great. 

Finally, this table may be considered as supplying evidence of 
the increased effect of higher or longer alcoholic dosage. The 
double male records which have usually been derived from ani- 
mals that have had longer or more treatment during the two 
generations are somewhat inferior to the one generation male 
treated records, and this inferiority is very much more decided 
for the female groups in the case of the higher-dosed two-genera- 
tion records. 


11. THE SEX-RATIO IN RELATION TO PATERNAL AND MATERNAL 
ALCOHOLISM AND TO THE TREATMENT OF MALE 
AND FEMALE ANCESTORS WITH ALCOHOL 


In the last group of table 3 it will be remembered that all of 
the non-inbred acoholic descendants were separated into three 
classes with only father alcoholic, only mother alcoholic, and 
both parents alcoholic. Again, in the last group of table 4, 
these 594 animals were rearranged into three classes, from only 
male ancestors treated, only female ancestors treated, and both 
male and female ancestors treated. The difference between 
these elassifications are made clear in the discussion of tables 3 
and 4. If we now record the number of males and females com- 
posing each of these six classes and express their sex-ratios on 
the basis of the number of males to every 100 females, a most 
peculiar result is obtained, and one for which it is very difficult 
to give a completely satisfactory explanation. 

The number of males and females and their mortality records 
in each of the six classes are shown in table 6. As a standard of 
comparison the 233 control animals are similarly recorded in 
this table. For further comparisons a total sex-ratio and the 
sex-ratios for animals born in different size litters are given be- 
low the table. The total sex-ratio calculated for about 1600 
animals is 109.6; that is, 109.6 males to every 100 females. Many 
of these animals were from alcoholic lines, so that this sex-ratio 
may not be exactly normal. Yet a further perusal of the table 


AND GEORGE N. PAPANICOLAOU 


STOCKARD 


R. 


CHARLES 


188 


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MODIFICATION OF THE GERM-CELLS IN MAMMALS 189 


will suggest a tendency on the part of the different alcoholic 
lines to level the sex-ratio to normal when they are combined as 
a grand total, yet we are not by any means comparing the sex- 
ratios from the alcoholic lines with an average alcoholic ratio. 
The sex-ratios of 194 animals born one in a litter was 113; of 
578 born two in a litter, sex-ratio 114.8; of 579 born three in a 
litter, sex-ratio 101.7, and of 248 animals born in litters of four, 
the sex-ratio was 106.6. 

The first column of table 6 shows that of the 233 non-inbred 
control animals, 120 were males and 106 were females. ‘The 
proportion of males to females is thus 113.2 to 100; that is, a 
sex-ratio of 113.2. The average-size litter in which these ani- 
mals were born is shown in parentheses in the sex-ratio space as 
2.77. The mortality record for the males was about the same as 
for the females, having only a very slight advantage. 

The third column of animals from only mother alcoholic are 
also in the majority of cases individuals from only female an- 
cestors treated, the sixth column, but not entirely so, as many 
of the mothers may have been alcoholic on account of a treated 
father or grandfather. In general, however, the third and sixth 
columns are rather the same in composition, the sixth being a 
purely female treated group while the third column is largely 
so but not entirely, and while not necessarily to be treated 
together they may be considered in connection with one another. 
A point of immediate notice is that the sex-ratios in both of 
these columns, 96.8 and 86.5, are very low. 

When only the father is alcoholic, second column, or only the 
male ancestors are treated, fifth column, the sex-ratios are higher, 
101.7 and 109.1. While if both parents are alcoholic, fourth 
column, or male and female ancestors are treated, seventh col- 
umn, the sex-ratios are very high, 121.1 and 123.5. It must 
also be noticed that these differences in sex-ratios are more 
-accentuated in the last three columns, giving the descendants 
from only female ancestors treated with alcohol the lowest sex- 
ratio in the entire table; those from only male ancestors treated 
a considerably higher ratio, and from both male and female 
ancestors treated the highest sex-ratio for all groups. A differ- 


190 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


ence of 37 between the number of males to 100 females in ani- 
mals from treated-female ancestors as compared with those 
from both male and female ancestors treated is indeed very 
creat. 

Are these differences in sex ratio a result of the direct influ- 
ence of alcoholism upon sex determination or sex differentiation? 
Or are they indirectly brought about by a difference between 
the early prenatal mortality rates of the two sexes in the sev- 
eral groups considered? Or are these merely chance differ- 
ences? There is no doubt that chance plays a large part in the 
make up of all sex-ratios, but to be consistent in six straight 
cases as the six groups show can scarcely be dismissed as a 
chance result. 

It is very peculiar that these different sex-ratios should coin- 
cide in a direct manner with differences in the early prenatal 
mortalities among the several groups of table 6, and thus suggests 
that the explanation for the sex-ratio differences may be in part 
at least along the line of the second of the above propositions. 
After considering this probability of differences between the 
early prenatal mortalities of males and females, we may then 
discuss the further possibility of the direct effects of the treat- 
ment on the sex-ratios. 

The groups having the lowest sex-ratios, female lines with 
ratios 96.8 and 86.5, also have, as shown in tables 3 and 4, the 
largest average litters, 2.69 and 2.66, or the lowest early pre- 
natal mortality. The lines having a somewhat higher sex-ratio, 
male lines with ratios 101.7 and 109.1, have correspondingly 
somewhat higher early prenatal mortalities, as indicated by the 
smaller average litters, 2.41 and 2.42; while the lines having 
the highest sex-ratios, double lines with ratios 121.1 and 123.5, 
have along with these the highest early prenatal mortalities as 
shown by the smallest average-size litters, 2.28 and 2.37. 

This is certainly a very suggestive parallelism. And if one 
now considers the fourth line of the table giving the total dead 
of each sex, in every column, with one exception, it will be seen 
that the female mortality is higher than the male. The ex- 
ception is in the column from both parents alcoholic; here the 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 191 


sex-ratio is very high and yet the late male mortality is higher 
than that for the females. The total mortality for the females 
is higher than that of the males, 31.71 per cent against 29.68 
per cent. It is further shown below the table that small litters 
have a higher sex-ratio than large litters, the sex-ratios for litters 
of one and two young being respectively 113.1 and 114.8. While 
for litters of three and four young the sex ratios are 101.7 and 
106.6. It has been pointed out before that the small litters are 
often due to an early prenatal mortality which has destroyed 
some of the original members, and since the sex-ratios of such 
litters are high the majority of embryos dying may have been 
females. We may see finally by a study of table 7 that female 
animals are generally smaller at birth than males in the same 
litter, and as their total higher mortality would indicate, they 
are probably also weaker. 


SWAB SS Vile, 
THE BIRTH WEIGHTS OF MALE AND FEMALE MEMBERS OF MIXED LITTERS 


Number of Totalweight |Total weighf | Total excess |Average excess |Percentof excess 
Litters Of males in|of {females | weightof males |weightof males|weightof males 
: Grams ingrams  joverfemales |over females |over females 


Litters of 105 736! 7525 336 
(lit (ES 


Litters of 136 
aleS 


Litters of 125 4428 
18 22 | (8856) 


9513 


Litters of 
i 
282 3G 2325 


Table 7 only includes mixéd litters; that is, those containing 
both male and female members. It shows that in 105 litters of 
two animals of opposite sex the total birth weight of the 105 
males was 7861 grams and of the 105 females only 7525 grams, 
or 336 grams less. The average excess weight of males over 
females in these litters of two was 3.2 grams, giving a percent- 
age of excess weight of 2.18 in favor of the males. 

One hundred and thirty-six litters of three, consisting of two 
males and one female, are recorded. The total weight of the 


192 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


272 males was 9513 grams; that for the 136 females was 4654 
erams. If this total weight be doubled for comparison with the 
total weight of the double number of males, we have 9308 
grams. The males again have a total advantage, amounting 
here to 205 grams. The average excess weight of the males is 
0.75 gram, or a 1.08 per cent excess weight of males over 
females. 

It will be noted in this table that the average weight of the 
individuals is very low. This is due to the fact that a number of 
abortions in which the sex could be distinguished, as well as 
premature still-births are included. ‘These small specimens 
have brought the average in some cases almost below the birth 
weight which permits survival. It is also noticed that the 272 
males in the second line only weigh about one-quarter more 
than the 105 males in the first line of the table, and this is due 
to the fact that there were many more abortions and early pre- 
mature births of litters consisting of three individuals than of 
two. While the males in the litters of one male and one female 
averaged almost 75 grams, the males in litters of two males and 
one female averaged less than 35 grams. 

The third line of the table shows 125 litters of one male and 
two females. The 125 males weighed 4428 grams which may be 
doubled to give 8856 grams for comparison with the total weight 
of 8790 for the 250 females. There is a total advantage of 66 
grams in favor of the males. The average excess of male weight 
is 0.26 gram, or 0.37 per cent over female weight. 

The last case of thirty-six litters, consisting of two males and 
two females each, gives a total excess of 85.5 grams to the males. 
The average excess weight of the males over females is 1.19 
grams, and the per cent of excess of males over females is 1.87. 

It is thus seen that the males born in litters consisting of 
both sexes possess a superiority in body weight over the females in 
every combination. We do not attribute this constant excess in 
favor of the males to a sexual dimorphism in size. In a group 
of guinea-pigs both young and adult females are often larger in 
size than comparable males, and no constant size difference be- 
tween the two sexes is known. It seems more probable that 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 193 


this advantage in weight on the part of the males, the majority 
of which are of alcoholic ancestry, is in line with the lower 
mortality records of the males shown in the various columns of 
table 6. And this may further bear on the explanation of the 
high sex-ratios in those lines with high early mortalities or small 
average litters. 

There is, therefore, much evidence to indicate that among 
aleoholic guinea-pigs the females very probably suffer a much 
higher early prenatal mortality than do the males, and it is 
shown that the female mortality is higher than that of the males 
at all other periods, table 6. 

Before proceeding further with our theoretical explanation of 
the different sex-ratios in the several groups, which leads finally 
to a consideration of views expressed in a previous communica- 
tion, still another important relationship may be pointed out 
between early prenatal mortality and the sex-ratio, on one 
hand, and the late prenatal and postnatal mortality, combined in 
table 6 under ‘total dead,’ on the other. Stated concisely, the 
higher the sex-ratio and the early prenatal mortality, indicated 
by the small average litter, the lower will be the total late pre- 
natal and postnatal mortality, and vice versa. The columns 
with the highest sex-ratios, 123.58 and 121.17, and at the same 
time the highest early prenatal mortalities or the smallest aver- 
age litters, 2.37 and 2.28, show the lowest late mortalities, 25.55 
and 27.12 per cent. In the opposite way the columns with the 
lowest sex-ratios, 96.8 and 86.51, and the lowest early prenatal 
mortalities, or the largest average litters, 2.69 and 2.66, have the 
highest later mortalities, 34.93 and 32.52 per cent. This is in 
line with what was brought out during the discussion of table 4 
showing that the higher the early prenatal mortality, or the 
smaller the average litter, the lower will be the late prenatal and 
postnatal mortalities. 

There is one very evident objection to the foregoing explana- 
tion of the peculiar sex-ratios as being due to a differential sex 
mortality during the early prenatal periods. That is, among 
the normal stock the sex-ratio is rather high, although the early 
prenatal mortality is probably very low as indicated by the 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, No. 1 


194 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


large average litter. With a large average litter the sex-ratio 
should be very low as in the female lines. We could only avoid 
this difficulty by assuming that the control lines are out of the 
consideration, since the other sex-ratios being discussed are all 
shown by modified alcoholic groups among which entirely dif- 
ferent conditions obtain from those existing in the control. 
Whereas there are reasons for such a position, it would seem 
preferable at present to admit that the case of the control is a 
real objection. And such an objection would serve to indicate 
that while a higher mortality on the part of the female embryos 
in the alcoholic groups might actually exist, yet it accounts only 
in part for the peculiar sex-ratios found. A recognition of the 
normal record also makes it difficult to account for the very 
low sex-ratios of the female lines. Here the early prenatal mor- 
tality was low on the basis of the average size litter, but if any 
early prenatal mortality did occur it could not have been partial 
to the female embryos, but must on the contrary have been 
confined almost totally to male embryos or else a sex-ratio could 
never fall 25 below the control.. Is it possible that wherever a 
treated male is concerned, as in the male columns and the double 
columns of table 6, there is a high early prenatal mortality among 
the female embryos, and on the other hand where only a treated 
female is concerned there is a high early male mortality? It is 
difficult to believe so, and therefore differences between the early 
mortalities of the sexes can, on our present data, only partially 
explain the sex-ratios found in table 6. This leads to a final ex- 
planation which may seem highly theoretical, yet it does have a 
basis of fact. 

In an earlier communication (Stockard and Papanicolaou, 716), 
we presented some evidence which seemed to indicate a possi- ° 
bility that the action of the alcohol treatment not only differed 
in its effects upon the two sexes treated, but also acted differently 
on the two groups of spermatozoa in the male, the so-called 
male-producing and female-producing sperm. 

We suggested that the action of the treatment was more se- 
vere on the germ cells of the male than on those of the female; 
in other words, that the spermatozoa were more susceptible 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 195 


than the ova. The inferiority of the column from male ances- 
tors treated as compared with that from female ancestors treated 
in the second group of table 4 seems to substantiate such a 
position. The possibility exists, however, that the treatments 
of the male and female ancestors may not have been equally 
severe, since they have been treated in different fume tanks. 
This question is now being studied. At any rate, we believe it 
is proved that the germ cells of the female are as definitely in- 
jured and modified by the treatment as are the germ cells of the 
male. This is the point of importance in the present connection. 

The female offspring from treated fathers were found in the 
report cited to be inferior as a group to the male offspring as 
regards their powers of existence and structural perfection. The 
opposite was indicated among the offspring of treated mothers, 
the males being inferior to the females. Our explanation of 
these conditions was that the two classes of spermatozoa which 
differ structurally also differ in the degrees of injury suffered 
from the treatment. We are further testing these suppositions 
by selected matings and hope to report on them in the future. 
For further details regarding the supposed differences between 
the behavior of the two classes of spermatozoa, the reader is 
referred to our 1916 paper. 

An explanation of the sex-ratios in table 6 may now be given 
along similar lines and the peculiarities found among these sex- 
ratios are exactly in accord with our previous theoretical consid- 
erations. Ifthe male guinea-pig does possess, as has been claimed 
(Stevens, *11), heteromorphic spermatozoa, one class with a 
small Y chromosome, the male producing, and the other class 
with a larger X chromosome, the female producing, the follow- 
ing may be assumed: In the treated-male lines the female-pro- 
ducing spermatozoa are more decidedly affected, possibly on 
account of their larger quantity of chromatin, and therefore, in 
the competition to fertilize the eggs they are not so successful 
as the less injured male-producing sperms. Consequently, more 
male animals are produced than female. Or, if the female- 
producing sperm are not in any or all cases actually prevented 
from fertilizing eggs, nevertheless the individuals produced by 


196 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


such a fertilization are inferior and more apt to die during early 
developmental stages, and thus a greater number of male em- 
bryos would survive and be born. 

When the alcoholic mother or early female treated lines were 
mated with untreated normal males, the sons were inferior to the 
daughters. Here again, taking into consideration the two 
structurally different classes of spermatozoa, the normal males 
paired with alcoholic females contribute a smaller amount of 
normal chromatin to the complex producing male offspring than 
to that giving rise to the female offspring. ‘The records of the 
males are hence inferior to those of the females. And in the 
present connection such males might be expected to suffer a 
higher early prenatal mortality and so give rise to the very low 
sex-ratios shown by the columns from ‘only mother alcoholic’ 
and ‘only female ancestors treated.’ 

Such reasoning from the present data is admitted to be highly 
speculative; nevertheless, if the morphological differences which 
have been found to exist between the two classes of spermatozoa 
in a number of animal species have any significance, they must. 
sooner or later be recognized as the underlying cause of such 
results as table 6 shows for sex-ratios in aleoholized guinea-pigs. 

These ideas also account for the fact that the sex-ratios of 
the normal animals is out of accord with the ratios of all the 
treated groups on the basis of the average litter size. This dis- 
cord was recognized as a possible objection to the purely dif- 
ferential sex mortality explanation previously discussed. In the 
present connection we may take the following position. 

The normal group has been subjected to no injurious action 
which has tended to modify the expression of the sex-ratio, 
while in the alcoholized groups there is evidence of a deviatien 
from the normal, in one direction or the other, depending upon 
the combination concerned. And this deviation is imagined 
to be due to a lower fertilizing ability on the part of certain 
spermatozoa. 

There is another question to be considered in connection with 
the differences in response on the part of the two classes of sper- 
matozoa; that is, the possibility of certain eggs being more sub- 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 197 


ject to fertilization by either the X or Y type of spermatozoa. 
Even though the egg might be practically equally accessible to 
both types under normal conditions, a peculiarly affected egg 
might become much more readily fertilized by one class of 
sperm than the other, and almost all male offspring might re- 
sult in one case and females in the second. .One might feel 
that these are large suppositions on the basis of the minute 
differences between the two groups of sperm. But it may be 
replied that the differences are only minute from the standpoint 
of the minuteness of the structure considered. Corresponding 
differences between great things would necessarily seem much 
more important, but with present powers of observation only 
very great differences between cellular structures are visible 
at all. 

There is evidence from a study of the control of sex-ratios 
in normal guinea-pigs to indicate that certain females have a 
very strong tendency to produce male offspring regardless of the 
male with which she is paired (Papanicolaou, 715). Other fe- 
males have as decidedly marked tendency to produce female 
offspring. Such females may be said to have either a male or 
female tendency, while other females are in this regard indif- 
ferent, producing as many offspring of one sex as of the other. 
These tendencies may be explained in accord with the above dis- 
cussion as due to a high affinity for one type of sperm on the 
part of the ova of one female, while the ova of another female are 
particularly susceptible to fertilization by the other class of 
sperm. The indifferent females are those with ova which are 
fertilized equally as well by one type of spermatozoa as the 
other. There are striking cases among the ascidians and other 
forms illustrating selective fertilization, and the above suggestions 
are by no means without foundation. 

Certain male guinea-pigs are also known to have a strong 
tendency to beget female offspring regardless of the females with 
which they are paired. Other males have a high male-producing 
tendency and still others are more or less indifferent in their 
sex-determining quality. This may be readily imagined to re- 
sult from a difference in the activity or fertilizing powers of the 


198 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


two types of spermatozoa in certain male animals. And there 
is evidence to show, as cited in our previous paper, that the 
fertilizing power of the spermatozoa may be modified in such a 
way as to render them much less capable of success. If this is 
the case, we may be justified in assuming that one class of 
sperm may often, even under normal conditions, be at a disad- 
vantage as compared with the other. It is even more prob- 
able that under modified conditions the two morphologically 
different classes of spermatozoa will not be affected to equal 
degrees. 

In conclusion, then, it seems highly probable that the peculiar 
sex-ratios shown by the several groups of treated animals re- 
corded in table 6 are in part due to differential sex mortalities - 
during early prenatal stages, on account of the close correlation 
between the sex-ratios and the average litter sizes. This differ- 
ence in early prenatal mortality between the sexes does not, 
however, completely satisfy the case. The sex-tendency of the 
animals considered and the possibility in the case of delicate 
treatment of affecting the two types of spermatozoa in different 
ways or degrees are certainly factors to be recognized in the 
production of the results obtained. 

Pearl found that for fowls treated with alcohol the relative 
proportions of the sexes produced were not significantly different 
from normal control series. Our results for the sex-ratio of the 
total alcoholic series agree with Pearl’s findings. The sex-ratio 
of the 594 alcoholic animals considered in the present paper is 
105.6, which, in view of the numbers involved, is not signifi- 
cantly different from the control series. Yet studying separately 
the several groups shown in table 6, we find strikingly wide dif- 
ferences in the sex-ratios and the arrangement of these differ- 
ences is decidedly consistent. From the standpoint of the 
above discussion it seems to us legitimate to consider the six 
groups individually, or at least as three classes, since there is a 
probability that different processes or conditions are affecting 
the results in the different cases. Several recent experiments 
on the modification of the sex-ratio would tend to strengthen such 
a probability. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 199 


12. THE BIRTH WEIGHTS AND RATE OF GROWTH IN THE NORMAL 
AND THE ALCOHOLIC SERIES 


In the present section the birth weights and ability to grow 
of the animals born in the normal and the alcoholic series may be 
compared. Here again comparisons must be made between ani- 
mals born in litters of the same size. It may be expressed gen- 
erally, as was done above for the mortality rate, that the birth 
weight of an animal, either normal or alcoholic, varies inversely 
with the size of the litter in which it is born. The average daily 
increase in weight during the first month varies in the’ same way. 
So that when one month old the weight of a guinea-pig also as a 
rule varies inversely with the size of the litter in which it was 
born. This condition holds up to three months, at which time 
the guinea-pig is mature. But the daily gain in weight during 
the second and third months after birth ceases to be greatest for 
the members of small litters. Yet the advantage in growth rate 
comes to the members of the large litters at so late a time that 
they are unable to make up their disadvantage sufficiently to equal 
in size the members of small litters within three months. All of 
these statements apply equally to both the alcoholic and normal 
series, and thus the influence of the litter size in general is the 
same in both cases. 

The question then arises whether there is an actual difference 
in birth weights and growth rates between the two series. Table 
8 contains the birth weights of 225 normal control and 531 ani- 
mals of the alcoholic series. This alcoholic group, as the fore- 
going tables show, not only includes F; animals, or offspring from 
directly treated parents, but also their descendants for several 
generations, F;, F;, and F,;. The animals of both series are 
arranged in table 8 according to the size litters in which they 
occur. 

A review of the table shows that the normal series is superior 
in the average birth weight of the individual and the average 
birth weight of the entire litter, as well as the average birth 
weight of the individual born in each of the five different-size 
litters. 


200 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


TABLE Vill 
BIRTH WEIGHTS AND RATES OF Ce OF 
NORMAL AND ALCOHOLIC YOUN 


Normal Alcoholic 


i 2 3 ca 5 i 2 3 4+ 5 
Weis b + 1035S 3719. 630 4125 Chal 4023 13867 1474! 5200 244 
ae lo 4G 37 GT 15 4! 163 225 92 Ss 
at birth (108.5) (82-15) (Jo-83) (61.56) (62-73) (48.42) (82.54) (65.5!) (56-52) (49-20) 
(Average 71- 1b) (Average 10-35) - 9.23% (ote mesn) 
Average productivity 197-12 ape Average productivity 170.0 Est 
; l D. 3 4 5 ! a 3 4 
Weight at 2228 9902 14288 6750 2302 2633 3o4b! 26398 6831 a 
the end of the 7 31 63 31 12 24 131 133. 0 z 
first TOA. (412-28) (240.59) (20-11) (122-43) (191.83) (247.63) (232-52) (193-48) (172-02) ({L4+0) 
(Average 228.64) (Average 213-94) —.6- 63% 
Averagedailyinuease] 6.24 San #64 402430 6.64 443 3-35 
inweight during the (Average 5.04) (Average 478) 4 


first month 


= ! 2 3 4+ 5 ! 3 > 5 
Weight at ihe Avs b4S4 25643 ssa abe fe Ast 7449 718 


end of the third 3 33 62 33 12 3! Jou 32 2 
menth (501,62) (433-0) (413.58) (330-36) (397.0) (460.12) “nes (404-32) (367-15) (354.0) 
(Average 425.11) (Averaze404.13)- 5.06% 


Average daily increase] 3.05 3.20 3.39 324 3.41 een 
inweight duying the 


2"4 and td months (Averages 3, 26) (Average 3.16) 


The average birth weight of the individual in the normal 
series is 77.16 grams against 70.35 grams for the alcoholic, and 
the average litter weight is 27.12 grams heavier among the nor- 
mal animals. The average weight of the individual in a given 
size litter is shown in parentheses below the litter number; this 
is obtained by dividing the total weight in grams of all such 
litters by the total number of animals composing them. For | 
example, in the alcoholic series there are 168 animals born in | 
litters of two and their total birth weight was 13,867 grams, | 
which gives an average weight of 82.54 grams per individual. | 
The average weight of the individual is lower in the large litters 
than in the small ones in both series. 
The second line of the table shows in a similar way the total 
weight at the end of the first month of all individuals in the sey- 
eral-size litters and below this the number of individuals con- 
cerned in each case. The quotient obtained by dividing the | 
total weight by the number of animals is given in parentheses | 
as the average weight of the individual in each litter at one | 
month old. At this age the average weight of normal animals | 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 201 


in litters of one was 318.28 grams against 297.68 grams for the 
alcoholic litters of one. The uence average weight at one 
month for the normal series was 228.64 grams against a general 
average of 213.94 grams for the alcoholics. 

The average daily increase in weight during the first month 
is given in the third line of the table. It shows a mean daily 
increase for normal animals of 5.04 grams and for alcoholic 
animals only 4.78 grams. Members of small litters in both 
groups gained more rapidly than members of large litters. 

The weights at the end of the third month, when the animals 
are about mature, are given in the fourth line of the table. Nor- 
mal animals born one in a litter average over 500 grams, while 
comparable alcoholic animals weigh only 460.12 grams. The 
average normal animal at three months old weighs 425.11 grams 
against an average of 404.15 for the alcoholic animal. 

The last line shows that the average daily gain in weight 
during the second and third months was about as great for the 
alcoholic animals as for the normals. A much greater selection 
or elimination has taken place previous to this time among the 
alcoholic series than among the normal, as a reference to any of 
the mortality tables will show. 

All in all, table 8 would seem to indicate that in every case 
the normal offspring weigh more and grow more rapidly silo 
after birth than do the young alcoholic specimens. 

The several points considered above and their general meaning 
may be much more clearly expressed in the diagram, figure 9. 
On the left side of the diagram are shown the records for the 
aleoholic series and the normal records are on the right. The 
shaded right-angle triangles represent the difference in average 
weight between the individuals in litters of one, two, three, four, 
and five at birth, at one month old, and at three months old 
from the two series. The altitudes of the right triangles measure 
the magnitude of the differences. 

Animals born one in a litter in the alcoholic and the normal 
series, as the bottom short triangle indicates, show a greater dif- 
ference in weight than those from any other size litter except 
that consisting of five individuals as the low long triangle repre- 


t 
} \ 
i | 
| | 
j | 
| | 


(a1) e2arlbj 
aT 


(LE) er agi 


| 


(sielzx9 
ADU ULS 


ADYI| UL ay 


! 
1 
(bs) 9s 19) 
1 
I 


(9) bs elt | 


a 
he) 
+ 


(£8) &8:0L 
Jatyi| Ul 


Aa] Ul? 


(3) tp 1ES 
AOU) ULI 


Normal lines 


Aa} Ul | ¢ 


bsp Lye 
' 


Aayl\ Ul 7 (891) 5° 
i 


Alcoholic lines 


1393 


AO |) Ul & (*9l) TE" bow Grt)| 959 
! ! 
do}y 4! +7 @e)si LIE eh) 15°9E 
U { 
Aayt| ug (e691 (S)0g8+ 


Fig. 9 Diagram illustrating the differences in weight between normal and 


alcoholic line animals born in litters of the same size. 


The weights are given at 


birth, at one month, and at three months old. Further explanations are to be 


found in the text. 


Ww) 


20 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 203 


sents. There is little difference between the birth weights of 
normal and alcoholic animals born in litters of two, three or four. 

When one month old the middle group of triangles represent- 
ing by their position the weights in grams again show the largest 
differences between alcoholic and normal animals in litters of 
one, the short triangle, and litters of five, the long triangle. The 
normal animals in litters of one have passed the 300-gram line 
in weight, while the average alcoholic member of a litter of five 
weighs only 169 grams. Members of the two series in litters of 
two, three, or four do not show very great weight differences. 

The top triangle shows a very large difference in weight at 
three months between normal and alcoholic animals born one in 
a litter. The triangles for two and three in a litter animals are 
almost flat at three months, indicating very little difference be- 
tween such normal and alcoholic animals. Alcoholic members 
of litters of four are somewhat smaller in average than normal, 
while alcoholic from litters of five are far below the normal in 
weight as the long triangle shows at three months. 

We have here an example of the influence of the alcohol effect 
combined with the action of a normal condition, the condition 
being the size of the litter in which the animal is born. From a 
consideration of the diagram we may, therefore, conclude, first, 
that normal-stock animals born one in a litter are so strong as to 
run far ahead of the one in a litter alcoholic animals, although 
the latter at birth, at one month, and at three months are much 
heavier than all normal animals born in larger litters at similar 
periods. Consequently, the advantage of developing alone in 
the uterus is sufficient, so far as birth weight and rate of growth 
are concerned, to overcome the disadvantages resulting from 
aleoholic ancestry to such a degree that these individuals are 
better than control animals developing in larger litters. Yet in 
birth weight and growth rate these singly born alcoholic animals 
are further behind the singly born control than are the alco- 
holies from any other size litters behind the control from the 
same size litters. Thus, although being born alone tends to 
overshadow the alcohol effect, nevertheless the effect is still 
shown by comparison with control specimens born alone. 


204 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


If we now reeall the fact that alcoholic animals produce more 
small-size litters than do the control, and recognize that mem- 
bers of small litters in all cases weigh more, grow faster, and are 
more apt to survive than members of larger litters, it becomes 
evident that the production of a high percentage of small litters 
is a fortunate provision tending to preserve the alcoholic stock 
by counterbalancing to some degree the magnitude of the 
effects induced by the alcoholism. 

Second, animals born in litters of two or three have a tendency 
to weigh the same at birth and to grow at a similar rate dur- 
ing the first three months, whether they are from the normal or 
aleoholie stock. In other words, being born in litters of this size 
gives no great advantage to the normal animals over the alco- 
holies, as does being born in litters of only one. Or stated re- 
versely, members of litters of two or three are not placed at a 
great disadvantage so far as birth weight and growth rate are 
concerned on account of their alcoholic ancestry, as is found 
below to be the case for the members of larger litters. 

In the third place, when animals are born in litters of four 
the aleoholic stock are at a disadvantage in birth weight when 
compared with the normal. The rate of growth of the alcoholic 
animals from litters of four is also slower than that of the com- 
parable control animals. 

Lastly, in the fourth place, alcoholic animals born five in a 
litter are very small and weak and only a few survive, yet these 
selected few fall far behind the normal animals from litters of five 
in their rate of growth. Thus at three months there is a greater 
difference in average weight between the alcoholic and control 
members of litters of five than between the members of any other 
size litters in the two series, except the animals born singly. 
The alcoholic animals as a group are at a disadvantage in birth 
weight and rate of growth, but when born in large litters of four 
or particularly five, this disadvantage is greatly exaggerated by 
the handicap which befalls the members of all large litters, the 
control as well as the alcoholic. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 205 


13. THE RECORDS OF NORMAL MALES AND FEMALES PAIRED SUC- 
CESSIVELY WITH NORMAL AND ALCOHOLIC MATES: THE 
CRUCIAL DEMONSTRATION OF THE EFFECTS OF 
ALCOHOLISM ON THE OFFSPRING 

When the records of any group of experimental animals are 
compared with the records of a normal group, the possibility 
presents itself that some selection either conscious or uncon- 
scious may have played a part in forming the groups. Such a 
source of error is no doubt practically eliminated by many well- 
known methods of choosing control and experimental animals 
from a given population. We believe such a defect is entirely 
insignificant in the foregoing records which have involved many 
animals through several generations from the same stocks in the 
case of both the experimented and the control. It is, never- 
theless, satisfactory to consider the records of the same nor- 
mal animals paired successively with control animals and with 
animals of the alcoholic lines. Table 9 presents all of the mating 
records of fourteen normal males and fifteen normal females 
that have been paired in this way. This table gives a-most 
perfect control and shows most clearly the alcohol effects. 


TEAS IS IRS 
ANORMAL MALES AND FEMALES PAIRED SUCCESSIVELY 
WITH NORMAL AND ALCOHOLIC MATES 


Individual matings of 14 normal] Individual matings of iSnormal | 
males, cach One mated succes] females, each One mated succe s- 
sively with sively with 
Normal! females} Alcoholic females| Normal males |AlcOholic males} 
Number of 3G 44 26 23 
matings 
Total number 3G 100 59 50 
Of young 
Negative 2 A | rr 
result (5.55%) (9.09%) (5.84%) (21.73%) 
7 . 
Lived ove Gis 58 51 30 
3 months 
Beene | a2 ee é a 
OS (24.41%) (42.0%) (13-55%) (A0.0 %) 
Defective fe) 6 fo) 5 
(6.0%) (10.0%) 


aa 


206 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


The fourteen male animals are in no sense selected; they are all 
of the normal males in our series of animals between the num- 
bers 613 and 1909 which have been mated with both normal and 
aleoholic females. The record numbers of these males are 665, 
666, 667, 669, 670, 676, 677, 679, 681, 682, 683, 854, 914, and 
1052. The fourteen males, as the table shows, have been 
mated in all eighty times. The fifteen females recorded include 
also every normal female among the animals considered in this 
paper that has been paired with both normal and alcoholic males. 
The record numbers of the females are 645, 646, 650, 652, 657, 
661, 662, 671, 674, 675, 703, 722, 760, 890, and 1043. These have 
been mated in all forty-nine times. 

There has been no selection or choice in mating these animals 
or in estimating the results, since it was only decided to arrange 
such a table after beginning the present study of the data. 

The first column of table 9 shows the results of thirty-six 
matings of the normal males with normal females. Two of the 
thirty-six matings failed to produce results, or 5.55 per cent, 
and the remainme™ thirty-four matings gave rise to eighty-six 
young. Sixty-five, or 75.59 per cent, of these lived to reach 
maturity, while 24.41 per cent died within three months. None 
of the eighty-six offspring showed any gross structural defects. 

When these same normal males were mated forty-four times 
with alcoholic females, the second column shows that four mat- 
ings failed, or 9.09 per cent, almost twice as ma®y as the failures 
with normal females. The forty successful matings produced 
one hundred offspring, only fifty-eight of which were capable of 
survival to maturity. Thus 42 per cent of the young animals 
died within three months against only 24.41 per cent of those 
from the normal mothers and same fathers. Six per cent of the 
young from the alcoholic mothers possessed noticeable structural 
defects. \, 

In every respect the matings of the fourteen norma! males 
produced greatly superior results when paired with normal fe- 
males, as compared with their records by alcoholic females. 
The numbers are comparatively small, but the differences 27e 
large and the inferior records are consistently in the same co] UMN. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 207 


The third and fourth columns contain similar records from the 
matings of the fifteen normal females with normal males and 
with alcoholic males. The twenty-six normal matings gave only 
one failure, while the twenty-three matings with alcoholic males 
failed to give results in five cases, or in 21.73 per cent of the trials. 
The alcoholic males always give a high percentage of mating 
failures even with normal females and, as this case shows, with 
females giving only a low per cent of failure by normal males. 

The normal matings produced fifty-nine young, fifty-one of 
which survived while only eight, or 13.55 per cent, died within 
three months. This is an unusually low mortality record and 
proves the ability of these females to produce strong viable young. 
None of the offspring from the normal matings were defective. 

The same females produced by alcoholic males fifty young, 
only thirty of which lived to maturity. Therefore, 40 per 
cent of them were non-viable, which is three times more than was 
the case with offspring from these females by normal fathers. 
Ten per cent of the fifty offspring were defective. The contrast 
between the two groups of results from the same females is so 
ereat that the possibility of the difference being due to the 
smallness of the numbers involved would seem to be completely 
eliminated. The records in the entire table are perfectly con- 
sistent and very clear cut. 

It would seem only proper to interpret such results, along 
with the mass of evidence in the foregoing pages, as showing that 
aleoholic guinea-pigs, whether directly treated or descended 
from treated individuals, have had their ability to produce 
strong, viable offspring definitely and decidedly lowered. And 
it may be added in this connection that evidence from purely 
male treated lines as well as that given by later generations from 
the female treated and mixed lines, points directly to the fact 
that the germ cells have been affected. The effects of this 
modification are transmitted through several generations, only 
to be lessened by the elimination through death and sterility of 
the weakest individuals from the mating records and the con- 
stant introduction of more and more normal germ plasm into the 
line by matings with the normal stock. 


208 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


14. THE CONTRASTED QUALITIES IN THE CONTROL AND THE 
: ALCOHOLIC SERIES 


The earlier reports on these experiments have given in the 
general text the various differences between the alcoholic and 
control lines; the case is made much clearer, however, if all the 
contrasted qualities be arranged together in summary fashion. 
In Pearl’s recent report on the influence of alcohol inhalation on 
the progeny of the domestic fowl, he has given a concise arrange- 
ment of the differences between the records of the experimented 
and control lines. The several qualities he has compared such 
as mortality records, fertility, abnormalities, etc., are the same 
as those considered in our previous papers. We have here con- 
structed a similar table to the one used by Pearl to show 
the qualities contrasted in the former sections of this paper. 
Definite numerical values have been presented for fourteen dif- 
ferent qualities studied in the two groups of animals. Several 
of these qualities are closely related, such as weights after dif- 
ferent periods of growth and the mortalities caleulated at dif- 
ferent periods, yet these are stated separately since they were 
measured in this manner and help somewhat to give a clearer 
analysis of the entire problem. 

Table 10 shows the qualities measured. The first column of 
figures are the records from the control, the second column are 
the alcoholic records. In the last column a — sign indicates 
that the alcoholics are inferior to the control for the given quality; 
a zero, that the two groups are similar in the given respect, and 
a + sign would show that the alcoholics are superior to the 
control. It is seen at once that the alcoholic series suffers by 
comparison in every case except one, and in this case the two 
series are equal on account of an earlier unusually large difference. 

The alcoholic guinea-pigs are less productive, giving litters 
of smaller size than the normal, their matings more often result 
in failure to conceive; associated with these two facts there is a 
higher early prenatal mortality which is the only quality in- 
cluded "in the table that cannot be numerically expressed for 
reasons brought out in previous pages. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 209 


Asie 
QUALITIES CONTRASTED BETWEEN THE 
NORMAL AND ALCOHOLIC PROGENIES 


c + 
eC ees 


Size of litter 

Failure to conceive 

Early prenatal death (sizeof litter, sailure,ete) 
Proportion late prenatal death 
Post-natal mortality 

Total mortality 


Abnormalities 
Oversize (+ 5004's. at 3 mos.) 
Undersize (— 3001's. at 5 mas.) 


. Late generations alcoholic improved, 
mortality index 


ie 
2. 
3. 
4. 
5 
6. 
te 
8. 
s) 
10. 


. Altered sex-ratios 
birth wt of fitter 
_ individual birth wt 
Y Wt. | month old 
-wt. 3 months old 


The aleoholies have a higher proportion of their total mortality 
occurring very early, so that there is a great elimination of weak 
embryos and fetuses; this lowers their later or postnatal mortality 
to about the normal record. In this case we have an elimination 
or selection of individuals or zygotes rather than a germinal 
selection. The total mortality record for the experimented 
group is far higher than for the control and a greater percentage 
of abnormal young are produced. The percentage of abnor- 
malities is lower than in our former records, as is also the total 
mortality rate. The improved mortality rate is partly due to 
better methods of breeding and caring for the animals. Yet the 
mortality record of the alcoholic group is very high, and when 
corrected for the normal rate on the basis of the size litters con- 
cerned it becomes 189 against the control as 100. Among the 


THE 1OURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, NO. 1 


210 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


normal animals of the same general stock as the alcoholies, not 
one grossly deformed individual has been born in over 400 cases, 
and, as stated above, this is a remarkable record which argues 
strongly for the perfection of the stock. In considering the de- 
fective young, one must also keep in mind the fact that these 
are not worse, but, on the contrary, are better organized than 
individuals which die during early stages of development. 

At three months old, as No. 8 in the table indicates, fewer 
alcoholic than control animals were larger than usual or over 
size, though some were, while the next line shows that more 
alcoholic animals were small or under size, weighing less than 
300 grams. 

The later generations of the aleoholie stock are improved by 
the continued elimination of weak and defective individuals 
which die or are unable to breed, and also by the introduction of 
more and more normal germ plasm from generation to genera- 
tion until a mortality rate of 42.4 per cent for the F, generation 
becomes only 17.14 per cent for the F, generation. This is a 
clear demonstration of the aleohol effect and may also serve to 
show the action of increased germ dosage. The earlier genera- 
tions being nearer the directly treated animals receive higher 
doses than do the later generations where in most cases the dose 
has been considerably diluted by a mixture of normal germ 
plasm. 

The sex-ratio in the alcoholic group seem to have been modi- 
fied in ways which we have attempted to explain. 

The average weight of the alcoholic litter is less than the 
normal and the average individual birth weight of an alcoholic 
specimen is also less than for the normal. The average weight 
of the alcoholic individuals at one month old is below the nor- 
mal and the average weight at the age of three months, when 
guinea-pigs are about mature, is still below the weight of the 
control animals. 

Therefore, in the fourteen measured points considered, the 
offspring of the alcoholic series are below the normal control in 
thirteen cases and apparently equal to the control in only one. 

The qualities are largely the same as those we have considered 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 211 


in former papers though analyzed in further detail. They are 
also very similar to those recorded by Pearl (’17) in his table 14. 
From a physiological standpoint it seems to us that these quali- 
ties are all closely associated and finally come down to the three 
related qualities: ability to develop normally, grow rapidly, and 
live to maturity. An animal possessing such qualities is usually 
termed a vigorous individual. At present it can only be stated 
that these properties are due to the vigor of the germ cells from 
which the individual arose. The qualities discussed might all 
involve a limited range of physiological factors so far as present 
knowledge permitsa separation of such factors and they only show 
on the part of the alcoholics a reduced capacity of development 
and growth. The same underlying cause may actually account 
for the abnormal sex-ratios, as has been pointed out in an 
earlier section. 

Leaving the environment out of account, the normal develop- 
ment, growth and length of life of a zygote varies with the 
perfection or vigor of the germ cells from which it originated. 
An experimental treatment may act upon the germ cells of an 
animal so as to modify them in some general way which lowers 
their ability to react normally in combination with germ cells 
from another individual. Thus zygotes are produced which 
tend to develop abnormally, grow slowly, or die during early 
stages of their existence, depending upon the degree of modifi- 
cation the treated germ cells have suffered. We are fully em- 
barrassed by the unsatisfactory nature of such statements, but 
have been unable to gather scientific facts that would permit 
any more definite estimate of the situation. 

All of our experiments on the modification of the germ cells 
have given results which express themselves in some such general 
fashion. Yet the germ plasm has been definitely modified and 
the subnormal condition is transmitted through a number of 
generations beyond the animals directly treated. This result is 
original on the complex material used, and is of primary impor- 
tance, although it may be disappointing in that it has not shown 
a modification in the mode of behavior of some particular char- 
acter known for its Mendelian inheritance. 


212 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


The experimental modification of the inheritance of definite 
characters by a treatment of the germ cells is a future possibility. 
It must be recognized, however, that one is able to produce 
grotesque monsters by a treatment of eggs or spermatozoa, and 
yet all of the known characters which Mendelize in such an in- 
dividual may be expressed in a perfectly normal fashion. This 
may be due to the fact that comparatively few such characters 
are known. Aside from the future definite modifications of 
inheritance, it would seem from the present study that the 
‘general qualities,’ for lack ef a more suitable term, of an or- 
ganism may be affected, on account of an experimental modi- 
fication of the germ plasm from which it arose. The modifi- 
cation may have taken place several ancestral generations ago. 
This is really the inheritance of pathological conditions which 
were induced upon and transmitted by the ancestral germ plasm. 
Such a type of inheritance is no doubt important in its relation 
to the normal processes of development and inheritance. 


15. GENERAL CONSIDERATIONS 


A discussion of the literature bearing on the influence of various 
chemical substances on the egg and spermatozo6n has been given 
in former papers of this series, particularly Stockard (712 and 
713). In all cases only the effects of the treatments on the zygotes 
immediately resulting from the modified spermatozoa or eggs 
have been studied. There has been no experimental investiga- 
tion of later generations arising from the affected specimens. 
And indeed, in almost all cases the developing individuals were 
lost during early embryonic stages as in the X-ray experiments 
of Bardeen and the radium studied of Oskar Hertwig which are 
the most satisfactory investigations on the direct injury of the 
sperm. These experiments really supplied no available material 
for an investigation of the inheritance or transmission of the 
induced defective conditions. 

Since the beginning of the present experiments other studies 
have been recorded which bear more directly on the results con- 
sidered in the foregoing pages. Of particular interest in connection 
with our supposed differential effects of the alcohol treatment on 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 213 


the behavior of the X and Y groups of spermatozoa is the ingenious 
double-mating experiment of Cole and Davis (14) with rab- 
bits. They found that when two male rabbits were mated with 
a single female, superfetation occurred in most cases, so that 
part of the resulting litter of young were sired by one male and 
part by the other. The males differed in their fertilizing abili- 
ties, so that one more often sired the majority of young of a 
given litter, and in the total number of competition matings he 
sired the greater number of young. This male with the fertiliz- 
ing advantage was then treated for a month or more with the 
fumes of aleohol by the inhalation method. As a result of this 
treatment his spermatozoa became affected in such a way that 
mated in competition with the same male he normally had 
beaten he now failed to sire any young. Yet when mated singly 
or alone with a female he still possessed the power to beget off- 
spring. This is a striking illustration of the debilitating effect 
of a short alcohol treatment on the physiological behavior of 
these spermatozoa, thus lowering their fertilizing ability below 
that of other spermatozoa which were formerly less potent than 
they. ; 

When it is seen how definitely and readily alcohol treatments 
affect the behavior of the spermatozoa, we are led to speculate as 
to whether the treatment might not affect the X and Y groups 
of sperm differently, and, thus be partially responsible for a dis- 
tortion of the sex-ratios, should such oecur. This responsibility 
may be due in the first place to a lowered fertilizing power on 
the part of one group of spermatozoa, thus giving rise to fewer 
individuals of one sex than of the other. Or, in the second 
place, even though both groups of spermatozoa should be equally 
capable of fertilizing the eggs, one group might be more affected 
as to its ability to produce viable zygotes in combination with 
normal ova, and thus an early differential sex mortality would 
occur causing a modification of the proportion of one sex to the 
other among the young born. We have elaborated somewhat on 
these possibilities in the section devoted to the sex-ratios of the 
alcoholic guinea-pigs. 

Cole and Davis originally devised their experiment as a cru- 


214 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


cial control for the influence of alcohol treatment on the male 
germ cells. In mating two males to a single female any defective 
condition that might arise among the offspring from one of the 
males, as compared with those from the other, could not be 
attributed to differences in developmental environment or in the 
qualities of the ova, as might possibly be the case where different 
females are used. 

Cole and Bachhuber (’14) have employed the same method in 
a study of the effects of lead on the germ cells of the male rabbit 
and fowl. Their conclusion in regard to the rabbit is ‘“‘that the 
offspring produced by male rabbits which have been poisoned 
by the ingestion of lead acetate into the alimentary tract have a 
lower vitality and are distinctly smaller in average size than 
normal offspring of unpoisoned males.’’ This conclusion is in 
exact accord with the conditions shown by our F;, generation of 
guinea-pigs sired by alcoholized fathers. Cole and Bachhuber 
have not reported on the transmission of the effects to later 
generations. 

Their results with fowls ‘‘are interpreted as indicating that 
in fowls also poisoning of the male parent with lead results in 
offspring of a distinctly lower average vitality.” This again 
accords with the results on the offspring when male guinea-pigs 
are treated with alcohol. 

A later more extensive report concerning the influence of lead 
as a substance producing blastophthoric effects is given by Weller 
(15). This investigator has treated both male and female 
guinea-pigs with commercial white lead. The lead is adminis- 
tered by mouth in gelatin capsules, the same method as was em- 
ployed by Cole and Bachhuber (’14). The effects from the 
lead poisoning on the guinea-pigs are very similar to those ob- 
tained by treating the rabbits and fowls. Weller has been 
careful not to overdose the animals and his precautions would 
make it seem probable that any effect from the treatment which 
might be shown by the offspring was actually due to the lead 
poisoning and not to impaired nutrition or other indirect causes. 

His conclusions are based on a total of ninety-three matings 
yielding 170 offspring. There were thirty-two control matings 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 215 


which produced only fifty-eight offspring. Whether or not every 
mating gave offspring is not definitely stated, but if so the aver- 
age-size litter was unusually small, being only 1.81. This would 
indicate either a stock of very low productivity or a high pro- 
portion of absorbed embryos and partial abortions, as a final 
result of which the litters would be small. In the foregoing 
tables where the numbers of matings and young are very much 
greater, not one group shows so small an average litter. From 
the thirty-four matings of lead-poisoned males with normal 
females, sixty-five offspring resulted, an average litter of 1.91, 
and from twenty-seven matings of normal males with lead 
females forty-seven young were born, an average litter of only 
1.74. 

The fact that among the few individual litters recorded there 
were three cases of litters of four, and five cases of litters of three, 
makes it seem as though there may have been a high proportion 
of mating failures, giving rise to the small average litters ob- 
tained when the total number of young is divided by the total 
number of matings. The distribution and cause of these mat- 
ing failures, as is pointed out in the text above, may be of con- 
siderable importance. 

Weller has analyzed his results in some detail. He takes into 
account the influence of litter size on the birth weight and gives 
several individual mating records which illustrate the effects of 
a treated sire on the birth weight of the young from a normal dam. 

Weller has also taken into account the relationship between 
lead dosage and birth weight of the offspring without finding 
very consistent correlations. The relationship between germ 
dosage and the condition of the offspring in our records may be 
calculated for every individual born in the alcohol experiments, 
yet the result is uninstructive so far as at present studied. 
There are a great number of confusing factors involved in this 
seemingly simple proposition. 

Weller’s final conclusions from the study of lead poisoning 
closely accord with our previous statements regarding the influ- 
ence of alcohol on the same animals. He finds that chronic lead 
poisoning in guinea-pigs produces a definite blastophthoric effect. 


216 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


This can best be demonstrated upon the male germ plasm, in which 
case the blastophthoria manifests itself in some instances by sterility 
without loss of sexual activity, by a reduction of approximately 20 per 
cent in the average birth weight, by an increased number of deaths 
in the first week of life, and by a general retardation in development 
such that the offspring of a lead-poisoned male remains permanently 
under weight. ’ 


These experiments with alcohol and lead on rabbits, fowls and 
guinea-pigs seem to their authors to modify the male germ cells 
in a definite manner. The offspring sired by treated fathers are 
inferior to those from control males. The transmission of the 
defects to subsequent generations has not been reported. 

In addition to the experiments on the direct treatment of the 
spermatozoa of lower forms, a few attempts have been made 
to treat the spermatozoa of higher animals directly with certain 
chemicals. Ivanov ('13) has given a short note on the effects of 
immersing the spermatozoa of several mammals in solutions of 
alcohol. He finds that when.fertilization is obtained after such 
treatments a normal development follows and normal offspring 
are produced. ‘To anyone who has studied the action of alcohol 
on the free swimming spermatozoa of lower vertebrates such re- 
sults are not surprising. The most probable explanation is that 
the spermatozoén has been entirely protected from the action 
of the alcohol of the strengths used. When any action is ob- 
tained the usual effect on the spermatozo6n is to render it im- 
mobile. To obtain a fertilization the motionless sperm must 
be activated by the use of some alkaline substance, such as 
NaOH. Following this activation the spermatozoa may often 
give normal offspring after union with normal ova, thus indi- 
eating that their chemical nature has not been disturbed. It is 
most difficult to treat the spermatozoén even of the very hardy 
fish, Fundulus heteroclitus, in such a manner as to injure it and 
afterwards obtain a fertilization. Dr. Wilson Gee (’16) experi- 
mented on the spermatozoa of fishes at Woods Hole for two 
seasons and found that the difference between an effective alco- 
hol dose and a fatal dose was so slight that it required the most 
delicate adjustment of solutions in order to injure the sperma- 
tozoa to such a degree that the development of eggs subsequently 


| 


a | 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 
° 


Oh, 


fertilized was rendered abnormal. Ivanov’s report is cer- 
tainly not sufficiently detailed to satisfy one that his results 
have any bearing on the problem of the modification of the germ 
cells by chemical treatment. 

There can be no doubt that if a spermatozoén is actually 
affected by a direct chemical treatment, the egg which it fer- 
tilizes will develop more or less abnormally. The radium and 
X-ray experiments of Bardeen and Hertwig, as well as fertiliza- 
tion by foreign spermatozoa give conclusive evidence on this point. 

The statistical research by Elderton and Pearson (’10) has 
frequently been quoted as if it shows that parental alcoholism 
was really to some degree beneficial to the human offspring. 
Their mathematical calculations were based on two series of 
statistics, the ‘‘Edinburgh Charity Organization Society Report 
and a manuscript account of the children in the special schools 
of Manchester provided us by Miss Mary Dendy.” ‘‘Sus- 
pected drinkers were included with drinkers,”. ‘‘the parents 
could be divided into two classes only, those who are temperate 
and those who are intemperate,’”’ and many other such state- 
ments make this biological data somewhat unsatisfactory to 
those interested in an experimental modification of the germ 
plasm. These authors, however, do not claim to find any 
effect, either good or bad, of aleoholism on the offspring, and 
finally state that 


On the whole the balance turns as often in favor of the alcoholic 
as of the non-alcoholic parentage. It is needless to say that we do not 
attribute this to the alcohol, but to certain physical and possibly mental 
characters which appear to be associated with the tendency to alcohol. 


Such a conclusion on the part of the authors themselves would 
searcely warrant anyone else in claiming that an effect of alco- 
holism on the parent had given evidence of its existence in the 
quality of the children produced. A number of English physi- 
cians interested in alcoholism largely from a social and senti- 
mental standpoint opened a bitter attack on the memoir by 
Elderton and Pearson, not because it claimed a beneficial effect, 


1Ttalies are ours. 


218 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 
. 


but merely because no harmful effect was shown. Such criti- 
cism is of little interest, yet one very serious point was cited 
against the data on which this study was based, and Pearson and 
Elderton (’10) in their reply failed to satisfy the objection. The 
children considered were in the neighborhood of nine years old 
at the time the statistics were collected and the fact that some 
parents were drinking at this time might not necessarily prove 
that they were drinking nine or ten years ago when the children 
were conceived. It is very evident that from our standpoint 
accurate data relating to this particular fact is most essential. 

This study really has no bearing in the literature on the chemi- 
eal modification of germ cells or the developing embryo, as 
Elderton and Pearson themselves state in the italicized portion 
of the quotation cited above. No one can confidently affirm 
that in their data alcoholics are being compared with normals or 
-really whether any alcoholics or normals as such are actually 
being considered beyond the chance probability that some in- 
dividuals of both classes creep into the statistics to be included 
in the two groups arranged. 

Very recently Pearl (17) has published a most thorough 
analysis of the influences of parental alcoholism on the progeny 
of the domestic fowl. He states (p. 285): 


that a careful study of the present results makes it impossible to assert 
that the treatment of the parents has had no effect upon the progeny. 

The offspring of the alcoholists, as a class, are indubitably 
differentiated from the offspring of the non-aleoholists. 


Such a statement agrees entirely with our results from the 
alcoholic guinea-pigs. In detail, however, Pearl finds that after 
treating fowls with alcohol the progeny produced are in some 
respects superior to the control. This, he believes, is brought 
about by an elimination of all weaker germ cells through the 
action of alcohol which thus serves as a selective agent to im- 
prove the race. At first sight this would seem to be entirely 
contradictory to our results, since the guinea-pig progeny is 
decidedly the worse for the experimental treatment. Yet*the 
treatment in both cases has affected the progeny through its 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 219 
action on the germ cells. This is the point of actual importance 
and the one of chief interest from the standpoint of these ex- 
periments. We are not here studying the alcohol problem from 
a social standpoint and it is immaterial whether the progeny be 
benefited or injured by the treatment of parental generations. 
Our interest lies in whether or not the germ cells are modified 
by the chemical treatment and whether the modification is of 
such a nature as to alter the qualities of the individuals which 
may compose the subsequent generations. 

Pearl, of course, fully agrees with such a position, and states 
(716 a, p. 258): 

Our results seem to me to be supplementary to those of Stockard, 
and to throw an interesting light on the need for caution in reac ing 


a correct interpretation of all experiments in which a mildly deleterious 
agent acts upon the organism. 


He also believes that his results are in no way contradictory to 
ours, but recognizes the fact that, although the same chemical 
substance may act upon the germ plasm of two different classes 
of animals, the visible response on the part of the animals need 
not necessarily be the same. In other words, one is not always 
within the realm of legitimate scientific speculation who assumes 
that since a given substance acts to induce a certain response 
on the part of one animal species that the same substance will 
call forth a like response on every other species. ‘‘What is one 
man’s food is another man’s poison.’’ With this we fully agree; 
it is dangerous to draw universal deductions from experiments 
on any one or two classes of animals. 

Another possibility also recognized by Pearl presents itself in 
considering the opposite effects of the alcohol treatment on the 
progeny of guinea-pigs and fowls. Small doses of many sub- 
stances, one of which is alcohol, may form a physiological stand- 
point produce a stimulating effect, while larger doses produce 
decided depression. There is a possibility that the same may 
be true of the action of such substances on the germ cells. Pear! 
has discarded such an explanation after very fair consideration, 
and is possibly right in so doing. The experiences, however, 
with the guinea-pigs makes our opinion decidedly prejudiced in 


220 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


favor of the possibility, that although a sufficiently large dose 
may have been used, yet it did not act solely to eliminate germ 
cells as such, but also caused the production of many zygotes 
which died during early developmental stages. 

The amount of dosage is very important. Treating female 
guinea-pigs with considerable doses of alcohol fumes only shortly 
before and during their pregnancies certainly does not injure 
the offspring to any noticeable degree. While the same dose 
of treatment, if administered for a number of months or years, 
will render these mothers almost incapable of producing vigorous 
young, even when mated with normal males. 

Pearl (17, p. 281) finds regarding his 1915 results which were 
obtained after the treatments had been running for only a few 
months that considering the number of animals in the experi- 
mental series the individual differences are not in every case 
sufficiently large to be significant in comparison with their prob- 
able errors. The control in this case was also not what Pearl 
had wished. He had originally chosen a carefully pedigreed 
control, taking as the one control male a half-brother of the three 
experimental males and using control females that were sisters 
of the treated hens as recorded in table 5, p. 158 (17). The 
only control male, No. 666, proved to be practically sterile and 
useless. This necessitated the use in paper No. III of an ordi- 
nary random sample control instead of the refined control 
originally planned in Part I of the series of papers, and nulli- 
fied the statement in the summary of Part I, p. 162, that “Full 
brothers and sisters of treated are used as control.” 

For certain qualities, such as the fertility and hatching records 
of the eggs, the control was not in all cases the same cross as the 
experiment, which was invariably between Barred Plymouth Rock 
hens and Black Hamburg cocks. The hatching weight and rate 
of growth of the experimental chicks on account. of want of con- 
trol data from the 1915 season were compared with chicks from 
a similar cross hatched and reared in 1913. Different keepers 
were in charge of rearing the chicks during the two different 
seasons. These unfortunate conditions, all of which are pointed 
out with conscientious fullness by Pearl, make it rather difficult 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 221 


to fully estimate the actual significance of the differences between 
the experimental offspring and the control groups used. 

Fortunately, however, the data from the 1916 season is avail- 
able (Pearl, *16 b) for comparison with the 1915 results. The 
aleohol treatments were continued throughout the time so that 
the 1916 chicks are derived from more highly alcoholized parents. 
Should the aleohol continue to improve the race by ‘‘completely 
putting out of commission all of the weaker germ cells,” the 1916 
results should in all respects show a further improvement in the 
qualities that had been previously benefited. 

The percentage of infertile eggs given in the 1915 table may 
be reversed to per cent of zygotes formed and compared with 
this column in the 1916 table. The percentage of zygotes formed 
in the several combinations of aleoholic mating should be less 
than in 1915, and they are. When both parents were alcoholic 
in 1915, 40.8 per cent of the eggs formed zygotes, while in 1916 
only 21.95 per cent produced zygotes; sire only alcoholic, 74.8 
per cent zygotes in 1915 and only 53.52 per cent in 1916. This 
is in line with the lowered fertility and increased number of 
mating failures from the alcoholic guinea-pig records. The more 
decidedly alcoholic the guinea-pigs become, the smaller the litter 
size from double alcoholic and sire only alcoholic matings, and 
the greater the number of failures to conceive. 

With the guinea-pigs, however, this is not alone due to a 
destruction of weak germ cells by the treatment, but is cer- 
tainly in part due to an increased very early prenatal mortality 
for which much evidence is given in the body of the present 
paper. The smaller number of zygotes formed by the treated 
fowls is probably also due in some cases to death in very early 
stages, as blastulae or gastrulae, before the egg is laid; or in the 
hen’s eggs these weakened zygotes may not be able to with- 
stand the developmental interruption following the: laying of 
the egg. Embryos dying during such stages could not be iden- 
tified except by a most minute study. 

It seems to us in keeping with what is known of biological 
reactions in general and the guinea-pig histories in particular to 
take the following position. The alcohol treatment acts on the 


222 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


germ cell populations of both fowls and guinea-pigs in such a 
manner that the weakest or least resistant ova and spermatozoa 
die from the effects of the treatment as germ cells without taking 
part in zygote formation. The somewhat more fesistant ova 
and spermatozoa are greatly injured though still capable of 
forming zygotes. The zygotes, however, are so defective as to 
be capable of only a short period of development and die during 
stages too early to be definitely detected by gross examinations of 
either the fowl’s egg or the mammalian mother. Still other em- 
bryos are capable of development to later stages and are actu- 
ally found dead, not only as the youngest embryos to be identi- 
fied, but from these early stages there occurs a continuous series 
of prenatal deaths up to the full-term still-births. Immedi- 
ately after birth the postnatal mortality is greatest and gradu- 
ally decreases until these specimens capable of reaching maturity 
may often enjoy a comparatively long life. 

At the present stage of the two experiments it w ould seem as 
though this elimination of defective germ cells and very early 
embryos was much more intense in the fowls than in the guinea- 
pigs as a group; so that the late prenatal and postnatal mor- 
tality among the fowl progeny was low and those specimens 
that hatched were the hardy survivors from this early vigorous 
process of germ cell and individual selection. The records from 
the double alcoholic and male treated lines among the guinea- 
pigs forms a second step. The size of the litters and failures 
to conceive in these lines indicates a.rather high degree of in- 
fertility or germ cell debility as well as early prenatal deaths, 
though this is not so extreme as among the fowls, and the late 
prenatal and postnatal mortality is higher. 

Finally the female treated guinea-pig lines produce large lit- 
ters and have few infertile matings, indicating a low germ cell 
and early prenatal mortality, and here the late prenatal and 
postnatal mortality is highest, not entirely on account of the 
action of the treatment on the developing individual in utero, 
since the same condition is found among other female gene- 
rations than the one directly treated. 

This presentation of the situation is somewhat similar to that 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 223 


which Pearl (’17) has illustrated in his diagrams, figures 5 to 7, 
pages 290 and 291. The chief difference being that we would 
decrease the proportion of eliminated germ cells and increase 
the proportion of defective and non-viable zygotes, and thus 
emphasize the selection of individuals rather than of germ cells. 

A further consideration of Pearl’s 1916 results as shown in 
table 1, p. 676 (16 b), may be used to argue in favor of our po- 
sition. The ‘prenatal mortality’ column of this table when 
compared with the ‘dried in shell’ column from 1915 records 
(table 1, p. 244, ’17) should show lower percentages according to 
our interpretation of Pearl’s expectation for an improved stock 
from the alcoholic lines. Instead of this, in only one combina- 
tion is the prenatal mortality lower. In both parents alcoholic 
it has been lowered from 26.9 per cent to 11.11 per cent, and 
here the postnatal mortality as we would expect is increased. 
In the other cases dam only alcoholic, none of which were re- 
ported for 1915 on account of the useless control male, gives 80 
per cent prenatal mortality sire only alcoholic increased to 47.08 
per cent from 36.6 per cent; sire and one grandparent, 46.84 per 
cent; one or more grandparents, 46.02 per cent; all alcoholic 
ancestry, 45.95 per cent, which is a considerable increase over 
the 1915 records. The control of 1916 also shows a higher 
prenatal mortality than that of 1915, though it is not stated 
whether the same breed crosses are used in the two controls. 

The postnatal mortality of the 1916 control is, on the con- 
trary, lower than the pestnatal mortality of the twenty-two 
‘random sample matings’ of 1915. 

While the total mortality for all the alcoholic groups is about 
the same, 17.6 and 16.5 per cent, for the two seasons, the indi- 
vidual combinations show wide variations. From both parents 
alcoholic the 1915 postnatal mortality was 10.6 per cent, while for 
1916 it rose to 25 per cent, sire only alcoholic fell from 21.1 per 
cent, 1915 record, to 13.79 per cent, 1916 record. Sire and one 
grandparent alcoholic gave a postnatal mortality of 28.38 per 
cent, while the non-alcoholic postnatal mortality was 21.2 per 
* cent. 

Considering the numbers involved, the records from the prog- 


224 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 


eny of the 1916 matings after longer alcohol treatment do not 
seem altogether improved as compared with the 1915 records. A 
comparison of individual lines in the tables frequently show 
disadvantages for the 1916 matings. This would seem as though 
some injured zygotes were present and all of the affected gern 
cells had not been completely eliminated by the treatment. The 
percentage of abnormal specimens among the 1916 alcoholies is 
about the same or slightly more than among the control, while 
Pearl had counted this point in favor of the alcoholics from his 
1915 records. 

It would thus seem, as Pearl (’17, 292) himself suggests, that 
“it might be supposed that with larger administration to the. 
fowls (higher germ dosage) or more years of drinking behind 
them in the case of Elderton and Pearson’s workingmen, the 
conditions shown in figure 7 would gradually pass over into those 
shown in figure 5.’ That is, that not only weak germ cells would 
be eliminated by the treatment, but that also a considerable 
proportion of defective individuals would arise to be eliminated 
during various developmental stages or persist as degenerate 
specimens. From these conditions we believe that there is a 
really close agreement between the results on the fowls and the 
guinea-pigs. 

These suggestions are advanced only in a spirit of the most 
friendly criticism. We have worked long enough in accumulating 
and considering evidence bearing on the various phases in- 
volved in this problem to highly appreciate the masterly manner 
in which Pearl has considered and analyzed his data; and we are 
thankful for many suggestions that have come to us through 
the contribution on parental alcoholism in the fowls. In the 
end our aims and objects are the same, to affect the germ plasm 
in so definite a manner as to be able to predict the quality and 
degree of the modifications subsequently expressed in the gen- 
erations to follow. 


MODIFICATION OF THE GERM-CELLS IN MAMMALS 225 


LITERATURE CITED 


Aruitt, A. H., ann Weis, H. G. 1917 The effect of alcohol on the repro- 
ductive tissues. Jour. Exp. Med. Vol. 26, p. 769. 

Barpreen, C. R. 1907 Abnormal development of toad ova fertilized by sper- 
matozoa exposed to the Roentgen Rays. Journ. Exp. Zodl., vol. 4, 
Dal: 

Coxe, L.J., anp Davis, C.L. 1914 The effect of alcohol on the male germ cells, 
studied by means of double matings. Science, N. 8., 39, p. 476. 

Coxs, L. J., anp Bacnuuser, L. J. 1914 The effect of lead on the germ cells 
of the male rabbit and fowl as indicated by their progeny. Proc. 
Soc. Exp. Biol. and Med., 12, p. 24. 

Exvperton, E. M., anp Pearson, K. 1910 A first study of the influence of pa- 
rental alcoholism on the physique and ability of the offspring. Eu- 
genics Lab. Mem. X, Univ. of London, pp. 1-46. 

Ger, Witson 1916 Effects of acute alcoholism on the germ cells of Fundulus 
heteroclitus. Biol. Bull. 31, p. 379. 

Herrwic, O. 1913 Versuche an Tritoneiern tiber die Einwirkung bestrahlter 
Samenfiden auf die tierische Entwicklung. Arch. f. Mikr. Anat., 82, 
Abt. IL. 

Ivanov, J. 1913 Expériences sur la fécondation des mammiféres avec le sperme 
mélangé d’aleool. Compt. rend. Soe. Biol. Paris, 74, p. 482. 

Meyer, A. W. 1917 Intra-uterine absorption of ova. Anat. Rec., vol. 12, 
p. 293. 

Papantconaou, G.N. 1915 Sex determination and sex control in guinea-pigs. 
Science, N S. 41, p. 401. 

Prart, R. 1916a On the effect of continued administration of certain poisons 
to the domestic fowl, with special reference to the progeny. Proc. 
Am. Phil. Soc., 55, p. 248. 
1916b Some effects of the continued administration of alcohol to the 
domestie fowl, with special reference to the progeny. Proc. Nat. 
Acad. Sci. 2, p. 675. . 
1917 The Experimental modification of germ cells, Parts I, II, and 
Ill. Journ. Exp. Zoél., vol. 22, pp. 125-186 and pp. 241-310. 

Pearson, K., anp Etperton, E. M. 1910 A second study of the influence of 
parental alcoholism on the physique and ability of the offspring. 
Being a reply to certain medical critics of the first memoir and an 
examination of the rebutting evidence cited by them. Eugenics 
Lab. Mem. XIII, Univ. of London. 

Stevens, N. M. 1911 Heterochromosomes in the guinea-pig. Biol. Bull. 21, 
ps 155: 

Srockarp, C. R. 1910 The influence Sf alcohol and other anaesthetics on em- 
bryonic development. Am. Jour. Anat., vol. 10, p. 369. 
1912 An experimental study of racial degeneration in mammals 
treated with aleohol. Arch. Internal Med., X. 
1913 The effect on the offspring of intoxicating the male parent and 
the transmission of the defects to subsequent generations. Amer. 
Nat.. 47, p. 641. 


226 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU | 


Srockarp, C. R. 1914 A study of further generations of mammals from an- 
cestors treated with alcohol. Proc. Soc. Exp. Biol. and Med. XI, 
p. 186. 

Srockarp, C. R., anp Crata, D. M. 1912 An experimental study of the influ- 
ence of aleoho] on the germ cells and the developing embryos of mam- 
mals. Arch. f. Entw’Mech. 35, p. 569. 

Srocxarp, C. R., anp Papanicotaou, G. 1916 A further analysis of the he- 
reditary transmission of degeneracy and deformities by the descend- 
ants of aleoholized mammals. Amer. Nat., 50, Part I, pp. 65-88, 
Part II, pp. 144-177. 
1917 The existence of a typical oestrous cycle B in the guinea-pig 
with a study of its histological and physiological changes. Am. Jour. 
Anat., vol. 22, p. 225. 

Wetter, C. V. 1915 The blastophthorie effect of chronic lead poisoning. 
Journ. Med. Res., 28, p. 271. 


AUTHORS’ ABSTRACT OF THIS PAPER ISSUED BY Reprinted from Tae AmerIcAN JOURNAL OF 
THE BIBLIOGRAPHIC SERVICE, DECEMBER 29 Anatomy, Vol. 24, No. 1, May 1918 


THE: DEVELOPMENT OF THE IDIOSOME IN THE 
GERM-CELLS OF THE MALE GUINEA-PIG 


GEORGE N. PAPANICOLAOU anp CHARLES R. STOCKARD 


From the Department of Anatomy, Cornell University Medical School, New York 
City 


TWELVE FIGURES 


PB IOTIROLUCULOTI Se ila etree aie crs aces sia viene slers¥are sina ealesisidl ectes SeGere alee wid ee 37 
Peateenera lemme thodranG materials seriereccicis siecle roles ereieiticieicie'o,efssniciesshe-* scejsswjoiere 38 
3. Description of the development of the idiosome.................-.+.+-++. 38 
A. The idiosome in the spermatogonia......................---+--=-:- 38 
B. The idiosome in the primary spermatocytes...............--5++-+++- 39 
CL Thelin Garin iynteseerae cess npcone Odo nam noe coEpactossgde Benodp 41 
D. The idiosome in the secondary spermatocytes............ ree Peeee 42 
Hee Hetidiosomecin the SPeErMAatldss.4.coee arc eras ee cies eee ctneeie= ele) stele 43 
F. The relation of the centrosomes to the idiosome................-..- 48 
Pe RHERDECIASLAIDIN SC MEUNOGS rls. feleisiec cleys/aier vets else «  clalato ei oeletclotes crete ile 49 
5. Review and discussion of previous studies.................2+sesecereeeee 52 
Pe@ione lmnians ald ie WaApOLU tS eet -ceteleieya sri ier vale erate) reel ole sielaie ole easrsveiave taerols 61 
fi, LOIGRD iS: CDG Goods gene de aonenecbrdedc.s Ge banacmennocucracscmbrortoon 62 


1. INTRODUCTION 


The idiosome was first described by la Valette St. George 
more than fifty years ago, and since that time a number of in- 
vestigators have studied the behavior of this structure during 
the spermatogenesis of many different animals. The various 
descriptions are by no means consistent, and in the mammals 
the idiosome has been reported both as a very simple and as a 
very complex body. This peculiar structure within the cyto- 
plasm of the cells during the stages of spermatogenesis is ex- 
tremely sensitive in its response to the many different ways of 
fixation and staining. The observations considered in the pres- 
ent paper have been made possible through the application of a 
new staining method, which seems to possess particular advan- 
tages for the study of the finer structure of the idiosome. These 

37 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 24, No. 1 


38 G. N. PAPANICOLAOU AND C. R. STOCKARD 


observations, we believe, add important details to our knowledge 
of the idiosome structure and the genesis of certain parts of the 
spermatozoon. 

The following pages describe the method in full and attempt 
to give a concise description of the idiosome and its developmental 
changes during the spermatogenesis of the guinea-pig. A brief 
consideration of the literature is also presented in order to place 
the new observations in their proper relation with previous ideas 
concerning this structure. 


2. GENERAL METHOD AND MATERIAL 


The general method consists of a combination stain with 
methylen blue and acid fuchsin following a fixation in Zenker’s 
fluid. The fixation with Zenker’s fluid is necessary in order to 
obtain a good differential stain for the idiosome. Other fixing 
fluids, corrosive acetic, Flemming’s fluid, Hermann’s fluid, and 
Bouin’s fluid, used for the same study have not been satisfac- 
tory, since the tissues fixed in them fail to stain in a clear dif- 
ferential fashion. 

The studies have been made on the testes of guinea-pigs, 
from both normal and alcoholic stock. The age of the animals 
ranged from six months to four years. The living animals were 
castrated and the testes from both sides were cut in thin, longi- 
tudinal pieces and fixed in Zenker’s solution for eighteen to 
twenty-four hours. The pieces were imbedded in paraffin and 
cut in sections 3 to 5 micra thick. The details of the staining will 
be described after considering the parts of the cells which are to 
be best demonstrated with different modifications of the method. 


3. DESCRIPTION OF THE DEVELOPMENT OF THE IDIOSOME 


A. The idiosome in the spermatogona 


The term idiozom, as proposed by Meves (’99), from té:os 
(own) and ¢aua (belt), should be better changed to idiosome, 
from ids (own) and caua (body), as suggested by Regaud 
(10), as will be evident from the descriptions of this structure 
which follow. 


THE IDIOSOME OF THE GUINEA-PIG 39 


The idiosome in the spermatogonia shows many variations in 
form. Its shape may be spherical, or oval and flattened and 
cap-shaped. Its boundary is usually clear and distinct, but 
sometimes seems to pass insensibly into the surrounding cyto- 
plasm. In some cells the idiosome presents a vacuolar structure, 
while in others there is a distinct differentiation into a peripheral 
and a central part. It is possible that the idiosome in the sper- 
matogonia may be divided into two zones, as is the case with the 
idiosome of the first spermatocytes described in the next sec- 
tion. This point may be decided by future studies. The 
present paper only attempts to give a description of the idio- 
some, beginning with the primary spermatocyte stage and 
passing through its subsequent phases. 


B. The idiosome in the primary spermatocytes 


The idiosome of the primary spermatocyte is differentiated 
into two distinct parts, a peripheral, which may be termed the 
idioectosome, and a central, the idioendosome.! The relation- 
ship of the two parts is that of one slightly oval body being 
enclosed within another. The endosome lies within a central 
cavity of the ectosome and is connected with the ectosome by a 
number of processes or prolongations from the periphery of the 
endosomatic mass. As these processes pass into the substance 
of the ectosome, a vacuolar aspect is produced, as shown in 
figure 1. The position of the endosome within the ectosome is 
generally a little eccentric, being nearer the side towards the 
spermatocyte nucleus. The idioendosome is the more essential 
part of the idiosome persisting through all stages of development, 
while the idioectosome is later to be eliminated and dissolves or 
disappears in the cytoplasm. 

During the preparation for division of the primary spermato- 
cytes, the idioendosome exhibits a peculiar transformation. Its 
periphery breaks up into a number of at first large and then 


1 These terms and others which follow are proposed by us not merely to burden 
the present confused terminology, but on account of an actual lack of technical 
words to sufficiently or exactly designate the structural details presented by the 
idiosome during its several developmental phases. 


40 G. N. PAPANICOLAOU AND C. R. STOCKARD 


smaller granules, as shown in figure 2. These we name the idio- 
granulomes. As they form the granulomes lose connection with 
one another while still lying within the idioectosome. Such an 
arrangement is to be seen during the prophase of the primary 
spermatocyte division. As the process of division progresses, 
the idioectosome loses its regular spherical or oval form to be- 
come irregular in shape and begins to break up. The pieces 
scatter in the protoplasm where they lose their identity. The 
idiogranulomes, after the breaking or disintegration of the 
idioectosome, are now set free in the protoplasm and become 
slowly dispersed throughout its substance. In this way the old 
idiosome is destroyed and its constituent elements, viz., small 
pieces of ectosome substance and idiogranulomes derived from 
the transformed idioendosome, are scattered throughout the 
cytoplasm. 

The staining reaction of the ectosome remains the same during 
all these phases, whereas the endosome changes its color reac- 
tion with its structural transformation. The idioendosome, as 
such, shows a violet color after the combined fuchsin-methylen 
blue stain, while its derivatives, the idiogranulomes, have a 
greater affinity for the red acid fuchsin stain, thus presenting a 
dark red color. In the beginning the idioendosome, therefore, 
resembles the idioectosome in its color reaction. 

After the end of the primary spermatocyte division, a new 
idiosome is reconstructed in each secondary spermatocyte. The 
idiogranulomes, which were dispersed in the protoplasm, migrate 
toward a place near the nucleus and close to the side of the old 
spindle remnant. 

Around these idiogranulomes a new idioectosomatic substance 
is slowly collected. This substance shows the same staining re- 
actions as the old ectosome, and is possibly the same substance 
being reformed or reconstructed. It seems improbable, yet 
another possibility is, that the new idioectosomatic substance is 
formed by the idiogranulomes. 

The concentration of new idiogranulomes and new idioecto- 
somatie substance progresses until a new idiosome is formed, 
having very probably been built from about half of the material 


THE IDIOSOME OF THE GUINEA-PIG 41 


of the old one in the primary spermatocyte. All of these stages 
are the same as those illustrated in figures 4 and 5, which repre- 
sent the corresponding phases in the division of the secondary 
spermatocytes. 


C. The karyogranulomes 


From the earliest stage of the spermatogonia down to the 
latest stage of the spermatids, the nuclei of the germ cells con- 
tain a number of granules which may be designated karyogranu- 
lomes. These granules are distinguished very clearly from all 
other constituents of the nucleus and they show the same stain- 
‘ing reaction and the same general structure as the idiogranulomes 
(figs. 1 to 11). The karyogranulomes seem to be independent 
of the chromatic substance in the nucleus and are the only ele- 
ments of the nucleus which show the red acid fuchsin reaction 
just as the idiogranulomes are the only elements outside of the 
nucleus which exhibit the same staining reaction. All other 
parts show a bluish or violet reaction. 

Are these karyogranulomes of the same origin as the idio- 
granulomes? Or, is there any relation between these two kinds 
of granulomes? These questions cannot be answered in a defi- 
nite way. That karyogranulomes come out through the nuclear 
membrane and go over to the idiosome or vice versa is very im- 
probable. It is possible, however, that, during the process of 
division when the limits of the nucleus are broken down, some 
of the idiogranulomes may pass into the nucleus and some karyo- 
granulomes may escape into the cytoplasm and later be incor- 
porated by the idiosome. The karyogranulomes during the 
division process may be seen among the spindle fibrils or 
on the chromosomes, while the idiogranulomes are dispersed 
throughout the protoplasm, see figure 4. Since no obstruction 
exists to prevent the mixture of the two kinds of granulomes 
during such a stage, it is very possible that some of the idio- 
granulomes may pass into the spindle and be later brought 
into the nucleus, or the opposite may occur and karyogranu- 
lomes may be detached from the spindle and left behind in the 
protoplasm when the division is over. Such a migration is al- 


42 G. N. PAPANICOLAOU AND C. R. STOCKARD 


most impossible to prove positively, as both kinds of granulomes 
show the same staining reaction and have the same structural 
appearance. 

The number and size of the karyogranulomes seem to differ 
in the different stages of the developing germ cells. They are 
greater in number and smaller in size in the stages represented 
by figures 3 to 6, during which stages the idiogranulomes are 
also small in size and very numerous. In the later stages, 
figures 8, 9, and 10, the karyogranulomes are generally less 
numerous and of larger size. This increase in size and decrease 
in number is probably the result of a fusion similar to that 
shown by the idiogranulomes in figures 7 and 8, where all of 
them have somehow run together to form a large spherical 
body. 

It is thus seen that karyo- and idiogranulomes show many 
analogies during ‘the different stages of their development. The 
finest granulation prevails in both karyo- and idiogranulomes 
during the stages illustrated by figures 3, 4, 5, and 6. This is 
what would be expected if one should suppose that the fine granu- 
lation represents a process to secure a distribution of the granu- 
lome material in the protoplasm and the nucleus during every 
division, as will be discussed beyond. 

The similar ways in which both kinds of granulomes react 
during the same stages strongly suggests some genetic relation- 
ship between them. Indeed it is probable that both sets of 
granules are the same things only located in different places. 

The karyogranulomes persist through all stages of the develop- 
ment of the germ cells as can be seen in the figures. In the 
ripe spermatozoa, however, they seem to be dissolved, as is the 
chromatic substance to disappear in the head of the spermatozo6n. 

It is also of importance to note that karyogranulomes may be 
occasionally seen in the nuclei of the Sertoli cells. 


D. The idiosome of the secondary spermatocytes 


The idiosome of the secondary spermatocytes, illustrated in 
figure 3, is a perfectly reconstructed, large, spherical, or slightly 
oval body, consisting of an idioectosomatice substance having the 


THE IDIOSOME OF THE GUINEA-PIG 43 


same color reaction as the idioectosome of the primary spermato- 
cytes and being filled with a great number of idiogranulomes. It 
seems that all idiosomes of the secondary spermatocytes have this 
granular appearance. The idiogranulomes have a tendency to 
be concentrated into one group of more or less circular outline, 
as if they were preparing to form a central sphere similar to the 
idioendosome of the primary spermatocytes. The idiosome in 
the secondary spermatocytes probably shows this constant 
granular type since the next division follows so quickly, little 
time being allowed for the idiogranulomes of this stage to fuse 
together as they do in all other more permanent stages. 

During the division of the secondary spermatocytes, the idio- 
some undergoes the same changes as the idiosome of the primary 
spermatocytes. These changes are illustrated in figures 4 and 
5. The idioectosome becomes irregular and begins to break 
into small pieces, while the idiogranulomes are dispersed in the 
cytoplasm. After the division a new idiosome is formed in the 
same way as was described during the corresponding stage fol- 
lowing the first spermatocyte division. The idiogranulomes 
flow together and a new idioectosome is slowly formed around 
them. During the reconstruction of the nucleus in the sperma- 
tids the number of the idiogranulomes increases and the idio- 
ectosome gradually becomes larger, assuming a regular spherical 
shape. In this manner the new spermatid idiosome is formed. 

In the nuclei of the secondary spermatocytes the number of 
karyogranulomes is large, corresponding to the great number of 
idiogranulomes. During the division of the secondary sperma- 
tocytes, these karyogranulomes are to be seen on the spindle 
fibrils or on the chromosomes, as illustrated in figure 4. 


E. The idiosome of the spermatids 


The idiosome of the spermatids presents, during its early 
formation, a type similar to that of the idiosome in the secondary 
spermatocytes, there being a great number of small granules 
enclosed in a large idioectosome (fig. 6). At the same time the 
nucleus contains a comparatively large number of karyogranu- 
lomes. 


44 G. N. PAPANICOLAOU AND C. R. STOCKARD 


This granular stage is of short duration in the spermatids, 
since the process of granular fusion begins very quickly after the 
reconstruction of the idiosome is completed. The small idio- 
granulomes fuse with one another to form first a smaller number 
of larger granulomes as shown in figure 7. This fusion process 
continues until ultimately a single large spherical body is pro- 
duced, which we have termed the idiosphaerosome. And it, 
like the granulomes from which it arose, exhibits a very intensive 
acid fuchsin reaction, as illustrated in figure 8. 

The idiogranulomes of the spermatids differ from those of the 
secondary spermatocytes in that each is contained within a dis- 
tinct small vacuole, the idiogranulotheca, the origin of which is 
very difficult to decide. It is possible that these vacuoles are 
formed by the idioectosome, but it is more probable that they 
are produced by the idiogranulomes themselves. These idio- 
geranulothecae flow together when the idiogranulomes fuse, form- 
ing larger vacuoles around the larger granules, until finally a 
single large vacuole that may be designated the idiosphaerotheca 
surrounds the final idiosphaerosome; the steps in this process are 
seen in figures 6, 7, and 8. : 

At times the idiosphaerosome or some of the larger idiogranu- 
lomes are connected with the wall or surface membrane of the 
idiosphaerotheca or of the idiogranulothecae, as the case may 
be, by one or more processes or prolongations. These prolonga- 
tions extend in different directions, sometimes towards the 
nucleus and sometimes away from it (figs. 7 and 8). The idio- 
ectosome in this stage is concentrated more and more on the 
upper periphery of the idiosphaerotheea, as shown in figure 8. 

The idiosphaerosome is a very changeable body. As soon as it 
arises it gives off a substance from its periphery mainly on the 
superior surface, which seems to have a different structure and 
different chemical qualities. This substance is distinctly vacuo- 
lar and its color reaction with the combined acid-fuchsin and 
methylen-blue stain is blue, thus presenting a striking contrast 
to the red color of the idiosphaerosome and the violet of the 
idioectosome. 


THE IDIOSOME OF THE GUINEA-PIG 45 


In this way the idiosphaerosome becomes differentiated into 
two distinct parts; one, an idiocryptosome, being more or less 
spherical in form, lies very close to the cell nucleus, while the 
other, the idiogalyptosome of our terminology, rests in the form 
of a cap over the idiocryptosome on the side away from the 
nucleus, as shown by figure 9. Both of these bodies, derivatives 
of the idiosphaerosome, are surrounded by the idiosphaerotheca. 

The idioectosome which, during the idiosphaerosome stage over- 
capped the idiosphaerotheca, begins now to assume amore concen- 
trated cap-like form and at the same time moves along the wall of 
the idiosphaerotheca, which it finally leaves to migrate along the 
surface of the nucleus to its posterior pole. This body is to be 
finally eliminated, and it perishes with the remains of the pro- 
toplasm during the metamorphosis of the spermatid as a sepa- 
rate spherical body, the idiophthartosome, shown in figure 10, 
id.phth. 

During all these changes the karyogranulomes are to be seen 
in the nucleus, but apparently in smaller number and of a 
somewhat larger size than in earlier stages, as shown by figures 
8 and 9. These karyogranulomes are most frequently found in 
close proximity to the nucleolus, but may also be seen in other 
places. 

In a later phase the idiocryptosome fits down closely upon 
the cell nucleus, and in so doing loses its spherical form to be- 
come somewhat discoidal or cap-shaped. The idiocalyptosome 
continues to increase in size, probably through some kind of con- 
stant reaction, and finally becomes a large body completely 
covering the cryptosome and a greater part of the nucleus, as 
is shown in figure 10. Its structure remains vacuolar. Some- 
times small pieces become detached from the cryptosome, erypto- 
granulomes, and are to be seen in the substance of the calyp- 
tosome, figure 10. -The idiophthartosome continues to move 
towards the posterior end of the nucleus, as figure 10 also shows. 

At this stage the karyogranulomes are generally very large 
and few in number. Exceptionally, however, they are small in 
size and more numerous. Some of them are to be seen in that 
portion of the nucleus immediately beneath the cryptosome, 


46 G. N. PAPANICOLAOU AND C. R. STOCKARD 


while others are nearer the posterior pole, as shown in figure 10. 
In some cases where great numbers of eryptogranulomes are 
present, as will be described later, the karyogranulomes which 
lie in that portion of the nucleus covered by the calyptosome may 
easily be confused with the cryptogranulomes contained within 
the substance of the calyptosome itself, since they are superim- 
posed. At other times the karyogranulomes come in such close 
apparent relation to the cryptosome that they seem to fuse with 
this body. It ishighly improbable, however, that any fusion of the 
karyogranulomes with the cryptosome or any migration of these 
eranulomes into the calyptosome through the wall of the nucleus 
ever takes place. The karyogranulomes later seem to dissolve 
in a fashion similar to the dissolution of the chromatic substance 
and are probably contained within the head of the spermatozoon 
in this dissolved condition. 

In its later development the calyptosome gradually attains an 
elongate shape until it forms a long cone which comes into con- 
tact with the prolongation from a Sertoli cell. At the same time 
it becomes more and more homogeneous, losing its original 
vacuolar condition. 

The cryptosome follows this change in shape of the calypto- 
some and forms a smaller cone enclosed within the conical calyp- 
tosome, while its wide base rests upon the nuclear membrane as 
illustrated in figure 11. At this stage the body of the erypto- 
some presents an irregular, granular structure (fig. 11). The 
idiophthartosome is now separated from the wall of the nucleus 
and passes into the cytoplasm with which it later disappears. 

As mentioned above, the calyptosome often contains within its 
mass small eryptogranulomes. This is probably due to a tend- 
ency on the part of the eryptosome to again break up into smaller 
granules. Such a tendency is not very strongly expressed in 
some animals, as, for example, the one which is taken as a type 
for the main description. Yet in other animals this tendency 
may be so strong that the cryptosome is broken up into a great 
number of eryptogranulomes, as shown in figures lla to lle and 
12a to 12d. All of these figures represent different degrees of 
cryptogranulosis observed in one and the same animal. 


THE IDIOSOME OF THE GUINEA-PIG 47 


This breaking up into granules begins very early, even during 
the formation of the calyptosome. When such is the case the 
calyptosome is, throughout its development, being filled with 
small eryptogranulomes, while a relatively small central crypto- 
some is left behind. In rarer cases this granulation begins even 
earlier at the stage when the idiosphaerosome is still present. 
The idiosphaerosome then consists of a number of granulomes 
which lie in a substance of semifluid appearance and are enclosed 
within the idiosphaerotheca. 

As mentioned above, this breaking-up process is only slightly 
expressed in some animals, while in others it is very prominent. 
Thus we may distinguish two different types of development, a 
massive, as in figures 11 and 12, and a granular type, as in fig- 
ures lla to 1le and 12a to 12d. Of the ten animals examined 
in this study, seven show the massive type and only three the 
granular. Of these three, two were treated with alcohol, one for 
four years and the other for three years, while the third was a 
normal but inbred animal (Stockard and Papanicolaou,’16). 
This merely suggests a possibility, and from present data it is 
only a possibility, that the granular type may. represent a dis- 
turbance of the normal type caused by the influence of some in- 
jurious factors, such as the alcohol treatment or inbreeding are 
found to be. It may be, however, that this deviation from the 
usual type is a normal variation due to some as yet unknown 
cause, and we have recently found a normal animal showing the 
granular type. 

During still later stages of development, the calyptosome 
loses its elongate shape and becomes more flattened, forming a 
cap over the upper part of the nucleus, which now appears 
almost homogeneous and is soon to form the head of the sper- 
matozo6n (fig. 12). The eryptosome, which is enclosed beneath 
the calyptosome, shows a tendency to form a unique homogeneous 
body. 

In the massive type the cryptosome changes its shape to form 
a smaller cap lying beneath the calyptosome cap, as seen in 
figure 12. Small granules on its surface soon disappear, and 
during the development of the spermatozo6n all granular struc- 


48 G. N. PAPANICOLAOU AND C. R. STOCKARD 


ture is lost and the eryptosome again presents an homogeneous 
appearance, as figure 13 will show., 

In the granular type of eryptosome the granules finally come 
to lie in a group at the base of the cap-like calyptosome and here 
fuse together, forming a body of the same conformation as in the 
massive type (figs. 12a to 12d). 

The heads of the ripe spermatozoa are thus covered by two 
caps, an inner, the cryptosome cap, and an outer or superior, the 
calyptosome cap. Without a special stain these two caps give 
the appearance of a single body, the spermiocalyptra. How- 
ever, with the staining methods to be explained in a following 
section, it is possible to differentiate the two parts of the calyptra; 
one as an intense red cryptosome cap and the other as a decid- 
edly blue calyptosome cap, as the figures illustrate. 

The spermiocalyptra is covered by a theca or membrane, the 
spermiocalyptrotheca, which is directly formed by the develop- 
ment of the idiosphaerotheca. This theca continues to exist 
through all stages of the transformation of the spermatids and 
becomes very large in size, covering the entire calyptra and a 
great part of the head of the spermatozo6n (fig. 13). 


F. The relation of the centrosomes to the idiosome 


Since the special methods used in this study of the idiosome 
do not stain the centrosomes, we have tried to study their evo- 
lution and especially their connection, if any, with the idiosome, 
by staining a number of the specimens with iron haematoxylin. 
The only stage during which the centrosomes are connected with 
the idiosome is that of the primary spermatocytes. In all pri- 
mary spermatocytes, at the stage illustrated by figure 1, the 
center of the idiosome is occupied by two dumb-bell-shaped cen- 
trosomes, as described by Meves (’99). We have never observed 
more than two centrosomes in one idiosome. As the centro- 
some stain does not furnish a clear differentiation between the 
ectosome and endosome, it is difficult to decide whether or not 
the two centrosomes are confined within the endosome sphere. 
In most of the cases, however, the two centrosomes appeared 


® THE IDIOSOME OF THE GUINEA-PIG 49 


to be enclosed within the endosome cavity, being usually in con- 
tact with its wall. 

When the endosome breaks up to form the idiogranulomes, the 
stage shown by figure 2, the centrosomes begin to migrate toward 
the cell nucleus, passing through the ectosomatic area and leaving 
the idiosome to perform their active réle during the division proc- 
ess of the primary spermatocytes. This behavior of the centro- 
somes and their later changes are described in detail by Meves 
(99), and our own observations agree very closely with his 
descriptions. 

The facts of particular interest in the present consideration 
are, first, that the centrosomes, on account of their specific 
staining reactions and their peculiar elongate slightly dumb-bell 
shape, should not under any circumstances be confused with the 
idiogranulomes; second, in no stage later than the primary sper- 
matocytes do the centrosomes show any connection with the 
idiosome. This temporary connection or association between 
the idiosome and the centrosomes and their later completely in- 
dependent and different activities throughout the process of 
spermatogenesis, along with their different staining reactions, 
suggest that the idiosome and the centrosomes, as well as their 
derivatives, are bodies of different natures with only early tem- 
porary topographical connections. Niessing (’96) has undoubt- 
edly confused the idiogranulomes with the centrosome, and this 
is probably the reason he sometimes finds more than two centro- 
somes. It also seems evident from his figures that what he has 
designated as a ‘Verklumpungsfigur der Centralkérpergruppe’ 
has nothing to do with the centrosomes, but is the endosome 
in process of transformation or granulation to form the idio- 
granulomes. Meves (’99) has also criticized this point in 
Niessing’s work. 


4. THE SPECIAL STAINING METHODS 


The manner of application of the fuchsin-methylen blue stain- 
ing method differs for the examination of the different parts of 
the idiosome in the various stages of its development. 


50 G. N. PAPANICOLAOU AND C. R. STOCKARD ‘ 


For the study of the idiogranulomes and of the karyogranu- 
lomes a satisfactory method is a single stain with acid fuchsin as 
follows: Method ‘A.’ Bring the sections through xylol and alco- 
hol into water, cover for a few seconds with Lugol’s solution, and 
then wash in water until the yellow color begins to fade out; 
then place in a saturated aqueous solution of acid-fuchsin for 
one-half to one minute, after which bring through the alcohols 
to earbo-xylol and mount in Canada balsam. 

With this method most of the cell structures stain a very light 
rosy tint, while the idio- and karyogranulomes have a decidedly 
dark red color. The idiosphaerosome and the cryptosome are 
also dark red, while the idioectosome and the calyptosome have 
the much lighter rose tone. The chromatin stains very lightly. 
When the chromatin does take on a dark color it indicates 
that the fixation is not very good. <A good fixation with Zenker’s 
fluid allows only the idioplasmatic substances to stain deeply 
with the above method. 

Method‘ B. After the staining with acid-fuchsin, as in method 
Ae? bring the sections into 80 per cent alcohol and then for a 
few seconds, ten to twenty, place in a saturated solution of 
methylen blue in 80 per cent aleohol. With this treatment the 
calyptosome takes on a blue color, while the eryptosome main- 
tains its dark red acid-fuchsin reaction. The karyogranulomes 
are not so prominent after this stain. 

Method ‘C.’ Bring the slide through xylol, absolute, 95 per 
cent and 80 per cent alcohol into a saturated solution of methylen 
blue in 80 per cent alcohol for several minutes. Then pass 
through the alcohols to water and leave for a few minutes in a 
2 per cent solution of iron-alum until a pronounced bluish tone 
is obtained. Wash thoroughly in running water until the blue 
tone begins to disappear and cover for a few seconds with Lugol’s 
solution. After this, place again in running water and leave for 
a few minutes, until the yellow color begins to fade; then stain in 
acid-fuchsin for from one to two minutes, and from this pass 
through the aleohols to carbo-xylol and mount in Canada 
balsam. 


THE IDIOSOME OF THE GUINEA-PIG 51 


With this method the granulomes, the idiosphaerosome, and 
the cryptosome are stained intensely dark red, especially the 
karyogranulomes and the eryptosome, which:take a very dark 
color, almost black. The calyptosome is light red and trans- 
parent, showing distinctly within it the granulomes and the 
eryptosome. The endosome and the ectosome of the primary 
spermatocytes are well differentiated and have a pronounced 
violet color. The ectosome of the spermatids and the phtharto- 
some are also violet. The chromatin has a light bluish or violet 
reaction, contrasting strongly with the dark red karyogranulomes. 

If a heavier chromatin stain be desired, the slides are left 
for a longer time in the methylen blue and less washed out in 
running water. In this way a blue chromatin stain may be ob- 
tained. The chromatoid Nebenkérper is also stained blue. 
The granulomes, however, are no longer so prominent and all 
other parts take a dark, heavier color. 

Method ‘D.’ ‘This method is the same as ‘C’ with the differ- 
ence that after the fuchsin stain the slides are brought into 80 
per cent aleohol and then to the methylen blue for a very short 
time, only a few seconds, and from this to carbo-xylol and 
mounted in Canada balsam. This method, like method ‘B,’ 
gives a good contrast between the cryptosome, which becomes 
dark red, and the calyptosome, which becomes blue.. The 
granulosomes show the same color as the eryptosome, but some- 
times are not so prominent. 

The two parts of the calyptra are.usually clearly differenti- 
ated by this method, as also by method ‘B.’ In the stages of 
the conical prolongation of the calyptosome, however, when the 
cryptosome and the cryptogranulomes are enclosed within the 
calyptosome, it is sometime difficult to see both parts clearly, 
since the calyptosome becomes very dark. For a better differ- 
entiation in this stage it is advisable to stain previously a shorter 
time in methylen blue, only a few seconds, or to use pure xylol 
instead of carbo-xylol for clearing the sections. All of these 
methods require a good fixation with Zenker’s fluid. If the 
fixation is not good, the different parts are not so nicely differen- 
tiated and one obtains a darker, rather diffuse stain. 


on 
bo 


G. N. PAPANICOLAOU AND C. R. STOCKARD 
5. REVIEW AND DISCUSSION OF PREVIOUS STUDIES 


The history of the idiosome begins with the studies of la 
Valette St. George (’65—’67), to whom is attributed the first de- 
scription of this body. La Valette St. George gave a simple 
description of the idiosome (Nebenkern) without going into the 
details of its transformations during the process of spermato- 
genesis. 

Merkel (’74) gave a description of the idiosphaerosome, Spit- 
zenk6érper, which he described as being formed through a partial 
condensation of the idiosome. The spermiocalyptrotheca, Kopf- 
kappe, derived as described in the foregoing text from the idio- 
sphaerotheca, was described by Merkel as being formed through a 
transformation of the nuclear membrane. 

VY. Brunn (’76) regarded the idiosphaerosome, Spitzenkoérper, 
as formed within the nucleus, probably from the nucleolus. The 
Kopfkappe was regarded by him as formed by the cytoplasm, 
and he believed it to be a temporary or perishable structure. 

Renson (’82) also described the idiosphaerosome, bouton ter-- 
minal, in rats and rabbits as a product of the nucleus. He de- 
scribed the formation and the disappearance of the idiophthar- 
tosome, corpuscule accessoire. The spermiocalyptrotheca was 
described by him also as formed from the separated nuclear 
membrane and was regarded as a transient structure. 

H. H. Brown (’85) gave a description of the idiosome in rats 
after the spermatogonial stage. He records also the changes of 
the idiosome, accessory corpuscle, during the division of the 
spermatocytes. Brown observed that the idiosome during the 
division of the cells broke into pieces and disappeared, and that 
a new idiosome was formed in the spermatids, probably from 
the remains of the old sphere. He also described the formation 
and the fate of the phthartosome, lunula stage, and the formation 
of the spermiocalyptra or cap from the idiosome. 

Hermann (’89) gave a clearer and more detailed description of 
the idiosome, Nebenkern, in the spermatocytes. He described it 
as being an uncolored oval body with another smaller body col- 
ored with gentian violet closely attached to one of its poles. It 


THE IDIOSOME OF THE GUINEA-PIG 53 


is very probable that this smaller body is nothing else than the 
chromatoid Nebenkérper. This body, which gives the same 
staining reaction as the nucleolus, is often in very close connection 
with the idiosome. Hermann believed that: the idiosphaetosome 
was derived from the nucleus. 

Benda (’91—’92) gave a detailed description of the develop- 
ment of the idiosome, Archoplasm, starting from the spermato- 
cyte stage. He described the dissolution of the idiosome during 
the last division of the spermatocytes and its reformation in 
the spermatids. He also described the formation of the idio- 
sphaerotheca, vacuole, and the appearance of the idiosphaero- 
some, kornartiger, stark farbbarer Kérper. He further recorded 
the formation and disappearance of the idiophthartosome, 
Archoplasma-rest. The spermiocalyptrotheca was correctly de- 
scribed by Benda as formed from the idiosphaerotheca, Vacuole. 

Moore (94) recorded in rats that the idiosphaerotheca, archo- 
plasmic vesicle, was formed by the fusion of many smaller 
vesicles. In each of these vesicles is formed a small central 
granule, archosome. All these archoplasmic vesicles and archo- 
somes flow together and finally form a single archoplasmic vesicle, 
our idiosphaerotheca, and a unique archosome, our idiosphaero- 
some. The idiosphaerotheca was regarded as a temporary for- 
mation. . 

In 1896 Niessing gave a rather detailed description of the de- 
velopment of the idiosome in guinea-pigs. He described the 
idiosome, Sphiire, of the primary spermatocytes, Mutterzellen, 
as consisting of a darker peripheral layer, Rinderschicht, and a 
central substance, Markschicht, in the midst of which lie the 
centrosomes, central Kérper, two to three or sometimes more. 
According to Niessing, the idiosome is traversed by a number of 
fibrils passing radially from the center to the periphery. In the 
peripheral layer Niessing observed a sharp and darker stained 
layer of granules, Kornerstratum. At times these granules were 
very numerous and formed in some cases as many as eight cir- 
cular layers within the peripheral portion. Sometimes the gran- 
ules extended into the cytoplasm beyond the limits of the idio- 
some. Niessing held that these granules were connected with 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 24, NO. 1 


54 G. N. PAPANICOLAOU AND C. R. STOCKARD 


the center by means of the radial fibrils, their positions being 
probably regulated through the contractions of the fibrils. 

Judging from this description by Niessing, the peripheral 
layer of the idiosome corresponds probably to our idioectosome 
and the central portion to our idioendosome. The granules ob- 
served in the peripheral part are the idiogranulomes and the 
radial fibrils are the prolongations from the endosome, as de- 
scribed above, page 39. All observers, who studied the idio- 
some before or after Niessing, failed to find any trace of these 
fibrils. We were never able to see such fibrils in the idiosome 
and think it probable that Niessing observed nothing more than 
the processes mentioned in our description as passing from the 
endosome through the ectosome. 

Niessing was not able to obtain a distinct stain for the idioendo- 
some, and for this reason he described the central portion of the 
idiosome as lighter than the peripheral, and thus failed to note the 
existence of the central body with clearly differentiated limits, the 
idioendosome. The description of Niessing leaves unsolved the 
origin and significance of the granules. He did not follow the 
distribution of the granules in the protoplasm during the divi- 
sions of the primary and secondary spermatocytes, nor their re- 
union in the formation of the new idiosome in the secondary 
spermatocytes and in the spermatids. 

The present description makes it clear, we believe, that the 
breaking-up process is probably a preparatory stage for the divi- 
sion of the idiosome and a means to carry out this division. The 
fine granulation secures a distribution of the idioplasmatie sub- 
stance in a way comparable to that by which the chromosomes 
serve to secure an exact distribution of the chromatic substance 
in every cell division. Fine granulomes accomplish a distribu- 
tion, but evidence is entirely wanting to show that the distribu- 
tion of the granulomes is equal in the daughter idiosomes as the 
chromosomal distribution is in the daughter nuclei. 

Niessing goes further with the deserrption of the idiosome, 
Sphire, in the spermatids, Tochterzellen. He states that the 
idiosomes immediately after the formation of the sperma- 
tids show all the elements described in the idiosome of the sper- 


THE IDIOSOME OF THE GUINEA-PIG 55 


matocytes, viz., centrosomes, radial fibrils, and idiogranulomes. 
He describes the idiogranulomes, Microsomenstratum, as being 
largely concentrated near the periphery. In our preparations the 
existence of radial fibrils is not demonstrated and the idiogranu- 
lomes not only occur near the periphery but also in the central 
part of the idiosome and very often close to the surface of the 
nucleus. We never have observed the centrosomes connected 
with the idiosome at this stage or at any other stage during the 
development of the spermatids. 

During the metamorphosis of the spermatids the idiosome 
undergoes the following transformations according to the de- 
seription of Niessing. The radial arrangement of the idio- 
granulomes, Microsomenstrata, disappears. The small idio- 
granulomes run together and thereby form fewer granulomes and 
finally a single large idiosphaerosome, Mitosom. At the same 
time the idiosphaerotheca, diinne glashelle Membran, is formed 
probably from the idiosome substance. The idiosphaerosome is 
still connected with the wall of the sphaerotheca by a fiber which 
extends towards the nucleus. Through the contraction of this 
fiber the idiosphaerosome comes into contact with the nucleus. 
As shown in the descriptive part of this paper, such prolongations 
of the idiosphaerosome may extend in all directions, sometimes 
directly away from the nucleus. 

The idiosphaerosome, Niessing believed, differentiated later 
into two parts, a central part which takes a black color with iron 
haematoxylin, probably our cryptosome, and an external part 
- which takes a gray color, probably our calyptosome. 

We say probably, because we are not altogether certain that 
the peripheral lighter,zone of the idiosphaerosome, as illustrated 
by Niessing and others, corresponds completely with the 
calyptosome. In our preparations the calyptosome is formed, as 
described above, in the shape of a cap resting on the superior 
pole of the idiosphaerosome (fig. 9), and is not of the nature of a 
theca or zone. It seems more probable that the outer lighter 
zone of Niessing is a part of the idiosphaerosome and not the 
first indication of the idiocalyptosome. Duesberg (’11) gives a 
figure showing the same differentiation into two zones in a large 


56 G. N. PAPANICOLAOU AND C. R. STOCKARD 


idiogranulome before the idiosphaerosome is formed through the 
complete fusion of all idiogranulomes, his figure 57. In our 
preparations, stained with the fuchsin-methylen blue method, 
the idiosphaerosome and the large idiogranulomes also show a 
differentiation in some cases into two zones; the outer one being 
generally somewhat darker in color, but always red. The blue 
calyptosome, with its distinctly vacuolar structure, is formed 
gradually from the anterior pole of the idiosphaerosome in the 
shape of a cap. In those of our preparations stained with iron 
haematoxylin, the differentiation into two zones was more ap- 
parent, with a central zone black and a peripheral zone gray, 
exactly as described by Niessing who used the same staining 
method. In some slides, stained with iron haematoxylin and 
differentiated with iron alum, we have observed certain places 
on a slide, where the differentiation with iron alum was 
slight, which shows the entire idiosphaerosome black; im other 
places, where the stain was further differentiated, the idio- 
sphaerosome is separated into two zones, as already described, 
a central black and a peripheral gray; while in still other places 
even more extracted the whole idiosphaerosome is gray. The ° 
same phenomenon may be observed in the staining and extract- 
ing of the idiogranulomes, some of them being black and others 
gray, onaccount of alessormore complete differentiation. This is 
probably one of the reasons that Niessing, as stated above, has 
confused the centrosomes with the idiogranulomes. The periph- 
eral gray zone of the idiosphaerosome in our iron haematoxylin 
preparations does not represent the first formation of the calypto- 
some. We cannot make this statement for the preparations of 
other investigators, but must point out the, possibility that the 
peripheral zone of Niessing may not be the early beginning of the 
calyptosome, but a differentiated part of the idiosphaerosome. 

This external part is described by Niessing as homogeneous 
and not as having a vacuolar structure. The cryptosome, 
dunkler Teil des Mitosoms, grows until it lies as a disk on the 
nucleus. The calyptosome, éiusserer teil, also grows until it is 
separated from the eryptosome. The development of the idio- 
sphaerotheca, helle Membran, into the spermiocalyptrotheca, 


THE IDIOSOME OF THE GUINEA-PIG 57 


Kopfkappe, is also described by Niessing. He further described 
the formation of the idiophthartosome, Sphirenrest, which mi- 
grates to the posterior pole of the nucleus and disappears with 
the cytoplasmic remains. 

Niessing deseribed very well the conical transformation of 
the calyptosome and he illustrated the existence of the crypto- 
some in this stage. Also in the transformed spermatozoén he 
showed the persistence of the same body enclosed in the calypto- 
some. He did not give sufficient importance to the separation 
of these two bodies, and for this reason he failed to designate 
them by different names. From his description one derives the 
impression that he did not regard the cryptosome and the 
calyptosome as two different things, just as he did not fully 
recognize the central and the peripheral parts in the idiosome 
of the spermatocytes as two separate bodies differing in structure 
and developing independently of one another. 

Niessing recorded the idiosome of the spermatocytes as a 
unique body showing a differentiation into two zones, an ex- 
ternal and an internal, in the same way as he described the 
spermiocalyptra, Spitzenknopf, as a unique body showing a 
differentiation into two zones, anexternal lighter gray in color and 
a central of a darker color. He did not observe that the calypto- 
some always presents a distinctly vacuolar structure in contrast 
with the homogeneous or granular structure of the cryptosome. 
He failed to notice the existence of the endosome as a separate 
body, its dissolution to form the idiogranulomes, the persist- 
ence and the role of these granulomes during the divisions, and the 
continuity of their development from the idioendosome to the 
spermiocryptosome. In a general way in spite of this Niessing 
gave a most detailed description of the idiosome, and his ob- 
servations mark an important step in its study. 

v. Lenhossék (’98) studied the development of the idiosome in 
rats and guinea-pigs. He also observed in the idiosome of the 
spermatocytes a differentiation into two zones, an external dark 
and an internal pale zone. This differentiation was not always 
distinct and sometimes the internal zone was eccentric in posi- 
tion. Although using the same method as Niessing, he was 


58 G. N. PAPANICOLAOU ‘AND C. R. STOCKARD 


unable to observe the radial protoplasmic fibrils or the concen- 
trie strata of granules described by Neissing. Lenhossék noted 
frequent granulations in the idiosome without giving their defi- 
nite distribution. He did not believe the idiosome to be of a 
fibrillar or granular structure, but to be a homogeneous concen- 
trated mass of protoplasm, and held that the granulations often 
observed in it are not regular or typical. He also observed the 
centrosomes in rats, invariably two in number, as is the case in 
guinea-pigs. 

Lenhossék found the idiosome appearing for the first time in 
the spermatogonia, in some of which it may still be absent, while 
in others only its early beginning can be seen. Only in the 
spermatocytes does it show a high development and a differ- 
entiation into a peripheral and a central zone. During the divi- 
sion of the spermatocytes the centrosomes come out of the idio- 
some, while the latter assumes irregular shapes and later breaks 
up to be dispersed in the cytoplasm. In the secondary spermato- 
cytes and in the spermatids a new idiosome arises from the 
remains of the old one. The newly formed idiosome of the 
spermatids is described by Lenhossék as homogeneous. At the 
border only he sometimes observed granules of different sizes 
staining black with iron haematoxylin and eventually uniting 
to form a continuous outline. He emphatically denies the exist- 
ence of any granules or fibrils, such as described by Niessing. 

Later on a vacuole is formed within the body of the idiosome, 
our idiosphaerotheca, and in this vacuole appears a central 
granule, the acrosome, our idiosphaerosome. ‘The central gran- 
ule appears suddenly, ‘wie durch einen Schopfungsakt,’ through 
a spontaneous concentration and differentiation of its substance. 
This acrosome later shows a differentiation into a peripheral 
zone and a central zone. 

Lenhossék further describes the formation and the fate of the 
idiophthartosome, the form changes of the calyptosome and the 
formation of the spermiocalyptrotheca, Kopfkappe, from the 
idiosphaerotheca, his vacuole. No further mention, however, is 
made of the cryptosome. The calyptosome through its entire 
development he described as homogeneous with no trace of 


THE IDIOSOME OF THE GUINEA-PIG 59 


differentiation. The spermiocalyptra he also described as an 
homogeneous mass resulting from the growth and transformation 
of the idiosphaerosome, Acrosoma, during whose growth the 
idiosphaerosome loses its affinity for the iron haematoxylin stain. 
He noticed also the existence of a sickle-shaped area between the 
spermiocalyptra and the head of the spermatozo6én, this area 
being filled by a pale substance, Kittsubstanz. It is possible 
that this area corresponds to our spermiocryptosome. 

From his description it is evident that Lenhossék, although 
using the same methods as Niessing, did not observe so many de- 
tails as the latter. The idiogranulomes seen by Niessing in the 
spermatocytes and the newly formed spermatids were not ob- 
served by Lenhossék. The formation of the idiosphaerosome 
and the idiosphaerotheca were incorrectly described as appear- 
ing suddenly, ‘wie durch einen Schépfungsakt.’ The differ- 
entiation of the calyptosome and the cryptosome he observed 
only during its first stage, while Niessing followed this differen- 
tiation to the ripe spermatozoén. In a general way the obser- 
vations of Lenhossék concerning the idiosome of the guinea-pigs 
confused, rather than advanced the subject. 

One year later, Meves (’99) also gave a description of the 
development of the idiosome in the guinea-pigs. Meves ob- 
served the granular structure within the idiosome of the pri- 
mary and secondary spermatocytes and in the spermatids. He 
found no granules in the spermatogonia nor in the cells of the 
growth period. In addition to the granulations he also ob- 
served in the idiosome of the spermatocytes the two elongate, 
slightly dumb-bell-shaped centrosomes. Meves described cor- 
rectly the formation of the idiosphaerosome, acrosoma of Len- 
hossék, through the fusion of the granules existing in the 
idiosome of the spermatids, as well as the formation of the idio- 
sphaerotheca by a fusion of the vacuoles, Blaschen, surrounding 
these granules. He also described correctly the formation and 
disposition of the idiophthartosome and the transformation of 
the idiosphaerotheca into the spermiocalyptrotheca, Kopfkappe. 
He observed the separation of the idiosphaerosome into two 
zones, an external, Aussenzone, and an internal, Innenkorn. 


60 G. N. PAPANICOLAOU AND C. R. STOCKARD 


Meves observed these two zones with different staining methods 
and expressed the opinion that they must represent actually 
different structural products. His staining methods, however, 
did not permit him to trace the persistence of the eryptosome, 
Innenkorn, through all the later stages to the ripe spermato- 
zoon. He, therefore, believed that the entire eryptosome, In- 
nenkorn, was transformed into the calyptosome, Aussenzone. 
The calyptra was described by Meves as being homogeneous, 
and he recorded the cryptosome as later disappearing completely. 

The descriptions of Meves are very accurate, but do not bring 
out facts other than those observed by previous investigators, 
especially Niessing. The granules in the spermatocytes and the 
spermatids, the formation of the idiosphaerosome and the idio- 
sphaerotheea, the formation and disappearance of the idiophthar- 
tosome, the separation of the calyptosome and the cryptosome, 
and the formation of the Kopfkappe had all been described before. 

Niessing is the only observer who followed the separate exist- 
ence of the calyptosome and the cryptosome up to the sperma- 
tozoon. 

The probable reason for so little essential progress after Nies- 
sing (’96) in the investigation of the idiosome is the fact that the 
major attention of investigators, studying the spermatogenesis 
in different animals, has been concentrated on changes in the 
nucleus and especially in the chromatic substance. The idiosome 
has been more or less considered in many of these studies, but it 
has not been made the central point of attack. It would be out 
of place to discuss here all the papers in which the development 
of the idiosome has been partly described in different animals. 
It is not our purpose to give a comparative review of the idio- 
some in the different animal classes, but only to consider the 
progress made in the study of this body in certain animals and 
especially in mammals, in order to convey some idea of the 
proper position of the present results. 

In the light of the above review, we may now state the case 
of the idiosome in the spermatogenesis of the guinea-pig in the 
following summary manner. 


THE IDIOSOME OF THE GUINEA-PIG 61 


6. CONCLUSIONS AND NEW POINTS 


1. The idiosome in the spermatogonia is not a fixed structure, 
but shows great variability in form and appearance. In some 
cases it is distinetly vacuolar and in others there is a beginning 
of a differentiation into two zones, a central and a peripheral. 

2. The idiosome in the primary spermatocytes has a definite 
structural division into two parts; an internal or central body, 
the idioendosome and a surrounding larger sphere, the idioecto- 
some. The endosome sends processes or prolongations of its 
substance into the surrounding idioectosome, giving the latter a 
vacuolar appearance. 

3. When the primary spermatocytes prepare to divide, the 
idioendosome breaks up into a number of granules, the idio- 
granulomes. As the division of the spermatocytes proceeds the 
idioectosome likewise breaks up into smaller pieces which are 
gradually dispersed in the cytoplasm. This liberates the idio- 
granulomes which now also become scattered in the cytoplasm 
of the spermatocytes. 

4. After the division of the primary spermatocytes a new 
idiosome is formed in the secondary spermatocytes through a 
reunion of the idiogranulomes and possibly of the original dis- 
persed pieces of the idioectosome. 

5. The idiosome in the secondary spermatocytes maintains at 
all times a granular structure, consisting of a number of idio- 
granulomes enclosed within the ectosome sphere. 

6. During the division of the secondary spermatocytes the 
idiosome again breaks up in the same manner as during the 
division of the primary spermatocytes. 

7. A reconstruction of the idiosome again takes place in the 
spermatids immediately after the division. The idiosome ex- 
hibits a number of idiogranulomes enclosed within the ectosome, 
but each idiogranulome seems now to be surrounded by a 
small vacuole, the idiogranulotheca. It is not certain whether 
these vacuoles are formed by the ectosome or by the idiogranu- 
lomes. 

8. The idiogranulomes and the idiogranulothecae run together 
and fuse into larger and larger granulomes enclosed in larger and 


62 G. N. PAPANICOLAOU AND C. R. STOCKARD 


larger vacuoles until finally one large granulome, the idiosphaero- 
some, is surrounded by one large vacuolar wall, the idiosphaero- 
theca. : 

9. The idiosphaerosome produces from its upper surface a 
new body in the form of a cap, having a vacuolar structure and 
different staining reactions. This is the calyptosome. The re- 
maining part of the idiosphaerosome is the idiocryptosome. 
These two bodies now develop separately and persist up to the 
formation of the spermatozoon. 

10. The idioectosome detaches itself from the idiosphaerotheca 
and migrates along the surface of the cell nucleus to its posterior 
pole and there passes into that part of the cytoplasm which is 
lost during the metamorphosis into the spermatozoén. 

11. The idiosphaerotheca persists through all later stages and 
develops into a membranous cover for the cap and head of the 
spermatozoon. 

12. There are two types of development shown by the erypto- 
some, one massive or concentrated and the other granular. 

13. During all stages of spermatogensis a number of granules, 
karyogranulomes, having the same appearance and staining re- 
actions as the idiogranulomes, are to be seen in the nucleus. 
During the last metamorphosis of the spermatids, when the 
nucleus is transformed into the head of the spermatozoén, the 
karyogranulomes seem to be dissolved within the sperm-head in 
the same way as is the chromatin. 

14. During the divisions of the germ cells the karyogranulomes 
are to be seen on the spindle fibrils or on the chromosomes, while 
the idiogranulomes are scattered throughout the cytoplasm. 
No membranes or structures separate the two classes of granules 
during division, and it is possible that some idiogranulomes may 
migrate into the spindle area and thus be earried to a daughter 
nucleus, or that karyogranulomes may be detached from the 
spindle and become free in the cytoplasm to form later part of 
the idiosome. A direct observation of such an exchange is al- 
most impossible, since the size variations, the appearance and 
the color reactions of the idio- and karyogranulomes are too 
closely the same. 


THE IDIOSOME OF THE GUINEA-PIG 63 


15. The breaking up into small granules may be considered 
as a process to secure a distribution of the idioplasmatic sub- 
stance during cell divisions. 


The new points brought out by the present study are the 
following: 

1. A deseription for the first time of the idioendosome. 

2. A description of the formation of the idiogranulomes through 
the breaking up of the idioendosome. 

3. The persistence of the idiogranulomes during the divisions 
of the primary and secondary spermatocytes, their distribu- 
tion in the cytoplasm, and their subsequent reunion during 
the reconstruction of the daughter idiosomes. Idiogranulomes 
have not been previously observed during the division of the 
spermatocytes. 

4. The existence of the karyogranulomes which had always 
been overlooked and their persistence through all stages of 
spermatogenesis. 

5. The exact manner of formation of the calyptosome and its 
vacuolar structure. This body had previously been described as 
homogeneous. 

6. The great variability in the development of the cryptosome, 
the formation of the cryptogranulomes and the granular con- 
sistency of the eryptosome during the stages shown by figures 11 
and 12. 

7. The double nature of the idiosome consisting of an ecto- 
somatic and an endosomatic substance, the development of the 
two substances being independent of one another. 

8. The réle of granulation in serving to distribute the idio- 
plasmatie substance during cell division. No theory until now 
has been advanced concerning the significance of the idiosome 
granules. 


64 G. N. PAPANICOLAOU AND C. R. STOCKARD 


7. LITERATURE CITED \ 


Brenpa, C. 1891-92 Neue Mittheilungen iiber die Entwicklung der Genital- 
driisen und iiber die Metamorphose der Samenzellen (Histogenese der 
Spermatozoen). Verhandl. d. Physiol. Ges. Berlin. 

Brown, H. H. 1885 On spermatogenesis in the rat. Q.J.M.S., 25, N.S. 

y. Brunn, A. 1876 Beitrage zur Entwicklungsgeschichte der Samenkérper. 
Arch. f. mikr. Anat., 12. 

DursperG, J. 1911 Nouvelles recherches sur l’appareil mitochondrial des 
cellules séminales. Arch. f. Zellforsch., 6. 

Hermann, F. 1889 Beitriige zur Histologie des Hodens. Arch. f. mikr. Anat., 
34. 

y. Lennoss&K, M. 1898 Untersuchungen iiber Spermatogenese. Arch. f. mikr. 
Anat., 51. 

MERKEL, Fr. 1874 Erstes Entwicklungsstadium der Spermatozoiden. Unters. 
aus d. Anat. Inst. zu Rostock. 

Meyes, Fr. 1899 Uber Struktur und Histogenese der Samenfaiden der Meer- 
schweinchens. Arch. f. mikr. Anat., 54. 

Moorg, J. E.S. 1894 Some pointsin the spermatogenesis of mammalia. Inter- 
nat. Monatsch. f. Anat. und Physiol., 11. 

Nresstne, C. 1896 Die Betheiligung von Centralkérper und Sphire am Aufbau 
des Samenfadens bei Saugethieren. Arch. f. mikr. Anat., 48. 

Recaup, Cu. 1910 Etudes sur la structure des tubes séminiféres et sur la sper- 
matogénése chez les mammiféres. Arch. d’Anat. Mier., 11. 

Renson, G. 1882 De la spermatogénése chez les mammiféres. Arch. de 
Biol., 3. 

y. LA VALETTE, St. Grorce 1865 and 1867 Uber die Genese der Samenkérper. 
Arch. f. mikr. Anat., 1 and 3. 

Srockarp, C. R.,anp Papanicotaou, G. N. 1916 A further analysis of the 
hereditary transmission of degeneracy and deformities by the de- 
scendants of alcoholized mammals. Amer. Nat., 50. 


PLATES 


All figures are camera-lucida drawings of the objects magnified by paired 
oculars No. 10 and oil-immersion objective 1/9 mm. of Bausch & Lomb. 


Abbreviations used: 
cal., calyptosome 
car.gr., karyogranulomes 
erypt., cryptosome 
crypt.gr., eryptogranulomes 
hd., head of spermatozoén 
id.ect., idioectosome 
id.end., idioendosome 


Figures 1 to 13 are from one animal and figures lla to Ile and 12a to 12d are 


from another. 


id.gr., idiogranulomes . 
id.gr.th., idiogranulotheca 

id.phth., idiophthartosome 

id.sph., idiosphaerosome 

id.sph.th., idiosphaerotheca 

n.id., new idiosome 

sp.th., spermiocalyptrotheca 


PLATE 1 


EXPLANATION OF FIGURES 


Fig. 1 A primary spermatocyte showing the idioendosome and the idioecto- 
some and with karyogranulomes in the nucleus. 

Fig. 2. A primary spermatocyte showing the formation of the idiogranulomes 
from the idioendosome. Two granulomes are to be seen in the protoplasm and 
four others in the nucleus. 

Tig. 3 A secondary spermatocyte showing the idioectosome, the idiogranu- 
lomes and a number of karyogranulomes. 3 

Fig. 4 A secondary spermatocyte in division showing a piece of the ecto- 
some filled with idiogranulomes, while other idiogranulomes are scattered in the 
protoplasm; karyogranulomes are seen on the spindle fibrils and on the chromo- 
somes. 

Fig. 5 A spermatid immediately after the secondary spermatocyte division, 
showing the reconstruction of the new idiosome, n.id. 

Fig. 6 A spermatid showing the reconstructed idiosome with the idiogranu- 
lomes enclosed in vacuoles, the idiogranulothecae. 

Fig. 7 A spermatid showing two large idiogranulomes formed by the fusion of 
smaller granulomes and another small idiogranulome. 

Fig. 8 A spermatid showing the large idiosphaerosome which resulted from 
the fusion of all the idiogranulomes; the idiosphaerosome is enclosed within the 
idiosphaerotheca, surrounding which is the idioectosome. 


66 


THE IDIOSOME OF THE GUINEA-PIG 
G, N. FAPANICOLAOU AND C. R. STOCKARD 


id.ect. 


car.gr. SS 


Cal. Sr. 
5 
cars! 
id. gr. 8 


Ff 
id.ect. , id. gt 
be — id. ar.th 
car. 3). se 


67 


PLATE 1 


id.ect TN id.gh. 
a 


¥< 
eae 


aa 7 sr. 
Yu 


8 


idect Gai, sph. 
~=__ id. Sph.th. 


PLATE 2 
EXPLANATION OF FIGURES 


Fig. 9 A spermatid showing the separation of the calyptosome, cal., and the 
cryptoson e. crypt., both are enclosed within the large idiosphaerotheeca. The 
idiophthartosome derived from the idioectosome begins to migrate towards the 
lower pole. ; 

Fig. 10 A later stage in the development of the calyptosome and the crypto- 
some. In the calyptosome are two small cryptogranulomes. The idiophtharto- 
some, id.phth., is near the lower pole. The chromatic substance begins to be 
dissolved, concentrating about the lower pole. Two large karyogranulomes 
are to be seen, one beneath the cryptosome and another near the lower pole. 

Fig. 11 A later stage showing the conical elongation of the calyptosome and 
the eryptosome. The eryptosome has a granular appearance. One cryptogranu- 
lome is near the apex of the cone. The karyogranulomes and the chromatic 
substance are dissolving in the nucleus. 

Figs. lla to lle Similar stages showing different degrees of cryptogranulosis. 
the granular type. 

Fig. 12 The head and ecalyptra of a spermatozoén in process of development 
as seen in a seminiferous tubule. The cryptosome still has a granular structure. 

Figs. 12ato12d Similar stages showing different degrees of cryptogranulosis. 
the granular type. in one and the same animal. 

Fig. 13 A mature spermatozoén head from the epididymis. The calyptra 
composed of the calyptosome and the cryptosome. as well as a large part of the 
head are covered by the spermiocalyptrotheca 


68 


THE IDIOSOME OF THE GUINEA-PIG 
G. N. PAPANICOLAOU AND C. R. STOCKARD 


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Cal. 
fais Ile 
Crypt or. = 
Ne 
a Call. 
Re i 
2b. I2c 
Cry pt. gr. Crypt.gr. ; 
: , cal. 


69 


PLATE 2 


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= —Cal. 


y eae 


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V7] 


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THE VAGINAL CLOSURE MEMBRANE, COPULATION, 
AND THE VAGINAL PLUG IN THE GUINEA-PIG, 
WITH FURTHER CONSIDERATIONS OF THE 
ESTROUS RHYTHM. 


“Reprinted from BIOLOGICAL BULLETIN, Vol. XXXVII., No. 4, October, toro } 


THE VAGINAL CLOSURE MEMBRANE, COPULATION, 
AND THE VAGINAL PLUG IN THE GUINEA-PIG, 
WITH FURTHER CONSIDERATIONS OF THE 
(ESTROUS RHYTHM. 


CHARLES R. STOCKARD anp GEORGE N. PAPANICOLAOU, 


CORNELL UNIVERSITY MEDICAL COLLEGE, NEW YorK CIty. 


Two years ago we recorded the results of a detailed study of the 
cestrous cycle in the guinea-pig. A rather full description of the 
histological and physiological changes which take place in the 
ovary, uterus and vagina during the “‘heat period’’ was presented. 
We emphasized particularly the importance of changes occurring 
in the microscopic composition of the vaginal fluid as indicative 
of the exact conditions in the uterine wall and ovarian follicles 
at corresponding moments. 

Since that time we have somewhat extended the analysis of 
these phenomena. It has been found that a membrane covering 
the orifice of the vagina furnishes a most valuable and simple 
means of diagnosing certain periods in the cestrous cycle. This 
we have termed the “vaginal closure membrane.’’ The exact 
moment of copulation and the conditions in the walls of the 
vagina and uterus at this time have been carefully followed, 
along with a consideration of the formation and significance 
of the vaginal plug. In the present paper a discussion of these 
several topics will be undertaken. 

Certain points in the literature will also be discussed, a more 
complete review having been given in the previous article. 


I. THE VAGINAL CLOSURE MEMBRANE. 


In the former communication attention was called to the fact 
that “the external vaginal orifice, which during the period of 
cestrous activity is more or less open, actually showing in many 
cases a little fluid or some blood, closes and becomes less accessible 
after the period.”” During ovulation the vagina is open, but the 
fact of its being open is not unmistakable proof of the time of 

222 


GESTROUS CYCLE IN THE GUINEA-PIG. 223 


ovulation unless the open vagina also contains what was described 
as second or early third stage oestrous fluid. 

At that time the method of closure of the vagina following the 
cestrus was not explained nor was its actual significance fully 
appreciated. The vagina is now found to be closed by a remark- 
able cellular membrane and in a very definite way. 

The external orifice of the vagina is crescentic in shape and 
the urethral opening lies in front of it in the mid line. The 
anterior and posterior lips of the crescent-shaped opening come 
together, and a delicate epithelial membrane grows over the 
opening and unites the lips. This occurs shortly after the heat 
period in females that have not copulated and in those that have 
copulated the closure follows the expulsion of the vaginal plug, 
a process to be considered beyond. The closure begins at the 
tips of the crescent-shaped opening and progresses toward the 
midpoint. The lips do not approximate so intimately at the 
midpoint and the membrane here seems to be under more 
tension than at other parts, even after the entire orifice has closed. 
The opening of the orifice by a tearing of the epithelial membrane 
begins at the strained middle part and extends from there 
laterally until finally the vaginal lips are freely separated. The 
midpoint is, therefore, the last to close, and the first to open as 
a general rule, although at times the opening may begin at either 
side of the midline. 

The epithelium completely unites the lips of the vagina so 
that nothing can escape from or enter into the vaginal lumen 
without tearing this closure membrane. Such a membranous 
closure of the vaginal orifice is unknown to us in any other 
mammal. In many species the sides of the vaginal opening 
may be approximated or cemented together by some hardened 
fluid or secretion so that the lips are not readily pressed apart, 
but a membranous growth closing the orifice after each heat 
period is apparently unique. 

This membrane also completely closes the vaginal opening 
throughout pregnancy and only becomes ruptured when the 
vulva swells shortly before parturition. 

Such an obstruction or closure of the vaginal lumen at once 
suggests the hymen of the human vagina. But this, of course, 


224 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU. 


is quite a different structure in its origin as well as in its later 
history. The closure membrane in the guinea-pig not only 
exists in the young immature animal but is regularly destroyed 
before and reformed after every heat period that takes place 
during the life of the female. The formation or growth of this 
membrane might also be compared in some respects to the 
membranous growths tending to extend across and close the 
pharynx and other canals under pathological conditions. 

The membrane is thin and delivate in structure and when 
stretched by slightly pressing apart the lips of the vagina with the 
fingers it is seen to be almost transparent, the outline of the 
vaginal lumen showing through. The closure membrane is of 
the same glossy appearance as is the surface epithelium covering 
the region of the vaginal lips with which it is continuous. It is 
composed simply of*stratified squamous epithelium which has 
grown from the borders of the lips over the orifice and contains 
no vessels or blood. 

When the membrane is torn or broken by accident during the 
dicestrum, or period of sexual rest, it reforms sometimes within 
a day, or within a few days, and remains until the beginning of 
the new period of heat or cestrus. A recognition of this mem- 
brane is then a great convenience in determining the onset of the 
cestrus in a group of female guinea-pigs. Daily smears of the 
vaginal fluid are not now necessary to find when the cestrus is 
about to begin in animals examined for the first time and whose 
rhythm is therefore unknown. 

Although the presence of the closure membrane is a definite 
aid in recognizing the condition of the cestrous cycle, it must be 
remembered that this membrane often persists up to the first 
stage of oestrus, at which time the lumen of the vagina is filled 
with a mucous fluid and first stage cells. This is actually the 
“heat’’ time and the normal moment for copulation as we shall 
explain below. When the closure membrane still persists until 
the vaginal lumen is so filled it may be distended and rounded 
out resembling a blister membrane on the point of bursting. 
Puncturing this the vaginal fluid oozes out through the break. 
As a general rule, however, the vulva becomes inflamed and very 
slightly swollen immediately before cestrus and the stretched 


(ESTROUS CYCLE IN THE GUINEA-PIG. ; 225 


membrane breaks. Thus the membrane has reached the break- 
ing point or has actually broken at just about the time the female 
is in heat and ready to copulate. 

While the presence of this membrane is a reliable index of the 
cestrous condition, the open vagina, or its absence, is by no means 
indicative of the cestrous state. Although the vagina is always 
open during what we have termed the second and third stages of 
cestrous, and, therefore, at the time of ovulation whether copula- 
tion has taken place during the first stage or not, it is nevertheless 
frequently open at other times. It is not permissible to assume 
that the open vagina indicates a state of heat or the time of 
ovulation in a guinea-pig. Only when the open vagina contains 
fluid showing on examination the cells described as second or 
early third stage is the ovary almost exactly in the condition of 
ovulation. It may be stated parenthetically that after long 
experience one is able as a rule to diagnose the stages of the 
vaginal fluid by slight difference in color and consistency without 
microscopic examination. 

Finally, then, when the vagina is open one may only be certain 
of the uterine and ovarian conditions by examining the contents 
of its lumen, but, on the other hand, if it be closed by this mem- 
brane one may be certain that the time of the new ovulation has 
not yet arrived. 


2. THE TIME AND MANNER OF COPULATION AND THE CONDITIONS 
IN THE REPRODUCTIVE ORGANS OF THE FEMALE AT 
THIS MomMENT. 


It is well known that female guinea-pigs in common with 
other animals of their class, and in fact most mammals, have a 
definite limited time during which they accept the male, the 
so-called “period of heat.’’ This period, very slightly revealed 
by external signs at the mouth of the vagina, but chiefly by the 
act of copulation has been the starting point in all previous 
studies on the reproductive activities of the guinea-pig. In 
order to prevent the modifying conditions of pregnancy following 
copulation, various operations have been resorted to, as in the 
case of some of Loeb’s experiments. Such operations might com- 
plicate or even vitiate the results which follow. 


226 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU. 


In the present account we wish to describe the exact moment 
at which copulation takes place during the sexual cycle and to 
show definitely the conditions of the vagina and uterus at this 
moment. From the condition of the vagina or uterus the ovarian 
condition is readily estimated, as we have shown in the former 
paper. After determining the exact cestrous condition of a 
female at the moment she is ready for copulation we may then 
recognize a corresponding moment in any female by an examina- 
tion of the vagina without the necessity of introducing the male 
or permitting copulation to occur. 

In order to designate the copulation time exactly, we must 
review briefly the characteristics of the four very clearly defined 
stages of the oestrus or “heat period”’ proper. During stage 
one the uterine epithelium swells, the cells becoming distended 
with an abundant mucous secretion which very soon pours into 
the lumen and reaches the vagina. At this time a desquamation 
of the epithelial cells from the lower part of the vagina also begins. 
The second stage shows a great accumulation of leucocytes 
below the uterine and vaginal epithelium with a slowly progress- 
ing desquamation of epithelial cells. The third is the stage of 
exodus of the leucocytes, myriads of them coming through the 
epithelial lining of the walls of the uterus and vagina, with an 
accompanying extensive destruction of the epithelium. During 
the fourth stage the broken down epithelium falls away in masses 
and at the same time a regeneration of epithelium takes place 
beginning from the mucosa of the uterine glands. 

The Graafian follicles of the ovary rupture and ovulation occurs 
at the end of the second stage or the beginning of the third stage, 
while during the fourth stage the recently ruptured Graafian 
follicles are already well under way in their development into 
new corpora lutea. 

A recent abstract by Long (’19) seems to indicate that four 
similar stages may be recognized during the cestrus in the rat, 
and that these stages agree almost exactly in significance with 
the comparable ones in the guinea-pig: Ovulation also occurring 
in the rat at the end of the second or beginning of the third stage. 

During our initial investigation we made no attempt to locate 
the exact moment of copulation and, of course, did not describe 


CGSTROUS CYCLE IN THE GUINEA-PIG. 227 


the corresponding vaginal or uterine stage. The description by 
Loeb (’14), however, of the great leucocyte migration in the 
wall of the uterus twelve to twenty-four hours after copulation 
would indicate that the true ‘‘heat’’ or copulation occurs before 
the beginning of the destructive changes in the uterus, and this 
we now find to be true. Here and in the following section we 
wish to point out just how the uterine changes seem to be 
associated with the act of copulation, the retention of the sperm 
in order to insure the fertilization of the eggs, and after this 
the means of ridding the vagina of the excessive seminal accumu- 
lation. 

A number of females have been placed with males while in 
one or another of the above mentioned four stages, as well as 
during different times of the dicestrum, or interval of sexual rest. 
The results show that a copulation is never accomplished except 
during the first stage of cestrus about twelve hours before the 
second stage begins. Long ('19), also finds copulation to take 
place in the rat during the first stage. At this stage in the 
guinea-pig the vagina contains a clear, foamy, saliva-like fluid in 
which desquamated epithelial cells of the first type are present. 
Differing from all other stages and times there are now no leuco- 
cytes to be found in the vaginal fluid,—compare our former Figs. 
1 and 2 with Figs. 5 and 6. Even during the resting period the 
vagina contains some mucus but this is very scant and filled 
with many leucocytes, being pus-like in appearance and con- 
sistency. 

To locate even more accurately the normal time of copulation 
the first stage may be subdivided into two shorter periods: a 
preparatory interval, the early beginning of the first stage, when 
the vagina is almost dry and contains only a scant amount of 
loose cells of the first type; and, second, what may be designated 
the true first stage, a more advanced period when the frothy 
mucous secretion has already begun to accumulate. Copulation 
takes place during this second phase of stage one and never during 
the first. 

During stage one the vagina is characterized by two important 
conditions, both of which contribute to the success of copulation. 
In the first place the existence of a mucous secretion evidently 


228 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU. 


facilitates the act of copulation. This abundant frothy mucous 
accumulation is limited to the first stage, particularly to the time 
when copulation takes place, and it never occurs in the vagina at 
other times. The second contributory condition is the complete 
absence of leucocytes in the mucous fluid. The leucocytes begin 
their migration through the epithelium of the uterus and vagina at 
the end of the second stage. They are extremely abundant in 
the lumen during the third stage, while from this time on they 
are found in the uterus and vagina in smaller or larger quantities 
up to the approach of the next first stage. Two days before the 
first stage begins leucocytes are still plentiful, but from this time 
first stage epithelial cells gradually become more abundant and 
the leucocytes decrease in number until finally when the first 
stage has actually arrived no leucocyte exists in the vaginal fluid. 
The mucous content of the vagina during the first stage hence 
lacks the pus-like appearance of the vaginal fluid of the “‘inter- 
menstrual” time and is clear and foamy. 

The absence of leucocytes from the vaginal lumen at the time 
of copulation is important, since, if present, they might by their 
dissolving powers or phagocytic action exert an injurious effect 
on the spermatozoa and thus interfere with their normal function. 
Later a special purpose of the leucocytes seems to be to destroy 
the excess of spermatozoa remaining in the uterus. This fre- 
quently occurs by an interesting process of phagocytosis. A 
leucocyte comes in contact with a spermatozoén which with its 
tail is longer than the leucocyte. The leucocyte by stretching 
and contracting finally takes into itself the entire spermatozo6n, 
the tail being wound in circular fashion within the cell body. 
The leucocytes, however, apparently accomplish most destruc- 
tion by their dissolving or disintegrating action. 

It seems that the migration of the leucocytes through the 
walls of the uterus and vagina, though not increased in extent, is 
accelerated by the act of copulation and the entire oestrous 
process is shorter than in non-copulated females. About six 
hours after copulation the third stage is in full development, 
while under virgin conditions a comparable stage is reached only 
after at least twelve hours from the time when copulation 
might have occurred. It may be said that copulation tends to 


GSTROUS CYCLE IN THE GUINEA-PIG. 229 


hasten ovulation, or that the act itself may facilitate the bursting 
of the Graafian follicles, which is.a very old conception. 

The act of copulation is short, lasting a few seconds only, while 
the preceding time of sexual excitement leading up to it is rather 
. long. The male becomes excited by the presence of the female 
some time before she reaches the proper condition for copulation. 
A male after long isolation from females becomes sexually excited 
by the presence of any female irrespective or her sexual condition, 
and he invariably attempts to copulate. Nevertheless, the 
excitement of the male is not so strong nor prolonged when in 
the company of a female during sexual inactivity as with one 
during her sexual season. When the female is nearing oestrus 
the male is extremely excited and tries again and again to 
copulate, while at other times he soon tires and loses interest 
and ceases his aggressive behavior. 

The male and female never fight during the long period of 
aggressiveness on the part of the male, which often lasts for 
many hours. The male tries to induce the female to copulate by 
irritation and excitement rather than by forcing her. The female 
may at times become nervous and attempt to bite the male, 
but an actual fight such as occasionally occurs between two 
males never takes place. No mating by force is observed; the 
consent of the female is necessary for the completion of copula- 
tion. Copulation is followed by a state of relaxation similar to 
that observed among mammals in general, and immediately 
afterwards both male and female may spend some time in cleaning 
their external genitalia. 


3. THE VAGINAL PLuG, ITs ForMATION, LENGTH OF EXISTENCE 
AND MANNER OF DISCHARGE. 

The spermatic fluid of the guinea-pig, especially that portion 
derived from the seminal vesicles, on entering the vagina of the 
female coagulates to form the bouchon vaginal, a rigid plug, 
filling the lumen of the vagina. This plug prevents the outflow 
of the sperm after every copulation. Such a vaginal plug has 
been described in many species of rodents and seems in general 
to be characteristic of this class of mammals. It was first 
observed in the guinea-pig by Leuckart in 1847. He correctly 


230 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU. 


described it as a Pfropf (plug), formed by the coagulation of the 
secretion from the seminal vesicles and serving to fill the vagina 
and prevent the flowing out of the sperm after copulation. 

Bischoff, in 1852, verified the observations of Leuckart and 
accepted his conclusions regarding the réle of the vaginal plug in 
the copulation process. Reichert, 1861, differed with these two 
original descriptions in failing to find the formation of a vaginal 
plug after every copulation, and concluded that its presence was 
not a general phenomenon. Later, however, Hensen in 1876 
brought new evidence confirming the observations of Leuckart 
and Bischoff. 

Landwehr, in 1880, examined the seminal vesicles and found 
their secretion to contain twenty-seven per cent. of fibrinogen to 
which its coagulation reaction is due. Coagulation may occur 
as soon as the secretion of the seminal vesicles comes in contact 
with a small amount of blood. 

Héron-Royer, 1881, observed the vaginal plug in Pachyuromys 
duprasi, but gave no satisfactory explanation of its formation. 
According to him the vaginal plug was formed in the vagina 
before copulation and was pulled out or loosened by the hooks 
on the penis during the act of copulation. These observations 
were entirely contrary to all earlier records, according to which 
the plug is formed after copulation and falls out some hours later. 
Blanchard made histological examinations of the vaginal plugs 
collected by Héron-Royer and found them to consist of two parts, 
a central, partie centrale, composed chiefly of great numbers of 
spermatozoa, and a peripheral part, couche corticale, formed of 
hardened mucus. 

Lataste, in 1882, after examinations of the vaginal plug in 
the same species, Pachyuromys duprasi, came to quite different 
conclusions. He states that the vaginal plug, bouchon vaginal, 
as he termed it, is not formed as Héron-Royer claimed, before 
copulation, but immediately after, and in the same way as was 
known for other rodents. Regarding its function he accepted 
the old opinion of Leuckart that it serves to prevent the spermato- 
zoa from flowing out of the vagina after copulation. He also 
mentions an instance in which a vaginal plug-like formation was 
found when there had been no previous copulation. From our 


CESTROUS CYCLE IN THE GUINEA-PIG. 231 


observation on the oestrous discharge in the guinea-pig it is 
probable that this plug-like structure was nothing else than a 
concentrated accumulation of such a discharge, it having become 
unusually dense or dried out. In fact, as will be shown beyond, 
the superficial portion of the vaginal plug is actually the sluffed- 
off vaginal epithelium surrounding the coagulated seminal fluid. 
Thus the plug is partly of vaginal origin. 

In later papers Lataste makes many contributions to the 
knowledge of the vaginal plug. In 1883 he described the vaginal 
plug in other rodents and pointed out that this formation was 
evidently not limited to a few species but was characteristic of 
the entire class. 

Regarding the function of the vaginal plug, he slightly modifies 
his former position and concludes that its réle is not only to 
prevent the sperm from flowing out of the vagina but rather by 
a filling up to push the sperm into the uterus. He extended the 
observation of Blanchard that the vaginal plug consists of two 
parts, differing in structure, a central core and a superficial en- 
velope. He described the central part as consisting chiefly of the 
coagulated secretion of the seminal vesicles and also of a quantity 
of mucus, 1888a, while the superficial portion, enveloppe vaginale, 
was formed of stratified epithelial cells. The enveloppe vaginale 
is produced in the female by a rapid exfoliation of cells from the 
uterine glands and the vaginal walls on account of the irritating 
presence of the coagulated core. (His conception of the cause 
of the exfoliation is entirely incorrect.) The envelopment of the 
core by loosened epithelium from the vaginal wall serves to 
make easy the expulsion of the vaginal plug. This epithelial 
production he thinks is probably of a pathological nature and 
may be compared to the condition in women known as vaginate 
exfoliante. 

These studies of more than thirty years ago by Lataste are in 
most respects surprisingly correct and it is only the nature of 
the process by which the outer epithelial envelope is formed with 
which we would materially differ. 

Tafani, in 1888, described the vaginal plug in the mouse and 
found it to fall out about thirtv hours after copulation. 

Steinach (’94) found that the removal of the seminal vesicles 


232 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU. 


from rats did not influence their sexual instincts or ability to 
copulate, but decidedly impaired the power of the male to fertilize 
the female. 

Sobotta (95) also has studied the formation of the vaginal 
plug in the mouse and found it present after every copulation. 
Histologically it. consists of an homogeneous mass which is 
surrounded by an envelope of vaginal epithelium. Spermatozoa 
are more abundant in the central mass at its upper end or that 
portion near the uterus as the plug lies in the vagina. He con- 
firms the observations of Tafani regarding the fate of the vaginal 
plug, finding that its surface gradually becomes soft and loose 
and the entire mass falls out of the vagina about twenty to thirty 
hours after copulation. Sobotta states that the vaginal plug 
in the guinea-pig falls out much sooner than in the mouse, being 
eliminated from the vagina within from four to nine and a half 
hours after copulation. The longer interval is approximately 
correct. He claims to have at times observed another copulation 
following the expulsion of the first vaginal plug. 

Camus and Gley (’96) studied the coagulation process in the 
formation of the vaginal plug. They claim coagulation to be 
due to the influence of a prostatic enzyme, “‘vesiculase,’’ upon 
the secretion of the seminal vesicles. The action of the prostatic 
enzyme is specific towards the seminal vesicle secretion of any 
rodent. The prostatic enzyme of a rat will coagulate the seminal 
fluid of a guinea-pig and vice versa. 

Rubaschkin (’05) returns to the old opinion of Reichert, 1861, 
in claiming that the vaginal plug is not a constant formation in 
the guinea-pig following copulation. His statements are as 
follows: Bei der Maus (Sobotta), und nach Bischoff und Hensen 
auch bei Meerschweinchen bildet sich nach dem Coitus ein 
‘Charakteristischer Vaginal-pfropf, der auf einen vorausgegan- 
genen Coitus hinweist. Ich muss hier die Beobachtungen von 
Reichert bestatigen, dass beim Meerschweinschen ein solcher 
Vaginalpfropf sich meistens nich bildet. Von aussen konnte ich 
einen klaren Pfropf in der Vagina niemals erkennen; in einigen 
Fallen liessen sich einige Schleimstreifen bemerken, die aber 
ganz unregelmassig und nicht immer zu Tage traten. In seltenen 
Fallen wurden nach dem Secieren Vaginalpfrépfe gefunden, 


GESTROUS CYCLE IN THE GUINEA-PIG. 233 


welche zum Teil aus verdichtetem Schleim, zum Teil aus Epithel- 
zellen bestanden. Unter diesen Verhaltnissen ist die Bedeutung 
des Vaginalpfropfs beim Meerschweinchen ganz nichtig, und am 
Anfange meiner Arbeit habe ich, durch diese Angabe Bischofi’s 
irregefiihrt, einige Tiere verloren, weil sie zu spat getétet wurden. 

Ko6nigstein (’07) described the vaginal plug in rats and agrees 
with the observations of Lataste, Tafani and Sobotta. He finds 
also the vaginal plug to consist of two parts, a central and a 
superficial. The vaginal plug contains in addition to the secre- 
tions of the male genital glands, mucus, detritus, many leucocytes, 
squamous epithelial cells in large numbers and a granular eosin- 
phil staining secretion. 

From this review the knowledge of the formation of the 
vaginal plug is found to be rather complete, although disagree- 
ments as to facts are expressed by several authors. It seems 
well established that the formation of a vaginal plug following 
copulation is a general phenomenon among the various species 
of rodents. The plug proper consists of a central core formed 
mainly by coagulated fluid from the seminal vesicles and this 
is surrounded or enclosed by a mass of flat epithelial cells, 
apparently derived from the vaginal wall. The coagulation of 
the seminal fluid may be due to the action of a prostatic enzyme 
although it is claimed that the coagulation occurs without the 
presence of such an enzyme. The vaginal plug as a whole 
falls out of the vagina a few hours after its formation. 

On the other hand it is not clear from the literature just 
how or why the peripheral part of the vaginal plug, enveloppe 
vaginale, of Lataste is formed. And the manner and cause of 
the separation of the epithelial lining from the wall of the vagina 
are also unknown. ‘These points could not be clearly understood 
without a knowledge of the changes occurring in the wall of the 
vagina and uterus during the cestrus, at which time copulation 
and the formation of the vaginal plug take place. 

As we pointed out in our description of the cestrous changes, 
there is a stage in the cycle when immense numbers of leucocytes 
accumulate immediately below the epithelium lining the uterus 
and the vagina. From this position the leucocytes attack the 
epithelial cells and at the same time dissolve or destroy the 


234 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU. 


connection between the mucosa and the subjacent connective 
tissue over extensive areas. This reaction is taking place a 
few hours after copulation during the latter part of stage one 
and throughout stage two of our description. A few hours later, 
during stage three, the leucocytes have made still further progress 
in their invasion of the mucosa and the destruction of its con- 
nection with underlying tissues. In certain sections of the uterus 
the entire mucosa filled with immense numbers of leucocytes is 
completely separated from the uterine wall and lies within the 
lumen, while in other regions the epithelium is loosely connected 
but still hanging to the wall. This disconnected and degenerat- 
ing mucosa loaded with leucocytes breaks into small fragments 
during the fourth stage and is expelled from the uterus and 
vagina, while a new mucosa begins to regenerate from the 
mouths and the regions about the uterine glands and from the 
deeper layers of the vaginal epithelium. This is the fate of the 
mucosa when no copulation has taken place. 

There is, then, no pathological “‘ vaginite exfoliante’’ due to an 
irritation of the vaginal wall by the seminal fluid as Lataste 
thought. But a simple periodic aestrous breaking down of the 
uterine wall under leucocyte invasion, entirely independent of 
whether copulation takes place or not. 

When copulation has occurred the loss of the epithelium 
follows a somewhat different course. Immediately after copula- 
tion the coagulated seminal fluid forms a mass within the lumen 
of the vagina and partly extending into the uterus. Around this 
mass the mucosa forms a close fitting envelope, thus preventing 
its early dislocation. The envelope serves to retain the plug in 
the vagina until the fourth stage of the cestrous cycle at which 
time the enveloping epithelium becomes completely separated 
from the vaginal wall by the dissolving effects of the ieucocytes. 
The epithelium is now expelled as one continuous tube forming 
the cover around the vaginal plug instead of sluffing off in smaller 
pieces as occurs during the fourth stage when a copulation has 
not occurred. However, the vaginal epithelium may occasionally 
be shed en masse without copulation. In one striking case the 
epithelium was pulled out of the vagina as a conical sheath, enclos- 
ing the speculum that had been introduced for examination. 


GSTROUS CYCLE IN THE GUINEA-PIG. 235 


It is clear, therefore, that what was termed by Lataste the 
“enveloppe vaginale’’ is the layer of epithelium separated from 
the underlying connective tissue by the dissolving action of the 
leucocytes which invade the walls of the uterus and vagina at 
this time. It is also readily understood how the plug, after its 
short sojourn in the vagina and cervix of the uterus, is finally 
separated from its adhesion or tight connection with the wall 
and expelled as a mass from the vagina. 

A possible function or effect of the vaginal plug in addition to 
those before mentioned has recently been suggested by Long 
(19). He states that a stimulation of the cervix of the uterus in 
rats, by merely inserting a glass rod during stage one of the 
oestrus, prolongs the next cycle, and suggests that the vaginal 
plug may also act in this mechanical way. We have not tested 
the prolongation of the cycle in guinea-pigs following copulation 
without conception as compared with its length in virgin animals. 


4. THE CEstrous RHYTHM. 


In our earlier review of literature it was pointed out that the 
knowledge of the actual time of ovulation in the guinea-pig was 
decidedly inexact. Nothing scarcely was known of the periodic 
recurrence of the cestrus stages in a given female. In short the 
moment of ovulation in the guinea-pig was not available for 
accurate experimental purposes and no definite .criterion or 
method had been devised for detecting the oestrous condition. 
And this was true in spite of a very long list of studies pertaining 
to the reproductive activities of these animals. 

Reichert, as long ago as 1861, had found that the Graafian 
follicles rupture about nine to ten hours after copulation. This, 
in general, approaches correctness, but in cases where copulation 
has not taken place, or failed to be observed, such knowledge is 
of little consequence. Rubasckhin (’05) had more recently 
claimed that the vagina was open and the vulva somewhat 
inflamed ten to twelve days after parturition, but this is certainly 
too short an interval to indicate an actual return of heat. It 
must be remembered that the female guinea-pig goes into ‘‘heat”’ 
and accepts the male almost immediately after the delivery of 
her litter. This fact makes the length of Rubaschkin’s interval 
still more improbable. 


236 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU. 


The most valuable and extensive investigations of the repro- 
ductive activities of the guinea-pig were those made by Leo Loeb 
(11, 14). But here the data were derived almost entirely from 
examinations of the uterus and ovaries after their removal from 
the body of the female. While such studies did give a means of 
comparing the conditions found among different individuals at 
different times, and made it possible to estimate approximately 
the length of the sexual periods, yet this estimate could not be 
transferred with certainty to any one living individual. We 
further objected to Loeb’s method of study since it failed to 
permit an investigation of the recurring cestrous periods in a 
number of unoperated females. The results of such an investiga- 
tion would be most important in determining the influence of 
any unusual or experimental conditions introduced with intent 
to modify the intervals between ovulations or other periods of 
the sexual cycle. These are just such problems as Loeb had 
under consideration. 

The entire literature showed that any such thing as a regular 
cestrous flow was completely undiscovered for the guinea-pig. 
It became necessary, however, for our studies to have an accurate 
knowledge of ovulation times, and to determine this, extensive 
investigations of the sexual cycle in the guinea-pig were under- 
taken. A simple method of examining the vagina of the living 
animal proved to be of the greatest value. Virgin females were 
selected and the fluid present in the vagine was taken daily by 
means: of a small nasal speculum and cotton swab. This fluid 
was smeared on slides, stained and studied microscopically. 
The method is fully described in the former paper. 

It very soon became evident that the vagina generally con- 
tained little or no fluid, but that periodically a great accumulation 
of mucus and cells was to be found. This excessive amount of 
mucus and cells is to be recognized as a typical cestrous flow. 
The constituent elements of the fluid change in their relative 
abundance in a definite manner from the beginning to the 
cessation of the flow.. Four clearly marked stages, as mentioned 
above, could be separated by microscopic examination of the 
fluid smears. 

These changes in the composition of the vaginal fluid were 


GESTROUS CYCLE IN THE GUINEA-PIG. 237 


found to be associated with comparable changes in the structure 
of the epithelial walls of the uterus and vagina. And not only 
was this the case, but the changes in the vaginal fluid proved to 
be most reliable indices of definite processes taking place in the 
ovaries in connection with the rupture of the Graafian follicles 
and the expulsion of the ova. It is, therefore, evident that by an 
examination from time to time of this fluid, one may know the 
exact condition. of the ripening follicles in the ovary and very 
nearly the exact moment of ovulation. 

The cestrous cycles in a group of guinea-pigs were fatlewed for 
a number of months in order to establish the normal periodicity 
or rhythm. The amount of variation that might exist in the 
length of the cycles in a given female was studied as well as the 
variations in cycle lengths among different individuals. An at- 
tempt was further made to discover any seasonal variations that 
might exist. 

Only slight time variations were found in the periodic rhythm 
of a given female. For example, in one animal the record of six 
consecutive periods shows the cestrous flow to begin on the 
sixteenth day five times and on the fifteenth day once. In 
another case of seven consecutive periods the flow began on the 
sixteenth day six times and on the seventeenth day once. For 
further cases the reader is referred to the table given in our 
former paper. 

There is only a limited variation in the length of the cestrous 
cycles among different individuals, ranging between fifteen and 
seventeen days in younger animals. In exceptional cases the 
period is slightly lengthened in older multiparee, sometimes reach- 
ing eighteen days. These limits of fifteen and eighteen days for 
the lengths of the oestrous cycles have never been violated under 
normal conditions during the several hundred observations which 
we have now recorded. The method of examining the vagina 
for the closure membrane above described, and, in the case of 
its rupture, for the composition of the fluid contained within 
the lumen, renders these individual variations of no consequence 
in determining the exact “heat period”’ and time of ovulation. 

Slight, if any, seasonal variations are shown by our animals. 
This may be due, however, to the uniformly warm temperature 
maintained in the breeding rooms during the winter months. 


238 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU. 


For a full description and photographs of the structural changes 
occurring in the cestrous fluid, the vagina, uterus and ovaries, 
the reader is referred to the original account. 

After the publication of our results it was found that one of 
the last papers by Leo Loeb (’14), bearing on a related subject, 
had unfortunately been overlooked. We regret this, since a 
discussion of his methods and results would have been somewhat 
clearer in connection with our full consideration of the oestrus 
given in the previous paper than in the present connection. In 
earlier papers Loeb (11) had completely failed to establish a 
definite length or periodicity for the sexual cycle in the guinea- 
pig. In the last paper, however, the length of the cycle was more 
nearly determined and a very thorough description was given 
of the microscopic changes taking place in the uterine wall during 
the heat period. Our independent account of the structural 
changes in the uterine wall fully confirms Loeb’s description. 
But we are unable to agree exactly with the lengths of the sexual 
periods as estimated from his examinations of the removed 
uteri. In a still more recent article Loeb (’18) repeats his 1914 
estimates and claims the lengths of the sexual cycles to vary 
between thirteen and a half and nineteen days. 

In all cases Loeb’s investigations had centered in a study of 
the sectioned uterus and ovary, thus necessitating their removal 
by operation or the death of the animal. Either procedure 
permits only one observation on a given female. No investiga- 
tion of the uterus and vagina in the living animal had been 
made and no continuous observations on the consecutive cycles 
of given individuals were carried out. 

As mentioned before, we recorded not only the structural 
changes of the uterus, but almost equally as marked changes in 
the wall of the vagina. And what we consider to be of still 
more importance from an analytical or experimental standpoint 
as a means of estimating the moment of ovulation, was the 
complete record of the changes in the microscopic composition 
of the vaginal fluid during the different stages of the sexual cycle. 
The removal and examination of this fluid is made without in 
any degree injuring the uterus or vagina and does not interfere 
with the further use of the female for ovulation and breeding 


GESTROUS CYCLE IN THE GUINEA-PIG. 239 


records. This knowledge of the definitely changing structure 
of the vaginal fluid made it possible to study the oestrous cycles 
in many living females and reduced the time element of ovulation 
in the guinea-pig to a certainty. 

We considered in a somewhat different manner the connection 
between the uterine reaction and the secretion of the corpora 
lutea, though essentially we share Loeb’s ideas of the functions 
of these bodies. It was concluded that the duties of the corpora 
lutea are probably about what Beard (’97) long ago argued in his 
monograph on “The Span of Gestation and the Cause of Birth.” 

The development and the degeneration of the vaginal and 
uterine mucosz were found to follow very closely the development 
and degeneration of the corpora lutea in the ovaries. The case 
was stated as follows: ‘‘The breaking of the Graafian follicles 
occurs during the cestrus as a result of congestion which began 
in the theca folliculi at about the same time as the congestion 
of the stroma of the uterus and vagina. And finally when the 
regenerative growth of the uterine mucosa sets in, the ovaries 
then possess new corpora lutea in an active state of differentia- 
tion which have been derived from these recently ruptured 
follicles.” The presence of the new active corpora lutea sup- 
presses the final steps in the development of the almost mature 
Graafian follicles in both ovaries, whether the corpora lutea be 
located in only one of the ovaries or both. When the corpora 
lutea become less active and their degeneration has proceeded 
to a certain extent, another ovulation may then take place. 
Therefore, the functions of the corpora lutea are probably, first, 
by their presence and activity to inhibit ovulation or to determine 
its time, and, secondly, to preserve the structure of the uterine 
wall and prevent its degeneration. 

Loeb has attacked the problems of corpora lutea function in 
the guinea-pig in a more direct experimental way than have other 
investigators. Yet while studying the effects of corpora lutea 
removal on the length of the ovulation period, he has been 
handicapped by the fact that his animals, after the initial 
operation, were later killed for examination and thus were no 
longer available for a continuation of the experiment. Only 
one observation was obtained from any particular female. The 


2 So ae 


240 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU. 


effects on the lengths of the ovulation periods of the removal of 
corpora lutea or the application of its extracts could be investi- 
gated to great advantage on guinea-pigs in which the oestrous 
cycles are definitely known and followed through a number of 
consecutive periods. This could readily be done by the method 
before described. This method is also of value in locating the 
early stage of developing eggs and in making exact matings for 
studies on fertilization, etc. 

Attention may be,called to further slight objections that might 
be raised in considering Loeb’s last paper. He studies the 
conditions in the structure of the uterine wall removed from 
females that had copulated shortly before, as well as, uteri from 
uncopulated females, and states, page three: ‘‘The sperm fluid 
present in the lumen of the uterus exerts a pressure on the surface 
epithelium and may thus contribute to the harmful influence of 
the leucocytes.’’ This idea is incorrect since it may be clearly 
shown that the action of the leucocytes is equally as harmful in 
the destruction of the uterine epithelium during the cestrous 
period of virgin females. 

In a similar connection Loeb also finds, page 11, that the 
number of leucocytes in the uterine mucosa is much smaller 
in animals that have not copulated. Again, page 16, “A few 
leucocytes can also be seen in the uterus of animals in which 
copulation had been prevented. . . . In such cases (non-copu- 
lated animals) also some degenerative changes occur in the 
uterine epithelium, but they are less marked than in animals 
which had copulated.’”’ These statements are not entirely in 
accord with our findings since there is no such marked dis- 
crepancy between virgin females and those that have copulated. 
Such conclusions are probably due to the fact that the uteri ex- 
amined were not removed from the non-copulated females at the 
maximum moment of leucocyte migration and degeneration of the 
uterine wall (our ‘third stage’). Loeb had no exact means of 
knowing the comparable stages in copulated and non-copulated 
females. 

The uterine mucosa of our virgin females may show leucocytes 
to be equally as abundant at comparable stages as the uteri from 
specimens after copulation. It must be recognized in this con- 


GESTROUS CYCLE IN THE GUINEA-PIG. 241 


nection that each stage in the condition of the uterine wall during 
the cestrous is of short duration and unless the uterus be removed 
at a given time, the abundance and position of the leucocytes and 
the condition of the uterine wall will be changed. Chance was 
against Loeb’s removing the uteri from the non-copulated females 
at the moment of maximum leucocyte migration, since he had 
no exact means of knowing when this would occur without 
having first observed copulation. 

Active migration and accumulation of leucocytes may be 
observed in the entire absence of sperm fluid. The réle of this 
fluid and the modification of the shedding or sluffing off of the 
vaginal and uterine epithelium in its presence was fully brought 
out in the discussion above of the vaginal plug. 

Loeb’s estimation of the ovulation times and uterine changes 
from microscopic examination of fixed specimens does not make 
it possible to know within a few hours, or even days, of the 
exact moment of ovulation in a given living individual. He 
states, however, on page 31, that to determine the effects of the 
removal of the corpora lutea on the duration of the sexual cycle, 
it ‘‘was necessary to determine the length of the cycle in the 
normal guinea-pig.’’ Not only is this necessary, particularly in 
view of the wide variations Loeb finds in the normal sexual cycle 
among different individuals, but it is better or even necessary, 
to know the actual length and variations of the sexual cycle in 
the given specimen experimented’ upon. As evidence of the 
correctness of the last statement, we may cite Loeb’s method 
and results in determining the normal cycle lengths. This was 
done ‘‘by observing the time of heat of a guinea-pig and by 
examining the uterus and ovaries at known intervals’’ (after 
removal from different individuals). Such examinations were 
made on many specimens that had to be either killed or operated 
upon. The following ovulation intervals were thought to be the 
normal sexual cycles, page 31, ‘‘ We found the length of the sexual 
period to be usually sixteen to eighteen or nineteen days; some- 
times the new ovulation may take place as early as fifteen days 
after copulation. In two exceptional cases we observed the 
new ovulation as early as thirteen and a half to fourteen and a 
half days.”” The sexual cycle, therefore, varies in length from 


242 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU. 


thirteen and a half to nineteen days, a range of almost six 
days. 

On this basis it is seen to be practically impossible to state 
within a day or so of when the next ovulation will take place 
in a female that has just passed the “heat period.’”’ And the 
“heat period’’ was very indefinitely known unless copulation 
had been observed. Thus, asa usual practice, in order to prevent 
pregnancy following a copulation used to prove the existence of 
heat, the oviducts were previously tied. Such a procedure might 
easily modify to some degree the sexual cycle and is inconvenient 
for further study of the animal. 

Any significant experimental modification of the ovulation 
intervals could be readily detected by a simple examination of 
the microscopic structure of the vaginal fluids collected from a 
guinea-pig. 

Finally, the “signs of heat’’ recorded from a report by Miss 
Lathrop, an animal breeder, are, according to our experience in 
examining and mating guinea-pigs, generally inaccurate and of 
little value for use in experimental studies. 


5. SUMMARY. 


1. The cestrous cycle in the guinea-pig is very definitely 
limited in length. Ovulations follow one another every fifteen 
to seventeen days in younger individuals, while in old females the 
period is slightly lengthened, in exceptional cases to eighteen 
days. 

2. These individual variations are readily controlled by the 
method of vaginal examination described in this and a former 
article, so that the actual moment of ovulation in any given 
female may be determined to within almost an hour of the 
rupture of the Graafian follicles. 

3. During the period of sexual inactivity, the dioestrum, as 
well as during pregnancy, the orifice of the vagina is completely 
closed by an overgrowth of epithelium which we have termed the 
“vaginal closure membrane.’’ This membrane ruptures just 
before or during the first stage of the cestrus in non-pregnant 
females and before parturition in the pregnant. It always re- 
forms to close the vagina shortly after the “heat period’”’ has 


(ESTROUS CYCLE IN THE GUINEA-PIG. 243 


passed. The presence of this closure membrane is therefore 
positive evidence that the time of a new ovulation has not been 
reached. When the membrane is ruptured and the vagina open, 
the ovarian condition may then be determined by examination 
of the fluid content of the vagina as described. A knowledge of 
this closure membrane greatly facilitates the examination of 
females in locating the beginning of the cestrus. 

4. Copulation takes place in the guinea-pig during stage one 
of the cestrous period and ovulation occurs, as we previously 
showed, at the end of the second stage or the beginning of the 
third. Long (’19) finds exactly the same to be true for the rat. 

5. At the time copulation occurs, the vagina is filled with a 
clear foamy mucus, and this is the only time at which no leuco- 
cytes are present in the fluid. Both the nature of the vaginal 
fluid at this time and the absence of leucocytes contribute to the 
success of copulation and fertilization. 

6. A vaginal plug is formed a few minutes after copulation, 
during the first stage, and remains in the vagina for only a short 
time, being expelled during the fourth stage of the cestrus. 

The core or center of the vaginal plug is composed chiefly 
of coagulated seminal fluid. This is enclosed within an en- 
velope of epithelial cells, being simply the sluffed-off mucosa 
from the wall of the uterus and vagina. This epithelial cover 
which is thrown off at every oestrous period is loosened or dis- 
solved away from the uterine and vaginal wall by an enormous 
invasion of leucocytes which progresses from the first to the 
third stage. The breaking away from the vaginal wall of this 
enveloping epithelium causes the plug to fall out of the vagina 
during the fourth stage. After the expulsion of the vaginal 
plug, the mouth of the vagina is closed by the growth of the 
vaginal closure membrane. 


6. LITERATURE CITED. 
Beard, J. 
’97 The Span of Gestation and the Cause of Birth. A Study of the Critical 
Period and its Effects upon Mammals. Fischer, Jena. 
Bischoff, Th. L. W. 
’52 Entwickelungsgeschichte des Meerschweinchens. Giessen. 
Camus, L., and Gley, E. 
796 ~Action coagulante du liquide prostatique sur le contenu des vésicules 
séminales. Soc. de Biologie, Juillet, pp. 787-788. 


244 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU. 


Heap, W. 
‘00 The Sexual Season. Quart. Jour. Mic. Soc., Vol. 44. 
Hensen, V. 
’76 Beobachtungen iiber die Befruchtung und Entwickelung des Kaninchens 
und Meerschweinchens. Zeit. f. Anat. u. Entwick., Vol. 1. 
Héron-Royer 
’81 Concrétions vagino-utérines observées chez le Pachyuromys duprasi. 
Zool. Anz., Vol. 4. 
82 A propos des bouchons vagino-utérines des Rougeurs. Zool. Anz., Vol. 5. 
Konigstein, H. 
’07_ Die Veranderungen der Genitalschleimhaut wahrend der Graviditat und 
Brunst bei einigen Nagern. Arch. f. Physiol., Vol. 119. 
Landwehr 
*89 Ueber den Eiweissk6rper, fibrinogene Substanz, der Vesicula seminalis der 
Meerschweinchen. Arch. f. Physiol., Vol. 23. 
Lataste, F. 
*82 Sur le bouchon vaginal de Pachyuromys duprasi. Zool. Anz., Vol. 5. 
83. Sur le bouchon vaginal des Rougeurs. Zool. Anz., Vol. 6. 
°88a Matiére du bouchon vaginal des Rougeurs. Compt. rend. Soc. Biol., 
8th Dec., pp. 817-821. 
?88b Enveloppe du bouchon vaginal des Rougeurs. Compt. rend. Soc. Biol., 
PP. 732-733- 
’88c Enveloppe vaginale et vaginite exfoliante des Rougeurs. Compt. rend. 
Soc. Biol., pp. 705-707. 
Leuckart, R. 
’47 Zur Morphologie und Anatomie der Geschlechtsorgane. Gdéttingen. 
Loeb, L. 
’r1a The Cyclic Changes in the Ovary of the Guinea-pig. Jour. Morph. 
~ Vol. 22. 
*1rb The Cyclic Changes in the Mammallan Ovary. Proc. Am. Phil. Soc., 
Vol. 50. 
*14 The Correlation Between the Cyclic Changes in the Uterus and Ovaries in 
the Guinea-pig. BroL. BULL., Vol. 27. ; 
*18 Corpus Luleum and the Periodicity in the Sexual Cycle. Science, N.S., 
Vol. 48. 
Long, J. A. b 
’19 The Osetrous Cycle in Rats. Proc. Am. Soc. Zoél., Anat. Record, Vol. 15. 
Reichert, C. B. 
’61 Beitrage zur Entwickluigsgeschichte des Meerschweinchens. Abh. D. 
Kgl. Preuss. Akad. d. Weissensch., Berlin. 
Rubaschkin, W. 
05 Uber die Reifungs- und Befruchtungsprocesse des Meerschweincheneies. 
Anat. Hefte, Vol. 29. 


Sobotta, J. 
’95 Die Befruchtung und Furchung der Eies der Maus. Archiv. f. Mikrosk. 
Anat., Vol. 45. 


Steinach, E. 
’94 Untersuchungen zur vergleichenden Physiologie der mannlichen Gesch- 
lechtsorgane, insbesondere der accessorischen Geschlechtsdriisen. Arch. 
f. Physiol., Vol. 56. 


(ESTROUS CYCLE IN THE GUINEA-PIG. 245 


Stockard, C. R., and Papanicolaou, G. 
‘17 The Existence of a Typical (strous Cycle in the Guinea-pig—with a 
Study of its Histological and Physiological Changes. Am. Jour. Anat., 
Vol. 22. 
Tafani, A. 
788 La fecondatione e la segmentatione Studiate nelle uova dei Topi. Acad. 
Med. fisic. Florence. 


© 


v 


Reprnted from the Proceedings of the Society for Experimental Biology and Medicine, 
1919, xvi, pp. 93-95. 


54 (1429) 
Developmental rate and the formation of embryonic structures. 


By CHARLES R. STOCKARD. 


[From the Department of Anatomy, Cornell Medical School, New 
York City.] 


The eggs of different species develop at specifically charac- 
teristic rates. These rates vary for a given species within certain 
limits. Variation in the direction of increased rate is far more 
limited and more difficult to bring about experimentally than is 
the opposite variation towards a decreased or slower rate. 

The slight acceleration of developmental rate that may be 
induced in early embryos does not seem to cause any marked 
deviation from the normal course of development, on the contrary 
such embryos are unusually well developed. It might be said 
that the ideal rate of development is probably somewhat faster 
than the so-called normal average usually followed. In other 
words, the normal conditions of development are not entirely the 
best possible conditions. 

The limits of retardation in developmental rates are extremely 
wide. The rate may be slowed down to almost zero or develop- 
ment may actually be to all appearances stopped for long or short 
periods of time in many species without noticeably injuring the 
embryo. Such an interruption normally occurs during the de- 
velopment of certain eggs as those of birds and some mammals. 
In eggs having a continuous development, such as those of fish, 
the rate of development may be slowed to apparent stoppage at 
many stages and held in such a condition for some time without 
injury to the embryo which results after the inhibiting influence 
has been removed. However, when the rate of development is 
retarded but not entirely stopped at certain critical periods and 
development is allowed to proceed at this diminished rate for some 
time, most serious structural anomalies are induced. 

Double monsters of varying degrees of doubleness may actually 


SCIENTIFIC PROCEEDINGS (98). 


Ny 


be produced by slowing the rate at a time when the primary 
embryonic bud should arise. Normally the initial appearance of 
the primary embryonic bud probably suppresses the appearance 
of other buds which potentially exist, but when the primary bud 
is delayed in its appearance it becomes possible for more than one 
bud to arise, usually two. The distance apart of these two buds 
on the blastodisc determines the degree of doubleness of the result- 
ing individual. Buds arising close together give two-headed 
monsters, while buds arising at opposite points on the periphery 
of the disk, 180° apart, each give rise to a complete individual, in 
such a case twins result. 

When the two buds arise simultaneously they have equal 
chances in development and symmetrical double monsters result. 
If, however, one bud obtains the start over the other bud this 
start constitutes a supremacy which almost invariably makes it 
possible for the leading bud to develop into a perfectly normal 
specimen, and invariably defeats the possibility of normal develop- 
ment on the part of the slower bud. 

An investigation of a large series of such double fish embryos 
lends strong support to the interpretation that the late bud is 
inhibited in its rate of development on account of the presence of 
the leading bud, just as the first bud to grow out from a notch on 
the leaf of Bryophyllum inhibits the growth of other buds as Loeb 
has so strikingly shown. The inhibited rate of development in 
the lesser component tends to suppress and interfere with the 
normal origin and development of certain organs, especially the 
eyes and other head parts. Organs may entirely fail to arise, or 
develop abnormally after they do arise. 

In the series of double fish when both individuals or both heads, 
as the case may be, are of equal size they are both normal, but 
whenever one component is larger than the other, the larger one is 
almost invariably normal and the smaller is invariably defective. 
This is not only true in the present series of specimens but also in 
all illustrations and descriptions of double monsters which I have 
been able to collect from the literature. 

These embryos furnish material for an analysis of the causes 
of many common structural defects about which there has been 
considerable discussion, a consideration of this phase of the subject 
will be given in the complete review of the experiments. 


AUTHOR'S ABSTRACT OF THIS PAPER ISSUED Reprinted from Tor ANATOMICAL RecoRD, 
BY THE BIBLIOGRAPHIC SHRVICE, May 1 Vol. 16, No. 4, June, 1919 


SYMMETRY REVERSAL AND MIRROR IMAGING IN 
MONSTROUS TROUT AND A COMPARISON 
WITH SIMILAR CONDITIONS IN HUMAN 
DOUBLE MONSTERS 


C. V. MORRILL 
Department of Anatomy, Cornell University Medical College, New York City 


BIGHT FIGURES (FOUR PLATES) 


In the extensive literature on the development of monsters 
there are frequent references to reversed symmetry and mirror 
imaging of unpaired organs. This latter condition is most fre- 
quently seen in double monsters of the dicephalous type, one 
component of the monstrosity often exhibiting a partial or com- 
plete situs inversus viscerum. As far as I am aware, mirror 
imaging in the viscera has only been found, or at least described, 
in human monsters, although there is no morphological reason 
why monsters in the lower vertebrates should not occasionally 
exhibit this condition. Recently an opportunity occurred to 
examine the question. In a collection of newly hatched trout! 
containing many double monsters a number were found in which 
the abdominal viscera of one component showed reversed sym- 
metry in some degree. The mirror imaging was practically per- 
fect in some cases, while in others it was only slightly indicated 
or irregularities appeared which made the interpretation diffi- 
cult. The conditions found in these fish, although not entirely 
novel, seem sufficiently interesting to merit a brief description. 
Their theoretical bearing will be considered in conjunction with 
similar conditions in higher forms. 


17 am indebted to Professor Stockard for the use of this material which he 
has under investigation from a somewhat different standpoint. 


265 


266 C. V. MORRILL 


SYMMETRY REVERSAL IN MONSTROUS FISH 


The collection of monsters under consideration exhibited 
various degrees of doubling. The less pronounced cases showed 
externally a double-headed condition with the rest of the body 
single. At the other extreme were duplicate twins attached face 
to face on a single yolk. Between were the varying degrees of 
anterior and posterior duplicity. In the figures (pls. 1 and 2) 
all the specimens are viewed from the ventral or ventrolateral 
surface with the yolk dissected away. 

The normal asymmetry of the viscera is shown in ue of the 
twin fish of figure 6. The stomach first bends slightly to the 
left, then sharply to the right, passing to the pyloric end where 
it turns posteriorly into the intestine. The swim bladder (S.B.) 
lies dorsal to and slightly to the left of the stomach. The 
liver (L.) lies to the right and in the hollow formed by the bend- 
ing of the stomach. The urinogenital system need not be 
considered here. 

In the monsters the doubling of the viscera corresponds in 
degree with the external duplicity. In the specimens shown in 
figures 1, 2, 4, and 5 where the head and a considerable part of 
the trunk are double, there are two stomachs, two swim bladders, 
and two livers. ‘The intestines are separate anteriorly, but unite 
posteriorly to form a common rectum which opens in the usual 
position through a single vent. Also, though not brought out 
in the figures, there are two hearts and two complete pairs of 
pectoral fins. In the specimen shown in figure 3, the duplicity 
does not extend as far posteriorly as in the foregoing. In this 
case there are two stomachs and two swim bladders, but the 
intestines are united immediately beyond the stomachs into a 
common bulbous enlargement (C.J.) from which a single tube 
leads straight backward to the vent. The liver (L.) is a single, 
irregular mass nearly twice the size of a normal liver and prob- 
ably formed by the union of two separate liver buds. It lies 
on the right side of the monster considered as a unit. The 
swim bladder corresponding to one of the components (lower 
in the figure) passes backward on the dorsal side of the liver 


SYMMETRY-REVERSAL IN MONSTERS 267 


and is almost concealed from view. The tip of it can be seen 
at S.B. The swim bladder of the other component is in the usual 
position. The specimens shown in figure 6 are twins which have 
been dissected away from a single yolk uniting them face to 
face. Each has the normal complement of organs. 

It will be convenient to designate the two components of a 
monster as A and B. The lower component in each figure is 
A and the upper is B. If one imagine the figures turned so that 
the heads are toward the top of the page then A is on the left of 
the observer as he faces the ventral surface of the specimen and 
B is on the right. The scheme then conforms to that which 
Wilder (’04, ’08, ’16) has adopted for human monsters. If 
figures 1, 2, and 4 are now examined, it will be seen that in each 
case the asymmetry of one component is the reverse of the other, 
as far as the principal abdominal viscera are concerned. Com- 
ponent B (upper or right-hand) has the normal asymmetry—the 
stomach bends first to the left, the swim bladder (S.B.) dorsal 
and slightly to its left, the liver (Z.) on the right side.2) Com- 
ponent A (lower or left-hand), however, has reversed asym- 
metry—the stomach bends first to the right, the swim bladder 
(S.B.) dorsal and slightly to its right, the liver (L.) on the left 
side. 

The transposition of viscera in one component is practically 
complete in the three specimens shown in figures 1, 2, and 4. In 
several other specimens (not figured) a partial reversal was 
indicated. Figure 3 shows a case in which the doubling involves 
only the head and extreme anterior end of the trunk. There are 
two hearts, as the figure shows, and four complete pectoral fins. 
In the abdominal cavity the two stomachs are crowded together 
and very much contorted, though both seem to bend toward 
the same side. There is a single large liver (L.), probably 
formed by the union of two liver buds. The swim bladder of 
component B (upper or left-hand) is the normal position. Its 
mate of the opposite side, for the most part hidden by the liver, 


2 The rights and lefts are used here with reference to one component, not to 
the entire monster. 


268 Cc. V. MORRILL 


does not exhibit the relations to be expected if mirror imaging 
were present, as a comparison with the position of the swim 
bladder in the corresponding component of figures 1 and 2 will 
show. It is difficult to draw any positive conclusion from this 
specimen, owing to the crowded condition in the abdominal 
cavity; but from the position of the stomachs and swim bladders, 
T am inclined to think that no reversed asymmetry is present in 
either set of organs. 

It is to be noted that the monsters showing complete reversal 
of symmetry on one side are all in the same stage of duplicity 
(figs. 1, 2, and 4). No sign of reversal was found in the more 
complete stages of doubling. In the twins from the same egg 
(fig. 6) each has the same (normal) asymmetry. In the case 
described in the preceding paragraph, where the degree of dou- 
bling was less marked (fig. 3), all indications were contrary to the 
idea of symmetry reversal. 

The mirror imaging described above, whether complete or 
partial, is not by any means the rule in these monsters. Even 
in the particular stage of doubling in which it occurs the major- 
ity of the specimens show the normal asymmetry in both sets 
of visceral organs. Figure 5 illustrates this latter condition. 
Here it is obvious that both stomachs bend first toward the left, 
forming a bay, which opens toward the right. The liver (L.) 
lies in the bay; that is, on the right side of the component to 
which it belongs. The swim bladders (S.B.) lie dorsal and 
slightly to the left of their respective stomachs. Summariz- 
ing the facts briefly, the abdominal viscera are mirror images of 
each other in some cases (figs. 1, 2, and 4) and not in others (fig. 
5), though most of the specimens present the same degree of dou- 
bling. Discussion of this point may be conveniently postponed 
until the conditions in human monsters have beep described. 

Despite the very simple condition of the gastro-intestinal 
tract in fish, the normal asymmetry is very well marked, and 
any change from this strikes the eye immediately upon opening 
the abdominal cavity. In view of this, it is curious that neither 
Windle (’95) nor Gemmill (’01, ’12) observed any reversal of sym- 


SYMMETRY-REVERSAL IN MONSTERS 269 


metry in their work on monstrous fish. Gemmill especially, 
in his study of the anatomy of double monstrosities in trout (’01), 
made a careful examination of the internal organs, but apparently 
saw no changes in symmetry, though some of his specimens 
exhibited the same degree of duplicity as those described in the 
present paper. Possibly reversal of symmetry is rare in fish, 
but the total number of specimens examined is too small to form 
any definite conclusion on this point. 


SYMMETRY REVERSAL IN HUMAN MONSTERS 


It is well known that some types of human diplopagi exhibit 
symmetry reversal and consequent mirror imaging in the un- 
paired viscera. At the time the present study was begun my 
attention was drawn to a double-headed human monster which 
had been kept in a museum jar for a number of years in our 
laboratory. It belongs to the dicephalous variety, which is 
the one most likely to show mirror imaging in the viscera, judg- 
ing by previous reports.* A photograph of the monster is shown 
on plate 4 (fig. 8). Using Fisher’s (’66) classification, it would 
be a dicephalus tribrachius dipus. The name is so descriptive 
that nothing more need be said of the external configuration. 
It is similar to the Barkow fetus, no 66 in Fisher’s list, except 
that in the present specimen the hands of the median arm are 
placed palm to palm and the heads are somewhat nearer to- 
gether. The sex, as in Barkow’s case, is female. 

The organs of the upper abdomen, with the exception of the 
liver, show complete mirror imaging (fig. 7). The stomachs 
are placed with the pyloric ends pointing toward each other. 
There are two spleens (Sp.), two gall-bladders (G.B.), two com- 
mon bile-ducts, and two pancreases (Pan.), each set showing 
the proper relations to the corresponding stomach. (The he- 
patic ducts are not shown in the diagram.) The liver forms a 
large compound mass with many irregular lobules (not figured). 
The small intestines are separate to within two feet of the 
caecum, at which point they unite to form a common ileum. The 


3 Fisher (’66); Eichwald (’70); Hirst and Piersol (93). 


270 Cc. V. MORRILL 


large intestine, including caecum (Cae.) and appendix, is single. 
The caecum lies in the right iliac fossa, from which the colon 
(A.C.) ascends in the usual way to the liver. From here the 
transverse colon crosses to the hypochondrium of the opposite 
side, the splenic flexure lying in relation to the spleen of the 
B-component (right-hand, as one faces the monster). The de- 
scending colon has the usual course and relations. There is 
a single pair of large, lobulated kidneys, each with a normal 
ureter. The uterus, tubes, ovaries, bladder, and rectum are 
normal in size and position for a single individual. 

The viscera are shown schematically in figure 7. It will be 
observed that the organs of the right-hand component (B) are 
more normal in shape and larger than those of the opposite side 
and that they have the normal situs. There is a disparity in 
size also in the thoracic organs (to be described below), again 
in favor of component B. The two heads, however, are prac- 
tically the same size though one, again the right-hand (compo- 
nent B) is placed a little more in the direct line of the compound 
body. 

The thoracic cavity contains two complete sets of organs. 
There are two hearts inclosed in a single pericardium. They were 
pressed close together, both apices directed forward and down- 
ward. In figure 7 the apices have been widely separated to 
show the medial surfaces of both hearts. It is obvious that 
there is transposition in the left-hand heart (component A). 
The left atrium in this case receives venous blood from the 
superior vena cava (S.V.C.) and hepatic veins (Vv.h) and de- 
livers it to the left ventricle from which the pulmonary artery 
(P.A.) springs. The pulmonary veins here empty into the right 
atrium and the aorta (A.) springs from the right ventricle. The 
reversal of symmetry is thus complete. The right-hand heart 
shows the normal symmetry and need not be described. The 
lungs consist of two pairs corresponding to the two tracheae. 
Those of the right-hand, or B-component, are normal in lobu- 


4 A detailed description of the vessels in this monster will appear in a forth- 
coming paper by Mr. H. B. Sutton, of our laboratory. 


SYMMETRY-REVERSAL IN MONSTERS 271 


lation and nearly so in size, while the other pair are much re- 
duced.’ There is a fair-sized thymus for each side. 

The diaphragm is extremely incomplete. Both stomachs with 
their adnexa are herniated high into the thorax, especially in 
component A, the fundus of whose stomach lies almost in the 
root of the neck. 

Summarizing the more important features of the monster: 
The viscera of the right-hand component (B) are the more nor- 
mal in size and shape and they have the normal situs. Those 
of the other component (A) are, generally speaking, reduced in 
size or irregular in shape and display situs inversus. Externally 
the head and neck of component B are more nearly in line with 
the axis of the trunk. Obviously it is always the same compo- 
nent (the left-hand or A-component according to the scheme 
adopted in the present paper) which exhibits transposition of the 
viscera whether in man or in fish (compare figs. 1, 2, 4, and 7). 
This point will be discussed in a later part of the paper. 

It has been frequently assumed, as pointed out above, that 
monsters of certain types, especially the dicephali and ischio- 
pagi, may be expected to exhibit mirror imaging. The number 
of cases on record where actual examination disclosed this con- 
dition are however, very few,’ and most of them are cited by 
Fisher (66) in his very comprehensive paper on diploteratology. 
From his list I have collected the following cases: ; 


Dicephali 


Case 50. Ritta-Christina, a dicephalus tetrabrachius dipus (fe- 
male) which lived about eight months. At autopsy it was found that 
the pericardium was single, but enclosed two hearts, which were right 
and left, touching at their apices. The stomachs, spleens, and pan- 
creases were right and left, and placed so that the pyloric ends of the 
stomachs faced each other, the adnexa conforming as in the specimens 
described above. ‘The livers, also right and left, were fused, and there 


*> It was impossible to determine the lobulation of this pair of lungs as they 
were partly destroyed when the corresponding head was removed from the body 
during delivery. 

° Bateson (’94) states that Eichwald (Pet. med. Zeitsch., 1870) found some 
transposition of viscera in thoracopagi, though to a varying extent. The original 
paper was not available to the writer. 


Zi2 Cc. V. MORRILL 


were two gall-bladders which occupied a median position. Other de- 
tails need not be given here. 

Case 60. Dicephalus tribrachius tripus (male). This specimen 
had two hearts, one right and one left, in a single pericardium; two 
aortae, one transposed, i.e., lying on the right of the vertebral column. 
The liver consisted of three portions, two lateral, each of which corre- 
sponded to the right lobe of a normal liver, one of them reversed, and a 
median lobe corresponding to the left lobes of normal livers fused. 
Each lateral lobe had a bile duct, gall-bladder, common duct, and 
portal vein symmetrically placed. There were two stomachs with 
pyloric ends turned toward each other; the fundus of that belonging 
to ‘A’ was in the right hypochondrium and therefore reversed, while 
that of ‘B’ had the usual position in the left. A spleen was connected 
with each. 

Case 71. Dicephalus dibrachius dipus (Gruber); sex not stated. 
There were two food passages; two stomachs with the fundus of each 
turned outward, and two intestines to within five inches of the lower 
end of the ileum. There was a large compound liver, two gall-bladders, 
and two bile-ducts; no pancreas; one spleen on left stomach; two hearts, 
the right small and imperfect; two sets of lungs and tracheae; uro- 
genital organs single and normal. 

Case 74. Dicephalus dibrachius dipus (Horner); sex, male. The 
thorax contained a compound heart. There were normal right and left 
lungs and a third compound lung due to the coalescence of adjacent 
lungs of different foetuses. The liver was single, but compound with 
increased number of lobes. . The gall-bladder was double with a com- 
mon duct which terminated in two orifices, one for each duodenum. 
There were two stomachs, one on the right, the other on the left, 
having their pyloric orifices pointing towards each other. The two 
small intestines, more or less aherent, finally blended into a single tube. 
The colon was single. There were two pancreases, but only one spleen, 
which was ‘attached to the larger left stomach. The kidneys were a 
single large pair; the bladder and genitals were single. 

Case 102. Dicephalus monauchenos (White); sex, female. There 
were two stomachs, the left in the usual place, the right reversed, its 
larger extremity towards the right. The two were united at the py- 
lorus and opened into a common duodenum. ‘The liver was single and 
very large. 

One further specimen may properly be placed with the foregoing 
five, namely, a dicephalus dibrachius dipus (female) described by 
Fisher (case 76) which possessed a single globular stomach with right and 
left fundus resulting from the fusion of two stomachs. An oesophagus 
from each mouth entered the compound stomach nearly at the same 
point. The liver and intestines were single. 


The cases cited above together with the one given in the 
present paper, are the only definitely described cases of mirror 


SYMMETRY-REVERSAL IN MONSTERS 273 


imaging in the dicephali. Unfortunately, the position of the 
viscera is not stated in the reports of Barkow’s fetus (tribrachius 
dipus) and Ruggles’ fetus (dibrachius dipus). Both of these 
had two stomachs, and it seems almost certain that one was 
transposed. We have, then, six certain cases of transposition 
and one indication of this condition in the fetus having a com- 
pound stomach with right and left fundus. 

Incidentally, one rather striking fact is brought out in look- 
ing over the various reports. In human monsters the amount of 
doubling in the viscera does not necessarily correspond with the 
amount of external doubling, as was the case in trout. Fisher 
cites one case (no. 64, from Benedina), a dicephalus tribrachius 
tripus (male) in which the gall-bladder, stomach, pancreas, 
spleen, and intestines were all single, although there were two 
hearts, two urinary bladders, and two pairs of kidneys. Com- 
pare this with the two cases of dicephalus dibrachius dipus (nos. 
71 and 74) in which the digestive systems were double as far as 
the lower part of the ileum. It must be admitted that Bene- 
dina’s case, if correctly reported, is very unusual. 

Among other classes of diplopagi in which the two compo- 
nents are more widely separated, it is difficult to find definite 
information on the position of the viscera. Bateson (’94) quotes 
Eichwald (l.c.) to the effect that the thoracopagous monsters 
examined by him showed, in almost every case, some transposi- 
tion of the viscera of one of the bodies, though to a varying 
extent. The pygopagous ‘Carolina twins,’ Millie-Christina 
(colored), were examined while living, and it was reported that 
“the apex of Christina’s heart is on her left side while that of 
Millie is distinetly felt in the right side.’”’”, Gemmnill (’02) reports a 
ease of ischiopagus tripus (human) in which modified transpo- 
sition occurred in the liver. His figure 14 seems to indicate 
transposition of the thoracic viscera of one component as well, 
but the author does not comment on it. Windle (’94) gives a 
report on the ‘Orissa sisters,’ Radica-Doodica, who were united 
in the thoracic region (xiphopagus or thoracopagus). Regard- 
ing the position of the viscera, he states that authorities differ 
as to whether one was situs inversus viscerum. In the case of 


274 Cc. V. MORRILL 


the famous Siamese twins, one of them is stated to have had a 
partial reversal of viscera. These few reports, meager as they 
are, show that some trace of visceral transposition or symmetry 
reversal may occur in monsters other than dicephali. 

In the syncephali, including Janus monsters, transposition of 
the viscera in one component apparently does not occur, though 
it seems to me in one case a slight indication was observed. 
Wilder (08), in describing a case of this kind (the ‘Baldwin 
synote’) makes the following statement:— ‘The common 
oesophagus leads into a common stomach, though evidently one 
formed of two components, since it presents two cardiac enlarge- 
ments one on either side of the oesophagus” (italics mine). ‘‘The 
outline of the stomach is thus heart-shaped, but is not quite 
symmetrical, since the cardiac lobe of component A is a little 
larger than that of Component B.” With regard to the re- 
maining organs the author states that there is no trace of ‘look- _ 
ing-glass symmetry.’ The stomach of this synote is thus simi- 
lar to that of Fisher’s dicephalus (case 76) mentioned above. 

Among other mammals a number of syncephali have been de- 
seribed: kitten, McIntosh (’68); cat, Reese (’11); pig, Carey 
(17), but none apparently showed any trace of mirror imaging. 
Kaestner (’07) has described in detail several syncephalous 
chick embryos, with especial reference to the heart region, but 
they were not far enough advanced in development to show the 
position of the abdominal viscera. Bishop (’08) gives an ac- 
count of the heart and anterior arteries in several dicephalous 
reptiles, but as no pronounced asymmetry of the heart is vis- 
ible in this class of vertebrates, there is little opportunity to 
look for mirror imaging. In cases where two hearts were pres- 
ent, both aortic arches developed on each side. It is unfortunate 
that among the large number of double monsters reported so 
much attention has been paid to external features and so little 
to the position of the abdominal viscera. 


SYMMETRY-REVERSAL IN MONSTERS 275 


DISCUSSION 


The question of symmetry reversal and mirror imaging has 
been discussed most recently by Wilder (’04, 716), Bateson (16), 
and Newman (’16, 717). It seems to be generally agreed that 
transposition of the viscera does not occur in human duplicate 
twins. In armadillo quadruplets Newman finds, after examina- 
tion of a considerable number of sets, that no symmetry reversal 
is present in the viscera. The same is true in the duplicate 
twin trout (fig. 6) described in the present paper. Some mirror 
imaging, however, does occur in human duplicate twins and 
armadillo quadruplets, but it is confined to the integumentary 
structures (friction-skin patterns in the former case, arrange- 
ment of the scutes and bands in the latter). The integument 
of young trout, unfortunately, does not present any regular pat- 
tern of asymmetry, at least none could be detected, and thus 
yields no information on this point. In double monsters, how- 
ever, it is admitted that a certain amount of symmetry reversal 
in the viscera is to be expected, although it may not occur in every 
ease. Fisher, in 1866, clearly expressed this opinion, and is 
quoted by Wilder (’04) to this effect. Wilder, though also quot- 
ing Bateson’s (’94) opinion, in agreement with Fisher, seems un- 
willing to admit the importance of this phenomenon and gives 
little space in his earlier paper (l.c.) to its discussion. In a later 
paper (716), however, he discusses a very interesting case of 
mirror imaging in the friction-skin patterns of a human diplopage. 

Newman, (16, 717) has given perhaps the fullest discussion of 
symmetry reversal, both in multiple births and in monsters. The 
relations of symmetry observed in armadillo quadruplets are, 
he considers, ‘‘the results of an intricate interplay of three 
grades of successively operating symmetry systems, the later 
tending to obliterate the effect of the earlier, but not always suc- 
cessfully.” This conclusion is based on the nature of the poly- 
embryonic development observed in these animals and is ex- 
plained by Newman as follows: ‘“‘When the primary outgrowths 
are formed (i.e., fission in the blastocyst stage), they are the 
product of the antimeric halves of the first embryo and should 


THE ANATOMICAL RECORD, VOL. 16, NO. 4 


276 Cc. V. MORRILL 


therefore show mirror-image relations. But a partial physio- 
logical isolation of the two halves permits a certain reorganiza- 
tion, or regulation of new symmetry relations, which tends 
more or less completely to destroy the original symmetry, yet 
often leaving a trace of the latter. Similarly, when the second- 
ary outgrowths arise between the primary ones a certain residuum 
of the primary symmetry may be carried over that frequently 
manifests itself in mirror imaging between twins derived from 
one-half of the original embryo. Finally, when each secondary 
outgrowth organizes its own bilateral symmetry, it tends to lose, 
partially at least, the earlier symmetry relations and to estab- 
lish its own mirror imagings of right and left sides” (third grade 
of symmetry). It must of course be remembered that in arma- 
dillos, mirror imaging between twins is confined to integumentary 
structures. In the case of duplicate twins and double monsters, 
there would be according to Newman’s conclusion, only two 
‘orades of symmetry systems.’ Any mirror imaging present in a 
monster would thus be evidence of the potency of a primary 
symmetry which had not been overcome by the secondary sym- 
metry acquired later by the separate components. If physio- 
logical isolation occurs in a comparatively early stage, there 
will be, he thinks, very little mirror imaging, as the secondary 
symmetry will have more time to operate. Conversely, if it 
appears somewhat later, there will be more mirror imaging. In 
consequence, double monsters probably arise somewhat later in 
ontogeny than duplicate twins, since the former more often show 
evidence of mirror imaging. 

Newman’s suggestion regarding primary and secondary sym- 
metry systems is to some extent supported by the conditions 
found in trout monsters. However, by far the greater num- 
ber of these monsters, of whatever degree of doubling, show no 
influence of a primary system of symmetry, that is a symmetry 
of the monster taken as a unit. On the contrary, each com- 
ponent develops its own system (secondary, according to New- 
man) as if it were entirely disconnected from its mate (fig. 5), 
and this symmetry (asymmetry), moreover, is the same as that 
of a normal fish. It is interesting to note that in the type of 


SYMMETRY-REVERSAL IN MONSTERS 277 


double monster known as autosite-and-parasite, a number of 
which occurred in the present collection, the parasite, whenever 
it was of sufficient size to possess a complete set of abdominal 
organs, always exhibited its own (secondary) symmetry and 
never appeared as a mirror image of the autosite. It is only 
in a small proportion of the monsters that the primary symmetry 
of the whole is still potent, in which case mirror imaging appears 
in the viscera (figs. 1, 2, and 4). 

Newman’s further suggestion that there is a direct relation 
between the occurrence of mirror imaging and the period in ontog- 
eny at which doubling takes place, does not accord with what 
seems to be the mode of origin of monsters in fish. In this form, 
the initial doubling probably always occurs at the same period 
of development, regardless of the degree of separation of the 
two components. This period corresponds with the first appear- 
ance of the embryonic anlage at the circumference of the blas- 
toderm, as Kopsch (’99) concluded in his analysis of the causes 
of fish monsters.7?. In the case of double monsters, two em- 
bryonic anlages are formed at the same time. The degree of 
doubling will then depend on how near the two anlages lie to each 
other. On this view, mirror imaging and the time at which 
doubling first appears cannot be causally related. Nor is there 
a very precise relation, it seems to me, between the amount of 
separation of the two components and the occurrence. of mirror 
imaging. It is true that there is a stage of doubling more favor- 
able than others for exhibiting symmetry reversal in one com- 
ponent, but only a small proportion of the monsters even then 
show any evidence of this condition (compare figs. 4 and 5). 
Furthermore, specimens showing less separation than in the 
stage just mentioned might be expected to exhibit more evi- 
dence of primary symmetry (symmetry of the monster as a 
whole) and therefore more mirror imaging, while in point of fact 

the contrary is true (p. 268). 

_ It was pointed out (p. 271) that in both fish and human mon- 
sters it is always the same component (the left-hand or A-com- 


7 This view apparently originated with Lereboullet. Kopsch has developed 
it in considerable detail in the paper referred to above. 


27. 


0 4) 


Cc. V. MORRILL 


ponent)’ which exhibits transposition of the viscera. In this 
the writer agrees with Eichwald, as quoted by Bateson (’94), 
except that the latter uses the term ‘right twin’ for what is here 
called left-hand or A-component. Bateson himself is in doubt 
on this point and quotes Kiichenmeister® to the effect that in 
xiphopagous twins it may not be possible to say which is the 
right and which the left. This objection, however, does not 
apply to dicephalous forms, whether fish or human. Here the 
undivided portion of the monster obviously has dorsal and 
ventral surfaces and these may be traced without interruption 
into the corresponding surfaces of the two components which 
usually face each other to some extent. The right-hand and 
left-hand components are thus easily distinguished. In cases 
where mirror imaging occurs, the arrangement of the two sets of 
organs is always the same’—the stomachs bend first toward the 
lateral borders of the monster (taken as a unit), then toward 
the median plane (plane of union) so that their pyloric ends 
face each other; the livers lie close together or are fused. It is 
difficult, however, to find an explanation for this fact, for even 
if the reverse arrangement occurred, there would still be mirror 
imaging—the fundus of one stomach facing that of the other, 
the pyloric ends pointing in opposite directions, the livers lying 
on the lateral borders of the monster. It has sometimes been 
assumed that in normal development the direction of growth 
taken by the liver bud determines the plan of asymmetry of the 
remaining viscera. In the case of monsters having either two 
livers or a composite liver, it might be further assumed that the 
two liver buds, having formed independently, were drawn to- 
gether by some sort of mutual attraction. If such a movement 
took place, the anterior ends of the two intestines together with 


8 Die. angeb. Verlagerung d. Eingeweide d. Menschen, Leipzig, 1888. The 
original was not available to the writer. 

® An exception to this appears to have been found in the famous Siamese 
twins where it was Chang, the left twin (right-hand or B-component of the 
present paper), in whose body there were indications of situs inversus (Kiichen- 
meister, quoted from Bateson). This would give the converse of the usual 
arrangement. The writer has not had access to the original description of these 
interesting twins. 


SYMMETRY-REVERSAL IN MONSTERS 279 


the pyloric ends of the stomachs would be drawn with the livers 
toward the plane of union. This would result in the arrangement 
found in practically all monsters in which mirror imaging occurs. 
While the above assumptions do, to some extent, account for 
the facts, there is some evidence to show, as will be pointed out 
below, that the factors controlling asymmetry are located in 
the primitive gut and become operative before the liver bud has 
developed. 

It must be admitted that we are still in the dark regarding 
the causal factors underlying the conditions of mirror imaging 
found in some types of monsters. The question here arises, 
why, in a certain stage of doubling, should mirror imaging 
occasionally appear and not always? One might assume that 
the rate of development in one component of a monster occa- 
sionally becomes a little slower than in its mate so that it tends 
to fall behind and is unable to develop or express an independent 
system of symmetry like that of a normal embryo. In this case 
the lagging component might be thought of as sharing with its 
more vigorous mate in a single system of symmetry, that is, the 
symmetry of the monster taken as a unit. The result would 
then be a mirror-imaged condition of the viscera. A suggestion 
of inferiority in one component was noted in the human monster 
described above where the transposed set of organs were found 
to be slightly smaller and more irregular in shape than those 
of the opposite side. In the fish monsters, however, no such in- 
equality between the two sets of organs was observed. Further- 
more, the assumption that a retardation of development in one 
component predisposes to transposition of viscera is rendered 
improbable by the conditions found in monsters of the autosite- 
and-parasite type. Here it may be fairly assumed that the 
parasite tends to be weaker than the autosite, and in fact is often 
defective; still whatever asymmetry exists in the parasite is 
that of a normal fish and never reversed. In other words, the 
parasite, even in its failing struggle for existence, retains the 
power to develop its plan of asymmetry as a separate individual. 

It is extremely difficult to formulate a theory which will 
satisfactorily account for a condition so casual in its appear- 


280 Cc. V. MORRILL 


ance as mirror imaging in the viscera of monsters, and further 
work on the early developmental stages of these forms is neces- 
sary before any definite conclusions can be drawn. The solution 
will, of course, involve the more fundamental problem of what 
determines the normal asymmetry of unpaired organs and why 
single individuals occasionally appear with transposed organs.!° 
Very little progress has been made in this direction. The most 
suggestive observations in the field are those of Pressler (’11), 
on experimentally produced situs inversus in Bombinator. The 
material was obtained from Spemann who performed the fol- 
lowing experiment: In the neurula stage, a four-sided piece 
of the medullary plate together with a portion of the roof of the 
primitive gut lying under it was cut out and replaced in reversed 
position, so that the anterior extremity of the piece was directed 
posteriorly, the posterior extremity, anteriorly. From these 
experimental embryos, tadpoles were reared which showed in 
many cases a complete situs inversus viscerum. It has some- 
times been assumed, as stated above, that the asymmetrical 
growth of the liver bud normally toward the right influences 
the position of the remaining organs. The question then arises, 
what determines the direction of growth of the liver bud? Spe- 
mann and Pressler’s work seems to indicate that the factors 
controlling asymmetry are located in the primitive gut and prob- 
ably arranged in such a fashion as to cause the gut in normal 
development to bend first toward the left, thus forcing the liver 
bud to grow toward the right. We may suppose that when the 
arrangement of these factors is reversed, as in the experiment, 


10 Bateson (’94, p. 560) points out that cases of this kind cannot be explained 
on the ground that one member of duplicate twins hassdied or failed to develop, 
since it has been shown that in duplicate twins neither member has transposed 
viscera. Conversely, Kiichenmeister (l.c.) collected 152 cases of transposition, 
of which only one could be shown to have been a twin. 

A somewhat similar suggestion has been made to account for cases of situs 
inversus in single individuals, namely, that this condition results from com- 
plete reduction of one component of a monster (autosite- and-parasite) in which 
mirror imaging occurred. The autosite in this instance must necessarily present 
the reversed asymmetry. In some cases it is thought that the parasite is taken 
into the body of the autosite during development, and gives rise to certain kinds 
of tumors. As far as I am aware, there is no evidence recorded that individuals 
with complete situs inversus have possessed tumors of this sort. 


SYMMETRY-REVERSAL IN MONSTERS 281 


transposition is produced. Pressler’s observations are, it seems 
to me, very important and indicate the direction along which 
further experiments should be made to determine the cause of 
asymmetry. They do not, however, throw any light on the 
cause of transposition in integumentary structures as found by 
Newman and Wilder. 

A very interesting suggestion as to the cause of asymmetry in 
the viscera is based upon the fact, first pointed out by Crampton 
(94), that in certain gasteropods the position assumed by the 
adult organs is correlated with the early segmentation pattern. 
In these snails the more usual type of asymmetry with dextral 
shell is associated with a right-handed spiral cleavage. Some 
forms, however, such as Physa (Crampton) and Ancylus rivu- 
larius (Holmes), have normally sinistral shells and reversed 
asymmetry in the viscera; this condition was found to be asso- 
ciated with a reversal of cleavage. These observations very 
naturally led to the view that in gasteropods there is a causal 
relation between cleavage pattern and the type of asymmetry 
found in the adult." It is very questionable, I think, whether 
this conception of the primary cause of asymmetry can be ap- 
plied to vertebrates. For in monsters, as has been shown, two 
sets of organs may develop as mirror images of each other, one 
with normal, the other with reversed asymmetry, though ob- 
viously both have arisen at the same period of development from 
a single blastoderm. It is difficult to imagine how changes in 
early cleavage pattern, if such occur in higher forms, could bring 
about the development of two types of asymmetry in the same 
embryo, as in the case just cited. From the evidence at hand, it 
seems probable that the primary cause of visceral asymmetry in 
vertebrates is to be sought for at the completion of cleavage 
rather than in the period of cleavage itself. 


11 This view, first expressed tentatively by Crampton (’94), was later more 
fully developed by Conklin (’97, Jour. Morph., vol. 13) and by Holmes (99, 
Amer. Nat.; 00, Jour. Morph., vol. 16) on the basis of additional evidence. 
Conklin more recently (Heredity and Environment, 2nd ed., 1917, p. 177) has 
expressed the opinion that the correlation between inversion of cleavage and in- 
version of symmetry observed in certain snails, will be found ‘‘probably in all 
animals showing inverse symmetry’’ (italics mine). I do not believe this latter 
generalization is warranted for the reasons given in the discussion (see beyond). 


282 Cc. V. MORRILL 


LITERATURE CITED 


Bareson, W. 1894 Materials for the study of variation. London (Macmillan 
& Co.). 

1916 Problems of genetics. New Haven (Yale Univ. Press). 

Bisnop, M. 1908 Heart and anterior arteries in monsters of the dicephalus 
group; a comparative study of cosmobia. Am. Jour. Anat., vol. 8. 

Carey, E. 1917 The anatomy of a double pig, Syncephalus thoracopagus, with 
especial consideration of the genetic significance of the circulatory 
apparatus. Anat. Rec., vol. 12. 

Crampton, H. E. 1894 Reversal of cleavage in a sinistral gasteropod. Ann. 
New York Acad. Sci., vol. 8. 

Fisner, G. J. 1866 Diploteratology. Trans. Med. Soc. State of N. Y. 

Gemmiy, J. F. 1901 The anatomy of symmetrical double monstrosities in the 
trout. Proc. Roy. Soc. London, vol. 68, no. 444. 

1902 An ischiopagus tripus (human), with special reference to the 
anatomy of the composite limb. Jour. Anat. and Phys., vol. 36. 
1912 The teratology of fishes. Glasgow (James Maclehose & Sons). 

Hirst AnD Pierson 1893 Human monstrosities, 4 vols. Philadelphia. 

Kanstner, A. 1907 Doppelbildungen an Vogelkeimscheiben. Arch. f. Anat. 
u. Phys. 

Korscu, Fr. 1899 Die Organisation der Hemididymi und Anadidymi der 
Knochenfische und ihre Bedeutung fiir die Theorien tiber Bildung und 
Wachstum des Knochenfischembryos. Internat. Monatsschr. f. Anat. 
u. Phys., Bd. 16. 

MclInrosa 1868 Notes on the structure of amonstrous kitten. Jour. Anat. and 
Phys., no. 2. 

Newman, H. H. 1916 Heredity and organic symmetry in armadillo quadrup- 
lets. II. Mode of inheritance of double scutes and a discussion of 
organic symmetry. Biol. Bull., vol. 30. 

1917 The biology of twins. Univ. of Chicago Press. 

Presster, K. 1911 Beobachtungen und Versuche iiber den normalen und 
inversen Situs viscerum et cordis bei Anurenlarven. Arch. f. Entw.- 
Mech., Bd. 32. 

Reese, A.M. 1911 The anatomy of a double cat. Anat. Rec., vol. 5. 

Wiper, H. H. 1904 Duplicate twins and double monsters. Am. Jour. Anat., 
vol. 3. 

1908 The morphology of cosmobia; speculations concerning the sig- 
nificance of certain types of monsters. Ibid., vol. 8. 
1916 Palm and sole studies, part II. Biol. Bull., vol. 30. 

Winptez, B.C. A. 1894 Report on ‘Radica-Doodica.’ Jour. Anat. and Phys., 
vol. 28. 

1895 On double malformations amongst fishes. Proe. Zool. Soe. 
London, pt. 3. 


a 


PLATES 


PLATE 1 
EXPLANATION OF FIGURES 


1 and 2 Specimens of monstrous trout showing complete mirror imaging in 
the abdominal viscera, ventrolateral view. S.B., swim bladder; L., liver; the 
stomach and intestine are not labeled. The position of the viscera in one com- 
ponent is the reverse of that in the other. 

3 Specimen in which doubling is less extensive than in the foregoing (1 and 2). 
There are two stomachs and two swim bladders (S.B.) one of which is almost 
concealed by the compound liver (Z.) The intestines unite immediately beyond 
the stomachs into a common enlargement (C.J.)._ Apparently no mirror imaging 
is present in this case. The two pear-shaped bodies anterior to the abdominal 
cavity are the hearts. 


SYMMETRY-REVERSAL IN MONSTERS PLATE 1 


Cc. V. MORRILL 


H. Murayama, del 


285 


PLATE 2 
EXPLANATION OF FIGURES 


4 Specimen showing complete mirror imaging similar to figures 1 and 2 
except that the two sets of organs are closer together, the livers (L.) almost in 
contact; ventrolateral view. 

5 Specimen showing the position of the viscera in the majority of monsters 
in this stage of doubling. The normal situs is present in both components; 
ventrolateral view. Compare with figure 4. 

6 Twins from the same egg. These two specimens lay on opposite sides of a 
single yolk mass. The position of the viscera is the same in both, the liver on the 
right, the stomach bulging toward the left as in normal fish (i.e., normal situs in 
both). 


286 


SYMMETRY-REVERSAL IN MONSTERS PLATE 2 
¢. V. MORRILL 


H. Murayama, del. 


PLATE 3 
EXPLANATION OF FIGURES 


7 Diagram of the viscera in the human monster shown in figure 8. The 
compound liver is omitted. The apices of the two hearts are widely separated to 
show the medial surfaces which were in close contact. A., aorta; P.A., pul- 
monary artery (origin); S.V.C., superior vena cava; Vv.h., hepatic veins; Sp. 
spleen; Pan., pancreas; G.B., gall-bladder; A.C., ascending colon; Cae., caecum 


288 


PLATE 3 


SYMMETRY-REVERSAL IN MONSTERS 
c, V., MORRILL 


H. Murayama, del. 


289 


] 
‘ 
I 
b 
a 
» 
, : 
a 
‘ae ! 
Pe 
PLATE 4 ij 
e EXPLANATION OF FIGURE a ae 


8 Photograph of the human dicephalus tribrachius dipus described in the 
present paper. =A , : 


~ 
any, d 
rhe 
. ay 1 
r 
i 
s | _ 


SYMMETRY-REVERSAL IN MONSTERS 
¢. V, MORRILL 


291 


PLATE 4 


Lal 


{Reprinted from THe JouRNAL or GENERAL PaysioLocy, September 20; 1919, 
Vol. ii, No. 1, pp. 49-68.] 


CHANGES IN PROTOPLASMIC CONSISTENCY AND THEIR 
RELATION TO CELL DIVISION. 


By ROBERT CHAMBERS. 


(From Cornell University Medical College, New York City, and the Marine Biological 
Laboratory, Woods Hole.) 


(Received for publication, July 29, 1919.) 


I. Periodic Changes in Consistency of the Egg Cytoplasm after Fertili- 
zation and during Cleavage. 


On fertilization an increase in the viscosity of the semifluid cyto- 
plasm of the sea urchin egg was noticed by Albrecht! and recently 
fully demonstrated by Heilbrunn.2 Heilbrunn based his conclusions 
on his observation that a greater centrifugal force is necessary to 
stratify the cell constituents of an egg after fertilization than before. 
I? have presented evidence, from microdissection studies on the sand- 
dollar egg and the egg of Cerebratulus, that the increase in viscosity 
is associated with the appearance and growth of the aster. 

Upon entrance of the spermatozoon into the egg a diminutive 
aster makes its appearance as a ball of a jelly-like consistency in the 
immediate vicinity of the sperm head. This aster, with the sperm 
nucleus, moves inward as it steadily increases in size until, when its 
center comes to lie in or near the center of the egg, its radiations 
extend throughout the whole egg. During this migration the sperm 
nucleus comes into contact with the egg nucleus. The aster then 
develops completely around the two nuclei, which fuse to constitute 
the cleavage nucleus. 

The development of the sperm aster in the sea urchin egg is at 
its height within 10 to 15 minutes after fertilization. This is the 


1 Albrecht, E., Untersuchungen zur Struktur des Seeigeleies, Sitz-ber. Ges. 
Morph. u. Physiol., 1898, xiv, 133. 

2 Heilbrunn, L. V., Studies in artificial parthenogenesis. II. Physical changes 
in the egg of Arbacia, Biol. Bull., 1915, xxix, 149. 

3 Chambers, R., Jr., Microdissection studies II. The cell aster: A reversible 
gelation phenomenon. J. Exp. Zool., 1917, xxiii, 483. 


49 


50 PROTOPLASMIC CONSISTENCY AND CELL DIVISION 


time when Heilbrunn informs me he found the egg substance to be 
of maximum viscosity. 

The increase in viscosity of the egg cytoplasm is produced by an 
influence spreading out in all directions from the center of the aster. 
While this occurs the central hyaline area of the aster (the hyalo- 
plasmsphere of Wilson) increases in size, and there is strong evidence® 
that this is due to the accumulation of a hyaline liquid which sepa- 
rates out of the semisolidifying cytoplasm and flows in very fine 
converging streams to the center of the aster. It is possible that 
this and kindred phenomena give to the aster the appearance of 
radiations from a common center. The consistency of the cytoplasm 
incorporated in the aster diminishes in firmness on passing from the 
interior of the aster to its exterior, being greatest in the region border- 
ing on the centrosphere and least at the periphery. 

The disappearance of the sperm aster, in the opinion of the writer, 
occurs through a process of liquefaction. During the liquefaction 
the substance of the centrosphere collects into two areas at opposite 
poles of the cleavage nucleus. The experiments to be described in 
this paper indicate that shortly before cleavage each of these areas 
becomes a center around which the cytoplasm commences again to 
pass into a semisolid state. The radial configuration about these 
areas constitutes the amphiaster. The comparatively firm consist- 
ency that the egg now attains for the second time since fertilization 
is due to two masses, the two asters, instead of to a single aster as 
was the case shortly after the entrance of the sperm. ‘The importance 
of this phenomenon in its bearing on cell division is discussed in the 
last part of this paper. 

Experiment 1—The consistency exhibited by the protoplasm of the 
sea urchin egg at various periods from the moment of fertilization 
until the completion of the first cleavage, was ascertained by careful 
probing with the microdissection needle. 

Immediately after fertilization the cytoplasmic granules readily 
flow by the moving needle. After the sperm has entered the egg, 
the sperm aster constitutes a comparatively firm mass which gradually 
increases in size as it moves to a central position in the egg. When 
the sperm aster is at its full development the highly viscous state 
of the cytoplasm is detected by the needle. Illustrations of this 


ROBERT CHAMBERS 51 


are given in a former paper.’ The cytoplasmic granules, instead of 
being readily dislocated by the moving needle, are held as in a jelly, 
and movements of the needle produce torsions of the entire egg 
substance. This condition is at its height 10 to 15 minutes after 
fertilization. 

15 to 20 minutes after fertilization, the radiations of the aster 
begin to fade from view, with a reversal in the cytoplasm of the semi- 
solid to a more fluid state. The cytoplasmic granules are now easily 
dislocated by the moving needle. The more prominent radiations 
disappear first, while the finer ones persist for some time, owing prob- 
ably to the viscid nature which the cytoplasm always maintains. 
The liquid substance of the central hyaline area now flows over the 
nucleus to its two poles, beyond which it often extends. This causes 
the appearance characteristic of this stage, of a hyaline streak plainly 
visible in the otherwise granular cytoplasm of the egg. Toward the 
end of this stage, which lasts for about 20 to 30 minutes, the hyaline 
substance finally collects into two semispherical masses lying at the 
two poles of the nucleus. 

Shortly before cleavage, about 40 to 50 minutes after fertilization, 
an increase in firmness sets in, spreading radially from each of the 
two centers situated at the poles of the nuclear spindle. This con- 
stitutes the amphiaster. The egg elongates, the long axis passing 
through the two centers of the amphiaster. The cleavage furrow 
now appears and the egg rapidly divides. The time of appearance 
of the amphiaster until completion of cleavage lasts from 10 to 15 
minutes. The increased viscosity of the egg during this amphiaster 
stage could be more easily demonstrated by the needle in the eggs 
of Echinarachnius and Cerebratulus than in those of Arbacia. 

After completion of the cleavage process, there are indications that 
the firmness of the cytoplasm persists in the two blastomeres while 
they are still more or less spherical. Within 10 to 15 minutes after 
cleavage the two blastomeres crowd up against one another, each 
assuming a more nearly hemispherical shape. At this stage their 
cytoplasm is again quite fluid. 

These observations demonstrate a pronounced periodicity in the 
physical state of the egg subsequent to fertilization and during the 
first cleavage process. In the immature egg the viscosity is high, 


52 PROTOPLASMIC CONSISTENCY AND CELL DIVISION — 


after maturation it drops. Upon fertilization it begins to rise again, 
to reach its maximum with the full development of the sperm aster. 
The viscosity drops again and continues low until the approach of 
cleavage. It thereupon rises again to drop only after completion of 
the first cleavage. Subsequent to the first cleavage the rhythmic 
appearance and disappearance of the asters within the blastomeres 
most probably indicate periodic successions of a process analogous 
to a jellying and liquefying of the cytoplasm. 

The segmentation process may thus be explained as consisting 
essentially in a growth within the egg of two bodies of material 
through a gradual transformation of the cytoplasm. ‘This transfor- 
mation is associated with a change in the physical state of the proto- 
plasm, two semisolid masses growing at the expense of the more 
fluid portions of the cytoplasm. 


II. Cutting Experiments on the Segmenting Egg. 


Tf it is true that the segmenting egg consists of two rather firm 
masses which are most fluid at their periphery, and if the physical 
state of the protoplasm is not affected in the process, one should be 
able to cut a segmenting egg into pieces without disturbing the 
cleavage plane. Cleavage should, therefore, proceed in such a manner 
as to complete the separation of what remains of the two bodies 
within each piece. This is what actually happens. Some experi- 
ments of Yatsu,‘ the results of which he made no attempt to explain, 
are in full accordance with mine and bear directly on this problem. 
Yatsu cut the eggs of Cerebratulus which were just beginning to 
segment (anaphase stage) into nucleated and non-nucleated frag- 
ments. He found that the cleavage furrow proceeded in its original 
plane irrespective of whether the fragments were nucleated or not. 
In Fig. 1, I have diagrammatically presented some of his results. 
Fig. 1 a represents a segmenting Cerebratulus egg being cut in a plane 
parallel to its long axis and to one side of the daughter nuclei. The 
original furrow persisted in the non-nucleated fragment (6) and 


*Yatsu, N., Some experiments on cell-division in the egg of Cerebratulus 
lacteus, Annot. Zool. japon., 1908, vi, 267. 


ROBERT CHAMBERS 53 
quickly completed its course in the nucleated fragment (c). Some- 


what later the cleavage of the non-nucleated fragment (d) was also 
completed. Fig. 1 e represents a segmenting egg in which the cut 


O 


yee 


a 


: 
| d 
C 

ee 

O ( O 

O 

‘i a 

e€ 


Fic. 1. A diagrammatic representation of Yatsu’s results* on cutting the 
segmenting eggs of Cerebratulus. The direction of the cut is shown in a. The 
original cleavage furrow completed its course in the nucleated fragment c at the 
same time that it persisted in the non-nucleated fragment 6. The furrow finally 
cut through the non-nucleated fragment in d. Ine a cut was made across one 
end of the segmenting egg. The original furrow completed its course in f re- 
sulting in two unequal blastomeres. 


was made at one end of the egg at right angles to its long axis. The 
original furrow persisted so as to divide the mutilated egg into two 
unequal blastomeres (f). 

My cutting experiments were carried out mostly on the starfish 
egg, as sea urchins were very scarce during the summer of 1918. 


54 PROTOPLASMIC CONSISTENCY AND CELL DIVISION 


The mature starfish egg averages 0.16 mm. (i.e. 160 ») in diameter. 
The needles used for dissection averaged 10 yw in thickness at about 
1 mm. from the tip and tapered gradually from there to a point far 
below 1 u. With such a needle one can make a puncture or a clean 
cut through the egg in any desired spot or plane without causing 
apparent disturbance in the protoplasm of the egg. For cutting 
purposes glass needles as shown in Fig. 2 were used.* As the egg 
lies suspended in a hanging drop the end limb of the needle (Fig. 2 a) is 
set in such a way as to push the egg against the cover-slip. Con- 
striction of the egg is produced by a continued upward pressure of 
the needle until the egg is cut in two. The operation does not neces- 


Cover: slip roofing moist Sia 
\ E ag in hanging dro 
_End limbof needle, 


a b 


Fic. 2. Methods used for cutting an egg in two. 4, side view of moist chamber 
magnified to show needle in position with its end limb so placed as to compress 
an egg between it and the cover-slip. Continued pressure of the needle cuts the 
egg in two. 6, a second method of cutting an egg by bringing the end limb of 
the needle down on the egg so as to press the egg against the lower surface of the 
hanging drop. 


sarily destroy the fertilization membrane which envelops the egg. 
The egg may also be cut in two on bringing it (Fig. 2 6) between the 
end limb of the needle and the lower surface of the hanging drop. 
Lowering the needle out of the drop in such a way as to give to the 
egg a rolling motion cuts the egg cleanly in two. This second method 
is not as satisfactory as the first for cases where one wishes to preserve 
the spatial relations of the egg contents, as the rolling motion pro- 
duces churning movements within the cell. 

Experiment 2——(Figs. 3 to 7.) An Asterias ovum just beginning 
to segment and with the amphiaster in full development was cut 


® Chambers, R., The microvivisection method, Biol. Bull., 1918, xxxiv, 121. 


ROBERT CHAMBERS 55 


in two in a plane diagonal to the cleavage furrow. ‘The fresh surfaces 
caused by the cutting form films which prevent reunion of the pieces. 
The egg was in this way cut into two pieces each consisting of egg 
substance lying on both sides of the cleavage furrow. 


4.20 


4.40 5.00 

Fic. 3. Effect of a diagonal cut through an A sterias ovum beginning to segment 
in which the cut did not disturb the physical state of the ovum. a, operation 
performed at 4.15 p.m. 0, 4.20 p.m., persistence of cleavage furrow in the original 


plane. c, 4.40 p.m., non-nucleated fragments pinched off. d, 5.00 p.m., 
nucleated fragments have segmented. 


On one occasion the operation was performed on twelve eggs. In 
nine cases the original cleavage plane was maintained so that each 
piece pinched off a non-nucleated fragment normally belonging to 
the other blastomere. Two of them are illustrated in Figs. 3 a to d 
and 4 a tod. 


56 PROTOPLASMIC CONSISTENCY AND CELL DIVISION 


In one case the cut was made at 4.15 p.m. (Fig. 3 a). 5 minutes 
later the cleavage furrow had progressed in the original plane (Fig. 
3 6). At 4.40 it had completed its course so that each piece was 
divided into a small non-nucleated and a large nucleated fragment 


Fic. 4. Similar operation to that shown in Fig. 3 except that the diagonal cut 
is more nearly perpendicular to the cleavage plane with the result that larger 
non-nucleated fragments are pinched off by the cleavage furrow. 


(Fig. 3c). At5 p.m. each of the two nucleated fragments or blasto- 
mere remnants had divided once (Fig. 3 d). 1 hour later they had 
divided once again. By the next morning the egg developed into a 
double blastula with the two non-nucleated fragments lying as inert 
masses within the fertilization membrane. 


ROBERT CHAMBERS 57 


Fig. 4 a to d illustrates a similar case in which the non-nucleated 
masses are considerably larger than those depicted in Fig.3. The 
similar behavior of one of the first two blastomeres in an egg is shown 
in Fig. 5 a and 6. 

In the remaining three cases the astral radiations faded out during 
the operation (Fig.6 a). The original segmentation furrow gradually 
filled up and disappeared (Fig. 6 6) and each piece assumed the ap- 
pearance of a normal blastomere. The nucleus then shifted so as to 
occupy a more central position in what one may term the reconstructed 
blastomere and further segmentation proceeded as if the ovum had 
not been operated upon (Fig. 6 c and d). This procedure always 


a 


Fic. 5. Effect of a diagonal cut through one of the first two blastomeres ofan 


Asterias ovum. a, egg showing direction of cut. 6, cut blastomere a few minutes 
later. 


occurred when the ovum was consciously rolled during the opera- 
tion so as to produce a disturbance evidenced by a churning move- 
ment of the egg constituents. 

A similar instance in the case of an Arbacia egg is shown in Fig. 
7 atoc. A piece was cut from one pole of the amphiaster egg. In 
the process the piece was cytolyzed. The amphiaster in the remain- 
der of the egg disappeared to reappear again in a new position with 
the result that two equal sized blastomeres were formed. 

That mechanical disturbances may cause a reversal of a solid to 
a fluid state has already been shown. This would make all the 


58 PROTOPLASMIC CONSISTENCY AND CELL DIVISION 


protoplasm on each side of the cut merge into a single fluid mass. 
The nucleus then comes to occupy a central position. Normal 
mitosis takes place with the formation of an amphiaster and cleavage 


4.00 A.25 


Fic. 6. Effect of a diagonal cut through an Asterias ovum in which the cut 
brought about a change in the physical state of the egg. a, operation performed 
at 3.30 p.m. 6, 3.35 p.m., original cleavage furrow beginning to be obliterated. 
c, 4.00 p.m., an amphiaster formed in each of the two pieces produced by the cut. 
d, 4.25 p.m., four celled stage in which one cleavage plane was produced by the 
needle and the other by normal fission. 


proceeds along the equator where the boundaries of the two asters 
are contiguous. 

In the nine cases, in which the original cleavage plane persisted 
after the cutting process, the semisolid state about the two astral 


ROBERT CHAMBERS 59 


centers was not disturbed. Each of the two pieces resulting from 
the cut, therefore, consisted of two unequal semisolid masses sepa- 
rated by a fluid area corresponding to the equator of the original egg. 
As this fluid area is incorporated into the two masses a furrow appears 


G: 


Fic. 7. Effect of cutting off a piece from one pole of an Arbacia ovum in the 
amphiaster stage. a, direction of cut. 06, the piece cut off cytolized. The 
original shape of the remainder of the egg persisted for some time as the ovum of 
Arbacia is less pliable than that of Asterias. c, the reappearance of a new 
amphiaster resulting in the formation of two equal blastomeres. 


which separates each piece into a larger nucleated and a smaller non- 
nucleated body. 

The operated eggs were kept under observation until the gastrula 
stage, indicating that the operation had not destroyed the capacity 
of the egg for further development. 


60 PROTOPLASMIC CONSISTENCY AND CELL DIVISION 


The following experiments are supplementary to the second. In 
all of them the results obtained are explicable on the basis of the 
existence, during cleavage, of reversible changes in the consistency 
of the cytoplasm. 


BE NG, 
WUANG 
NOSE IRS 


oi 
WSWZZ LAX e7 
ie 


ce 
= > 


540 


Fic. 8. Development of an Asferias ovum manipulated with a needle so as 
to suppress the first cleavage furrow. a, 5.00 p.m., disappearance of the amphi- 
aster and obliteration of the cleavage furrow. 0, 5.35 p.m., appearance of two 
amphiasters. c, 5.40 p.m., change in shape of the ovum with appearance of 
second cleavage furrow ahead of the first. d, 5.45 p.m., ovum cleaving into 
four blastomeres. (The ovum developed into a normal embryo.) 


Experiment 3—(Fig. 8.) In this case the first segmentation furrow 
was prevented from forming by tearing at the equator whenever it 
made its appearance. The progressive changes within the egg were 


ROBERT CHAMBERS 61 


undisturbed. As soon as the amphiaster disappeared there was no 
longer a tendency for the furrow to form (Fig. 8 a). The two nuclei 
now lay ina fluid cytoplasm. Within half an hour after the suppres- 
sion of the first segmentation furrow, an amphiaster developed about 
each nucleus preparatory to the next division. The two amphiasters 
lay side by side but remained distinct from one another, no connecting 
radiations being formed (Fig. 8b). The formation of the two amphi- 
asters resulted in the transformation of the egg substance into four 
semirigid bodies, the four asters. Cleavage furrows now extended 
into the fluid regions between the asters and divided the egg almost 
simultaneously into four blastomeres. The furrow corresponding to 
the second cleavage started to form and cut through the egg about a 
minute ahead of that of the first (Fig. 8c and d). 

This experiment may throw light on the nature of the segmentation 
in ova in which several nuclear divisions follow one another with no 
outward manifestation of the segmentation of the egg. After a 
certain period the ovum breaks up simultaneously into as many 
blastomeres as there are nuclei. This is the normal method in certain 
Actinozoa and can be artificially produced in many eggs by exposing 
them to various reagents, notably hypertonic solutions.®7 

The solidification associated with the aster formation divides the 
egg cytoplasm into a number of bodies each surrounding a nucleus. 
Between successive divisions the cytoplasm reverts to a more fluid 
state but its viscid nature may suffice in preventing the merging of 
neighboring areas. After a varying number of nuclear divisions with 
accompanying solidification periods furrows suddenly appear between 
these bodies and the ovum tends to break up at once into separate 
blastomeres. A difierentiation of this type may possibly have taken 


5 Loeb, J., Investigations in physiological morphology. III. Experiments on 
cleavage, J. Morph., 1892-93, vii, 253. Norman, W. W., Segmentation of the 
nucleus without segmentation of the protoplasm, Arch. Entwcklngsmechn. Organ., 
1896, iii, 106. Wilson, E. B., Experimental studies in cytology. I, ibid., 1901, 
xii, 529. Lillie, R. S., Fusion of blastomeres and nuclear division without cell 
division in solutions of non-electrolytes, Biol. Bull., 1902-03, iv, 164. 

7 Wilson, E. B., Experimental studies in cytology, II and III, Arch. Ent- 
weklngsmechn. Organ., 1902, xiii, 353. 


62 PROTOPLASMIC CONSISTENCY AND CELL DIVISION 


place in the unsegmented Cheloplerus embryos experimentally pro- 
duced by Lillie.® 

Experiment 4.—(Fig. 9.) Fig. 9 a to e depicts the case of an egg 
with the cleavage furrow just beginning in which the diagonal cut 
was incomplete so that the two pieces remained connected at one 
end of the cut. The original furrow persisted for a time during which 
it deepened considerably. 30 minutes after the cut had been made 


AAS 5.00 

Fic. 9. Effect on an Asterias ovum of a deep cut which did not persist. a, 
operation performed at 4.10 p.m. 0, c, and d show the egg respectively at 4.24, 
4.40, and 4.45 p.m. Both the cut and the cleavage furrow disappear together 
with a reversal of the ovum from a semisolid to a more fluid state. e, 5.00 p.m., 


the ovum has divided into four normal blastomeres. (The ovum developed into 
a normal embryo.) 


no sign of astral radiations were present and both the original seg- 
mentation furrow and the cut produced by the needle were being 
obliterated (Fig. 9 c and d). At 5 p.m. the egg had divided into 
four apparently normal blastomeres (Fig. 9 e) and was only slightly 


8 Lillie, F. R., Observations and experiments concerning the elementary phe- 
nomena of embryonic development in Chetopterus, J. Exp. Zool., 1906, iii, 153. 


ROBERT CHAMBERS 63 


behind the normal controls. By the next morning it had developed 
into a swimming blastula not to be distinguished from the normal 
controls. 

The obliteration of the cut and of the furrow is consequent to a 
reversal of the egg cytoplasm from a semirigid to a more fluid state. 
The film projecting into the egg gradually merges into the liquid 


Fic. 10. Successive stages of an Asterias ovum showing persistence of a puncture 
made below the first cleavage furrow as it is beginning to form. 


cytoplasm surrounding it and surface tension forces finally overcome 
the deformation of the egg. The egg now proceeded to divide into 
four blastomeres as in Experiment 3. 

Experiment 5—(Fig. 10.) This experiment demonstrates a pecu- 
liar property of the equatorial region during the formation of the 
cleavage furrow. A tear was made through the egg below the seg- 
mentation furrow (Fig. 10 a). The hole produced by the tear 


64 PROTOPLASMIC CONSISTENCY AND CELL DIVISION 


remained open. The cleavage furrow continued its course beneath 
the hole leaving an outer margin as a bridge of protoplasm which 
connects the two blastomeres (Fig. 10 6, c). After several divisions of 
the egg the bridge thinned down in its middle until it broke through 
and the resulting strands were gradually drawn into the blastomeres 
from which they had projected. 


Cina 


6.00 6.12 7.10 


8.00 410.00 


Fic. 11. Effect on an Arbacia ovum of a deep cut which persisted. For des- 
cription of the results see text. Pigment granules collect in plane of original 
furrow. 


Experiment 6.—(Fig. 11.) This experiment was performed on an 
Arbacia egg. An incomplete cut was made almost perpendicular to 
the cleavage furrow but to one side of the daughter nuclei. The furrow 
on the side away from the daughter nuclei became obliterated (Fig. 
11a). On the other side it continued its original course resulting in 
the pinching ofi of the nucleated Blastomere a (Fig. 11 6). The 
nucleus in the remainder of the egg shifted its position only slightly and 


ROBERT CHAMBERS 65 


the amphiaster (Fig. 11 c), forming about it, resulted in a second un- 
equal cleavage with the formation of Blastomere 6 (Fig. 11 d). The 
projecting piece of the egg above the obliterated furrow remained 
quiescent during these divisions and not until after the third unequal 
cleavage resulting in the formation of Blastomere y in Fig. 11 e, did 
it become incorporated in Blastomere 6. 

In this experiment the cut was probably made in the egg when the 
process for the first cleavage was too far advanced for the egg to 
retrace its course. The gash was therefore not obliterated and a 
very peculiar condition resulted in a succession of advances of the 
cleavage process about the gash. Blastomere a, being the earliest 
formed, segmented ahead of its fellows (Fig. 11 d). Blastomere 8 
came next (Fig. 11 ¢). Unfortunately before Blastomeres y and 6 
divided the egg died. 

It is significant that Blastomere 6 is larger than y as evidently the 
former finally incorporated the hitherto inactive part of the egg that 
lay above that part of the original first cleavage furrow which lay on 
the right side of the gash (Fig. 11 0). 


III. Concerning the Mechanism of Cell Division. 


The changes in shape that an echinoderm egg undergoes during 
cleavage can be in part understood on the assumption that the astral 
formation is a solidifying process. It has long been known that at 
the time of cleavage the eggs of echinoderms, many worms, mammals, 
etc., become elongated,’ the cleavage furrow forming in a plane at 
right angles to the long axis of the egg. As the furrow deepens, each 
resulting blastomere tends to assume the shape of a sphere (Fig. 12 a). 

Nobody, however, has thus far been able to explain the cause of 
this elongation. The observations recorded in this paper may ex- 
plain this phenomenon. The two spheres of solidification grow at 
the expense of all but possibly a small peripheral part of the fluid 
egg substance. The combined diameters of the two fully formed 
semisolid spheres are greater than the original diameter of the egg, 


® Hertwig, O., Beitriige zur Kenntniss der Bildung, Befruchtung und Theilung 
des thierischen Eies, Morph. Jahrb., 1876, i, 347. Gurwitsch, A., Morphologie 
und Biologie der Zelle, Jena, 1904. 


66 PROTOPLASMIC CONSISTENCY AND CELL DIVISION 


and hence the egg must elongate. After elongation the surface of 
the egg seems to tear in the plane separating the two semisolid spheres. 
The periphery of the two asters of the amphiaster stage never becomes 
so firm as their interior. This may account for the observation of 
von Erlanger,!° confirmed by Spek,'! who described peripheral currents 
in the rapidly dividing nematode egg. In this egg peripheral currents 
flow from the two poles toward the equator and from there inward 
to the center of the egg. Spek suggests that such currents exist in all 
dividing eggs, and that they are easily visible in the nematode egg 
because of the great rapidity with which it segments. Conklin” de- 
scribed an inward flow of granules at the equator of the dividing Crepid- 


Fic. 12. Change in shape of an A sterias ovum (a) before and (6) after completion 
of the first cleavage furrow. 


ula egg, and I3 have observed a similar current, although a very slow 
one, in the sand-dollar egg. 

Immediately after cleavage both of the two blastomeres are more 
or less spherical; but later, when they become more fluid, they are 
pressed against each other so as to be flattened at the plane of contact. 


10 von Erlanger, R., Beobachtungen tiber die Befruchtung und ersten Teilungen 
an den lebenden Eiern kleiner Nematoden, Biol. Centr., 1897, xvii, 152, 339. 

1 Spek, J., Oberflichenspannungsdifferenzen als eine Ursache der Zellteilung, 
Arch. Entwcklngsmechn. Organ., 1918, xliv, 5. 

12 Conklin, E. G., Protoplasmic movement as a factor of differentiation, Marine 
Biol. Lab., Biol. Lect., 1899, 69. 


ROBERT CHAMBERS 67 


Wilson,’ in producing binucleate eggs by artificially obliterating 
the first cleavage furrow, noted that when this was caused by shaking, 
the resulting binucleate eggs retain the elongated shape (Fig. 13) 
characteristic of the egg in cleavage. During the ensuing pause 
(corresponding to the completion of the first cleavage and when 


Fic. 13. Copy of Fig. 58 from Wilson’ of Toxopneustes ovum immediately after 
shaking which caused obliteration of the first cleavage furrow. 


Fic. 14. Copy of Fig. 34 from Wilson’ of Toxopneustes ovum in which obliter- 
ation of the first cleavage furrow was produced by exposure to ether. 


the astral radiations fade out preparatory to formation of a new 
amphiaster system) the egg becomes more nearly spherical. Evidently 
the shaking does not necessarily produce a reversal of the semisolid 
astral system to the more fluid state. As soon, however, as this 
occurs (in the ensuing pause) the egg resumes its spherical shape. 


68 PROTOPLASMIC CONSISTENCY AND CELL DIVISION 


Wilson noted that the suppression of the cleavage furrow can also 
be produced by placing eggs, during their anaphase stage, in a 2.5 
per cent ether solution. The astral radiations disappear and the 
resulting binucleate egg at once resumes the shape of a sphere (Fig. 14). 
This phenomenon may be comparable to the experiments illustrated 
in Figs. 6, 8, and 9 where the obliteration of the astral radiations 
follows a precocious reversal of the cytoplasm to the more fluid state. 
The suppression of the furrow in these cases seems to be primarily 
efiected by the change in the physical state of the egg substance 
which, on reverting to a more fluid state, merges into a single spherical 
mass. 


CONCLUSIONS. 


1. The development of the amphiaster is associated with the for- 
mation of two semisolid masses within the more fluid egg substance. 

2. The elongation of the egg during cleavage is possibly produced 
as a consequence of the mutual pressure of these two growing semi- 
solid masses. 

3. The division of the egg into two blastomeres consists essentially 
in a growth, within the egg, of two masses of material at the expense 
of the surrounding cytoplasm. When all the cytoplasm of the egg 
is incorporated in these two masses cleavage occurs. 

4. After a certain period of time the semisolid masses revert to a 
more fluid state. In the eggs studied this normally occurs after the 
cleavage furrow has completed the separation of the two blastomeres. 
The formation of the furrow, however, may be prevented in various 
ways, upon which the egg reverts to a single spherical semifluid mass 
containing two nuclei. 

5. An egg-mutilated during its semisolid state (amphiaster stage) 
may or may not revert to a more fluid state. If the more solid state 
is maintained, the cleavage furrow persists and proceeds till cleavage 
is completed. If the mutilation causes the egg to revert to the more 
fluid state the furrow becomes obliterated and a new cleavage plane 
is subsequently adopted. 

6. The nuclei of eggs in the semifluid state are able to alter their 
positions. In semifluid mutilated eggs the nuclei tend to move to 
positions which may assure symmetry in aster formation and cleavage. 


SEcTION IV, 1918 (41] TrRAns. R.S.C. 


‘ 


A Report on Results Obtained from the Microdissection of Certain Cells. 


By RoBert CHAMBERS, CORNELL UNIVERSITY MEDICAL COLLEGE, 
NEw York Ciry. 


Presented by J. Playfair MeMurrich, F.R.S.C. 
(Read May Meeting, 1918.) 


INTRODUCTION. 


Cytological research has hitherto been confined largely to observa- 
tion through the microscope ‘at a distance’ as it might be said. It 
has been clear to investigators that such a method may lead to errone- 
ous conclusions and it is true that a great deal of confusion has resulted 
from misinterpretations of optical appearances and the description 
of artifacts as if they existed normally in the cell. This confusion 
is largely responsible for the fact that the true significance of cell 
anatomy is in danger of being ignored by many physiologists. 


As long ago as 1859, Doctor H. D. Schmidt of Philadelphia, 
attempted to dissect cells by means of a ‘microscopic dissector,’ 
consisting of a base to be fastened on the stage of a microscope with a 
number of clamps to hold instruments, each clamp possessing three 
movements controlled by screws. A lever fastened in one of the 
clamps holds the tissue in place. Fine scissors, knives or steel needles 
are fastened in the other clamps. By turning the various screws, the 
instruments can be brought into place and be operated with remark- 
able accuracy. Doctor Schmidt worked with the tissue, the instru- 
ments and the lower lens of the objective immersed in water or diluted 
alcohol. 

The principle introduced by Schmidt, viz., the use of screws 
to control movements of instruments lying in the focus of a micro- 
scope objective seems to have been for a long time lost sight of. It 
was revived in 1907 and elaborated in 1914 by M. A. Barber, lately 
of the University of Kansas, in his construction of an instrument to 
manipulate micro-pipettes. With this instrument Barber was able 
to isolate single micro-organisms and to inoculate living cells with 
bacteria. Barber’s instrument was soon applied to the dissection of 
cells (Kite and Chambers '12) and a new field of endeavor was 
opened for the study of the structure of protoplasm and cell mechanics. 


42 THE ROYAL SOCIETY OF CANADA 


THE INSTRUMENT. 


The apparatus used in cell dissection is shown in the accom- 
panying figure. The moist chamber, which is open at one end and 
with sides from 8 to 12 mm. high, is placed on the microscope so that 
it may be moved about with the mechanical stage. The chamber is 
roofed over with a specially cleaned coverslip, on the under surface 
of which, the specimen is mounted in a hanging drop of Ringer’s or 
lymph fluid and held in place by surface tension. The dissecting 
needle is made by drawing out one end of a piece of hard glass tubing 
which is then bent at right angles, two or three millimeters from the 
pointed tip. The needle-holder, a mechanism allowing of three 
movements, is clamped to one side of the miscroscope stage, and the 
needle is adjusted so that.it projects into the moist chamber with its 
tip pointing up into the hanging drop. By proper adjustment the 
cell to be dissected and the point of the needle can be brought into 
the same focal field. The three movements of the needle permitted by 
the needle-holder and the two movements of the moist chamber by 
the mechanical stage give the experimenter ample opportunity to 
carry on dissection under the highest magnification of the microscope. 
The dissecting needle-points can be made stiff and yet so fine that 
their size bears about the same relation to that of a human red blood 
corpuscle as an ordinary knitting needle does to the palm of the hand. 

Through the courtesy of the Biological Board of Canada I was 
given in July 1917 the opportunity of continuing some microdissection 
work at the Atlantic Biological Station, St. Andrews, N.B. I haye to 
thank Dr. Clara C. Benson for allowing me to take some material from 
lobsters she was using for experimental purposes. 


EXPERIMENTAL. 


The ganglion cells of the Lobster. The ventral nerve cord was laid 
bare and pieces of a nerve ganglion excised and placed on a thin 
coverslip in a drop of lobster blood serum. This liquid is expressed 
during a preliminary clotting of the blood and does not itself clot 
for a considerable length of time. The nerve cells are carefully 
isolated by teasing with needles under an ordinary dissecting micro- 
scope. The coverslip is then inverted and placed on the moist chamber 
so that the nerve cells lie ina hanging drop ready for microdissection. 
The cell bodies lie among the closely interlaced nerve and neuroglia 
fibers. When the fibers are torn away, the cell body may be isolated 
with ease. : 

The cell cytoplasm is very viscid in consistency and allows of 
considerable tearing without disintegrating. Highlyrefractive spindle- 


[CHAMBERS] MICRODISSECTION OF CELLS 43 


shaped bodies, the mitochondria, imbedded in the cytoplasm are 
very prominent. The cytoplasm is very extensile and exhibits a 
certain amount of rigidity throughout its substance. It can be pulled 
out into long, viscous threads and the imbedded mitchondria are 
drawn in the direction of the pull. 


Gide to side 
moye ment 


Up and down 
zmeve ment 


Figure 1. Barber’s Three-Movement Pipette Holder, Glass Needle 
and Moist Chamber arranged to illustrate method of dissecting 
cells in a hanging drop under the highest magnification of the 
microscope. (Substage of microscope omitted in drawing.) 


On exerting a pull on the cytoplasm in a direction away from the 
nucleus, a triangular space appears in front of the nucleus and persists 
fora few minutes. This is due to the viscosity of the cytoplasm which 
prevents an even flow of material around the nucleus as should occur 
if the cytoplasm were liquid. 

There is a limit to the amount of mechanical injury which the 
cytoplasm can bear without completely changing its normal prop- 
erties. When this limit is passed the viscid plasma sets into a 
coagulated, non-viscous mass which may be broken into non-glutinous 
pieces. 


44 THE ROYAL SOCIETY OF CANADA 


The cell nucleus is an optically hyaline sphere occupying about 
one-fourth of the cell. Within the hyaline substance of the nucleus 
lies a small body, the nucleolus, which is visible because of its high 
refractivity. - 

The extremely sensitive nature of the nucleus is evidenced by the 
fact that, on the slightest mechanical injury, certain changes occur 
which cause the nucleolus to fade completely from view. The nucleus 
may be pushed about in the cell without apparent injury. If the 
surface be torn the contents flow out and the nucleus disappears. 
If care be taken the nucleus may be cut in two, each portion at once 
assuming the shape of a sphere. This indicates a high power of 
extensibility and regeneration in the surface film. On pulling the 
nucleus out of the cell the nucleus immediately begins’ to swell and 
fades from view. 

The Egg Cell of the Flounder. The immature egg of the flounder 
of about half a millimeter or less in diameter was selected for this 
study. The nucleus is a liquid sphere similar to that of the nerve cell. 
It, however, possesses a more persistent surface film or nuclear mem- 
brane. This may be caught by the tipof the needle and a consider- 
able strand pulled out. The nucleus is easily cut into several pieces 
which immediately round up. On touching one another the portions 
fuse indicating the absence of a morphologically persistent nuclear 
membrane. 

Considerable injury is necessary to bring about dissolution of 
the nucleus and the surface film is the last to disappear. Injury 
apparently causes this film to set into a definite membrane, so that 
when torn it often wrinkles and a fluid (apparently the nuclear sap) 
collects between the cytoplasm and the partially collapsed membrane. 

The flounder egg is surrounded by a closely fitting tough egg 
membrane. This rather interferes with an adequate comprehension 
of the consistency of the cytoplasm, especially of that on the cell 
surface. Results obtained from studies made in other marine ova 
are more satisfactory. 

The egg cell of Asterias. Work done at St. Andrews on Asterias 
confirms the views already published (Chambers ’17*, 17°) on the cell 
protoplasm of Echinoderm ova. The protoplasm consists of a hya- 
line fluid matrix in which are imbedded granules of various sizes. 
The fluid offers no perceptible resistance to the needle and an indica- 
tion of its very slight viscosity lies in the fact that, when the needle is 
moved through the fluid, the only granules displaced are those in the 
immediate vicinity of the needle. The protoplasm coagulates with 
ease. Mere compression will sometimes cause an egg to coagulate into 
a solid mass. 


[CHAMBERS] MICRODISSECTION OF CELLS 45 


The surface layer of the egg cell is dense in consistency when 
compared with the cell interior into which it merges insensibly. In 
the unfertilized egg, the cell granules are imbedded in it up to the very 
line of division between the egg and surrounding medium. With the- 
needle the surface may be pulled out into long strands without other- 
wise disturbing the contour of the cell. On being released the strands 
tend to curl and retract slowly till they disappear. If a more rapid 
tear be made, and if the cell be under compression, the spot torn 
bulges out as the internal cytoplasm presses on the weakened surface. 
The surface layer of the swelling protuberance is very easily broken, 
upon which the interior may pour out. The cytoplasm then either 
disintegrates entirely in the surrounding water or, if remaining normal, 
reestablishes a film on its surface. When left undisturbed the new sur- 
face film gradually strengthens into a definite ectoplasmic layer and the 
protuberance slowly retracts until the original contour of the egg is 
reestablished. If the point of attachment of the protuberance be 
small, the protuberance may be pinched off to form a spherule of 
cytoplasm which to all appearances is normal. 

In summary, we may say that the surface layer is a highly exten- 
sile, contractile and viscous gel capable of constant repair. Its 
establishment and maintenance is a property essential to protoplasm. 
With the film intact the mass of protoplasm maintains itself and the 
life of the cell is assured. When the film is destroyed the cytoplasm 
flows out, the cell granules swell and disappear, the whole mass com- 
pletely disorganizes and disappears in solution in the surrounding water. 

The egg cell of Solaster. The Solaster egg is very large when 
compared with other Echinoderm eggs, being well over 1 mm. in dia- 
meter. This is partly due to the fact that it is heavily laden with 
yolk. The nucleus, however, is also very large, so large in fact that 
it is visible to the naked eye and can be easily isolated with needles 
under an ordinary dissecting microscope. Its enveloping surface 
film exhibits a distinct resistance to compression. Tearing the surface 
allows the fluid contents to escape and the nuclear wall collapses. 
The Solaster egg appears to be the only case on record of a Metazoon 
cell of which the nucleus is large enough to be actually handled and 
dissected with ordinary needles. 


CONCLUSION 


In conclusion, one may make the following statements with 
regard to the consistency of the living cells which were dissected: 

1. The cytoplasm of an egg cell consists of a semi-liquid interior 
enclosed in a jelly-like and highly viscous surface layer. The surface 


46 THE ROYAL SOCIETY OF CANADA 


layer is very extensile and contractile and is readily regenerated upon 
injury. Tearing of this surface, if unrepaired, results in the pouring 
out of the internal cytoplasm and dissolution. 

2. The cytoplasm of the nerve cell exhibits, throughout its sub- 
stance, the properties of the surface layer of the egg cell, viz., it con- 
sists of a highly viscous, extensile, jelly-like hyaline substance. 


3. The resting nucleus of all the cells studied is a liquid sphere, 
the external surface of which may form a more or less temporarily 
rigid membrane. 


4. The production and maintenance of a limiting membrane 
appears to be one of the properties essential to protoplasm. 


LITERATURE. 
BARBER, M. A. 


1914. The pipette method in the isolation of single micro-organisms and 
in the inoculation of substances into living cells. The Philippine Jour. of 
Sc., vol. 9, Sec. B., Tropical Medicine, p. 307. . 


CHAMBERS, ROBERT. 


1917a. Microdissection Studies, I. The visible structure of cell proto- 
plasm and death changes. Am. Jour. Physiol., vol. 43, p. 1. 

1917b. Microdissection studies, II. The cell aster, a reversible gelation 
phenomenon. Jour. Exp. Zool., vol. 23, p. 483. 

1918. The microdissection method. Biol. Bull., vol. 34, p. 121. 


Kite, G. L. AND ROBERT CHAMBERS. 


1912. Vital Staining of chromosomes and the function and structure of 
the nucleus. Science, N.S., vol. 36, p. 639. 


ScumiptT, A. D. 


1859. On the minute structure of the hepatic lobules, particularly with 
reference to the relationships between the capillary bloodvessels, the 
hepatic cells, and the canals which carry off the secretion of the latter. 
The Amer. Jour. of the Med. Sci., N.S., vol. 37, p. 2. 


SEcTION IV, 1918 [145] TRAns. RS.C. 


A Report on Cross Fertilization Experiments, 
(Asterias x Solaster) 


ROBERT CHAMBERS AND BEssIE Mossop 


Presented by J. P. McMurrich, Ph.D., F.R.S.C. 
(Read May Meeting, 1918) 


During the first two weeks of August, 1917, a number of adult 
Solaster endeca (Forbes) were obtained by dredging over a rocky 
reef in the vicinity of Joe’s Point near the Biological Station, St. 
Andrews, N.B. At this time mature Asterias forbesii (Desor) were 
to be had at St. Andrews. The extent of the breeding season of 
Asterias in the vicinity of St. Andrews may be estimated from the 
occurrence of Bipinnaria in the plankton of Passamaquoddy Bay, 
which is being investigated by Professor J. P. McMurrich. During 
1916 the first Bipinnaria observed occurred on July 20th. They 
were present in all subsequent tows till August 31st. In 1917, none 
appeared till August 8th, after which date they appeared throughout 
the remainder of the month. 

The Asteriide and the Solasteride are comparatively closely 
related families, both belonging to the order Cryptozonia in the 
Asteroidea. The Solaster possesses a heavily yolk-laden ovum (1 mm 
or over in diameter) which undergoes a somewhat incomplete meta- 
morphosis, the free swimming larva not having a completely formed 
alimentary tract. The Asterias, on the other hand, undergoes com- 
plete metamorphosis, the larval form being a typical Bipinnaria. 

Because of this and because of the fact that the spermatozoa of 
the two species are very much alike, although the ova are very dis- 
similar in size, the possibility suggested itself that interesting results 
may arise from attempts at cross-fertilizing these two species. 

Among the specimens of Solaster procured, the females contained 
large numbers of apparently mature ova. Repeated attempts at 
fertilizing them with spermatozoa of their own species as well as with 
those of Asterias proved unsuccessful. The breeding season of 
Solaster, according to Gemmill,! is normally in March or early 
April, at least for those on the British Coast. This may account for 
our failure with the Solaster eggs so late as July and August. On the 


1912, Gemmill, J. F., The Development of the Starfish, Solaster endeca, Forbes, 
Transactions Zoological Society, London, Vol. 20, p. 1. 


. 
; 
. 


_ 


SEcTION IV, 1918 [145] Trans. R.S.C. 


A Report on Cross Fertilization Experiments, 
(Asterias x Solaster) 


ROBERT CHAMBERS AND BEsSIE Mossop 


Presented by J. P. McMurrich, Ph.D., F.R.S.C. 
(Read May Meeting, 1918) 


During the first two weeks of August, 1917, a number of adult 
Solaster endeca (Forbes) were obtained by dredging over a rocky 
reef in the vicinity of Joe’s Point near the Biological Station, St. 
Andrews, N.B. At this time mature Asterias forbesii (Desor) were 
to be had at St. Andrews. The extent of the breeding season of 
Asterias in the vicinity of St. Andrews may be estimated from the 
occurrence of Bipinnaria in the plankton of Passamaquoddy Bay, 
which is being investigated by Professor J. P. McMurrich. During 
1916 the first Bipinnaria observed occurred on July 20th. They 
were present in all subsequent tows till August 31st. In 1917, none 
appeared till August 8th, after which date they appeared throughout 
the remainder of the month. 

The Asteriide and the Solasteride are comparatively closely 
related families, both belonging to the order Cryptozonia in the 
Asteroidea. The Solaster possesses a heavily yolk-laden ovum (1 mm 
or over in diameter) which undergoes a somewhat incomplete meta- 
morphosis, the free swimming larva not having a completely formed 
alimentary tract. The Asterias, on the other hand, undergoes com- 
plete metamorphosis, the larval form being a typical Bipinnaria. 

Because of this and because of the fact that the spermatozoa of 
the two species are very much alike, although the ova are very dis- 
similar in size, the possibility suggested itself that interesting results 
may arise from attempts at cross-fertilizing these two species. 

Among the specimens of Solaster procured, the females contained 
large numbers of apparently mature ova. Repeated attempts at 
fertilizing them with spermatozoa of their own species as well as with 
those of Asterias proved unsuccessful. The breeding season of 
.Solaster, according to Gemmill,! is normally in March or early 
April, at least for those on the British Coast. This may account for 
our failure with the Solaster eggs so late as July and August. On the 


1912, Gemmill, J. F., The Development of the Starfish, Solaster endeca, Forbes, 


_ Transactions Zoological Society, London, Vol. 20, p. 1. 


146 THE ROYAL SOCIETY OF CANADA 


other hand, the breeding season of Solaster may be much later at 
St. Andrews than in Great Britain (as is the case with a number of other 
forms) and the sperm may have ripened earlier than the ova, as was 
observed in the case of Asterias in 1917. 

The Asterias sperm and ova were normal and ripe. The ova. 
matured within 30-40 minutes after being placed in sea water. As 
the spermatozoa were rather sluggish, a few drops of ammonia were 
added to the water. Sperm thus ‘treated became very motile, and 
when added to the ova of its own species induced a development of 
from 90 to 100 per cent. 

On July 22, 1917, at least two weeks before Bipinnaria were 
found in the plankton, male Asterias were procured whose testes 
were swollen and large and had every appearance of being mature. 
The spermatozoa, however, when introduced into sea-water remained 
motionless. On adding enough ammonia to the water to raise the 
NH; concentration to 0-0055 grams in 100 c.c., the sperm became 
motile and capable of fertilization. A lowering of the concentration 
to 0-0045 grams in 100 c.c. stopped all movement. Fresh sea water 
was found to contain normally 0-002 per cent NH;. Later in the 
season (early August) spermatozoa were active in normal sea water. 
It is known that increase in the hydrogen ion concentration will inhibit 
the movement of spermatozoa and cilia. Possibly the testis is acid 
owing to the presence of a relatively great amount of COs produced 
in the active metabolism of the developing sperm. The mature 
sperm would thus be existing in an anesthetized condition. The 
addition of ammonia neutralizes the inhibiting effect of the CO: and 
renders the mature sperm motile. In a fully ripened testis, the de- 
veloping process has largely ceased and the acidity has had a chance 
to be dissipated. For such a testis the alkalinity of normal 
sea water is sufficient to activate the spermatozoa. The Solaster 
testes, according to this assumption, were not fully ripe, since the 
spermatozoa of Solaster were quite motionless in sea water and 
were activated only by being placed in alkaline sea water. 

Solaster spermatozoa, which had been thus activated, were 
poured into bowls containing mature Asterias ova. In one and one 
half hours fertilization membranes appeared in about 20 per cent 
of the eggs. Their polar bodies were all outside the fertilization 
membrane. In the case of the control (Asterias sperm x Asterias 
ova) the polar bodies were all inside the fertilization membrane. 

The difference in the position of the polar bodies is 
due the difference in time of the initiation of fertilization. 
In the cross-fertilized ova this is delayed well beyond an hour during 
which the polar bodies are extruded. When the membrane forms it 


[ CHAMBERS-MOsSopP] REPORT ON CROSS FERTILIZATION 147 


lifts up from the surface of the ovum and pushes the polar bodies 
ahead of it. In the self-fertilized ova the membrane forms within a 
few minutes, the polar bodies are produced later and, therefore, come 
to lie within the membrane. 

Development proceeded regularly in the cross-fertilized ova, but 
more slowly than in the self-fertilized Asterias ova. Six hours after 
mixing the sperm with the ova the Solaster sperm x Asterias ova had 
developed into the 16-cell stage while the Asterias sperm x Asterias 
ova had developed into the 32-cell stage and some even into later 
stages. Except for this delay in rate and for the very much de- 
creased percentage of developing eggs, no difference was discernible 
on comparing the living self and cross-fertilized Bipinnaria. 

To exclude the possibility that the alkalinized water alone might 
have caused the development, Asterias ova were placed in alkaline 
water without sperm. None developed. Also, to exclude the pos- 
sibility of Asterias sperm being present with the Asterias ova before 
introducing the Solaster sperm, all. the water used was carefully 
heated to boiling point and then cooled. The starfish used were 
thoroughly rinsed in such water before removing the gonads. 

We know that fertilization involves two sharply separated proc- 
esses; first, an impetus which activates the hitherto quiescent egg 
so that it will segment and undergo embryonic development; and 
second, a fusion of the male pronucleus, introduced into the egg by 
the spermatozoon, with the female pronucleus, a part of the original 
nucleus of the egg. The second process involves the inheritance, in 
the offspring, of the paternal and maternal characters. 

The first process has been independently produced in the labor- 
atory by a variety of so called ‘‘parthenogenetic”’ agents. The Asterias 
ova are very easily induced to this sort of development. The appli- 
cation of heat and mere shaking occasionally suffices to start cortical 
changes resulting in the throwing off of a fertilization membrane 
followed by segmentation. It is possible that the Solaster sperm 
may have acted on the Asterias eggs only in so far as to induce them 
to parthenogenetic development. This would explain the purely 
maternal appearance of the larve resulting from the cross. Further 
investigation of this problem is being conducted. 


Sec. 1V, Sig. 11 


REPRINTED FROM 


ANNALS OF SURGERY 


227 South Sixth Street, Philadelphia, Penna. 
Marcu, 1918. 


REGENERATION OF BONE 
By Apert A. Bere, M.D. 


SURGEON TO MT. SINAI HOSPITAL, NEW YORE CITY 
AND 
Wiiuiam TrHaruimer, M.D. 


ASSISTANT IN THE PATHOLOGICAL LABORATORY OF MT. SINAI HOSPITAL, NEW YORK CITY 


(From the Anatomical Department of Cornell University Medical College, 
New York City) 


INTRODUCTION 


THE transplantation of bone is one of the very important branches of 
reparative and conservative surgery. The cardinal principles governing the 
successful transplantation of bone were laid down by Ollier, but there still 
are divergent views both as to the best technic for bone transplantation and 
the vital processes by means of which the reproduction of bone takes place. 

The reproduction or regeneration of bone is the basic phenomenon upon 
which a successful transplantation depends, and it is in the hope of shedding 
more light upon this process that the experiments recorded below were per- 
formed by the authors. 

A summary of the normal development, growth, and structure of bone 
along with a brief review of the literature on bone transplantation will be 
given before proceeding to the results of our work. 

There are two types of bone found in man, cartilage bone which develops 
on a cartilaginous basis, and membrane bone, which develops directly from 
connective tissue without an intervening cartilaginous stage. The mode 
of development of cartilage bones only, and not of membrane bones, will be 
described here, because most of the skeleton is composed of bones of this 
type and most of the experimental research on transplantation has been done 
with cartilage bones. Previous experiments have yielded divergent results 
both as to the fate of the transplanted bone and the regenerative power of 
its various constituents. 


THE DEVELOPMENT, GROWTH, AND STRUCTURE OF CARTILAGE BONE + 


A description of the development of one of the long bones will serve as 
an illustration of the embryonic development of cartilage bones. 

The site which is later occupied by a mature, fully developed bone is first 
filled in by embryonic connective tissue with closely packed cells. This 
tissue is transformed into cartilage. The cartilage is non-vascular, but is 
surrounded by a vascular, fibrous membrane, the perichondrium, which de- 
velops into periosteum. 


This description is taken in the main from Cunningham’s “Text-book of 
Anatomy,” Fourth Edition; and Quain’s “Anatomy,” vol. ii, Part 1, “A Text-book 
of Microscopic Anatomy,” by E. A. Schafer. 


331 


BERG AND THALHIMER 


Next, two changes occur simultaneously: (1) The cells at the centre of 
the cartilage become enlarged and flattened and pile up in longitudinal 
columns radiating from the centre towards the ends, and the matrix becomes 
hardened by a granular calcareous deposit. (2) The cells of the inner lining 
of the perichondrium assume a flattened or cuboidal shape and become osteo- 
blasts, constituting the osteogenic or cambium layer, and form a bony layer 
on the surface of the cartilage. 

Bone is formed from osteoblasts by the deposition of inorganic material 
about each cell. The osteoblasts are thereby included in small spaces or 
lacune and are then called young bone cells. These cells are not surrounded 
by a solid calcified wall, but small channels called canaliculi, which arise 
from the lacune, radiate irregularly through the surrounding matrix, and 
anastomose with canaliculi from neighboring lacune. The cells within these 
spaces also send filamentous processes into these canaliculi, thereby giving 
the cells a spidery appearance. 

The next step is a migration of the subperiosteal vascular and osteo- 
blastic tissue into the centre of the cartilage, followed by an absorption of 
the calcified cartilage and by formation of marrow spaces. These spaces 
are filled by jelly-like embryonic marrow and are lined by osteoblasts. This 
lining of osteoblasts is known as endosteum, but it should be noted that it 
arises from periosteum. Bone is deposited around these marrow spaces by 
an advancing line of osteoblasts. The osteoblasts follow and surround the 
blood-vessels and deposit bone in layers about them. As layer after layer of 
bone accumulates about the vascular structures, ramifying, communicating 
trabeculz of bone are formed, and the large spaces between them become 
narrowed into intercommunicating channels. While this process is taking 
place layers of bone are also being deposited beneath the periosteum, but 
this cortical deposit is penetrated by numerous blood-vessels which com- 
municate with those in the medulla. 

At about this time large cells, which are usually multinuclear, appear 
along the edges of the bone which has been formed. Their action is the 
absorption of bone and they are termed osteoclasts. In performing their 
function they excavate hemispherical pits known as foveole of Howship. 

Bone absorption is necessary for the growth of bone. The absorption 
occurs most actively in the centre, or medulla, of the bone, though to some 
extent it also occurs in the cortical portion. By this means the marrow 
spaces which have, in the meantime, been filled with cancellous bone are 
reformed as the secondary marrow space. The cortical bone is being re- 
moved from the centre, and while this is occurring bone is being deposited 
in lamellz upon the outer cortical surface, thus bringing about lateral growth 
or increase in width. Growth at the ends occurs by an advancing deposit of 
bone in the growing cartilage. 

At about this stage additional centres of ossification develop in each end 
of the bone. These are called epiphysial centres and are situated between 
the end, or epiphysis, and the shaft, or diaphysis. The epiphysial line is 
formed by blood-vessels or vascular loops advancing into the epiphysial carti- 

332 


REGENERATION OF BONE 


lage and carrying with them endosteum or osteoblasts. This osteogenetic 
tissue then becomes completely separated from the osseous shaft by a portion 
of cartilage which remains. This cartilage persists in some places until after 
puberty, and continually grows, and by being continuously replaced by bone 
causes the longitudinal growth of bone. 

The intercommunicating channels, referred to as arising in the marrow 
spaces and in the bone deposited by the periosteum, are lined by osteoblasts 
and deposit concentrically at their periphery successive lamellz of bone. By 
this process the channels become reduced to very narrow canals, called 
Haversian canals. These Haversian canals therefore contain blood-vessels 
and are lined by osteoblasts. The systems of concentric bone lamelle are 
joined together by irregular systems of lamelle. Upon the surface of the 
shaft, completely around all these systems, additional concentric lamelle are 
deposited beneath the periosteum by the cambium, or osteogenic layer of 
periosteum. 

Eventually the marrow spaces become converted into a central canal 
filled with mature marrow * and containing only a few trabecule of bone. 
Around this central canal is a layer of interlacing bone trabecule forming 
cancellous bone, and all of these trabecule are covered completely by a layer 
or layers of osteoblasts. Outside of this is found the compacta, or cortex, 
the Haversian systems and lamellz of which have been described above. The 
surface is covered by the osteogenic or cambium layer of osteoblasts, which 
can be considered as lying between the cortex and the fibrous periosteum, or, 
one might say by way of comparison, that the cambium layer lies between 
the tree and its bark. It is this similarity which has given rise to the name 
cambium layer of the periosteum. 

The blood supply of the bone is furnished (1) by numerous minute 
nutrient arteries (mentioned above) which penetrate the cortex from the 
periosteum through the Haversian canals; (2) by one or more larger nutrient 
arteries which penetrate the cortex usually at about the centre of the 
shaft and enter the medullary canal. 

The important points to be emphasized are: 

(1) All cartilage bone is produced by cells arising from the osteoblasts 
lining the periosteum and is deposited in preformed cartilage, the latter being 
absorbed. 

(2) The endosteum is formed of osteoblasts which arise from those 
lining the periosteum, and osteoblasts also extend from the endosteum 
and the osteoblastic (cambium) layer of the periosteum into the Haversian 
canals and line them. 

(3) Cartilage which is about to be ossified undergoes certain changes, 
among which is an enlargement and flattening out of its cells, with their 
arrangement into columns at right angles to the plane of bone growth. 

(4) Bone cells (not osteoblasts) are enclosed in bony lacunze which inter- 
communicate by means of canaliculi. 


? The development of marrow will not be discussed, as it has nothing to do with 
the growth of bone. 
333 


BERG AND THALHIMER 


(5) None of the bone cells are in immediate contact with blood-vessels 
or capillaries and their only nourishment must be plasma obtained through 
the canaliculi. 

(6) The above described method of bone growth is known as growth by 
absorption. 

In addition, several other things of importance may be mentioned. 
Young lacunar cells which have just been formed from osteoblasts may 
divide and form a very limited amount of bone. When bone develops in 
this manner immediately in apposition to older bone, the older bone may be 
absorbed and replaced by this young bone. The method by which bone is 
absorbed in this instance is not known, but it is not accomplished by the aid 
of osteoclasts. Apparently it occurs in some direct manner which appears 
somewhat like solution of the bony structure. This type of bone growth or 
bone substitution is called creeping replacement. 

Most bone, however, arises directly from osteoblasts. Fully developed 
bone cells, in the accepted sense of this term, that is, cells within well calci- 
fied lacune, have never been shown by microscopical observation to have 
divided and formed new bone. Undoubtedly most normal bone is formed 
by the endosteum and cambium layer of the periosteum and only to a less 
degree by osteoblasts lining the Haversian canals. 


REVIEW OF LITERATURE 


The earliest experimental work bearing upon bone transplantation was 
done by that great master of bone surgery, Ollier, in 1867. He did not make 
a histological study of his transplants, nor did he have the aseptic and anti- 
septic methods of operating that make the work of to-day so sure of suc- 
cessful issue. His conclusions on bone transplantation were all based upon 
macroscopical observation, and up to the point where microscopic study is 
necessary they have been found absolutely true. Briefly stated, they are as 
follows: 

1. For the transplantation of bone there is a fundamental difference in 
the use of autogenous, periosteum-covered grafts, on the one hand, and every 
other kind of bone material, on the other. 

2. Only the former manifests an increase of thickness after a rapid 
fibrovascular connection with its bed, and it is the increase of thickness which 
is a sure indication of continued vitality of the transplanted bone. 

A true graft of bone (i.e., a graft that retains its vitality) is possible only 
after transplantation of living, autogenous periosteum-covered bone, and 
this is in virtue of its living periosteal covering. This latter, after trans- 
plantation, remains alive, and thereby maintains the life of the transplanted 
bone. It is, therefore, the most important factor in bone transplantation. 

3. Every other kind of bone when transplanted dies, if it is not already 
dead at the time of its implantation. All of these varieties of transplants 
become foreign bodies, and either remain intact and encapsulated at the site 
of implantation, or they are resorbed, the latter process often being hastened 
by the blood-vessels which penetrate into them. If such material is im- 


334 


REGENERATION OF BONE 


planted in a bed that is itself bone-producing, it is possible, under favorable 
condition, for it to be replaced by new bone formed from this bony bed. 

4. Where it is desirable to restore bony continuity after the removal or 
destruction of bone, one must make use of living, autogenous, periosteum- 
covered bone grafts. 

These conclusions were accepted and remained classical until the work 
of Radzimowsky, 1881, and Bonome, 1885. Both these experimenters con- 
tended in contradistinction to Ollier that all transplanted bone tissue dies 
even when it is autogenous and covered by living periosteum, but that the 
periosteum lives. As proof of their contention they instanced the death 
of the bone cells as shown by the change of the latter in morphology and 
staining qualities. 

Radzimowsky demonstrated in the cranial and long bones of birds and 
mammals that bony union between bone fragments and adjacent bony tissue 
easily takes place irrespective of whether the periosteum is preserved or 
not, over such fragments. Such bony union, however, may be considered 
no evidence of enduring life in the fragments of bone, and further, the 
presence of blood-vessels in such fragments is no evidence of life therein, 
inasmuch as dead bone may also be permeated by blood-vessels from its vi- 
cinity. Microscopic examination of such bone fragments showed that they 
were dead, for the bone cells were dead. On the other hand, he demonstrated 
that the periosteum lived and produced new bone. Therefore, he concluded 
that when living periosteum-covered bone is transplanted, the bone tissue 
proper dies, but the periosteum lives and produces new bone that is deposited 
not only on to the surface of the transplanted dead bone, but also into its 
lacune and enlarged Haversian canals. 

Bonome, working with rats, reached conclusions similar to those of 
Radzimowsky, and also showed that when fracture of the bone occurred 
the bone in the immediate vicinity died, as was shown by the staining qual- 
ities of the bone cells. As to the ultimate fate of the dead bone he con- 
cluded that this is resorbed and replaced by new bone which is formed from 
the osteogenetic layer of the periosteum. 

The work of these two experimenters established, with the aid of micro- 
scopic study, the first great advance after the pioneer investigations. 

No new advances or contributions to the subject of bone transplantation 
were made until Barth, 1893, reported the results of his experiments. He 
likewise found that when autogenous, living, periosteum-covered bone is 
transplanted the bone tissue dies, and in addition he makes the important 
assertion that he could not convince himself that the periosteum or marrow 
fared any better than the bone tissue proper. He deduced from his experi- 
ments, which were performed on the skull, that the transplanted periosteum 
also died, and was replaced by the growth of periosteum from adjacent bone 
which extends over and covered the graft. He concluded, therefore, that 
inasmuch as all the parts of a living periosteum-covered bone graft died, it 
is immaterial whether we use, for transplantation purposes, bone covered 
or uncovered by periosteum, or decalcified or macerated bone. He thought 

335 


> 


BERG AND THALHIMER 


that the transplant was a sort of splint, to be gradually replaced by new bone 
formed from the surrounding bony tissue. Briefly stated, the conclusions of 
Barth were as follows: 

1. Fragments of any kind of bone like other foreign bodies can be im- 
planted into the living tissues. 

2. All varieties of bone material are similar in the process of their im- 
plantation and of their bony replacement. 

3. When living bone with periosteum is transplanted, all of its integral 
parts die. 

4. Therefore, all varieties of bony substance are foreign bodies at the 
outset, or become so, and are gradually replaced by new formed bone from 
the adjacent bone-producing tissue. 

These conclusions, radically different as they were from those of Ollier 
and his followers, were accepted, and for the next decade surgeons gave 
up altogether bone grafting and used macerated or decalcified bone or other 
foreign material to fill gaps in the continuity of the skeletal system. The 
lack of success of these procedures was brought out in the German surgical 
congress of 1902, when the subject of bone transplantation was brought up 
for discussion. It was the general opinion at this meeting that though the 
experimental conclusions of Barth might be true, the same did not hold in 
practice. The opinion expressed seemed to be unanimous—that the best 
results were obtained when living, autogenous, periosteum-covered trans- 
plants were used. Even Barth had to concede this in the light of his subse- 
quent experience. 

In order to gain more information on the subject Axhausen took up the 
work anew, and by his experiments and histological examinations placed 
the whole subject on a firm basis. His conclusions were as follows: 

1. The conclusions of Barth as to the relative equal value of all varieties 
of bony material for transplantation cannot be upheld. 

2. The first law of Ollier, namely, that there is a fundamental difference, 
as regards transplantation, between the use of living, autogenous periosteum- 
covered bone, and every other kind of material, is true in other animals as 
well as in the human subject. 

3. The difference lies not, as Ollier thought, in the survival of the life 
of the transplanted bone tissue proper, for most of this dies, only a few cells 
persisting, and is replaced by new bone, but exists in the periosteum which 
survives. 

4. This surviving periosteum produces the new bone. When the trans- 
plantation is made into a bone-producing bed the new bone formation from 
the periosteum is not marked, for this bed is alone sufficiently able to fill the 
gap in its substance. But in the case of transplantation into a defect of the 
long bones this periosteal new bone formation, together with that from the 
marrow, are the only means for filling the gap. 

5. The survival of the bone-producing periosteum established a rapid and 
intimate vascular connection between the transplant and its bed, and in 
virtue of its bone resorbing and bone forming power this surviving periosteum 

336 


REGENERATION OF BONE 


forms an intimate connection with the underlying transplanted dead bone. 
This is in marked difference to the loose fixation of the transplant in the 
tissues when bone uncovered by periosteum is used. 

6. The survival of the marrow has no dependence upon whether the 
graft is covered or uncovered by periosteum. 

These experimental conclusions were in accord with the empirical ones 
reached by practical surgeons, and since these classical labors of Axhausen 
the surgical world has accepted, until recently, the fundamental law of Ollier, 
namely, that for a real bone graft we must use an autogenous portion of bone 
covered by living periosteum. 

The truth of this law of Ollier has been disputed by William MacEwen 
in a monograph entitled “The Growth of Bone,” published in 1912. 

MacEwen claims that periosteum is merely a limiting membrane, which 
has not the capacity to form bone, but serves only to confine the bone within 
bounds and prevents its overproduction. He believes that bone has the power 
to reproduce itself, if it receives proper blood supply, and is the only tissue 
from which new bone can grow. 

There are two important criticisms which can be made of MacEwen’s 
work: 

1. In spite of his large number of bone transplantation experiments 
there is an almost complete absence of microscopical study of the grafts. 
Therefore, evidence, which is only revealed by the microscope, that the bone 
cells of the transplant are alive and that regeneration has originated from 
them and them alone, is lacking. 

2. He concludes that transplanted bone is alive if it has contiguous con- 
nective tissue adherent to it and gives with the X-ray a shadow almost as 
dense as that of the original living bone. This, however, has been disproved 
by Kiittner, who showed that this occurred in homogeneous human trans- 
plants of large portions of bone, such as the head and upper third of the 
femur, and still on microscopical examination all of the bone cells were dead 
and no osseous regeneration of any sort had taken place from the transplant. 

Since MacEwen’s monograph several important contributions have ap- 
peared, in the main contradicting his conclusions. 

Mayer and Wehner, from a carefully planned series of experiments of 
transplants of periosteum, subperiosteal resections, cap implantations, and 
bone transplants, conclude that all of the results ““ combine to emphasize the 
osteogenetic function of the specific osteoblastic cells (of periosteum, endo- 
steum, and lining Haversian canals) and the inability of the adult bone cells 
to form new osseous growth. 

Their cap experiments are especially conclusive. 

They note the part played by the osteoblasts lining the Haversian canals 
in regeneration of bone, but do not assign to these cells the importance they 
probably deserve. They demonstrate, however, that it is the outgrowth of 
these cells from the Haversian canals which forms new bone about free 
transplants of cortex devoid of endosteum and periosteum, and that this is 

337 


BERG AND THALHIMER 


not due to the metaplasia of the surrounding connective tissue according 
to Baschkirzew and Petrow. 

Mayer and Wehner also have shown that in the replacement of old bone 
by new bone the process described by Marchand and Barth as “ creeping 
replacement ” is of equal, if not greater, importance than the well recognized 
one of absorption and substitution. 

Phemister’s investigation adds additional experimental evidence to that 
of Mayer and Wehner. In some very well selected experiments he demon- 
strated that in artificial or acquired fracture in the middle of a transplant of 
a portion of the entire shaft of a long bone, new growth of bone occurs at 
the fracture, whereas the bone between this fracture and the ends of the 
transplant (which are in contact with the original shaft) is dead. This new 
bone arises mainly from endosteum and periosteum. Phemister believes, 
however, that a few bone cells of the compacta do proliferate and form 
new bone. A careful perusal of his protocols does not. reveal any definite 
evidence of this. It would seem that here he is dealing with osteoblasts of 
the Haversian canals as noted by Mayer and Wehner and not with bone 
cells. The healing of these fractures in the transplant, of course, com- 
pletely disproves the idea that bone reproduction occurs only through “osteo- 
conductivity,” which was held originally by Barth and at present by Murphy 
and by Davis and Hunnicutt. 

Smith also reached the conclusions from his experiments that mature 
bone cells are end products and that osteogenesis is limited to the osteoblasts. 


OUTLINE OF EXPERIMENTS 


The object of the experiments to be reported was to determine the fate 
of the various component tissues which make up bone, when these were 
transplanted either singly or in different combinations, and also to find out 
under what circumstances these transplants produced new bone, and which 
element or elements were capable of generating bone. 

The following experiments were performed on twenty-two full-grown 
cats; the transplants were autogenous, and the material was taken from the 
tibia. In all cases primary union was secured at the wound over the tibia 
and at the site of transplantation. A few of the transplants were placed 
on the surface of, and within the substance of, the spleen, or subcutaneously. 
Most of them, however, were placed upon the costal cartilages, after these 
were either scraped bare of perichondrium or else after removing a wedge 
from the cartilage or cutting away its outer half. An attempt was made at 
first to lift up the perichondrium and insert the transplant between it and the 
cartilage. The perichondrium was found so intimately bound down that it 
was impossible to do this without tearing it severely, so this method had to 
be abandoned. The transplants were held in place by two black silk ligatures 
which were placed at the two ends and around the cartilage. With a sharp, 
full curved needle, the ligature could be passed around the cartilage without 
entering the pleural cavity. In order to determine the nature of the material 
transplanted, pieces of tissue were removed from each transplant as soon 

338 


REGENERATION OF BONE 


as it was secured, placed in ro per cent. formalin and examined micro- 
scopically. A series of control experiments was performed by injuring 
the cartilage in a number of ways, in order to determine if any impetus or 
tendency to form osseous tissue could be given cartilage by manipulations 
similar to those necessary in using it as a site for transplantation. 

Cartilage was chosen as the site of most of these experiments for several 
reasons. It has been shown that tissues differ in the degree of their capacity 
for serving as a satisfactory medium or soil for the implantation upon them 
of other types of tissue. Certain structures, such as the spleen, serve in this 
capacity very poorly, and in fact the spleen seems to have a destructive action 
on grafts placed within it. Subcutaneous or intramuscular situations have 
been the ones most often and most successfully used in transplantation experi- 
ments. There are also certain special affinities which exist between tissues, 
as, for instance, with testes and ovaries. Stockard has shown that these 
organs serve well as a base for transplantation of grafts taken from each 
other, whereas grafts from either do very poorly on other tissues. Conse- 
quently because of the intimate relationship which exists between bone and 
cartilage, both during the process of growth and in the stage of full develop- 
ment, it was thought that it would be interesting to note in what manner 
cartilage would serve as a base for transplants of different elements of fully 
developed bone, and whether it still retained its ability to readjust its own 
elements in the peculiar manner which occurs in the laying down and growth 
of enchondral bone. 

Also one of the requisites for the successful transplantation of tissue is 
that the graft should be rapidly vascularized. For this reason transplants 
are usually made subcutaneously, intramuscularly, or into parenchymatous 
organs. Cartilage, however, is nonvascular, and it was thought that trans- 
plantations upon this would prove a severe test of the regenerating power 
of the transplanted bone, which could not get its vascular supply from the 
cartilage but would have to obtain it from only one side which would lie in con- 
tact with the connective tissue. In addition, because of the freedom of 
the cartilage from blood-vessels, if the bone graft did grow in the direction 
of the cartilage, it would furnish a good opportunity to observe just which 
element or elements of the graft proliferated. 


MATERIAL AND METHODS 


The material for the transplants was obtained in the following manner: 

The anterior surface of the right tibia was exposed and all tendons and 
muscular attachments cut away, care being taken not to injure the periosteum. 
The periosteum was obtained from the entire surface exposed, by outlining 
a long quadrilateral piece with the scalpel, then lifting it up at one end with 
a fine forceps, and teasing it away from the bone with the handle of the 
scalpel. It always peeled away very easily and no macroscopic bone came 
away with it. The question as to the nature of the cambium (osteogenetic) 
layer, or portion of this layer, which was lifted off with the periosteum is 
discussed later. Next, with a gouge chisel, thin layers of cortex, from I to 3 

339 


BERG AND THALHIMER 


mm. thick, were removed, care being taken not to enter the medullary cavity. 
It was assumed that a portion at least of the cambium layer remained upon 
the cortex, and for convenience the pieces of the cortex taken in this manner 
are called cortex plus cambium. To obtain pieces of cortex alone, the sur- 
face was first thoroughly scraped with a heavy scalpel, and then the pieces 
were removed with a gouge. Cortex covered by periosteum was obtained by 
outlining with a scalpel an area of periosteum about .5x 1 cm. Then the 
cortex beneath this, with the periosteum still adherent, was removed with a 
gouge chisel. Care was taken not to enter the medullary cavity so as not to 
include endosteum. 

The transplants called cortex plus endosteum are simply pieces of cortex, 
where the gouge was allowed to enter the medullary cavity and remove in 
addition a portion of the medulla. 

Portions of these different types of transplants which served us as con- 
trols showed uniform microscopical pictures, and need not be described re- 
peatedly under the different experiments, but can be taken up collectively 
here. 

Periosteum Control-Serial Sections——Sections are made up of the usual 
connective and fibrous tissue, but along the edge corresponding to the cam- 
bium layer, the connective tissue is rather compact and the cells are arranged 
parallel to the surface. A portion of the surface which faced the cortex is 
lined by cells which resemble the adjacent connective-tissue cells, but at the 
same time present an appearance somewhat similar to endothelium. In other 
places, these surface cells are slightly larger and more rounded and seem to 
differentiate themselves from the connective-tissue cells immediately beneath. 
This special layer of cells which constitutes the cambium layer is found 
only over portions of the surface, occupying about half of its extent. The 
remainder of the surface is made up of bare connective tissue. 

Cortex Control.—This is made up of typical dense cortical bone. The 
Haversian canals are small, most of them the size of capillaries. (Much 
smaller than in any of the transplants described below.) The edges are 
everywhere devoid of connective-tissue cells or any cells similar to the cam- 
bium layer of the periosteum described above. Also the edge to which the 
periosteum was attached, and where the cambium was scraped away, is slightly 
uneven, as though the surface of the bone had been scraped away. 

Cortex plus Cambium Control—The edge from which the periosteum has 
been removed is smooth, and in places the cortex is bare, whereas in other 
places there is adherent to the cortex a small amount of loose areolar tissue. 
At these latter places, the cells immediately next to the cortex are drawn 
out and flattened, and have somewhat the appearance of endothelial cells, 
and are similar to those of the cambium layer found lining the periosteum 
described above. Immediately beneath this layer of cells the superficial 
cortex does not exactly resemble the deeper portions, staining somewhat dif- 
ferently, taking a slight diffuse hematoxylin stain, and its lacunar cells are 
closer together. The nuclei of the lacunar cells immediately beneath the 
cambium layer are drawn out and stained densely, and are identical with 


340 


REGENERATION OF BONE 


those of the cambium; whereas the nuclei of cells further away are round or 
oval in outline. A number of nutrient vessels spring from the cambium 
layer and enter the cortex at an angle, and their canals are sparsely lined by 
the same type of cells which are immediately adjacent to the cortex and 
which form the cambium layer. 

Cortex plus Periosteum Control.—Shows cortex covered by periosteum, 
these structures being identical with those described above. The cambium 
or osteogenic layer is similar to that described under cortex plus cambium, 
only it is thicker and made up of more of the same type of cells. In one 
series of sections, whereas the lacunar cells immediately beneath the cambium 
are similar to the cells of the cambium layer, after a distance of one or two 
cells into the cortex the lacunar spaces are larger and their cells are also 
larger and more oval and more vesicular, so that these cells, taken altogether, 
have the appearance of young lacunar cells. (These are somewhat similar to 
those seen in the new-formed bone in the transplants to be described below.) 

Cortex plus Cambium plus Endosteum Control.—These transplants are 
made up of cortex and cambium identical with those described above, and 
in addition a portion of the medulla of the bone made up of marrow spaces 
filled with marrow and subdivided by trabecule of bone. The marrow 
spaces are lined by flattened cells somewhat endothelial in type, identical with 
those forming the cambium layer. These cells are seen dipping down into 
the Haversian canals and lining them. 


GENERAL DISCUSSION OF RESULTS 


Experiments with Transplants of Periostewm.—Twenty-five periosteal 
transplants upon costal cartilage were examined at periods from 7 to 389 
days. All showed active bone formation. One subcutaneous transplant after 
77 days showed bone, one on the surface of the spleen showed bone after 82 
days, but one in the depth of the spleen for the same period showed nothing 
but a scar. This is in accordance with the experiments of others with dif- 
ferent tissues, and shows the tendency of spleen to absorb foreign tissues. 

The process of growth of bone from these periosteal transplants has 
therefore been studied at such intervals that a definite conception may be 
formed of the manner of its occurrence. 

In the youngest transplants (7 days) there is found in the midst of granu- 
lation tissue, an extremely young osteoid tissue which is in contact with the 
osteogenic layer of the periosteum. The next stage is found after about 28 
days, where young bone is seen as branching columns, covered by a con- 
tinuous line of osteoblasts. Here the lacunar cells are just about formed 
and are large, with large, oval vesicular nuclei. From this stage on, there 
occurs in older transplants well formed bone, with all of the characteristics 
of bone. The lacune are fully developed and are grouped concentrically 
around Haversian canals, and also irregularly disposed between these con- 
centric systems. At a distance from the periosteum instead of canals are 
found larger spaces which are irregular in shape and are first filled with 
blood-vessels and delicate granulation tissue, and lined by osteoblasts, and 

341 


BERG AND THALHIMER 


are later occupied by genuine hematopoietic marrow (sometimes admixed 
with fatty marrow), composed of myeloblasts, myelocytes, megalokaryocytes, 
megaloblasts, etc. When this new-formed bone is in contact with cartilage it 
almost invariably invades it in the same manner as enchondral bone invades 
its cartilaginous matrix, in the normal development of bone, i.e., in the man- 
ner described in the protocols as epiphyseal line formation. It would seem 
from this that cartilage is an excellent medium to accommodate the growth 
of bone, and that even though it is nonvascular, bone will grow into it and 
carry its nourishment along with it. 

The older the transplants the more calcified they are and the more the 
lacunar cells assume the appearance of adult bone cells, their spaces and 
nuclei being smaller and more elongated, and the latter also staining darker. 

There is one observation of importance concerning the young lacunar 
cells which are not yet in a bony matrix but are away from any layer of 
osteoblasts. The same process of amitotic division and growth is found 
as was described by Mayer and Wehner. It seems definite that these young 
cells proliferate in this manner and so account for some of the production of 
bone. They are found crowded together in areas, in the midst of well 
developed bone. Here the lacunar spaces are somewhat larger than else- 
where and in some of the areas are in the midst of a matrix which partakes 
of the properties of both bone and cartilage, for with the hematoxylin and 
eosin stain it has the appearance of bone but stains bluish, like cartilage. 
Nuclei which are much elongated, indented, or figure-eight in shape, are 
relatively common in these lacunar spaces, but two nuclei in a single space 
are seldom seen. Young lacunar cells which lie close to one another or are 
barely separated by a very thin partition and which appear to have just 
divided are relatively frequent. Therefore, taking all these facts into con- 
sideration, it must be recognized that this is one manner of bone growth. 
This growth of bone is from young, immature lacunar cells which have just 
developed from osteoblasts and whose lacune are not yet formed by calci- 
fied osseous material. It is this type of growth which occurs in bone trans- 
plants and in the development of bone and causes the “‘ creeping replacement ” 
of Marchand and Barth. We have never observed this or any other manner 
of growth proceeding from adult bone cells. 

The source of origin of the hematopoietic marrow and its method of 
development is another point of interest and needs further study for its 
elucidation. The transition has gradually progressed from (1) Haversian 
canal containing a blood-vessel with a wall formed of a single layer of 
endothelium and a layer of osteoblasts between the vessel and bony wall, (2) 
a larger space containing in addition delicate connective tissue, and, finally, 
to (3) a completely developed marrow. Whether this process of the develop- 
ment of marrow has to do with the histocyte or the endothelium or the osteo- 
blasts is a problem for further investigation. 

The manner in which cartilage serves as a medium for bone is remark- 
able in that it is identical with the embryologic development of bone in 
cartilage. The reason why the cartilage cells arrange themselves in columns 

342 


ee 


le Maplg 


REGENERATION OF BONE 


at right angles to the line of advancing bone has never been explained. ‘This 
method might serve as a means to attack the problem. 

In the older transplants bone absorption is taking place as well as bone 
growth, for osteoclasts are found at various places along the periphery of 
the new bone and also of the marrow spaces. 

Many of these transplants could not be identified macroscopically as bone, 
and certainly most of them would not have thrown a shadow with the 
X-ray. Therefore, it follows that in all experiments of this kind the end 
result must be carefully controlled by microscopical examination, and where 
this is not done the conclusions arrived at are based on incomplete evidence. 

It is inconceivable that after the careful microscopic examination with 
negative findings of control pieces of periosteum any bone cells could have 
been adherent to these transplants. Therefore, the conclusion must be 
drawn that the bone grew from osteoblasts which form the osteogenic 
layer of the periosteum and did not arise from bone cells. The fact that 
the control pieces of periosteum were lined only in places by osteoblasts 
and not over their entire extent explains why bone did not always spring 
from the entire surface of the periosteum. 

Transplants (of Cortex minus Cambium, Cortex plus Cambium, Cortex 
plus Cambium plus Endosteum, Cortex plus Periosteum).—Although some 
of these transplants were in place in the cats as long as 250 days, and were 
comparatively small pieces, not more than 2x 3x IO mm., none were com- 
pletely absorbed in that time. Many of them which showed microscopically 
either well formed bone or growth of bone were too small to have cast a 
shadow with the X-ray. 

The bone or cortex of all the different types of transplants showed the 
same process, and the nature of this would explain the discordant results 
which have been reported about transplantations and the osteogenic power 
of bone. 

The transplants removed after the shorter intervals show that most, 
but not all, of the bone cells are dead and therefore unstained. Those 
cells which are stained, however, are situated near sources of nourishment 
(by diffusion), such as the edge of the transplant, the periosteum, or 
Haversian canals. After a somewhat longer interval there are fewer stained 
bone cells, but even these have small, irregular, darkly-stained nuclei. In 
the oldest transplants the tracing of these cells is interfered with by other 
processes which are going on, but always a certain number of these cells are 
seen, which are undoubtedly the original cells transplanted, and can be 
differentiated from the young bone cells present. Therefore it must be 
as Axhausen contended that, whereas many, if not most, of the bone cells 
transplanted die, some persist and live. Although carefully examined, none 
of these adult cells which persist were ever found in any place in the 
transplant either dividing or giving rise to new bone. 

The blood-vessels in most of the Haversian canals evidently, very soon 
after transplantation, join with surrounding blood-vessels, for their walls 
are made up of living cells which stain normally, and they contain blood- 

343 


BERG AND THALHIMER 


cells which are normally stained and alive. The cells of the vessel walls 
must have been kept alive in the meantime by the blood which was in them or 
by plasma which diffused in from the tissue next to the transplant. Some 
canals are empty or contain abnormally-stained blood-vessels and blood- 
cells which are dead. Very soon after transplantation the Haversian canals 
enlarge and are found lined by osteoblasts, though a few osteoclasts are also 
found at this stage. But the absorption of the bone around the canals seems 
to occur largely in a direct manner and not by the aid of the osteoclasts. 

Almost simultaneously with the enlargement of these canals is found a 
formation at their periphery of a ring of young bone composed of young 
bone cells. Soon this ring is several cells thick and is found progressing 
into the bone originally transplanted, the canal becomes progressingly larger. 
These young cells then proceed more or less irregularly, and, where the 
canal is near the surface of the transplant, they gain the surface and spread 
out over it. 

This process of growth from the osteoblasts lining the Haversian canals 
is a very active one and is seen replacing all, or almost all, of the bony 
transplants and spreading out beyond. These young lacunar cells are also 
seen dividing just as they were in the bone formed from periosteal transplants. 
As there are no osteoclasts between them and the original bone which is 
being absorbed, the absorption must be accomplished by them, perhaps 
through a biochemical action, and they replace the old bone by “ creeping 
replacement.” It is conceivable that they might slip into the empty lacunar 
spaces of the original bone, but this was never observed. 

The transplant, cortex without periosteum, removed after an interval of 
53 days (see Fig. 7) is an excellent illustration of this type of bone growth. 
It also shows that bone cells of the original cortex transplanted, even 
though they are well nourished by being in apposition with living granula- 
tion tissue, do not grow, whereas osteoblasts lining the Haversian canals do. 

In order to follow these two types of cells carefully, full grown and not 
young cats were used in these experiments so as to be able to observe the 
behavior of adult bone cells and not confuse the study with young bone. 
Consequently the results of this study carry with them the firm conviction 
that adult, differentiated bone cells are sufficiently specialized, in the same 
manner that nerve cells are, to be unable to reproduce themselves. 

In the oldest transplants practically none of the original bone is left, 
but its place is taken by this new formed bone. Osteoclasts are also found 
here and there along the edges of the transplant, and at the periphery of 
enlarged Haversian canals. 

As the Haversian canals enlarge they first form spaces, thinning out the 
intervening bone into trabecule, and later forming larger marrow spaces 
filled with hematopoietic marrow. 

The new bone arising from the Haversian canals grows from the edges 
of the cortex transplant just as vigorously as does that arising from the 
cambium layer of periosteal transplants, and invades cartilage in the same 
manner with epiphysial line formation. 

344 


——— 


aE 


eee 


4 


Fic, 1.—Control: Regeneration of cartilage only, after slivers of cartilage were lifted up and tied back 
again in place ( ¥ 60) 


Fic, 2.—26-day transplant of periosteum upon the surface of cartilage denuded of perichondrium; showing 
the growth of osteoid tissue from the periosteum (A) ( X 60). 


Fic. 3.—53-day transplant of periosteum upon surface of cartilage denuded of perichondrium; showing 
growth of young bone from the periosteum (A) ( X 60). 


O18 (gq) UOvUIO; au 
qAvo JO dovyjins uodn u 


sAydida yy (7) auoq 


BUIMOYS !UNIpUuoL ysoliod jo juvjdsuvs Avp-She—b ‘oly 


SAMEEW 


Fic. 


5 


Cig 


.—High power of Fig. 4. 


A, bone; B, marrow; C, epiphyseal line formation ( X 100). 


i 


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yiMors st aroyy *Aydura ere saoeds reunoR] ayy pur (P) peap si auo0q pozuLjdsuey ayy “ydess030yd oyy ur uMoYs jou st 
ase|Igseo ayy ynq ‘ase[yIVO Jo sovjIns uOdnN st juL[dsuvsy “wMazsoliad ynoy4yrM X9}109 auOg Jo JuL[dsursy Avp-gz—-9 “DIY 


There is pr 


eration 


o 
a 


ation of bone, the 


where there is prolifera 


he conditions of growth, such as v 


sue just asitisat B, 


At X, where there is no prolifer, 
urred at X, this section would indicate that adult bone cells 


d from adult bone cells t 


rent to the surrounding connective tis 
Since growth has not oce 


ant, same as Fig. 7 and showing the same. 


, if the growth of bone occurre' 


-day transpl 


FyS 


Consequently 


nourishment, are as good at X as at B. 


8. 


Fic. 
surface of the transplant of bone is adhe 


are incapable of growth and proliferation (X 60). 


of bone. 


Fic. 9.—High power of same as Fig. 8, showing double nuclei in a young lacunar cell (A) (X 200). 


“(001 X) (y7) S[BURO URISIOAR]] punoie uOIZeI1OJI[01d QUO Os|v puB aUOG pap Jo svoIe 


JayjO quaseid ore asoyy *(37) 2UOq pajyuL[dsuv1, pRep JO vaiv uv pure ((7) Wne4solied usaMgoq (gq) AUC JO UOTZBIAz[O1d 


SMOYS SIV], “WUiNtIpuoyoliod jo papnuop ase] 41" Jo vovyins uodn uMojsoriod snjd xoq109 jo yuL[dsuvsy Aep-SE—'‘Ol “OIyy 


REGENERATION OF BONE 


Those transplants which are covered by periosteum show a growth of bone 
between it and the cortex which is more advanced than that proceeding from 
the Haversian canals and so undoubtedly is formed from the intact cambium 
layer. 

Where the transplants are made up of cortex plus cambium, the growth 
from the Haversian canals, which breaks through on to the surface, is as 
active as the growth from the cambium layer. 

The subcutaneous transplants show this same group of processes. One 
series of transplants into the spleen shows also the same findings, but another 
series left for even a longer interval showed less growth of the transplants, 
but persistence of them. This must be attributed to either a difference in 
action of similar transplants in different animals, or to a difference in the 
action of the spleen in different animals. 

The few transplants which included endosteum, though not enough to 
allow any definite conclusions to be formed, showed an even greater growth 
from the endosteum than from any other transplants, even including 
periosteum. 

It must also be noted that the transplants of cortex plus periosteum re- 
tained their vitality longer than those devoid of periosteum and more of their 
bone cells persisted and less of their bone was absorbed. 

To summarize: Especial emphasis must be placed on the activity of the 
osteoblasts lining the Haversian canals in forming new bone. These cells 
are always transplanted along with bone and consequently play a con- 
siderable rdle in bone formation under these circumstances. This point 
has been noted by Mayer and Wehner, but its significance and importance 
has not yet been fully emphasized. Many workers have reported new 
growth of bone as occurring from freely transplanted pieces of cortical 
bone. As these pieces have been devoid of periosteum, cambium layer, and 
endosteum, the new bone production has been considered as arising from 
the bone cells. Most of these specimens have not been carefully examined 
microscopically, and consequently the exact source of osteogenesis was not 
determined. 

We believe, therefore, that it has never been shown conclusively that 
an adult bone cell can divide and produce new bone cells and new bone— 
and by an adult bone cell is meant a fully developed bone cell within a lacuna 
formed of completely calcified osseous tissue. The adult bone cell must 
be carefully differentiated from the osteoblast within a lacuna surrounded 
by uncalcified matrix. This latter is not a bone cell in the accurate 
sense of the term, but has frequently been erroneously so called. This young 
cell is the active one in creeping replacement. Mayer and Wehner indicate 
that they have also reached this conclusion, © 

The study of this point in the literature is difficult because of a con- 
fusion arising from a loose use of the term bone cell. An instance of this 
can be noticed in the excellent recent article by Phemister, where he de- 
scribes bone formation from the osteoblasts lining the Haversian canals 

345 


BERG AND THALHIMER 


and apparently considers the osteoblasts to be bone cells. Of course, in 
regenerating bone where replacement of the osseous tissue is occurring by 
both lacunar absorption and substitution and by creeping replacement, the 
young bone cells are in apposition with the old cells and differentiation is at 
times difficult. Still, we believe that this differentiation is possible by careful 
microscopical study. The identification of periosteum, endosteum, and osteo- 
blasts lining Haversian canals, and the bone which arises from them, is in 
reality easily accomplished. It has been definitely proved that osteoblasts 
in all of these locations can produce bone and that those in the Haversian 
canals can assume considerable osteoproductive activity when they are prop- 
erly nourished. This accounts for their activity in transplants of small frag- 
ments of bone where the revascularization of the Haversian canals takes 
place rapidly and the lining osteoblasts are kept alive till this occurs by 
plasma from surrounding tissues. In large transplants more time is required 
to establish a new source of nourishment for these particular osteoblasts 
and their osteoproductivity is correspondingly reduced. Nevertheless, in 
transplants of cortex devoid of periosteum and endosteum these cells are 
responsible for whatever new intrinsic bone formation takes place. It 
naturally follows that any factor which aids in the nourishment of these 
cells also aids bone growth in the transplant. Since the periosteum covering 
the cortex is of material assistance in this nourishment, it can readily be seen 
how transplants of cortex covered by intact living periosteum are more 
probable of success, aside from the osteogenic function of the cambium layer, 
than are transplants devoid of periosteum. 


CONCLUSIONS 


1. Periosteum, devoid of adherent bone cells when transplanted into 
foreign tissues, produces bone. 

2. Endosteum and osteoblasts lining Haversian canals in bone transplants 
produce bone very actively. 

3. The cambium layer when adherent to transplanted cortex produces 
bone. 

4. Some bone cells in the transplants are able to persist for almost a 
year, but most of the bone is absorbed. 

5. Fully developed adult bone cells, although they may remain alive for 
a considerable time, do not reproduce themselves and form bone. 

6. Very young lacunar cells (frequently erroneously called bone cells) 
can reproduce themselves and form bone. 

7. Transplanted bone is absorbed not only by osteoclasts, but also by a 
direct action (biochemical?) of growing, young bone, and the transplanted 
bone is replaced either by a creeping forward of the new bone or a gradual 
extension or expansion of the new bone into the transplant. 

8. Marrow spaces and hematopoietic marrow are formed in the bone 
which develops from transplanted periosteum. The source of these 
hematopoietic cells was not determined. 

346 


REGENERATION OF BONE 


g. Bone, when it grows into cartilage, does so in the same manner char- 
acteristic of the normal embryonic development of enchondral bone, including 
also epiphysial line formation. 


REFERENCES 


Axhausen: Archiy f. Klin. Chirurgie, 1908, Ixxxviii; Deutsche Zeitschrift f. 
Chirurgie, 1907, xci; Archiv f. Klin. Chirurgie, 1911, xciv. 

Barth: Ziegler’s Beitrage zur Pathologischen Anatomie, 1896, xvii; Uber Histologische 
Befunde nach Knochen Implantation. Arch. f. klin. Chir., 1803, xlvi, 400 

Baschkirzew and Petrow: Deut. Zeitschrift f. Chirurgie, 1912, cxiii, Heft 5 and 6. 

Bonome: Virchow’s Archiv, 1885, c. 

Davis and Hunnicutt: The Osteogenic Power of Periosteum; with a Note on Bone 
Transplantation. An Experimental Study. Johns Hopkins Hospital Bulletin, 
Ig15, vol. xxvi. 

Kiittner: Verhandl. d. Deut. Gesel. f. Chir., 1913, vol. xlii, 353. 

MacEwen: The Growth of Bone, Glasgow, 1912. 

Marchand and Barth: Prozess der Wundhheiling, Stuttgart, 1901. 

Mayer and Wehner: An Experimental Study of Osteogenesis. American Journal of 
Orthopedic Surgery, 1914. 

Murphy: Journal of American Medical Association, 1912, vol. lviii, p. 985, et seq. 
Ollier: Journal de la Physiol., 1859, T. I1; Mem. de la Soc. de Biol., 1859; Traite 
Experimental et clinique de la Regeneration des Os, T. I and II, Paris, 1867. 
Phemister: The Fate of Transplanted Bone and Regenerative Power of Its Various 

Constituents. Sur., Gyn. and Obstet., 1914, xix, pp. 303-333. 

Radzimowsky: Ueber Replantation und Transplantation de Knochens, Diss. Diew. 
(Russian), cited by Marchand, pp. 457 and 467. 

Smith: Periosteal Regeneration of Bone. Sur., Gyn. and Obstet., 1915, vol. xx, pp. 
547-552. 

Stockard: The Fate of Ovarian Tissues when Planted on Different Organs. Archiv 
fiir Entwicklungsmechanik der Organismen, I91I, xxxii, p. 298. 


347 


we 
¥ 


PUBLICATIONS 


OF 


Cornell University 


WEE IORI CCOLICEG LE 


ae? Ge apots S 


Department of Anatomy 


(> 


VOL OME, VIET 
1920-21 


NEI YORK CITY 


on 


6. 


~ 
(. 


CONTENTS 


Being reprints of studies published in 1920-1921. 


DEVELOPMENTAL RATE AND STRUCTURAL EXPRES- 

SION: AN EXPERIMENTAL STUDY OF TWINS, ‘DOUB- 

LE MONSTERS’ AND SINGLE DEFORMITIES, AND 

THE INTERACTION AMONG EMBRYONIC ORGANS 
DURING THEIR ORIGIN AND DEVELOPMENT. 

By Charles R. Stockard. 


Am. Jour. of Anatomy, Vol. 28, 115-278. 


A PROBABLE EXPLANATION OF POLYEMBRYONY IN 


THE ARMADILLO. 
By Charles R. Stockard. 


Am. Naturalist, Vol. LV., 62-69. 


EFFECT OF UNDERFEEDING ON OVULATION AND THE 
OESTROUS RHYTHM IN GUINEA-PIGS. 
By George N. Papanicolaou and Charles R. Stockard. 


Proc. Soc, Exp. Biology and Medicine, Vol. XVII., 143-144. 


SOME STUDIES ON THE SURFACE LAYER IN THE LIVING 


EGG CELL. 
By Robert Chambers. 


Proc. Soc. Exp. Biology and Medicine, Vol. XVII., 41-43. 


DISSECTION AND INJECTION STUDIES ON THE AMOEBA. 
By Robert Chambers. 
Proc. Soc. Exp. Biology and Medicine, Vol. XVIII., 66-68. 


DISTURBANCES IN THE DEVELOPMENT OF MAMMALIAN 
EMBRYOS CAUSED BY RADIUM EMANATION. 
By J. F. Gudernatsch and H. J. Bagg. 


Proc. Soc. Exp. Biology and Medicine, Vol. XVII., 183-187. 


A REVIEW OF THE CHROMOSOME NUMBERS IN THE 
METAZOA. 
By Ethel Browne Harvey. 


Jour. of Morphology, Vol. 34, 1-68. 


AUTHOR'S ABSTRACT OF THIS PAPER ISSUED Reprinted from Tue AMERICAN JOURNAL oF 
BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 6 Anatomy, Vol. 28, No. 2, January, 1921 


DEVELOPMENTAL RATE AND STRUCTURAL EXPRES- 
SION: AN EXPERIMENTAL STUDY OF TWINS, 
‘DOUBLE MONSTERS’ AND SINGLE DEFORMITIES, 
AND THE INTERACTION AMONG EMBRYONIC 
ORGANS DURING THEIR ORIGIN AND DEVELOP- 


MENT! 
CHARLES R. STOCKARD 
Cornell University Medical College, New York City 
THIRTY-TWO TEXT FIGURES AND SIX PLATES 
CONTENTS 

i, Ueto GhiCUGRs 5 nop ones bes aaooede socce bans DOOee DAOC AD AGata Sh OCC an rE 116 
2. The specific rate of development in a given species.................+-:. 118 
3. Continuous and discontinuous modes of development................... 120 

4. Experimentally changing a continuous into a discontinuous mode of 
Gl yer NA Boone SoCs Gad One CORR ADULT ao nano Eee eae or cree 122 
C@mLbewnethod: of eExperiment....< 2 cjosie ie .5 sce Seiten vas Sbusless ase teeveielsc oe 122 

b. Stopping or retarding the progress of development at stages of 
apparent indifference to such interruption..................... 125 

c. Stopping or retarding the progress of development at stages of 
critical susceptibility to such interruption.....................- 138 

d. Differences in effect between greatly reducing the developmental 
rate and actually stopping the process temporarily........... 154 

e. The types of arrests or deformities following a stop in development 
OM anslowin gral bhearaterecmav.w tose a eee ee 160 

5. Experimental production of twins and ‘double monsters’ by an early 
arrest on the developmental rate... .--casemer come etree eerste. 162 

a. Arresting development by low temperature and the production of 
double embryos and twins in Fundulus........................ 166 

b. Arresting development by reducing the oxygen supply and the 
occurrence of double individuals in the trout and Fundulus.... 173 
IPR eSsuLbs withee in GULusseyras seem cpeeteerny ee tie eee crits cere sie 173 
2 Doublevembpryos-n toute gesteeee eee eee cee econ: cane 177 

c. An explanation of the frequent occurrence of twin and double chick 
itl aigo) -eaMeee cnt caie eter cad 6b ce Roe ee OO OR RO Lo nana 186 


1 This investigation has been aided by the Memorial Foundation of St. Bar- 
tholomew’s Hospital for Diseases of the Alimentary Canal, of New York City. 
The author expresses his appreciation for this valuable support. 


115 


118 CHARLES R. STOCKARD 


moment will produce a double monster or identical twins and at 
another moment slowing by the same method will give rise to 
the cyclopean defect. In fact, the same thing which causes 
the double monster may later in development induce one of its 
heads to be cyclopean. 

Thus there is no longer any ground for considering certain 
defects as specifie responses to particular treatments. And there 
is as little reason for further descriptions of individual monsters, 
since all belong to the same class and the individual differences 
simply result from the different moments during which the devel- 
opmental interruptions have acted. 

The important consideration then arises as to what internal 
and external factors may tend to introduce the developmental 
arrests. Does one growing part in any way inhibit the activity 
of other developing organs? We shall devote a section to a con- 
sideration of the interaction among the developing and growing 
organs within the embryo. The study of the growth influences of 
one embryonic organ on another is one of the most important 
problems in the analysis of structure. 

Finally, the interaction among growing parts and the inhibit- 
ing effects of one rapidly proliferating region over other regions 
will be very briefly considered in connection with abnormal and 
malignant growths. 


2. THE SPECIFIC RATE OF DEVELOPMENT IN A GIVEN SPECIES 


It is a generally known fact that the eggs of different species 
do not progress at the same rate of development even during 
comparable stages. The lengths of time between fertilization 
and the first cleavage and the rates at which the early cleavages 
follow one another may differ decidedly among the eggs of 
even closely related forms. These differences in developmental 
rate are probably fundamentally connected with differences in 
chemical structure of the egg substances, and in particular with 
the different rates of oxidation of certain stuffs. It is a well- 
known chemical fact that very slight differences in composition 
between substances may cause very great differences in their 
oxidation capacities. 


STRUCTURE AND DEVELOPMENTAL RATE 119 


The efforts on the part of numerous embryologists to associate 
the differences in rate of cleavage and time required to attain 
certain stages of development with the size of the egg, the amount 
and position of the yolk substances, or even the types of cleavage 
have not been satisfactory. Certain meroblastic eggs develop 
much faster than certain holoblastic ones, while other holoblas- 
tic eggs have a rate of cleavage far more rapid than the mero- 
blastic types. All of the so-called laws of cleavage rates based 
on morphological differences among egg types have been found 
to fail so decidedly when applied in general that one is forced 
to seek more deep-seated causes for the differences in develop- 
mental rate. 

At the present time we can only state that such causes probably 
reside in the differences in chemical make-up of the several 
species of eggs. The rate of development certainly depends, 
particularly during later stages, on the amount of food avail- 
able, but the supply of oxygen and the degree of temperature at 
which development is taking place have a far more striking influ- 
ence on the rate. Cessation of development also occurs much 
more promptly from absence of oxygen or sudden changes in 
temperature than from any other natural modifications which 
happen in the environment. These facts point decidedly to the 
rate of development as being dependent upon kind and rate of 
chemical change, most particularly upon rate of oxidation. The 
egg probably has a definite coefficient of metabolism dependent 
upon the interaction of its specific chemical structure and the 
given environment in which it normally develops. The rate of 
development results from both the internal qualities of the egg 
and the nature of the surrounding environment. 

The present extremely crude state of our knowledge of the 
chemistry of development will permit of no more satisfactory 
statements of the principles underlying differences in develop- 
mental rate than those which have been attempted above. The 
inadequacy of such statements is as keenly appreciated by the 
writer as by the critical reader, but this inadequacy concerns 
chiefly the absence of the details involved, while the statements 
in general I believe are correct. 


120 CHARLES R. STOCKARD 


Although there is a definitely normal rate of development for 
a given egg, this rate is frequently subject to wide variations, usu- 
ally as a result of variations in the surrounding conditions. The 
two chief, or most frequent, modifying causes are a change in 
oxygen supply or a change in temperature. An acceleration of 
the usual rate only takes place to a limited degree under natural 
conditions and but slight increases in developmental rate have 
been experimentally obtained. On the other hand, a very wide 
range of decrease in developmental rate is readily brought about. 
Slight changes in the surrounding temperature or reduction in the 
oxygen supply will readily tend to slow the rate of development 
to a marked degree. Finally, the entire progress of development 
is frequently stopped in nature by removing the supply of oxy- 
gen or by sufficiently lowering the surrounding temperature, as 
will be discussed in subsequent sections. 


3. CONTINUOUS AND DISCONTINUOUS MODES OF DEVELOPMENT 


Although, as stated in the foregoing section, each egg has a 
more or less characteristic rate of development, this rate is not 
uniform throughout the different developmental stages. All eggs 
develop with rythmical changes in rate, going alternately faster 
and slower from stage to stage. Certain stages are passed very 
rapidly, almost suddenly, while others are slowly attained in a 
tedious manner, yet the process of development is as a whole con- 
tinuous. That is, development begins with fertilization which 
is soon followed by cleavage, and then continues without inter- 
ruption until a free living larva or young embryo is formed. 
This then proceeds to grow and change until the adult structure 
is attained.- Such a continuous mode of development is most 
common, indeed so common, that it is often carelessly consid- 
ered to be universal, while a discontinuous mode is looked upon 
as something very strange or unusual and not as a phenomenon 
extremely important in an understanding of the more common 
continuous type of development. 

The continuous mode is found among the great majority of those 
animals in which the eggs develop in a uniform or homogeneous 
environment, such as the sea-water. The general conditions of 


STRUCTURE AND DEVELOPMENTAL RATE 121 


moisture, oxygen supply, and temperature are comparatively 
uniform, and although the eggs may develop faster or slower 
under slightly different conditions of temperature, etc., yet the 
variations in the medium are rarely sufficient to inhibit or stop 
development entirely, and when they are the eggs usually die. 

On leaving the sea the fresh-water and land-living inverte- 
brates and vertebrates show most varied and complex methods 
and arrangements for insuring an environment of sufficient uni- 
formity to permit an uninterrupted development. Many forms, 
as is also the case in certain sea-living animals, have evolved 
a method for the development of the embryo within the body of 
the mother. Such an internal environment tends to control very 
effectively the conditions of moisture and in mammals also the 
temperature, but at times, as we shall see beyond, the oxygen sup- 
ply is not properly adjusted and the continuity of development 
may be interrupted or interfered with on this account. 

The land-living animals have not always succeeded in obtain- 
ing an ideal developmental environment, and there are many 
examples of a discontinuous mode of development as a result of 
environmental breaks in the strictest sense. That is, the egg 
begins to develop and attains a certain stage, when a more or 
less sudden change or break in the environment occurs and devel- 
opment stops completely and may remain at a standstill for vari- 
ous lengths of time—days or possibly weeks. Another alteration 
in the environment then occurs which again permits development 
to start and continue until the fully formed animal is obtained. 
Such a discontinuous mode of development is universal among 
one great class of vertebrates, the birds. Among the birds devel- 
opment, as far as studied, is invariably interrupted when about 
the stage of gastrulation, at which time the egg is laid or passed 
out of the warm body of the mother. The fall in temperature 
causes development to stop and the egg remains in the gastrular 
stage until incubated by the heat of the parent’s body or until 
artificially incubated at a similar temperature. 

The means of interrupting development seem to reside entirely 
outside the egg itself, they are properties of the environment. 
As far as is known, all eggs having once begun to develop will pro- 


122 CHARLES R. STOCKARD 


ceed in a continuous manner from stage to stage until the larva 
or free living embryo is formed, the environment permitting. 
Stops in- development take place through lack of oxygen, 
unfavorable temperature, insufficient moisture, or shortage of 
available nutriment, but the egg itself is wound or set for de- 
velopment so as to continue through if possible. Thus experi- 
ments on discontinuous development must apply as methods 
various means for modifying the environment, and the results 
will depend upon the power of the egg to adjust itself to or with- 
stand these changes. Being unable to meet the situation, abnor- 
mal or unusual developmental productions may arise. 

The question then presents itself as to whether the develop- 
ment of any egg may be interrupted for definite lengths of time 
and later be allowed to finish or proceed. What would be the 
consequences of such interruption in the case of a normally con- 
tinuous mode of development? Would the effects of the manner 
of development be the same following interruptions at different 
stages, or would the effects vary depending upon the stage of 
development at which the interruption occurred? In other 
words, are there indifferent and critical moments of develop- 
mental interruption? Would a complete stop in development 
have an effect similar to a decided slowing of the rate, or 
would the one be more effective than the other? The experi- 
ments recorded in the following sections were devised in order to 
answer these and other queries. 


4, EXPERIMENTALLY CHANGING A CONTINUOUS INTO A DISCON- 
TINUOUS MODE OF DEVELOPMENT 


a. The method of experiment 


The continuous mode of embryonic development is the more 
common type in nature. We are, therefore, warranted to some 
extent in assuming that the discontinuous mode is nature’s ex- 
perimental modification of the continuous. What methods of 
modification has nature employed that may be artificially imi- 
tated? The simplest, commonest, and most evident natural 
method is change in temperature which causes the interruption 
of development in the eggs of all birds. 


STRUCTURE AND DEVELOPMENTAL RATE 123 


Changing the temperature of the environment and, therefore, 
of the egg, is the method employed in most of the present experi- 
ments in order to interrupt or make discontinuous a normally 
continuous development. 

There are several definite natural cases of discontinuous devel- 
opment among mammals, the significance of which will be con- 
sidered in another section of this paper. But in the present con- 
nection we may be certain that nature has here employed 
another method than temperature change in causing the interrup- 
tion. The temperature of the maternal body in which the mam- 
malian embryo is developing is sufficiently uniform never to 
interrupt the progress of the egg. For reasons to be more fully 
cited beyond, changes in the supply of oxygen would seem to be 
the most probable cause of interrupted development in the rare 
cases of this phenomenon among mammals. Lack of oxygen or 
excess of CO, has also been resorted to in the present experiments 
as a means of interrupting or retarding the rate of a normally 
continuous development. 

Neither of the two methods is new. A number of experi- 
menters have studied the influence of temperature changes on 
the manner of development of different eggs. The effects of 
abnormally high and low incubator temperature on the develop- 
ment of the hen’s egg have been recorded by Dareste and many 
others, most recently by Miss Alsop (719). The development 
of amphibian eggs under unusual temperature conditions has 
been considered by O. Hertwig (’96), King (’04), and ‘others. 
The influences of low temperatures on the development of the 
fish’s egg have been investigated by Loeb (’16) and Kellicott 
(16). 

These studies on temperature, however, are of interest in the 
present connection only in so far as they almost all show how 
readily abnormal development of the embryo may be induced by 
unfavorable temperature conditions. The attempted explana- 
tions of the deformities which were given in only a few cases, as 
by Kellicott, entirely disregard or dismiss the real point of fun- 
damental importance; that is, the induced change in the rate of 
development resulting from the modified temperature. Kellicott 


124 CHARLES R. STOCKARD 


attempted to refute the slow rate as a cause of structural modi- 
fication in discussing my assumption of arrested development. 
The present experiments differ from the previous temperature 
experiments in that they were undertaken with an almost 
completely different problem in view. The former experiments 
will be considered only as they bear on the specific questions in 
the discussion to follow. 

Numerous studies on the behavior of eggs deprived of oxygen 
as well as in the presence of various reducing and anaesthetic 
substances have been conducted. All of these oxygen studies 
have little or no bearing on the immediate problems and are not 
treated in this connection. 

The material used in the present experiments were the eggs 
of the common minnow Fundulus heteroclitus. I have studied 
and experimented with these eggs for a number of years and am 
familiar with a great many common deformities which they may 
be induced to present. The exact method of experimentation 
with temperature change was as follows: the eggs were taken 
from the female and fertilized in a ‘dry bowl.’ Aboutfifteen 
minutes later they were rinsed free of foreign material with sea- 
water and left standing under water. ‘The first cleavage takes 
place after about two hours, varying a little with the season and 
the temperature. The next cleavage follows after another hour, 
and development proceeds in a continuous fashion from then on 
until the fully formed fish hatches from the egg membrane and 
swims freely about within from eleven to eighteen or twenty 
days, depending again upon the season and temperature. There 
is a wide variation in the rate of development of these eggs, yet 
under all usual conditions after development once starts it is 
continuous. 

The eggs were placed during different stages of development in 
compartments of a refrigerator at temperatures of 5°, 7° and 9°C. 
and left for varying lengths of time, from one to five days. At 
the lowest temperature development was almost if not completely 
stopped, while in the other two compartments it was slowed 
down to from one-twentieth to one-fiftieth of the normal rate. 
The responses shown in the manner of development are so differ- 


STRUCTURE AND DEVELOPMENTAL RATE 125 


ent in eggs stopped or slowed at different stages that the exact 
time of treatment will be considered in connection with the dif- 
ferent effects obtained. The difference in effects between slowing 
and actually stopping development will also be considered. 

Other eggs were crowded close together in bunches and de- 
veloped in bowls at room temperature. The eggs near the center 
of the masses or bunches obtained much less oxygen and were in 
a higher concentration of CO: than the more superficial ones. 
These were slowed in their rate of development. Sea-water was 
boiled so as to drive out most of the air and afterward kept stag- 
nant. Egg masses were developed in this water and the inner 
eggs of the mass were almost completely stopped in many cases. 
In all such arrangements the rate of development was so retarded 
that many abnormal and deformed embryos resulted. 

These in general are the methods employed; the different 
times of application and the results will be discussed in the par- 
ticular cases below. 


b. Stopping or retarding the progress of development at stages of 
apparent indifference to such interruption 


In order to successfully change a continuous into a discontinu- 
ous mode of development, without producing ill effects on the 
resulting embryos, it becomes necessary to locate certain indiffer- 
ent periods during embryonic development at which the inter- 
ruption may be induced. Certain of these indifferent periods 
are those moments at which the interruptions of development 
occur in nature. Should the stoppage naturally take place dur- 
ing a sensitive period, the species would readily be eliminated 
on account of the high proportion of abnormal embryos which 
would result. 

When the eggs of Fundulus are placed in low temperatures 
after having passed through the earliest active stages of devel- 
opment, cleavage, gastrulation, the formation of the germring 
and early appearance of the embryonic shield, they may be 
stopped for several days, or caused to develop at an extremely 
slow rate, without marked injury to the resulting embryos. In 
fact, when such eggs are returned to room temperature after 


126 CHARLES R. STOCKARD 


being in the refrigerator for three or four days, they may often 
resume development at such a fast rate, probably as a result of 
the stimulation of raising the temperature, that they may hatch 
only a day or so later than control embryos. The percentage 
of such eggs that do hatch may also be equally as high as that 
from the control. 

These statements may be illustrated best by a somewhat de- 
tailed consideration of the records from experiments. A large 
number of experiments have been performed and are recorded 
in my notes, but only a few of these may here be selected as 
typical examples of the series in general. 


Experiment 905. A group of eggs, 23 hours after fertilization, with 
high segmentation caps just beginning to flatten on the yolk-sphere, 
were carefully selected, being certain that every one was developing, 
and arranged as follows. 

Lot C, was placed in the refrigerator at 5°C., Cz at 6°C., C3 at 8°C., 
Cy, at 9°C., and C; was placed in the top compartment of the refrigerator 
which ranged from 9.5° to 10°C. 

When 27 hours old, the control group showed the germ-disc somewhat, 
further flattened on the yolk-sphere, but there was no visible indication 
of a germ-ring and the dise had not begun to descend over the yolk. 
This experiment was being conducted during the early June season, and 
normal development at this time was unusually slow. 

At 27 hours old, three other lots were placed in the refrigerator as 
follows, D, at 5°C., De at 6°C., and D; at 8°C. 

When 48 hours old, the control showed the germ-ring about one-fourth 
over the yolk-sphere with the embryonic shield clearly forming. The 
C and D series had become arrested and were still in much the same 
condition as when placed in the low temperatues on the previous day. 

The control at 3 days, or 72 hours old, showed the embryos well 
formed, though the germ-rings were not yet entirely over the yolk-sphere 
(fig. 1). 

Lot C,, having been 49 hours at 5°C., was still in high segmentation 
stages much the same condition as when placed in the refrigerator 
(fig. 2). These were now returned to room temperature. 

Lot C, showed much the same condition as C; and were also removed 
from the refrigerator. 

Lot Cz; seemed as completely stopped as the other two and was 
returned to room temperature. 

The members of the C, group were also in about the same stage as 
when placed in the refrigerator, though their temperature was 9°C. 
These remained in the refrigerator. 

The C; lot in about 10°C. had developed slowly, the caps had flat- 
tened and the embryonic shield had just become visible, though the 


STRUCTURE AND DEVELOPMENTAL RATE 127 


germ-ring had scarcely begun its descent over the yolk (fig. 3). These 
also remained in the refrigerator. 

Lot D, after 45 hours at 5°C., was still in about the same stage of 
development as when placed in the low temperature at 27 hours old. 
These are now placed at room temperature. 

Lot D» was in a closely similar condition to D;, but remained at the 
reduced temperature. 

Lot D; had also failed to make noticeable progress during the 45 
hours at 8°C., but was allowed to remain at this temperature. 


3 


Fig. 1 A control embryo 72 hours old, the body is well outlined and the 
germ-ring almost completely over the yolk. 

Fig. 2 An egg 72 hours old that had spent the last 49 hours at a temperature 
of 5°C. Development had been practically stopped in this high segmentation 
stage. 

Fig. 3 A specimen 72 hours old that had been during the last 49 hours at a 
temperature of 10°C. Development had progressed slowly, the germ-dise being 
flattened and the embryonic shield, indicated by stippling, has just become 
visible. 3 


128 CHARLES R. STOCKARD 


When four days old, the control embryos were fully formed with 
prominent optic vesicles, hearts were formed, but not yet pulsating. 
Thus they were not more than up to a midsummer 72-hour stage, since 
the heart beat had generally begun about this time. However, all of 
these embryos were normal and well, as is shown by their later records, 
even though the cool season had thrown them about 24 hours behind 
within four days. 

Lot C,, now having been at room temperature for 24 hours, were all 
going very well. The germ-rings varied in position from one-quarter 
to one-third over the yolk-spheres. Only a few had failed to resume 
development and the eggs in general were about up to the condition 
of the present control when they were 50 hours old. These C; eggs 
had now actually developed at room temperature for about 47 hours, 
the first 23 hours after fertilization and the fourth day. 

Lot C2: was also after similar periods of experience in a uniformly 
good condition with the germ-rings all about one-third over the yolk- 
spheres. Thus subjecting to low temperature after 23 hours of develop- 
ment is decidedly less injurious than similar treatment during the 
early cleavage stages, as will be seen from the records beyond. 

In lot C; the germ-rings had all descended about half way over the 
yolk-sphere. 

The D series showed somewhat the same response. Lot Dj, after 24 
hours at room temperature, were developing normally with the germ- 
rings from one-half to two-thirds over the yolk-spheres and the em- 
bryos well formed. Thus stopping for 48 hours after 27 hours of de- 
velopment, when the segmentation caps were flattened over the top of 
the yolk, showed no ill effects on their present development except to 
render them almost exactly two days behind the developmental stage 
of the control. 

The control at 5 days old had a vigorous heart beat, but the circula- 
tion was just beginning to be well established. 

Lot C,, almost all of the embryos were full length, the optic out- 
pushings were just beginning, but not fully formed, thus about in the 
condition shown by the present control at 72 hours. These were 
still about two days behind the control, or had practically lost the time 
spent in the refrigerator. There’ were a few with the germ-rings not 
entirely covering the yolk and with the body of the embryo short and 
poorly formed at the caudal end. 

Lot C2 were about in the same condition as C;. 

Lot C; seemed on the average a little further along, though closely 
similar to the two foregoing lots. 

Lot C., now four days in the refrigerator at 9°C., seemed in good 
condition, with the germ-rings well formed and descended about one- 
half over the yolk. These specimens had thus continued their de- 
velopment at this temperature, although very slowly, and had ad- 
vanced about 12 hours in development within the 4 days. They were 
now returned to room temperature. 


STRUCTURE AND DEVELOPMENTAL RATE 129 


Lot C; at about 10°C. for four days, were possibly a little further 
along then C,, though in general they showed a similar condition. 
These were also returned to room temperature. 

Lot D; contained full-length embryos, some with the optic processes 
already formed and others without. These specimens were about 
one and a half days behind the control or about in the stage of the two 
and a half day control. 

Lot Dz, after four days at a temperature of 7°C., introduced after 27 
hours of normal development, were still in about the same stage as 
when placed in the refrigerator. The segmentation caps were flat with 
early germ-rings forming and the embryonic shields just beginning. 
The descent of the germ-ring has been considerably prevented. All 
of the specimens were living and seemed well. They were now returned 
to room temperature. 

Lot D3, all seemed in good condition with germ-rings from one- 
quarter to one-half over the yolk-sphere and with well-formed embryonic 
shields. Thus this slightly higher temperature of 8°C. had given the 
D; group a considerable advantage in progress over the D, lot. These 
were now also returned to room temperature. 

When six days old, the black and red chromatophores were fully 
expanded on the yolk and embryonic bodies of the control specimens. 
The embryos were now occasionally twitching and moving their 
bodies. 

Lot C;, after being out of the refrigerator for 3 days, had embryos 
comparable to about a usual midsummer 70-hour stage, or about the 
condition of the present control when 4 days old. The heart beat had 
not begun. 

Lot C:, embryos were also in a stage just prior to the heart beat, 
and the C3; group was about the same. 

Lot C, were now out of the refrigerator for one day after having 
been at a temperature of 9°C. for 4 days. The embryos were well 
formed and the blastopore was about closing, so they had madea 
considerable advance from the condition of the previous day when the 
germ-rings were only one-half way over the yolk. The C; group are 
still further advanced with the optic outpushings prominently shown. 

Lot D, now showed chromatophores both on the yolks and on the 
embryos’ bodies, yet no heart beat could be detected in any of those 
examined. 

Lot Ds, when one day at room temperature after being at 7°C. for 
four days, showed the germ-rings*two-thirds over the yolk-sphere, 
with the embryonic axis well formed in the shield. 

Lot D3 contained long embryos with the optic outpushings just 
beginning, so these were still ahead of Dp. 

At seven*days the control embryos were actively moving and the 
yolk vessels were now clearly mapped out by the pigmented arrange- 
ment. 

Lot C;, now out of the refrigerator for 4 days, showed many em- 
bryos, with good circulations and pigment migration, some had a 


130 CHARLES R. STOCKARD 


heart beat, but had not established a circulation and others had not 
yet developed a heart beat. 

Lot Cy showed a good circulation in almost all. 

Lot Cs presented a majority with good circulation, there were, how- 
ever, many with imperfect circulation or no circulation, although the 
heart was pulsating. 

When 9 days old, the control presented a perfectly normal condition. 

Lot C; showed practically every specimen normal and _ strong, 
apparently just as good as the control, though somewhat behind. 

Lot Ce. were in equally as good a condition. 

Lot C3 was much the same as the other two groups. 

Lot Cy, also seemed to contain all normal embryos. 

Lot C; were further advanced than C,, since they had continued to 
develop slowly while in the refrigerator at the higher temperature of 
about 10°C. They had, therefore, developed slowly for 4 days, and 
after having been out for 4 days were practically perfect in their 
development. 

Lot D, were all normal at 9 days old and as perfect as the control 
except for the fact of being behind in developmental time due to the 
few days stand-still spent in the refrigerator. Thus development 
can be discontinued for 3, 4, or 5 days at the stages used in this 
experiment (27 hours old, just after gastrulation has started) with no 
subsequent ill effects on the development and structure of the early 
embryos. 

Lot D, contained specimens further behind in development than the 
D, group, since they remained in the cold longer, but all appeared 
perfectly normal at this time. 

Lot D3 were all normal. 

At 12 days old, the control seemed about in the condition to hatch. 

The C series which had been subjected to developmental interrup- 
tions after being 23 hours old now presented perfectly normal con- 
ditions. In lot C, three specimens had not developed and sixty were 
normal This is as good a record as is usually found under ordinary 
conditions. Lot Cy. contained about 100 specimens, which were all liy- 
ing and normal. Lot C; had about the same number in similar con- 
ditions. LotC,also contained about 100 normal specimens, so that the 
numbers examined were sufficiently large to furnish a very reliable 
index of the reactions. 

Lot C; contained a few more than 100 normal specimens and a 
single individual that was abnormally small, yet even this one was 
sufficiently normal to have a free blood circulation. 

Lot D,, which was put in the refrigerator 27 hours after fertiliza- 
tion, contained six specimens that did not develop out of a total of 
seventy-five eggs. The other sixty-nine specimens were normal. The 
D, lot were all normal, and so was the D; group, yet all were behind the 
control in their developmental stage corresponding to about the 
length of time they had spent in the refrigerator. 


STRUCTURE AND DEVELOPMENTAL RATE 131 


When 19 days old the control were almost all hatched actively free 
swimming young fish. The few yet unhatched seemed normal and 
ready to hatch at any time. 

Lot C; contained a majority hatched and all seemed normal. 

In lot C. there were not quite as many hatched, but all were in 
good condition. 

Lot C3 were about the same in hatching record, so there was little 
effect to be noticed at this time resulting from the two days spent in 
the refrigerator following their first 23 hours of development. 

Lot Cy had remained longer in the refrigerator, 4 days, and at this 
time none had hatched, though they seemed fully ready. In lot C; 
also none had hatched. 

Lot D; contained a majority hatched, almost as large a proportion 
as the control. These had remained in the cold only two days. Lots 
D, and D; had remained in cold for 4 days, and only one specimen in 
the two groups had hatched. All appear normal and ready to hatch. 

When 20 days old, the first one in C, had hatched. In lots D». and 
D; many had now hatched, so these are not very much later than the 
control in spite of their 4 days’ arrest. 

In lot C; none had yet hatched, although during the next 24 hours 
many of them did hatch. 

When 22 days old, a few of the control were still unhatched, though 
they were normal. Lot C, had 12 unhatched and 50 hatched. Lot 
C. contained 18 unhatched and about 80 hatched. Lot C; had 29 un- 

. hatched, one with a deformed body, and about 70 normal ones hatched. 
This record was about as good as a usual control. 

About half of the C, lot had hatched, and all seemed normal, though 
they remained in the refrigerator twice as long as C;, Cs, and C; had. 

Lot C; also showed about half of the specimens hatched. 

Lot D, had 7 unhatched and about 60 hatched, all of them seemed 
normal. 

Lot D, contained 29 unhatched and about 40 hatched, all of which 
were normal. 

Lot D; showed 20 unhatched and about 30 hatched. 

When 25 days old, every egg in the control had hatched. 

Lot C,, only 4 were unhatched, one of these had abnormally small 
defective eyes and no blood circulation. So these are a little behind 
their particular control in quality at this stage, but very little, and 
probably their disadvantage is of no significance, since such a single 
specimen might occur in any group of eggs. 

Lot C2, every specimen hatched. In lot C; only 3 failed to hatch. 
One of these was grossly deformed and the other two had slightly 
abnormal eyes. So this group is somewhat inferior when compared 
with the control record. 

Lot Cy, contained 12 specimens still unhatched. One hatched speci- 
men was bent and unable to swim. One of the 12 unhatched was 
abnormal, so this record also was a little worse than the perfect control. 


132 CHARLES R. STOCKARD 


Lot Cs; contained 10 unhatched, one of which was abnormal, the 
others were all normal. 

Lot D,, only one unhatched, all seem fine. 

Lot D. contained 2 unhatched, and lot D; had 3 unhatched, though 
all of these seemed normal. 


This experiment shows very clearly that stopping or arrest- 
ing the development of Fundulus eggs after about twenty-four 
hours of development, when gastrulation has definitely begun, 
produces very slight or no ill effects on such specimens up to 
the time of hatching and becoming free swimming little fish. 
Whether during later stages of growth these fish might show some 
disadvantages following the developmental interruption we have 
not attempted to determine. It is probable, however, that these 
specimens were interrupted in their development during a particu- 
larly passive period and that no later disadvantages would 
accrue. This would seem further probable since it is at just such 
a stage in development that the eggs of birds are normally inter- 
rupted, and clearly without ill effects on the group. 

These experiments not only show that stopping development at 


this stage, just after gastrulation has started, is not noticeably. 


injurious in effect on the development of the young fish, but 
further, that after gastrulation has commenced the rate of devel- 
opment of the embryo may be slowed to a most extreme degree, as 
occurred in the upper temperatures of the refrigerator, without 
serious injury to the structure of the young fish. 

To further establish the correctness of the above results, we 
may record one other similar experiment in brief detail. 


Experiment 906. Bs.:. A group of eggs when 24 hours old containing 
all normal fine specimens were placed at a temperature of 5°C., and 
later compared with a selected control from the same parents. 

At 46 hours old, the control were developing rapidly, with the germ- 
rings almost completely over the yolk and the embryos well formed. 

The B,.2 lot now in cold for 22 hours showed the same condition as 
when placed in the refrigerator except that the segmentation cavities 
were distended so that a vesicle appeared below each disc. These 
eggs were now moved to an upper compartment of the refrigerator to 
allow them to develop slowly at a temperature of about 9°C. 

When four days old in the 9°C. temperature they were developing 
slowly but normally, with the germ-rings about one-half over the 
yolk-spheres and with embryonic shields in which the axis of the 
embryo was beginning to form. 


——— 


STRUCTURE AND DEVELOPMENTAL RATE 133 


At five days old these eggs were still developing remarkably well 
although very slowly. The germ-rings were a little further over the - 
yolk. They were now returned to room temperature after having 
spent 4 days in the refrigerator, 24 hours at 5°C. and 3 days at 9°C. 

One day later, all of the eggs were developing and almost every one 
presented a well-formed embryo normal in appearance. 

When ten days old, all were living with a fine circulation of the blood 
and otherwise apparently normal. 

When 17 days old, 18 of these embryos had hatched and 24 were 
unhatched. 

After 24 days, 12 were still unhatched, one of these being very ab- 
normal. All of the embryos had seemed normal when ten days old, but 
at this time it was readily seen that the 12 unhatched specimens were 
really far behind the control. While showing no gross deformities 
they were smaller and not so well developed as the control. 

Although these early arrests do not give marked effects on the very 
young fish, it is certainly possible that many later symptoms might 
develop if their existence was observed through longer periods of time. 

When 29 days old, 4 embryos were still unhatched, one had died and 
3 seemed normal and ready to hatch. Thus the record of this group for 
the length of time it was followed does not compare unfavorably with 
the ordinary control records of Fundulus embryos up to a comparable 
period. As might be expected, however, eggs after being 24 hours old 
which were stopped or retarded in development for 4 days are not 
able to hatch on schedule time with the control; but are several days 
late in reaching the hatching stage. 


Such results will be found to differ entirely from those con- 
sidered beyond as obtained when the eggs are stopped during 
more critical developmental stages or at times when rapid cell 
proliferation and developmental changes are occurring. There- 
fore, it may be stated in general that certain indifferent mo- 
ments in development do exist during which time the rate of 
development may be slowed to almost stopping, or development 
may be actually stopped, and later resumed at a normal rate 
without causing structural anomalies or unusual conditions in 
the resulting young fish. 

It is also shown by the above experiment that development 
may be stopped at certain indifferent periods, in a temperature 
of 5°C. and then resumed at an extremely slow rate in 9°C. for 
several days, and later increased to a normally rapid rate at room 
temperature without injury. 

Thus it is not always necessary that development be promptly 
resumed at a normal rate in order to avoid structural defects. 


134 CHARLES R. STOCKARD 


The next experiment is cited to show the behavior of eggs 
arrested in still later periods of general indifference. 


Experiment 907.—Eggs with germ-rings one-quarter to one-third 
over the yolk sphere and with embryonic shields well formed, a stage 
acquired after 48 hours of development during the early cool June 
season, were placed in the refrigerator in two groups, Es at 6°C. and 
E; at 8°C. 

After 24 hours in the refrigerator they had advanced only slightly 
beyond the condition of the day before. The E; group had advanced 
somewhat more than the E, lot particularly in the formation of the 
embryonic line, or axis, in the shield. 

When 5 days old, and after having been in the refrigerator for 3 days, 
the E, group at 6°C. have advanced the germ-ring to about two-thirds 
over the yolk sphere. They were thus not as completely stopped by 
this temperature of 6°C. as were eggs placed in the same temperature 
during early cleavage stages, as will be seen beyond. These eggs 
were now, after 3 days of extremely slow development, returned to 
room temperature. 

The E; lot at this time showed the germ-ring almost completely over 
the yolk-sphere, and the embryonic body was well formed in the ma- 
jority of the eggs. These specimens at a slightly higher temperature 
had developed somewhat further than those above. They were now 
also returned to room temperature. 

After being at room temperature for 24 hours, the rate of develop- 
ment had greatly increased in both lots. The E, group now showed 
long embryos with the optic outpushings well begun in many. The 
E; lot showed optic outgrowths well formed in all, and were thus a little 
ahead of the E, ones in development. 

At 9 days old, the specimens in both lots seemed behind the control 
to the extent of their 3-day stay in the refrigerator. 

When 12 days old, they were closely examined for slight anomalies. 
The E, lot showed one abnormally-small embryo with no blood cir- 
culation, 4 had stopped, and did not develop after removal from the 
refrigerator, and 45 specimens seemed to be in normal condition. 

The E; lot all appeared to be normal except that they were about 
3 days behind the control in their development. 

Thus subjecting the embryos to a severe reductionin developmental 
rate after they were 48 hours old had only slight, if any, detrimental 
effect on their ability to resume a normal developmental rate and to 
form apparently normal young embryos. Very probably, however, 
minor effects are produced which would be indicated in the later struc- 
tural or physiological history of the specimens could they be studied 
through a longer season of their existence. 

At 19 days old, when a large majority of the control had hatched 
and were free swimming, none of the E» or E; lots had hatched. But 
when 21 days old, a number were hatched in both lots. 


—— UCU lee 


STRUCTURE AND DEVELOPMENTAL RATE 135 


When 22 days old, the E. group contained 25 hatched and 20 un- 
hatched. Three of the latter were abnormal with no blood circulation, 
two being small and inactive, and the third was grossly deformed. 
The E; group had 25 hatched and 11 unhatched, all of which seemed 
normal in structure. 

At 25 days old, 4 of the E, group were still unhatched, but all of 
the E; lot had hatched. They were kept until 34 days old, at which 
time many had died on account of the difficulty in feeding them, but 
the 4 specimens in lot E, never succeeded in hatching. 


When these records of late arrests are compared with those 
from eggs arrested during early cleavage stages, one will be struck 
with the low mortality following removal from the refrigerator in 
the case of the former. The complete absence of double monsters, 
ophthalmic deformities, etc., among the specimens arrested dur- 
ing late stages also contrasts with the common occurrence of such 
conditions among specimens arrested during cleavage stages. 
The general nature of the circulatory disturbances, etc., which do 
occur after late arrests is also characteristic. A contrast is 
further noted by considering this experiment in comparison with 
the specimens described above which were introduced into the 
cold after one day of development—there again the advantage in 
subsequent development is on the side of those specimens caused 
to develop very slowly during the later developmental stages. 
But of the specimens almost completely stopped in development, 
those stopped very soon after gastrulation seem to have an ad- 
vantage over specimens stopped when one day older, or further 
advanced in development. The stage immediately following the 
first rapid changes of gastrulation would seem to be an extremely 
indifferent period. 

Two other sample experiments will be reviewed in brief to 
illustrate the gross reaction following still later developmental 
interruptions. It must be realized that in all of these experi- 
ments we are at present simply recording the outward gross 
appearance and behavior of the specimens. A closer microscopic 
examination of the young fish in section might show a consider- 
able depression in the development or expression of certain inter- 
nal organs, for example, the conditions in the branchial regions, 
digestive glands, ete., while observation of the living specimen 
had given no indication of its inner defective condition. 


136 CHARLES R. STOCKARD 


Experiment 908. Specimens 72 hours, or three days old, with the 
optic cups already invaginated and formed, but just before the begin- 
ning of a heart beat, were carefully selected, so that every individual 
was normal and good, and arranged in two groups. Group F;, consist- 
ing of 62 vigorous specimens, were placed in the refrigerator at 5°C. 
and group Fs, containing 36 normal embryos, were subjected to a tem- 
perature of 8°C. 

When 6 days old and after being 3 days in the refrigerator the F; 
lot were in much the same condition as when put in the cold, the 
hearts had not begun to beat and the general structural appearance 
had not changed. The F; lot were a little further advanced, but 
there was still no heart-beat. The control embryos at this time 
have, of course, a vigorous circulation of the blood, they are well pig- 
mented and the yolk vessels are mapped out by the chromatophores. 

At 8 days old, the F, group were still in the same condition as when 
put in the 5°C. temperature 5 days before. There was no heart beat 
and the embryos appeared as if about 3 days old. They were now 
returned to room temperature. 

The F; lot, after 5 days at 8°C., were further advanced, their hearts 
were pulsating feebly and very slowly, blood-cells were formed on the 
yolk-sacs and masses of blood were frequently observed in the tail 
regions. These embryos were also now returned to room temperature. 

After being at room temperature for 3 days, with a total age of 
eleven days, the F, lot seem recovered and are developing well, though 
about 4 or 5 days behind the control. All of this lot were living. 
The F; lot were also all alive and in apparently perfect condition. 

When 18 days old, almost all of the control embryos had hatched. 
The F, lot all seemed normal, but none had hatched, and the same was 
true of the F; group. Two days later, however, many had hatched 
in both lots. Thus they were 3 or 4 days later than the control in 
hatching, which was a little less than the time they had spent at low 
temperature. 

Finally, when 27 days old, none of the embryos in the two lots had 
died, which indicates that they were all unusually good specimens. 
Every one of the 36 in the F; group hatched, and but 2 in the F; group 
failed to hatch, although these appeared normal in structure. 


A complete stop or an arrest in developmental rate of as much 
as five days after the optic cups are already formed and just before 
the beginning of a heart beat does not exert an injurious effect 
upon any organ that would prevent the normal development of 
the body form or the capacity to hatch and swim freely. 


Experiment 909. Embryos 6 days old, with fully vigorous blood 
circulation over the yolk-sac and within the embryonic body, with 
chromatophores fully migrated and expanded, and with their bodies 
moving and twitching, were placed in a temperature of 7°C. After 


STRUCTURE AND DEVELOPMENTAL RATE 137 


24 hours the hearts were still beating, but much slower than the con- 
trol, and they had fallen about 20 hours behind the control in devel- 
opment. 

After 3 days in the cold these embryos had fallen far behind the 
control in size and development. The heart was beating slowly and 
the blood was circulating in all. 

Two days later, when the embryos were 11 days old, they were 
still in about the 6-day condition, although all were living at a slow 
rate during the 5 days in the refrigerator. 

When 13 days old, and after being 7 days in the low temperature, 
the embryos were all alive. They had a slow heart beat and a circu- 
lation which in many was so sluggish as to allow large sinusesin the 
yolk-sac to remain distended with blood, although the circulation 
within the embryonic body was complete. At this time they were 
returned to room temperature, and after 24 hours the heart beat had 
regained a normal rate and the blood was circulating freely and fast in 
each of the specimens. All seemed fully recovered from the depres- 
sion caused by the low temperature. 

At 19 days old, almost all of the control embryos had hatched, but 
none of these that had spent 7 days at 7°C. were yet up to the point 
of hatching. 

At 22 days old, still none were hatched. But when 23 days old, 16 
had hatched and 38 were unhatched. They were thus 5 days behind 
the control in beginning to hatch as a result of their 7 days of slow 
development at the low temperature. 

On the 25th day only 2 were still unhatched, and finally, on the 
27th day, these two had not hatched, although they seem normal in 
structure. 


There is, therefore, no evidence that any harm was done by 
subjecting advanced embryos with blood freely circulating to low 
temperatures. Although under the cold conditions the heart 
rate was greatly reduced and the circulation rendered extremely 
sluggish for a period of seven days. On return to normal tem- 
perature recovery was rather prompt and seemed on superficial 
examination to be complete. 

A number of similar experiments to those reviewed above are 
recorded in my notes, and in all cases the results are in close 
accord. If we consider them entirely from a standpoint of the 
external evidence of injury produced, a fair comparison may be 
made with the results of further experiments in which the eggs 
were stopped and arrested at other developmental periods or 
moments. It will be readily shown that periods very close to 
some of those used above are decidedly dangerous moments at 


13 


19/4) 


CHARLES R. STOCKARD 


which to stop or interrupt the progress of development. From 
such experiments one seems justified in classing these moments 
in development as indifferent at which arrests may be induced 
without causing subsequent high mortality among the embryos 
and without a considerable percentage of gross structural de- 
formities resulting. The eggs treated in the above experiments 
were all stopped at comparatively indifferent moments in the 
course of development so far as their gross structure and behavior 
up to the newly hatched free swimming stage of life would indi- 
cate. In the section following a review of experiments with 
decidedly different results will be considered. 


c. Stopping or retarding the progress of development at stages of 
critical susceptibility to developmental interruption 


From facts we know of development in nature, as well as, from 
the experiments discussed in the preceding section, it becomes 
evident that the course of embryonic development need not nec- 
essarily progress in a continuous manner, but may be stopped 
entirely for a considerable length of time or may be decidedly 
reduced in rate without necessarily injuring the end result. On 
the other hand, it is equally well known in a general way, and 
even more widely believed, that when a developing egg is injured 
in such a manner as to cause its development to stop, it is usu- 
ally incapable of resuming development at all, or if it does start 
again to develop it will only continue for a short time and often 
in a very abnormal fashion. 

These two apparently contradictory statements are equally 
true. This is due to the fact that the way in which a developing 
egg responds after having had the progress of its development 
stopped or arrested by any unfavorable condition depends 
entirely upon the stage in development at which the interruption 
occurred. In the first case stated above, the interruption is 
introduced at a stage in development when no unusually rapid 
changes are taking place, a comparatively quiescent moment 
during which all parts are developing, but during which no par- 
ticular or important part is going at an excessively high rate. 
Such a time we may term a ‘moment of indifference.’ 


STRUCTURE AND DEVELOPMENTAL RATE 139 


In the second case, the interruption occurs at a time when 
certain important developmental steps are in rapid progress or 
are just ready to enter upon rapid changes, a moment when a 
particular part is developing at a rate much in excess of the rate 
of the other parts in general. Gastrulation is an important 
developmental step which apparently cannot be readily inter- 
rupted without serious effects on subsequent development. 
Many of the chief embryonic organs seem also to arise with ini- 
tial moments of extremely high activity, processes of budding or 
rapid proliferation and growing out. During these moments a 
given organ may be thought of as developing at a rate entirely 
in excess of the general developmental rate of the embryo. Such 
moments of supremacy for the various organs occur at different 
times during development. As is well known, a certain organ 
arises much earlier or later in the embryo than certain others. 
When these primary developmental changes are on the verge of 
taking place or when an important organ is entering its initial 
stage of rapid proliferation or budding, a serious interruption of 
the developmental progress often causes decided injuries to this 
particular organ, while only slight or no ill effects may be suffered 
by the embryo in general. Such particularly sensitive periods 
during development I have termed the ‘critical moments.’ 

That we may analyze the responses of embryos in which devel- 
opmental interruptions have been introduced during some of 
these critical moments, resource may again be had to the records 
of the experiments. Here also a large number of experiments 
have been performed, but we shall only attempt a review of cer- 
tain typical examples from the entire series. 


Experiment 901, B Series. Eggs were fertilized at 11 a.m., and 
three hours later, immediately before the first cleavage, they were 
divided into four lots, one for control and three others which were 
placed in a refrigerator at temperatures of 5°, 7°, and 9°C. 

When 24 hours old, the control had reached a high segmentation 
stage, the germ-discs in only a few had flattened down on the yolk 
sphere, but in none had the cap begun to descend over the yolk or to 
form the germ-ring. The night had been unusually cool and the 
control was thus developing far more slowly than the normal sum- 
mer average rate. At 24 hours old, the germ-ring is usually well 
formed and has descended about one-third to one-half way over the 


140 CHARLES R. STOCKARD 


yolk-sphere. The inhibition resulting from the cool nights of the 
early season very probably accounts for the almost uniform inferiority 
of embryos developed at this time as compared with those developing 
during early July, the height of the spawning season for thus locality. 

Lots B; and Be, in temperatures of 5° and 7°C., respectively, for 19 
hours, were all in either 2- or 4-cell stages. They were thus almost 
completely stopped in development. The 2-cell stage was about 
reached when they were placed in the low temperatures, and probably 
some were dividing the second time before the surrounding water had 
cooled to the temperature of the refrigerator (all dishes contained 60 
cc. of sea-water). 

Lot B; at 9°C. contained after 19 hours fairly regular 16- and 32-cell 
stages. At this temperature cell division had been able to continue, 
although at a greatly reduced rate, accomplishing only three or four 
divisions in the 19 hours. 

The control eggs 48 hours after fertilization showed the germ-ring 
only one-quarter over the yolk sphere, with the embryonic shield be- 
ginning to form (fig. 4), a stage that should be attained within 24 
hours during the warmer part of the season. 

Lot B,, after 45 hours at 5°C., was in first-, second-, or third-cleavage 
stages. The arrangement of the cell groups was often very irregular 
and many cells contained large vacuoles. There were a very few 
almost typical 2- and 4-cell groups. In some of the ‘2-cells’ a large 
central vacuole seemed to almost divide each of the cells (fig. 5). 

These eggs at 5°C. have thus only in rare cases divided more than 
once during 48 hours. This lot was now removed from the refrigerator 
and returned to the room temperature after being 45 hours in the cold. 

Lot Bs, at 7°C., was in much the same condition as lot Bi, except 
that some eggs had undergone one or two further cleavages. There 
were many irregular cleavage patterns and a few almost regular 16- 
or 32-cell stages. A number of the germ-dises consisted of irregular 
partly divided masses (fig. 6). 

Lot Bz, at 9°C., had developed very slowly but fairly well, and now 
after 45 hours in the low temperature contained germ-discs composed 
of from 64 to about 128 cells. The cell arrangements and shapes of the 
dises were almost uniformly regular. Therefore, at this temperature 
development progresses, though very slowly, and none of the cell masses 
had yet begun to flatten down to cap the yolk-sphere. 

When 3 days old, the control embryos were well formed, although 
the germ-ring was not yet entirely over the yolk-sphere, much the 
same stage as shown above in figure 1. 

Lot B,, after being at room temperature for 24 hours, had passed 
from the 2-, 4-, and 8-celled conditions and had reached a high seg- 
_mentation stage. The discs had not fully flattened on the yolk- 
spheres, but were beginning to descend. There was no gross indication 
of germ-ring or embryonic-shield formation. Many eggs had promptly 
recovered their ability to develop on return to higher temperature and 
had progressed during the 24 hours about as far as the control had gone 
during the first 24 hours of their development. 


STRUCTURE AND DEVELOPMENTAL RATE 141 


Lot Bz had now been for 70 hours at 7°C. These showed many 
irregular germ-dises, but some were fairly regular 16- and 32-cell stages. 
Their condition was thus much the same as on the day before and 
they had scarcely progressed at all during the 24 hours. These eggs 
were now returned to room temperature. 


Fig. 4 A control embryo 48 hours old, the germ-ring only one-quarter over 
the yolk, far behind the usual stage on account of the cool season. 

Fig. 5 A group of cleavage patterns 48 hours after fertilization and after 45 
hours at a temperature of 5°C. Development is practically stopped. In many 
of the two-cell stages large vacuoles, V, occupy the entire center of the cells. 

Fig. 6 An irregular partly undivided protoplasmic mass with blastomeres 
at its ends, 48 hours old after 45 hours at 7°C. 


Lot B; still had, after 70 hours at 9°C., high segmentation discs 
about the 128-cell stage. The dises were normal in general appearance. 
Thus at this temperature development continues, but at an extremely 
slow rate. This lot was now also returned to room temperature. 

When the eggs were 96 hours, four days old, the control embryos 
were fully formed with prominent optic vesicles, the embryonic heart 
was not yet visible, and there was no pulsation. These embryos were 
thus searcely up to the midsummer 72-hour stage, since the embryonic 
heart beat is often fully established before such a time. The cool 


142 CHARLES R. STOCKARD 


weather of early June had caused this control to fall about 24 hours 
behind in the four days. Although such embryos appear to be nor- 
mal, many of them are inferior in size and general appearance when 
compared with more rapidly developing specimens of the later warmer 
season. This advantage is no doubt due to the retarded development 
primarily resulting from the cooler temperature, and not to a poorer 
quality of the eggs, since the midsummer eggs will fare in a similar 
fashion when caused to develop at the same temperature. Such a 
retardation, however, is too slight to produce gross defeats in any 
average lot of eggs, yet the embryos very probably are somewhat below 
par as their physiological responses would indicate. 

Lot B; had now been for 2 days, 48 hours, at room temperature after 
having spent 45 hours at 5°C. The germ-caps were about one-half 
over the yolk-sphere, the germ-rings and embryonic shields were well 
formed in most of them. They presented the condition of a midsum- 
mer 24-hour stage, or were about up to the condition of the present 
control at 48 or 50 hours. Thus during the 48 hours at room tempera- 
ture these eggs had developed about as rapidly as did the control during 
their first 48 hours of development. 

The embryonic shields with the embryo in outline appeared normal, 
although some were considerably behind others and a great many 
failed to resume development after being removed from the refrigerator. 

The lot Bo, after 24 hours at room temperature following a stay of 
70 hours at 7°C., showed disc-like caps flattened down, but no germ- 
rings were yet formed and the disc had not begun to descend over 
the yolk-sphere. Some caps were still high or mound-like and many 
were irregular, containing cells of different sizes (fig. 7). A large 
number of eggs failed to resume development and there were many 
dises with vacuoles in their centers, ete. 

The mortality resulting from this exposure was, therefore, high and 
many embryos were rendered abnormal during these early stages. 

The lot B;, after 24 hours at room temperature, were in an even 
worse condition than those in Bs, although a single individual had a 
germ-ring one-fourth over the yolk-sphere and was thus the most 
advanced specimen of the two lots. The majority, however, presented 
high germ-dises with a peculiar vacuole occupying about half of the 
dise and distorting the position of the cells (fig. 8). 

Vacuoles similar in appearance are frequently present in eggs slowed 
by other methods, such as solutions of LiCl, ete. But in this case the 
vacuole differs somewhat in not being a simply distended segmentation 
cavity. 

It will be recalled that these eggs developed very slowly at 9°C. for 
70 hours, so that they had progressed much beyond the lots B; and B: 
when removed from the cold. Yet after 24 hours at room temperature 
they were at a disadvantage rather than an advantage when compared 
with B. at this moment. The extremely slow progress during the 70 
hours would seem to be more detrimental at this stage than the almost 
complete cessation of development in lot Bs. In later stages, however, 


STRUCTURE AND DEVELOPMENTAL RATE 143 


those eggs which have been subjected to the higher temperature will 
gain a decided advantage as compared with the lower-temperature 
groups. 

At 5 days old, the control showed the heart beat just beginning, 
but no circulation. Lot By, after 3 days at room temperature, con- 


VTA 


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Fig. 7 Three specimens 4 days old, having been 24 hours at room temperature 
following a stay of 70 hours at 7°C. The upper outline shows a disc-like cap 
flattened down on the yolk-sphere; the middle one, a high segmentation cap; 
and the bottom specimen has a cell mass comparable to a normal 12-hour stage. 

Fig. 8 Top and lateral views of 4-day specimens, having been 24 hours at 
room temperature following 70 hours at 8°C. These segmentation masses are 
very abnormal and are distorted by the presence of a huge vacuole, V. 


tained short embryos on the surviving eggs, but the majority of eggs 
failed to develop at all after being removed from the cold. Lot B» 
had germ-rings only about one-half, or a little more, over the yolk- 
sphere. Thus the one day longer in the refrigerator had caused these 
to be far behind B). 


144 CHARLES R. STOCKARD 


The Lot B; had germ-rings also a little more than half over the yolk, 
though here again a great many were not developing at all. 

The 6-day-old control presented black and red chromatophores 
fully expanded on the yolk-sac and the embryo. The circulation was 
completely established both within the embryonic body and on the 
yolk-sac. The embryos had begun twitching and moving their bodies. 

Lot B, had now been at room temperature for 4 days after having 
been arrested for 45 hours at a temperature of 5°C. The embryos 
were small with no circulation, almost all seemed abnormal at the 
head end and many were short; the tail region was not properly formed. 
They were thus far behind a usual 4-day embryo. 

Lot Bo, after now developing at room temperature for 3 days, con- 
tained many small cyclopean and otherwise defective embryos, but the 
majority of eggs had stopped and did not develop beyond the condition 
shown by them after the 70-hour stay at 7°C. 

Lot B; contained some fairly regular 3-day embryos, but with no 
circulation, and many of these were deformed. 

Seven days after fertilization the blood-vessels of the control 
embryos were well mapped out by the alignment of pigment and the 
embryos themselves were vigorously active. 

Lot B; contained at this time many well-formed embryos with good 
circulation, pigment migration, ete. Others had a sluggish and poorly 
established circulation, some showed a heart beat, but no circulation, 
and many more had stopped in development and the cells had wan- 
dered apart to le over the yolk surface. Some eggs presented simply 
yolk-saes with blood-spots scattered over them, but without an em- 
bryo. A few of the apparently well-formed embry os were abnormal 
in various ways. 

Lot Bz showed no circulation, many eggs did not develop, and almost 
all were readily seen to be abnormal. The lot B; also showed no cir- 
culation, but contained some well-formed embryos just about in con- 
dition for the heart beat to begin. 

When 9 days old, the control contained all fine vigorous embryos. 

Lot B, still showed those with only blood and pigment on the yolk-sae, 
with noembryonic body present. Others still had the cell-mass confined 
to the upper yolk-pole and there were a few abnormal embryos, some 
with and others without a circulation. The majority of the living 
specimens were now normal in appearance with a vigorous circulation, 
as if some degree of regulation and recovery had taken place 

Lot B, contained many apparently normal embryos with a good 
circulation, while some were small and some were abnormal without a 
circulation. Some eggs showed the old mass of early cleavage cells 
at the upper yolk-pole still alive after 9 days, though not developing; 
the cell-masses were irregular and the individual cells spherical in form. 
Several yolk-sacs also contained blood-cells and a few pigment cells, 
although no embryo was present. 

Lot B; contained a few eggs with early cell-masses similar to those 
in lot Bs. The large majority of the surviving individuals now seemed 


STRUCTURE AND DEVELOPMENTAL RATE 145 


_ normal with a good circulation; very few were slightly defor med with 
poor or no circulation. 

The majority in all B lots were now normal in appearance with a 
good circulation. In the B; lot 47 seemed normal out of 61, so that 
14, or about 25 per cent, were abnormal, and of these 6 show ed the 
early cell-mass condition or were not developing. Thus only 8 
embryos were smaller or slower than normal. Yet it must be recalled 
that many dead eggs had been removed during the first few days 
following return to room temperature. In the control, however, there 
were no abnormal ones and there had been no unusual mortality. 

When 12 days old, the control were all normal and about in the 
condition to hatch. 

In lot B,; 6 showed that development had stopped during an early 
stage, 4 showed yolk-sacs with blood and pigment but no embryos, 10 
were deformed embryos with no circulation, 4 were also deformed, 2 
being eyeless, but with a circulation. Of all the survivors in this lot 
24 were affected and 45 were apparently normal at this time, thus over 
34 per cent were bad. 

In lot By 14 failed to develop beyond the cell-mass stage, 4 pre- 
sented only yolk-sacs with blood-spots and pigment cells, 8 were 
abnormal with no circulation, and 3 were abnormal with a circula- 
tion, while 31 appeared-to be normal. Thus 15 of those that continued 
to develop, or about 33 per cent, were abnormal and 25 per cent of the 
total number that lived were unable to resume development after their 
stay at 7°C. 

In lot B; 6 stopped development early, though continuing to live, 
3 were deformed and possessed a circulation, 5 were deformed without 
a circulation, and 47 individuals were apparently normal. Here, then, 
only 14 per cent were deformed of those that developed. Such a 
record is twice as good as that attained by either of the other groups. 
Thus the 9°C. temperature, at which an extremely slow rate of de- 
velopment is possible, is not so injurious to the later development of 
those individuals which survive it as are the more severe temperatures 
of 5° and 7°C., which practically stopped the progress of development 
entirely. 

The control when 15 days old had not yet begun to hatch, on account 
of the cool season. In lots Bi, Bs, and Bs one or two more of the 
abnormal embryos in each had died and all of the individuals were 
behind the control in their developmental condition, though, as stated 
above, many in all groups now appeared normal. 

When 19 days old, a large majority of the control were hatched 
and swimming about in a typically active fashion. In lot B, none 
had hatched and several more had died. In Bs none had hatched and 
a few more also had died. In B; none had hatched, many still seemed 
normal, and many were deformed, showing distinctly typical eye 
anomalies, cyclopia, etc., and there were many types of head and 
caudal end deformities. 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 2 


146 CHARLES R. STOCKARD 


When 21 days old, in lot B; 2 more had died and 3 had hatched, - 
in B, 2 had hatched, and in Bs many had hatched. 

The control at 22 days old showed 47 hatched and 18 unhatched, 
although all were normal. In lot B, 5 had hatched, and 52 were 
unhatched, the majority were normal in appearance, but 13 were 
grossly deformed in the head region and possessed small ill-formed 
bodies. In lot B. 4 had hatched and 36 were unhatched, of these 11 
were grossly deformed and 25 seemed normal in structure. In lot B; 
15 were hatched and 39 were not, of the latter 7 were grossly deformed, 
one a typical cyclops and one a monophthalmia. Four others had 
slightly underdeveloped eyes in addition to the 7 actually deformed. 

When 23 days old, only 2 of the control were still unhatched. In 
lot B; there were 35 hatched and 20 unhatched. Lot Bs contained 27 
hatched and 12 unhatched. In lot B; over 40 had hatched and only 
9 were unhatched. One had died and 2 of those that had hatched 
showed their bodies so badly twisted that they were unable to swim. 
One of these had a badly deformed body and one eye was abnormally 
small with the lens protruding. 

At 25 days old, every individual in the control lot had hatched. 
Lot B; had 16 unhatched and 4 of those that had hatched showed 
deformed bodies and could not swim in a straightforward manner. 
Thirty-seven of those hatched were normal in appearance, 3 of the 
unhatched had died. The following deformed conditions existed: One 
was a double-headed specimen, many had no eyes, monophthalmia, 
abnormally small eyes, short bodies, etc. Thus at this time after the 
great number of specimens had died there were still over 20 per cent 
deformed. 

In lot B, 11 were unhatched and 29 had hatched. Two of those 
hatched were so deformed and twisted as to be unable to swim. The 
11 unhatched ones were all grossly deformed, so there were 13, or 33 
per cent, of the total living specimens deformed at this time. 

Lot B; showed 5 unhatched and about 40 hatched. Four of those 
hatched were so deformed as to be unable to swim in a normal fashion. 
One of these presents the peculiar condition of a heart beat, but no 
circulation in a hatched fish with a long normally shaped body. There 
was a large accumulation of blood-cells within the sinus venosus and 
the median vein in the region of the anus was filled with red cor- 
puscles. This specimen could swim poorly from place to place, had 
fairly regular respiratory movements, and waved its fins without a 
circulation of its blood. 

When 34 days old, the B, lot finally had 9 specimens which were 
unable to hatch, all of them were deformed. 

Lot B, showed 6 unable to hatch, all deformed and without a blood 
circulation. In lot Bs; 4 failed to hatch. It must be recognized that 
a great many specimens in each of the lots B;, Bo, and B; had died 
during the preceding 20 days. The weaker and actually most defective 
individuals are eliminated as shown by the early mortality records. 


STRUCTURE AND DEVELOPMENTAL RATE 147 


The above 6 unhatched embryos in lot Bz were kept in order to deter- 
mine how long such specimens might be able to survive. When 52 
days old, these specimens were still alive, although the yolk-sphere 
had become very small, being almost absorbed. The small monsters 
were practically at a stand-still as to their life processes and were not 
kept after this time. 


These experiments are here considered in a general way with- 
out going into the details of the deformities concerned. They 
demonstrate the fact that a normally continuous development 
may be modified into a discontinuous one by stopping its course 
during a very early cleavage stage. The fact is also shown that 
this stoppage is followed by a too slow resumption of the devel- 
opmental rate and results in about 33 per cent of gross anoma- 
lies among those specimens able to survive the treatment. The 
mortality induced by stopping at such periods is high, the major- 
ity of eggs in all cases dying after return to normal temperature. 
Great variation in ability to withstand such treatment is shown 
by these hardy Fundulus eggs. The weakest ones succumb with- 
out resuming development on removal from the cold. Stronger 
specimens may undergo a few further divisions and live for some 
time in a high segmentation stage without being able to continue 
or progress further in their development. Other eggs continue 
development, but in such extremely abnormal fashion as to fail 
completely to form the embryonic body and only differentiate 
certain tissues scattered irregularly over the yolk-sac. Still more 
hardy specimens succeed in forming the embryonic body, but 
many organs requiring a high degree of cell proliferation and 
growth for their development, such as the eyes, other brain diver- 
ticula, mandibular, hyoid, and branchial pouches, ete., are unable 
to form in a normal fashion, and numerous defects in these parts 
are to be found. 

Finally, the most resistant or hardiest eggs withstand the stop- 
page due to the low temperature and are able to resume develop- 
ment at an almost normal, though slightly retarded rate. ‘These 
individuals may seem typically normal in structure, and often 
develop into hatched free-swimming fish, yet even these not in- 
frequently show some indication of a subnormal condition in 
having their bodies slightly twisted or bent, and in being unable 


148 CHARLES R. STOCKARD 


to swim in a perfect fashion. Very probably the best of these 
specimens would present various ill effects from their early arrest 
could they be kept and observed throughout a longer life period. 
There are only a few simple performances to be observed in the 
actions of a newly hatched fish. Whether they are later capable 
of feeding and digesting food, reproducing, and performing other 
functions in a normal fashion is unknown for such individuals. 
The probable later effects as well as the classification of the de- 
formities following stoppage at various developmental moments 
will be more fully considered in the subsequent sections. 

One other similar series of experiments may be briefly recorded 
to further make clear the results which follow various degrees 
of interference with the rate of development during its early 
stages. A careful consideration of these records also brings out 
some of the differences between the effects of completely stopping 
and of slowing to a decided degree. The significance of the very 
varied types of deformities which result from early interrup- 
tions will be considered in connection with the records in the 
following sections of the discussion. 


Experiment 902, B, C series. Three hours after fertilization, when 
in the 2-cell stage, eggs were placed in the refrigerator in the following 
arrangement: B,; and C; at 5°C., By at 7°C., and B; at 9°C., with a 
control from the same groups of eggs kept at room temperature. 

When 24 hours old, the control were all developing in a perfect man- 
ner, but again somewhat slower than the maximum midsummer rate. 
The germ-caps had flattened on the yolk, but there was neither germ- 
ring nor embryonic shield formation yet visible. The B; and C, lots 
had all divided once or twice before cooling down to the 5°C. tempera- 
ture. Every egg in both vessels was alive and in the 2-, 4-, or 8-cell 
stage. In the C; lot almost all were 8 cells. In many the 8 cells 
were arranged into two groups of four (fig. 9). 

Lot Bz were, as a rule, in the same condition, all eggs being alive, 
the great majority in the 8-cell stage, with a few showing the 4-cell 
stage. 

The B; lot, after 24 hours at 9°C., were practically all developing 
at a very slow rate and had reached about the 64- or 128-cell stage. 
They seemed normal and in good condition other than for their very 
slow progress. 

Forty-six hours after fertilization, the control showed every egg 
developing, the germ-ring having grown almost completely over the 
yolk-sphere, the embryonic body was well formed, but the optic out- 
pushing had not yet arisen. 


STRUCTURE AND DEVELOPMENTAL RATE 149 


In lots B; and C, the eggs had divided once during the last 24 hours 
and were now almost all in the 8- and 16-cell stages, while a few were 
irregular 32-cell stages. Much cellular disorganization had taken place 
and the cell groups were broken and irregular, often with large unseg- 
mented protoplasmic masses. 

Lot Bz were in somewhat similar conditions, all showed more or less 
irregular 8- and 16-cell masses. Many also showed large unsegmented 
protoplasmic areas with a few cells around the periphery (fig. 10). 

Lot B; at 9°C. were all developing somewhat faster than the above, 
and now presented well-arranged high-segmentation caps. They were 
normal in appearance up to this time. 

When 4 days old, the control showed a perfect condition with not 
one egg having failed to develop. There was a vigorous heart beat and a 


9 10 


Fig. 9 A cell group 24 hours old, having been in a temperature of 5°C. since 
three hours after fertilization. The 8-cells are peculiarly arranged into two 
groups of 4 each, such specimens may give rise to ordinary single individuals. 

Fig. 10 A large unsegmented protoplasmic mass with blastomeres around 
the periphery. A frequent specimen in lot B2, experiment 902, when 46 hours old 
after 43 hours at 7°C. 


good circulation fully established. They were thus developing consid- 
erably faster at this time of the season than did the control of experi- 
ment 901, which was fertilized 10 days earlier. These 901 embryos 
had not developed a heart beat or established a circulation when 4 
days old. 

Lot Bi, after 4 days at 5°C., showed a few regular cleavage caps of 
about 64 cells. The majority, however, exhibited very irregular 
cleavage arrangements and some were almost amorphous protoplasmic 
masses, although all were translucent and alive. The eggs had, there- 
fore, developed at an extremely slow rate, but had not completely 
stopped. These specimens were now placed at room temperature. 

In lot C, the majority had, after 4 days at 5°C., rather regular 
64- or 128-cell caps. This lot was from a different group of eggs 
than the B series, and its control was going in a manner exactly similar 


150 CHARLES R. STOCKARD 


to the B control. These eggs, however, may be individually more 
resistant. This lot was also now returned to room temperature. 

Lot Bs, after 4 days at 7°C., showed some eggs with regular cleay- 
age caps of 64 cells and more, but the majority showed caps of irregular 
cell masses. These were now placed at room temperature. 

Lot Bs, after 4 days at 9°C., had all reached a high segmentation 
stage comparable to about the condition of the control at 18 or 20 
hours old. All of these eggs had a distended bubble-like segmenta- 
tion cavity similar to that described by me (’06) as resulting from 
treatments with LiCl solutions. Every egg was developing and fur- 
nished a particularly fine lot for an experimental test of this sort. 
These also were now placed at room temperature. 

At 5 days old, the B and C controls were perfect with all embryos 
developing well. In lot B,; the great majority had failed to resume 
development after being 24 hours at room temperature. The seg- 
mentation caps were breaking down and becoming disorganized. The 
few specimens that had resumed development showed the germ-ring 
formed and about one-half over the yolk-sphere. 

The lot By were in very nearly the same condition as By. 

In B; the great majority were developing and the germ-rings were 
here also about half over the yolk-sphere. ; 

Lot C, showed many stopped in development, but here the major- 
ity seemed well and showed the germ-ring about one-quarter over 
the yolk. 

When 6 days old, the treated groups had been at room temperature 
for 2 days, the B, lot presented the following condition: Eight embryos 
had formed, there was one yolk-sac with scattered cells, and 33 eggs 
had died or failed entirely to resume development. All eggs in this 
lot had originally begun development and the control from the same 
group of eggs was perfect, thus the low temperature for 4 days had 
caused a very high mortality. Only about 22 per cent of the eggs 
resumed development. 

In lot Bs 49 had formed embryos, 6 of these were short, lacking a 
complete formation of their caudal ends, the others were well-formed 
specimens with optic vesicles present. Fifty-eight did not resume 
development, although all had begun before being placed in the cold, 
thus there was a mortality of 54 per cent in this group. 

In lot Bs; practically all formed embryos which now showed optic 
vesicles and body somites clearly formed. This lot was about as good 
as the control in respect to the number of eggs developing. Thus a 
4 days’ sojourn at 9°C., with an extremely reduced developmental rate 
did not prevent the possibility of again resuming a development of 
normal rapidity. This extreme slowing at a slightly higher tempera- 
ture is not nearly so fatal or injurious to later development as the almost 
complete stop caused by the lower temperatures of 5° and 7°C. 

Lot C, similarly treated at 5°C., but consisting of eggs from another 
parental pair, contained at this time 21 embryos with optic vesicles 
forming, 7 short embryos with the germ-rings not completely over the 


STRUCTURE AND DEVELOPMENTAL RATE 151 


yolk, and 13 had died or failed to resume development. ‘Therefore, in 
this lot 66 per cent were able to resume development, which is a some- 
what better record than the B series. This difference may easily be 
due to individual variations between the two lots of eggs from the two 
different pairs of fish, yet both lots of eggs were unusually fine, as was 
shown by the perfection of the B control as well as the C. 

When 10 days old the controls were going perfectly and seemed 
about at the point of hatching, having grown long with the tails curved 
around to cover the side of the heads, yet the yolk-spheres were still 
rather large. 

In lot B, 7 of the 9 living eggs showed embryos almost normal in 
appearance with good circulations, one was badly deformed and had a 
pulsating heart, but no circulation, while the one yolk-sac without an 
embryo had not progressed in development. 

Lot B, showed 36 strong embryos with good circulation, though one 
of these was slow, with eyes abnormally close together. Four speci- 
mens were badly deformed, one with a circulation of the blood and 
three without. There were two yolk-sacs with blood and pigment 
cells present and two others did not develop. Thus 42 eggs were still 
alive, of which 7, or 163 per cent, were grossly deformed. 

All eggs in lot Bz; seemed normal and well, although far behind the 
control. 

In lot C, 15 specimens seemed normal in structure, though two of 
these were slower than others in development. Ten specimens, or 40 
per cent of the total, were deformed, 8 showed grossly malformed heads 
and bodies, one embryo being represented by an amorphous mound of 
tissue on the yolk-sac, and two other specimens had only deformed heads 
with a fair circulation of the blood. Thus in this lot where the mor- 
tality following removal from the cold was low, the percentage of de- 
formed specimens is two and one-half times greater than from the By 
lot that had suffered a high initial mortality. 

When 16 days old, the majority of both control lots had hatched, 
though none of the inhibited ones had. When 17 days old, one in lot 
Be and 3 in lot By had hatched, though none in B, and C). 

At 18 days old, the controls still had a few unhatched. 

In lots B; 6 were hatched and 2 were not; in Bs 21 were hatched and 
20 were unhatched; in B; 33 were hatched and 27 were not; in C, 13 
were and 12 were not hatched. 

When 24 days old, lot B, contained one badly abnormal specimen 
still unhatched. In lot B. 17 were still unhatched, 5 of these were 
grossly deformed. In lot Bs; 12 were unhatched, though seemingly 
normal in structure. These were all far behind the control in time and 
manner of hatching. In lot C; 8 were deformed and unhatched, and 
one, slightly abnormal in gross appearance, partially succeeded in 
freeing itself from the egg membrane. Thus really 9 of these were 
deformed and unhatched. 

When 29 days old, one individual in the B control had not hatched 
though the others had been free swimming for 10 days. This was the 


152 CHARLES R. STOCKARD 


only lack of perfection in this control of more than 50 individuals. In 
B, there was one unhatched monster. In By 10 were still unhatched, 
though 6 seemed normal and ready to hatch; therefore, the cold treat- 
ment greatly reduces the strength and delays the hatching moment 
of these embryos. In B; 3 were unhatched. In C, 8 were unhatched, 
7 of these were deformed, one being a twin specimen and one almost 
normal. 


This series of experiments further shows the possibility of 
almost stopping, or reducing to an extreme degree, the rate of 
development during the earliest cleavage stages and again resum- 
ing a more or less normal rate on the part of a few individuals. 
An almost complete stoppage at an early cleavage stage results 
in a very high mortality ranging from as great as 78 per cent and 
54 per cent, down to 34 per cent. However, the reduction in 
rate brought about by a less severe temperature of 9°C. does not 
cause so great a mortality and does not prevent the resumption of 
development of almost normal rapidity. 

It is clearly shown, however, that although certain specimens 
may resume a fairly normal developmental rate after such treat- 
ments, the early arrests have had injurious effects upon the 
quality of the resulting embryos. A considerable percentage of 
gross abnormalities occurs in all of the groups, and even those 
embryos which appear on close examination to be normal in 
structure are extremely slow in hatching and are not in all cases 
capable of typical swimming reactions and perfect behavior as 
young fish. 

A point of particular importance is that in such a series as this 
which had been arrested during an early cleavage stage, the 
monsters resulting are not limited to any particular type, but 
exhibit, in a series of sufficient extent, almost all known types. 
There may occur double monsters of varying degrees, from sepa- 
rate twins, fused but with complete bodies and tails, to double 
bodies and single tails, and finally different degrees of double-_ 
headedness on single bodies. There are specimens exhibiting 
anophthalmia, monophthalmia, microphthalmia, cyclopia, and 
all types of malformed eyes. The brains may be slightly asym- 
metrical, irregular, tubular with no primary ventricles, or de- 
formed in various ways. The mouth and branchial region may 


STRUCTURE AND DEVELOPMENTAL RATE 153 


exhibit almost any known defect. The fins may be poorly 
developed and the bodies ill-shaped and twisted. The tails may 
be short, bifed, and undeveloped due to a slow or arrested descent 
of the germ-ring. And finally there may be such minor defects 
as would escape observation until the hatched embryos were: 
found to be unable to right themselves and swim. These are the 
defects to be seen on simple external examination, the internal 
structures are as frequently abnormal. The latter fact is borne 
out by numerous examinations of these monsters in sections. 
I have studied a great many of the sectioned specimens during 
the past number of years. 

The reason for this great variety of monsters following arrests 
during cleavage stages is that the development of all organs 
or parts must subsequently take place and all may thus become 
arrested and deformed. When eggs are treated at later stages, 
as at the beginning of gastrulation, no double monsters will 
occur, their moment has passed, though the various brain, 
branchial, and other defects mentioned may exist. When treated 
after the embryonic axis is visible, it is most difficult to get any 
gross eye defects and so on. 

Thus it may be said that the earlier the arrest the more numer- 
ous will be the type of defects found and the later the arrest the 
more limited the variety of deformities, since there are fewer 
organs to be affected during their rapidly proliferating primary 
stages. 

The same treatment that causes a gross deformity when ap- 
plied during an early stage, will during a later embryonic stage 
often give only a minor effect. 

The further records of experiments will render these statements 
more fully certain. Here I wish simply to call attention to the 
great variety of gross deformities resulting from these early 
arrests. The contrasts in detail between these and the later 
treatments will be shown in the following pages. 

I hasten, however, to caution any experimenter who may in 
the future find a double monster or cyclopean monster, for ex- 
ample, in a group of eggs arrested or treated during late develop- 
mental stages, not to assume that this is due to the late treat- 
ment or that it disproves the standpoint stated above. For 


154 CHARLES R. STOCKARD 


such an occurrence is simply accidental and due to the fact that 
the specimen was already arrested or defective in an early stage 
as might by chance happen in any normal lot of eggs. It is 
clearly true, as I shall show beyond, that only very early and 
carefully regulated treatment can artificially produce twins and 
double monsters, a phenomenon which must happen about the 
stage of gastrulation. Therefore, the treatment must be applied 
much before this time. Cyclopia may be induced by slightly 
later treatments. but only during a rather limited time, and 
quite early at that. 

Other experiments will later be considered in order to illustrate 
the difference in response on the part of these eggs following 
treatments similar to those above, but applied as nearly as pos- 
sible at certain particular developmental periods. 


d. Differences in effect between greatly reducing the developmental 
rate and actually stopping temporarily the process 


In the foregoing review of experiments attention was fre- 
quently called to the fact that in certain of the low temperatures 
employed an almost complete stop in development was actually 
obtained, while at the somewhat higher degrees the progress of 
development was reduced to an extremely slow rate, but not 
actually stopped. A more specific comparison between the ef- 
fects resulting from actually stopping and greatly slowing the 
rate of development may now be made. 

Three groups of Fundulus eggs when in the two-cell stage were 
placed in temperatures of 5°, 7°, and 9°C., respectively, as 
reviewed under experiment 901, B series. The first two temper- 
atures were sufficiently low to almost completely stop develop- 
ment, so that after twenty-four hours of such exposure the eggs 
were still in the two- or four-cell stage. The group at 9°C., 
however, developed very slowly and attained either sixteen- or 
thirty-two-cell stages within the first twenty-four hours. \ In 
other words, at this temperature three or four cell divisions occur 
per day. When all had remained for three days in these low tem- 
peratures, they were removed from the refrigerator and the fol- 
lowing results ensued: 


STRUCTURE AND DEVELOPMENTAL RATE 155 


The two groups that had been completely stopped in develop- 
ment suffered very high mortalities. In each a considerable 
majority of the eggs failed to resume development at room tem- 
perature, and during the early days of development very many 
of the survivors appeared abnormal in structure. These, how- 
ever, later showed some ability to recover, but finally at an ad- 
vanced stage about 33 per cent of them were still deformed. 
In contrast to this, the eggs that had developed slowly at 9°C. 
suffered only a low mortality on return to ordinary temperature 
and there was not nearly so high a percentage of abnormalities. 
At a late stage only 14 per cent were deformed as against over 33 
per cent in the two other groups. The slowed group also hatched 
earlier and with a better record than the two stopped groups. 

Similar differences in records between such groups of eggs were 
often even better shown, as is indicated in the results of experi- 
ment 902. In this case three lots of eggs in the two- and four- 
cell stages were placed at 5° and 7°C. for four days, after which 
interval they had divided four or five times and were all in about 
the sixty-four-cell stage. They were almost, though not actu- 
ally stopped, accomplishing only one cleavage per day. On re- 
turn to room temperature, one of the lots from 5°C. suffered a 
mortality of 78 per cent, only 22 per cent of these eggs being able 
to resume development, although every one was developing when 
first placed in the cold temperature. The lot from 7°C. showed 
a mortality of 54 per cent. 

Another group of eggs from the same parents and accompanied 
by the same control were placed at 9°C. at the same time and for 
the same interval as the above lots. These eggs developed 
slowly at 9°C., so that after their four-day sojourn they presented 
high segmentation caps, similar to the condition of the control 
after eighteen or twenty hours of development at normal tem- 
perature. On return to room temperature, these slowly develop- 
ing eggs resumed a normal rate and practically all formed em- 
bryos. Thus, in respect to the number of embryos that were 
developed, their record compared favorably with the control and 
contrasted acutely with the only 22 per cent which resumed 
development after the 5°C. interruption. The number of de- 


156 CHARLES R. STOCKARD 


formed embryos was decidedly less from the 9°C. slow lot than 
in the groups from 5° to 7°C. which had been almost stopped 
in their development. The 9°C. group also hatched earlier and 
somewhat better than the other inhibited lots. 

It is thus seen that during the early rather critical stages of 
development an almost complete stop is much more severe in 
effect than a decided slowing, on both the resumption of develop- 
ment and its later progress. An egg developing very slowly but 
still continuing the process during the early cleavage stages ap- 
parently possesses sufficient powers of adjustment or regulation 
to take up a much more rapid development either gradually or 
rather abruptly. When, as a result of low temperature, devel- 
opment actually stops during the cleavage or pregastrular stages 
on raising the temperature, it is frequently stimulated to start 
again, but the start is so irregular and so out of normal rhythm 
that many specimens are unable to continue development. 
These undergo a cellular disorganization followed by death. A 
considerable percentage of the specimens that do succeed in re- 
establishing development, still fail to obtain a proper adjustment 
and balance of developmental activities among their parts. 
Thus numerous arrested and defective organs are found. This 
lack of developmental balance among the various parts and the 
resulting defects are again not so common with eggs that have 
maintained a continuous development, although for a time it 
may have been slowed down to an extreme degree. In nature 
development rarely or never stops during the active early cleay- 
age stages, though slight temperature changes may frequently 
cause considerable slowing. The natural interruptions usually 
occur later, as among the birds, just after gastrulation has been 
well established.. The experiments in previous sections also con- 
tain data bearing on the effects of stopping and slowing during 
these later developmental moments. 

Experiment 905 shows the record of two series of eggs both 
stopped and slowed when twenty-three and twenty-seven hours 
old, respectively. In both cases the germ-rings were about 
formed and gastrulation was well on its way. The lots Ci, 
C., and C; after being twenty-three hours old, or in gastrula- 


STRUCTURE AND DEVELOPMENTAL RATE 157 


tion, were almost completely stopped for two days. Their con- 
dition when returned to room temperature was about the same 
as when placed in the refrigerator. Development was very 
promptly resumed at room temperature and only a slight mor- 
tality resulted from the stopping. Only a few of the embryos 
showed slight defects, but they were behind the control in time 
of hatching, on account of the two days’ arrest. 

Lots C; and C; at twenty-three hours old were placed at tem- 
peratures of 9° and 10°C. in which they continued their develop- 
ment at very slow rates, so that after four days the germ-rings 
had descended over about one-half of the yolk-sphere. During 
these four days they had advanced in development to a stage 
usually attained in about twelve hours or were going approx- 
imately at a developmental speed of one-eighth of the control 
rate. On return to room temperature these lots quickly resumed 
the normal rate, suffered no mortality on account of the retarda- 
tion, and developed into normal specimens which hatched some- 
what later than the control. These slowed embryos possibly had 
some real advantage over the completely stopped groups Ci, C2, 
and C;, though it was only slight if any. 

The D series stopped and retarded at twenty-seven hours 
old, when in gastrular stages, gave exactly similar records. 
There was no noticeable excess mortality and no later injurious 
effects. It may be generally stated that stopping or slowing the 
development of Fundulus eggs after the gastrular stage, with the 
temperatures here employed, have no appreciable effect upon the 
quality of the young fish up to the time of hatching. The mo- 
ment after gastrulation is established seems generally to be a par- 
ticularly passive stage at which neither stopping nor slowing 
the rate of development is followed by injurious results. 

The question now arises whether after stopping development 
during the gastrular stage there is any difference in result if it 
recommences rapidly or slowly. When twenty-four hours old, 
eggs were stopped for one day by cooling to 5°C. They were 
then allowed to resume development very slowly by being brought 
into a temperature of 9°C. They developed at a very slow rate 
for three days and were then brought into the room temperature 


158 CHARLES R. STOCKARD 


and resumed a normal rate of development. Such a procedure 
introduced at this given developmental stage seemed to have no 
effect other than to throw the lot of eggs several days behind the 
control in their degree of development and time of hatching. 

In order to determine the ability of the embryo to initiate 
certain functional reactions when developing at an extremely 
gradual rate, specimens three days old, just prior to the establish- 
ment of a heart beat, were placed in the refrigerator at 5° and 8°C. 
These temperatures do not seem to inhibit changes in the late 
embryo to the same extent as they do the early cleavage processes. 
The group in 5°C. still had no heart beat after being chilled for 
five days, so these specimens may be said to have been almost 
completely stopped. After five days the lot at 8°C., however, 
had developed a very slow and feeble heart beat. Thus these 
had definitely progressed at such a temperature and had estab- 
lished the functional activity of the heart muscle. Both groups 
on being returned to room temperature recovered completely 
and hatched in a normal fashion. Therefore, neither stopping 
for five days nor slowing to an extreme degree the development 
of these three-day-old embryos produces noticeable effects on 
their subsequent development and hatching ability. 

If abnormal development is simply the result of developmental 
arrests, why should not eggs which have been decidedly slowed 
in their developmental rates by lowering their temperature give 
rise to monsters as frequently as do those eggs which have been 
actually stopped in development at critical stages? When eggs 
are treated with alcohol, other anaesthetics, or a great variety of 
chemical substances, their development is not necessarily entirely 
stopped in order to induce monstrous results. These specimens, 
however, act during later development in a manner much more 
comparable to that shown by eggs actually stopped by refriger- 
ation than like specimens in which the developmental rate was 
simply greatly reduced. The explanation of this fact is probably 
as follows: 

Specimens which are caused to proceed at a greatly reduced 
though continuous rate of development by simply lowering their 
temperature apparently adjust the developmental progress of 


STRUCTURE AND DEVELOPMENTAL RATE 159 


their several parts to the slow rate in such a manner as to main- 
tain the normal differences in rate of activity among the several 
parts. The developmental rhythm of the parts is retained and 
the proper system of balance is unchanged. On resumption of 
the normal rate the parts all respond in their usual accord. 
After a complete interruption in development at a critical stage, 
on resuming the process those parts or organs that were formerly 
developing at a rate in excess of the parts in general are unable to 
start up again with their original excess or advantage and other 
parts have an opportunity to compete equally with them and 
may thus cause their reduced or arrested expression. That 
organ developing at the most rapid rate or having the highest 
degree of metabolism or oxidation at the time of the stop is less 
able to initiate its original rate when the moment of resuming 
development occurs than are those parts that were developing 
more slowly. 

The production of abnormal development and malformation of 
organs by treating eggs with strange chemical materials is brought 
about in a similar manner to the abnormalities following stopping. 
The part or organ developing at the most rapid rate is inhibited 
more decidedly by the treatment than are less rapidly developing 
parts and is, therefore, most affected or modified in its develop- 
ment. For example, at certain stages, the formation of the optic 
outpushings from the neural tube is the most energetic process 
taking place in the embryo. Any injury to the egg at this time 
works to the particular disadvantage of this process and results 
in underdeveloped or deformed eyes. If the injurious element is 
then removed, all other parts may continue their development 
normally, since they were not sufficiently active at the time of 
injury to be affected in particular. In other words, all.of the 
other parts were affected similarly and no one was any more 
inhibited than another. 

The results of slowing and stopping development may be 
stated very concisely as follows: On slowing development, all 
parts and organs lower their rates in a somewhat relative fashion, 
the faster-going parts, even though more decidedly slowed, are 
still progressing at a faster rate than the slow-going parts. On 


160 CHARLES R. STOCKARD 


resuming a normal rate, the more rapidly developing parts still 
maintain their necessary supremacy. 

On completely stopping development at a critical stage, that 
is, when certain parts are progressing at excessive rates, as com- 
pared with the rate in general, the rate of all parts is reduced to 
zero or equality. On resuming development from such a con- 
dition, the differential rates are not again established with suf- 
ficient promptness and certain parts or organs are suppressed, 
poorly expressed, or deformed in structure. On stopping devel- 
opment at an indifferent stage, that is, when important inequali- 
ties in developmental rate of the different parts are not occurring, 
it matters not if the entire rate be reduced to zero. On resuming 
development the parts all begin at about equal rates without the 
necessity of a prompt establishment of Set wees and no par- 
ticular arrests or suppressions occur. 


e. The types of arrests or deformities following a stop or slowing in 
the rate of development 


Only a general statement of results from a few experiments 
have been given in the previous pages without going into par- 
ticulars regarding the variety of deformities occurring. At this 
juncture I should like to enumerate in a very brief way the kinds 
of abnormalities which have occurred in all of the experiments 
where development has been stopped or slowed by a reduction 
in temperature. 

In the first place, there were produced a number of double- 
headed, double-bodied, and twin individuals which will be fully 
considered in the following section. Along with these were single 
individuals with all varieties of eye defects, anophthalmia, micro- 
~phthalmia, monophthalmia, cyclopia, etc. These defects were 
present in heads with either structurally normal or variously 
malformed brains. The mouth and branchial arrangements were 
frequently deformed. The otic vesicles were occasionally sup- 
pressed to various degrees or developed abnormally during the 
later stages. A number of specimens were short bodied, some 
with bifed caudal ends. The general body form and the shape 


STRUCTURE AND DEVELOPMENTAL RATE 161 


of fins showed frequent peculiarities. Extreme cases arose in 
which amorphous masses of embryonic tissue were present on 
the yolk, but no definite embryo was formed. There were simple 
yolk-saes with blood-cells and chromatophores scattered irregu- 
larly through them. Along with these variously defective indi- 
viduals were almost invariably certain specimens which in gross 
structural appearance were normal and succeeded in hatching 
and swimming freely about. Others were almost normal with a 
pulsating heart, but without a circulation of the blood. Further 
more detailed conditions need not be mentioned. 

This list of defects is sufficient to show that the types and actual 
individual conditions resulting from a simple interruption of 
development by reducing the temperature are all identical in 
character with those induced by treating Fundulus eggs with 
various chemical solutions (Stockard, ’07, ’09, ’10, 715, ete.) dur- 
ing their early developmental stages or actually with the results 
of certain mechanical operations upon these (Lewis, ’09) and 
other eggs (Stockard, 713). Furthermore, deformed hybrids 
resulting from crosses between distantly related species also 
present exactly the same structural peculiarities (Newman, 715). 
And finally the progeny derived from male guinea-pigs that have 
been chemically treated for long periods of time occasionally 
exhibit exactly similar deformities of their eyes and other parts 
(Stockard, 713; Stockard and Papanicolaou, 715 and 717). 

It seems difficult to imagine that the deformities occurring 
among the eggs that have been merely interrupted by being 
placed in a refrigerator temperature could be interpreted as other 
than simple arrests in development resulting from the slow prog- 
ress which had taken place at certain critical times. It seems 
equally as certain that the comparable conditions following the 
other experimental procedures have resulted from a similar cause, 
simply a lowering of the developmental rates of certain parts at 
critical moments in their origin or developmental history. In 
the several sections to follow I shall give much crucial evidence 
bearing on such an interpretation. 


162 CHARLES R. STOCKARD 


5. EXPERIMENTAL PRODUCTION OF TWINS AND ‘DOUBLE MON- 
STERS’ BY AN EARLY ARREST OF THE DEVELOPMENTAL RATE 


One of the earliest accomplishments in experimental embry- 
ology was the production of two embryos, or twins, from a single 
egg (Driesch, 92; Wilson, 93; Morgan, ’93; Zoja, 795; Loeb, 
95; Schultze, ’95, and others). This phenomenon was first pro- 
duced by separating the two primary blastomeres so that they 
were no longer in their usual intimate relation, each then devel- 
oped independently and produced a complete individual. In the 
light of this striking experiment, the occurrence of twins and 
double monsters under natural conditions was readily explained 
as being the result of an undue separation of the two blastomeres 
during the first cleavage. Such a separation might have been 
caused in a mechanical way, the two cells being pressed or 
squeezed apart, or something unusual in the chemical nature of 
the environment may have reduced the normal degree of cohe- 
sion between the first two blastomeres, allowing them to fall 
abnormally far apart and finally to become entirely separated 
from one another. — 

This clean-cut experimental production of twins and its ready 
application and acceptance as an explanation of the modus oper- 
andi for a well-known natural phenomenon, has undoubtedly 
held back our real understanding of the phenomenon and strik- 
ingly illustrates the dangers of directly interpreting occurrences 
in nature on the basis of results from experiments. 

Almost at once evidence began to accumulate which questioned 
the general application of the separate blastomere explanation of 
twin formation. Such evidence was not always appreciated in 
this connection, but from our present point of knowledge its 
bearing is more readily seen. The discovery was very soon 
made (Wilson, ’04; Conklin, ’05, and others) that on separating 
the primary blastomeres in certain species of eggs complete twin 
embryos do not result. Yet there is no reason to believe that 
in nature twins and double monsters do not at times arise from 
the eggs of such species. Twin formations are certainly not due 
to the separation of the first two blastomeres in these particular 
species, since each of these blastomeres developing independently 


STRUCTURE AND DEVELOPMENTAL RATE 163 


gives rise to a partial and not an entire embryo. Such eggs have 
an early differentiation and localization of ‘organ-forming stuffs’ 
and these stuffs are unequally distributed to the blastomeres even 
at the first cleavage. The individual blastomeres are, there- 
fore, not totipotent, but only capable in their later development 
of giving rise to certain parts of the embryo and not the whole. 
The eggs of a number of worms and molluscs present this very 
early localization of differential stuffs, yet in some of these vari- 
ous types of double individuals are not uncommon. These 
double individuals I believe, in the light of evidence contained 
in the literature along with that presented here, are the results 
of a simple process of budding. 

Again it was shown by Enders and later by Spemann (’03) 
that double specimens not only resulted from the separation of 
blastomeres, but the late blastular and gastrular stages could 
mechanically be caused to develop into double instead of single 
individuals. The degree of duplicity depended somewhat upon 
the extent to which the eggs were constricted in a given plane. 
This was evidently a case of dividing or separating into two 
parts the growing region of a single individual and thereby estab- 
lishing two new growing points instead of the original one. The 
division of a single growing bud into two may be illustrated on 
plant buds, embryonic animal limb buds, ete. The interpreta- 
tion of the two separated regions as being the exact derivatives 
of the two original blastomeres, as Wilder has suggested, is in 
many cases entirely implausible. 

Doubleness in nature is probably due to a modification of a 
budding process, and double monsters and actually identical 
twins, like all other abnormalities, may result from an arrest or 
inhibition in development. To state that twins and double indi- 
viduals are induced by a developmental arrest seems at first 
thought almost absurd; for how could an arrest serve to give a 
formation structurally exceeding the normal in extent? One 
might accept developmental arrests as explanations for many 
deficiencies in structural expression, but such an explanation of 
excessive conditions or double-headed and twin individuals would 
scarcely be suggested. In the present consideration, however, it 


164 CHARLES R. STOCKARD 


will be very conclusively shown that double conditions and twin- 
ning in nature are the result of an unusual budding process pro- 
duced by an early interruption of developmental rate, and are not 
connected with a separation of the primary blastomeres except 
under experimental procedure. 

Before entering into the particular points of the present experi- 
ments, it may be well to explain in some detail the writer’s con- 
ception of embryo formation and the general process of budding 
in plants and animals. 

It has long been known that the notches around the border of 
certain plant leaves, such as Bryophyllum, have the power under 
certain conditions to bud and give rise to an entire new plant. 
It is observed, however, that the new shoots, as a rule, arise from 
only one or two notches instead of from many. Loeb (’16) has 
performed most elucidating experiments on the budding phenom- 
ena in these leaves. In the first place, although in nature only 
a few notches on any one leaf send out shoots at any one time, 
yet Loeb has shown that there is a potential ability present in 
every notch to form a shoot. This fact is demonstrated by cut- 
ting the leaf into parts in such a way as to isolate each notch. 
Following such an operation a tiny shoot grows from every one 
of the isolated notches. It becomes evident, therefore, that not 
only does each notch possess the potential ability to form a 
shoot, but under ordinary circumstances this shoot-forming abil- 
ity is suppressed in most of the notches by ths erowule of shoots 
from only one or a few notches. 

It was further found that almost any notch on the leaf could 
be selected and forced to bud at the expense of the other notches 
by simply suspending the leaf so that the selected notch dipped 
into water. This suggests, of course, that ordinarily the condi- 
tions for bud formation are not equally favorable in all notches 
and, therefore, only a few shoots arise from a leaf instead of one 
in every notch. These few then tend to suppress the origin of 
buds from other notches. Does any such set of comparable con- 
ditions exist in a developing egg or blastoderm before the initial 
line or axis of the embryo arises and begins development to 
form a complete animal? 


STRUCTURE AND DEVELOPMENTAL RATE 165 


The periphery of the blastoderm in the eggs of the bird and 
mammal or the germ-ring in a teleost’s eggs is probably in some 
sense comparable to the notched order of the budding leaf. Ata 
certain place along the germ-ring in the fish’s egg a peculiarly 
rapid cell multiplication begins and the embryonic shield with 
the axis of the embryo buds away from this place. There is 
already evidence for believing that more than the one place may 
be capable of embryonic axis formation, and much is added to 
such evidence by the experiments now to be presented. There 
are many potential points around the germ-ring at which an 
embryonic axis might arise. Here again, as in the plant, 
when one bud or embryonic axis has arisen, it tends to suppress 
the potential ability of other points to form an axis, and nor- 
mally only one individual is developed from the egg. 

We are entirely unable to state the reasons why a certain 
point along the germ-ring should form the bud and not another. 
One can only imagine that this point has some peculiar advantage 
of position which gives to it a higher power of oxidation and a 
temporarily more rapid rate of cell proliferation than is possessed 
by other points, just as the notch which is dipped below the 
water surface possesses a budding advantage over the other 
notches around the leaf. Can the advantage of position pos- 
sessed by a particular point on the germ-ring be reduced so as to 
equalize the budding tendency of several points and thus allow 
them all to express their ability to form embryonic axes? Could 
such a condition be brought about double embryos, twins, 
triplets, etc., would be produced. 

The use of the word bud or budding in connection with double 
embryo formations as employed by Patterson (714) has been criti- 
cised by Assheton, who suggests fission as the better word for 
the process. Such a discussion seems devoid of value and I 
employ the word bud to mean what is indicated above. 


166 CHARLES R. STOCKARD 


a. Arresting development by low temperature and the production of 
: double embryos and twins in Fundulus 


A number of years ago I occasionally found a double embryo 
or a twin condition in Fundulus eggs that were arrested in their 
development by being kept in solutions of MgCl, (Stockard, ’09, 
figs. 22,56,and 57). Such specimens, however, were so extremely 
rare that their occurrence was never associated with the experi- 
mental procedure. Chidester (’14) also found a twin among 
Fundulus eggs arrested in ether solutions, and reported one 
other in an egg which had developed in a crowded condition. 

The eggs of Fundulus heteroclitus are extremely hardy and 
twins or double monsters are practically never found among 
these eggs developing under ordinary conditions. During four- 
teen spawning seasons many hundred control embryos have been 
examined and I have not found among them a twin or double 
specimen. While on the contrary trout eggs are known to be 
rather sensitive, and must be developed under very carefully 
regulated conditions. In the trout hatcheries double embryos 
and twins are very often found and have at times been collected 
and studied in large numbers (Windle, 95; Gemmill, 00, and 
others). 

Recently I have found strong evidence of a causal relation 
between slowing development and the formation of twins in 
trout, this will be discussed beyond. The evidence led me to 
experiment with Fundulus eggs in order to determine whether 
here also there was a direct connection between arresting devel- 
opment or slowing its rate and the origin of double individuals 
and twins. During the past three spawning seasons, a number of 
experiments have been performed and the general results of these 
may be reviewed. 

Two methods of slowing the rate of development have been 
employed; lowering the temperature and reducing the oxygen 
supply. The latter method will be considered along with the 
occurrence of duplicities in trout eggs. 

It was soon learned that double embryos and twins could be 
induced, but only by treating the eggs during a limited develop- 


STRUCTURE AND DEVELOPMENTAL RATE 167 


mental period. Either stopping development or greatly reducing 
its rate during cleavage stages and before the germ-ring has 
formed, that is, at periods preceding gastrulation, frequently 
serves to cause doubleness in the subsequent embryo formation. 
Specimens subjected to any degree or kind of treatment after 
the gastrular period never produced double or twin embryos. 

Subjecting Fundulus eggs to low temperatures during early 
cleavages, the four-, eight-, or sixteen-cell stages, not only arrests 
the cleavage process, but on later resuming development many 
eggs fail to establish a normal rate and balance for some time and 
the early processes of gastrulation would seem to be disturbed. 
The majority of eggs after a stoppage of cleavage are completely 
unable to resume development and may live for a few days in an 
almost stationary condition and then die. Other arrested cleav- 
age caps undergo a breaking-down or falling apart of the indi- 
vidual cells before the death of the eggs. A small minority of 
these hardy eggs after an arrest during cleavage stages succeed 
in finally readjusting their development to a sufficient extent to 
give rise to apparently normal free-swimming young fish. The 
individual variations in resistance and developmental ability 
shown among Fundulus eggs are remarkable in all experiments 
performed on them. Our present consideration is to be centered 
on that group which is sufficiently viable to continue develop- 
ment, but not so resistant as to be able to completely readjust 
its developmental processes following the early interruption. 

Not only does the entire experimental lot become divided into 
the three above crude classes, but the members of our selected 
group which is not completely capable of normal readjustment 
by no means all develop in a similarly defective fashion. These 
discrepancies again are due to individual variations in the man- 
ner of resuming development. 

Certain specimens after removal from low temperatures 
resume their cleavages with a fairly normal rhythm and form a 
typical embryonic shield, but later the larger diverticula from 
the interior parts of the central nervous system fail to arise in a 
usual manner, or other processes requiring a high degree of devel- 
opmental energy are not sufficiently expressed and various de- 


168 CHARLES R. STOCKARD 


fects become evident. Other individuals resume their cleavage 
processes, form a typical blastoderm and begin the formation 
of a germ-ring, which indicates the commencement of gastrula- 
tion, but just here the degree of energy necessary for normal de- 
velopmental processes is insufficient and a single embryonic bud 
is not formed with that normal rate of growth which sup- 
presses the appearance of other embryonic buds. Therefore, 
instead of the one point proliferating at a disproportionate rate 
to form the embryonic shield, two such points are established with 
more or less equal rates of proliferation, both of which may be some- 
what less active than the single one should be. The formation 
of two embryonic shields or the initiation of two points of rapid 
gastrulation away from which will grow the axes of the embryos 
is in fact the initial or primary step in double formations. The 
phenomenon is exactly the same as when two buds arise from two 
notches on the leaf border instead of one bud growing from a 
single notch. Every notch is a potential bud-forming point, and 
in the same way many potential invagination points exist on the 
blastoderm, and when more than one such place begins to grow 
we have double formations. In this sense it may be appreciated 
that the intrinsic conditions which give rise to double monsters 
or twins exist in all eggs and are not produced by the experiment. 
The experimental modifications of the external conditions simply 
serve to allow more than the one growing point to express itself. 

The actual results of several rather typical experiments may 
be given as better illustrating the occurrence of the double 
individuals. 


Experiment 903. A particularly fine lot of eggs was obtained from 
a large female and fertilized by a single male on July 5, 1919, during 
the height of the spawning season. Three groups of these eggs were 
selected, one serving as a normal control and the two others, A; and Ags, 
at three and one-half hours after fertilization when dividing into the 
8-cell stage, were placed in temperatures of 6° and 8°C., respectively. 

The outside temperature was unusually warm and the control eggs 
developed at a vigorous rate. When 22 hours old the germ-rings were 
from one-third to one-half over the yolk-spheres in all the specimens 
the embryonic shields were well formed with the embryonic axes already 
indicated in the midline. Every egg in the control lot was developing. 


STRUCTURE AND DEVELOPMENTAL RATE 169 


The two lots in the refrigerator at 22 hours old had as a rule under- 
gone only one cleavage further than when placed in the cold. All 
were in a rather typical 16-cell stage. The low temperatures had not 
quite completely stopped development. 

At 48 hours old, the control embryos were in a very advanced con- 
dition. They were large in size with fully formed optic cups and 
lenses, about 10 to 12 pairs of somites, the pericardium distended and 
the heart formed, although not yet pulsating. Chromatophores were 
present and though small had already differentiated into the red and 
black types. Five hours later the hearts were pulsating, but the blood- 
vessels were not fully connected and there was no circulation. One 
familiar with these embryos will realize that such a condition of develop- 
ment is rarely attained in less than 70 hours, thus this control group 
was developing with unusual rapidity. 

The eggs composing the A; lot when 48 hours old at 6°C. were in 
about 32- or 64-cell stages. Many of the blastoderms were discs of 
irregular cell arrangement and some presented large uncleaved proto- 
plasmic portions. The A» lot were in a closely similar condition. 

The control specimens when 72 hours old had a vigorous blood 
circulation, with the vessels already mapped out by the migrating 
chromatophores. During the cooler, earlier part of the season a similar 
condition was not reached in less than four days of development. 

The eggs in lot Aj, after being 69 hours at a temperature of 60°C., 
all showed irregular segmentation caps, the cells of which seemed to 
be in a large vesicle or bubble-like formation. The caps appeared to 
contain approximately 64 to 128 cells loosely arranged and in every case 
located within the bubble-like area, which seemed to prevent the normal 
flattening down of the cap upon the yolk-sphere. 

There seems to be a clearly marked surface film between the yolk 
and the region containing the cleavage mass. It is as if the cleavage 
mass existed in a drop of more transparent highly refractive fluid. 
The drop is not in a segmentation cavity, but probably consists of 
accumulated fluid such as normally exists in the cavity, but here located 
between the cell mass and the yolk, poss-bly on account of some 
peculiar osmotic effect. 

The specimens of group A, kept at 8°C. were at 72 hours old in a 
closely similar condition to those of A;. Both groups were removed 
from the refrigerator and placed at room temperature after this 69-hour 
exposure to the low temperatures. 

After being out of the refrigerator for two days, many eggs in the 
A; and A» lots had failed to resume development and had died. 

When 8 days old, or 5 days after removal from the low temperature, 
many more, 41 of the remaining 99 eggs in lot A,, were dead and many 
of those living were grossly deformed. In lot A, a few more were 
dead, many were not developing, a number were grossly deformed, yet 
some were apparently normal. 

When 10 days old, the eggs were all very carefully examined to 
determine as nearly as possible the exact nature of the abnormalities 


170 CHARLES R. STOCKARD 


which had occurred. The control consisted of 114 eggs, each of which 
contained a normal well-formed fish. In the A; lot 4 more had died, 
and thus the total mortality in this group after removal from the cold 
was very high, a little over 70 per cent. In all 54 individuals had 
survived to develop embryos, and of these 16, or 30 per cent, showed 
gross abnormalities. Five of the 16 abnormal ones showed double condi- 
tions. One was a complete twin, two were double-headed and two 
had double anterior halves with single tails, Y embryos. Thus 9.3 per 
cent of all surviving embryos were specimens exhibiting some degree of 
doubleness, and 33 per cent of the deformities which occurred were 
duplicities. When we consider the very delicate degree of arrest and 
the particular developmental moment that must be affected on the 
basis of our explanation of double monsters, the above result is a remark- 
ably significant one and is as good as any I have obtained by this 
method during the past three seasons. 

In the As group at 10 days old 2 others had died and 88 were now 
alive. Among the 88 survivors eleven individuals, or 12.5 per cent of 
all, were grossly deformed and many others were pale in color and far 
behind the average in their degree of development. Two of the 11 
grossly deformed specimens were double, one showed a slight degree 
of anterior duplicity and the other was a twin with the two embryos 
180° apart on the yolk. One of the twin components was large, well 
developed and normal in structure, the other was a short embryo with 
almost no body but with a well-formed head containing eyes and a 
pulsating heart and good blood circulation. In this group only 2.3 per 
cent of the surviving embryos were double specimens, but almost 20 per 
cent of those actually deformed were of this type. 

When 25 days old, many of the normal specimens in both the A, 
and A» groups had hatched, although all of these were far behind the 
control, which had begun hatching when 12 days old. 


The actual percentage of double individuals induced by this 
experiment is not really large, yet it is comparatively very sig- 
nificant. From a long experience with these eggs I would ven- 
ture to believe that under normal developmental conditions there 
is only a small chance for finding one double specimen among a 
thousand. During the past three spawning seasons a great num- 
ber, certainly many thousand, of Fundulus eggs have been ar- 
rested in their development by being placed in low temperatures 
after the germ-ring had begun to form. These specimens were all 
examined with such care in connection with the various problems 
being studied that no double specimen could have escaped record. 
Yet among all these late arrests not one double individual 
existed. 


STRUCTURE AND DEVELOPMENTAL RATE 171 


In comparison with such facts, the occurrence of 9.3 per cent 
in lot A, and even the 2.3 per cent of doubleness in group A» would 
searcely warrant any other interpretation than that such condi- 
tions had in some way been induced by the experimental treat- 
ments. There can be little doubt that the embryonic axis is 
initially expressed during a very critical and comparatively brief 
developmental moment. When the axis is once expressed, com- 
mon observation teaches us that in some way it prevents the oc- 
currence of other axes or other embryos on the same blastoderm. 
Doubleness very probably, as will be more fully discussed below, 
results from the almost simultaneous occurrence of two embry- 
onic shields instead of one, and this is further due I believe to the 
probability that neither of the axes possesses the advantages 
which normally suppresses the expression of other potential 
budding points. 

To further illustrate the occurrence of doubleness in Fundulus 
following treatment with low temperature, we may briefly sum- 
marize one other experiment. 


Experiment 890. These eggs were developed during the early cool 
part of the season and the control itself progressed rather slowly. 
The lot B, was placed in a temperature of 5°C. 3 hours after fertiliza- 
tion when in an early 2-cell stage. 

Twenty-four hours later the control had develored high segmentation 
discs which had not yet flattened to cap down upon the yolk-sphere. 
The night had been unusually cool and these eggs were thus consider- 
ably retarded in their development. This amount of retardation is not, 
however, particularly injurious, as is shown by the later development 
of the eggs. It would seem that Fundulus eggs were sufficiently resist- 
ant not to be noticeably deformed by the retardations in development 
induced by the degrees of low temperature which might occur during their 
spawning season in this climate. Nevertheless, embryos developed dur- 
ing the early cool part of the season are not so large in size or vigorous 
in behavior at the time of hatching as are those being developed during 
the warmer days to follow. 

The eggs of lot B,; after 20 hours at 5°C. are in 2- and 4-cell stages, 
they are, therefore, almost completely stopped, having divided only once 
during this time. 

When 2 days old, the control had the germ-ring only about one- 
fourth over the yolk sphere, with the embryonic shield beginning to 
form, a stage not more than one-half as advanced as is usual for this 
age. Group B; contained eggs in the first, second, and third cleavage 
stages with many very irregular arrangements of the cells. These 


172 CHARLES R. STOCKARD 


eggs were now returned to room temperature, and many of them very 
soon began again to develop. 

The control at 3 days old showed the embryos well formed, 
although the germ-ring was not entirely over the yolk-sphere. The By 
lot, after being out of the refrigerator for 24 hours, had high seg- 
mentation dises which had not begun to flatten down upon the yolk- 
sphere. There was no indication of the germ-ring or embryonic-shield 
formation. After 24 hours more the germ-caps had flattened and 
grown about one-half over the yolk-sphere, the embryonic shield was 
well formed in most of the specimens and the line of the embryo was 
visible in the shield. Thus within the first 48 hours after removal 
from the low temperatures many of these eggs have attained about 
the same condition as was shown by the present control specimens 
when 50 hours old. Many of the eggs after refrigeration failed to 
recover, and died during the first two days at room temperature. 

When 6 days old, the control embryos were twitching and moving 
their bodies and were in all respects normal in condition. The B, 
group contained small embryos without a blood circulation, many of 
them were abnormal at the head end, and many were short. Thus after 
developing for 4 days at room temperature they are far behind a usual 
four-day embryo. 

The B, group were carefully surveyed for deformities when 9 days 
old. Four eggs had yolk-sacs containing blood-cells and chromato- 
phores, but without formed embryos. Six eggs still had an early cell 
mass at the upper pole which had not developed, although even at 9 
days it was translucent and alive. There were 10 deformed embryos 
without a circulation, and 4 deformed but with a circulation. The 
majority, 45, of all living specimens seemed normal, with vigorous 
circulations. Thus more than 34 per cent of the specimens which 
survived the low temperature were grossly abnormal. ‘Three of the 10 
eggs which contained abnormal specimens with circulating blood 
showed double embryos. One was two-headed, and two were double 
throughout their anterior halves, each having two heads and two 
bodies with a single caudal half. 

The control embryos were with two exceptions all fine normal speci- 
mens. Two of the 86 individuals were small and considerably behind 
the others in their stage of development, although their structures 
were normal and they later succeeded in hatching several days after 
their fellows. 


This experiment again shows a pronounced difference between 
the modes of development in the normal control lot of eggs and 
in a similar lot which had been inhibited by lowering their tem- 
perature before the time of gastrulation. More than 4 per cent 
of the eggs which survived the inhibition contained double 
embryos, and one-eighth of all the gross abnormalities was of this 


STRUCTURE AND DEVELOPMENTAL RATE 173 


nature. Here again it would seem to be strongly indicated that a 
connection of primary importance existed between the retardation 
of development and the origin of the double specimens. 

A number of similar experiments with low temperature arrests 
could be reviewed, but they would differ little in their general 
results from those above. We may, therefore, pass to an analy- 
sis of another type of experiment before undertaking a general 
consideration of the significance of the results. 


b. Arresting development by reducing the oxygen supply and the 
occurrence of double individuals and twins in the 
trout and Fundulus 


Cellular proliferation which is so important an element in 
development is a great energy-consuming process. No doubt the 
interruptions in cell proliferation which were described in the 
preceding section as due to low temperatures are actually caused 
by a lower rate of oxidation which takes place at such tempera- 
tures. In nature development is not only interrupted at times 
by indirectly lowering the rate of oxidation through temperature 
changes, but also by directly reducing the oxidation rate through 
a lack of free oxygen. In the present section we may review 
some of the consequences of lowering developmental rate by 
directly reducing the available oxygen supply. 

The methods employed have been extremely crude, just such 
methods as nature might frequently use. With such methods the 
results are, of course, more variable than might be obtained from 
highly refined manipulations, yet the variations themselves are 
quite instructive. Experiments with Fundulus eggs may first 
be considered. 

1. Results with Fundulus. The eggs of Fundulus are demersal 
and are supplied with long thread-like processes which normally 
serve to entangle them on the blades of sea-grass or other objects 
among which they are deposited by the female. This arrange- 
ment serves to keep the eggs near the surface, and to insure 
contact with a better oxygen supply than might be obtained 
should they lie in the sand or silt of the bottom. When these 
eggs are developed in the laboratory they are kept in small glass 


174 CHARLES R. STOCKARD 


dishes, ordinary ‘finger-bowls,’ containing about 60 ce. of water. 
The thread-like processes from the egg membranes become en- 
tangled and cause the eggs to cluster together in bunches of from 
a few to even as many as one hundred or more. 

It is a well recognized fact that in such clusters the conditions 
for development of the individual eggs are not equal, and the 
egg group fails to present a uniform mode of development. The 
common practice is to separate the eggs in a dish so that they lie 
apart and are not clustered together. The permanent separation 
of the eggs requires care and attention, since they may again 
become bunched by the agitation of the water. When they are 
properly kept apart the entire lot in a dish will develop with 
remarkable uniformity. 

No control group of Fundulus eggs should serve as a standard 
for development unless the individual eggs are kept completely 
free from contact with one another. In my experience, under 
such conditions only the most insignificant percentage of develop- 
mental abnormalities ever occur. I am convinced that the high 
percentage of abnormalities recorded by certain experimenters 
among their control sets are due to a failure to properly separate 
the eggs. The clustered condition also vitiates the results ob- 
tained from experimental groups of eggs. 

Advantage was taken of this tendency to become entangled 
into clusters in order to study the developmental reactions of 
eggs with more or less access to a free oxygen supply. The eggs 
about the outside of such a cluster are in contact with fresh sur- 
rounding water and a sufficient amount of oxygen for normally 
rapid development. Those specimens lying deeper and deeper 
in the cluster are more and more removed from a freely changing 
water supply, and, therefore, experience various degrees of a 
stagnating environment. Such eggs not only lack a constant 
oxygen supply, but no doubt exist in an environment containing 
an excess of waste products, such as the CO, given off by their 
neighbors. The developmental perfection attained varies directly 
with the distance from the center of the egg cluster, the further 
removed from the center the more perfect the development. 


STRUCTURE AND DEVELOPMENTAL RATE 175 


In many of the experiments the available oxygen supply was 
further reduced by first boiling and driving the air out of the 
sea-water into which the eggs were to be placed. The central 
eggs of large clusters in this boiled water frequently had their 
development stopped in various stages, while other specimens 
progressed at an extremely slow rate. One group of such experi- 
ments will be briefly reviewed as illustrating the general results 
from all. 


Experiment 915. A large number of eggs, from three females, was 
fertilized by a single male. After 3 hours they were almost all devel- 
oping and presented the typical 4-cell stage. About 75 of these eggs 
were placed in ordinary sea-water and separated apart on the bottom 
of a dish to be developed as a control. The other eggs were divided 
into three lots. Two lots were placed in dishes containing sea-water 
that had been boiled, and the third lot was put in ordinary sea-water. 
The eggs in the three dishes were then moved gently around until 
they became clustered into large groups of about 100 or more. 

After 2 days of development, the control contained well-formed 
embryos with the optic vesicles prominently shown and with 8 to 10 
pairs of somites present. Many eggs on the outer parts of the clusters 
in the unboiled sea-water were equally as far along, while others 
near the center of the clusters were still in segmentation stages, and still 
others were in various degrees of arrested development. The two lots 
in boiled sea-water were in closely similar conditions. 

When 8 days old, the entire experiment was carefully examined and 
the following conditions found. The control eggs all contained normal 
embryos except for the fact that 3 specimens were smaller than the 
others and somewhat delayed in development. These, however, later 
succeeded in hatching. 

The clusters in unboiled sea-water contained many dead eggs. The 
more superficial eggs of the cluster contained in general normal embryos, 
though some were behind the control in their degree of development. 
Almost all of the more centrally placed eggs of the group were several 
days slower than the control in their developmental stages. These 
embryos were small and pale with poorly expanded chromatophores, 
and 15 of them, or 13 per cent of the small embryos, showed gross 
abnormalities. They possessed narrow undeveloped heads, defective 
eyes, deformed hearts with no circulation, and other common defects, 
while 2 of the larger better developed specimens were double-headed 
embryos. This dish contained a few more than 200 eggs, thus only 
about 1 per cent developed double conditions. 

The first group that had been clustered in the boiled sea-water 
showed a somewhat better record than the preceding. “Here also 
many of the eggs had died. There were again a number of normal 
embryos in the superficial regions of the cluster. The more centrally 


176 CHARLES R. STOCKARD 


located specimens were small and far behind the control in their rate 
of development, but here only about 10 per cent of them were actu- 
ally grossly deformed, and there were no double conditions at all. 

The second group in boiled sea-water was more decidedly affected 
than any. Many of the eggs died. Many superficial ones were 
almost up to the control in their state of development. But the great 
majority of specimens were small, pale, and poorly developed, being 
several days behind the control. Almost 16 per cent of these small 
specimens were considered to show gross defects. Twelve specimens 
had no circulation of the blood; 10 had decidedly defective eyes, minute 
in size and poorly developed or deeply buried in the head, and two 
were cyclopean. 

Four specimens that were near the surface of the clusters and very 
well developed presented double conditions. One egg contained sepa- 
rate twins, both embryos being fully developed. The 3 other eggs 
showed different degrees of anterior duplicity. Therefore, more than 
2 per cent of the entire number of specimens developing in the dish 
were double; this is much the highest record that was obtained among 
ten similar experiments. 


The other experiments with low oxygen supply gave closely 
comparable results to the three above, and need not be reviewed 
in detail. Only a very small number of double specimens oc- 
curred in any of them. In all cases, the double individuals were 
among those of fairly normal development and were not extremely 
small and highly defective specimens. This, in my opinion, is 
a fact of considerable importance, and is to be explained some- 
what as follows. 

The origin of two embryonic axes or growing points on the 
germ-ring of the fish probably results from a rather mild or 
slight reduction in the normal developmental rate at the time of 
gastrulation or embryonic-shield formation. It is probably more 
important to obtain the reduction in rate at an exact and very 
limited moment than to have a definite degree of reduction. 
That is, the reduction in rate may be little or much, but it must 
occur during a very limited time and not continue for long after 
the doubling has once been accomplished. Should the arrest 
continue, it is possible that one of the buds, even though it had 
begun to develop, might be suppressed, and the more vigorous 
or more favorably placed one might later continue as an appar- 
ently single individual. 


STRUCTURE AND DEVELOPMENTAL RATE 177 


Certain delicate or sensitive eggs will probably respond more 
readily and give double conditions more frequently than hardier 
eggs. The eggs of Fundulus are very hardy, and it may be that 
a treatment when acting in a delicate manner affects favorably for 
our purpose only the more sensitive eggs, while the large majority 
are too resistant to respond. Should the conditions be more severe: 
they would act too harshly to obtain a double response from any. 
These speculations will appear to have a stronger foundation 
after we have reviewed the very remarkable tendencies on the- 
part of the delicate eggs of the trout to give double and twin 
embryos. 

2. Double embryos in trout eggs. The eggs of the trout un- 
questionably possess a stronger innate tendency to form double 
and twin individuals than do those of Fundulus. Twinning and 
double formations, like all other unusual developmental phenomena, 
are not simply and entirely due to the action of an ususual environ- 
ment, but also depend upon the internal structure of the given egg 
and its peculiar manner of development... An environmental stim- 
ulus which would frequently induce double formations in one 
type or species of eggs might be completely ineffective in its 
action on the eggs of another species. The burden of evidence 
for the cause of twin formation as well as the means of artificially 
inducing it indicate an accessory budding or double blastopore 
formation as the primary step, and it is obvious that the early 
morphology of certain eggs more readily lends itself to the estab- 
lishment of accessory blastopore formations than does that of 
others. 

Not only is this morphological difference to be expected, but 
from what we know of the physiology of budding, it is also 
logically probable that in certain eggs the bud for the embry- 
onic axis will-arise with higher powers for dominating the entire 
budding region than in others. Different degrees of dominance 
of the apical bud in different plants is a well-recognized fact. 
In some plants the terminal bud grows to form a long slender 
stalk, without producing axillary or lateral shoots, while the 
terminal shoot of other plants grows to a limited extent only 
before axillary buds and branches make their appearance. The 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, No. 2 


178 CHARLES R. STOCKARD 


embryonic axis in the vertebrate egg may be compared with the 
terminal shoot of a plant. A second embryonic axis formation is 
roughly comparable to the occurrence of an ordinary lateral or 
axillary plant bud. A better comparison, however, is made be- 
tween the animal egg and the budding leaf, such as Bryophyllum. 
In both of these, the egg and leaf, we may recognize an area of 
potential budding capacity, and we know that not only one, but 
several equal buds may arise simultaneously from either of these 
two stocks. There are doubtless certain kinds of budding leaves 
which are more prone to form multiple buds than others. From 
such leaves instead of one shoot arising in a certain notch, several 
notches are equally capable of budding and several shoots are 
formed. It would seem that certain animal eggs also normally 
possess a disposition to produce several buds. Such eggs develop 
more than one embryonic axis and give rise to several individu- 
als instead of the usual single embryo from a single egg. This is 
probably the case in the Texas armadillo. 

The eggs of Fundulus and those of the trout, very probably 
illustrate two different degrees of capacity to form several in- 
stead of one embryonic bud. This much of the twinning pro- 
cess is truly inherited, and variations in the tendency may occur 
not only among the eggs of different species, but probably also 
exist among the individual eggs of the same species. For ex- 
ample, certain mothers may produce eggs highly inclined to give 
rise to two embryonic buds or twins, and such an inclination may 
be transmitted or inherited by her daughters or even through her 
sons. Davenport (’20) has recently found in a study of human 
twins that the males of a family carry or transmit the twinning 
tendency in equally as evident a manner as do the females. Double 


and twin individuals are also of much higher frequency in certain . 


human families than in the community as a whole. - All this indi- 
cates that the eggs of certain individuals are more inclined to 
form twins than are those from others. In the human cases 
the present consideration refers, of course, only to so-called iden- 
tical and not fraternal twins. The latter, truly speaking, are 
not actually twins. 


= ep 


STRUCTURE AND DEVELOPMENTAL RATE 179 


There can be little doubt from the experiments recorded above 
and the results to be given below that the environmental condi- 
tions or external factors are of greater importance than the in- 
ternal tendencies in twin formation. It is evident that eggs, 
although capable of producing more than one embryo, rarely ever 
do. The number of twin formations in a given lot of eggs may 
experimentally be increased so greatly in excess of the natural 
occurrence of such individuals, that we are forced to believe 
even in the cases before mentioned of the armadillo and the exces- 
sive occurrence of twins in certain families; as cited by Daven- 
port, that the environment may in these instances also be the act- 
ually direct cause. A peculiar uterine reaction may be inherited 
in the armadillo and in certain human families which prevents a 
ready or rapid placentation and thus primarily brings about an 
initial slowing of development. There is much evidence of a 
slow placental formation and a peculiar uterine condition in the 
armadillo which will be considered more fully beyond. 

This brief estimate of the internal and external developmental 
factors concerned in twinning has been given just here in order 
that the reader may appreciate more fully the very different reac- 
tions shown by Fundulus and the trout. He may form for him- 
self some idea as to whether this is due to a difference in morpho- 
logical pattern of the germ-rings or potential budding regions in 
the two species or to differences in the physiological reactions 
to the environment or finally to a combination of both. 

Double and twin trout are classical objects, they often occur 
in the hatcheries in various parts of the world and have been 
frequently figured and describéd since the early studies of Lere- 
boullet by Rauber, de Quatrefages, Klaussner, Gemmill, and 
others. : 

All of the double individuals and twins recorded have been 
surprisingly well developed and normally formed. From figures 
and descriptions, it would seem as though the trout egg possessed 
a rather normal tendency to form double embryos, and the causes 
necessary to give expression to this tendency were so slight as 
not to be further injurious to the development of the individual 


180 CHARLES R. STOCKARD 


embryos. In other words, some very small and simple chemical 
or physical irregularity in the developmental environment is suf- 
ficient to cause two embryos to grow from the germ-ring, but is 
not so injurious as to induce a deformed or abnormal develop- 
ment in the young fish. When either component of these double 
specimens is deformed, the cause of such deformities may be 
more reasonably attributed to conditions other than the sur- 
rounding environment (see beyond). 

Several years ago I obtained a large number of young trout, 
many of which were twins and others presented different degrees 
of doubleness. Since then I have visited several trout hatcheries 
and have found in all that double specimens very frequently 
occur. The practical fish culturists in two of these hatcheries 
thought that such abnormal double specimens were caused by 
early development under too crowded conditions, or in sluggish 
water where the eggs did not obtain sufficient aeration. Such 
views are very probably correct, since all of my experimental 
studies with fish eggs has indicated that some retardation in rate 
or interruption of development was the simple cause of unusual 
structural responses in the embryo. Only recently, however, 
could a satisfactory explanation of double conditions be worked 
out on this basis, and the trout specimens gave the key to the 
situation. The foregoing experiments with Fundulus were then 
conducted to further substantiate the conclusions. 

The artificial production of double trout embryos is no doubt 
rather difficult to bring about, since evidently only a slight slow- 
ing of the rate of cell proliferation at a particular moment is 
favorable. 

Plates 1 and 2 illustrate a series of double trout which are 
selected from the large number of such specimens that have been 
obtained. The series shows the various degrees of double forma- 
tion, beginning with a partially double-headed condition, and 
passing through the double anterior regions on single bodies, to 
double bodies with single tails, and on to the condition of com- 
plete doubleness but with the two components jomted more or 
less intimately together. The final specimen shows two com- 


STRUCTURE AND DEVELOPMENTAL RATE 181 


pletely developed twins attached to the common yolk-sac. It 
will be noted at once that each of the final twin individuals is 
equally as large and perfect in form as is the single specimen at 
the beginning of the series. This fact is of importance in showing 
that up to this stage of development and growth there is no 
question of available food, since the amount to be had in each egg 
is here demonstrated to be sufficient to form two full-size perfect 
young trout instead of the usual one. 

In studying the graded series of duplicities illustrated by plates 
1 and 2, the question immediately presents itself as to why the 
two components in the several specimens show the different 
degrees of separation? What conditions or arrangements deter- 
mined that the specimens in the upper part of the series should be 
double headed, while those at the end of the series are com- 
pletely double bodied? Gemmill (’12) in his monograph on the 
teratology of fishes, considered these propositions and gave an 
explanation for the varying degrees of doubleness which I believe 
my studies completely confirm. On the other hand, Gemmill 
failed to give any explanation of the initial or actual cause of 
doubleness. 

In accordance with the view that has often been suggested, 
the germ-ring was recognized by Gemmill “‘as a stock, able to 
give rise vegetatively, so to speak, to more than one embryo.” 
The embryonic axis or body begins to form in the embryonic 
shield which arises from certain places along the germ-ring. 
When two shields arise, the degree of duplicity of the resulting 
double fish “varies directly with the original distance between 
the two centers of embryo-formation.’”’ When the centers of 
embryo formation are close together, only 5° to 10° apart on the 
germ-ring, the embryonic axes very soon become united so that a 
double-headed specimen with a single body finally develops. 
It may be stated generally that when the original buds are less 
than 90° apart the specimens formed will exhibit various degrees 
of double anterior halves on single posterior parts. When the 
distance between the initial buds is greater than 90° and on up 
to 180°, the resulting specimens will show the double condition 


182 CHARLES R. STOCKARD 


not only involving the anterior half, but extending into the pos- 
terior part of the body. Finally, at 180° apart, the two embry- 
onic shields give rise to two completely separate twin individuals. 

The accompanying diagram may serve to illustrate the man- 
ner in which such processes operate. In figure 11 the diagram 
on the left shows two early embryonic shields arising about 20° 
apart. When the germ-ring has descended further over the 
yolk-sphere, the dotted line indicates how thé two embryonic 


Tigi 


Fig. 11 A series of diagrams illustrating the manner in which the degree of 
duplicity in embryos is determined by the original distance apart of the two 
embryonic shields on the single germ-ring. The solid lines indicate the early 
germ-rings with the two embryonic shields, and the broken lines show the re- 
sulting body outlines of the former embryos. The figure on the left has the 
embryonic shields less than 90° apart on the germ-ring and the dotted outline 
of the resulting embryo indicates it to be a double-headed specimen. In the 
central figure the embryonic shields are a little more than 90° apart, and the 
resulting duplicity extends throughout the upper half of the body. In the 
figure on the right the embryonic shields are 180° apart, or opposite ope another, 
and two complete twin individuals result, as the dotted lines indicate. 


> 


axes become united or common in the body region, and such a 
condition would finally give rise to a single-bodied fish with two 
heads. The middle diagram in figure 11 illustrates similar steps 
in the history of a ‘Y monster,’ or individual with two heads and 
bodies and a single tail. The right diagram of figure 11 shows 
two embryonic shields arising 180° apart, or opposite one another 
on the yolk-sphere, each of these has an entire half of the germ- 
ring to develop from, and complete twins are produced. 


STRUCTURE AND DEVELOPMENTAL RATE 183 


In studying the early embryos of Fundulus these several steps 
have actually been observed. An observation of further import- 
ance in this connection has also been made, but unfortunately at 
present on very few specimens. In attempting to discover the 
earliest stages of doubleness from great numbers of eggs, I have 
selected all specimens seeming in any way to possess two early 
embryonic shields. On two occasions a fair number of such 
specimens were apparently found, one lot of seven such eggs and 
another of five. These seemingly double-embryo formations were 
isolated and observed during later stages, with the result that 
from among the seven specimens only two double-headed indi- 
viduals arose, while the remaining five formed typically single 
embryos. The second group of five seemingly early double 
shields gave rise to five perfectly single specimens. There would 
appear to be only one interpretation for such a phenomenon: 
two initial buds may sometimes appear, but later one is com- 
pletely suppressed by the other, or the two possibly fuse com- 
pletely and only one normally single individual is developed. 
Therefore, it would seem that initial multiple buds are much 
more common than the resulting double specimens indicate, and 
that many secondary buds are suppressed or lost during early 
development. A comparison of the two components in older 
double monsters which is undertaken in a further section of this 
paper makes still more probable such deductions. 

I wish to present these observations on the early double speci- 
mens with the chances of error fully in mind. In the first place, 
I succeeded in isolating a very few such probable early specimens, 
twelve from the many hundred eggs examined, and from these 
twelve only two actually showed double conditions during their 
later stages of development. The early embryonic shields were 
irregular and not strongly expressed. On the other hand, it 
seems to me significant that from the twelve specimens which 
were isolated two of them definitely developed double embryos, 
while it is recalled that among the great numbers of Fundulus 
eggs experimented on extremely few double specimens actually 
occurred. However, when one selects early specimens thinking 
them to be of a definite type and they later develop into indi- 


184 CHARLES R. STOCKARD 


viduals of another type, his confidence is considerably shaken in 
the validity of the selection. I have had similar experiences in 
attempting to isolate from large numbers of eggs those showing 
the earliest indication of the cyclopean defects. There are no 
doubt processes of regulation which may tend to correct and 
obliterate an early unusual arrangement, yet in spite of the rec- 
ognized probabilities for mistake, I nevertheless feel that the 
foregoing indication of suppression of early buds has some real 
value, since two actually double specimens were certainly selected, 
as later development showed. 

Kaestner (’98—’07) has figured very early double primitive 
streaks in the chick and Assheton (’08) double embronic shields 
in the sheep. Kopsch (95) has described in Lacerta agilis, the 
European lizard, one blastoderm with two blastopores, and thus 
showed that a double gastrulation had taken place. From this 
observation he agreed with O. Hertwig that all twin formation 
as well as all anterior duplication arose from a double gastrula- 
infolding or proliferation. This position leaves, as Kaestner (’99) 
has stated, the question of doubleness or twins merely moved 
back to an earlier stage before the origin of the two blastopores, 
it remained to be answered why the double infolding takes place, 
and why it is so rare? In the present study it is felt that both 
questions are answered. A developmental arrest does away with 
the normal advantage of the usual growing point and permits a 
double gastrulation; the condition is rare for the same reason 
that the apical or dominant bud rarely fails to grow. 

Returning to the consideration of the actual case in the trout, 
we may judge indirectly by the degree of separation of the two 
components in the several individuals as to the probable distance 
apart of the original embryonic shields or embryonic axes on the 
‘germ-rings. Gemmill (’01), has found a rather high propor- 
tion of complete twins among something more than seventy 
double trout specimens that he examined, while Windel (795), 
‘had found only nine complete twins among 117 eggs containing 
double trout, or a proportion of one to thirteen. Among my 
double trout specimens there is one case of complete twins for 
every eight. From these observations it may be concluded that 


STRUCTURE AND DEVELOPMENTAL RATE 185 


when two embryonic shields arise from the germ-ring they occupy 
positions about 180° apart, or are opposite one another on the 
yolk-sphere, in nearly 10 per cent of the cases. 

A further question bearing on the relative position of the em- 
bryonic shields on the germ-ring suggests itself. If the germ- 
ring is actually a potentially budding stock, why does not trip- 
let and quadruplet formations appear almost as frequently as the 
double or twin condition? This question can at least be an- 
swered with a probable explanation, if not with a completely 
satisfactory one. In the first place, the extreme tendency of 
the eggs to form only a single rather than a double individual is 
important in this connection. There is certainly only a slight 
chance to form double specimens. The single bud is almost 
always capable of suppressing further expressions in the blasto- 
derm. When this capacity is in any way lowered and a second 
bud arises, the stock is then still further dominated or pre- 
empted by the presence of the two, and the chance for still a 
third embryo formation is decidedly reduced. Yet among a 
hundred multiple cases one triplet may be found. 

Gemmill found one case of three embryos in a trout egg as 
against over seventy doubles. This specimen had one almost 
perfect embryo and the other two were very abnormal and poorly 
developed. Among something less than 150 double fish embryos 
seen during the past few years I have observed only one triple 
specimen. This arose from a Fundulus egg that had been inhib- 
ited during early development by a weak solution of alcohol. 
One embryo, almost normal, was on the same blastoderm with a 
double-headed specimen. 

I have never observed, nor found record of a fish’s egg contain- 
ing more than three embryos. 

The conclusions seems warranted that one point of gastrulation, 
or embryo formation, has an extremely high tendency to prevent 
or suppress the existence of any other such point of excessive pro- 
liferation. When a second point is capable of expression the two 
almost without fail completely dominate the growth capacity 
of the entire germinal region and triplets are the rarest exception. 


186 CHARLES R. STOCKARD 


The relative conditions of the individual components in the 
double trout are of considerable importance and will be diseussed 
in connection with their several particular bearings in the pages 
beyond. 


c. An explanation of the frequent occurrence of twins and double 
chick embryos 


It is extremely rare among birds for a double-headed or other- 
wise double individual to hatch from the egg; a few such irregular 
cases have been recorded. I have never, however, found record 
of complete twins hatching from the hen’s egg. On the other 
hand, when the earlier stages of chick development are studied in 
the laboratory, one rarely fails, even in a limited experience, to 
meet with double and twin embryos. The prevalence of these 
early specimens has long furnished material for studies on twin- 
ning in the chick. Among many such investigations are those 
of Gerlach (’82), Burckhardt, Dareste, Klaussner, Erich Hoff- 
man, Mitrophanow, five somewhat more recent studies by Kaest- 
ner (’98, ’99, ’01, ’02, and ’07), and most recently the description 
of several double chick embryos by Tannreuther (719). 

The almost abundant occurrence of double specimens among 
the limited numbers of eggs developed in the laboratory and the 
well-known high mortality among incubating eggs of the poultry 
farm, makes it highly probable that double and deformed em- 
bryos are not uncommon under natural conditions, but that they 
usually die during the early days of development. 

From a survey of the literature on double monsters in the 
various vertebrate classes, it would be impossible to form any- 
thing like a correct estimate of the comparative frequency of such 
individuals in these several groups. It would be simply specula- 
tion to claim that doubleness was more frequent among the em- 
bryos of birds than among those of mammals. Yet the double 
condition in birds is just here of particular interest as probably 
being due to a somewhat definite and uniform cause arising out 
of their peculiar mode of development. The double bird em- 
bryos are very probably the result of a rather easily followed 
natural experiment. 


STRUCTURE AND DEVELOPMENTAL RATE 187 


It is a well-known fact, as mentioned in the early pages of this 
discussion, that the eggs of birds normally have a discontinuous 
mode of development. Fertilization takes place in the upper 
part of the oviduct and the egg begins its development in the 
high temperature of the maternal body and continues to develop 
as it travels down the uterine tube and becomes surrounded by 
its several accessory coats. Finally, at the time of laying, the 
blastoderm has passed the gastrula stage. The fall in tempera- 
ture experienced on leaving the body of the mother causes devel- 
opment to stop in this early postgastrula condition, and the egg 
remains quiescent until the temperature is again raised to about 
that of the bird’s body. 

From the evidence given in preceding sections regarding the 
developmental time of inducing double embryo formations, it is 
seen that the bird’s egg at laying has just passed the critical 
moment for causing double invagination or double blastopore 
formation. Since these double invaginations may be brought 
about by either interrupting development or slowing its rate 
before gastrulation, it would seem that the bird’s egg had been 
piloted beyond this danger period within the body of the mother. 
How, then, is the frequency of double and twin chicks in the bird’s 
egg to be accounted for? 

In studies on the early stages of development in the bird’s egg, 
it has been found by Patterson (’09) and others that the process of 
gastrulation takes place very close to the actual moments of lay- 
ing. The time relationships between the moments of laying and 
finished gastrulation are, however, in general slightly variable, 
and the eggs of certain females, as I learn in conversation with 
Professor Patterson, differ decidedly from others in their tendency 
to be deposited at an unusually early stage. There would thus 
seem to be a strong probability that all eggs of the bird have not 
reached or passed the gastrulation process before the time of 
laying. This is a most important probability, and is believed to 
be true by some of those who have studied these early stages very 
extensively. 

On the basis of my own experimental results, this probable 
variation in the moment of laying is entirely sufficient to account 


188 CHARLES R. STOCKARD 


for the double individuals and twins among the chick embryos. 
It also accounts most satisfactorily for the apparent frequency 
of such occurrences. ‘The interruption of development following 
a fall in temperature at laying and before gastrulation has begun 
prevents the single gastrulation process from beginning at a rate 
sufficient to dominate the growth conditions of the entire blasto- 
derm as it normally does. A second gastrular infolding or blas- 
topore formation is established and thus two embryo formations 
are begun. 

The usual interruption in the development of the bird has, 
with slight variations, been introduced at a most fortunately pas- 
sive stage, just following gastrulation. This is a moment at 
which developing fish eggs may be stopped with impunity for 
considerable lengths of time and injurious results rarely ever 
follow. It is a moment following which no important embryonic 
structure need arise for a considerable length of time. After 
gastrulation only the linear growth to establish the embryonic 
axis immediately occurs. None of the highly energetic folding 
processes resulting from a localized excessive or unequally rapid 
proliferation take place until after a considerable interval of slow. 
growth has passed. This interval of slow cellular proliferation 
following gastrulation is the fortunate occurrence that has pre- 
served the birds as a class among present-day vertebrates. Had 
birds been so constructed that the egg was laid and allowed to 
discontinue its development before gastrulation had taken place 
it is conceivable that this condition could have eliminated them 
from the animal kingdom. There would have followed such a 
high proportion of deformed and defective specimens from eggs 
interrupted before gastrulation, that the individuals of a class 
having its eggs stopped at this time would very soon become so 
generally deformed as to be unable to maintain their existence. 
The important matter of a few hours’ difference in egg-laying 
time lies between the successful class of birds and a hopelessly 
unfit monstrous condition. 

Obviously, the evolution of the developmental environment 
has been of equally as great importance in the survival of a spe- 
cies, as has been its constant structural fitness. Nature’s experi- 


STRUCTURE AND DEVELOPMENTAL RATE 189: 


ment of temporarily lowering the surrounding temperature and 
stopping the developmental progress of the bird’s egg has not 
proved fatal simply on account of the fortunate fact that the 
development is usually stopped during a very passive stage. 

The slight individual variations in egg-laying time which cause 
certain eggs to be interrupted before gastrulation very probably 
furnish the material for the many descriptive studies of double 
avian embryos. On the other hand, it is a most significant fact 
to note that in spite of the many experimental studies on devel- 
oping hen’s eggs by Dareste, Fere, the writer, and others no 
double monster or twin conditions have been produced. This 
absence of double productions would naturally be expected, 
since the eggs were experimentally treated only after having been 
laid.’ They had thus passed gastrulation or the time after which 
double conditions cannot be induced. 

Gerlach (’82) long ago thought that he had probably induced 
experimentally double anterior ends in chick embryos. His re- 
sults were most uncertain, and have been interpreted as acci- 
dental by subsequent writers. He made injections over the blas- 
derm so as to get fusions with the overlying shell. With such 
experiments he obtained double indications at the forward end 
of the embryos in two cases out of sixty eggs. Gerlach realized 
that conclusions could not be drawn from these meager results, 
but believed that if this method were perfected, it would yield 
more convincing results. Such experimental efforts to produce 
doubled conditions in hen’s eggs are very probably futile, since 
the evidence at hand would indicate that there is only the rarest 
chance of the experimenter’s striking an egg in the proper devel- 
opmental condition to make possible the production of twin or 
double individuals. Should such specimens be obtained among 
the eggs employed in an experiment, there would always be the 
possibility that the natural interruption in development occur- 
ring in an egg laid at an unusually early stage was the cause of 
the doubleness, and not actually the experimental procedure. 


190 CHARLES R. STOCKARD 


d. An explanation of polyembryony in the armadillo 


On examining the uterus in two pregnant specimens of a South 
American armadillo, von Jhering in 1885 discovered that each 
contained eight fetuses enclosed within a single chorion. He cor- 
rectly concluded that all of the fetuses in each mother had been 
derived from a single egg by some process of division into separate 
embryonic rudiments. After this valuable discovery and inter- 
pretation, the study of the armadillo’s development lapsed and 
nothing of importance was added for almost twenty-five years. 
Two series of investigations were then begun simultaneously, one 
on the South American species by Fernandez (’09) and the other 
on the Texas armadillo by Newman and Patterson (’09). The 
growth and expansion of these twin studies has brought our 
understanding of the phenomenon of polyembryony in the arma- 
dillo to a considerable state of maturity. 

These authors readily agreed that in most species of armadillo 
the individual members of a litter, usually four in the Texas 
species and eight in the common South American form, are all 
derived from a single egg. It required considerable effort, how- 
ever, to obtain the material that would furnish the morphological 
stages of the proccess by which this polyembryonic development 
was accomplished. We are finally indebted to Patterson (713) for 
the very thorough and satisfactory manner in which he has col- 
lected and studied the early embryonic conditions; and particu- 
larly for having shown the first stages of the budding process 
through which the single blastocyst gives rise to four distinct 
embryonic areas, each exhibiting a typical primitive streak region. 

In connection with the idea constantly advanced in the present 
study that twins and double vertebrate embryos arise from ac- 
cessory growths or invagination points around the blastoderm, 
it now becomes important to ascertain exactly what degree of 
development has been attained by the armadillo blastocyst at the 
time the budding process begins. And since, according to our 
interpretation, these buds should arise at the time of gastrula- 
tion or blastopore formation, it becomes necessary to consider 
very briefly the germ-layers and gastrulation in mammals. The 


STRUCTURE AND DEVELOPMENTAL RATE 191 


decidedly precocious and highly modified method of forming the 
primary germ-layers in the mammalian blastocyst is not strictly 
comparable to gastrulation or the method of germ-layer forma- 
tion found among the other vertebrates. On the other hand, 
the embryonic line or primitive streak of the mammalian egg is 
exactly comparable to the blastopore and embryonic process 
formation in the simpler forms. 

The blastocyst of the armadillo has already, by a process of 
cell migration and delamination, separated off the primary ento- 
derm from the ectoderm and further modified these layers before 
the budding which forms the embryonic primordia has begun. 
But it is in the primordia that the invagination of the entoderm 
forms the secondary entoderm of the gut and the embryonic 
mesoderm arises from a typical primitive-streak region much as 
in lower vertebrates. The precocious cell migration and _ split- 
ting into layers in the mammal’s egg is associated with the early 
implantation of the embryo upon the uterine wall of the mother, 
and the later primitive-streak formation may be interpreted as 
related to the actual gastrulation or blastopore formation away 
from which the line of the embryo always develops. 

Whether the validity of the above briefly outlined interpreta- 
tion of the germ-layer formation is admitted or not, we have in 
the armadillo a process of budding taking place from the blasto- 
derm and associated with accessory or extra blastopore formation 
in much the same way as are the accessory embryos along the 
germ-ring in the egg of the bony fish. These buds also accord 
with Kopsch’s (’95), description of a double gastrular condition 
with two blastopores in a blastoderm of Lacerta agilis, from which 
he concluded that twin formation as well as anterior duplica- 
tion arises from a double gastrula—Einstiilpungen. And, further, 
Assheton has described a similar condition in a blastodermic 
vesicle of the sheep. He, however, imagined the condition to 
have been due to a splitting during the morula stage. 

The double primitive streaks in the hen’s egg and other forms 
_ all lend themselves to strengthen the interpretation that double 
embryo formation first asserts itself by a double gastrulation 
or blastopore formation, which is initially a process of double 


192 CHARLES R. STOCKARD 


instead of single bud formation. Patterson’s description of the 
origin of the quadruplet buds in the Texas armadillo furnishes the 
most striking case in the study of these conditions. And we may 
conclude that the budding or accessory embryo formation in the 
egg of the armadillo is exactly the same developmental process 
as that which gives rise to twins and double individuals in other 
vertebrate eggs. 

However, the very important question yet remains to be an- 
swered: Why does this accessory bud formation occur so con- 
stantly in the Texas armadillo in contrast to the single embryo 
formation of mammalian eggs in general? Patterson (713) failed 
entirely to answer this question, but he supplied some very sig- 
nificant data which Newman (’17) has appreciated as being inti- 
mately connected with the occurrence of polyembryony. 

In connection with the collection of material Patterson (’13) 
discovered a ‘period of quiescence’ of the embryonic blastocyst. 
Regarding this he states: ‘“‘The fact was first made apparent in 
1911, when, after I had started collecting two weeks earlier than 
in the preceding year, I failed to obtain the cleavage stages, al- 
though judging from the condition of development in the vesicles 
collected in previous years, one would naturally expect to find 
these early stages during the period of my first collection in 1911.” 
The following year be began collecting still two weeks earlier 
and again had a similar experience. ‘‘Practically all of these 
vesicles lie free within the uterine cavity, either in the horizontal 
groove or in the region of the attachment zone (placental area).”’ 

“Tt is evident from these data that the embryonic vesicle re- 
mains for some time lying free within the uterine cavity. Just 
how long this period lasts, I am unable to state; for practically 
every old female taken at the earliest date (October 15th) at 
which I have collected, possesses a free blastocyst. How long 
such blastocysts have been in the uterine cavity it is, of course 
impossible to determine; but I should judge not very long, be- 
cause two vesicles taken from the fallopian tubes show a develop- 
ment almost as far advanced as that of some vesicles taken from 
the proximal parts of the horizontal grooves. Taking all the facts 
into consideration, I estimate the ‘period of quiescence’ to last 


STRUCTURE AND DEVELOPMENTAL RATE 193 


about three weeks; that is, from about the middle of October to 
the third or fourth of November. . . . Of the thirty-four 
free blastocysts obtained in 1911 and 1912, twenty-eight of them 
were secured within this period.” 

In a study of sections no mitotic divisions were found to occur 
in the blastocysts during the ‘quiescent period.’ 

The only point of interest cited by Patterson in connection 
with this peculiar phenomenon of interruption in development, 
was the fact that in no other mammal except the deer, had such 
a condition been found. Bischoff (54) had long ago reported a 
‘period of quiescence’ lasting for some weeks during a so-called 
morula stage of the deer embryo. 

Newman (’17) has recognized the importance of Patterson’s 
discovery of the ‘period of quiescence’ during the early develop- 
ment of the armadillo, and states in a discussion of twin formation 
that this ‘period of quiescence’ probably “‘holds the clue to the 
physiological explanation of polyembryony.” In this position 
Newman is, in my opinion, largely right, but this is as far as the 
data led him, and he finally remarks: ‘‘The problem is to locate 
the factors responsible for the slowing down of the develop- 
mental rhythm. Whatever these factors may be, and we have no 
definite knowledge of them, the result of retardation is polyem- 
bryony.” 

Newman thus fails to appreciate the second point in Patter- 
son’s discovery, and that is, that the blastocysts always lie free 
in the uterus during the ‘period of quiescence.’ This fact en- 
ables us to go one step further since the lack of attachment and, 
therefore, lack of oxygen supply are very probably “the factors 
responsible for the slowing down of the developmental rhythm.” 
The armadillo egg, like that of most mammals, undergoes its 
early development in the Fallopian tube and. is, therefore, capable 
of reaching the blastocyst stage on its initial oxygen supply. After 
this time however, it must become attached to the uterine wall 
for a further source of oxygen. For some reason, in the armadillo 
the reaction between the blastocyst and the uterine wall is post- 
poned and the blastocyst is incapable of further developmental 
progress until this reaction is established and the necessary supply 


194 CHARLES R. STOCKARD 


of oxygen becomes available. In exactly the same way the de- 
velopment of the blastoderm in the fish’s egg is experimentally 
retarded or stopped by reducing the available oxygen and is again 
made to resume its development by supplying oxygen. In the 
ease of the fish egg, the supply of ordinary nutriment is not in- 
volved, and reactions similar to those of the armadillo egg are 
only obtained as responses to changes in temperature or rate of 
oxidation. 

I do not believe the retardation in the armadillo egg is of the 
nature of a starvation phenomenon, since we see nothing of the 
kind in other forms. Temperature changes are ruled out, since 
the temperature of the uterus is more or less constant. The ab- 
sence of oxygen necessary for the energetic process of cell divi- 
sion is, therefore, in all probability the arresting cause, and the 
retardation results in polyembryony. 

Thus Patterson has found the developmental interruption to 
exist, and he has also shown the blastocyst to be disconnected 
from the uterine wall and its netessary oxygen supply during this 
time. However, he has furnished no data bearing on the reason 
for the delay in uterine reaction and the consequent failure of 
immediate implantation of the blastocyst such as normally occurs 
in other mammals. 

The consideration of the armadillo egg up to this point has 
taken account only of the external factors influencing its mode of 
development. It must now be remembered as a fact of serious 
importance that the production of quadruplets from the single 
egg of the Texas armadillo is an almost constant occurrence, 
while the experimental attempts to produce twins and double 
individuals in fish eggs and other forms have given at best only 
small percentages of such individuals among the large groups of 
eggs treated. It is also recalled that all eggs do not furnish 
equally favorable material for artificial twin production. The 
eggs of the trout seem unquestionably more disposed to give rise 
to twin formations than do the eggs of Fundulus. Thus some 
eggs would seem to have an hereditary or truly innate predisposi- 
tion toward polyembryonic formations. There is much reason 
to believe that, aside from the external factors discussed, the 


STRUCTURE AND DEVELOPMENTAL RATE 195 


armadillo egg itself is highly disposed toward the formation of 
* accessory embryonic buds. 

There is the possibility, of course, that this natural experiment 
with the armadillo egg has become so exactly regulated as to 
influence the developmental processes precisely the same way 
each time, yet this is highly improbable for several reasons. 
The armadillo egg is not a case of simple twin growths from the 
blastoderm, but, as Patterson finds, there are primarily two buds, 
and then very promptly two secondary ones arise making the 
four, and after this the budding process ceases. In the South 
American species, however, it would appear as though a tertiary 
budding occurred giving the usual eight embryos; and in rare 
cases still another budding occurs from a few of the existing buds, 
giving a total of as many as twelve. It would certainly seem as 
though the blastoderm in these species passes through a stage of 
agametic reproduction or budding of a nature unknown among 
other higher vertebrates. But the possibility for such expression 
might only exist on account of the delay in implantation of the 
blastocyst and consequent shortage of the oxygen supply neces- 
sary for the rapid formation and growth of the single embryo. 

It is important to keep in mind that there are species of the 
armadillo which produce only a single offspring from one egg. 
It is not known whether their embryos have a ‘period of quies- 
cence, but if they have the period either occurs at a different 
developmental stage or the egg does not possess the inherent 
budding tendency of the other species. 

It remains now to account for the fact that although the egg 
of the deer has a ‘period of quiescence’ during its development it 
does not give rise with any degree of frequency to twin indi- 
viduals. In the first place, it is entirely uncertain from the scanty 
accounts as to what time in development the quiescent period 
occurs. Assuming that such a period does exist, it might occur at 
some indifferent stage when no peculiar result would be expected, 
for example, after gastrulation as it does in the bird with no 
subsequent effect. In the light of the experimental production 
of double individuals, it is readily understood that even though 
the egg of the deer is interrupted in its development at an early 


196 CHARLES R. STOCKARD 


stage, it might still be capable, on resuming development, of 
giving a normal single embryo. The egg of the deer may possess 
only a very slight tendency toward accessory embryo formations. 
A study of the experimental production of twin and double indi- 
viduals among fish leads one to be surprised at the case of the 
armadillo and to expect the reaction found in the deer. The 
constant interruption occurring in the development of the birds 
and other animals at indifferent developmental moments with 
no subsequent ill effects renders commonplace the fact that the 
deer successfully withstands an interruption during its develop- 
ment without noticeable modifications in structural response. 

In conclusion we may summarize the cases as follows. The 
development of the armadillo is interrupted on account of a 
failure to become promptly implanted on the uterus and a con- 
sequent exhaustion of available oxygen supply. The interrup- 
tion occurs at a critical period just preceding the primitive- 
streak and embryonic-line formation. The internal qualities of 
this egg gives to it a decided tendency under conditions of arrest 
to form accessory embryonic buds. As a result of the inter- 
action of these external and internal forces polyembryony is 
produced. 

In the case of the deer only one probable fact is known, and 
that is that a ‘period of quiescence’ occurs. It is uncertain at 
what stage the arrest takes place, but it is probably due, as in 
the armadillo, to a delayed implantation of the blastocyst. Hither 
on account of the stage of arrest or a lack of tendency to form 
accessory embryo buds, a typically single individual arises 
from this egg. The external factors may be the same as in the 
case of the armadillo, but they interact with different internal 
factors or different developmental moments to give a very differ- 
ent result. 


STRUCTURE AND DEVELOPMENTAL RATE 197 


e. ‘Alternation of generations’ and twins in vertebrates 


Among plants and lower animals, particularly the coelenterates, 
there commonly exists a so-called alternation of generations. A 
given species at one time reproduces sexually by the union of 
gametes, egg and sperm cells, and the individuals derived from 
such gametes then give rise to a number of other individuals by 
a growth and fission or a budding process. Finally, sexually 
mature individuals again occur to reproduce another generation 
from germ-cells. In general this phenomenon is thought to be 
limited to these lower forms. 

The suggestion has frequently been made but without sufficient 
emphasis that the blastoderm may be looked upon as a stock 
able to give rise asexually to more than one embryo. Since the 
natural process of budding to form four or more embryos in 
the armadillo is recognized, and accessory individuals may be 
produced experimentally from other vertebrate eggs, it becomes 
evident that even man and the highest animals may actually at 
times exhibit an alternation of the sexual and asexual processes of 
reproduction. 

In a subsequent section of this paper the origin of various or- 
gans of the individual’s body will be considered as arising initi- 
ally through a budding process exactly comparable to the ini- 
tial embryonic axis bud on the blastoderm. These buds may 
also be suppressed or inhibited in their expression in much the 
same way and by similar experimental methods as was described 
above in the case of the embryonic axis or initial embryo bud. 

From a general biological standpoint the adult body of higher 
animals may be very correctly considered to be derived from a 
sexually produced embryonic axis the stock which gives rise by 
an asexual method of budding to the various special organs. 
The vertebrate body is thus composed of a group of different 
zooids, the organs. There are seeing, hearing, excretory zooids, 
and so on, comparable to the zooids of a siphonophore colony. 

Alternation of generations is here considered a phenomenon, 
not limited as is generally taught to lower forms, but occurring 
throughout the animal kingdom. 


198 CHARLES R. STOCKARD 


6. STRUCTURAL DIFFERENCES BETWEEN THE TWO COMPONENTS 
IN CONNECTED TWINS AND DOUBLE INDIVIDUALS 


As illustrated in plates 1 and 2, the components in connected 
twins and double individuals exhibit various degrees of separate- 
ness from partial double-headedness to completely double indi- 
viduals. It has also been brought out in the previous section 
that the degree of doubleness shown by any such specimen de- 
pends upon the original distance apart of the two embryonic 
shields along the germ-ring of the fish’s egg, as illustrated in the 
diagrams of figure 11. As Morrill (’19) has pointed out, the dif- 
ferent extents of doubleness are in no way connected with differ- 
ent times of origin of the condition as was suggested by Newman 
(17, p. 17-18), since every extent of doubleness is shown in this 
fish series and the time of origin from the developmental stand- 
point is the same in each case. 

Irrespective of the degree of doubleness or the distance apart 
of the two components, there is a most significant competition, 
so to speak, between the components themselves, just as exists 
among several buds growing from a common stock. It is the 
results of this interaction or competition between the two com- 
ponents which we wish to consider in the present section, and 
their bearings, of very general importance, will be analyzed in the 
sections following. 


a. Double individuals with identical or equal-size components 


The two components in each of the specimens photographed 
in plates 1 and 2 are practically of equal size. The first plate 
illustrates the young trout from a dorsal view and the second 
plate shows the same individuals arranged in the same order from 
the ventral aspect. On comparing the two views of every speci- 
men, it will be found that all heads are perfectly normal in appear- 
ance, each having two fully developed eyes, a perfectly formed 
mouth and branchial structures and a perfectly developed bilat- 
eral brain with its general contour clearly visible below the skin. 
On further comparing the two views in a given specimen, the body 
regions of the components are also found to be about equally 


STRUCTURE AND DEVELOPMENTAL RATE 199 


developed, except that in one or two of the cases one component 
is more decidedly twisted than the other. This twisted condition 
in some cases causes. one component to appear considerably 
larger than the other. This, however, is only an appearance, 
and examination of the actual specimen shows the components to 
be very closely equal in size. 

Correctly speaking, none of these components are structurally 
deformed. The application of the term ‘double monster’ to such 
individuals as these is actually a misnomer, since there is nothing 
whatever deformed or monstrous about their structures. The 
condition of being double is a perfectly normal result of the growth 
of two buds from a single stock. However, these individuals 
have arisen from unusual conditions acting on the developing egg 
during a particular interval and exhibit, therefore, unusual and 
modified developmental results. Similar conditions affecting 
other developmental periods are responsible for the production 
of all types of structural deformities and so-called monsters. 
The double series is, therefore, similar in so far as its causal origin 
goes to the ordinary monstrous forms, yet one could scarcely 
term two perfectly developed identical twins such as those shown 
by the last specimen of the series as monsters. 

A study of the series here illustrated in addition to a large num- 
ber of similar double specimens not only of fish, but of other ani- 
mals as well as man, leads to the general conclusion that, When 
the two components of a double individual are equal in size they are 
both normal in structure. This means simply that such compo- 
nents are as strongly inclined to be normal as is a single individual 
and not that they are never deformed. All figures of double 
specimens in the literature further illustrate this point. One 
may deduce from these facts that if there was a competition of 
any kind between two such components, the advantages of each 
in the struggle have been equal. When the advantages are un- 
equal, it will be found that a very different state of affairs results. 


CHARLES R. STOCKARD 


STRUCTURE AND DEVELOPMENTAL RATE 201 


b. Double individuals with unequal components 


In every extensive collection of. double specimens we not only 
have those with components of similar size, but also a number of 
double individuals presenting two components of different size. 
The discrepancies in size between the two components may be 
arranged in a graded series beginning with only a slight size dif- 
ference and finally ending with a very small mass attached to 
the larger component. Figures 12 to 17 illustrate such a series 
in cases of anterior duplicities, and figures 20 to 27 show various 
size differences between the components in completely double 
specimens. 

Associated in all cases with these size differences are strikingly 
noticeable and important structural differences between the 
components. 


Figs. 12 to17 A series of double-headed trout specimens some time after 
hatching, and illustrating the fact that when the two components of a double 
individual are unequal in size the larger component is normal in structure and 
the smaller component is invariably defective. 

Fig. 12 The two heads in this individual are equal in size and both are 
structurally normal. 

Fig. 18 The left head is slightly smaller than the right, and the right eye of 
the smaller head is defective with a wide coloboma. The right head is entirely 
normal. 

Fig. 14 The difference in size between the two heads is more marked than in 
figure 13 and the smaller head is also more decidedly deformed. Its right eye is 
entirely absent and the left eye is extremely defective, being only a small choroid 
body with a protruding crystalline lens. The mouth and gills are unopened 
with considerable structural distortion. The larger left head is in all respects 
perfectly normal. ‘ 

Fig. 15 The left head is normal in size and perfect in structure, while the 
smaller right head is completely deformed with a twisted irregular shape and no 
definite outer indications of mouth and gills. The right eye is absent and the 
left eye is defective. A somewhat different view of the smaller head is shown 
immediately below the entire figure. 

Fig. 16 A double specimen with the left head still smaller in size and more 
completely deformed. It has a cyclopean eye, and a narrow tubular brain, and 
the branchial parts are entirely distorted. 

Fig. 17 Completes the series with a perfectly formed larger component, 
while the smaller left head is represented by an amorphous mass as seen from 
surface view. Should this specimen have attained adult size, it would probably 
have been a normal trout with a small nodule representing the lesser component 
projecting from its body wall. 


202 CHARLES R. STOCKARD 


1. Condition of the larger component. Whenever the compo- 
nents of a double individual are unequal in size, the larger com- 
ponent, with one exception in more than seventy such specimens 
that I have studied, is invariably normal in structure. <A careful 
examination of a large number of illustrations of such specimens 
through the literature, without exception confirms the above 
fact. It would seem to be a rule, that the larger component of a 
double individual is no more likely to be defective in form or structure 
than is a single individual of the same species developing under a 
similar environment. 

2. Condition of the smaller component. Whenever the compo- 
nents of a double individual are unequal in size the smaller compo- 
nent, in all cases examined, is always abnormal in form and structure. 
A survey of the figures in the literature also shows this to be 
constantly the case. 

A study of the types of deformities and defects exhibited by 
these smaller components is most instructive, and is further 
extremely suggestive in an analysis of the causes underlying all 
abnormal development. 

Examining first the cases of anterior duplicities, figure 12 shows 
two heads of equal size, both structurally normal. In figure 13 
the left head is only slightly smaller than the right. The right 
head is normal, but the right eye in the left head is small and de- 
fective in form, with a ventral coloboma and a protruding crystal- 
line lens. The size difference between the two heads is slight 
and the abnormalities shown by the smaller are not of an extreme 
type. : 

In figure 14 one head is decidedly larger than the other, the 
larger head, as usual, is normal, the smaller is very abnormal. 
There is only one minute deeply buried eye, #, and the structures 
of the mouth and branchial arches are peculiarly distorted. Fig- 
ure 15 shows a still more marked size difference between the two 
components, and the smaller one here is decidedly twisted, with 
two poorly developed eyes almost in apposition on the ventral 
surface of the head. Mouth and gill formations are superficially 
suppressed, but there are certain contorted structures represent- 
ing these parts. The brain lacks its usual bilaterality and has a 


STRUCTURE AND DEVELOPMENTAL RATE 203 


twisted tubular shape. The small figure immediately below the 
defective head represents the opposite view of this head. 

In figure 16 the smaller component presents a typical cyclopean 
eye beneath the anterior tip of a narrow, almost solid brain. 
Here again the mouth and gill structures are grossly deformed. 

Finally, figure 17 shows only an amorphous mass representing 
the smaller head on a perfectly normal larger component. This 
head mass contained no ophthalmic structures at all, the brain 
was entirely distorted and the mouth was completely absent, 
with the gill structures greatly deformed. Behind this head mass 
the pectoral fins were fairly developed and a short anterior body 
portion representing the rest of the component is shown in the 
figure. 

Figures 18 and 19 give two views of the only case observed 
among these individuals in which the larger component was 
also deformed. In figure 18 the larger left head is seen, in.dorsal 
view, to have a left eye, but no right. The ventral view shown 
in figure 19 illustrates the normal left eye and also shows the 
normally well-formed mouth and gill arrangements in the superior 
component. The right head, or smaller component, is shown 
from both views to be much more decidedly deformed than the 
left. It is completely anophthalmic and the brain, mouth, and 
gill structures are clearly abnormal. 

Since in all the other specimens the larger component is normal, 
we may claim with justification that the larger component in 
this specimen simply happens to be deformed as any single 
individual might chance to be. But the smaller component is 
more decidedly deformed than the larger, and the deformity in 
this instance no doubt results from the same reasons which have 
brought about similar deformities in all other smaller components 
of the entire group of double specimens studied. It is only to be 
expected that the larger component developing under somewhat 
modified conditions, such as those necessary to induce the initial 
doubleness, will occasionally be further affected and present 
some structural deficiency. Such abnormalities are not uncom- 
mon among those members of the experimental group which are 
not induced to double formations, but continue to develop as single 


204 CHARLES R. STOCKARD 


individuals. In other words, monophthalmia, cyclopia, anoph- 
thalmia, deformed brains, and branchial structures occur among 
single specimens developing along with the double ones. 

We may now consider the condition of the smaller component 
in double specimens in which the components are two complete 
individuals, or conjoined twins. 


JA 


1 


Figs. 18 and 19 Two views of a rare double specimen in which the compo- 
nents differ slightly in size yet both components are deformed. The left larger 
head has only one eye, the left; it is otherwise perfect, as the figures show. The 
right smaller head is completely eyeless and grossly deformed in the anterior 
portion. In this case the larger left head is by chance defective just as any 
single individual might be. 


Figure 20 illustrates normal equal-sized identical twins at- 
tached to a common yolk-sac. The development of a teleost 
embryo on a large yolk-sphere and the structure of its yolk-sac 
prohibits a free separation of identical twins and they always 
remain joined as shown in this figure. 


STRUCTURE AND DEVELOPMENTAL RATE 205 


In figure 21 the lower component is somewhat smaller than 
the upper and there is a complete absence of one eye. A drawing 
- of the opposite side of this head shown below figure 21 represents 
the other eye with an extreme coloboma, its entire ventral part 
being deficient. This component seems otherwise normal. 

Two views of another double individual are illustrated by 
figures 22 and 23. The larger component. is perfectly normal 
except for the fact that its tail is somewhat unusually bent. 
The smaller component is completely anophthalmic and _ its 
brain presents a very abnormal contour. 

In figure 24 the smaller component is still more reduced in 
size aS compared with the larger normal member. Here also 
the extent of deformity is still more marked than in the two 
foregoing specimens. There is one small deeply buried eye in 
a more or less shapeless head. The mouth and gills are dis- 
torted and poorly developed and the brain is deformed. The 
body is small and abnormally developed. 

The specimen shown in figure 25 carries the condition a step 
further. A normal well-formed trout has attached to its ventral 
surface a greatly coiled and twisted twin. This small component 
shows a minute almost buried eye, H, and the head is in many 
ways, grossly deformed. But for the extreme coiling, the body 
would present almost as good an appearance as that of the smaller 
component in figure 24. 

In figure 26 the small twin has a still more malformed head 
with no eye, but a more or less anteriorly protruding crystalline 
lens just benéath the skin. The body here is shorter than in 
the figure above and has only a single twist. 

Finally, in figure 27, the last of the series, the large component 
is a splendidly developed young fish with little more than a nodu- 
lar twin attached to the ventral portion of its yolk-sac. The 
little component has one small eye deficient ventrally, no external 
mouth or branchial formations, the brain is tubular and the entire 
head knob-like in shape. The middle body portions are sup- 
pressed and only a conical stump-like tail end is shown. The 
entire growth of the lesser component has been but a small 
fraction of that attained by the larger member. One might 


CHARLES R. STOCKARD 


STRUCTURE AND DEVELOPMENTAL RATE 207 


readily imagine that if this specimen had grown from its present 
length of 3 em. up to a size of 30 cm., the small component would 
have been so outgrown by the larger as to appear a tiny almost 
unnoticeable nodule on the ventral surface of the large fish. 
The little component might possibly have become entirely 
included within the ventral body wall of the larger one. A 
twin inclusion would thus be formed. 

8. The small component and the frequency of double or twin 
individuals. The frequency of double and twin individuals is 
probably much greater than realized. No doubt such speci- 
mens as the last one considered in the foregoing section might 
often attain the adult state without being suspected of their 
twin nature. It is also likely, in view of the fact that a graded 
series of reductions in the size of the smaller components in 
double specimens can be arranged down to the conditions here 
illustrated, that still more decided reductions exist. There 
probably are specimens with merely a trace of the smaller compo- 
nent, or it is possible that the small component might entirely 
disappear. Thus an individual appearing as a typically single 
specimen might in truth partake of the qualities and nature of 
the major component of a double individual. 

In connection with such probabilities the condition of situs 
inversus viscerum is of interest. Morrill (19) has found in an 
examination of certain of these double fish that a reverse arrange- 
ment of the viscera occurs in one of the components with a far 
greater frequency than has ever been known to occur among any 
group of single vertebrate individuals. The reverse arrange- 


Figs. 20 to 23 A series of united twin trout, some time after hatching, further 
illustrating the principle that in double individuals with components of different 
size the larger one is normal structurally and the smaller is deformed. 

Fig. 20 Twin trout, both of equal size and normal structure. Each twin is 
fully as large as a single specimen of the same age. 

Fig. 21 The upper individual is the larger and is structurally normal, the 
lower specimen is slightly smaller with no eye on the right side and the left eye, 
shown in the small accompanying figure, is deformed with a decided coloboma. 

Figs. 22 and 23 Two views of the same united pair. The upper larger indi- 
vidual is structurally normal, and the lower smaller twin is eyeless and some- 
what further deformed, with a twisted caudal region which also causes a twist 
in the tail of the larger specimen. 


208 CHARLES R. STOCKARD 


STRUCTURE AND DEVELOPMENTAL RATE 209 


ment of the viscera in one component, though it by no means 
always occurs, would seem in some manner to be associated with 
the double condition. This reversed visceral arrangement also 
occurs very rarely among man and other mammals in single 
individuals. Its remarkable frequence among these double speci- 
mens would lead one to suspect very strongly that when a reversal 
of the visceral arrangement occurs, the apparently single indi- 
vidual is in reality a twin. All such specimens should be care- 
fully examined for twin or embryonic inclusions as positive evi- 
dence of their double nature. Failure to find such inclusions 
would not, however, disprove the above suspicion, since the 
inclusions might be represented by structures so minute as to 
be readily overlooked. 

4. The small component and certain theories of teratoma. An- 
other much-debated problem may be somewhat illuminated by 
this study of double specimens. I refer to the various ideas of 
the possible origin of so-called teratomata or embryonal tumors. 
Such formations occur with greatest frequency in the lower 
abdominal or pelvic region. Certain pathologists have thought 
them to arise from a development of misplaced or arrested blasto- 
meres, others have thought it possible that they might arise 
through some form of parthenogenetic development, and still 
others have looked upon them as a type of twin inclusion. The 


Figs. 24 to 27 A continuation of the twin trout series shown in figures 20 to 
23. In this group the smaller member is still more inhibited in size and more 
completely defective in structure. The larger component is perfect in all. 

Fig. 24 The smaller twin is little more than half the size of the larger with 
an amorphous head containing one defective eye and the body is twisted. 

Fig. 25 The smaller twin is here greatly twisted or coiled, its head is de- 
formed, possessing a large defective left eye, and the right eye consists of a tiny 
choroid vesicle indicated by the dark spot, EL. 

Fig. 26 The lesser component is still smaller in size, short and twisted with a 
considerably suppressed eyeless head. 

Fig. 27 The larger twin is a beautifully normal specimen, while attached to 
the opposite surface of the yolk-sac is a small individual represented by a badly 
deformed head with no mouth or gills and one defective eye. Almost the entire 
body of this component is absent and the tail is represented by a conical mass 
with no caudal fin. Should such a specimen attain adult size the smaller indi- 
vidual would be attached to the ventral abdominal wall of the larger as a nodular 
twin. 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 2 


210 CHARLES R. STOCKARD 


frequent occurrence of teratoma in the pelvic regions was in 
line with any of these explanations. Misplaced blastomeres 
might readily be in this portion of the body and twin inclusions 
or partially deformed twin bodies are frequently connected with 
the pelvic region. 

The parthenogenetic theory which has received considerable 
support would necessitate the occurrence of all such formations 
in close proximity to the gonads and therefore would practically 
limit their occurrence to the pelvic region. It so happens, how- 
ever, that teratomata occur with considerable frequency in the 
head and neck regions. This is most difficult to explain on the 
basis of a parthenogenetic origin. It might be possible, though 
not so probable (for the blastomere theory), that misplaced 
blastomeres arrested in the early egg might develop in such 
cephalic positions. 

On the other hand, if teratoma arises from early twin inclusions, 
one would, on the basis of our series and a general survey of 
recorded double individuals, readily recognize that the head and 
neck regions should be places of frequent occurrence for these 
structures. The double specimens arrange themselves roughly 
into two groups, the anterior duplicities or double-headed lot, 
and the completely double specimens. 

Should the smaller components of the first group become 
greatly inhibited and included within the larger components, we 
should have the inclusions in the head region. Should these 
then give rise to teratomata, such growths would not be expected 
to contain tissues found in the caudal regions of the body, such, 
for example, as nephric, gonadal, lower intestinal structures, 
etc. The rather frequent occurrence of teratoma in the head 
and neck would be what one would expect. 

The inclusion of the smaller component of the completely 
double specimen would in most cases occur in the lower abdominal 
region. This would account for the great frequency of pelvic 
teratomata. Such teratomata in contrast to those of the neck 
region, may be found to contain tissues characteristic of any 
portion of the body, since these are inclusions of a possibly 
complete twin and are not limited to structures of either the 


STRUCTURE AND DEVELOPMENTAL RATE 211 


anterior or posterior regions. Nevertheless, from what we have 
seen of the tendency on the part of the smaller component in 
the completely double individuals to possess poorly developed 
head ends, it would not be surprising to find that ophthalmic 
tissue and other cephalic structures were frequently absent from 
pelvic teratomata, though such structures might in certain cases 
be particularly evident. 

5. Types of defects exhibited by the smaller component. We may 
now return to a brief consideration of the types of deformities 
shown by the smaller components in the unequal pairs and 
decide whether these defects are similar in kind to those which 
occur in nature, as well as those experimentally induced, among 
single individuals. 

In the first place, it is noted at once that ophthalmic deformities 
are particularly frequent. The illustrations show complete 
anophthalmia, monophthalmia, and typical cyclopean conditions 
as well as various degrees of imperfection in the individual eye, 
such as coloboma and reduction in size of the retinal region. 
Duplicities produced by any method such as the mechanical 
constriction employed by Spemann, as well as those occurring 
in nature, show in the smaller component the same ophthalmic 
defects as are found among these double specimens induced by 
development in low temperatures or with insufficient oxygen 
supply. 

The brain in the smaller components shows various abnormal 
contours or may be simply tubular in shape without a normal 
expression of bilateral diverticula or hemispheres. 

The mouth is often deformed and frequently absent and the 
operculum and branchial arches are distorted in shape. The 
fins are often small and underdeveloped. The general body 
shape may be variously modified, the caudal end being short 
and stumpy or absent. The heart may be poorly developed 
and pulsating feebly so that the blood fails to circulate and 
becomes massed in various regions of the body. 

It is unnecessary to do more than enumerate these defects 
and examine the illustrations to convince anyone familiar with 
the commonest developmental anomalies that the structural 


212 CHARLES R. STOCKARD 


modifications and defects of the lesser components are in every 
sense identically the same as the defects which have been recorded 
and illustrated as occurring in single individuals. 

The types of defects most commonly found, such as those of 
the eyes, are also the most frequently observed anomalies in 
single individuals. Therefore, not only the kinds of defects, 
but the frequencies of their occurrence are the same among these 
lesser components as among single deformed specimens. 

The fact that these malformed components are developing 
in intimate union with larger normally formed components makes 
it evident that the causal factors for the malformations are to 
be sought in some difference that exists between the develop- 
mental processes of the two. And, further, since the malforma- 
tions of the one component are identical with those in single 
specimens, the difference in conditions found between the larger 
and smaller component may also furnish the clue to causes of 
malformed structures in general. We shall attempt beyond, 
in section 7, to give a logical explanation of abnormal structure 
from this standpoint. 


c. The components in double human specimens 


In order to demonstrate that the conditions above described 
as existing in double specimens of fish are in no way limited to 
this class of vertebrates, I wish briefly to consider several very 
interesting degrees of double development in human specimens 
that have recently come into my laboratory. 

All of these specimens have been examined by my colleague 
Doctor Morrill for conditions of situs inversus viscerum, and the 
left component in one case of anterior duplicity, as reported by 
him (719), shows a reversal in the position of the viscera just as 
was found in certain of the components among the double fish. 

The three specimens seen in plates 3 and 4 to form again a 
graded series. The series begins with a double individual pre- 
senting two heads and anterior portions on a single pelvis, seen 
in the upper photograph, plate 3, and passes on to a completely 
double specimen with the components strongly united through 


STRUCTURE AND DEVELOPMENTAL RATE 213 


their ventral surfaces, the lower figure, and finally ends with the 
entirely separate unquestionably identical twins illustrated in 
plate 4. 

The case of separate twins differs in many ways from the other 
two and will be considered alone. 

The first two specimens are in general alike. In each the two 
components are practically of equal size, and all of the four 
components present entirely normal structures. These speci- 
mens follow exactly the rule stated in reference to the double 
trout; that is, when the two components of a double individual 
are equal in size they are both normal in structure with almost 
the same frequency as a single individual would be. 

In the first specimen, of plate 3, each head shows a perfectly 
formed face with all the sense organs fully developed. <A dis- 
section of this body shows the vertebral columns to be separate 
down to the sacrum. ‘The pelvic skeleton is single with the nor- 
mal single pair of lower extremities arising from it. The median 
arms of the two components have their soft parts fused or pecu- 
liarly arranged, a synbrachium, the details of which are being 
studied by Mr. H. B. Sutton. Each arm possesses a complete 
skeleton. Further details of the visceral structures are of no 
importance in the present connection. This specimen is in 
general comparable to the fourth case of anterior duplicity shown 
in the equal component series of trout (plates 1 and 2). 

The second human specimen, in plate 3, is a case of full-term 
united negro twins. There are two completely formed compo- 
nents with a very wide ventral union into which the two livers 
and other viscera are drawn. The babies are females and each 
would weigh more than 5 pounds. A careful examination shows 
all organs and parts to be perfectly formed and of normally 
large size. This specimen is comparable to the last ones shown 
in the equal component series of trout (plates 1 and 2). 

Here again we are warranted in attributing the different 
degrees of doubleness to the different distances apart of the two 
original embryonic lines or axis as they appeared on the blasto- 
derms. In the first case the primitive streaks were not far 
apart and in the second case they were in positions almost 180° 


214 CHARLES R. STOCKARD 


apart on the blasto-dise. Such an interpretation would certainly 
seem proper in the case of the fish and the bird. 

The third case, that of the separate twins, plate 4, differs from 
the others in that both babies are deformed. Yet the deformities 
and other peculiarities of this case make it unique in value. It 
has been seriously questioned, on the basis of psychological and 
other studies (Thorndyke ’05), whether actual cases of human 


0 OM 


28 


Fig. 28 Drawings of the ventral surfaces of the hands and feet of the iden- 
tical human twins shown in plate 4. A, from one individual, and, B, from the 
other. The four hands are all polydactylous, having an accessory finger on the 
ulnar side, and the four feet are similarly polydactylous, all having an accessory 
toe on the fibular side. The polydactylism is practically identical in the two 
individuals. 


identical twins do exist. The structural conditions of the two 
male twins in this case renders it practically certain that they 
arose from a single fertilized egg. There are six fingers on each 
of the four hands, as shown in plate 4, and more distinctly in 
figure 28 A and B; there are also six toes on each of the four 
feet, as the illustrations show. Such a polydactylous condition 
is known to be derived from a peculiar germinal complex and is 
not produced by the developmental environment. The chance 
is one against thousands that two fertilized eggs carrying exactly 


STRUCTURE AND DEVELOPMENTAL RATE 215 


the same capacities for polydactylous development should occur 
at one time in this mother. The expression of polydactyly in a 
family in which it is hereditary, is most variable. Neither the 
father nor the mother of these twins were said to have polydac- 
tylous hands or feet, and it was claimed that they had another 
child with an ordinary number of fingers and toes. This, how- 
ever, was a case of a ‘seven-month’ stillbirth in a New York 
hospital, and it proved impossible to obtain a family history of 
positive value. 

These twins show further deformities that are almost identical 
in the two. All of the four kidneys are cystic and the left kidney 
in each individual shows the cystic condition in a more exag- 
gerated form than does the right. The two heads have posteri- 
orly protruding meningoceles, one being slightly larger than the 
other. The meningeal hernias are probably due to the action 
of some environmental influence that produced closely similar 
responses in these two individuals of identical germinal composi- 
tion. In this case both members of the pair are malformed in 
addition to their unusual characters of genetic origin. 

In every way these twins are structurally about the same with 
the exception that the one on the left is smaller than the one on 
the right to just the extent carefully shown in the two figures. 

This is a most positive case of identical human twins and 
would certainly seem to leave no reason for question as to the 
occurrence of such individuals. 

From these examples it is probable that the two equal compo- 
nents in double human individuals are in about the same relation- 
ship to one another as are the equal components of other double 
vertebrate specimens. 

The double human specimens seen in many museums as well 
as those illustrated in the literature in which the two components 
differ considerably in size also follow the rule found for similar 
fish specimens. In double human specimens the larger compo- 
nent is usually normal in structure and the smaller component 
is always deformed. Extreme cases of this type are exhibited 
among the freaks in ‘side shows.’ In these living specimens 
the larger component has a well-formed human body with the 


216 CHARLES R. STOCKARD 


smaller component represented by a malformed partial body, 
attached to or protruding from it. 

The cause of doubleness and twinning in these human speci- 
mens is in all probability the same as in the other cases dis- 
cussed. The rate of development of the egg was probably 
arrested during an early stage, and perhaps on account of some 
interference or delay in implantation on the uterus. Very 
recently I have obtained a specimen through the kindness of 
Dr. Frank Erdwurm, of New York, which is of great value in 
an understanding of twin development in man. 

The specimen secured by Doctor Erdwurm is shown by photo- 
graph in plate 5. A living female baby weighing 6} pounds was 
enclosed within the upper membranes seen in the photograph. 
The cord from this baby is connected to the upper placenta near 
its lower border. After delivering the child, a second chorionic 
sac ruptured and discharged its fluid to the surprise of the 
observer. Later the two dead fetuses seen in the picture were 
delivered along with the placental mass. The fetuses proved 
to be identical twin girls enclosed within a common chorion and 
attached by their cords to a common placenta. The photograph 
clearly shows the single membranous sac in which they were 
enclosed and the positions of attachment of their cords to the 
placenta. 

The size and other conditions of the fetuses indicate a stage 
of about six months’ development. They had evidently been 
dead for a long time, probably about three months. The heads 
and bodies were somewhat macerated and shriveled and the 
blood-vessels had broken down in their placenta so that this no 
longer had any circulatory communication with the uterus. 
The two umbilical cords had become so wound around each other 
and knotted, as to completely cut off the connection of the fetal 
bodies with the placental circulation. The two fetuses were no 
doubt asphyxiated after six months of development. 

This structural evidence is substantiated by the behavior of 
the mother. She had passed through the first six months of 
pregnancy in a normal fashion and then became greatly dis- 
turbed, so that it was feared that her pregnancy might be inter- 


STRUCTURE AND DEVELOPMENTAL RATE 217 


rupted entirely. The severe condition gradually subsided and 
she was able to carry the living child to full term. The reaction 
of the mother was doubtless due to the death of the two fetuses 
and the cut-off in the placental circulation. The uterus was able 
to adjust itself to the condition and the fetuses remained 
aseptically enclosed within their membranes. 

This was the mother’s second pregnancy; exactly twelve 
months before, lacking one day, she had given birth to a single 
normal child. 

My interpretation of this triplet condition is as follows: The 
mother liberated from the ovary two eggs, both of which became 
. fertilized and began development. One became implanted 
slightly before the other and developed into the single living 
girl The second egg was not so favorably implanted as the 
first; this is indicated in the specimen by the lower placenta 
riding up over the larger one. The delay in implantation, due 
to the presence of the first egg, caused a slow rate of develop- 
ment at an early stage in the second and two embryonic buds 
arose instead of one, just as was described on the germ-ring of 
the fish. In this human specimen there is fortunately present 
the physical cause that might have produced the delay. 

The woman gave birth to triplets, two of which were female 
identical twins derived from one egg and the other was a single 
sister individual derived from another egg. 

Doctor Erdwurm furnishes a further very important record. 
‘This woman’s mother had eleven pregnancies, nine of which 
resulted in living single births and two in abortions during the 
first half of pregnancy. One abortion, the tenth pregnancy, 
consisted of twins, and the eleventh pregnancy resulted in the 
abortion of triplets. The nature of these twins and triplets, 
unfortunately, is not known. 

Evidently there is here a family tendency to ovulate more 
than one egg at a time. This may be due to simultaneous 
ovulations from both ovaries, from two follicles of one ovary, or 
from the rupture of a single follicle containing two or more ova. 


218 CHARLES R. STOCKARD 


7. THE DOUBLE INDIVIDUAL WITH UNEQUAL COMPONENTS AND 
AN UNDERSTANDING OF THE CAUSE OF ALL 
MONSTROUS DEVELOPMENT 


The most valuable material that falls into the possession of 


the investigator attempting to analyze the causes of abnormal ~ 


development is furnished by the united twins and anterior dup- 
licities where one component is fully developed and perfectly 
normal in structure, while the other component presents in a 
series of such forms various degrees or combinations of mal- 
formation and deformities. There are practically numberless 
attempts, from the time of Aristotle until now, at theoretical 


explanations for the cause of monstrous development, but none - 


of these, as far as I know, have recognized as crucial the con- 
dition presented by the combination of a larger normal twin 
developing in actual union with variously deformed lesser indi- 
viduals. Certainly, all theories that conflict with this condition 
of fact may be discarded as being inadequate in general. And, 
as mentioned before, the explanation of abnormal development 
probably les in the differences between the factors operating 
on the development of the two connected individuals. 

In the first place, one could scarcely state in the presence 
of these specimens that the abnormalities of the lesser compo- 
nent are of germinal origin. Yet similar deformities in single 
individuals have been interpreted in Wilder’s (’09) theory of 
‘Cosmobia’ as always being of such origin. The larger and 
smaller members of the double complex have both arisen from 
a single fertilized egg. There is no trace of either direct or col- 
lateral evidence to indicate that the hereditary factors are not 
equally distributed in the cells of both components. The germinal 
origin of one component could in no way be different from that 
of the other, since the entire specimen was a single individual up 
to about the stage of gastrulation. Further, when the two 
components are of equal size their identical genetic composition 
and character is evident. Obviously, then, defects similar to 
those enumerated as occurring in the smaller component are 
not in general of germinal origin. 


ice 


STRUCTURE AND DEVELOPMENTAL RATE 219 


It is further evident that ‘identical twins’ and the components 
in double individuals need not necessarily exactly resemble each 
other as is commonly thought. The two members of the pair 
may be structurally very different; in extreme cases one may be 
normal and the other actually deformed. 

What influences could act on the smaller component that do 
not also act on the larger? Evidently there can be nothing in 
the external environment that would not come in equal contact 
with both components, since they are intimately united and 
enclosed within the same egg membrane. There can be no case 
of injurious substances inducing a ‘blastolysis’ in the one com- 
ponent and not acting on the other, or causing an early ‘cellular 
disorganization’ (Kellicott, ’17), which would not affect both com- 
ponents. There is also no question of an insufficient supply of 
nutriment of the ordinary type, since it is shown by many speci- 
mens that twonormal embryos equally as large as the usual single 
one may develop on the yolk-sac of the fish’s egg, compare the 
first and last specimens shown by photograph in plates 1 and 2. 

There must, however, be some sort of competition between 
the two components other than a competition for the appropria- 
tion of ordinary yolk material. Much evidence suggests that 
an interaction exists between the components similar to, if not 
identical with, the interaction between two plant buds growing 
from a common stock. When the growing tip is cut from the 
shoots of certain plants, e.g., the ordinary privet, Lagustrum, as 
a rule the axillary buds of the two leaves immediately below the 
cut give rise to growing shoots. In many cases two shoots grow 
at equal rates and are about equal in size, in other cases one of 
the shoots evidently possesses some advantage and grows much 
faster and becomes larger than its companion. Finally, in a few 
cases a single vigorous shoot arises from one of the resting buds 
and the opposite bud is entirely unable to grow. There is here 
involved a factor in addition to available food material, just as 
Loeb has found in the Bryophyllum leaf, and whatever this fac- 
tor may be, through it the growing parts exert an inhibiting influ- 
ence on one another. In the first case cited for the plant, the 
growing impulse was balanced between the two upper axillary 


220 CHARLES R. STOCKARD 


buds and they grew equally, in the second and third cases one 
bud occupied a position of advantage over the other; this advan- 
tage may have been due to a slightly more favorable exposure 
to sunlight, heat, or moisture, or to a better flow of sap material 
on its side of the stem. Its more rapid rate of growth in some way 
imposes an inhibiting influence on the expression of the other bud, 
causing it to be smaller, sometimes ill-formed or suppressed 
entirely. If the larger bud be pinched away in any of the cases, 
the smaller immediately improves its condition and grows large, 
provided it has not been held back too long (figure 29 A, B 
and C). 

The advantages of certain positions on a stem over ethene is 
strikingly shown by privet branches growing in dense shade. 
These branches are slender shoots with long intervals between the 
pairs of leaves until finally they reach the sun. After a certain 
length of the stem has grown into the sunlight, thé axillary buds 
of a particular pair of opposite leaves grow into shoots. Later, 
when still further in the sun, the two axillary buds immediately 
below those that grow first, now grow to form the second pair of 
shoots. Still later the axillary buds from the leaf pair immediately 
above the first shoots send out the third pair. I have observed 
this exact order of growth in nine cases of shaded stems. The 
first shoots to appear have an advantage of position over the 
second and third on account of a proper exposure to sunlight and 
at the same time occupying a certain distance away from the 
growing tip. The second buds then come into sufficient sunlight 
and grow out despite the inhibiting influence of the first pair, and 
finally the third pair of buds now grow on account of having be- 
come more mature and further removed from the growing tip. 

The two embryos developing from a single blastoderm compete 
in a comparable way, and the results of the competition are also 
similar to the case of the plant buds. In the present state of our 
knowledge, it is impossible to say what the primary cause is that 
gives one of the growing parts an advantage over the other. We 
may merely express this in a non-committal way as an ‘advantage 
of position.’ It would in no sense relieve our ignorance of the 
situation to state the likely probability that one of the growing 
points has a higher rate of metabolism or a more rapid oxidation 


STRUCTURE AND DEVELOPMENTAL RATE 221 


than the other. No doubt this is a fact, and should it be demon- 
strated, we still have the question: why is the rate of metabolism 
or oxidation higher? Why does this difference in rate of oxida- 
tion exist in some instances and not in others? What is there in 
these apparently similar points around the germ-ring that brings 


22) 


Fig. 29 Outlines of branches from the common privet in which the terminal 
portions had been cut away, as indicated at the upper ends of the stems. In the 
first and usual case, A, following removal of the tip each of the upper axillary 
buds have given rise to equal-size shoots. In B the shoot from the right axillary 
bud is large and strong, while the shoot from the left bud is slow-growing and 
small. In C the right shoot is normally expressed, but the left upper bud has 
failed to grow entirely, yet if the right shoot were pinched away the left bud 
then readily grows out. In A the advantage of position in the two upper axil- 
lary buds was equal, but in B the right bud had an advantage in growth posi- 
tion over the left bud, and in C this difference in growth advantage was still 
greater. In both of the latter cases the growing shoot exerted an inhibiting 
influence over the opposite bud. 


222 


CHARLES R. STOCKARD 


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STRUCTURE AND DEVELOPMENTAL RATE 223 


about this higher affinity for oxygen at one point than at another? 
Certainly, we do not at present know! 

This unknown factor acting between the growing buds or em- 
bryos when out of equilibrium, inhibits to an unusual degree the 
rate of oxidation and through this probably the rate of cell- 
multiplication, and certainly the rate of development in one of 
the components. All the defects observed in the inferior com- 
ponent are simply due to a slowing of its developmental rate or 
are strictly what I have always termed developmental arrests. 
This problematical factor, then, simply tends to lower the rate 
of development in the one component and thereby does what the 
experimenter is able to do in various ways with any developing 
single individual. I (’06, ’07, 09, 10 ab, 713, etc.) have experi- 
mentally produced in single embryos all of the deformities seen 
in the smaller components by arresting the developmental rate 
of eggs with a large number of different chemical and physical 
treatments. Newman (’17) has observed exactly the same types 
of monsters among slowly developing hybrids. As Newman very 
correctly points out, one obtains similar monsters by any method, 
either treating the eggs with injurious chemicals, strange physical 
conditions, or by heterogenic hybridization. Each of these 
methods simply lowers the rate of metabolism and the rate of 
development. Newman, in agreement with my position, rec- 
ognizes all of the monsters as being primarily due to a lowering 
of developmental rate and, therefore, generally speaking, they 
are actually developmental arrests. From an extensive study of 
monstrous individuals, Dareste (’91) long ago believed that all 
developmental abnormalities were in general arrests, yet he 
lacked proof for such a position. 

The present study, however, enables me to state the case in 
far bolder and more definite terms than it has been possible to 
do before. In the first place, every type of developmental monster 

Fig. 30 The terminal portion of a long privet stem which had grown upward 
in a shaded position. On reaching direct sunlight the axillary buds three leaves 
from the bottom of the figure grew into lateral shoots. Very soon after this the 
axillary buds of the pair of leaves immediately below the first shoots give rise to 
the second pair of lateral shoots, and finally the buds of the leaf pair immediately 


above the first shoots grow into a third pair of lateral branches. In nine cases 
this budding sequence was invariably followed. 


ee 


224 CHARLES R. STOCKARD 


known in the literature may be produced by one and the same experi- 
mental treatment. For example, simply by lowering the surround- 
ing temperature or by treating with a weak ether solution all 
monsters may be produced. Secondly, the same structural abnor- 
mality may be induced in the embryos of various species by a great 
number of different experimental treatments. Thirdly, in all cases 
the initial effect of the experimental treatment is a lowering of the 
developmental rate, and the resulting deformity is always second- 
arily due to this slow rate of development. Fourthly, the type of 
monster or deformity is determined by the developmental period dur- 
ing which the slowing in rate is experienced. An early slowing 
will induce the growth of accessory embryonic axes or duplicities, 
while a similar reduction in rate at later periods may produce 
anophthalmia or cyclopia, simple tubular brains, malformed otic 
structures, deformities of the mouth or branchial arrangement, 
etc., depending upon the time at which the rate of development 
was slow. Slowing by a number of different ways if done at 
the same developmental time will give closely similar defects. 

We may finally state a common law of both normal and abnor- 
mal development as follows: Structural quality may be affected by 
many things, but always depends directly upon the rate of develop- 
ment of the part or of the individual. I have many reasons for 
believing that this law equally applies during postnatal growth 
and change in higher animals, as well as during their prenatal 
development. 

In a study of a number of different embryos it will be observed 
that a particular structural modification is more common in 
one species of egg than in another. With trout eggs for example, 
duplicities more readily occur than in the egg of Fundulus. While, 
on the other hand, cyclopia and certain eye defects are more 
common among deformed specimens of Fundulus than among 
those of the trout. It would seem as though particular moments 
and localities were more susceptible to modifications in one egg 
as a result of slow development than in another. Further, cer- 
tain eggs are in general much more sensitive than others and, as 
is well known, more frequently deformed. Their developmental 
tate is less strongly regulated than is the case in the more resist- 


STRUCTURE AND DEVELOPMENTAL RATE 225 


ant types. These facts are due to the different hereditary back- 
grounds on which the modifying conditions act. 

Finally, if one admits the above generalizations to be true, 
studies and descriptions of individual monsters and deformities 
lose much of their interest and so-called value. It is evident 
from the present standpoint that a single deformed specimen, 
whether human or lower vertebrate, must be considered asresult- 
ing from an arrest or slowing of its developmental rate during a 
particular period. Observing the nature of the deformity or the 
parts involved enables one to estimate the developmental period 
during which the arrest was effective. Such individual monsters 
in no way supply evidence to determine what the initiating cause 
of the deformities may have been, since we know that the same 
type of deformity may be experimentally caused by many differ- 
ent treatments. We can estimate simply that the exciting cause 
acted to lower the rate of development during a definite interval. 

The great number of descriptions in the literature of isolated 
monsters have added very little to our understanding of the 
causes of abnormal development. ‘The writer believes after a 
prolonged study of this subject that the only benefit to be derived 
from examinations of such isolated specimens is possibly to ob- 
tain aid in studying the normal sequence of development. This 
of course is valuable and only to this extent are such descriptions 
worthy of record. Detailed descriptions of monsters occupy the 
same level of scientific value as do records of ordinary structural 
anomalies observed in the dissecting room. 

Having stated the above conclusions derived from an exten- 
sive study of abnormalities in single individuals as well as the 
double specimens under present considerations, it is not deemed 
necessary to enter into a general discussion of the various views 
regarding abnormal development with which morphological lit- 
erature abounds. Many of these positions have been discussed 
in my previous papers. I shall here only attempt to consider 
briefly the last contribution by Mall (’17) who devoted so much 
study and masterly consideration to this subject. The inves- 
tigations by Mall are far the most valuable that have been 
made on human material. 


226 CHARLES R. STOCKARD 


In his last study on the frequency of localized anomalies in 
human embryos and stillborn infants, the data from a thousand 
specimens are recorded. 

Mall’s method of arranging this material may be considered 
in brief in order to attempt fitting it into our above conclusions. 
The material was primarily divided into normal and pathological 
specimens. Some of the ‘normal’ may possess localized anomal- 
les, such as cyclopia, ete., and a study of the pathological group 
Mall believes justifies this inclusion of localized anomalies among 
the normal embryos. 

The pathological group was then subdivided into seven classes 
of specimens: first, those consisting of only degenerate chorionic 
villi; second, of only the chorion with the extra embryonic coelom; 
third, of the chorion and amnion; fourth, of the embryonic mem- 
branes containing a nodular embryo; fifth, of cylindrical embry- 
os; sixth, stunted embryos, and, seventh, dried and deformed or 
soft and macerated specimens. The series thus begins with the 
‘most degenerate conditions and passes on to those specimens 
which maintained their integrity fairly well, though evidently 
malformed and dead for some time before being aborted. Un- 
questionably, all members of such a series have suffered develop- 
mental arrests of the severest types. In some cases the arrest 
has come at an early stage and been followed by a disorganiza- 
tion or cytolysis and subsequent absorption of the embryonic 
material. In guinea-pigs one frequently finds similar stages of 
embryonic degeneration in utero, and here also the placenta and 
membranes are the last parts to disappear. In almost all of these 
cases portions of the pregnancy must have remained in the uterus 
for some time after the embryo died before being discharged. 

If these pathological specimens are primarily due to develop- 
mental arrests, what, if amy, evidence is there that conditions 
may have existed which could probably have induced such 
arrests? Or, is there evidence that human embryos are affected 
very readily by strange conditions? Very valuable data bearing 
on both of these questions are supplied. In the one thousand 
specimens considered, about 33 per cent of the ova and embryos 
from the uterine lot were pathological, while as many as 66 per 


STRUCTURE AND DEVELOPMENTAL RATE PpPatl 


cent of the ectopic specimens were of this nature. The double 
frequency of pathological specimens in ectopic pregnancies 
shows at once the influence of unfavorable environment. These 
facts are of primary importance and Mall discusses them in a 
most instructive way. Hestates, to account for human monsters: 
“Tt would have been quite simple to conclude that the poisons 
produced by an inflamed uterus should be viewed as the sole 
cause, but when it is recalled that pathological ova occur far more 
commonly in tubal than in uterine pregnancy, such a theory be- 
comes untenable.”’ It is then stated further: ‘‘For this reason 
(meaning the records from ectopics) I have sought the primary 
factor in a condition buried in the non-committal term, ‘faulty 
implantation.’ ’’ The faulty implantation acted to injure the 
development, in Mall’s opinion, on account of supplying insuf- 
ficient nutriment. I should be inclined to accept the faulty 
implantation as the primary factor, but the injurious effects of 
such an arrangement are due to an insufficient oxygen rather than 
food supply. This difference in interpretation is only of aca- 
demic value. Malnutrition effects developing individuals in a 
general way causing a condition of undersize, while insufficient 
oxygen decidedly slows the rate or may completely interrupt 
development and thereby induces various structural deformities. 

Mall in this paper is inclined to drop the cruder term ‘nutrition’ 
and admits that, ‘‘ Probably it would be more nearly correct to 
state that change in environment has affected the metabolism 
of the egg.” This would be entirely in accord with the interpre- 
tation of arrest as being due to lowered oxygen supply. 

Again, Mall reaches significant conclusions when considered 
in connection with the foregoing general principles of abnormal 
development. For, on page 72, he states: “Accordingly, when 
an embryo through changed environment is profoundly affected, 
the development of one part of the body may be arrested, while 
the remaining portion may continue to grow and develop in an 
irregular manner. In very young embryos, tissues or even entire 
organs may become disintegrated, as can easily be recognized by 
the cytolysis and histolysis present, and the resultant disorgan- 
ized tissue cannot continue to produce the normal form of an 


228 CHARLES R. STOCKARD 


embryo. If this process (evidently meaning disintegration) is 
sharply localized, for instance, in a portion of the spinal cord or 
in the brain, spina bifida or anencephaly results. To produce 
a striking result, as in cyclopia, a small portion of the brain must 
be affected at the critical time.” 

The one position with which we are entirely unable to agree is 
that the arrested development must so constantly be “‘associated 
with the destruction of tissue.” This tissue destruction is not at 
all essential to the production of such defects as spina bifida or 
anencephaly. It may be demonstrated in many experimental 
cases that the tissues fail entirely to arise or differentiate without 
there being any indication whatever of a previous destruction. 

As stated in the beginning, Mall included localized anomalies 
among his normal specimens, yet such anomalies occurred about 
twice as frequently among the pathological individuals as among 
the normal. This is closely in accord with what has been found 
for the abnormal fish embryos. 

After this review of monstrous development in general, and 
an analysis of its causes from the conditions found in the smaller 
components of double individuals, we may consider in the fol- 
lowing section the interaction, if such can be observed, among the 
early organs in the single individual. It may be possible that 
these organs are related to one another in their development in 
somewhat the same manner as are the components of double 
specimens. A further test, therefore, of the correctness of my 
interpretations for abnormal development may be had in an 
analysis of the relationships among the developing organs in the 
single individual. 


8. THE DOUBLE INDIVIDUAL WITH UNEQUAL COMPONENTS AND 
AN ANALYSIS OF THE DEVELOPMENT OF ORGANS 
IN THE SINGLE INDIVIDUAL 


By way of introduction, we may further consider certain condi- 
tions in developing plants on account of their apparent simplicity 
and also their very striking suggestiveness in connection with an 
analysis of the origin and growth of organs in the vertebrate 
embryo. The imaginary elements involved in comparisons of 


FP 


STRUCTURE AND DEVELOPMENTAL RATE 229 


plant conditions with animal development I very fully recognize. 
There is, however, evidence of certain actual similarities which, 
along with deductions from my experiments on embryos, may 
serve to elucidate the problem of organ formation to a consider- 
able extent. Particularly suggestive is an examination of— 


a. The growth influence of the apical or primary bud over the 
secondary and potential buds in plants 


It is commonly observed that when a number of beans or other 
seeds are planted in a row under similar conditions of soil and 
moisture, the initial bud from each seed sprouts upward and 
grows to a definite extent and then temporarily stops. On exam- 
ining the row of young shoots, each with two horizontally spread 
terminal leaves, it is generally found that all are very nearly of 
the same height. Should a certain part of the row occur in a 
more favorable environment than another the sprouts in this 
part may grow higher than in the others, or should certain seeds 
have been defective or their environment in the row unfavor- 
able, the sprouts in such cases are lower and smaller than the 
average. These low small plants seem as a general rule unable 
to overcome their inferior condition during later growth and either 
die or form very poor specimens. The small sprouts would appear 
to have suffered an arrest during their early development in 
consequence of which they generally fail to be normally large 
fruiting plants. 

The original shoot is entirely formed from the food contained 
within the cotyledons and the water of absorption. After attain- 
ing a definite length, it stops or slows its progress until the roots 
become sufficiently established to obtain further food and mois- 
ture from the soil. On becoming properly rooted the apical 
bud then grows upward from a point between the two original 
leaves and from this the development of the plant proceeds. 
We thus have an interruption, after the formation of the original 
sprout, similar to that found in the development of many verte- 
brates and from a somewhat similar cause. Here the plant could 
not continue to grow until certain substances were supplied by 


230 CHARLES R. STOCKARD 


the roots, through the assimilation of which, cell multiplication 
* was made possible. In the birds and in the experiments with 
fish eggs, the initial development is interrupted by a sudden 
lowering of temperature and through this the chemical processes 
necessary for cell multiplication are slowed or stopped and devel- 
opment ceases. Although the stuff is available, the conditions 
prevent its use. 

The case of the mammal is more closely analagous to that of 
the plant. Here the fertilized ovum within the Fallopian tube 
begins to develop and continues until it exhausts its initial supply 
of oxygen, though there may possibly be here also an exhaustion 
of nutriment as in the plan. Following this, the development of 
the embryo is either stopped for a considerable time, as in the 
extreme cases of the deer and armadillo, or it is temporarily inter- 
rupted or slowed until the membranes have become established 
or embedded in the uterus of the mother and a further source of 
oxygen and nutriment is thus acquired. The placentation of the 
mammalian ovum and the rooting of the plant in the earth as a 
mother, are comparable processes. Any lack of perfection in 
the process is either fatal or lowers the supply of necessary stuffs 
and thus causes an abnormally slow rate of development and 
growth with a resultant imperfection in structural formation. 

After the original linear sprout of the plant has rooted, and a 
certain extent of linear growth has taken place from the apical 
bud, growth in length gradually slows as if the apical bud had 
passed beyond the point at which it could dominate the growth 
activities throughout the length of the plant. When this time is 
reached, the axillary buds at the base of the leaves are able to 
express their growth capacities and the plant develops its lateral 
branches. Though all the branches of a plant have a more or 
less similar function, yet each may be looked upon as an organ, 
and their origin and subsequent competitive growths are in many 
respects similar to the origin and growth of organs in the verte- 
brate embryo. 

Such a statement of the situation in plant development is 
rendered further justifiable by a very common experiment. If, 
instead of allowing the apical bud to gradually exhaust its suprem- 


STRUCTURE AND DEVELOPMENTAL RATE 231 


acy by continuous growth, it be injured or pinched away at an 
early stage, the lateral buds very quickly grow out, showing their 
liberation from some controlling influence possessed by the apical 
bud. In other words, each growing bud (also true of the embry- 
onic organs) exerts a depressing influence on the growth of all other 
buds in the individual plant. As a shoot gradually ceases to grow 
its depressing influence also gradually ceases. 


b. The initial linear growths, subsequent lateral buds, and the 
interactions among the organs of the vertebrate embryo 


When the first trace of the embryonic body begins to express 
itself in the blastodermic matrix it appears as a linear growth, the 
head process extending forward from the blastopore or primitive 
streak. This very soon becomes surrounded by, or associated 
with the linear outline of the arising neural folds, the beginning 
central nervous system. 

The neural folds indicating the early nervous system are origi- 
nally of more or less straight outline and their first growth is largely 
a growth in length. When in a given species the neural groove 
has attained a certain length, it then begins a series of lateral 
outgrowths, or branches. ‘The first and largest of these are the 
two optic outpushings and after them follow in a general way, a 
series of bilateral outgrowths designated the three primary brain 
ventricles. 

The initial linear origin and growth of the nervous system is 
very probably due to an equal rate of cellular proliferation along 
the entire extent, with perhaps a somewhat more rapid rate 
at the tip. The lateral outgrowths arise on account of an exces- 
sively high rate of proliferation occurring in a given region during 
acertain time. For some unknown reason the rate of metabolism, 
or actually the rate of oxidation becomes disproportionately high 
in a particular group of cells, and these begin to multiply rapidly 
as compared with the multiplication rate of neighboring regions, 
and thus a folding or outgrowth occurs to produce, for example, 
the optic vesicles. Since other portions of the brain seem not to 
be proliferating so rapidly at the same moment, it may be that 


232 CHARLES R. STOCKARD 


the growing optic vesicles exert a depressing influence over the 
growth of other parts. There is indirect experimental evidence 
for such a statement. 

The initial moment of high cell multiplication for a particular 
organ outgrowth is a most critical instant in the development of 
this organ. Thus, if the general developmental rate of an em- 
bryo be reduced by exposure to low temperature or cutting off 
the oxygen supply at the time when the rapid proliferation of 
the optic anlage should occur, the disproportionate growth of this 
region is prevented, and the result of such an experiment will be 
either the complete suppression of eye development, anophthal- 
mia, cyclopean eyes, monophthalamia, or some other degree of 
defective eyes. This result ensues in spite of the fact that after 
the critical moment for eye origin has passed the embryos may 
have been again developing at the usual rate in a normal environ- 
ment. The eye has only one favorable period for its origin, its 
moment of supremacy so to speak, and when it is unable to ex- 
press itself at this time, the opportunity is largely, if not entirely, 
lost. This is probably due to other organ anlagen having arrived 
at their controlling moments, the optic inhibition being no longer 
sufficient or capable of suppressing them, but they, on the con- 
trary, now suppress the optic bud. 

The arrest in development necessary for suppression of the 
optic vesicle must be induced in the early embryo, before the 
embryonic shield stage in the teleost, or before the optic anlage 
is at all visible in the neural plate. This I (’09, 13) have shown 
by a number of different experiments, and now also find to be 
true in case of treatments with low temperature and scant oxy- 
gen supply. 

I (09) have reported a number of experimental cases of fish 
embryos in which the eye was absent or was cyclopean, while the 
general brain structures were as usual bilateral and normal. 
Such specimens are viable and swim actively about. It is evi- 
dent in these cases that the arrest was limited in its effect to the 
optic outgrowths and was no longer effective when the primary 
brain ventricles were forming. — 


STRUCTURE AND DEVELOPMENTAL RATE 233 


Specimens have again been recorded from my experiments, 
and these also may be induced in a great number of ways, show- 
ing either two poorly formed eyes, cyclopia, or anophthalmia, 
accompanied by a narrow tubular brain. Here the arrest or 
slowing in developmental rate has affected the optic outgrowths 
only slightly im some cases, in other cases severely, but in all 
cases it has persisted or continued to act for a longer period and 
has thereby also suppressed the outgrowths which normally form 
the series of primary bilateral brain ventricles, hence the final 
narrow tubular brain. Depending, then, upon the rate of devel- 
opment at a given moment, we may obtain: first, as is normally 
the case, optic vesicles on a brain with three bilateral primary 
ventricles; second, no optic vesicles, yet a brain with the bilateral 
primary ventricles; in the third case, we may or may not obtain 
optic vesicles on a brain with no growth of the bilateral ventricles 
—a simple tubular brain (fig. 31). 

We may describe the development of the central nervous sys- 
tem in the vertebrate embryo very simply and schematically as 
follows. At first a more or less straight linear growth takes 
place until a given length for the given species is attained, then 
the linear growth possibly becomes slower in rate and lateral 
branches or outgrowths begin to appear, first the optic vesicles 
and then the first, second, and third primary brain ventricles. 
A competitive element is involved in the origin and growth of 
the lateral outpushings so that should one of these fail to express 
itself during the usual time for such expression, it is later unable 
to grow out normally or may not grow out at all (fig. 31). 

We know from experimental demonstration (Lewis, ’04; Sper- 
mann, the writer, Leplat, 719, and others) that the optic vesicles 
are derived from a definitely located group of cells in the neural 
plate of the embryo. When they do not arise from this group of 
cells no other cells are capable of forming optic vesicles and they 
do not appear at all. 

In addition to our knowledge of this definitely located optic 
anlage in the embryonic brain, I have now to contribute a fact of 
equal importance in the development of the eye which may be 
stated thus. When the optic vesicle does not grow out from the 


234 CHARLES R. STOCKARD 


brain at a definite developmental moment, it is subsequently 
unable to grow out and develop normally or it may be unable to 
grow out at all. I have definitely inhibited development during 
this period in a large number of experiments and have either sup- 
pressed or modified the development of the eye. It may be con- 
‘cluded that, such an organ as the eye is not only derived from u 


31 


Fig. 31 A series of diagrams indicating modifications in the lateral outgrowths 
or budding processes of the anterior region of the central nervous system. A, 
outlines the normal case with the optic-outgrowths shown above and followed 
by the first, second, and third primary ventricular outgrowths of the brain. B, 
shows the outline of a brain in which the optic outpushings were suppressed, but 
the three brain ventricles succeeded in their lateral expansion. C, indicates the 
opposite case in which the optic outpushings were expressed, but the three brain 
ventricles were suppressed. This is a narrow tubular brain with eyes developed 
from it. D, outlines the condition of complete suppression of all lateral out- 
growths, there being neither eyes nor bulging brain ventricles. A simple tubular 
brain. 


definitely located primordia, but must also be derived during a limited 
moment of development. 

This time-limited opportunity for origin is probably due to a 
growth competition between organs. The eye, not attaining a 
maximum growth rate at its proper moment, may permit an 
excessive growth to commence in a neighboring part and such a 
growth may then further prevent the initial growth of the eye. 


STRUCTURE AND DEVELOPMENTAL RATE 235 


There is also a possible chemical interpretation of the limited 
moment. The great activity and high oxidation rate of a given 
group of cells might result from the formation of certain specific 
compounds of a highly labile nature within these cells. Should 
the available oxygen be insufficient or the temperature be too 
low at the moment of origin of such molecules, they would be 
unable to produce the usual cellular activity, and on account of 
their labile nature would soon become changed. The oppor- 
tunity for unusual growth activity of the specific kind on the part 
of the given cellular group would be lost. No doubt some such 
peculiar chemical process must be taking place during the different 
stages of cellular growth and differentiation in a complex ver- 
tebrate embryo. When such labile compounds do break down 
we may also imagine that a more generalized chemical condition 
of the cell is produced. And such cells may subsequently take 
part in the formation of the more general tissues and may not nec- 
essarily be lost on account of not having succeeded in giving rise 
to the specific tissue intended. Certainly, one does not find 
necrotic and disintegrating cells in all brains of anophthalmic 
embryos. 

Ralph Lillie (’17) has described structures simulating organic 
growths arising from electrolytic local action in metals. He also 
shows the formation of filaments from one metal to be inhibited 
by contact with another metal. ‘The inhibitory influence of 
zine upon the formation of ferricyanide filaments from iron may 
be shown as follows: a straight piece of thin bright iron wire 
some centimeters long, one end of which is wound with a small 
strip of zine, is placed in a 2 per cent K;,FeCy, solution in dilute 
egg-white. Filaments put forth rapidly from the zinc, especially 
near the iron, but the iron itself remains perfectly bright and 
bare, and may show no development of filaments for hours. If 
then the wire be cut in two by scissors, the part remaining in 
connection with the zine remains unchanged, while the isolated 
part quickly develops the characteristic blue-green filamentous 
growth of ferrous ferricyanide. Evidently this growth had Bue: 
ously been repressed by the influence of the zinc 
Or when the zine becomes completely covered by a al ot 
zinc ferricyanide the growth of ferrous ferricyanide will begin. 


236 CHARLES R. STOCKARD 


Such reactions resemble in general the inhibiting effects of one 
growing bud or organ over the growth of other buds in the plant 
or organs in the embryo. 

The consideration up to this point has been limited to the 
developing nervous system and its organs. Does a similar rela- 
tion of linear and lateral growths and evidence of a similar com- 
petition among organ buds exist in other systems of the embryo? 
And, further, is there any evidence of a wider competition between 
the different systems of the embryo? 

The development of the foregut from which is derived a large 
portion of the alimentary tract in the vertebrate embryo is closely 
similar in many ways to that outlined above for the nervous sys- 
tem. The initial anteriorly directed conical evagination of the 
entoderm first undergoes a linear development or growth, simply 
becoming longer. When a certain length has been attained by 
this early tubular foregut, here again lateral outgrowths begin to 
appear, and a series of them is formed in order from the anterior 
end backward in much the same way as the early neural tube 
gives off the optic vesicles followed by the three primary brain 
ventricles. The first and largest of the early foregut outgrowths 
is the pair of mandibular pouches, in association with which the 
mandibular arches arise to form the lower jaw. This pair of 
outgrowths is soon followed by the hyoid pair and this by the 
series of branchial pouches associated in later development 
with the several gill arches. An outline scheme of these growths 
is simply represented by the three accompanying diagrams in 
figure 32. 

The further development of the alimentary canal also shows 
in a very definite way a continuation of this process of lateral 
outgrowths or buds to give rise to other organs. The lungs in 
higher animals bud away from the floor of the entodermal canal 
immediately behind the branchial pouches. And again in the 
branchial region the thyroid and other glands arise by a definite 
budding process from the epithelial wall. 

The development of the stomach itself is due to an excessive 
proliferation or diffuse budding in a limited region, giving 
finally the local sacculation in the otherwise narrow tube. Fol- 


STRUCTURE AND DEVELOPMENTAL RATE 237 


lowing closely behind the stomach, the canal buds off its most 
striking secondary growth. This begins as an evagination follow- 
ing rapid cell multiplication, the excessive growth becomes too 
great to be longer retained by. the wall and the liver pushes out, 
always maintaining the original connection through the bile-duct, 
its old stalk. This large liver bud generally contains some cells 


52 
Cc 


Fig. 32 A series of outlines indicating the primary linear, or cephalad, growth 
of the foregut, and the subsequent lateral branches or outgrowths from it. A, 
outlines the simple forward growths of entoderm to form the foregut. B, lateral 
outgrowths have begun from the forward end to form the mandibular pouch. C, 
a series of lateral branches, following the mandibular, now grow out to form the 
hyoid and branchial pouches. 


not exactly of its own kind, and these later begin to increase and 
again bud away from the wall of the bile-duct as the ventral pan- 
creas. Other cells of a similar kind are left in the wall of the tube, 
and these now grow out as the dorsal pancreas. This is the 
behavior of the pancreas in higher forms, while in lower animals it 
may arise from more than two separate buds or may fail entirely 
to grow away from the tube, and remain as scattered masses of 


238 CHARLES R. STOCKARD 


cells in the gut wall of this locality. The pancreas in different 
vertebrate groups illustrates the phylogenetic steps in the devel- 
opment of a budding outgrowth from the wall of a linear canal. 

The entire alimentary tract in the lowest vertebrates was no 
doubt originally a simple tube and the lateral outgrowths or buds 
are the highly specialized organs that have become so excessively 
developed as to necessitate their separation from the tube. Thus 
in phylogeny as well as ontogeny of the vertebrate gut it would 
look as though the primary growth was linear and its complexity 
has been added by lateral buds and offshoots. 

The above being the general state of affairs in the development 
of the foregut, we come now to the point of experimental impor- 
tance in the dynamics of these organ-forming processes. And 
that is, that each of the organs derived from the entodermie wall 
is in its critical or sensitive stage at the moment of its outgrowth, 
or at the time of the excessive cell proliferation in its region of the 
wall. Should any condition be introduced which would lower the 
general developmental rate, that organ will be most affected which 
happens at the time of the arrest to be in or nearest its critical 
moment. Thus an arrest during very early development will 
inhibit the growth of the mandibular pouch and through its mal- 
formation distort the formation of the mandibular arch, causing 
deformed and strangely developed mouths (see figures of the 
deformed fish). Since the hyoid and other branchial pouches 
arise so nearly at the same time as the mandibular pouch, they, 
with later accompanying structures, are likewise almost invaria- 
bly deformed along with deformities of the mandibular structures. 
Such deformities as these may, however, exist in individuals with 
perfectly normal stomachs, livers, ete. In these cases, a normal 
or fair rate of development had again been established before the 
critical moment in the origin of the latter organs had arrived. 

It would thus seem possible that an experimenter might inhibit 
at will the rate of development during particular intervals and 
thereby succeed in suppressing and deforming the mouth and 
branchial structures and leave the more caudally situated organs 
uninjured. Or, reverse the experiment and obtain normal mouth 
and gill structures in an embryo with suppressed and underde- 


STRUCTURE AND DEVELOPMENTAL RATE 239 


veloped liver and pancreas. I have repeatedly succeeded in per- 
forming the first experiment with Fundulus embryos by early 
arrests. The second experiment is more difficult and is not yet 
completely perfected, though among a large number of cases 
certain specimens arise in the experiment with underdeveloped 
livers, but normal mouth and branchial regions. 

The experiments with the alimentary organs are more difficult 
than those on the eyes and brain, since the former are more diffi- 
cult to observe and are not all so decidedly expressed in the young 
embryo. The study of the liver and pancreas must also be 
largely limited to examination of microscopic sections, while the 
mouth and branchial arrangements and the eyes and brain are 
very readily examined in total specimens after some experience. 

The experiments on the nervous and alimentary systems as 
they now stand make very probable the correctness of the follow- 
ing proposition. The organs arise as a series of buds which bear 
a relationship to one another very similar to that existing among 
the buds of a growing plant. A given bud is dominant or has an 
‘advantage of position’ for a limited time during which its rate of 
oxidation and cell proliferation is higher than that of other poten- 
tial buds in the system. It grows at this moment and continues 
to dominate the situation until it has exhausted the advantage, 
' when its proliferation rate decreases and another region attains 
the advantage and begins to bud to form another organ. If 
the entire embryo be depressed or has its developmental rate 
reduced at a moment when a certain bud is proliferating at its 
height, this bud is more decidedly reduced in its rate than any 
other portion of the embryo. On resuming a more rapid rate the 
other slow-going parts are able again to attain their ordinary rates, 
but the bud in question is unable to regain its extraordinarily 
high rate and therefore loses its exceptional advantage. This 
bud may be subsequently unable to express itself, since other 
parts now arrive at the stage of advantage. 

The problem is then to locate the given critical moments for 
the several developing organs. By depressing development dur- 
ing a period which would cover a definite moment one might be 
able to suppress any given organ at will. With sufficiently re- 


240 CHARLES R. STOCKARD 


fined technique we could get not only embryos otherwise normal, 
but without eyes, without normal brain hemispheres, without 
normal mouth and branchial structures, and without ears, as can 
now be done, but also simply without a liver, without a pancreas, 
ete. 

In many of the arrested embryos it would seem highly probable 
that the total number of red blood-cells in the yolk-sae and body 
was greatly reduced. The red cells may be considered as a diffuse 
organ, and this organ seems at times reduced in size as a result 
of arrest. It cannot be positively stated that the entire embryo 
in such eases is not proportionately reduced. Thus the proba- 
bility of having specifically arrested the early blood formation is 
still questionable. 

The development of one other organ, however, may be fre- 
quently interfered with by arresting the rate of development of 
Fundulus embryos for a time immediately following the early 
embryonic-shield formation. Specimens so arrested by low tem- 
peratures, treatment with alcohol solutions (Stockard, ’10), or by 
reduced oxygen supply, often show various abnormal condi- 
tions in the development of the otic vesicle. ‘The vesicle on one 
side may be absent and the other normal or poorly formed. Or 
the semicircular canals may not arise and only the ampullae or 
small cysts may represent the entire ear. 

In such eases, as I pointed out and illustrated in 1910, the 
cartilaginous capsule representing the hard parts of the inner ear 
forms immediately around and exactly fits the defective mem- 
branous arrangement. The cartilage development would seem 
to be regulated by the membranous portion of the ear. The 
details of these experiments may be more fully presented when 
a larger number of these critical moments in organ origin are 
more exactly located by a further refinement of the experimental 
method. 


It is most difficult to apply a treatment that is not permanently — 


injurious in such a way as to have the rate of development very 
low at a given brief interval of time and again restored to the 
normal rate shortly following. The crudeness of the experiment 
necessitates bringing on the arrest some time before the particu- 


STRUCTURE AND DEVELOPMENTAL RATE 241 


lar moment desired and it later continues until further critical 
stages are interfered with. In this way, as a rule, we obtain 
specimens with several regions or organs deformed and rarely 
secure a specimen simply defective in respect to the state of a 
single organ or part. 

In plants the conditions are far more simple, and it is possible 
to suppress or bring out a given bud at will. In spite of the fact 
that we may not understand exactly how it is accomplished, it is 
definitely the growth of one bud in a plant that prevents the 
growth of another particular one. Similarly in the embryo prob- 
ably the growth of a given organ holds back the initial growth 
of another organ until the first organ has exhausted its power of 
suppression. 

The two components of a double individual interact on one 
another in a way which would strongly support the foregoing 
interpretations. When the components arise in positions of equal 
advantage on the germ ring their interaction is balanced and both 
develop normally and are equal in size. When one component 
possesses an advantage over the other, its growth tends constantly 
to suppress the growth and development of its fellow, and the 
inferior component is, therefore, deformed and arrested in its 
development. 

When a growing shoot of a plant, such as the common privet, 
has finally exhausted itself, the terminal bud goes into a dormant 
or resting stage and stands only a little above the axillary buds 
of the two uppermost leaves. After a certain interval of rest 
the shoot may again begin to grow, and then one of several possi- 
bilities may occur. In the first place, the terminal bud generally 
possesses an advantage or occupies a more advantageous growth 
position. It again shoots up continuing the line of the original 
shoot. Its advantage is so complete that the uppermost axillary 
buds are’unable to express their growth potential and remain dor- 
mant (fig. 1,plate5). In thesecond place, the terminal bud may 
again shoot up, but its growth is not so pronounced and it fails 
to completely suppress the two uppermost axillary buds. One 
of these being in some way more favorably located than the 
other, also begins to grow a shoot in a direction at an angle to 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, No. 2 


242 CHARLES R. STOCKARD 


that of the shoot from the terminal bud. The other top axillary 
bud, however, was not so fortunate as its fellow and was not 
capable of overcoming the inhibiting influence of the terminal bud 
and so remained dormant, as is shown in figure 2, plate 5. 

Finally, the third possibility very rarely occurs and all three 
of the uppermost buds are able to grow. In this case both of 
the axillary buds had a potential or tendency of growth sufficiently 
strong to successfully compete with the inhibiting influence of the 
terminal bud (fig. 3, plate 5). 

We may imagine that the growth of the terminal bud in the 
second and third cases was not normally vigorous. For some 
reason the advantages of the two or three potential buds were 
equalized and we find twin and triplet shoots growing out. 
Similarly, we may imagine the potential buds around the germ- 
ring of the fish’s egg to vary in their degrees of supremacy, ordi- 
narily only one grows and suppresses the growth tendency of all 
other potential points. But if the developmental rate be slow, 
the one bud fails to suppress all other points on the blastoderm, 
and twin or triplet buds may become capable of expressing them- 
selves. 

In conclusion, these experiments and observations make it 
seem highly probable that influences similar to those acting be- 
tween a growing plant bud and its resting buds, or between the 
stronger component in a double vertebrate embryo and the 
smaller component, are also acting between a rapidly growing 
organ bud and other potential organ buds in the embryonic indi- 
vidual. Such a conception has the decided advantage of being 
of practical scientific value. Since on this basis the experimenter 
has a logical working scheme for a study of the abnormal and 
through this, the normal development of a given organ. Sucha 
method in an analysis of the development of the eye, for example, 
has been most valuable. 

Summarizing the present status, I have succeeded in locating 
more or less definitely the critical moment of origin in the follow- 
ing individual developmental processes: 

1. The growth of the primary embryonic axis: If this be slowed 
by arresting early cleavage stages or pregastrulation stages, the 


STRUCTURE AND DEVELOPMENTAL RATE 243 


usual single axis does not arise with sufficient influence to suppress 
the origin of other axes, and twins or double individuals result. 
Twins and double monsters are, therefore, types of developmental 
arrests. 

2. The suppression of the eyes or modification of their struc- 
ture: When an arrest is induced later than the above, but before 
the origin of the embryonic shield in the Fundulus embryo, all 
known modifications in the structure of the eyes may result in 
otherwise normal individuals or in further deformed specimens. 

3. Suppression of the primary brain ventricles inducing sub- 
sequently deformed or tubular brains: Arrests induced before 
and just about the time of first appearance of the embryonic 
shield result in malformation of the embryonic brain ventricles. 
These do not express their usual bilateral outgrowths and are 
frequently of a simple tubular outline. 

The periods of arrest necessary to induce the eye and the brain 
modifications are so close together or so nearly the same, that 
one generally finds combinations and mixtures of the defects 
among the same experimental group of embryos. Arrests at the 
earlier moment give a majority of eye conditions, many without 
brain involvements, while arrests at the slightly later stage give a 
majority of brain modifications, a few with fairly well-formed eyes. 
The individual variations in developmental moments among the 
embryos of a group also tend to contaminate the results and give 
mixtures of the two classes of deformities. 

4. Modified or contorted mouth and branchial systems: Ar- 
rests during the embryonic shield stage, and earlier, frequently 
cause deformities of the mouth and branchial regions of the Fun- 
dulus embryo. In a few cases these deformities have existed in 
individuals otherwise normal. They, therefore, must possess a 
critical moment occurring at a time more or less distinct from 
that of the other organs. The close association of mouth abnor- 
malities with those of the eyes is probably not due to identical 
critical moments of origin in the two cases, but more likely to 
the fact that when a slow rate of development exists during the 
eye moment it is rarely completely overcome and the normal 
rate reestablished before the critical moment for the bilateral 
outgrowths of the mandibular pouch is reached. 


244 CHARLES R. STOCKARD 


There is also probably some overlapping between the critical 
moments of origin for the ectodermic organs, for example, those 
of the central nervous system and the entodermic organs of the 
alimentary canal. There is, further, the possibility that the 
interaction or inhibiting influence existing among the buds of 
one system may not extend to the organ buds of a different sys- 
tem. I believe, however, that this is not the case, and it is more 
probable that the growth of each organ affects to a degree the 
growth of all other parts in the entire embryo. The degrees of 
effect exerted by one growing organ over the others may differ, 
for example, the growth of the optic vesicles very probably affects 
the primary brain ventricle growth more strongly than it does the 
growth of the branchial organs, etc. 

5. Modifications in size and structural outline of the inner ear: 
Arrests during a closely similar stage to that in case 4 sometimes 
show their effectiveness on a different bud. In such cases the 
ear as well as the mouth and gills becomes affected. The otic 
vesicle may be completely suppressed or develop into a simple 
tiny cyst with no outgrowth for the semicircular canals. 

6. Faulty development of the liver and pancreas: Later arrests, 
after the embryonic line is distinctly seen, in the Fundulus em- 
bryo may cause an abnormally small outgrowth representing the 
liver. Such conditions make it appear as though the primary 
liver bud had been inhibited in its outgrowth from the intestinal 
wall or the later rate of multiplication of liver cells had been 
reduced. The pancreas evidently arises and has its critical mo- 
ment at a somewhat later instant than does the liver. Yet the 
two moments are so close together that it would require a very 
delicate difference in time of arrest to affect the one and not the 
other. 

The findings in these six attacks on the problem make evident 
this very important fact: That each organ arises at a definite 
moment during embryonic development and not during widely 
different moments just as truly as that an individual organ arises 
from a very definite embryonic area or anlagen and from no other. 

The organ defects found in nature further confirm the results of 
experiments on the Fundulus embryos. It is well known from 


EE EE 


STRUCTURE AND DEVELOPMENTAL RATE 245 


the studies by Mall (17) and many others that localized anom- 
alies are quite frequent in both normal and pathological human 
specimens. The localized anomaly may involve only the eye, 
only the bilaterality of the brain, only the ear, only the mouth 
structures, only the kidneys (I have dissected two fetuses at 
term neither of which possessed any evidence of a kidney, but 
one of which was otherwise structurally normal), only the geni- 
talia, ete. It is evident that such anomalies could not occur 
unless there was a certain moment of specific and peculiar sus- 
ceptibility on the part of each organ during which any unfavor- 
able condition would act on it in a selective way. Of course, the 
specific action of certain substances on certain embryonic buds 
would give a possible explanation, but there is so much strong 
positive evidence in the present study as well as from other sources 
against this once attractive possibility that it can well be dis- 
carded. A strong point of evidence is the fact that a typical 
defect of one organ can be induced by a great number of treat- 
ments, and different defects of many different organs can all be 
induced by one and the same treatment. In every case the result 
depends upon the developmental time during which the treatment 
acts and not upon chemical or physical properties of the sub- 
stance used. As repeatedly shown, the primary effect in all in- 
stances is simply a slowing in the rate of development. 


c. Developmental rate and postnatal changes 


The relations between the rate of growth and particular de- 
velopmental moments found in the embryo probably continue to 
be of importance during postnatal development as well. The 
numerous studies on inanition in rats and other mammals bear 
on the same general problem as that considered in the present 
report. The one important effect that inanition might have on 
the subsequent history of a developing individual would be the 
malformation and arrest in development of certain tissues or 
parts. After birth a number of important organs and tissues 
still have a considerable degree of growth and differentiation to 
accomplish, and very probably the same rules apply to this activ- 


246 CHARLES R. STOCKARD 


ity during the postnatal period as are found to apply in the 
embryo. The important question at once arises as to whether 
there are periods during which starvation might produce no sub- 
sequent ill effects, alternating with more or less critical periods 
during which a similar treatment might be followed by consider- 
ably more serious results. For example, if a given treatment be 
administered to one group of animals from the second to the fifth 
week after birth, and to a similar group from the fourth to the 
seventh week, the results might be the same or they might be 
very different. The results would depend very largely upon 
whether significant tissue changes susceptible to variations in 
developmental rates had occurred during the time intervening 
between the two experiments. 

The interaction or competition among the growing and devel- 
oping organs found in the embryo certainly continues during 
postnatal development. The suppression of development in 
certain organs and tissues by the activity of another organ is 
splendidly illustrated by the glands of internal secretion. The 
further development of certain secondary sexual characters, such 
as hair and plumage, after subdued activity of the gonads is a 
case in point. : 

The difference in importance between two developmental 
moments in the postnatal individual is, however, of far less sig- 
nificance than in the early embryo, just as arrests during early 
cleavage stages are of more far-reaching consequence than similar 
arrests after gastrulation hasoccurred. Differencesin developmen- 
tal rate during postnatal periods incline to affect the finer features 
or the type of the individual rather than cause actual malformation 
or pathological deficiencies in the tissues. Such effects are read- 
ily observed in many of the arrested, status, and infantile human 
types. A fuller conception of the significance of developmental 
rate and rhythm in the determination of human types and ap- 
pearances will be given in a separate communication. 


STRUCTURE AND DEVELOPMENTAL RATE 247 


9. CONTINUITY OF THE SERIES FROM MONSTRA IN DEFECTU 
THROUGH THE SINGLE NORMAL INDIVIDUALS TO MONSTRA 
IN EXCESSU AND FINALLY IDENTICAL TWINS 


It has long been recognized that certain types of monsters ex- 
hibit their characteristic defect to varying degrees. The cyclo- 
pean series, for example, may present individuals not only with 
a single median eye, but with a bilaterally wide eye, hour-glass 
eyes, and finally closely approximated separate eyes. The series 
of diplopagi likewise exhibit all degrees of doubleness, as illus- 
trated in plates 1 to 4. 

In studying monsters bélonging to these groups, Wilder (’08) 
went a step further and called attention to the fact that the so- 
called series of defective monsters passed by degrees up to the nor- 
mal individual and continued from there through the excessive 
series on to identical twins. He was impressed by the ‘orderly 
development’ of the members in such a series and termed these 
individuals ‘Cosmobia.’ The treatment of the series as variations 
about the normal as a standard was a most important advance in 
an analysis of their structural conditions. Wilder further empha- 
sized the important fact that monstra in defectu and monstra in 
excessu are both due to the same kind of cause and should be 
considered together in any general treatment of the subject, 
especially concerning cause. 

However, after enunciating this clear arrangement of the prob- 
lem, Wilder was entirely misled in his interpretation of the cause 
of these individual anomalies. The fact of their ‘orderly’ and 
symmetrical structure, and the further evident fact that normally 
formed identical twins represent the termination of the diplopage 
series, led him to consider all such forms as due to a definite germ- 
inal variation. It seemed to him more probable that orderly 
deviations from the normal would arise in the germ-plasm than 
that they should occur as a result of some modification during 
individual development. The burden of evidence, however, is 
unfortunately against such a proposition, and weighs decidedly 
more at the present moment than when Wilder published his 
account. 


248 CHARLES R. STOCKARD 


From what we know of germinal variations and mutations, they 
do not necessarily give rise to individuals that gradually grade 
away from the usual type. There may be wide structural breaks 
between the parent stock and the mutant. On the other hand, 
we now know that unusual environmental conditions tend to 
modify the normal course of structural development to varying 
degrees and give rise to the exact series of defects on which Wild- 
er’s conceptions were based. The present contribution clearly 
demonstrates the underlying factors and the very probable cause 
of this orderly series of beings deviating from the normal indi- 
vidual as monstra in defectu and monstra in excessu. 

The idea is entirely correct that double monsters and twins are 
due to the same cause as cyclopia. And both may be experi- 
mentally produced by an identical physical change in the envi- 
ronment, lowering the temperature. Both conditions also result 
from a slowing of developmental rate, but one differs from the 
other because of the difference in the developmental periods dur- 
ing which the slowing in rate was effective. 


10. THE NECESSITY OF A CONTROLLED OR REGULATED ENVIRON- 
MENT IN WHICH TO DEVELOP HIGHLY COMPLEX INDIVIDUALS 


From the foregoing considerations it has become evident that 
normal development of the vertebrate embryo depends acutely 
upon the stability of certain factors in the environment. Changes 
in the conditions of moisture, temperature, or oxygen supply are 
the most frequent causes of embryonic death as well as monstrous 
development. Any degree of actual dryness is fatal to the ver- 
tebrate embryo, and sudden lowering of the surrounding tempera- 
ture and reductions of the oxygen supply interrupt development 
with the significant consequences discussed above. A normal 
amount of ordinary food materials is not, however, so acutely 
necessary for perfect-structural expression. The rate of develop- 
ment under malnutrition is slow, but the depression does not 
come on suddenly nor is it often sufficiently complete to cause 
serious structural anomalies. 

Vertebrate animals are faced with the problem of the necessity 
of a regulated environment in which to develop their eggs into 


CC 


STRUCTURE AND DEVELOPMENTAL RATE 249 


the free living individual. The lower vertebrates are almost 
entirely aquatic and their eggs undergo only a short embryonic 
development before reaching the swimming larval stage. The 
birds and mammals, however, at the moment of birth or hatching 
have, as a rule, attained a complexity of structure greater than 
that of the adult stage in fishes and lower forms. The period of 
their prenatal development is extremely long, offering far greater 
opportunity in time for changes in the environment and, there- 
fore, necessitating some means of control on the part of the parent 
generation. - 

The marine and fresh-water fishes live in a more or less homo- 
geneous medium which rarely undergoes sudden or marked 
changes during the spawning seasons. ‘Their eggs are deposited 
in the water in instinctively chosen places during definite times 
when the conditions of oxygen and temperature are generally 
favorable for the given species. This developmental environ- 
ment may in unusual cases fail in one or all respects. The water 
may become so stagnant as not to supply oxygen, or it may 
suddenly become either too hot or too cold for the welfare of the 
developing eggs, or in a dry season it may become evaporated or 
carried off, allowing the eggs to dry. The instinct of the fish 
helps to guard against such accidents, and the eggs are deposited 
at a season when the temperature changes are least likely to be 
harmful, and localities are chosen where the water is properly 
supplied with oxygen and is sufficient in amount to escape rapid 
drying. 

The higher land-living vertebrates have no such surroundings 
in which to develop their eggs. In becoming terrestrial, these 
animals must have evolved not only appendages for locomotion 
on land, but also some means of controlling or providing an envi- 
ronment in which their long embryonic development could take 
place. 

The eggs of reptiles and birds, as is well known, are provided 
with comparatively enormous amounts of food-yolk surrounded 
by layers of other food and enclosed in protective membranes and 
shell. These arrangements not only supply food, but insure a 
moist environment essential to all development and permit free 


250 CHARLES R. STOCKARD 


access of oxygen from the surrounding air. The one element es- 
sential for development of these eggs, not yet provided, is a con- 
stant high temperature. The reptiles are largely confined to 
warm regions and deposit their eggs during the hottest periods 
of the year in sand or other heated places, and in this way the 
proper temperature is usually provided. The birds, however, 
with the extremely high temperature of their own bodies, supply 
in a more definite way the proper amount of heat for the incuba- 
tion of their embryos. Lack of moisture and oxygen very rarely 
causes the death or abnormal development of the eggs of reptiles 
and birds. But failure to maintain a uniform temperature and 
unfavorable degrees of heat and cold are the chief causes for 
embryonic mortality and deformity in these animals. 

The mammals have advanced a step further in perfecting a con- 
trolled developmental environment. The internal development 
of the embryo not only insures a properly moist condition, but 
the high temperature of the maternal body is sufficiently uniform 
never to cause interruption of the normal progress of develop- 
ment. The supply of oxygen is derived from the blood of the 
mother through the placental circulation, and this is the one 
element in the mammalian developmental environment which 
most frequently becomes deranged. Faulty placentation cuts 
down the supply of oxygen to the mammalian embryo and lowers 
its rate of development, producing as a result prenatal death and 
all varieties of malformation. Yet we may well believe that the 
long and highly complex development of the mammalian embryo 
could not take place unless it was protected by a fairly well 
regulated environment. Abnormal development in the embryos 
of birds may very rarely result in nature from poor ventilation on 
account of a coated egg shell, but more frequently it results from 
failure to maintain a uniform temperature. While in mammals 
the temperature changes are eliminated by the internal mode of 
development, the one great danger to normal development still » 
not completely controlled is the chance of a low oxygen supply 
brought about by a delayed or poor implantation of the pla- 
centa. The great majority of monsters in mammals are very 
probably due to an insufficient oxygen supply during develop- 
ment, and this results as a rule from faulty placentation. 


STRUCTURE AND DEVELOPMENTAL RATE 251 


The ready manner in which the structures of the developing 
individual are modified by changes in temperature and oxygen 
supply makes it evident that the existence of the species often 
depends upon some means of regulating the developmental en- 
vironment. We may readily believe that species have been lost 
during evolution not only on account of failure of their adult 
structures to fit them for existence, but equally often as a result 
of failure to obtain an environment in which their embryonic 
development was possible. 

No developmental environment in nature is constantly per- 
fect, and this fact is the underlying cause of the frequently occur- 
ring malformations and monstrous productions. 


11. GROWTH COMPETITION BETWEEN THE TWO COMPONENTS IN 
DOUBLE INDIVIDUALS AND THE TIME OF OCCURRENCE 
OF TERATOMA IN MAN 


It has been clearly seen that in cases where one component of 2 
double individual is larger because of a more favorable location, 
the smaller has been inhibited in its growth and development by 
the presence of the larger. In plants this inhibiting influence is 
readily demonstrated, since on pinching away a growing shoot the 
suppressed buds immediately spring into growth. There is much 
evidence to indicate that a similar interaction exists between two 
developing organs in a single individual. The alternating mo- 
ments of rapid growth among the several organs of the embryo is 
a case in point. 

With the preceding discussions of these propositions in view, 
if it be now admitted that teratoma in man often originates as a 
twin Inclusion, we may expect an antagonistic growth reaction to 
exist between the teratoma and the host. In other words, while 
the host individual is rapidly growing, the teratoma will be sup- 
pressed and when the rate of growth of the host individual be- 
comes slow, the teratoma will tend to grow more rapidly. If 
such an opinion be correct, there should be a marked correlation 
between the postnatal growth curve and the time of enlargement 
or recognition of teratomata. When the individual is growing 
very rapidly during the first year and a half of infancy, few tera- 


252 CHARLES R. STOCKARD 


tomal enlargements would be expected; following this period 
there is a decided fall in growth rate and the teratomata of early 
childhood may occur. The alternating periods of fast and slow 
growth should then continue to correspond with periods of few 
and many recorded teratomata. Dr. H. E. Himwich has under- 
taken a careful survey of the teratomata as recorded in the lit- 
erature in order to ascertain whether any apparent relationship 
does exist between the time of occurrence of a teratoma and the 
periods of fast and slow growth rate in man. The results of his 
investigation are soon to be published. 


12. CANCEROUS GROWTHS AND THE GENERAL CESSATION OF ALL 
NORMAL GROWTH IN THE OLD INDIVIDUAL 


In an interpretation of the cause of cancer the fact that the 
condition is so much more frequent in the adult and old individ- 
ual than in the young is to be recognized as of deep significance. 
The fact that there is an interaction and especially a growth- 
inhibiting effect exerted among proliferating tissues in the indi- 
vidual is a second point of great importance. 

In the young rapidly growing and developing person almost 


all organs and tissues are increasing in amount through multi- - 


plication of their cellular constituents. The liver, for example, 
grows in actual mass until it reaches the adult size. This size, 
although decidedly variable in a group of individuals, has rather 
definite limits. The normal human liver is never indefinite or 
unlimited in its growth. Almost all other organs are similarly 
of limited size. Thus growth in general tends to cease as the 
body approaches its adult- proportions. Finally, in the old indi- 
vidual, the only remaining cell proliferation becomes almost 
entirely confined to the germinative layer of the skin, the lining 
epithelium of the alimentary tract, the testes in the male, and 
the production of red blood-corpuscles. Even these proliferation 
processes become feeble with increase in age and new cells are 
not abundantly supplied. This is the normal course of events. 

The size and proportion of parts are largely determined by 
heredity, but may be seriously interfered with by irregularities 
in the environment. 


STRUCTURE AND DEVELOPMENTAL RATE 253 


A slowing of the developmental rate at particular times may 
largely suppress the growth of certain organs, rendering them 
abnormally small in size and insufficient in their function. The 
normal proportion of things becomes distorted. Again it may 
rarely happen that one organ takes on an excessive growth and 
attains a size entirely out of normal proportion. There is thus a 
frequent lack of proper balance and adjustment among the several 
organs of the developing body. 

The properly regulated balance among the organs is to a great 
extent due to the inhibiting and controlling effects of one growing 
region or part over other parts. This is readily demonstrated 
by the modifications which result in size and proportion of certain 
parts of the body following the experimental removal of other 
parts. All parts may be thought of as having more or less to do 
with the ultimate growth results of the whole. 

On becoming adult, a state of apparent balance is maintained. 
Growth is considerably reduced and largely confined to the repair 
of natural loss and the maintenance of this state of adult balance. 
Under such conditions there still remains considerable regenera- 
tive powers following injuries of various kinds. Yet these regen- 
erative processes are not so perfectly accomplished or so well con- 
trolled in the adult animal body as they were in the larval or 
immature condition. This fact may in some way be associated 
with the absence in the adult of general growth and the well- 
expressed regulatory processes which are necessary in the devel- 
oping individual. 

The regenerative growth following injuries to the adult animal 
may become morbid in degree and without regulation, thus giving 
rise to malignant conditions. Such a growth might rarely occur 
in the immature body, but in this case one would expect to find 
the growth proportions among the tissues in general to be abnor- 
mal and distorted. Thus, juvenile cancer conditions are rare and 
are probably associated with other deformities. 

Cancer in the adult would be expected to occur more fre- 
quently in certain families, since the growth balance and propor- 
tions are hereditary characters, and on the state of these, the can- 
cerous growth largely depends. Families or persons derived from 


254. CHARLES R. STOCKARD 


similar cytological complexes show more nearly similar growth 
and tissue reactions than do random groups of individuals derived 
from non-related parentage. 

In the old individual with but little normal growth still in exist- 
ence, there can be, on the basis of my interpretation, but slight 
inhibition to any regenerative process that might be set up. 
Such animals naturally on account of their old condition usually 
regenerate very slowly, but following continued trauma, active 
regenerative growths are frequently begun, and not being under 
the inhibiting control of any other active growth processes, this 
regeneration attains an excessive, distorted, and malignant con- 
dition. 

All very old animals no doubt experience a considerable amount 
of trauma, and if they lived long enough almost all of them might 
possess some cancerous growths. The truth of this statement is 
well illustrated by comparing the frequency of reported cancer in 
rats and mice with similar growths in guinea-pigs, all constantly 
used laboratory animals. Rats are very old after three years of 
life, and actually at two years old may properly be compared, 
according to Donaldson (’15), with a man at sixty. Mice attain 
old age even earlier, and at two years are very old. This being 
the case, it frequently happens that the rats and mice used in 
laboratories have actually become old individuals, having been 
kept by the breeders and the laboratory for as long as two years. 
Cancerous growths are common in these animals. 

The guinea-pig under favorable conditions does not become 
old until it has lived for about five years, and we have frequently 
kept these animals for more than seven years; at this age, how- 
ever, they are extremely old. Thus, as a rule, the guinea-pigs 
used in laboratories are really young individuals, generally less 
than three or four years old. Consequently, cancerous growths 
are said to be uncommon among these animals. However, among 
the old individuals in our stock a considerable percentage of can- 
cerous ones have occurred. So it might be inferred that if as 
great a number of really old guinea-pigs were observed as of old 
rats and mice, cancer might be found to be almost as common 
among guinea-pigs as among rats and mice. And finally it may 


pw 


STRUCTURE AND DEVELOPMENTAL RATE PAD} 


be supposed that every mammal would develop some form of can- 
cerous growth should it chance to live until extreme old age. 
The increased length of life in man may be associated with the 
increased frequency of cancer. 


13. GENERAL SUMMARY 


In considering the results of the present study it is necessary to 
recognize the fact that a given animal species passes through its 
embryonic stages at a specific rate of development, probably 
dependent upon the rate of oxidation in the protoplasm of the 
species. This developmental rate varies within certain normal 
limits; should variations in rate extend beyond these limits, the 
developmental result frequently becomes modified and distorted. 

The rate of development is not uniform throughout the entire 
process, but periods of rapid progress alternate with moments 
of slow rate or almost quiescence. In spite of these changes in 
rate, development in most forms does not actually stop after it 
has once started, but progresses in a continuous manner until the 
fully formed animal is produced. 

There are certain animals in which the continuous mode of 
development has become modified. In these forms development 
begins and attains a definite stage and then stops completely, to 
remain at a standstill for days or even weeks, until a change in 
the environment again permits the resumption of the develop- 
mental processes and the completion of the fully formed animal. 
Such a discontinuous mode of development is universal among 
the birds and is known to occur in several mammals. 

With these points in mind, the results of the present study may 
be summarized as follows: 

1. The continuous mode of development may be experimentally * 
changed into the discontinuous by two very simple methods, tem- 
porarily lowering the surrounding temperature and thereby re- 
ducing the rate of oxidation and by directly cutting off the sup- 
ply of oxygen. 

The effects on subsequent development of interruptions caused 
by these methods depends upon the stage during which the inter- 
ruption is introduced. There are stages of apparent indifference 
toa stop in development. Shortly after gastrulation is completed, 


256 CHARLES R. STOCKARD 


the development of the fish’s egg may be stopped for a considerable 
length of time with impunity, no ill-effects resulting. This is the 
developmental moment at which the bird’s egg is normally stopped 
on account of the fall in temperature experienced after passing 
out of the mother’s body. 

There are other stages during which a temporary interruption 
of the developmental processes will be followed by most disas- 
trous effects. These critical stages are usually moments during 
which marked inequalities in rate of cellular proliferation are tak- 
ing place in different portions of the blastoderm orembryo. ‘The 
period preceding the process of gastrulation is Just such a critical 
moment. 

2. There are considerable differences in effect between greatly 
reducing the rate of development and actually stopping the proc- 
ess temporarily. The development of certain eggs may be 
slowed down to one-tenth or one-twentieth of the usual rate and 
be maintained in such a slow condition for days without the major- 
ity of specimens losing their power of regaining the normal rate 
and giving rise to structurally perfect individuals. If at similar 
stages the development of the same eggs be completely stopped 
instead of slowed down, they are in many cases unable later to 
resume the process and die, in other cases they may resume devel- 
opment in a most abnormal fashion, or finally a few may be 
capable of resuming the apparently normal process. 

This difference in results between a severe reduction in devel- 
opmental rate and an actual temporary stop is to be explained as 
follows: Slowing does not completely eliminate the normal in- 
equalities in rate of developmental change existing among the 
several parts. Those parts that were in states of rapid develop- 
‘ment are depressed in the same proportion as other parts that 
were developing more slowly and inequalities in rate still exist in 
the slow-going embryo. When such specimens are allowed to 
resume a faster development the several portions of the embryo 
are able again to maintain normal differences in developmental 
rate and a proper balance is assured. 

A complete stop in development reduces the rate of all parts to 
zero and eliminates normal inequalities. On resuming develop- 


ee 


STRUCTURE AND DEVELOPMENTAL RATE 257 


ment from such a state, parts that should progress at a dispropor- 
tionately fast rate are unable to attain such supremacy and all 
portions of the embryo start at about the same rate. The usual 
developmental balance and inequalities in rate among the parts 
are lost and thus the typical form of the individual which actually 
depends upon these inequalities in rate of growth becomes modified. 

3. The types of deformities following a stop in development as 
well as those occasionally resulting from a slowing of the rate 
are similar to the defects produced by all experimental methods. 
Practically any deformity recorded in the literature other than 
those resulting from germinal variations or mutations may be 
induced by lowering the temperature and thus modifying the 
developmental rate. 

4. By an interruption of development during late cleavage 
stages a considerable percentage of twins and double individuals 
may be produced. When the eggs of the sea-minnow, Fundulus 
heteroclitus, are subjected to temperatures of 5° or 6°C. during 
cleavage stages, development is almost stopped. On returning 
such eggs to a summer temperature, after several days’ sojourn 
in the refrigerator, there will follow a high mortality, but many 
specimens will resume development producing a significant per- 
centage of twins and a number of variously deformed conditions 
along with a good proportion of normally formed young fish. 

Arresting or stopping development of the same eggs during 
the same developmental stages by diminishing the available sup- 
ply of oxygen will be followed by closely similar results. - 

The eggs of the trout are naturally much more inclined to 
develop into double individuals than are those of Fundulus. 
When the oxygen supply during early development is not abun- 
dant, a great many twin and double trout specimens are frequently 
found to occur. 

All of these double conditions result from arrests during very 
early stages of development, invariably before the process of blas- 
topore formation has in any way begun. No: duplicities or 
twins have been found to occur among the great numbers of fish 
eggs which have been arrested during postgastrular stages of 
development. 


258 CHARLES R. STOCKARD 

5. The bird’s egg is usually laid, according to investigations 
on this subject, after the process of gastrulation has commenced. 
Yet double chick embryos are not uncommon among the develop- 
mental stages observed in the laboratory, although in nature such 
specimens almost never exist at the time of hatching. 

The cause for the double chick embryos is the same, I believe, 
as that indicated above in the case of the double fish. Although 
the great majority of hen’s eggs are laid and their development 
stopped by the fall in temperature after gastrulation has begun, 
still it is recognized by those who have investigated the subject 
that there is considerable variation in the developmental stages 
of the eggs at the time of laying, and a minority of eggs are laid 
before gastrulation has begun. When an egg in this stage is 
stopped by the fall in temperature following laying, it would be 
expected from the experience with the fish that just such eggs 
would frequently give rise to two points of gastrulation and two 
embryonic fundaments instead of one. The interruption in the 
process of development at this critical time and the resumption 
of development at an equally slow rate in all regions of the blasto- 
derm, permits more than one potential embryo-forming region 
to.express itself. The interruption at this particular moment is 
the very probable cause of twin and double specimens. 

6. Polyembryony in the armadillo is in all probability explain- 
able on a similar basis to the cases above. Development begins 
as in most other mammals in the fallopian tubes and continues 
until theegg passes down: into the uterus as an early blastocyst. 
Development then stops in the armadillo for a period of several 
weeks with the blastocyst lying free in the uterus, as Patterson 
(13) has reported. The stop here is not due to a temperature 
change, since none has occurred, but is very probably on account 
of an exhaustion of the original oxygen supply derived from the 
ovarian blood. The uterus fails to react immediately to the pres- 
ence of the blastocyst, implantation is delayed, and no means of 
obtaining oxygen necessary for continuing development is pos- 
sible until the egg becomes implanted. After the delayed implan- 
tation has taken place, development is slowly resumed in a way 
which gives rise to multiple embryo formations or budding, as 


STRUCTURE AND DEVELOPMENTAL RATE 259 


has been fully considered above. The ‘quiescent period’ in the 
armadillo egg is probably the result of lack of oxygen and thus 
the cause of polyembryony. 

Twinning or polyembryony may be considered a typical method 
of asexual reproduction, and its occurrence in mammals and 
other vertebrates makes the phenomenon of so-called ‘alterna- 
tion of generations’ universal among animals. 

7. The degree of duplicity in double individuals depends upon 
the original distance apart of the embryonic buds on the blasto- 
derm. 

The relative sizes of the two components in double specimens 
vary widely. In many double individuals the two components 
are practically equal, while in others one component is of normal 
size and the other component in a series of specimens varies from 
slightly below normal size down to a very small mass. This size 
difference between components is in no way associated with the 
degree of duplicity. 

8. In double individuals in which the two components are 
equal in size they are both normal in structure. When the two 
components of a double specimen are unequal in size, the larger 
component is almost always normal in structure, and the smaller 
component is always deformed. The degree of deformity in the 
smaller component varies directly with the extent of difference 
in size between the two components. 

9. As the large component reaches adult size the lesser com- 
ponent may have become so relatively small as to be represented 
by a nodular mass on the body of the larger, or it may be lost to 
sight entirely as a twim inclusion. Such conditions make it evi- 
dent that doubleness and twinning are actually more frequent 
than records would indicate. 

10. The types of defects and the degree of deformity exhibited 
by the smaller component are exactly similar in kind and degree 
to the deformities found among single individuals. This fact 
renders the double individual with unequal components a most 
valuable key to an understanding of the cause of all monstrous 
development. The two components are from identical germinal 
origin and are developing in organic connection in exactly the 


260 CHARLES R. STOCKARD 


same environment, yet one is structurally perfect while the other 
smaller member presents all types of deformities. The difference 
between the two is in their developmental rate, the larger having 
a normal rate and the smaller progressing more slowly and in 
an arrested fashion. The depressed state of the one component 
is the result of an inhibiting influence exerted by the other. 

11. The deformities of the small component in the double 
individuals and the similar defects induced by stopping the devel- 
opment of single individuals make it evident that all develop- 
mental monstrosities are the results of simple arrest. During 
my experiments with Fundulus eggs it has been possible to induce. 
a single type of defect with a great variety of different experi- 
mental treatments. The reverse is also true; all varieties of 
defects may be induced by subjecting the embryos to one and 
the same experimental treatment. 

The primary action of all the treatments is to inhibit the rate of 
development, and the type of deformity that results depends simply 
upon the developmental moment at which the interruption occurs. 
All monsters are the result of the same cause, and the type of 
monster depends upon the time at which the cause was in opera- 
tion. 

Several developmental moments have been located at which 
rather definite defects of particular organs may be induced. 
These are the moments during which the organs are in their most 
rapidly proliferating condition. Arresting the rate at such a 
moment gives decidedly injurious results. When an organ is 
developing at a slow rate the arrest fails to affect it. 

12. The development and growth of organs in the single indi- 
vidual are interrelated in a way similar to the interrelations be- 
tween the components of a double specimen. When one organ 
or one component has a higher rate than another, it develops at 
- this rate for a limited time and tends to inhibit development on 
the part of other organs. This is readily demonstrated by the 
inhibiting effect of the growing shoot over all the potential buds 
of a plant. When the growing tip is pinched away, the inhibited 
buds immediately express their capacity to grow. There is 
much evidence to indicate that a similar interaction exists among 
the developing parts of an animal embryo. 


STRUCTURE AND DEVELOPMENTAL RATE 261 


13. The initial growth giving origin to an embryonic system, 
such as the brain and spinal cord, is linear in type, until a definite 
length is attained when linear growth subsides. This is followed 
by a series of lateral outgrowths in consecutive fashion. These 
lateral outgrowths from the central nervous system may be 
experimentally suppressed by slowing development at definite 
times, and when all are absent a simple tubular brain is the end 
result. The same plan of development holds for the foregut and 
its lateral outgrowths to form the mandibular pouch, etec., and 
the development of this system may also be modified in a manner 
similar to that mentioned for the brain. 

14. Monstra in defectu and monstra in excessu, which have 
frequently been treated as such distinctly different classes of con- 
ditions, are as a matter of fact closely similar. Both classes of 
anomalies are due to a common cause and may actually both 
exist in the same specimen. For example, an arrest of develop- 
ment before gastrulation may cause a blastoderm to form two 
embryonic processes which later develop into a double-headed 
individual—a typical monstrum in excessu. Ata very early stage 
one of these embryonic processes may become inhibited and later 
form a cyclopean eye instead of the usual two lateral eyes; this 
head is then a typical case of monstrum in defectu. The fact 
that the normal individual stands between these two arbitrary 
classes of monsters has no other significance than that the mon- 
sters themselves are simply modifications of the normal condi- 
tion resulting from an unusual reduction in the rate of develop- 
ment during certain critical periods. 

15. The great importance of developmental rate in influencing 
the type and quality of structure is not confined solely to embry- 
onic development, but postnatal development, and structures are 
similarly influenced by the rate at which the processes are accom- 
plished. This phase of the subject is to be presented in a sub- 
sequent communication. 

16. In view of experimental results, it becomes evident that 
normal development of the vertebrate embryo depends acutely 
upon the stability of certain factors in the environment. Changes 
in the conditions of moisture, temperature, and oxygen supply 


262 CHARLES R. STOCKARD 


are the most frequent causes of embryonic death as well as mon- 
strous development. ‘The existence of the species may frequently 
depend upon some means of regulating the developmental envi- 
ronment. Species may be lost during evolution not only on ac- 
count of failure of their adult structures to fit them for existence, 
but equally as a result of failure to obtain an environment in 
which their embryonic development is possible. ‘The highly com- 
plex forms, such as birds and mammals, with a long embryonic 
period have partially succeeded in controlling their develop- 
mental environment. But inno case is the regulation constantly 
perfect and this fact is the underlying cause of frequent malfor- 
mations and monstrous productions. 

17. The double fish specimens with unequal components and 
the growth reactions between these components are important 
in connection with certain teratomal conditions in man. If 
teratoma in man frequently originates as a twin inclusion, we 
may expect an antagonistic growth reaction to exist between the 
teratoma and the host. While the host individual is rapidly 
growing the teratoma will be suppressed and when the host 
slows its growth the teratoma should tend to grow more rapidly. 
There should thus be a correlation between the postnatal growth 
curve and the time of enlargement or recognition of teratomata. 
Dr. H. E. Himwich has undertaken a survey of this subject which 
will soon be published. 

18. The interaction between the growing organs of a develop- 
ing individual has been discussed in its relation to regeneration 
and cancerous growths of old age. In the old individual with but 
little normal growth still present there can be but slight inhibi- 
tion to any regenerative process that may be set up following a 
continued trauma. 


STRUCTURE AND DEVELOPMENTAL RATE 263 
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Harper, BE. H. 1904 The fertilization and early development of the pigeon’s 
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Hertwie, O. 1892 Urmund und Spina Bifida. Arch. f. mikrosk. Anat., 
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264 CHARLES R. STOCKARD 


Karstner, 8. 1898 Doppelbildungen dei Wirbelthieren. Ein Beitrag zur 
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1899 Neuer Beitrag zur Casuistik der Doppelbildungen bei Hiihner- 
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1907 Doppelbildungen an Vogelkeimscheiben. Fiinfte Mitteilung. 
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Korscu, Fr. 1895 Ueber eine Doppel-Gastrula bei Lacerta agilis. Kel. 

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Lepiat, G. 1914 Localisation des premiéres Ebauches oculaires. Patho- 

génie de la Cyclopie. Anat. Anz., Bd. 46. 
1919 Action du milieu sur le développement des larves d’amphibiens. 
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LeuckarT AND ScHroHE 1891 Die Organisation der Hemididymi und Ana- 
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Lewis, W. H. 1904 Experimental studies on the development of the eye in 
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1909 The experimental production of cyclopia in the fish embryo 
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1915 The blindness of the cave fauna and the artificial production of 
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1916 Further experiments on correlation of growth in Bryophyllum 
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1918 Chemical basis of correlation. I. Production of equal masses 
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1918 The law controlling the quantity and the rate of regeneration. 
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1919 The physiological basis of morphological polarity in regenera- 
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STRUCTURE AND DEVELOPMENTAL RATE 265 


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1895 Half-embryos and whole-embryos from one of the first two 
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266 CHARLES R. STOCKARD 


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PLATE 1 
EXPLANATION OF FIGURES 


A series of young trout that started development with a slightly insufficient 
supply of oxygen. The series begins with an ordinary single individual and 
passes through increasing degrees of anterior duplicity, shown in the two upper 
rows. It then continues with specimens showing step after step of completely 
formed double bodies and tails and finally ends with perfectly formed identical 
twins, in which both members of the pair are equally as large and perfect in 
structure as is the first single individual. 

The photographs were all made at one magnification and show as nearly as 
possible the dorsal aspect of each specimen. On careful examination it will be 
found that in every specimen the two components are practically identical in size, 
and when the anterior halves are considered all heads are found to be normal in 
structure. 


PLATE 2 
EXPLANATION OF FIGURES 


The same series of trout specimens and photographed in exactly the same 
order as illustrated in plate 1. The individuals are here shown from as nearly as 
possible the ventral aspects. Selecting any given specimen and comparing its 
dorsal and ventral surfaces, as shown in plates 1 and 2, it is clearly seen that in — 
all cases the two components are equal in size and both are structurally normal. 
These are not ‘double monsters,’ but perfect individuals. The condition of 
doubleness is unusual, but not deformed or monstrous. The identical twins could — 
not be considered monsters, and they only differ in degree of doubleness from the © 
other members of the series. 


PLATE 3 
EXPLANATION OF FIGURES 


Two degrees of duplicity in human individuals. The upper photograph 
illustrates a doubled condition extending superficially only to below the shoulders, 
but internally the doubleness extends to the sacrum in the skeleton and to the 
lower ileum in the intestine. The lower photograph shows two complete babies 
extensively united by their ventral walls. 

In both of these specimens the components are of equal size and their struc- 
tures are normal throughout. Jn all specimens of human duplicities examined or 
found recorded in which the components were of equal size they were both struc- 
turally normal. . 


270 


DEVELOPMENTAL RATE AND STRUCTURAL EXPRESSION PLATE 3 
CHARLES R. STOCKARD 


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PLATE 5 
EXPLANATION OF FIGURES 


Photograph of a specimen delivered by Dr. Frank Erdwurm of New York. 
A living female child was attached to the cord leading away from the upper 
placenta. This baby was enclosed within its own chorion, the upper membranes 
shown in the picture. The two fetuses of about six months’ development shown 
below were enclosed in a common chorion and their cords are attached to a com- 
mon placenta. These are identical twin girls. About three months before 
birth their cords became so twisted that the placental circulation was cut off 
and they died. The mother was disturbed for a time until the uterine situa- 
tion became adjusted and this placenta shunted off. The fetuses remained 
enclosed within their membranous sac and at birth were considerably shriveled 
and somewhat macerated. 

Probably the implantation of the single individual in some way delayed im- 
plantation of the other placenta and caused the arrest which resulted in the 
twinning of the second egg. 


DEVELOPMENTAL RATE AND STRUCTURAL EXPRESSION 


CHARLES R, STOCKARD 


PLATE 5 


PLATE 6 
EXPLANATION OF FIGURES 


Outline drawings of terminal branches from the common privet. 

Fig. 1 A branch in which the terminal bud is in a resting state. The common 
condition following a limited period of growth. \ 

Fig. 2. A new shoot grows from the apical bud, all of the axillary buds at the 
base of the leaves remaining in a resting state. This is the usual manner of 
resuming growth. 

Fig. 3. An unusual case in which two shoots grow out after the resting period, 
the usual one from the apical bud and the second one from the upper right axil- 
lary bud. The potential impulse to grow on the part of the axillary bud was as 
great as that of the apical bud and twins developed from the two potential growth 
points. 

Fig. 4 The very rare case in which not only the apical bud grows into a 
shoot, but shoots also grow from both upper axillary buds. Here all of the 
upper potential growth points produce individual shoots, and ‘triplet’ branches 
arise. 


_ DEVELOPMENTAL RATE AND STRUCTURAL EXPRESSION PLATE 6 
CHARLES R, STOCKARD 


{Reprinted from Tar American Narurauis?, Vol. LV., Jan.-leb., 1921.] 


A PROBABLE EXPLANATION OF POLYEMBRYONY IN 
THE ARMADILLO : 


PROFESSOR CHARLES R. STOCKARD 


CorRNELL University MepicAn CoLurGr, New YorKk Criry 


By arresting the development of the fish’s ege during early 
stages double individuals and twins are frequently induced. 
The interruption or arrest makes it possible for more than one 
potential growth point along the germ-ring to give rise to an 
embryonic shield. In other words, accessory invaginations or 
blastopore formations occur as the initial structural step in 
doubleness. The interruption in the development of the fish 
embryo must be introduced during the cleavage stages and be- 
fore gastrulation in order to produce such phenomena. Among 
hundreds of eges arrested during later developmental stages no 
double monsters or twins ever occurred. A complete account of 
these experiments is soon to be published but for our present 
purpose two facts are important: First, accessory embryo forma- 
tions result from arrests in the developmental process; and 
second, the arrest must occur before gastrulation has taken 
place. 

In the light of these experiments it has seemed possible to 
interpret somewhat more clearly than has formerly been done 
the remarkable phenomenon of multiple embryo formation in 
the armadillo. 

On examining the uterus in two pregnant specimens of a 
South American armadillo von Jhering, in 1885, discovered that 
each contained eight fetuses enclosed within a single chorion. 
He correctly concluded that all of the fetuses in each mother 
had been derived from a single ege by some process of division 
into separate embryonic rudiments. After this valuable dis- 
covery and interpretation, the study of the armadillo’s develop- 
ment lapsed and nothing of importance was added for almost 
twenty-five years. Two series of investigations were then be- 
gun simultaneously one on the Texas armadillo by Newman and 
Patterson,* and the other on the South American species by Fer- 

1H. H. Newman and J. T. Patterson, Jour. Morph., Vol. 21, p. 359, 1910. 

62 


63 THE AMERICAN NATURALIST [VoLALy » 


nandez.* The growth and expansion of these twin studies has 
brought our understanding of the phenomena of polyembryony 
in the armadillo to a considerable state of maturity. 

These authors readily agreed that in most species of armadillo 
the individual members of a litter, usually four in the Texas 
species and eight in the common South American form, are all 
derived from a single egg. It required considerable effort, how- 
ever, to obtain the material that would furnish the morphologi- 
cal stages of the process by which the polyembryonie development 
was accomplished. We are finally indebted to Patterson,* for 
the very thorough and satisfactory manner in which he has col- 
lected and studied the early embryonic conditions; and particu- 
larly for having shown the first stages of the budding process 
through which the single blastocyst gives rise to four distinet 
embryonic areas, each exhibiting a typical primitive streak 
region. ’ ' 

In connection with the fish experiment it now becomes im- 
portant to ascertain exactly what degree of development has 
been attained by the armadillo blastocyst at the time the bud- 
ding process begins. And since, according to my interpretation, 
these buds should arise at the time-of gastrulation or blastopore 
formation, it becomes necessary to consider very briefly the 
germ-layers and gastrulation in mammals. The decidedly pre- 
cocious and highly modified method of formine the primary 
germ-layers in the mammalian blastocyst is not strictly com- 
parable to gastrulation or the method of germ-layer formation 
found among the other vertebrates. On the other hand, the em- 
bryonic line or primitive streak of the mammalian egg is ex- 
actly comparable to the blastopore and head process formation 
in the simpler forms. 

The blastocyst of the armadillo has already, by a process of 
cell migration and delamination, separated off the primary ento- 
derm from the ectoderm and further modified these layers be- 
fore the budding which forms the embryonic primordia has 
begun. The primordia are first formed by a thickening of the 
ectodermal layer of the blastocyst. The primary entoderm then 
invaginates into the primordia to form the secondary entoderm 
of the gut. The precocious cell migration and splitting into 
layers in the mammal’s egg is associated with the early implanta- 


2M. Fernandez, Morph. Jahrb., Bd. 39, p. 302, 1909. 
3 J, T. Patterson, Jowr. Morph., Vol. 24, p. 559, 1913. 


No. 636] POLYEMBRYONY IN THE ARMADILLO 64 


tion of the embryo upon the uterine-wall of the mother, and the 
later primitive streak formation may be interpreted as related 
to the actual gastrulation or blastopore formation away from 
which the line of the embryo always develops. 

Whether the validity of the above briefly outlined interpreta- 
tion of the germ-layer formation is admitted or not, we have 
in the armadillo a process of budding taking place from the 
blastoderm and associated with accessory or extra blastopore 
formation in much the same way as are the accessory embryos 
alone the germ-ring in the egg of the bony-fish. These buds 
also accord with Kopsch’s description of a double gastrular 
condition with two blastopores in a blastoderm of Lacerta agilis, 
from which he concluded that twin formation as well as anterior 
duplication arises from a double Einstiilpungen. And further, 
Assheton has described a similar condition in a blastodermie 
vesicle of the sheep. He, however, imagined the condition to 
have been due to a splitting during the morula stage. 

The double primitive streaks in the hen’s ege and other 
forms all lend themselves to strengthen the interpretation that 
double embryo formation first asserts itself by a double gastrula- 
tion or blastopore formation, which is initially a process of 
double instead of single bud formation. Patterson’s description 
of the origin of the quadruplet buds in the Texas armadillo 
furnishes the most striking case in the study of these conditions. 
And we may conclude that the budding or accessory embryo 
formation in the egg of the armadillo is exactly the same develop- 
mental process as that. which gives rise to twins and double indi- 
viduals in other vertebrate eggs. 

However, the very important question yet remains to be an- 
swered. Why does this accessory bud formation occur so con- 
stantly in the Texas armadillo in contrast to the single embryo 
formation of mammalian eges in general? Patterson failed to 
answer this question, but he supplied some very significant data 
which Newman,* has appreciated as being intimately connected 
with the occurrence of polyembryony. 

In connection with the collection of material Patterson® dis- 
covered a ‘‘period of quiescence’’ of the embryonic blastocyst. 
Regarding this he states: 

The fact was first made apparent in 1911, when, after I had started 
collecting two weeks earlier than in the preceding year, I failed to 

4H. H. Newman, ‘‘The Biology of Twins,’’ Univ. of Chicago Press, 1917. 


65 THE AMERICAN NATURALIST [Vou. LV 


obtain the cleavage stages, although judging from the condition of devel- 
opment in the vesicles collected in previous years, one would naturally 
expect to find these early stages during the period of my first collee- 
tion in 1911. 


The following year he began collecting still two weeks earlier 
and again had a similar experience. 


Practically all of these vesicles lie free within the uterine cavity, 
either in the horizontal groove or in the region of the attachment zone 
(placental area). 

It is evident from these data that the embryonie vesicle remains for 
some time lying free within the uterine cavity. Just how long this 
period lasts, I am unable to state; for practically every old female 
taken at the earliest date (October 15) at which I have collected, pos- 
sesses a free blastocyst. . . . Taking all the facts into consideration, I 
estimate the “period of quiescence” to last about three weeks; that is, 
from about the middle of October to the third or fourth of November. 


In a study of sections no mitotic divisions were found to occur 
in the blastocysts during the ‘‘ quiescent pericd.’’ 

The only point of interest cited by Patterson in connection 
with this peculiar phenomenon of interruption in development, 
was the fact that in no other mammal, except the deer, had 
such a condition been found. Bischoff had long ago, 1854, re- 
ported a ‘‘period of quiescence’’ lasting for some weeks during a 
so-called morula stage of the deer embryo. : 

Newman‘ has recognized the importance of Patterson’s dis- 
covery of a ‘‘quiescent period’’ during the early development of. 
the armadillo, and states in a discussion of twin formation that 
this ‘‘period of quiescence’’ probably, ‘‘holds the clue to the 
physiological explanation of polyembryony.’’ In this position 
Newman is, in my opinion, largely right, but this is as far as the 
data led him, and he finally remarks: 


The problem is to locate the factors responsible for the slowing down 
of the developmental rhythm. Whatever these factors may be, and 
we have no definite knowledge of them, the result of retardation is 
polyembryony. 

Newman thus fails to appreciate the second point in Patter- 
son’s discovery, and that is that the blastocysts always lie free 
in the uterus during the ‘‘period of quiescence.’’ This fact en- 
ables us to go one step further since the lack of attachment and, 
therefore, lack of oxygen supply are very probably ‘‘the factors 
responsible for the slowing down of the developmental rhythm.”’ 


No. 636] POLYEMBRYONY IN THE ARMADILLO 66 


The armadillo ege like that of most mammals undergoes its 
early development in the fallopian tube and is, therefore, cap- 
able of reaching the blastocyst stage on its initial oxygen supply. 
After this time, however, it must become attached to the uterine 
wall for a further source of oxygen. For some reason in the 
armadillo the reaction between the blastocyst and the uterine 
wall is postponed, and the blastocyst is incapable of further de- 
velopmental progress until this reaction is established and the 
necessary supply of oxygen becomes available. In exactly the 
same way the development of the blastoderm in the fish’s egg 
is experimentally retarded or stopped by reducing the available 
oxygen supply and is again made to resume its development by 
supplying oxygen. In the case of the fish egg, the supply of 
ordinary nutriment is certainly not involved, and reactions simi- 
lar to those of the armadillo egg are only obtained as responses to 
changes in temperature and rate of oxidation. 

In the armadillo egg I also do not believe the retardation is 
of the nature of a starvation phenomenon, since we see nothing 
of the kind in other forms. Temperature changes are ruled 
out, since the temperature of the uterus is more or less constant. 
The absence of oxygen necessary for the energetic process of 
cell division, is, therefore, in all probability the arresting cause, 
and the retardation results in polyembryony. 

Thus Patterson has found the developmental interruption to 
exist, and he has also shown the blastocyst to be disconnected 
from the uterine wall and its necessary oxygen supply during 
this time. However, he has furnished no data bearing on the 
reason for the delay in uterine reaction and the consequent fail- 
ure of immediate implantation of the blastocyst such as normally 
oecurs in other mammals. However, from what is known of the 
dependence of uterine reactions on conditions in the ovary (Leo 
Loeb,*> Stockard and Papanicolaou® and others) it may very 
probably be that some peculiarity in corpora Iutea formation 
is primarily responsible for the entire series of reactions leading 
to polyembryony in the armadillo. 

The consideration of the armadillo ege up to this point has 
taken account only of the external factors influencing its mode 
of development. It must now be remembered as a fact of serious 

5 Leo Loeb, Jour, Morph., Vol. 22, 1911, . 


6C. R. Stockard and G. N. Papanicolaou, Am. Jour. of Anat., Vol. 22, 
1917. 


= 


61 THE AMERICAN NATURALIST [ Vou. LV 


importance that the production of quadruplets from the single 
ega of the Texas armadillo is an almost constant occurrence, 
while the experimental attempts to produce twins and double 
individuals in fish eggs and other forms have given at best only 
small percentages of such individuals among the large groups 
of eggs treated. It is also a fact that all eggs do not furnish 
equally favorable material for artificial twin production. The 
egos of the trout seem unquestionably more disposed to give rise 
to twin formations than do the eggs of Fundulus. Thus some 
eggs would seem to have a hereditary or truly innate pre- 
- disposition towards polyembryonie formations. There is much 
reason to believe that aside from the external factors discussed, 
the armadillo ege itself is highly disposed toward the formation 
of accessory embryonic buds. 

There is the possibility, of course, that this natural experiment 
with the armadillo egg has become so exactly regulated as to 
influence the developmental processes precisely the same way 
each time, yet this is highly improbable. The armadillo egg is 
not a case of simple twin growths from the blastoderm, but as 
Patterson finds, there are primarily two buds, and then very 
promptly two secondary ones arise making the four and after 
this the budding process ceases. In the South American species, 
however, it would appear as though a tertiary budding occurred 
giving the usual eight embryos; and in rare eases still another 
budding occurs from a few of the existing buds giving a total of 
as many as twelve. It would certainly seem as though the 
blastoderm in these species passes through a stage of agametic 
reproduction or budding of a nature unknown among other 
higher vertebrates. But the possibility for such expression might 
only exist on account of the delay in implantation of the blasto- 
cyst and consequent shortage of the oxygen supply necessary for 
the rapid formation and growth of the single embryo. 

It is important to keep in mind that there are species of the 
armadillo which produce only a single offspring from one egg. 
It is not known whether their embryos have a ‘‘period of quies- 
cenece’’ but if they have, the period either occurs at a different 
developmental stage or the eggs do not possess the inherent 
budding tendency of the other species. 

We have further to acknowledge the fact that although the egg 
of the deer has a ‘‘period of quiescence’’ during its development 
it does not give rise with any degree of frequency to twin indi- 


No. 636] POLYEMBRYONY IN THE ARMADILLO 68 


viduals. In the first place it is entirely uncertain from the 
scanty accounts as to what time in development the quiescent 
period occurs. Assuming that such a period does exist, it might 
occur at some indifferent stage when no peculiar result would be 
expected, for example after gastrulation, as it does in the bird 
with no subsequent effect. In the light of the experimental 
production of double individuals it is readily understood that 
even though the egg of the deer is interrupted in its development 
at an early stage, it might still be capable, on resuming develop- 
ment, of giving a normal single embryo. A study of the experi- 
mental production of twin and double individuals among fish 
leads one to be surprised at the case of the armadillo, and to 
expect the reaction found in the deer. The constant interrup- 
tion occurring in the development of the birds and other animals 
at indifferent developmental moments with no subsequent ill 
effects, renders commonplace the fact that the deer successfully 
withstands an interruption during its development without 
noticeable modifications in structural response. A full considera- 
tion of the different results following interruptions at critical 
and indifferent developmental moments will be published in a 
forthcoming number of the American Journal of Anatomy. 

In conclusion we may summarize the eases as follows: The 
development of the armadillo is interrupted on account of a 
failure to become promptly implanted on the uterus and a con- 
sequent exhaustion of the available oxygen supply. The inter- 
ruption occurs at a critical period just preceding the primitive 
streak and embryonic line formation. This ege appears to have 
a decided tendency under conditions of arrest to form accessory 
embryonic buds. As a result of the interaction of these external 
and internal forces polyembryony is produced. 

In the case of the deer only one probable fact is known, and 
that is that a ‘‘period of quiescence’’ occurs. It is uncertain at 
what stage the arrest takes place but it is probably due as in the 
armadillo to a delayed implantation of the blastocyst. Either 
on account of the stage of arrest, or a lack of tendency to form 
aecessory embryo-buds a typically single individual arises from 
this egg. The external factors may be the same as in the case 
of the armadillo, but they interact with different internal factors 
or different developmental moments to give a very different 
result. 


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Reprinted from the Proceedings of the Society for Experimental Biology and Medicine, 
1920, xvii, pp. 143-144. 


77 (1537) 


Effect of underfeeding on ovulation and the cestrous rhythm in 
guinea-pigs. 


By GrorGE N. PAPANICOLAOU and CHARLES R. STOCKARD. 


[From Cornell University Medical College, New York City.] 


Under well-regulated food conditions the cestrous cycle in the 
guinea-pig is almost uniformly 16 to 17 days in duration. 

Underfeeding with a diet of 20 grams of carrots per day pro- 
duces a prolongation of the dicestrum and, at the same time, a 
congestion in the ovary and uterus and a degeneration of develop- 
ing graafian follicles. 

The extent of prolongation of the dicestrum depends upon the 
stage at which an animal is underfed. 

Underfeeding during the first 5 to 7 days of the dicestrum has 
only a slight effect, postponing the next cestrus for one or two 
days, while underfeeding during the later part of the dicestrum 
gives much more marked results. 

When an animal is underfed for 5 days, from the 12th to the 
17th day after an ovulation and cestrus, the next ovulation and 
cestrus is delayed for about 7 days, being expressed at the 23d to 
25th day instead of at the 17th. 

Should an animal be underfed for 7 days, from the roth day to 
the 17th day after cestrus, the next ovulation and cestrus is post- 
poned for to to 11 days, arriving at the 27th to 28th day, instead 
of the 17th day. 

This variation in the effect of the underfeeding when applied 
at different periods of the dicestrum is associated with the fact that 
the conditions of the ovary differ at the different times. 

Shortly after an ovulation the ovary contains almost entirely 
small primary follicles. These follicles are not so unfavorably 
affected by food conditions as are the large graafian follicles, 
which begin their growth and development during later stages of 
the dicestrum. 


tN 


SCIENTIFIC PROCEEDINGS (107). 


A large follicle at the height of its development seems to require 
much better nutrition than a small primary follicle, and the lack 
of proper food arrests its progress very readily. Thus a late 
underfeeding has a more injurious effect than an early one, and 
the postponement of the next oestrus is correlated with a post- 
ponement of the development of new ripe follicles in the ovary. 
The entire cestrus activity depends chiefly upon the conditions 
prevailing in the ovary. 

The fact that following a late and long underfeeding the next 
ovulation is delayed about 11 days after the underfeeding has 
been stopped is in accord with the results of operation experi- 
ments which Papanicolaou has performed on the corpora lutea in 
guinea-pigs. 

These experiments show that after removal of all young corpora 
lutea following an ovulation, the next ovulation arrives in about 
II days instead of 16 to 17 days as would be expected. This 
acceleration of 5 to 6 days is due to the absence of the corpora 
lutea, which if present evidently inhibit the maturation, or prolong 
the time necessary for the development, of ripe follicles in the 
Ovary. 

These experiments all demonstrate the sensitiveness of the 
follicles within the ovary to environmental conditions and when 
considered in more detail than is here possible, they throw light 
on many peculiar reproductive phenomena observed in nature. 
The extreme variations in the oestrous cycles recently recorded for 
the rat by Long and Evans (Proc. Am. Ass’n of Anatomists, 
Anatomical Record, April 1920) may be in part, at least, due to 
the variations in the diet taken by the individuals. When rats 
are fed a mixed diet no doubt certain individuals receive a ration 
quite different from that eaten by certain other members of the 
colony. 


Reprinted from the Proceedings of the Society for Experimental Biology and Medicine, 
IgIQ, xvil, pp. 41-43. 


23 (1483) 
Some studies on the surface layer in the living egg cell. 
By ROBERT CHAMBERS. 


[From Cornell University Medical College.] 


‘ 


The results recorded here were obtained through the use of 
Barber’s mechanical pipette holder somewhat modified for micro- 
dissection purposes. 

The cells experimented upon were the egg cells of the starfish 
and of the sea urchin. The eggs, which are somewhat over 1/10 
of a millimeter in diameter, were placed in a drop of sea water 
hanging from the roof of a moist chamber. The microscopically 
fine tips of the glass dissecting needles projected into the moist 
chamber and up into the hanging drop. By manipulation of the 
screws of the mechanical pipette holder the cells in the hanging 
drop could be dissected with considerable accuracy and an esti- 
mate ascertained of their physical consistency. Detailed accounts 
of Barber’s apparatus and its application to microdissection have 
already been published.! 

The egg cells studied consist of a decidedly fluid interior sur- 
rounded by a more solid surface layer of appreciable thickness. 
This surface layer is most solid on its external surface. Internally 
its consistency seems to merge insensibly into that of the fluid 
interior. The inner surface of this layer adheres to the touch. 
This is demonstrated by introducing a microdissection needle into 
an egg and pushing the needle through until its tip comes into 
contact with the inner boundary of the surface layer on the side 
of the egg opposite the puncture. On withdrawing the needle the 
layer adheres to the needle tip and strands are drawn into the 
interior of the egg. 

If the surface layer be torn while the egg is kept under compres- 


1 Barber, Philippine Journal of Sc., Vol. X, Sec. B, Tropical Medicine, 1914; 
Chambers, Biol. Bull., Vol. 34, 1918. 


2 SCIENTIFIC PROCEEDINGS (102). 


sion the fluid interior will bulge out through the tear. The 
cytoplasm, on coming into contact with the surrounding water, 
tends to establish a definite surface film which prevents the cyto- 
plasm from mixing with the water. If the internal pressure be not 
too great this film persists and, in time, strengthens into a definite 
ectoplasmic layer. The bulge then slowly retracts until the orig- 
inal contour of the egg is reéstablished. If the neck of the pro- 
truding mass of cytoplasm be small it may pinch off a spherule of 
cytoplasm which to all appearances is normal. If the internal 
pressure be too great a succession of films may form as, one after 
the other, they succumb while the escaping cytoplasm disperses 
and disintegrates in the surrounding water and the film which 
finally holds out may enclose only a fraction of the original cell 
but what it encloses will be normal protoplasm.! 

Churning of the contents of a mature unfertilized sea-urchin 
egg causes the ectoplasmic layer to revert to the fluid condition of 
the interior. The surface film of such an egg is very thin and very 
easily tears upon which the entire egg disintegrates. On standing, 
however, the surface film steadily strengthens until the normal 
condition is reéstablished. 

That the distribution of substances throughout the egg cell is 
not uniform can be demonstrated by the following experiment on 
the starfish egg: If the surface of a mature unfertilized egg be 
torn while the egg is kept under compression almost all of the 
internal cytoplasm may be made to flow out to form a spherule of 
cytoplasm which pinches off from the rest of the egg. What is left 
behind is a collapsed remnant consisting mainly of protoplasm 
which originally enveloped the egg. This remnant consisting 
largely of the more solid ectoplasm tends only slowly to round up. 
The extruded mass, which is very fluid, immediately assumes a 
shape approximating that of a sphere. This may be termed an 
endoplasmic sphere. The remnant containing the original ecto- 
plasmic substance of the egg is readily fertilizable and undergoes 
segmentation. The endoplasmic sphere is unfertilizable. If, on 
the other hand, the endoplasmic sphere remains for some time 
connected by means of a bridge of protoplasm with the remnant 
containing the original ectoplasmic substance it is fertilizable. 


1 Chambers, Amer. Journ. Physiol., Vol. 43, 1917- 


SURFACE LAYER IN THE Livinc Ecc CELL. 3 


The ability of the endoplasmic sphere to approximate normal 
conditions of segmentation is a function of the length of time that 
it remains in organic continuity with the original ectoplasmic 
mass. Possibly there exists a substance necessary for develop- 
ment which normally accumulates in the surface layer of an egg. 
This substance is diffusible and will distribute itself over new 
protoplasmic surfaces. If a bridge of protoplasm connects the 
ectoplasmic remnant with the endoplasmic sphere this substance 
will diffuse into the sphere thereby rendering it fertilizable. 

The nature of the surface film produced by cutting an egg cell 
differs in an unfertilized egg from one which has been fertilized. 
Before fertilization the needle may be pushed vertically into the 
side of the egg and moved through the egg from one side to the 
other without cutting the egg in two. The cytoplasm closes 
behind the needle thus obliterating the furrow. Shortly after 
fertilization, however, such a procedure cuts the egg cleanly in 
two. The sides of the furrow produced by the needle do not fuse 
although contiguous. The character of the surface film which 
forms over a cut is thus changed upon fertilization. This change 
prepares the egg for the ensuing segmentation process by causing 
the formation of a type of surface film which prevents contiguous 
blastomeres from fusing with one another. 


Reprinted from the Proceedings of the Society for Experimental Biology and Medi- 
cine, 1920, xviii, pp. 66-68. 


30 (1612) 
Dissection and injection studies on the Amceba. 
By ROBERT CHAMBERS. 


[From the Department of Anatomy, Cornell University Medical 
College, New York City.] 


The species used was Ameba proteus. By means of a micro- 
pipette liquids of various kinds were injected and the effect noted. 

Oils form spherical droplets which are carried about in the 
cytoplasmic currents. A large drop is usually expelled. Imme- 
diately on being extruded the drop tends to flow over the surface 
of the Ameba thus partially engulfing it. 

Distilled or spring water diffuses through the granular endosare 
diluting it. The dilution is followed by a contraction of the endo- 
sarc and the massing of a hyaline fluid between the endosarc 
and the external pellicle of the Amewba. This dilates the area 
usually termed the ectosarc. The fluid soon accumulates on one 
side of the Ameba in the form of a blister which is ultimately 
pinched off. 

A number of acid indicators were injected. The color reactions 
showed that the protoplasm of the Ameba is more acid than its 
environment. Upon death the colors change to those character- 
istic of the surrounding medium. 

The difference in behavior of living protoplasm to ‘‘basic”’ 
and to ‘‘acid” dyes is striking. The “‘basic’”’ dyes used were all 
chlorides of colored basic radicles and the ‘‘acid’’ dyes, potassium 
or sodium salts of colored acid radicles. In every case the ‘‘basic’”’ 
dyes had a coagulating and the “‘acid”’ dyes, a liquefying effect on 
the protoplasm. 

In the case of the “‘acid’’ dyes, when the effect is local, the 
healthy non-colored portion of the endosare shrinks away from 
the colored liquefied area. This liquid accumulates under the 
pellicle in the form of a blister and is ultimately pinched off. 


2 SCIENTIFIC PROCEEDINGS (TIO). 


If the ‘“‘basic”’ dye be relatively nontoxic its injection results 
in a coagulated area which is localized as a colored lump of inert 
material. This lump is carried about in the protoplasmic currents. 
The color gradually diffuses out of the lump and stains many of 
the cytoplasmic inclusions in the Ameba. 

Dissection indicates that the granular endosarc is capable of 
easily reverting from a fluid to a solid state and vice versa. 
Peripheral to the endosarc is a hyaline liquid zone, the ectosarc, 
which is bounded externally by a very thin, extensible, pellicle. 
The extosare can be enlarged by a hyaline liquid extruded from 
the endosare. 

In the formation of a pseudopod a localized area of the pellicle 
softens. The accumulation of liquid in the ectosarc immediately 
under this area produces a bulge. The more jellied endosare at 
the base of the bulge liquefies and a liquid suspension of granules 
streams into the bulge and up to its tip where it spreads out and 
flows back peripherally in the manner of a fountain flow. The 
granules heap up around the base of the bulge where, by means of 
a jellying process, a semisolid wall is built about a central liquid 
channel. Retraction of a pseudopod is accompanied by a reversal 
of the jellied to a liquid state. 

An undisturbed Ameba usually forms numerous pseudopodia. 
Upon continued agitation a broadly lobate pseudopod is formed. 
The jellying process of the backward flowing endosarc is di- 
minished. The base of the pseudopod, consequently, broadens 
more and more until all of the endosarc reverts to a liquid state 
and the entire body of the Ameba becomes transformed into what 
one may term a single pseudopodium within which vortical 
currents occur analogous to those of a chloroform drop creeping 
along a bed of shellac under water. 

The motile activities of an Ameba depend upon a delicate 
balance between the liquefying and solidifying tendencies of its 
protoplasm. The most recently solidified regions are the ones 
that most readily liquefy. In this way a gradient exists with a 
definite antero-posterior axis. The posterior end consists of a 
heaped up mass of jellied material which is more resistant than 
other parts to the liquefying process necessary for the formation 
of pseudopodia. In an actively moving Ameba the amount of 


ee 


DISSECTION AND INJECTION STUDIES ON AMG:BA, 3 


such material is very small and pseudopodia may form on either 
side thus tending to mask its presence. Exceptionally the pos- 
terior end may be made to liquefy but usually the inert pos- 
terior end compels an Ameba, in order to retrace its path, to turn 
about. 


Reprinted from the Proceedings of the Society for Experimental Biology and Med. 
cine, 1920, xvii, pp. 183-187, 


98 (1558) 


Disturbances in the development of mammalian embryos caused 
by radium emanation. 


By J. F. GuDERNATSCH and H. J. Bace (by invitation). 


[From the Department of Anatomy and the Memorial Hospital, Cor- 
nell University Medical College, New York City.] 


As has been shown by various observers, the exposure of living 
tissues to the influence of radium rays leads to a severe injury and 
ultimate destruction of these tissues. In our work an attempt 
was made to study this destructive influence on mammalian 
embryos in utero, in the hope that a partial or complete destruc- 
tion of one or more tissues might lead to definite abnormalities 
or malformations in these fetuses. 

Bagg had lately used a method of applying radium, which 
was described in the Journal of Cancer Research, Vol. V, 1920. 
Radium emanation, carried in a very small amount of saline solu- 
tion, was injected in measured quantities into adult rats, either 
subcutaneously or intravenously. This solution contained all 
the properties of the radium metal itself, and, no doubt, the re- 
sulting physiological changes were due mainly to the activity of 
a-rays. Such an injection produced peculiar destructive changes 
in the inner organs of the animals. 

The same method was used in our experiments. After long 
experimentation we found that a dose of 5 mc. (= milli-curies, a 
standard unit in radium experimentation) was about the optimal 
dose. Such an amount was injected into female rats, pregnant 
and non-pregnant, with the purpose of either injuring the ovarian 
or uterine tissues, or, in case of pregnancy, the embryonic tissues. 
While the results were not those which we expected, viz., the 
production of various types of monstrosities, yet a definite in- 
fluence of radium on the fetal and placental tissues was noticeable. 


2 SCIENTIFIC PROCEEDINGS (108). 


Radium-treated rats were killed at different periods of pregnancy, 
so as to procure a series of fetuses of various ages. 

The most destructive results of radium emanation, injected 
subcutaneously, were seen in a number of pregnant females, in 
which the embryos were killed in the uterus and, instead of being 
aborted, remained attached to the uterine wall and were gradually 
absorbed (group I). Whether the embryos were killed primarily, 
or their death was due to the destructive influence of the radium 
on the maternal, placental tissues, cannot, of course, be deter- 
mined. Probably the first assumption is correct, since other 
findings (group II) showed, that the toxic agent does pass the pla- 
centa and affects the embryos directly. 

A number of such partially absorbed embryos were found, the 
age of which, naturally, could not be determined. Judging from 
the sizes of their respective placentz, however, development must 
have proceeded to some extent before the radium was applied. 
The remnants of the embryos were small, nodular bodies attached 
to the placentz (figures were shown) and had lost all resemblance 
to properly developed fetuses. 

In one case a small, ovoid shaped sac was found, attached by 
a thin stalk to the uterine wall (figure shown). This apparently 
represented the remnants of a former embryo and placenta, 
although neither one could be recognized any longer. In the 
sac extravasated blood and cell detritus were found. A great 
many large cells of an epithelioid nature probably belonged to 
the former embryonic syncytium. The wall of this cyst was 
formed by fibrous connective tissue.» 

In a number of other cases (group II), the fetuses were not 
killed by the radium emanation, but peculiar macroscopic lesions 
appeared in their skin vessels. 

When the fetuses were removed from the uteri, peculiar hemorr- 
hagic areas were noticeable, in some cases just along the dorsal 
midline, in other cases, spreading over the entire body with the 
exception of the ventral surface. These extravasations took place 
in the vessels of the subcutaneous connective tissue and along 
the meningeal sinuses. In all cases, one or more hemorrhage. 
appeared in the midline, mainly in the head and thoracic regions 
It seems that the vessels in this dorsal median zone are especially 


——" 


MAMMALIAN Embryos. 3 


liable to injury. In one instance, there was a large area of hemor- 
rhage extending over the thoracic and lumbar region. Its outline 
was just symmetrical to the dorsal midline (figure shown). In 
other cases, a great number of such hemorrhagic areas, some 
extremely small, were found over the lateral aspects of the head 
and body. Probably these affected fetuses would have died, if 
left longer in the uterus, and would have undergone absorption. 
In many animals which we killed in the early parts of the experi- 
ments we failed to find any fetuses, although we definitely believed 
that these animals had been pregnant before. We probably waited 
too long after treatment, so that the embryos were completely 
absorbed, when the animals were opened. 

Not all of the fetuses of one litter are affected in the same degree. 
In one case, for instance, we found among 7 fetuses 3 showing 
hemorrhagic lesions, 2 beginning to macerate and 2 in the process 
of absorption. This difference in resistence may be due either 
to the higher or lower vitality of the embryos themselves or to 
the amount of radium which passes the placenta. In another 
case the fetuses, although injured, were carried to full term and 
among 6 young of one litter we found two normal and four showing 
hemorrhagic spots on head, face and along the dorsal midline. 

In one very remarkable instance the female had been treated 
22 days previous to conception and yet the fetuses, approximately 
16 days old, showed areas of extravasation (one of considerable 
size shown in figure). These lesions were much more widely 
distributed than in previous cases, extending over both lateral 
and dorsal surfaces (figure shown). These results cannot be ex- 
plained at present. It would seem as if the treatment of the 
mother previous to conception had lessened the faculty of the 
later embryos to form proper endothelial walls. The wide distri- 
bution of the lesions would seem to substantiate such a view. 
This is in accordance with findings in adult animals treated with 
radium in which the extravasations in the organs are due not only 
to increased blood pressure, as would seem at first, but to the 
actual breaking down of the endothelial tubes. In other words, 
the effect of radium on endothelium might be selective. 

When the radium was injected intravenously (group III) 
instead of subcutaneously, the same lesions resulted along the 


4 SCIENTIFIC PROCEEDINGS (108). 


vascular channels. Females of about 19 days pregnancy were 
injected intravenously and the young, born dead 24 hours later, 
showed the hemorrhagic lesions along the dorsal midline (figures 
shown). In one case we found a striking difference in the size of 
the placente of different fetuses. One fetus, for instance, had 
a markedly enlarged placenta completely filled with blood, so 
that it had the appearance of a large hemorrhagic sac. This 
fetus did not show any hemorrhagic lesions, while their pla- 
centze were of normal size and moderately filled with blood. 
It would seem as if in the first case the placenta functioned as an 
effective ‘‘shock-absorber,” while in the other cases the radium 
emanation passed through the placente to the fetuses. 

Lately Bagg exposed pregnant females, near full term, directly 
to the action of y-rays (group IV). This radiation of the fetuses 
in utero, through the abdominal walls produced hemorrhagic 
lesions of the same nature as described above. However, the 
lesions did not appear until about 10 days after exposure. The 
young were born 2 days after treatment and appeared normal. 
After about a week they began to fail considerably, hemorrhagic 
areas appeared along the mid-dorsal line, especially in the head 
region and death followed. The hemorrhages in these animals 
were mainly along the meningeal sinuses (figures shown), in some 
cases frontal and occipital hemorrhages were just beginning, in 
others they extended considerably over the cerebral hemi- 
spheres. Additional lesions on the dorsal side of the thorax were 
found. 

The interval of 10 days after treatment strictly corresponds 
to the time at which a primary skin erythema develops in radium 
treated patients. Again it seems as if the endothelial walls had 
been injured at the time of exposure and gradually gave way to 
the blood pressure. 

In the course of the experiments, we also found numerous 
hemorrhagic areas in the uteri and especially in the ovaries 
(figures shown). Congestion of the uterine vessels always was 
pronounced. 

While in experiments on adult animals reported by Bagg 
before, the injection of radium emanation led to considerable 
injuries in the internal organs, in our experiments the weaker 


MAMMALIAN EMBRYOS 5 


doses did not produce any macroscopically visible effects on the 
maternal tissues. However, the embryonic differentiating tissues 
were easily affected. This fact might be of some biological signi- 
ficance, when one remembers that radium rays have a decided 
effect on fast growing tumor and cancer tissues. 


A Review of the Chromosome 
Numbers in the Metazoa 
Part U 


ETHEL BROWNE HARVEY 


Reprinted from JourNAL or Morpuo.ocy, Vol. 34, 
No. 1, June, 1920 


ETHEL BROWNE HARVEY 


Dee ee ee 


£14 ‘1g “1907 “axq“f | 
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(S16I-S161) LNANATddAS “I LUVd 
SUAANWOAN ANOSONOUHO FO NOILVTNAVL ‘IL 


CHROMOSOME NUMBERS IN METAZOA 


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penuizuoy—VdOdOUH Luv 


‘Il 


CHROMOSOME NUMBERS IN METAZOA 


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ETHEL BROWNE HARVEY 


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Z ‘duo “sn TIN OT, ‘Yorue 48] urejod 07 x P21 ‘IT P2I ‘IT LSI ads ¢z “-snUsBUT X19}0,0UAIY J 
2] aBpigiz407, X1}}0} 818g 
s | dapun 9a8 | xX19jeJOWION 
2 erg d ‘62 ‘“qdioy “mor LI, ‘SuniOow LZ1 ds 97 sereessessss 708 BLIQUIIO TA 
g payoor 
= -109 ¥UNO008 JAI 
2 | -18 5} “48ST Ur ajo 
= | 0} ‘ulo1yo Jey4O (FZ=)U0Ss 677 
3) 61g “4 ‘6z “dso “anoy | LI, ‘dunpoow | -uB of payouyye KX} (2r ‘It=) SUT] (21 ‘TT=) SIT (G1=) OTT (g¢=) 3ds zz | °° 8} B4ALATG BeUIEyy 
|euue au0 
| ul quaseid Aresour 
-nusedng ‘sired 
ut pasny aq ABur 
su014o 19430 
48] ul aod 0% (aI (8 [+21 10 
61S “4 ‘6z “Ydsoy “Ino LT, ‘AUNTHoW | ‘eer1} 10 payourys Xx ‘TI=) L2I-01 | t=) SZI-6 | (es=) Bdsec-6T | ** “Stprata x199070I0d8a 77 
sbLicnerod snsoroads x1340}010d60: 
0 ‘wosyo x0y90 | (er ‘Tt=) Ott | @r‘tt=) © (r=) Or | (ee=) Bds ede eth ea 
61g 'd ‘az yduoyy anor LT, ‘Banpooyq | -we 03 peqownie x t ic a=) ) “is {steudyesa x19}0} 010080 FH] 


EE EO 


wos 60g 
00 0g 
? UI08 67 
OT6T ‘OdQrT 91, ‘IGOW 48] ur ejod 07 x OST ‘FT POT ‘FT Lat ads 67 To" * * BUIISSIPTITA B38N100'T 
THe UW 
Joriq ur pazenqel pesny oq Avur ‘ 
gor 4 ‘gz “TING “org GI, ‘AesjooM | suro1go jo sited Z ads og ‘gg “****JO[OOLUN BUBOTBUIBS 
posny 
e297 4 ‘gz “TINE “Lore ST, ‘Aesjooy | eq Avur suroryo 7% &8T ‘LT Bdsce‘pe | "8ye49NAQGns BuvoreurEs 
sor d ‘92 “TING 108d | ST, ‘Aosqooy, 48] ul efod 04 x O8T ‘LT PST “LT 8 ads og “BARR BUBOTUIUL 
(6g ‘d ‘] q4Bq) DBPI}snoo7T ‘a 
al 287d 'g¢ “ydioyy “mor | OT, ‘oudBg Pi8 ads cy LE SOPOT C(I 
g MOI; SUUBS[NA BdlByoT[AIH 
> | 
5 ised ‘gz “ydsoy “anor | 91, ouAeg AX 9 ads Z] cosenseees SINGol Ty 
| | WOdy SIRNA BATBIO[[AIH, 
| | syuemeya Z=x BOO $7 
= 182d ‘gz “ydsoyw “unor 9, ‘euABy | “48ST ul sojod 03 XX PZI ‘IT PZ ‘Tl LUI 3ds $7 “s+ -siwal0q Bdyeqzo]] Arn) 
5 (g¢ °d ‘] qaBq) OBpIAIH “P 
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5 WOS 6 $% { “BSN YyNs psidoazosourts 
= cpp ‘gz “ydsoqy “anor LI, ‘saeyqoing 48] ul ojod 0} x¥ PZ ‘TL PZ gids ¢z -xulpey ystdosjor9unts 7, 
& B00 FI 
eal 611 0 ‘27 “yao “anoge OT, U0szI0 077 PL dds gy *-oyeuued 
BraUoarACel vapiaiy4ay, 
4ay40 24} JO x SB 
ajod aures ‘487 ur 
ajod oy, “sj[ao 
pus wos ur yeu 
o1z dd ‘Or -uB | Ul quesaid HOO FI 
[Ng 98 “Aluy) susuBy LI, ‘UOszs9qoYy Axyeiouinuseadng ULOS OST 
6L1 ‘22 “qdaoyy “snoe OT, ‘UOKqIOqOY  4s~ ur ajod 04 x OLD OL ads gy] stuuediAred vapiaiqyay, 
ponulju00—(eBpIploy 
Jo ATTUIBI Ng) eBpIgyIaT, 
OMAN 
oO FONSUTAAY ua AUNaSAO SuuVNGH at- GLKO- ANZ G0GA0- 18] |-ONGHIUVa ANY sa10aas 
a a10 Tad 


penayu0O—VGOdOUHLUyY ‘Il 


b coe | 
re 


NUMBERS IN METAZOA 


CHROMOSOME 


L¥¥°d ‘gf get ‘Uopuo'y 
00g *AOY ‘suBry, [Yq 90. ‘aqnyo | 300 4g | +eeeeeeeompytq Uopayy 


VAGIONIUD “9 


poe “d ‘¢ “y[[9Z “Youy OI, ‘wepsor W0s gf BO |" “BJ UE;OOUINFues Bpaaqua,y 
jo ed 6 
19 81 . 
608 “4 ‘er “TIME “Ole « 20, ‘FueuUET, | Ms8equoy “-y aq AVY 6 6 6 3d8 8] i SHBA[NA SBLIO}SV 
L1¢°d '9 “y19Z “ory IT, ‘seugong = 9g =93a-+'q “d puz ost ost ost Jo ed gg “ “SIBIOB]T SBLIOISV 
(uor7eyndor 

egz ‘d ‘6 [OA ‘J TT ‘408 -09N®B) [oO Vd gT 
“uaz yo “dxa "[007 ‘yory 10, ‘ed8[aq 1 81 teres ess RTTBIOB[T SBLIOISY 
oe d ‘{1 “ydsoyy “snore 66, ‘PIP i uoid 96-8 Pisisieins “"“SUBIOBIZ SBLIOISY 

68 “4 ‘ZOr 
“qng “4suy elsoureD | 80, ‘UBpso¢ 
6f “d ‘Te “2uy “yeay 20, “UBpsrof ost der ost (hy ile | aida Wseqi0} ser1098y, 
[9 ud gy] “Bo 
60g “4 ‘er “IN [org | 10 ,{QuUeuuey,  sireZ[NAa ‘y oq AB 12 9¢ TISaqJO} SBIIOISY 
| 90, ‘onzo0xy 
Lg “d ‘g “1907 “dxq “¢ pus yueuey, ost ost TOOT i reek HeoqlopeBIIO2 By) 
96, ‘so 
61g 4 ‘OT “qdsoyy “inor | -4yB PUB UOSTTM S2LI CPHAt BLT ||) i ecieisgea lisaqi0} SBLIOISy 
Vadloudisy “Dd 
VLYWHYAGONIHO’" ‘AI 
(8161-8281) II LUVd 
e6e d'ga“ydioy “anog | 11, VIRIEH VL “D | | a RR Ses eo Be sen arp wqyUTpay 
(g9 “d ‘T y1Uq) evunlyooiy -¢ 

gg ‘42 “ydioyy “amor | OT, ‘HIB LO | Sar | Crd | dt | ie vee eees py Ags0} dey BABID 


(79 ‘d ‘J q18q) oBsnpeuoqyuy “e 
avuyojdeTy *] 
yozouaxy ‘p 


VLIVUALNATAOO “IIT 


ETHEL BROWNE HARVEY 


12 


Ce es oo wie 


06 
“d ‘g ‘SinqzinAy ‘sar S81 
peu Aq q “iaqssunzz1g SI, ‘1929, 8q SyusMaBIy 
L6F dd *g “SEZ “qory OT, ‘19z3]8q | ET, Ut WO.arI00 ade poyeeyo [9 Bd gy 
BFS 4G “ITZ “ory 60, ‘19z7;8q | WOT “O Ur AX | -nue ur pst 198 [Pea SUS[BAIG ‘IBA BNF 
~B[NoJaqnyzOIOIUL SNUIqOR 
syueusBIy 
ar 379 pozBeo 
‘d ‘oy “yorngug “yory 20, '819A9}9 -nuo ul Pst 681 ost 19 98 vo 8s" * SUe]BATG “IBA 813 
-B[NOLaqnyOsOLUA SNUITOoT 
1% 
“d ‘Zz ‘ransquaday "480,75 96, ‘SIMqIOFT “YY 
Th a ‘Ty 
‘ueyounyy ‘“jorsdAqq 
“n “ydioyy lenoa mus $6, ‘BIMWOH “Y | 
66d *f 
‘ueqounyy ‘yorsAqq sutuqoAI48 [9 Bd gT-9] 
“n “ydioyy “jased “21g 88, ‘JIMqIaH YW | YA pazvesz sBF0 vq S8I-91 yo-F0e eee sua[BAI “IBA 8NQ 


~B[NoJeqnzoOsoTU SNUTYOR, 
(111 warIpnyg-ueT[az) 

Fg “d “LT “spez “vor 06, ‘taaog Sasuo Moy UU] 3sr dst Peres caine (Gp anid 
-Og) SUd|BAIG “IBA sny 
-B[No1aqnN 4O.1OLUL snuryony 


1d ‘qe ‘uopuoT 


‘00g “AOY “suBLy “1d £1. 190K 1° 8 RAs snquefnose snuryooy 
€L, ‘Avip 
esh ‘d ‘gg "Ss Wie ‘O | pus —_ sey svou0g 
Sip 'd ‘OT TT, ‘ABIn 
“208 [Yq “Que oo1g | puw = LojSvou0g 1 88 Pease snquelnose snUulyouy 
L104 ‘96 “SW LO £0, ‘eoArg ; O91 69T 391 1 ze oes Eng UelMos SNUTYOR 
eI, ‘Atin | 
esp da ‘ges W “f° | pus dJoysBouod 
SIF “od ‘OT TT, ‘ABig 
“208 [Yd “QUO ‘oo1g | puv = JojSBOUOG | TREES 9 eesoue “* sngnowe snuryouy 
L6F 4 ‘g “S197 “Yyouy * OL, ‘4923;8q 1° 0F oe selina esoinysnd Bioequy 
16g 4 ‘zr “[o07 “dxq -¢ ZI, ‘uspsor 19 OF “Bo sees paeingound Bioeqiy 
YAdIONIHOW °9 
OMLaNaAD 
aONaugAgay uqAuasao suuvNay aii- ALXO- ANZ GLXAO- LST -ONTHLUVd ONY sa10gds 
aioTdia 


penayuojO—VLVYNUAGONIHOU “AI 


13 


NUMBERS IN METAZOA 


CHROMOSOME 


L6F 4“ “319% “Yory | 


1G} 
d ‘Zz ‘inequasay “4sa,7 


OL, “1929 Ey 


96, ‘StIMqI0H “UY 


Td ‘oT 
‘aedunqonsiezul) “[Olg OL, ‘8N1z,9a77 


89rd ‘12 TIM “lore IT, ‘Aouurg 


11d ‘ee “ydsoyy “anos | 
| 


ZI, ‘yueuuey, 


sora ‘Te “TIN “TOE | TL, ‘Aeuurg 
68d ‘zor | 
“qng “ysuy ardausBy | 80, ‘UBpi0r 
ogz ‘6 ‘TIT 
“ua 4a “dxa [007 “qo1y 10, ‘ea8[aq 
i] 
| 81, ‘ABID 
esp d ‘gg''s W “£°O | pus —_ra}svou0g 
STF ‘OT TT, ‘Abin 
“009 [Yd quiep‘s01g | puBw = taysBouog 
892 
‘d ‘% “yormjug “qory og, ‘UBdIOW 
| 
649 | 


“d ‘9g “yorgag “yory | 90, ‘PISMe_poH 
6F “d ‘G0, ‘ataoo 
-B1Q 9p saousINg sap 


*pBow,| 9p “UsazUI “ING 60, ‘BYS[ayBIy 


leh 
“d ‘cy “yorajuq "qory 


(A werpnys-ueT197) 
ohh 4 ‘Ze “ey107 “uer 
(IIT werpnys-ueT]97Z) 
1g ‘d “21 “sz1ez “uor 


Z0, ‘8U9A949 


$0, 'HeAog 


06, ‘eaog 


£ a AX 10X 


602-91 


queulsesy 
59 payeajon 
-w8 Ul PZT-01 


SpyUSUIIBIY 
330 pazeao 
-nue Ul PZT-6 


8} ueUur 

-301j 340 

pe}sejonue 
ur 6 66 


66 


681-91 


56 


(Aqq3 
-qo1d) 19 OF 


[0 ed gy 
1 9F 

19 #£-28 
12 428 


[9 81 


19 F£-08 


DBZ Ley 

SyUoUIdBIy 

339 poy 
-ajonue ur 
‘PII-8 “1 2 


[19 81 


19+02 


1? 81 


19 81 


StuBpnuwid snurqoorasydg 


SLIB[NuBis snuryoorseydg 
snjze[no1eq 
“nyoro1ur ~snuryomy sep 
-un 998 ‘snurqoauIUesg 
“*'STRITIUL SNUTqOIIB 
sapeiaie’eiepars ***s0d0.178 B110 


“*-"*-equaynose aouoddryy 


tees Byue[nose souoddryy 
Si aietete 8} Ua[Nose souoddryy 


sereesssspignyds SNUIYOR 


Seer SLIBI[IU SNUTYOR, 


sscsiesiainge SIIBIIUT snUryooy 


“**“Sua]7BATUN “IBA 8N4 
~B[NoLaqNyoroUT SNUTTOHT 


eNO Te (snurqooururesg 
=) sua]BAIUN “IBA sNy4 
~B[NoreqnyzOosoTUL snUIqogy 


“"*"*“sue[BAIUN “IBA 8NQ 
-B[NosreqnyosoTM sNUIyoR, 


evecare sese9(g9, Stuu9A 
-Og) SUdTBATUN “IBA sNy4 
~a[Noseqnyos01m snUrqogy 


OO OE OEE 


ETHEL BROWNE HARVEY 


14 


| 


11d “gz “yduoyy “¢ €I, ‘jueuuay, | 
| 


a, qouuusy, | 


| 
IT, ‘Aounrg — 
| 
OF, ‘19uyeH | 


Tee “4 ‘z1 “1907 “dxq “fp 
sora ‘1g TIM “Tor | 
cor “4 “er SIME “10Le 

ece_ | 


d ‘gp “yornqugy “qory 10, ‘WOSTTA\ 
626 9 | 
“d ‘gy “yorgug “yory TO, “WOs|t Ay 
erp a ‘TT “qduoyy “nor 96, “WOSTIAL 
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IBA “XOJT, TOA 
Salg] Sap Bunzyonsjog,, 82, BxUaTIS 
iat 
‘d ‘Te “yornjag “yory TT, ‘®[PUrE 
erg “dd 
‘og, “Qeuy “4yIUT “yory IT, ‘Jexeyog 
06d ‘9 ain 
“ZINA “[A8A5H) “pay | 
Add *raqszunszig SL, ‘1924 8q 
L6F 0 ‘e187 “Yory | Or, ‘1023/8 q 
BFS “A'S “STTAZ “YOI\ 60, “1929/8 q 
DST ae #0, 


*jddns -quysp *[0o7 “YyospIMeyunszeg 


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“d ‘og ‘dunqgzin\y *[98 
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gz “d ‘6 “[ I] “ues 
ya ‘1adxe "[907 “Yyouy 
ese "d *y “TIT “ues 
qe “4edxe "]907 “youy 


10, ‘e38[oq 
66, ‘9dBlaq | 


i | | 
‘d ‘Z ‘inequedean “48a | 96, ‘AIMqIeFT “Y | 


96 | 


‘d ‘eg “yorngug “qory | GI, “PYSMeTPOH | 


siz0 
poyvojonue 


P ul AX IOX | Ul PGT JO gy 


(26 UW) AX 


GONGuaAg uUGAUTSHO 


£8 
€[, Ul Worjoed100 o8t 
wong “P Ul AX f8T 
dsr 
8ST 
$6 
eurmyoArys 
YIM po}eesy S300 UT 
SUUVNaU ait- ALXO- ANZ 


penuygu0oo—VLVNWUAGONIHOU “AT 


681-91 


ALO IS] 


[9 ed gy 
19 9€ 


[9 bo-FI 


]o ed gy 
19 9€ 


19 9 


[9 9 


[9 98 


[9 9€ 


(worgelnzar 
-03N8) [o wd gy 
19 81 


[o ed gT-91 
1o-+08 


Jo ed gy 


oOILANGD 
-ONFTHLUVd GNV 
alotdia 


“snqvwonva soysnoudoxo 7 


he 


“sngedouva saysnoudoxo 7 


Ni 


“sngezoriva soysnoudoxoy, 


“snjewarva soysnoudoxoy 


ob 


“snqedoeaA saysnoudoxoy, 
= isis aielsjeinit +++ gnqeand 
-ind snjo1}u900[A3u0130 


tieeeeeereree een 
-IAT] Snyoryua00;ATU0149 


“snp 
-IAI] sngor}ua00;AdU0199 


see osnp 
IAT] S8nyorzUa00[ 4900146 


ferereeereeeengnp 
-IAT] 8Nyo1jzma00[AFu014g 


Fite eeeereeerrngnp 
-IAT] 8nyorzua00]Adu0149 


tereeeeeeeeeregnp 
-IAI]T 8njo1}zme00[A3u0145 


SlIB[nuBid snutyoo1sey dg 


sa10gds 


15 


CHROMOSOME NUMBERS IN METAZOA 


Lee A ‘Or “qaqep yoog 16, ‘puBpegouyy | [ord fe) 1 cma iene a Po. ‘Bolu 
| -10j1]eo ‘Brpryy|Aydouneyg 
(III warpnys-w9[197) : | uord gq 
Fg “d “zt “szWeZ “vor 06, ‘Moeaog | oor $91 391 19 ze “s+ epeydaong soyry[sy 
| 
“d'eo -ydiow ‘poo: ~-**-eqeyid BnsezU0 
ig-d'ee“amqer-ydioy | £0, ‘poomyrentg | 391 391 Pigeaeed eerste oaerieercor 
rite 5 | Fi | 
ig d ‘gg “qayep qduoyy 80, ‘pooatreurg | 591 591 he oe ee te BPBIq SOC, 
270 4 ‘or “ayer [O07 | 26, ‘PUBLIBPV]y | rat ++ “sisuaZarIpues B[N[NVBICT 
BIqousIqIpnN “Tt 
BIqouBIGoOYISIdG “8B 
vinauhying *T 
VaodOudsy’) “9 
7 dé 
“SOIOTUL ones, Te 69 | pete "+" “sreuroyyo Bidag 
VaodO1vHaa, “D 
VOSO TION ‘IA 
é _ ie | 
“dig “9 Ay ctege “tes | 80, ‘297 
qa ‘dxa “]007 “yoy | -[BAB] 99 Alay[neD fon fon fons Pp9 asuooo1ydo winyedoyyy 
LIF | 
“d ‘6g ‘[00Z “SSIA “9107 | 80, ‘[e1s0q wos-+-00T ung euIae WOOZ0[dBTT 
1 'd‘y ‘(,f) ‘onbisjag | 
ap ‘Aoy “pBoy “99 
“Pp [OQ aed ‘qnd “wayyy | 10, ‘UUBUyTe yy COSI HW eH ajlowis vouuoaAdI 
VOZOSANW “A 
1d ‘zor ; 
“qng “ysuy erseuIeD 80, “spr0r SiSEE0) || Releinee AR ee ee vjruind wmov01ydG 
VaqdIOUNHdQ) “9 
goa °d ‘TT ydsoyg “anor $6, ‘PION 
usp “d ‘g “zuy ‘yeuy £6, ‘PIO 6-8 P8I-91 Bds gg-sg | ****,,Suapoutyog 10490 


pus,, sijeser sndoyong 


VAGIOMAHLOIOY “Pp 


JOURNAL OF MORPHOLOGY, VOL. 34, NO. 1 


a, 


i 


$0 “4 ‘1 “1988 | (e104 
“jooz "yos}nep "YyIOA. Il, ‘Ytuaez = pug urejod 07 x pozeys Jou) gz | BVUEpOdOsU BIUUBUOpAT J, 
| 
| oyu 
-10usz0p OT 
WTA SHOUT 
906 4 ‘1G 1282 pug ar “£2  (pasny) oe (o10yM 
*JoOZ =“ yoRynep “qieA IT, ‘Yruwaz aod 09 (pasnj) XZ} X & + OF ‘OF | (PAsNy) XZ+0T Pal paqzuys you) #% | *****BPBINAS BI|AOO[ GAH] 
| 
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*JOoZ “yosynep “qioA TT, ‘HPa1aZ pug ur ejod 03 x poyezs yOu) FZ |" **** * BPBPUEpII} voBlBATY 
69g °d ‘of ‘pawaseyy 
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=) BIBp[OS BoUTUTETT 
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S *JooZ ‘qos}ynep “q19A IT, ‘Yrasez | poyeys you) gg |****** *aosed winquisD 
5 £16 ‘d | 
i ‘ey “9eay “yur “yory 60, ‘JossBraN 
6IT “od ‘pg “zuy “Quay £0, ‘JOS8BIHON | oor 19 & ee" stored BrnqurAD) 
jo vd oF 
601 19 02 
| (jeaorouny W108 0% 
° coz “4 ‘Tz “119885 jou 6 Gym 501 401 B00 02 
a *JO0Z “yosynep “Pp “q19A TT, ‘yturez = pug ur ajod 07 x | a8043) LOT ‘6 £01 POT ads 0% toe “aie oe OOTOS'STOBST. 
| 
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| “UBT 908 ‘BITBZI[08 BNET 
| tec °d ‘¢ 
: “quia 9 “4BU8 [BT YOLY | G0, ‘t2zBreE | (qsvo] 48) [9 pz |*°**** ByBZoOUN wBIsA[dy 
70, ‘UOpFuIL 
cre ‘dd ‘Tz ‘aINT[9D BT -[q pus suossuvy | d9T 691 691 sereseeeonamgound Biss[dy 
19g ‘d ‘4 | 
“quia 2 °FBUB "[BzT YOry QO, ‘1ZZ2818—-) (4so] 48) [0 #Z | BULOBUNT] BISATdy 
99g ‘4 ‘eget ora 
200BID “OS “PRY IME | 66, ‘Youeyoor, Coa) 691 sos --supridep visk[dy 
See Oe ee Ee ee eS SS 
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a1o1dia 
tS | ——S . $———————————  —————————————————S 


2 paenuryuopD—VOSN TION “IA 


17 


CHROMOSOME NUMBERS IN METAZOA 


or “d ‘ge “sztez “us | 


60, ‘MOUTeL Sy 
$0. 


} 
| 
| 


ele “d ‘6g ‘oneH “Weuy | ‘MoxruMosseqosy, | 


68e “4 “6T “Org “Gory 
6FI 
‘d ‘Ty “yeay “asorqrd | 
Ler a‘ TT qayer [007 
eg “d ‘22 ‘anyaD BT 
LF ‘97 ‘anyTaD BT 


161 “dl ‘et ‘amny[aD BT 
£26 “A “TT ‘ana BT 


Ist “4 'TT “HIPEZ “Wory 
opF “dl ‘ge “sytaz ‘ue 
cry d ‘gg “sy1ez “uo 


OL A ‘ZI “zuy “007 


Fe A '2 “TPZ “Yory 


Tez “d ‘g ‘toresun yy 
48N ‘Sn, se[suuy 

Ld ‘fou 'g-4'z 

0G “We “8 “P10 
‘enbizjaq “AOI “pBoy 


OT “4 ‘ZT “zuy “[o0z | 


wed 


£0, ‘TeouVy 

20, ‘]a0uV 

86, ‘Aviinyy 
IT, ‘99°T-se]]0q 
66, ‘99°[-82] [9g 


16, ‘90'T-SaT[0gq 
96, ‘99'T-8919q 


SI, 102918 
60, ‘enreyy 


60, ‘Q4aureTy 
68, ‘3]NBUIE) 


IT, ‘Yosean gy 


OT, ‘S908 


Ol. ‘sulv'T] 


68, ‘4[neares) 


Ik, 
UL UOIZI01109 UOT 


peur 
-layapun ELA § 
| "FZ 10430 sapis 


99/48, Ur septArp 
moIyO18jay [[BUIg 


98, ‘LouqeIq | 


PF 


PF 


OFS 


OFS 


PFE 


OFZ ‘qoid 


OFS 


PF 


PF 


OFS 


PRG 


PF 


Palit 


-8% poy [ear 
-10B 9U0 ‘977 


PF 
OF 


502-91 


PRG 


PFT 


SOT 
602-91 


602-91 


ads gf 


m08-+08 


19 8F 
(g1qnop 
Fo=) dds gf 


ads gf “Bo 


aids gp 


(8-06) 3ds gp 
(8¥-0F) 2s gf 


(SF 
*qoid) 4ds+-0F 


Ads gf “Bo 


19 Gf 


Fees -gueTBA 
-1q “IBA BIysuIOd xI[a}{ 


sua[BA 
-Iq ‘“I8A BI}yeUIOd TTey] 


++ -guayea 
-1q “IBA BiyeUIOd xI[ayT 


suayea 
-1q “IBA wyyBUIOd xI[o}] 


“*° (96, ‘ABqunyy) suayRA 
-1q “IBA BIyBIOd xI[a}T 


wpeeoe ***SI[BIOUIOU XI[AFT 


plotsisteies aisi2 SIPBIOUIAU XI[OFT 


: “Cy BaqouL, 
=) sismazi0y = - XT] FT 


viene tress spsr9dse XI]OFT 


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penaydop—VOSNTION “IA 


19 


CHROMOSOME NUMBERS IN METAZOA 


| &l. 


lez ‘Or s19Z “Yory | ‘ospIMayBYosnyy 


Log 4 ‘ZT “S19 “YoIV | FL, ‘Z}UWy UO, | 


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£% 4 ‘1061 | 
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cor d ‘gz “spez “ues 96, ‘Youqieny 


peed ‘gy “zuy “yeuy | 
14 ‘ar “epg, 


96, “Youn yo 


9S “JEN “Poy “nor 20, “UIP{LOD, 
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ss eleje eve “"SesID SNJaulIa A 


a a “sijeurosid BYCA[BA 


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penuydoO—SHHININTGHLVYWAN “IIIA 


23 


CHROMOSOME NUMBERS IN METAZOA 


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ETHEL BROWNE HARVEY 


24 


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THA 


METAZOA 


CHROMOSOME NUMBERS IN 


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SIZ “$1 “9 “TTT 
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ETHEL BROWNE HARVEY 


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“Nn “ydioyy "[[a805 *Zz}1g 18, ‘Maaog pus wornuruid uoid 97 
216, ‘unig 
19 °c ‘gy ‘ainyjaD By | -ey pus Aouiwy 
666 
pus 1 “dd ‘g ‘anyjap By 98, ‘Aouiny uoid 67% 
Td ‘Z ‘arnypeD BT 9g, ‘Aoursy uoid 97 
agoNauaaaHN uaAMASHO SuuVWaY ai- 


2% 


6 yoo 
Z jo sdnois 7 


(19 t=) 62 


6 yova 
Z jo sdnoid 7 


6% 


6 youa 
Z Jo sdnois z 


ed 
LPT 


6 Yyoua 
p Jo sdnous 7% 


(19 8=) dz 


6 qovo 
~ Jo sdnois 7 


6% 


6 youe 
p~ jo sdnois @ 


$n 


GLAO- ANG 


ALXO- LA] 


B00 F 
ads seetessss <“SUOTBAIG “BA 
Brpeydacojeszeur = Stuwasy 
PP teres sss SUaTBAIG “IBA 
BBydavojesaur == stuwosy 
Pr settee ss “BUQTBAIG “IBA 
B[Bydaoojeseu —stuwosy 
Pr terres sss SO9TBAI “IBA 
Beydacojesaur —st1Bosy 
beeeeess ss suaqparq “aBA 
Beydeacojeseu =~ StBosy 
PP teeeeess ss BUQTBATG IBA 
Bpeydaoojesaur — stuwosy 
PF teeeveeess SUO[BAIG “IBA 
epeqdooojeweu —sstunosy 
OILANGY 
-ON@HINVd ANV sa10das 
a1oldta 


penuyuo)—SHHLNINTAHLVAGN ‘IITA 


Woo x98 B 
410 UOleyUaUIZBIy 
| 0} anp ‘surroma 
qSOUuL jo sax0 OUIOS o¢ 
Oc “4 ‘b ITEZ “Youy 60, ‘Wuwog | at UIOLYO [[BUIs BUC) ads ¢ ees “SUO]BAIG ‘IBA 
A ‘Bey doooyesour Sileosy 
) 
d‘g ‘qst[BingeN o14O 90, ‘834115 6% ord eid ss STeTBAIG “IBA 
‘Bey daoojezour SLIBosy 
1, £88 pus .gce “dd F ois . 
a sg “Qeuy “yr “qory 80, ‘BOrBhaLL LF PF ap a “*sua[BAIq “IBA 
‘Bey davoyes 
g 99 °d ‘2 HJeZ “Gory gO, ‘Atoutosyu0p; uoid 7 Wa Gia ae ple  r f 
=< 2e1 4 ‘9 “TIME [Ore $0, ‘Axouromy U0 uoid 67 6z 6z Pr ures sss" “SUa[BATG "IBA 
3 gse -d “a[eydaoojexour * srvosy 
= ‘6g “4euy “TYME “yoly | ZO, ‘P{SMoyzs0y xa 6a sreeees =" * “SUOTBATG "IBA 
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= . Bleqdooojeaour SLIBosSy 
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s | -HBIy SoUlty 
ecb 4 ‘gt ‘yorg ‘qory | C6, ‘8/19 ~“aWOS 9 F | suayearq “8A 
2 | Bpeydacoyeseur ~ stuvosy 
= | 
3 | +21 
fe | -8 DUA 
‘s[ja0 Ulied 
3s “uitidut uo1y 
-B}UDUITB LY 
. SOUIIQAUIOG 
9 soulry 
GhY | “autos: ale 
d py “qpeiquaZ “[O1g £6. ‘YEU 0A (9,qnop (AOD); 2 RR IO *SUO[BAIG “IBA 
aca Pe Bey doooyesour SlBosy 
“Tp oRUy “YIU “Yory  ‘LISMOPIISEAA UOA S[[99 Xos F verses? ss SUaTBAIG “IBA 
pata ‘leq deoojexour SliBosy 
‘op ‘ yeuy DW “Yyory £6, ‘onvigy LZ nerd held ads 7 soreesssss-SUaTBAIq “IBA 
Bpeydooojesen ~ stuvosy 


ETHEL BROWNE HARVEY 


28 


sso d ‘er “31197 “Yory 


Tog “4 ‘gz ‘AINTT9D BT 


1d ‘qeog ‘wopuoT 
‘90g "AoY ‘suBly [Gd 


cep d ‘oT 


“rylul -yeuay,p “ory 


6FT 4 “6 HIPZ “Yoty 


(an) 
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1 ‘d ‘deig “uasst A, 
"esa “Wyo “Buoy 


Iz ‘oT 
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werd ‘Go “WP “Yoty 
FI¢ “d ‘Tg ‘aoual0g 


12 F=X Aporey 
*soAiquie urT]a g= 
X “mH0q Ul SaptA 
=Ip 10 ‘pug 10 48] Ut 
ajod 03 (e[qnop) 
X ‘seus sayy, 


Sp¥B1}0} JO pvazsur 


GT, ‘z}tulay = spBAq ‘[vurruB auG 


£1, ‘Teejapevg ep 


£1. “Y99W 


fli 
‘Jao. JINBT 


moiyo 
TOYZOUB YALA 
ZI, ‘BMO[OI ==pasny 10 aywredes 


“([BUIIUe 80) 


ZL, ‘Yoszyney = "9 UL sarqtpeuu0Uqy 


ZI, ‘AYsAoplan 


IT, ‘snizg0yy | 


(SULIOM §) Pug 


OL, ‘BPABApHT 
410 48[ UL aod 03 x 


OL, ‘SPIBA PFT 


FONGHA AAU 


UAAUASHO SMUVAGU 


(z9-89) 
quia 409 "B90 
(FS-6F) 
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(q ‘d { Ajao 
pus xX) PL 
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uo = =|-sB1jyozenp 9 -O1q w1oryo 
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“‘eyeqdoooyenoen SIBOSy 
52 sees ss ss < STOTBAIG "IBA 
‘apeydeoojeseur ~~ strwosy 
Cod 300 F eee ess* 9 “S0Q]BAIG “IBA 
‘epeydooojeaour = siiBosy 
PF 
fir ord 6% 300 F 
LPG LG herd ads 7 aie “‘SU9[BAIG “IBA 
‘ayeqdooojeseu ~~ SLiBosy 
Pp 
uoid OF 10 Z FIO(LS) PD 
G10 6€ Z10 O€ G10 68 (4) 19 9 ‘as BOOT BAIG "IBA 
‘a[Bydooojeseur = sLIBosy 
‘quia (2) 39€ 
“que ({) P22 
wo1d OZ-T [9 8-1 reese sss BUST BAIG "IBA 
‘ayeqdooojezoul = stwosy 
uoid 4% 
uoid 9% (orp! suc “-“Sua]BAIq “IBA 
‘apeydaoojesour = slimosy 
uo0id 7% 
6% 66 OG PF “ 8" "“SUaTBAIG “IBA 
tapaydeoopeneut SIIBOBY 
Pe 'S BZ 10 OF okey sds ¢ ig 8" **"“SUaTBAIG “IBA 
‘ayeydaooyssour = stiwosy 
OMLANGD 
at- DAO- ANG OALO- DS] -ONWHIAVd ONY SaLOd As 
a1oTdta 


ponayynog— $01 CM ENIWTSHLY WAN 


“THIA 


29 


CHROMOSOME NUMBERS IN METAZOA 


179° 
‘ge “yRuy “yr ‘gory | 


19 “4 ‘ey ‘eINTT9D BT 


ech “4 ‘eT “Trg “Yory 


ect ‘d 
‘te “4eUYy “yu “gory 


bee 
‘Tp “yeuy “1yrUr “qory 


611 “4 ‘6 
“WatM\"3SU] “[007 “qiy 


1‘d 
gg “QBUy YIU “yory 


199d 
‘Te *9eay “rytur “gory 


949 “d ‘g “zuy 4euy 


Ist “d “¢ “31197 “Yory 
Buer ,/SurexT127 “P| 
zuBysqng “WIO014o "Pp 
MOIyNAYSUOYy “qazsiny,, 

£88 
‘d ‘raydny uo, “4807 
¥II “d 
it} veqoun *porsAy 
"n ‘ydiop ‘[a8an “zg 

(II varpnys-aoy[az,) 
ggg “d ‘oy “s}ez “uo 

(J werpnys-weT[97) 
cap “A “FI “87107 “uar 


| 


| 
| 


SIZ 
“d ‘ee “qyBaquay [orgy 


20, ‘wNBqssnNy | 


16, ‘un.1q 


[© WI0s UI suIOIYo 
| Jo uous UOUIABIy 
pues uonnurmitq 


-9JT pues AoursD | 


C6, ‘B10 
€6, ‘Tenvig. 


£6. 
‘DYSMOSISUA, TOA, 


16, ‘tepreuyog 


06, ‘JIMq10 7 

88, ‘AxzZ4rYOSz[N yy 
98, ‘AYsMmo10jsoq, 
60, “Meaog 


f0, ‘aaog 


66, ‘WoAog 


ZG, ‘Moaaog 
88, ‘HoAog 


28, “HOAog 


ZL, ‘SBLByougy 


| 


s]jeo Wos ut 
SUIOIYO JO UO1VRy 
-TOUIDBIy PUB UOTy 
“NUTMIp U1ZBUIOL YO 


uloIyo “890 

-08 [[BUIS SOUITJANIOG 
“Alun xX 

“AIG Sso10 A[quqorg 


uoid 4] 
uoid Py 


uoid P| 
oT 


LT 


PT 


LT 


uoid 4] 
uoid | 


word OT 
ol 


uoid (3) 5% 
uoid (4) ©] 


5197 


LT 


él 


S19 t 


OT 


DO 


él 


12% 
S]]29 Xos 7 


19G 


ids 7 


S[[90 XO8 7 


PG 
300% 
dds 7% 


300% 
Sds Z 


Ps 


PZ 


Pe 


Pe 


verses se esnaTBAran “IBA 
‘gjeydeoojesem = stiwosy 


rs ‘“suo/BATUN “IBA 
fara denoyatet sIIBosy 


sesees ss" “SuO[BATIUN “IBA 
‘eeydeoojeseur + siivosy 


sees ss ST@TBATUN “IBA 
‘aeydeoo[eseu = stiBosy 


sess ss STQTBATUN "IBA 
‘speydooojezeur =sLivosy 


POC an SUdTBAIUN “IBA 
‘epeydeoojeseur = sireosy 


SUs[BAIUN “IBA 
epeyleoopaeut SIIBOsy 


POC SUeTBATUN “IBA 
tepeudeoopseou SLIBOSy 


vests sss -STOTBAIUN “IBA 
‘geydooojeseur = struosy 


(06, ‘SIAN 
-}19}) SsuayBaran “IBA 
‘ayeydaoojusout = st1woBYy 


eieidieisisisicie'e inisie)s (aq, ‘sui 
-BYOBZ) SUA[BAIIY “IBA 
‘ueqdaoojesour =stiBoRy 


ee eer ae 


10¢ 
“d "py “(007 “SsiM “4107, £0, ‘Ta1}IB 19 21 “"" “suegoja snueiinon 
i I Hen 
£9 uoid OF 
pus] “dd ‘g ‘ainyjap BT 9g, ‘Couey x4 oF és P8 "Fuga:  mmeee ren SIBNG 
-O1 10) BlODT]]108 BI[IUOI0L) 
6g d‘g*[oviseisg “nog QT, ‘UOIEM | §[]99 X98 ZZ seers esse igonbil} siiBosy 
“U UINUIOJSsOLsy 
908 ‘BSOUDAOIZIU SlItOSy 
| siuBo 
| | ‘y oos ‘xujshur sitosy 
og puv er “dd "2] OI 
al ‘uoDUNYONssiazUs) [OIE] FI, ‘8N1z}0 37 rom 300% sete sss SUOTBAIUN “IBA 
>} | ‘gjeydaoojesour = strwosy 
ima 
i=} cep “d ‘oT | 300% 
=< “aryl “gBuYy,P “Youy |g], ‘palmer 91n BT al ads Z sores -SUaTBATUN “IBA 
ja | Gecrecarinn SliBosy 
cred uoid 61 
fea} ‘9g “[asay “yeuy “YyloA ZI. ‘PION : uoid Py] (re eeobes **-suayeatun “IBA 
7 ‘ejwydooojesour = stuosy 
= uoid 4] 
ecg “d ‘7p “zuy “yeuy ZI, ‘SBIBYORZ uoid Sy ““SuO]BAIUN “IBA 
2 ‘gjeydaoojesoul staBosy 
oa Td ‘9 “saz “yory OL, ‘Z}49y0uR[ (19 t=) dT BOO'7 ee @ eros ss Suo[BALUN “IBA 
=] ‘epeydooojesou = slreosy 
e zp d ‘e “y[9Z “Youy OL, ‘Sprvapoy | UIOMaUG *Y ULX TONG Wyirhebeeerstetayeees SUd[BAIUN “IBA 
a ‘ayeydaoojeseu = sLiBosy 
5 (sae z 10 
J Ul) WoIYO xos B 
410 UOIPE}UOUABIY 
O21 4 ‘p “3TTaZ “Yory 60, ‘Bunog | 0} enp woiyo [jeug Pes O90 “‘suo]BAIUN ‘IBA 
Moentereemet SlBosy 
oz “4 *Z “S]9Z “Yory | 80, ‘etAouu0g OL. Cle fem hae “*""suo/BAIUN “IBA 
‘ejeydaoojes0ur siivosy 
ese “d 
‘og “Quuy “yl “yoy G0, ‘yoxslqory, LI YT LT ads Z sess “SuaTBAIUN “IBA 
“‘gpeqdeoopssen Slluosy 
OlLANGD 
GONTUaGATY uaAUASAO SUUVWAY ai- GLXO- ANZ GALO- LS] -ONGHLUVd GNV sa10aas 
a1o1dia 
Oo | 
of panuyuoO—SHHLNINWTHHLVWHN ‘IIIA 


51 


METAZOA 


IN 


CHROMOSOME NUMBERS 


16L-4 “TT 394V 
‘eg “Ruy “1ylur “qo1y 


£8 
d ‘Tp ‘dinqgzin A "]]28 


-arny “pou “sXyd “yiaA 


48 
“d ‘egy “ [007 “SSiM “4107, 


£e¢ “d ‘op ‘*Zuy “(007 


cued ‘y “yoissyq | 
n-geuy ‘syBuo]yy “1ojUyT | 
#9g d‘g‘jorssyq | 
n yeuy ‘s}yeuoy “19}uT 


£9 
pus 7 ‘dd ‘g ‘apnyjap BT | 


Bee ‘9 “WYaZ “Youy | 
628 d ‘9 “W987 “Yory 
6ge ‘9 ST1EZ Yory 
ger “4 “s “SfleZ “Youy 


£9 
pus {dd ‘¢ ‘anyjag wT 


ot“ “Ty 
‘28 “Jeuy “1ytur “yory 


| pug 
$1, ‘24qny | 10 ysT ut ajod 07 x 


| 


gjod youe 0} Xx 
Io pug urajod 03 x % 
uol}eidueD 
o1giporyd Bure yy 


4s] ur ajod 03 x 
IT, ‘MaAog suoiBioues oyBindog 


iadi0¥ 
-jsoyy Ul papiBo 
-sip WOiy9 xos aU 
Aj[BuoIswo0Q, * 
pug ur sajod 03 AX 


ed Al[[Bol 
€], ‘tosnay | ‘so Ul opBIOUeZap 
ZI, ‘tomnay | witedg -q-d] AlUO 


06, ‘TByFUEMOT 


68, ‘[BYQUeMOT 


9s, ‘Aourep 
i HONS) 4sq ur ood 03 Xx 
I. SND 4s] ur ajod 04 x 


TT, orn 4sq ur ajod 04 x 


G0, ‘uaaog 4s] ut ajod 03 x 


9g, ‘Aourep 


GT, ‘S0A0L 


9 'S 


69 
(ayeiouadep 1 
yqtm urszeds) 


PL a) ‘¢ 
69 
(jeuoouny 


jou ¢ YIM 
uriads) 99 ‘¢ 


é¢ 
oF 


é¢ 
Oo '> 


uoid Ff *qoig 
5% 


g's 


69 
£9'S 


59 
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69 
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0 


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69 


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“Alun OU “ATG 


6 A[PIBY 
(Arun G++ 
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6-1 


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68 


69 


B00 


nds [] 


d wos 7] 
500 Z] 


(6) 19 aI 
(4) 19 IT 


UIOS gT 
[19 8T 


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{ ‘ores]NA wNu0ZsOIE[9g 
WINUIMba WNUIOJSOIO|IY 
“tingequape (snpAz 
| -W014g =) WINUTOSOIB]Og 

u 


UINMOSOInUW 9as ‘Beso 
-WdAOATIU BULEUOPgBI yy 


“+ "BSOUBAOIFLU SIPIPGBYY 


ess -SURLIOG® SIZIPqBq yy 


presse seen BIQure SLINAXO, 


“ung 
-Buoronut wIMUWOJsOIydGO 


***" SLIB]NOISOA STB10}OTT 

notes ss “BXOUUT SI] BI9IOFT 
seseeess sTBdsIp SIYBIOJOPT 
vreeeeeee suds srBsaqoRT 
** UINAB[OISNUL SAaplolB] Ly 


il “nsojpidud BIBL] 


JOURNAL OF MORPHOLOGY, VOL. 34, No. 1 


ETHEL BROWNE HARVEY 


32 


022 “4 ‘2067 BtA0o8ID 


‘Og “PROV “soUY "][Ng 20, Eouvysoy 6or 591 Rely “sNPBUIFIBU SN[NzBIqarar) 
oz dd ‘ar “qayer “1007 66, ‘909 d9r fon) é oor rd “SNFBUIFZIBUI BN[NZBIGa1a—) 
ecg “d ‘oz “ydsoyy “inor 60, ‘NSIBA 
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| 
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VINVANIUT, “9 
SIL 
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£05 * 
‘op “jeuy “ry ‘yory | 16, ‘AYsutpeq eT roid fear oF *SN[NOLUIIOA BUIULA}SBIZAT, 
VINVAWIC 'D 
VANILYUAWAN “XI 
16g “d ‘zz “s}eZ ‘uar 96, ‘IaA0W 39 “sn yzUBIBIZ07 sN|ABUOI4S 
6g “a ‘9 “S97 “Gory Tl. XomNyH pug ur ajod 03 x £9 ‘¢ 9 9 sores" sine} BN[AZUOI;ZG 
619 ZI 
1 IT 
69 69 69 B00 @T 
6ge “4 ‘9 “397 “qory IL, “49ND 48] ut ajod 03 x 9‘? 9's 9 ads IT “***snxopsied snjAzu0i4g 
lug "d ‘zz “qaqep [ooZ | 90, ‘uuBuryonayg 69 **snxopsisd snjAsuoi4g 
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a1oldia 


penuyu0)—SHH LNIN TAH LYWAN 


“IIIA 


33 


NUMBERS IN METAZOA 


CHROMOSOME 


14° ‘6 


worjyedey Bporose, 908 
‘umnoryedey uwnu0ysTCy 


“sOIDIUI "FBUY,P "Gory 90, ‘AndouueyyT 88-9 “unorzedey UInu0zSIC 
c6g “d ‘gz “quyer "007 60, ‘A18pD “ad 103'*q ‘d aug 691 Jo Bd oT **snye10dure} snostpojdiq 
G19 “dd “F “3T19Z “YOIV OF, ‘191401 £01 £01 ads 0% pa erage eae Seca Cay 

-8[O90UB] LINUIOISIC] =) 
UIN}Be0UB] WANTPAOOIOICT 
eo 4 *T S18Z “UV | 80, “FPHAYOSpIOH 601 601 601 300 07 fone eee ee (UTD, 
-8[090UB] UINUIOZSIC] =) 
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19 02 
601 300 0G 
OLF 4 ‘OT “3I9Z “YOAV | CL, ‘2ztUWEy UOA POT ads 0% *** Q]00IssB10 “| =) aBIP 
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vayjauatiq *] 
VaOLVWAUT, “9 
$39 (91-81 
*d ‘1g “[00Z “sSIM “4197 | 20, ‘Pporusr uo, “qoid) 691-9 ede as BYBIIOS BINT], 
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med '9g"s we} | IL, ‘ygnoy b uF ByYBPUNAIIZU9) BULLOPIAW 
VaoisaD ‘D 
SAHLNIN TAH LV Td “Xx 
sep “d ‘21 “qayer ‘[007 | 66, ‘20D 69r 19 é were ee 80880 BINION 
8L £1, ‘10UxO uoid 3 
‘d',0] “[00Z sam "g1ez | pus uneqssnyy 68 (19 2=) 68 [9 OL sees" Joqni sneury 
“Ld ‘qe0z wopuoT : 
‘00g “AOY “suBIY, [14d £1, 129W 6 291 PP 2ce ‘ snez0u] sneury 
1d ‘g0u dinqsiajag 
"49°78N dup 00g ‘Avr, | 66, Ploury 58 58 pus eae Sisualassed snoury 


ETHEL BROWNE HARVEY 


34 


1°4 ‘TT -34V 

‘eg “guy “pyrur “yory 
Lyd 

‘98 “Tsay “yeuy "GIA 


zr “d 22 
‘ueqounyy  “porsdqg 


n "ydioyy "pasay “Zig 


ee “d ‘gg ‘a[ny[aD BT 


Td ‘o:ou‘y “aangun 
“WB “Brawysiyd 
“ASPS-[su9prA “IAS 


sre dz y1aZ “Youy 
209 “4 ‘Tz “quyer “007 


org d ‘er “197 “Yory 


1d ‘644A ‘40g 
“uaa ya “xo "1007 "youy 

err d ‘9 “31197 “Yory 
Tas 4 "T2 “qzysr “1007 


, ser 4 ‘11 44 
sg “9uuy “xyTUr "Yory 


GONANAAGH 


1, ‘UuBUTessU AY 


ZI, ‘UuUBINIOSSE A, 


IT, ‘WuvurIosse 


60, ‘Loder 


80, ‘1oureryog 


80, praryospjon 


$0, ‘3prartospjop 


FI, ‘oupury 


IT, ‘eusoyoq 


IT, ‘Broquoyjeyag 


go, ‘wuBurqnyos 


PT, ‘AAoT 


ugANASAO 


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(d00 
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9fod youa 04 g “Atp uoid (pg 300 Of 
pug ur uomonpayy 6g SOL SOL ads oy vreesesss STATUE SNUOZOOZ 
$8 
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-OJBULOBY WNUIOSO4sSTYOg 
“AIP 48] UL 6 (sured) oy 00 (sured) Oy 
ajod yous 0) sured g | (saved) ¢ (sured) ¢ © (sured) gy | dds evecs OL | woryedoy Borosuy 
mory 
-onpeal ou 
“SZ. sosuo 1 ZI 
39 59 eumos ‘49 500 ZI ‘ “woryudoy BlOIosEyy 
uoid 6F 
uoid pF - Ps “= "(uino1yedey wunw04s 
-Iq =) Boryedoy vos] 
66 66 66 1? 81 7) UNPIBING UNO SIC] 
cunyBeo 
-UB] UWINTTIODOI0TC] 908 
‘UIN}B]Oo.UB] WNUIOSIC] 
—— =; = 
OLA NAD 
SUUVWaN an ALXO- ANZ ULAO- LS] |-ONGHLAVd aNV SU10Mas 
aloldia 


penuyjuo)—SHHLNINTWHLVId “xX 


CHROMOSOME NUMBERS IN METAZOA 


ugg -d 16, uoid 9 
‘Sp QBUY “INI “yory | ‘Uul01ZsMOHOUITYy 69 39 
£92 “d ‘or ‘9S uoid POT 
“pwoy “UuOH “suvIy 66, ‘OWEN UA 601 601 
GT, ‘WBUTAT AY 
112° ‘gz “TING “org. pus wosio}4tq 401 
Lor ‘d ‘6 “ydsoyy “anor #6, ‘T9]90UM 301-6 
Ss 
*d ‘eg ‘anbidjag “Ao0yy 
‘pwoy “noD ‘Way 16, ‘9},00UBL YT 
Ss 
*d ‘cg ‘anbisjag “Ao0yy 
‘pwoy ‘inopD “Way 16, ‘27; 00uB.1 yy 68 
£9¢ “d ‘or 9g uoid POT 
“‘peoy “uu0D ‘suBIy, 66, ‘AUlBNY uvA uoid & 0] S01 
6st “d 
‘9 “3 “TIT ‘40g ‘ued 
ya “1adxe “]007 “yo1y 86, ‘2}}00UB. YT 
S 
“d ‘eg ‘anbisjag *Aoxy | 
“pwoy “Moy “ney 16, ‘2};000B1 7 58 68 
apprpyphpog *] 
VINVTIGaNOY, “9 
oie 168 
“d ‘Ty “O07 “ssi. “4197 | 70, ‘FPIUTyosploH oF 68 
162 “4 ‘gt “org “yoy 20, ‘UPA 601 
uoid 9g 
StF 4 ‘or “H1eZ “Yory FT. P1tD sop ao Arey 69 69 
erga uoid PF 
*, “Iddng -qayer "1007 #0, ‘Loureyyey pug ur worjonpey oF 68 
1¥z ‘dd ‘9g “zuy "1007 £0, ‘Eyotuee UO A So} I9UIOAIB YT 68° qoIg 


poyauabouopw *g 


69 


601 


60r 


68 


58 


fot) 


58 


58 


fo) 


39 


38 


[9 OT 


1? 02 


[9 91 


Ps 


12 0¢ "v0 


21 
3ds ZI 


(6 saurty 


-aul0s) [90 


**sN4Bq4IA snavia00q4S01g 


“*'"Bs0[nqou Biao0uT, gy 


tts BoTinbur Bia0uR, gq 


tess BuTTinbur Bi900uR, gq 


vere ss BNQLING sapBpoalO 


***stB][auier} BuBl[doydaT 


*-snorzdiyja snqooyAysn 


“*-“snsoyidud sniodojaAp 


bee eeee eee eres es eT UT 
-leda}zUr = wn UIOWSA[OT 


-l11oZa}Ur =uINUIO}SA[OT 


*** -suBgaja sn[AjowporAr) 


“suBdoja sn[AyowporAr) 


g |" ‘suBdoja sn[AjowporAry 


ETHEL BROWNE HARVEY 


p04 
*T ‘Moosoyy “83197, “101g, 


ey ‘d ‘oy “qduioyy “nog 
gop 4 ‘2g “ydsoyy “inoe 
62% 
“dg °2 ‘AT “zag “ues 
qe ‘sedxe *[007 “ory 
FI 
d ‘251 “og “pBoy "YO 
6ST “4 ‘ST “3IeZ “Yory 


£1¢ 
“dg “[007, “881M “9107, 


nesag ,sungyonsy 
“og oules pun tq seq, 


SLT d '2e “TINE “TOL 


Get “4 ‘81 ‘ANID BT 
681 4 ‘9 3 “TTT “40g 
“uad yo “dxo *[007 ‘qory 

Ger “4 ‘QT ‘AINT]9D BT 


681 “4 ‘9 “9 ‘TIT “199 
“ued ya “dxe "1007 "qo1y 


¢ 
“d ‘eg ‘anbisjag "Aoy 
“peoy “NOD “Woy 
DONDE AGH 


AID pug MOET, 
OT, ‘Uryqosadey | urasny suioryo OMT, Rela Rey 4 ads § 
86, ‘Jeurprey PT 
OT. ‘ea oF Ps 
80, ‘22112 
80, ‘29118 (I2F=) 6% (198=) 3% PF 
uoid OG 
PI, ‘880A UOA 8g és 3 (sired) ¢ 300 OT 
#0, ‘NBlssorg, a3 Iau1uNg 6g 19 OL 
OBBABOIO 
£8, ‘Iaprauyog | 0} siojor A[qeqorg yo 2 "Bo 
ZI, ‘wosi0}}8q oF P8 
DI20IOPgDY Ye 
10, ‘prBIeH 66 
86, ‘8},00U81 68 38 58 1? oF 
10, ‘Pavey 69 
86, ‘e},00uB1T 
16, ‘a},00uRBI uoid 69 59 69 
OLaANGD 
uqAUuasao SxUVWHU an- ALKO- ANZ G@LX0- 18] |-ONGHIUVd GNV 
aro ldia 


be eeeeeee sss SIDTITA XO}IOA 


**-sngupnuo snsa0qoA[og 


+958 Blgyer5) 
wiedijjaued xo}1OABIUT 


ress ss" TID IB) X9PIOABIBT 
“*“1HTaquerIys BULOJSOSa TV 
“HIaqualyo UWINUTOSOSapY 
“IH19QUdIYO UINUIOJSOSO TY 


‘a XOPIOAVIB 
9as ‘BiBdi|[ouled Bp YyeIy 


“+ eredi|[auled BI] Weary - 


“***-umiptped snyao[44g 


Cite eeeeeeereeee sgn 


*"Sn}B44LA snevio00q4801g 


**sngB44IA snowsio0ayysorg 


sqi10gds 


ponujuoO—SHHLNINIGHLVId “X 


37 


IN METAZOA 


CHROMOSOME NUMBERS 


1d ‘27 ‘aM 119D BT 


298 “4 ‘CT “11g “Gory 
od 

‘TT ‘825 ‘Jeay “ysaA 

Td ‘gz “Jorg “gory 

621 “4 ‘Fz “Quyer {007 
Lz 


*d ‘py, “007 “Sst "}107 
FE “d ‘yg “Zuy "1007 


80z “d ‘gc “BIG 
“IQ4BN “pBoy “001g 


LZ 
*d *yp “007 “Sst °3187 
FE “d ‘yz “Zuy [007 


Ter “dg “yT19Z “gory 
621 “4 ‘Fz “qayer ‘[007 
ise “d *gz “qayep *[007 

1° “$1 S197 “Yory 


T¢°d ‘TT “31197 “qouy 
621 “4 “Fe “quyer “007 


PLE 
‘d ‘11 “1007 “astm “3107 


be “d “7g “au “[007 


G0, ‘3408 90q9S 
Z0, ‘JAeByooyoy 


86. 
‘WQ914g Joep UBA 
16, 
‘qyoljg Joep uBA 
20, ‘S1mayor 
20, ‘dtaqyos 


0, ‘WOsEI}4 BV 
#0, ‘WOsory4eW 


#0, ‘SUPAR}G 


FO, ‘WOSaty7B WV 
0, “WOSE147B]Y 


60, ‘ploury 


20, ‘drepqog 
90, ‘drerqos 


cy, ‘gaodeddey 


€T, ‘1919D 
20, ‘diepqos 


#0, ‘WOSOI7 BY 
#0, ‘UOSAI7 BY 


66 
uoid ¢§ 
“Ip 
puz pus 4sq urejod 
yose 07 F ‘saseyde 
SUL Ur paTanopsONy oF 
poonpal 9 puw 
¢ ‘Spury omg aq Av 
iATD. 
pug pur 4sy uraejod 
yove 03 F:soseyde 
COLOR algo p iON oF 
8 
8 
pATP 
pug put 4s] urajod 
yore 02 p tosvyd 
-ejour pug pue 
98] Ul pa[qnop “ON oP 


56 66 
56 
66 £6 
9 09 
8 8 
(aseyd esuydoid 


-o1d ul ¢) 68] UrP (8-4) 8 


5 9-£ 
Py IOE 


(aseyd 


-o1d ut 7) 68 | ur F ‘(8-F) 68 


39 'F's 
PP 10g 


aseydoid 


8 
8 68 
és 
8 8 
éL 
8 £8 
(oseqd aseydoid 


-o1d ut #) 68 | UP ‘(8-h) 68 


el 


Dprpypru4y, “§ 


1? 81 


281 
ads g] 


WIOS ZT 
Sds G1 


[9 9 


ul0s 9 


P9 
ds g 


19 9F 


300 OT 
500 OT 
ads 9] 


Dds $2-02 


ads FT 


[9 9T 


““'1yo001q uoozouBsAqy, 


“***""1u9901q UoOZoUBsAT,T, 


**** "19 OB]103 S9pOIIVOI 


***uidru stjaoA[og 


****BAI0} BIIBUB[ 


*** BULISST[CUIIS BILBUB[ 


sess -noryosod BiBuel 


uinay 
~O8] Wwinya0o0I1puacy 
aos = “BaZQOBL «= BINBUL] 


*(aIn9zoB] WIN;IOI0Ip 
-uaq =) BojoR] BIIBULT 


Bey daoouos viivuLy 
ween re *eurdye BIIVUB] | 


¥9}08| BIUBUB]Y 998 
‘unoezoR] UInaooo1puecy 


“uInazoR] UINaoooIpuaqy 


“uInayoR] UUN[IOoOIpPUa(T 


**mINa}OB] WNPa0oo1pudcy 


ETHEL BROWNE HARVEY 


38 


626 “A '2e “TOL “YAY 
suomeg "Eto 
-d *g9, ‘aouat9S ep "IO 


gp, ‘SUrByUOjaD 


‘anbidjag “AOI “puoy GO, ‘AULBzyUOJIIH fort $71 dal ZOO % “su yupoaouy] snxorgdiny 
gtd (a1 
‘og “yeay “ay1w ‘qory 16, ‘873090 “qoid) I-01 6621 1 ba “-snqupoaouny snxorqdury 
96, 
69p A ‘FT [Ole “Yory | ‘WYoMAg dep uv 
SBOUIByY "6Eo 
“d ‘og “9 ‘g “tag ‘onbia £6, 
-Pq “Aoi ‘pwvy [Ng | ‘FYING Jap uBA qunoo [ 5 401 “*snqu[qaouR] snxoryduy 
(VQUOHOOTVHdHO) VINVUOV I 
VLVGYOHOOLONd “4 
(3.4194) 
sq°dz BAe 19}UIM fp] 
6, q dG SH0 ed PET-1L 
zat d 'g “1907 “xo “¢ 60, “Aougry A, ‘q-ay dda wd 6 67-78 cs “-Bquas BUIFBPA FY 
§,q ‘dz “qolg Ada JAYUIM FZ] 
L119 ‘d ‘1z “zuy "[oo7 86, ‘uessuo'T ‘qd ado vd pe 
Bo 
6rF “4 ‘FT ‘ANID BT 96, ‘uossueT sq ‘don wd SZ. 10 OL uyuas BUlpEpA TT 
VUAALLOU ITX 
[2 9T 
e201 4 ‘p ITIPZ “YOty | 60, ‘Uasuez10¢ 68 58 68 300 8 “ss -snupydes tapuBoAg 
062 ‘d ‘9, “zuy “‘yeuy 66, ‘SBBY 591 TOUS) aaa snuBydes vipuworg 
ZOO OI-8 
sie d ‘oo “SW LO $1, ‘Apued 601-8 C5) 8 |e BssaId 10) BIFUVI 
OT, “UryoulW 8] [99 
19 d‘es*s "Ww Lb pus w0s}104 0% eyjoo gy, “Ba jo” BIIBIIOD BULIYZBI) 
OMaNaD 
GONGuada uagAuasao SNUVNGHY ait- G@LAO- ANZ ALAI- LST -ONTHLUVd GNV Su10dds 
aio1dia 


VuUadIHOd 1X 


39 


NUMBERS IN METAZOA 


CHROMOSOME 


TOT “4 ‘9 3‘ A “49g 
‘uaz jo ‘dxe “[007 *“qouy 


TOT ‘9 "9 ‘A “49g 
“uel ya ‘dxe "007 "yory 


Td ‘ET “TOA ‘eT 

‘ou ‘vg “iog ‘1oduNy 
Ip ‘9g "pRoy “UW WV 
Gre “4 ‘6T ‘AINT9D BT 
66T 4 “LT ‘AINT19D BT 
69g 4 ‘or “ydsoyy “anor 

are 'd 

‘, “quuy “anor ‘waMy 
Tp d ‘1g “zuy “yuuy 


TOT “9 ‘9d “A “was 
“uaz qa 'dxa [007 “youy 
Sie “4 ‘GT ‘ANTI BT 


Ist “d ‘9g ‘a[nyeD BT 


OL, WOT HINE 


OF, “OTLB A RET 


40. UPA BIO 
TO, ‘Unaqa'y 


00, ‘Unaq 
-9T pus Aouwiwy 


80, ‘Buryy 
“10, ‘Bury 
20, ‘uty 
OF, WOrTEyRET 


10, ‘uniqa'y 


60, ‘SUrey 
/-[IM 30 Suossuny 


-— 


re ee eee 


£6 °d ‘eg ‘onbia 
“PY 39 “Ay “os “TNE 
cre “d ‘98 “SW LO 


ply d ‘yornsdy “og 
“APY “oOssy “glig¢ “doxy 


1g ‘d ‘eg ‘pavarnyy 
*joog ‘duos ‘sn yy "[[Ne 


(U1 werpn4yg-uai[9Z) 
Fg “d ‘ZT ‘syez ‘uo 


(HT warpnys-wor[az) 
HE “A UT “eyez “vor 


£6, Urn 
96. TH 
$6. 1H 


66, ‘JJoroungy 


06, ‘Marog. 


06, ‘OAOg 


69 “+s -anguqgound soyApojag 
56-8 teeeeeeee es *SrBR[NA Of ET 
300 FZ-81 peeeees ees sBTIVA[NA ON ET 
68 501-8 sertesees ss SLIMAINA ON ET 
da ford! HOO f% 
bat ZI pera | dds $7 sores e es USOULTIFUOT OFN ET 
é6+21 reser ees ss saa raTBluo ON Ey 
61-9 sees snus! LOPBUIY Og, 
LOT ads 7g : *SUDITI}OISGO SOPATY 
VUONY "D 
VIHINANYV I 
VIVUEALUAA “9 
“q4oj ULOM4 UTOPIA 
-1p_ S]quqoad py rod oF $8 00 f 
-wuriods Jo SuLOIyD PT ‘ LZ OF ads + BLiB[Nssord sisdoposqy 
(6 
410 g) uoid Og (91-81) 
(6-4) 628 68 68 1 OL oes BA RT [LUBUL BISN [BY 
Cras +++ stuzuaptov0 BId[BqsIq, 
uoid & 36 
word P46 [9 81 sees ss BITBUIQSOPUL BILOID, 
66 “B[NQUOUL BIPlOSy 


VauOHOOUN IL 


ee 


ETHEL BROWNE HARVEY 


40 


09 "d ‘F 
“og "pwoy “YBN “001g 


eort “d ‘zcT 
‘stuBg “0S “PHY “YO 
SFI “4 ‘GT 
‘suueg “0S "PBwOy “UO 
ee “A ‘9 “zay 4uay 
og “d ‘}T 
‘tg “yey “AyIW “yory 
ete 4 “6r ‘9INT19D BT 
661 “4 “LT ‘2INT12D BT 
60F 
‘d ‘gr ‘‘syeuoyy “Jozuy 
02 “4 ‘ge “TIM “19rd 


“Lee “4 ‘9% “1org “UorV 


Tot “d ‘9 °3‘A “19g 
‘uaa ja “dxo [007 “yoy 


sor d 
‘op “qBUYy “YIU “qory 


og -d ‘TT 
‘og “quay “aytur “Yyory 


&1 “d ‘2g 
“a8 4a “dxa [007 ‘yoy 


gor d 
‘oF (QBuy “Ayr “yory 


9zp 
‘Te “yay “Ty “youy 


02 4 ‘ge “TINE “1Ore 


ST, ‘qa0T 


OT, ‘eusr0yoq 
16, ‘uazAnyyoq 


eT, ‘AAaT 


10, ‘unaqa’y 
00, “anaq 
-aT ye ourwDp 


96, ‘Ta1yooRzlog 
LT, ‘e]3urng 

TI, ‘}9qoBIg, 
OF, ‘UOTTVEyVE 
$6. ‘UVEY WO, 
cy, ‘AAT 

et, ‘Adureqo 

C6, ‘WIBY WO, 
88, ‘Jopug]}04oS 


LT, ‘e13utMg 


GONGUasaH 


ugAugasao 


IT, ‘euroqoq | 


qprayos 
-PlOH JO uo1jvasos 
-qQ “doy wd 4[npy 


8a} fo000no'] 


481 ur ofod 07 x 


‘ou [eur 
-4ou 0} UOTZE|NFey 


48] ur ejod 07 x 


| SuMUVNA 


39 
£9 
601 
L8T ‘ZT 
PU Pera 
PEI ‘ZI PEI ‘ZT 
at GLXO- ANZ 


PET 


ds ud-+oz BOBGOCIICOISS faa piedoeyT, 


uros vd 9 
mos (sired) ZT 
UIOS OT 


jo ed gy “Bo 
19 #2 


00 97 
aids 67 


wos vd +97 
jo ed ZT 
Bds 77 
Bds G7 "80 
3ds 9 
(arey 
p27yB4s JOU) FZ 
ULOS FZ 


300 9% 


GLAI- LST 


OILANAD 
-ONTALYVd ONY 
alo dia 


EODOUIAEISN ayer (ohsChto yy 


Viteeeerereeeess uogonT, 
Buty 


teeeerereeee(uosny 
10) ‘wrieiodtme, suBy 


veeeesssverrelodulo} BUBYY 
sreessssoprrp10dule} BURY 


sreeeeeeeeescsugidid BuBy 


“BOSNy BUTT 


“BOBNy BURY 
salsa eleisisiziai(i})) wosny suey 
voeeeee seepage BUBYY 
veeeeesess-pamgMmosa BUBY 


cteesesese-paTginosa BURY 


*"qosoiy Jouniy), 
BJ Ua|Nose BURY 


esses BIBIGSI}) BUBY 


sa10ads 


penuyuoyj—VvIqIHdWNV ‘I 


41 


CHROMOSOME NUMBERS IN METAZOA 


1d ‘21 “y119z “yoy | 


Td ‘or “quid 


a "yeuy ‘"[B}I “qory | 


‘Ie “1@3T “1007 “4100 W 


T “o ‘0g ‘ain T19D ®T | 


Td ‘*g 1INg “10rd 

esc “4 *F TING TOE 
66d 

‘| “qeuy “iInof “lowly 

g0z “4 *Z “TIN “1907 

19 “d ‘gg “ydioyy “inoe 


11e °4 ‘ZZ ‘9INTL2D 1 | 


61F 4 ‘0G ‘PIN19O BT 

TdT qaaonsanof 
T1¢ “4 ‘TT 

“Qn BIMIOF TED “ATU !) 
gg “d 

*jddng ‘et ‘“ydiojyy “anor 


611 “4 ‘6 
“‘TIhg “99 “Aluy) sveuLyy 


Tez “4 “gz ‘OINTPPD BT 


Loy 4 ‘8h “SIN LO 


629 
d ‘9¢ “Joo “sstm “4197 


.ueayos 
-Uay_ Sap a1qaTqaaen,, 


FI, “WUIaL 
TI, ‘ta19L, 
OL, ‘Tus8L 


ZO, ‘uniqoT 
Z0, ‘unigaT 


g0, ‘Araul0syUOT, 
G0, ‘Ainqssury 
66, ‘Aanqssury 
or, “UWS 
cg, ‘Suessuur 

€0, ‘Zour 
-nq je suassuee 
00, “Wostg 


an 
‘Zu07T pue yooug 


66, ‘1OSIINH oI 


FL. OPW 


€], ‘UUBUTIexON 
70, ‘wosuryuar 
£6, “Ald 


68. *TOxTLOM 


pera 


PST 
pera | 


LC 


yiIaaoun “4 


LZ 


6 @I-01 


21 
pera § 


Para | 


(QI-#1) SST 


68 


pera t 


(GI-01) ST 
LEI 


LI 
(qoid) 621 


LET 
LEI 
(ST 


soso Zul) OFT |(Og-E7) Bde 


LEI 


(9I-#1) 6ST 


(g 
*qoid) S0I-F 


Sds #7 


ads $7 


UIOS $5 


3ds $zZ 


WLOS FZ 


UIOS $Z 


1? 0g "80 


1p 91 “80 


1? 


++ eesrsssenosny UOqTI}0aN) 


****"-sn8010} SNpIpOAUIOT, 


****snosny snyqyeusoursaq 


“***snosnj sngyBusoursaqy 
snes eesivessis * -sisuoru 

-ayZo][8 snyousiqozydArZy 
“snyenue}}e sdesoyoviqeg 


“snyenue}je sdasoqorieg 


amonsnRUoooD oneor “(cep 
-ojny) sliqndn, sepreuy 
bagescnnsies + -gumniqdury 
rereretevetetatotar et emi0qss{qury 
spon annbade sees (our 


-oysA[qury) woparig, 
ees pasSdeaane arayarae 


te teseerereeess OIOxy, 


BoqoD steseseees uopatg, 
: seuro}sA[quy 


ETHEL BROWNE HARVEY 


Ee _——— — — oa rm x 
e213 "4 ‘IT 
‘LL yeuy “IyLUT “yoy TT, ‘Sao 
1d UIOS $% 
‘gp “Buy “1ylur “yory 16, ‘SeAQT JOO $7 
ogg “d ‘oy “Zzuy “quay G6, ‘SAARI PZ PU adds 5% “*BSO[NOVUL BAPUBUILE[RS 
(e1qnop 
OFF at Sout, 
“d “FL “qpeaqueg “org 76, ‘YIVY WO, | syuour -all08) WO FZ 
16 -3J9 ayuindos = do00 (a[qnop) ZI 
“d ‘yg “[OOZ “SSIM “}107 £6, ‘YB WOA | spudp puy spurqay, Pera (12 #2=) OZT | (19 BF=) Lit | dds eames GL | UsO[NOBUE BApPUBUAE[ BS 
| EEMEICA 
| -[0} a9 puv 
1g d ‘5 “zuy “yuuy 68. “TAVU | urnijeq? 
$1Z | ~1d9s1980} 9] Bo 
‘d ‘or “qayere “yduoyy os, ‘1QBY LUOS p% *MSO[NOBUL BAPUBULR[ERS 
6sed | 
‘6g “quay “LytuL “qory 18, ‘Suara oy 
SJunpeyyyjez pun 
uly ‘ZUBSQNS|[IZ, 28, ‘SUrururay yy 
1d | 
‘0g “}BUY “TALI “Yyory G8, ‘SULUIUTI| YT | LZ LI nora | UWO8 4 “COT BRO[NOBUT BIPUBUIE| ey 
et d ‘zg “uez 
qo ‘dx "JooZ “qo1y er, ‘Adueyy | ads 9] ceeees ss 8198 BIPUBULE[ES 
6cz 4 ‘p1INg [Olg | go, ‘Areuos} UO; PZI ads 57 “7 **“snotauld UOPOY yl q 
4sI 
UL paynqiysip Ay 
-]enboa ‘Aiwiounw 
-sadns = sulo1yo 
w101y sevard 
paqovjoq ‘pug ur 
payonyze 10 ddI 5] 
“qs—T ut ajod 04 
‘uO4o LOYZOUT OF 
cop d‘g‘ psovay ‘quuy ZI, ‘duty | poyouyye AK IO x LZI “+ SN SO[NOBUT SiLIN409 Ny 
182 “dl ‘92 ‘a[N] [9D BT [, ‘UuUBUrtexon JY Dds $7 “ess gigsED0y Ad oF[OW 
| 
OILANGD 
SONGUAIAY UGAUASHO SuNVWaY aig- ALAO- ANZ ALKO- LS] -ONTHLUVd ONY Sa10ad8 
a1oTdia 


penuyu0)—VIEIHdWV ‘I 


43 


METAZOA 


I} 


CHROMOSOME NUMBERS 


gcd ‘9 
*SOIOIUE “YRUY,P “"Qoy 


$F9 “4 ‘pz “zuy 4uuy 
6cl “A ‘1g “zuy “yuy 

gd “GT ‘9INT19D BT 
0zg “d ‘yz, “2uy yuuy 
£0c “4 ‘OT ‘AINT[ID VT 


er “d ‘2g 
*uod ya “dxa [007 "qo1y 


879 ‘d ‘pg “zuy ‘jeuy 
6cI “4 ‘1Z “zuy “qvuy 

Gd “6 ‘OINTTED BT 
oce *d “yy “zuy yeuy 
ste 4 ‘6E ‘AINTT9D BT 


£02 4 ‘OT ‘ANTI BT 


Toy “dl ‘6T “Zuy “[oog 


Tec “dl ‘92 ‘OINTPPD BT 


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JOURNAL OF MORPHOLOGY, VOL. 34, NO. 1 


BROWNE HARVEY 


ETHEL 


48 


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49 


CHROMOSOME NUMBERS IN METAZOA 


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53 


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56 ETHEL BROWNE HARVEY 


Ill. HISTORICAL AND CRITICAL 


The first attempt to count chromosomes in the Metazoa was 
made in 1878 by Selenka, in his “ Befruchtung des Eies von Tox- 
opneustes.’’ He gave the number in the cleavage cells as vary- 
ing between 14 and 24, a rather wide range and not very close to 
the mark (36). Retzius? in 1881 gave the number for Triton 
punctatus as between 12 and 16 in the somatic cells, also some- 
what far from correct (24). The next observation, by Flemming? 
on the salamander in 1882, was accurate and correct, and to him 
therefore belongs the credit of establishing a definite number of 
chromosomes for a definite species. He was also the first to at- 
tempt a count on human cells, given in the same publication. 
Very shortly after this, Strassburger,! ’82, gave definite and cor- 
rect numbers for several species of plants. Then came three 
papers giving the chromosome numbers in Ascaris, Anton Schnei- 
der’s ‘Das Ei und seine Befruchtung’ in 1883, Nussbaum’s® 
paper in 1884 and the very thorough and brilliant work of Van 
Beneden® which was published in 1883, although it did not ap- 
pear till April 1884. Soon after, still in the ’80’s, came Carnoy’s 
and Boveri’s papers on the nematodes and other works on the 
nematodes, molluscs and vertebrates. Since then, chromosome 
counts have been made by many observers on about 960 different 
species of animals. 

Several lists of chromosome numbers have appeared previously, 
the first by Wilson in ‘The Cell’ in 1900, a partial list which in- 
cluded about fifty species of animals and a few plants. In 1905, 
Enriques’? gave an incomplete list of numbers in animals, ex- 
pressing the different numbers in mathematical formulae, as 
powers.of 2 and 3. Montgomery® in 1906 gave a list which was 
supposed to be very nearly complete, but there are many omis- 
sions and a good many inaccuracies in the list. Montgomery’s 


2G. Retzius. 1881. Biol. Untersuchungen, p. 109. 

3 W. Flemming. 1882. Arch. mikr. Anat., 20, p. 1. 

4. Strasburger. 1882. Arch. mikr. Anat., 21. 

5M. Nussbaum. 1884. Arch. mikr. Anat., 23, p. 155. 

6H. Van Beneden. 1883. Arch. de Biol., 4, p. 265. 

7 Paolo Enriques. 1905. Archivio di Fisiologia, 2, p. 258. 

8T. H. Montgomery. 1906 Trans. Amer. Philos. Soc., 21, p. 97-162. 


CHROMOSOME NUMBERS IN METAZOA 57 


general conclusion was that chromosome number should be con- 
sidered as an important factor in taxonomy and that animals 
having widely different numbers should be placed in different 
genera. McClung has also been a strong advocate of the value 
of chromosome numbers in taxonomy. Della Valle’s® list in 
1909 is of little value, as it is a prejudiced one, given entirely 
with the object of showing that chromosome numbers are incon- 
stant and of little importance. ‘Two comprehensive lists of chro- 
mosome numbers in plants have appeared recently, Tischler’s!® 
and Ishikawa’s" in 1916. The latter is exclusively a list of 
numbers, his general conclusions being reserved for a further 
publication. ‘Tischler’s list is accompanied by able discussions 
and criticisms, his general conclusion being that it is still too 
soon to solve any large phylogenetic problems on the basis of 
chromosome investigations. It may be of interest as a compari- 
son with the work on animals to give some of his statements 
concerning numbers in plants. The Asco- and Basidiomycetes 
have very small numbers, the mosses and Gymnosperms in gen- 
eral small numbers, whereas the Algae, Pteridophytes and Angio- 
sperms have species with both small and large numbers. The 
Magnoliaceae and Nymphaceae (Angiosperms) and the Ophio- 
glossacea, Equisitaceae and Lycopodiales (Pteridophytes) have 
very high numbers, although not a great many species have been 
studied cytologically. Finally Winge” in 1917 has given an 
additional list in plants and has concluded from that and from 
Tischler’s list that the numbers in related species are in arith- 
metical progression,—e. g. the chrysanthemums with 9, 18, 27, 
36, and 45,—these arising by hybridization of species with lke 
numbers; and that in general numbers occur in factors of 2 and 3 


12 (2.2.3) occurring most frequently. 
A eursory survey of Tischler’s or Ishikawa’s list of numbers in 
plants and of my own list in animals is sufficient to show that very ~ 


9P. Della Valle. 1909. Archivio Zologico, 4, p. 1-177. 

1G. Tischler. 1916. Progressus Rei Botanicae, 5, p. 164-260. 

1 Mitsuharu Ishikawa. 1916. The Botanical Magazine, Tokyo, 30, p. 404- 
448, 

120. Winge. 1917. C. R. Travaux du Laboratoire de Carlsberg, 13. 


58 ETHEL BROWNE HARVEY 


closely related species may have widely different numbers, and that 
the numbers in related species are not usually in arithmetical ratio 
although occasionally they are, especially in plants. It is also 
apparent that numbers which are resolvable into factors of 2 and 
3 are of frequent occurrence, as one would expect since nearly half 
of the numbers between 2 and 20 (the most frequently occurring 
numbers) are resolvable into these factors. However, it is equally 
apparent that other numbers not resolvable into these factors are 
also of frequent occurrence. 

In using the present tabulation for any generalizations or con- 
clusions, several facts must be taken into consideration. Many 
of the observations recorded are of too early a date to be of much 
value. Other observations are contradictory and in many cases 
it is impossible to judge which is correct; this is largely due to 
difficult material and is especially true for the mammals, where 
for man the number of chromosomes varies between 8 and 48 
(diploid) according to different authorities. 


IV. CHROMOSOME NUMBERS 


In looking over the fore-going list, there can be no doubt to 
an unprejudiced mind that the constancy of chromosome 
numbers for a species is a fact, and that any variation in number 
for a definite species is an exception to the general rule. Such 
variations occur regularly in Notonecta insulata, Jamaicana uni- 
color and J. subguttata, and Hesperotettix viridis where two or 
more chromosomes may be united or separate; in species with 
supernumeraries (see p. 66); in cases where multiple groups occur 
(e. g. Culex pipiens, Notonecta, Anasa), and where fragmenta- 
tion has taken place (e. g. Ascaris, pig). A few sporadic varia- 
tions occur in certain species owing to the lack of conjugation of 
two univalents (e. g. Lygaeus turcicus, Coenus delius, Euschistus) 
and a few which have not been explained (e. g. Trichopepla, 
Lygaeus reclivatus which is now under investigation). When a 
range of numbers is given instead of one definite number, it is 
usually due either to the early date at which the observation was 
made or to difficult material rendering accurate counting 
impossible. 


CHROMOSOME NUMBERS IN METAZOA 59 


There is however a great range of numbers among the different 
forms of animals. As is well known, there is only one chromo- 
some in the haploid groups of Ascaris megalocephala univalens; 
some species of Gordius (Nematode) also are reported as having 
only one chromosome in the reduced groups and Styelopsis (As- 
cidian) as having only one in the spermatid. Indeed, according 
to Moore, ’93, there is only one chromosome in the oogonia of 
Apus (Phyllopod crustacean). Animals having only two chromo- 
somes in the haploid groups are: Ascaris megalocephala bivalens, 
Cyclops viridis brevispinosus, Pediculopsis graminum (arachnid), 
Icerya purchasi (Homoptera), Tetrastemma vermiculus (Ne- 
mertean), Vortex viridis and Paravortex cardii (Rhabdocoels). 
At the other end of the series are: two species of Cambarus (Dec- 
apod), with 104 and 100 (reduced), Artemia (Phyllopod) with 
84, Cancer and Hippa (Decapods) with 60, Astacus (Decapod) 
with about 58 and Nyssia (Moth) with 56. The number occur- 
ring most frequently among the forms investigated is 12; other 
numbers occurring very frequently are 6, 7, 8, 9, 10, 11 and 16. 

There is also often a considerable range in number among dif- 
ferent forms belonging to the same class, e. g. Nematoidea (1-24 
reduced), Aphidae (8-20), Copepoda (2-17). The classes show- 
ing the greatest constancy are the Acrididae (Orthoptera) and 
the Urodeles (Amphibia). The Diptera and the Nematodes 
have, in general, low numbers whereas the Decapods and Lepidop- 
tera have high numbers. 

A chromosome is really a compound structure, carrying many 
characters or genes which are themselves the elements of he- 
redity. However genes may arise, it is conceivable that in some 
cases one or more new genes may be placed in a chromosome 
without disturbing its integrity, the number of chromosomes in 
related species thus remaining the same. On the other hand such 
additional genes may disturb the existing complex and cause the 
whole mass of genes to be entirely redistributed, thus causing a 
change in chromosome number in nearly related species. Should 
a certain group of genes be placed in one chromosome in one 
species and in two in another, there would not be necessarily 
any difference in these two species. It would seem, however, 


60 ETHEL BROWNE HARVEY 


that there might be a tendency in any large group of related 
animals for the genes to segregate out according to some definite 
pattern. 

If, therefore, we make a list of all the chromosome numbers 
which have been reported for all the species of a certain class" 
of Metazoa, leaving out of account results which are conflicting 
or are too old to be accurate, we find that a certain number of 
chromosomes is characteristic of that class; that is, there are con- 
siderably more species having that number of chromosomes than 
any other number. This I will call the ‘type number.’ The 
type number of a class of animals is the most frequently oceur- 
ring number and may be considered tentatively as the funda- 
mental chromosome group. One or more chromosomes of this 
group or of a group derived from it may split into two (or more) 
parts, or they may fuse, thus causing the differences in number 
which occur in related forms. Whether the double groups which 
occur in closely related forms in many plants (e. g. Oenothera 
gigas and O. lamarckiana, Drossera longifolia and D. rotundi- 
folia, Spiranthes cernua and 8. gracilis ete.) and in some animals 
(e. g. the bivalens and univalens varieties of Ascaris megaloceph- 
ala, Helix pomatia, Echinus microtuberculatus, Artemia salina; 
Cyclops viridis and C. gracilis, Anopheles sp? and Anopheles 
punctipennis ete.) are derived in all cases by a splitting of all the 
chromosomes of the simple group, it is difficult to say. It may 
be, as suggested by Gates and supported by Strassburger that 
the double groups are derived in some cases at least, by a failure 
of cell division after the division of the chromosomes. A slight 
change in number may also be obtained by the disappearance 
of a whole chromosome, but this must be rare. All numbers 
referred to hereafter are the haploid numbers, and X, when 
present, is counted as one chromosome, even when it consists of 
several elements. 

The type number for the Coelenterates cannot be determined 
yet, as the data are too scanty and the results conflicting (e. g. 
Hydra). Possibly it is 12. For the Nemathelminthes, the type 


13 The term ‘‘class’’ is used loosely to include related families, orders or classes 
of ordinary classification. 


CHROMOSOME NUMBERS IN METAZOA 61 


number is 6, for the Echinoderms 18, for the Amphibia, the only 
class of Vertebrates satisfactory for generalizations, itis 12. For 
the Plathelminthes, the type number is 8, for the molluscs 16, 
for the Annelids 16. As one might expect in a group with so many 
distinet subgroups, the Arthropods have several type numbers. 
For the Crustacea it is 8 (the Malacostraca have higher num- 
bers), for the Hemiptera 7, Orthoptera 12, Coleoptera 10, Diptera 
6, Lepidoptera 31. It is of interest that the type numbers in the 
enterocoelous series are all multiples of 6 or 6, whereas those of 
the teloblastic series (except the tracheates) are multiples of 8 
or 8. It is also of interest that the molluses and Annelids which 
are so closely related have the same type number 16. The sub- 
groups which are degenerate or highly modified (e. g. Trematodes, 
Acanthocephala) usually do not have the type number of their 
groups. The data for the insects are the fullest and the most 
reliable and these offer the best study of changes in chromo- 
some numbers. 

The type number for the Hemiptera is 7 (haploid), including 
an XY pair or an X. Other numbers occur, all of which can be 
attributed to the fusion or splitting of chromosomes of the type 
group. The two Thyantas have been a puzzle, owing to their 
likeness in form and the wide divergence in chromosome number. 
Thyanta custator has a diploid number of 16, which would mean 
8 haploid including X or Y. Thyanta calceata has 28 in the 9 
diploid, 277 (owing to X being of two parts, Y of one), which 
would mean a haploid number of 142, 13. These numbers 
may be explained on the supposition that in Thyanta custator 
one chromosome of the type group has split in two, whereas in 
Thyanta calceata all of these have split except Y. Evidence of 
this splitting is given by X, which is of two parts, not always sep- 
arate in T. caleeata, each about the same size as Y and about 
half as large as the X in T. custator. This explanation may be 
expressed as follows: 


TYPE THYANTA CUSTATOR T. CALCEATA 


9 6+X 6+1+X 6+6+2X 
of 6+Y 6+1+Y | 6+6+Y 


62 ETHEL BROWNE HARVEY 


The same explanation holds for the two Banasas, one of which, 
B. dimidiata has a haploid number of 8 (like Thyanta custator) 
and the other, B. calva has a haploid number of 13 (like T. 
calceata, except that X is single). Euschistus crassus differs 
from five other species of the same genus which have the type 
number, in having one less. A comparison of Foot and Stro- 
bell’s figures of this species with their figures of the other species 
would indicate that a union of two large chromosomes has taken 
place. That the chromosome number does change by the fusion 
or splitting of chromosomes is shown by the Notonectidae. In 
three species (Browne, 716) there are 13 chromosomes, including 
two small ones, and in two species there are 12 chromosomes 
including only one small one. In a sixth species, N. insulata, the 
second small chromosome may be seen attached to another chro- 
mosome in the first division of some cells, while in other cells it 
is free, whereas in the second division it is permanently fused 
with the other chromosome. The d-chromosome of Nezara may 
also represent a stage in splitting or fusion, as suggested by 
Wilson. The fact that the X chromosome may consist of two 
or more parts, as in Syromastes, Phylloxera and some Reduvioids, 
would indicate that other chromosomes whose identity is not so 
easily established, may also split into two or more parts. 

The type number for the Diptera is 6, including XY which 
are, however, not always distinguishable. There is a decided 
tendency for the chromosomes to fuse, especially among the 
Drosophilas, most of which have 4 chromosomes, and the Culi- 
cidae, most of which have 3. In Anopheles punctipennis, (Ste- 
vens, 711), the X and Y are seen to be attached toanother pair 
in the diploid groups. Metz, ’16, has made a careful study of 
the chromosomes in the genus Drosophila, and has shown that 
many groups having a larger number of chromosomes contain 
rod shaped ones which are represented in groups with a smaller 
number by half as many V-shaped ones, two rods uniting to 
form aV. When the linkage groups in other species of Droso- 
phila have been worked out as.extensively as in D. melanogaster, 
it may be possible to establish the relation between chromo- 


CHROMOSOME NUMBERS IN MBTAZOA 63 


somes of different species and to determine whether fusion of 
chromosomes has actually taken place. 

The Orthoptera have been carefully studied by McClung and 
his students, and they have found a great constancy, especially 
among the Acrididae. The type group for the Orthoptera is 12 
including X. In Stenobothrus (Chorthippus), which has 9, 
Robertson has shown that three of these are really compound. In 
Chortophaga, there is a union of chromosomes in the diploid 
groups (McClung, ’14). In two species of Hesperotettix (Mc- 
Clung, ’17), X is fused with another chromosome, while in an- 
other species, H. viridis, it may be fused or free, and fusion may 
occur among other pairs, correspondingly decreasing the number 
of chromosomes. Also in Mermeria bivittata (McClung, °17) X 
is fused with another chromosome, while in other species it is 
free. Among the Locustidae, Jamaicana (Woolsey, ’15) shows 
steps in change of number. Some individuals of J. unicolor have 
31 rod-shaped chromosomes and 2 Vs. in the diploid groups; 
some individuals of J. subguttata have 33 rods and 1 V; other 
individuals of these two species and all of J. flava have all rods 
and a diploid number of 35. Robertson (716) has suggested that 
the 2 V-shaped chromosomes of Steiroxys are represented by 4 
rods in Decticus, giving a total of two more in the diploid group 
of the latter. An unpublished account of Mohr agrees with this 
in showing the two Vs in Steiroxys and he also shows them in 
Locusta viridissima (diploid number 29) whereas no Vs are pres- 
ent in several other genera whose diploid number is 31. Robert- 
son also points out that several Vs are present in Gryllus do- 
mesticus, which, if counted as twice as many rods, would give 
the number of chromosomes in G. assimilis; as no figures are 
given of the latter, this cannot be verified. 

There is therefore considerable evidence from the Orthoptera, 
Diptera and Hemiptera that chromosome numbers change by 
the splitting and fusion of chromosomes. The splitting of the 
sex chromosomes, which occurs in many other groups than the 
insects (see p. 66) indicates the probability that a similar proc- 
ess may take place among the other chromosomes. 


JOURNAL OF MORPHOLOGY, VOL. 34, No. 1 


64 


ETHEL BROWNE HARVEY 


Sex chromosomes in insects 


SEX CHRO- 
MOSOME 


GROUP 


FAMILY 


EXCEPTIONS 


TO 
POLE 
IN 


EXCEPTIONS 


LA 


Orthoptera 


Acrididae 
Blattidae 
Gryllidae 
Locustidae 
Phasmidae 


Gryllotalpa; XY 


Forficulidae? 


1st 


Hemiptera 
homoptera 


Aphidae 
Cercopidae 
Fulgoridae 
Jassidae 
Membracidae 


Enchenopa bi- 
nottata (X or 
XY?) 


1st 


Enchenopa bi. 
nottata (X, 
2nd; or XY, 

1st?) 


Hemiptera 
heteroptera 


Coreidae 
Hydrometridae 
Pyrrochoridae 


Some Metapo- 
dius (XY) 


Coleoptera 


Elateridae 
Lampyridae 
One Carabidae 
Some Chryso- 
melidae 
One Silphidae 


Diptera 


Anthomyidae 
Asilidae 
Bombyliidae 
Culicidae 
Drosophilidae 
Muscidae 
Sarcophagidae 
Statiomyidae 
Syrphidae 


Hemiptera 
heteroptera 


Belostomidae 
Galgulidae 
Lygaeidae 
Nabidae 
Nepidae 
Notonectidae 
Pentatomidae 
Reduviidae 


Oedancala (X) 


2nd | Archimerus (1st) 


1st 
Photinus, 2 sp. 
(2nd) 


1st 


2nd 


Tingis (Tingiti- 
dae) (1st) 


CHROMOSOME NUMBERS IN METAZOA 65 


Sex chromosomes in insects—Continued 


° 3 GROUP FAMILY EXCEPTIONS POLE EXCEPTIONS 
4s IN 
Fy 
XY | Coleoptera Buprestidae Ist 
Cerambycidae 
Cincindellidae 
Coccinellidae 
Lucanidae 
Melandryidae 
Meloidae 
Scarabaeidae 
Staphylinidae 
Tenebrionidae 
Some Carabi- 
dae 
Some Chryso- 
melidae 
One Silphidae 


V. HETEROCHROMOSOMES 


The most conspicuous heterochromosomes are the sex chromo- 
somes, an unpaired X or an unequal XY pair, which occur most 
characteristically in certain groups of insects. As may be seen 
from the accompanying table, an unpaired X occurs in practically 
all the Orthoptera and Hemiptera homoptera and in some families 
of the Hemiptera heteroptera and Coleoptera; an XY occurs in 
practically all the Diptera and in some families of the Hemiptera 
heteroptera and Coleoptera. In some families of the Coleoptera, 
an X is found in some genera and an XY in others. The sex 
chromosomes undergo their differential division in the /irst 
maturation in the Orthoptera, Hemiptera homoptera (except 
Enchenopa?), Diptera and Coleoptera (except Photinus), and in 
the second maturation division in the Hemiptera heteroptera (ex- 
cept Archimerus and Tingis). Sex chromosomes have not been 
described in any Hymenoptera, and are not of general occurrence 
in the Lepidoptera, though here an equal XY in the @ has been 
described in several species, an XY ini the @ in Phragmatobia and 
an X in the @ in Abraxas. Among the other Arthropods, an X 


66 ETHEL BROWNE HARVEY 


has been described in some Myriapods, most Arachnids (Araneida) 
and a few Copepods, but in no other Crustacea and not in Peripa- 
tus. Sex chromosomes have not been described in any Annelids, 
Coelenterates, Nemertines, Porifera, Rotifera, Protochordates or 
fishes and for only one Plathelminth. A few cases of their 
occurrence have been reported in the Echinoderms, molluses, Am- 
phibia, Birds, Reptilesand mammals. In the Nematodes they are 
of frequent occurrence. In all cases it isthe # which is hetero- 
zygous except in the Lepidoptera and birds and the genetic evi- 
dence agrees with the cytological. Although X and XY are typ- 
ically single elements, the X consists in some cases of two or 
more elements closely or loosely associated. An unpaired X of 
two elements has been described for Syromastes, two species of 
Phylloxera, Leptinotarsa, Agalena (spider), Hyalocylis (molluse), 
Schistosomum (Trematode), Rhabditis, fowl, and man and pig: 
an X of two elements accompanied by a single Y for Thyanta 
calceata, Gryllotalpa borealis, Cincindella and some of the 
Reduvioids. In other Reduvioids, Galgalus, Notonecta indica 
and Ascaris incurva, the X of an XY pair consists of three 
or more parts—of 8 in the last named, and in Ascaris lumbri- 
coides and A. canis, an unpaired X consists of five and six ele- 
ments respectively. A Y consisting of several elements has 
been described for Phragmatobia, and a Y of two elements in 
one testis of Odontota (Coleoptera). In a few cases the sex 
chromosomes are attached to another chromosome during part 
at least of their history: Ascaris megalocephala, Leptynia, Dix- 
ippus, Hesperotettix, Mermeria bivittata, Anopheles punctipen- 
nis and Necturus. 

Another set of heterochromosomes are the supernumerary 
chromosomes, which typically accompany sex chromosomes and 
divide in only one division, but are distributed irrespective of 
X and Y; they are constant in number in an individual, but differ 
in different individuals of the same species. They have been 
found in Metapodius, Euschistus variolarius, Banasa calva, 
Diabrotica, Ceuthophilus, Drosophila ornatipennis, Circotettix, 
Trimerotropis, Hesperotettix viridis, Tettigidea parvipennis, some 
spiders and Necturus. 


CHROMOSOME NUMBERS IN METAZOA 67 


A third set of heterochromosomes are the m-chromosomes, 
small chromosomes which remain condensed in the growth period 
and conjugate late. These are characteristic of the coreid Hem- 
iptera and occur in some of the Lygaeidae; they accompany an 
unpaired X except in Metapodius and Ichnodemus where they 
accompany an XY. 

Finally there are those chromosomes which are normal in beha- 
vior but consist of unequal parts. To this class belong the d- 
chromosome of Nezara hilaris and the compound chromosome 
of Notonecta insulata, which divide equally in the two divisions, 
and the unequal tetrads of some of the Orthoptera which divide 
into unequal parts in one division: Schistocerca (Hartmann, 
13); Acridium granulatus, Tettigidea parvipennis (Robertson, 
16); Arphia, Dissosteira, Brachystola (Carothers, 713); and Phry- 
notettix (Wenrich, 716). 


Princeton, N. J. 
December, 1918. 


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WUD UTI AG RSID RE AA eae alias, Diy, A, 


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PUBLICATIONS 


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Department of Anatomy 


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NEW Y ORK CITY 


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CONTENTS 


Being reprints of studies published in 1921-1922. 


HUMAN TYPES AND GROWTH REACTIONS. 
By Charles R. Stockard. 


Am. Jour. of Anatomy, Vol. 31, 261-288. 


MORPHOLOGY OF CYSTIC GROWTHS IN THE OVARY AND 
UTERUS OF THE GUINEA PIG. 
By George N. Papanicolaou and Charles R. Stockard. 
Proc. Soc. Exp. Biology and Medicine, Vol. XIX, 401-402. 


EXPERIMENTAL RESULTS BEARING ON THE ETIOLOGY OF 
CYSTIC GROWTHS IN THE OVARY AND UTERUS OF 
THE GUINEA PIG. 
By George N. Papanicolaou and Charles R. Stockard. 
Proc. Soc. Exp. Biology and Medicine, Vol. XIX, 402-403. 


STUDIES ON THE GONADS OF THE FOWL. III. THE ORIGIN 
OF THE SO-CALLED LUTEAL CELLS IN THE TESTIS OF 
HEN-FEATHERED COCKS. By José F. Nonidez. 

Am. Jour. of Anatomy, Vol. 31, 109-124. 


THE FORMATION OF THE ASTER IN ARTIFICIAL PARTHENO- 
GENESIS. By Robert Chambers. 
Jour. of General Physiology, Vol. IV, 33-39. 


STUDIES ON THE ORGANIZATION OF THE STARFISH EGG. 
By Robert Chambers. 
Jour. of General Physiology, Vol IV, 41-44. 


THE EFFECT OF EXPERIMENTALLY INDUCED CHANGES IN 
CONSISTENCY ON PROTOPLASMIC MOVEMENT. 
By Robert Chambers. 
Proc. Soc. Exp. Biology and Medicine, Vol. XIX, 87-88. 


MICRODISSECTION STUDIES, III. SOME PROBLEMS IN THE 
MATURATION AND FERTILIZATION OF THE ECHIN- 
ODERM EGG. By Robert Chambers. 

Biological Bulletin, Vol. XLI, 318-350. 


MEROGONY EXPERIMENTS ON SEA-URCHIN EGGS. ’ 
By Robert Chambers and Hiroshe Ohshima. 
Proc. Soc. Exp. Biology and Medicine, Vol. XIX, 320-321. 


t 
APPARATUS FOR MICRO-MANIPULATION AND MICRO- 
INJECTION. By Robert Chambers. 
Proc. Soc. Exp. Biology and Medicine, Vol. XIX, 85-87. 


13. 


14. 


15. 


16. 


17. 


NEW APPARATUS AND METHODS FOR THE DISSECTION AND 
INJECTION OF LIVING CELLS. By Robert Chambers. 
The Anatomical Record, Vol. 24, 1-19. 


A MICROMANIPULATOR FOR THE ISOLATION OF BACTERIA 
AND THE DISSECTION OF CELLS. By Robert Chambers. 
Jour. of Bacteriology, Vol. VIII, 1-5. 


A MICRO-INJECTION STUDY ON THE PERMEABILITY OF THE 


STARFISH EGG. By Robert Chambers. 
Jour. of General Physiology, Vol. V, 189-193. 


A NOTE ON THE ENTRANCE OF THE SPERMATOZOON INTO 
THE STARFISH EGG. By Robert Chambers. 
Proc. Soc. Exp. Biology and Medicine, Vol. XX, 137-138. 


DISTURBANCES IN MAMMALIAN DEVELOPMENT PRODUCED 
BY RADIUM EMANATION. By Halsey J. Bagg. 
Amer. Jour. of Anatomy, Vol. XXX, 133-161. 


A SIMPLE APPARATUS FOR DELICATE INJECTIONS. 
By Alice L. Brown. 
The Anatomical Record, Vol. 24, 295-297. 


CHAMBERS’ MICROMANIPULATOR FOR THE ISOLATION OF A 
SINGLE BACTERIUM. By Morton C. Kahn. 
The Jour. of Infectious Diseases, Vol. 31, 344-348. 


AUTHOR'S ABSTRACT OF THIS PAPER ISSUED Reprinted from Tar AMPRICAN JOURNAL OF 
BY THE BIBLIOGRAPHIC SERVICE, JANUARY 15 Anatomy, Vol. 31, No. 3, January, 1923 


HUMAN TYPES AND GROWTH REACTIONS 


CHARLES R. STOCKARD 
Cornell University Medical College, New York City 
THREE FIGURES 


The following article is presented as an introduction to a series 
of studies now under way which bear on the origin and develop- 
ment of certain well marked types found among the mammals. 

In a previous communication, ’21, the significance has been 
pointed out of the influence of modifications in developmental 
rate upon embryonic structure. It has also been shown in the 
case of chemically treated male mammals how certain questions 
of inheritance are involved in analyzing modified structural 
reactions. These considerations may now be extended to many 
problems of fetal and post-natal growth and to an interpretation 
of several peculiar structural patterns and conditions seen in 
man and other animals. In order to make clear the situation 
extreme structural peculiarities may be briefly reviewed and 
from these we shall attempt to determine the factors concerned 
in regulating the less marked and more usual human types. 
The growth rate of the individual depends upon both internal 
and external factors—hereditary composition and functional 
activities—the latter being modified largely by surrounding 
conditions. 

To illustrate the problem we may begin with the familar 
condition presented by the thyroid cretin. Is the absence of the 
thyroid hereditary or due to an arrest in development or to both? 
Such individuals so far as is known may apparently arise from a 
parentage with subnormal or poorly functioning thyroid glands. 
However, no one has yet analyzed this condition sufficiently to 
determine exactly its genesis. It is not known, for example, 
whether the subnormal thyroid of either parent affects the germ- 


261 


262 CHARLES R. STOCKARD 


cells so as to modify those genes which determine thyroid develop- 
ment and structure. We may have reasons for believing that 
the thyroid of the embryo and fetus is capable of developing 
independently of the thyroid hormones in the blood of the 
mother, but there has not been sufficient experiment to make it 
certain that the mammalian embryo does not need the thyroid 
stuff in the mother’s blood for its proper growth functions. 

We may simply state that the cretin is a thyroid dwarf which 
has attained an incomplete degree of development and cannot go 
further without the application of thyroid stuff. This we know, 
but why the condition arises we have yet to determine. It is 
obvious that the origin of the condition is the more important 
thing to know and the mere treatment of the abnormal end pro- 
duct can supply little knowledge of its cause. 

The one instructive thing to us at present is that the human 
child without a thyroid gland can only develop to the stage 
shown by the typical cretin. This stage might be called the 
early larval condition of man. The most definite and clear cut 
experiments done on the influence of the internal secretions in 
development are those which show that the thyroid is essential 
for the metamorphosis of the amphibian larva into the adult stage. 
The human cretin without thyroid will not metamorphose or 
develop into an adult. Also the human individual with a sub- 
normal amount of thyroid may be expected to be more child-like 
and less adult than the individual with a normal supply of the 
thyroid secretion. There is a great bulk of evidence, impossible 
to mention within the limits of this introductory article to show 
that the amount or quality of thyroid secretion present in the 
developing individual is an enormously important element in 
determining the rate of its growth. The thyroid effects the 
growth rate by primarily determining or affecting the rate of 
metabolism. The significance of this we have pointed out in 
the articles cited above by showing that the rate of development 
is the most profound factor in determining the quality and type 
of structural production. 

The cretin is an abnormal or pathological individual. Its 
conditions would preclude the breeding of a race of such speci- 


HUMAN TYPES AND GROWTH REACTIONS 263 


mens. It is not in itself a type, it is an arrested child stage but 
the cretin furnishes an extreme growth condition which is most 
helpful in fully appreciating the influence of the thyroid gland on 
the growth of the so-called normal types of men. 

If space permitted we might review a number of peculiar 
specimens that could in some manner be attributed to the unusual 
action of several other glands of the body which modify growth 
by effecting the rate of metabolism. Many striking pathological 
growth reactions are familiar to all, so we may proceed at once 
to consider certain strange and unusual individuals constituting 
real or actual types, which should not properly be classed as 
pathological. 

The various dwarf types of man are instructive in a study of 
growth reactions and structure. Many of the dwarfs are in 
most respects normal and they simply differ from the common 
type as the small breeds of several domestic animals differ from 
the large. The cause of such types we can determine by con- 
sidering both the genetic and developmental modifications 
possibly concerned in their production. The statement that 
this may be the same large problem as the origin of any species 
or type should not be discouraging since certain peculiar types 
may be more likely to reveal their causes than the commoner 
long existing ones. 

The simplest dwarf type of man from our present growth 
standpoint is the small African pygmy. These are» childish 
Africans of very low intelligence as well as small size. When 
one examines pygmies closely, particularly their physiognomy, 
a striking similarity of features is found between them and the 
thyroid cretin, in both the mouth, nose, forehead and brow are 
closely similar in form and proportions. On administering 
thyroid to the cretin these features become rapidly modified by 
progressive development and approach the normal picture. 
One can scarcely resist the temptation to suggest the experiment 
of administering thyroid to the young pygmy. The pygmy may 
be looked upon as a not fully metamorphosed large negro, a mild 
and slow growing condition of cretinism. They are not true 
dwarf types but certainly people with general growth arrests. 


264 CHARLES R. STOCKARD 


They are quite cretinous in their behavior which is certainly 
unlike the fully grown African of the coast countries surrounding 
them. The pygmy is the most primitive of African savages in 
his behavior, building no huts and having only the crudest tribal 
organization. The fact that they are capable of reproduction 
does not argue against their partial cretinism since arrests are 
so commonly known in which all organs are not equally affected. 
With our present knowledge it is impossible to say whether the 
pygmies are a true genetic breed or whether they simply live in 
an environment unfavorable to complete development. On 
removal to the coast regions they might within a short time 
become taller and better developed. 

There are other true human dwarfs, however, about which we 
may speak with a greater degree of certainty since they have been 
studied for a long time and a considerable mass of data exists 
regarding them. Much of the data has been well presented by 
Rischbieth and Barrington of the Francis Galton Laboratory 
for National Eugenics in England. These workers, however, 
discarded one of the most valuable means of understanding 
such human types by deciding that they show only an apparent 
resemblance to the similar types found among lower animal 
breeds. This attitude I believe is a vital mistake since certain 
animal breeds not only closely resemble the human dwarfs 
but very probably arise and developed in an exactly similar way. 
The understanding of the origin and development of the one 
is certainly of the highest value in a biological consideration of 
the other. 

Among the true dwarfs the achondroplastics may be discussed 
first since other dwarfs also often show some degree of achondro- 
plasia. The typical achondroplastic dwarf is very short and 
stocky, the head and trunk are large being often as big as those 
of an ordinary individual, but the extremities are short and some- 
what twisted, on account of the peculiar development of the 
long bones. The muscles are short and thick, standing out in 
a knotty fashion, particularly on the extremities. They are 
very active, often acrobatic and quite bright and intelligent. 
Their head growth and face shape is characteristic. The base 


HUMAN TYPES AND GROWTH REACTIONS 265 


of the skull is short. The ossification and cessation of growth in 
the basal cartilage between the occipital and sphenoid bones fre- 
quently occurs before birth, instead of after twenty-two years as is 
the usual case. This failure to continue the growth of the skull 
base after birth renders the head short and consequently dispro- 
portionately wide. Such dwarfs are, therefore, brachycephalic. 
The lack of growth at the base of the skull also causes a failure 
to push forward the nasal septum. The nose bridge remains 
low and often actually sunken in below the overhanging forehead. 
The upper jaw likewise is not carried the usual distance forward 
so that the fully developed mandible projects beyond the maxilla, 
the teeth do not properly lock, and the ‘undershot’ jaw condition 
prevails. The entire face is flat, vulgarly termed a ‘dish-face.’ 

Achondroplastic dwarfs have been accounted for in many 
ways on the basis of disturbance in the glands of internal secretion 
which regulate growth. Several have attempted to associate 
their entire peculiar make up with an unusual action of the 
hypophysis. But many of these dwarfs seem to show some 
peculiarity in the structure of the thyroid and for this reason 
they have been thought to be the result of unusual thyroid 
function. Sir Arthur Keith, ’22, in his very suggestive Herter 
Lectures at Baltimore, for instance, has referred to them as a 
“minus-thyroid” condition. 

Be the condition of the thyroid as it may, the primary cause for 
these dwarfs is more fundamental and deep-seated than the 
glands and the probable effects of the glands on growth are 
secondary even though their secretions may induce the peculiar 
forms. We know from the monograph by Rischbieth and 
Barrington that the condition is already present far back in the 
early fetus. The extremities are originally far too short for 
the trunk size and length and the early bones are typically 
peculiar, not as in rickets or any other known condition. This 
definitely peculiar growth continues throughout development 
until the adult dwarf condition is attained. The question may 
arise as to whether their dwarf condition may be due to peculiar 
glands in the mother. That this cannot be true is shown clearly 
in the pedigree tables of Rischbieth and Barrington where the 


266 CHARLES R. STOCKARD 


condition is transmitted by the father.. The male could only 
transmit such a state to its offspring through the spermatozoon, 
and if this be done the condition is truly hereditary. Rischbieth 
and Barrington record the case of a typical achondroplastic 
dwarf man married to a normal woman. ‘Two children resulted 
from this combination, a boy and a girl that lived to become 
adult, and both were completely achondroplastic dwarfs closely 
resembling the father. There are other such cases on record as 
well as similar dwarfs produced by achondroplastic mothers. 
Ordinary parents may sometimes produce a typical achondro- 
plastic dwarf along with several normal sisters and brothers. 

One strange fact regarding these dwarfs is that they frequently 
die at birth from no known cause. This and their sporadic 
occurrence in families make it seem possible that this complex 
may arise as a Mendelian dominant that is only viable in the 
heterozygous form. When there is a double dose of the dom- 
inant and the zygote is, therefore, homozygous a lethal expression 
follows and the child is incapable of living after birth. Morgan 
and others have pointed out several cases of this homozygous 
dominant lethal in their genetic studies. Such cases show why 
although a character may be dominant it is unable to establish 
itself in a homozygous condition and may never become abundant 
in the race. 

There are only isolated facts from human cases of achondro- 
plasia but in the light of our present knowledge of inheritance and 
development they leave little doubt that the fundamental or 
primary cause of the complete achondroplastic dwarf condition 
is a germinal mutation or sport and the condition is definitely 
hereditary. The expression of this condition is probably due 
to an inherited modification of a gland regulating growth, the 
hypophysis. 

Turning from man to the domestic animals, we have on hand 
an abundance of material for the study of achondroplastic dwarf 
conditions. Achondroplasia occurs in various degrees among 
several of the domesticated species, but the most varied and 
remarkable examples of the condition exist among the fancy 
breeds of dogs. With these animals nature and man have per- 


HUMAN TYPES AND GROWTH REACTIONS 267 


formed an enormous experiment lasting in some cases for hun- 
dreds of years and today they present the investigator with 
material of remarkable value for a real analysis of the processes 
of inheritance and growth concerned in mammalian achondro- 
plasia. There are breeds of dogs, such as the small French bull, 
which show a complete achondroplastic condition exactly dupli- 
cating the typical human achondroplastic dwarf. The head 
form, extremity and trunk conditions are all closely comparable. 
Other dogs show a marked skull and head type without fully 
developed achondroplasia of the extremities. Still others show 
the most pronounced condition in the extremities with the 
usual head form, as seen in the Dachshund, Scottish terrier, 
Bassett and others. Finally there are several dog breeds, 
Pekingese and some peculiar terrier forms, combining various 
degrees of achondroplasia with an ateliotic or midget condition. 
This combination with achondroplasia is also very often seen 
among human midgets. 

If we choose either the best type British or French bull-dog we 
find on close study and comparison point for point a resemblance 
to the stocky human dwarf. All have the short wide head with 
flat muzzle giving the sunken ‘dish-face.’ The base of the skull 
has failed to attain its usual length through failure in growth of 
the basi-sphenoid and more especially the basi-occipital bone. 
A shortened disproportionately wide condition necessarily fol- 
lows. The nasal septum is not carried fully forward and the 
root of the nose is flat or actually sunken giving a marked depres- 
sion below the forehead. The maxilla is also for associated 
reasons in an unusually posterior position and the unaffected 
mandible, therefore, projects in front of the maxilla. The teeth 
fail to meet in the normal biting position and the common under- 
shot jaw condition exists. The entire facial expression and head 
carriage of the bull-dog and the stocky human dwarf are strik- 
ingly the same and are due to the same structural background. 
The condition of the extremities in these dogs and in the dwarf 
are also structurally alike. The proximal segment in all four 
extremities is very short and somewhat bowed or bent being the 
most modified segment of the limb. The fore-arm and leg seg- 


268 CHARLES R. STOCKARD 


ments are also short and somewhat bowed though not so short- 
ened as the arm and thigh. The hands and feet are very much 
of the ordinary shape and proportions and are about as large 
as those of the common large breeds of the species. The muscles 
of the extremities are short and knotty and the entire animal 
makes a most muscular and stocky appearance. The trunk 
as well as the head may be about as large as in the ordinary ani- 
mal. These points are more evident in the British than in the 
French bull, the latter being more of a terrier. The external 
genitalia are generally of normal size but the extremely short 
thighs give the male genitalia by contrast an abnormally exag- 
gerated appearance. 

A most striking and convincing comparison is obtained on 
holding a fine pointed French bull dog up on its hind feet by the 
side of a human achondroplastic dwarf, or the identity of the 
types is equally well shown by placing both the man and the dog 
down on all fours. 

Breeders of various achondroplastic varieties of dogs often 
admit that a considerable mortality occurs among the pups at 
birth and also recognize that they are difficult to feed and rear 
during the first few weeks after birth. Following this period 
the survivors are strong, long-lived and hardy. These observa- 
tions scarcely warrant in themselves scientific consideration, but 
they suggest the desirability of obtaining comparative early 
mortality records, since the high birth mortality of human achon- 
droplastic dwarfs seems well established as well as the record of 
hardy long lives for those that do survive. As mentioned before 
such records may indicate that the survivors in these breeds are 
possibly heterozygous for a dominant achondroplastie condition 
and that homozygous achondroplastics with the two dominant 
doses have some lethal complex that causes their elimination or 
death about the time of birth or before. 

It may be possible, however, that the double dominant condi- 
tion in man would give a lethal effect, while among the achon- 
droplastic dogs this would not necessarily be the case, and the 
bull dogs might thus be a pure homozygous breed. Yet their 
out-crosses do not indicate them to be homozygous, provided 
achondroplasia is a dominant condition. 


HUMAN TYPES AND GROWTH REACTIONS 269 


The general attempt to explain achondroplasia as due to 
peculiar actions of the glands of internal secretion, either the 
hypophysis, thyroid or any other, leaves out of account the 
numerous examples of partial or localized achondroplasia so com- 
monly seen in man and:the lower animals. If the internal 
secretions in the blood produce these peculiar growths, why do 
they not always act on all similarly growing parts in a similar 
manner? How is it possible that one humerus may be typically 
short and twisted and all other long bones unaffected? How 
does it happen that the dachshund and the bassett hound have 
the most pronounced achondroplastic legs and yet the heads of 
these dogs are like the ordinary head of the common large hounds. 
Numerous other examples of partial and localized achondroplasia 
could be cited which make it difficult to account for the condition 
as simply due to unusual internal secretions. Yet we might 
imagine that certain tissues or parts such as one humerus could 
inherit a peculiar sensitiveness to certain internal secretions 
which would cause this one bone to respond differently from all 
others, though such an hypothesis is difficult to conceive. 

All that we know from the breeding records of dogs of the 
above varieties and the fragmentary data from human dwarfs 
indicates most strongly that these are true varieties or breeds of 
animals that probably have arisen by some mutation or sport 
from the ordinary form. They breed fairly true to type but not 
perfectly so since great variations in the condition constantly 
arise and for this reason most careful selection is necessary to 
maintain a high standard stock. Such selection is of course 
entirely unnecessary in maintaining the stock of a wild species 
of the wolf or fox, for example. 

The point of much importance to recognize is that these 
strange forms, in spite of some apparent evidence to the con- 
trary, may be transmitting peculiarly acting glands of internal 
secretion. This type of gland is being inherited, and possibly 
causes the production of the strange structural type. The types 
are definitely the result of clean cut growth reactions and these 
might be secondarily brought about by the primarily strange 
type of internal gland. There is much argument both for and 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 31, No. 3 


270 CHARLES R. STOCKARD 


against such a position on the basis of apparent facts available 
at present. It must be recognized that these problems are still 
largely unsolved and the remarks on the cases above, as well as 
what is to follow, are given as a conception of the situation, a 
conception which we hope to either prove or modify by the 
detailed data now being accumulated in our studies. 


ATELIOSIS 


A final dwarf type of interest from the standpoint of growth 
and structure is the tiny ateliotic midget. Some of the most 
celebrated dwarfs have been of this type. They differ from the 
achondroplastics in having the bodily proportions and general 
outline of the normal large individual. Their trunk and arms and 
legs may all be of the proper proportionate length. The head 
is small and the body is often delicately and gracefully formed. 
A sexual infantilism with poorly developed genitalia is very 
common in the ateliotic, yet many are normally developed 
sexually and there are numerous records of their having produced 
offspring. 

A midget full grown may resemble in head and body size a child 
of six. Yet they are frequently bright and very intelligent. 
They seem to grow normally for five or six years after birth and 
then stop. The wrist and ankle cartilages fail to ossify, the 
epiphyses of the long bones do not fuse with the shaft and the 
skeleton is about like that. of a child of seven. In nature this 
is clearly a genetic condition. One or more midgets may be 
born from perfectly full grown parents along with full size 
brothers and sisters. We are investigating a case of three midget 
sisters who claim to have two large brothers and two large sisters 
who were born alternately with them from large parents, the 
father being Dutch and the mother German. 

Although the ateliotic condition may occur in the simple form 
it very frequently has associated with it a certain degree of 
achondroplasia. When one examines a great number of midgets 
it will be found that many of them have unusually short arms, 
the fingers reach only to the hip joint or great trochanter instead 


HUMAN TYPES AND GROWTH REACTIONS 271 


of striking almost half way down the thighs. The faces of these 
are closely alike and usually sunken at the nasion just as in the 
achondroplastic, though not so pronounced: but the undershot 
jaw condition is rarely present in the midget. 

Again we find dogs of exactly similar type, for example, the 
head and face of the King Charles Spaniel is in shape, outline 
and expression almost a picture of the human midget. The 
psychology of the two is much the same for general behavior 
reactions. The extremities and head of the King Charles 
Spaniel are also quite achondroplastic as is so frequent in the 
human midget. There are several other tiny breeds of dogs 
which are slender and have a sharp muzzle, real ateliotics, with 
no achondroplasia. Many of these tiny dog breeds also seem to 
have some infantilism and individuals are frequently sterile. 

Bantam chickens and other miniature animal forms are 
probably the same in origin and type as the human ateliotic 
dwarf. 

All of these dwarf forms give us valuable suggestions for an 
interpretation of many usual types of growth and development. 
And when a study of them is associated with an investigation 
of the opposite extreme, the giants, a remarkably complete series 
of growth and structural conditions is supplied. 


EXCESSIVE GROWTHS 


The giants and acromegalic individuals are so well known from 
many recent studies that only passing mention need here be 
given. I simply wish to point out certain features of these types 
before attempting a final estimate of what may be considered 
the usual human types from a growth and development 
standpoint. 

The fine youthful giant is a properly proportioned lithe and 
active individual often with a well chiseled prominently featured 
face. This is simply what might be recognized as a splendid 
human specimen of gigantic size. The giant forms of lower 
animals are frequently of this type of supernormal growth with 
proper proportions. Among the dogs, the Great Dane is a 


272 CHARLES R. STOCKARD 


splendid example of this kind, alert and youthful with no acro- 
megalie symptoms. 

Just as most human midgets show some achondroplasia, so do 
most human giants present some degree of acromegaly. Achon- 
droplasia and acromegaly are apparently opposite reactions in 
bony growth and both conditions are probably associated with 
definite hypophyseal states. It may safely be added that the 
usual or so-called normal bone growth and the ordinary hypo- 
physeal condition stand just between the two. This strikingly 
illustrates the delicacy of the growth balance in the normal 
individual,—if the scales waver in one direction a countenance 
mildly suggesting acromegaly accompanies a large framed body, 
whereas a tip the other way gives a flattened face on a small 
person with a somewhat long body and short extremities. 

The usual giant is particularly apt to become acromegalic as 
he grows older. It should be recognized, at the same time, that 
most normal people, as well as other animals, also show various 
slight degrees of acromegaly as they approach middle life. The 
general thickening up and increased body weight after the age 
of thirty-five is something of this nature though complicated 
in other ways. Many pronounced acromegalies were short and 
stocky as young men. The diseased condition of the hypophysis 
may come on gradually after the long bones have ossified their 
growth cartilages so that no further increase in height or length 
can then take place. The bones merely become larger and 
heavier and the features thicken to greatly exaggerated size. 
Early photographs of patients taken before acromegaly had 
developed generally show a face which, of course, would and did 
pass as normal but which has a very decided touch of those 
symptoms which later develop in so pronounced a fashion. In 
other words, as a rule an uncertain hypophysis that is later to 
become deranged fails originally to give an extremely delicately 
modeled face. 

There are certain human types presenting what might be 
called anatomical or normal rather than pathological acromegaly. 
These types may be pronounced in a certain family or even in 


—— 


a a 


HUMAN TYPES AND GROWTH REACTIONS 273 


a localized region where pathological acromegaly is not unusually 
abundant. This is also true of lower animals. There are some 
really acromegalic species. Several of the large dogs, the St. 
Bernard, the mastiff and others, show recognized symptoms of 
acromegaly along with gigantism. But in the dogs as in man 
acromegaly may exist apart from gigantism. The blood-hound 
is a splendid example of the acromegalic type without gigantism 
and his facial expression and general appearance is closely 
similar to the human acromegalic. 

Many breeds of dogs have been selected and perpetuated for 
centuries. The breeders selected those specimens having certain 
well pronounced characters and features that they desired to 
preserve. They also thought that these characters were the 
things primarily inherited but we may be certain for many 
breeds at least that the visible marks for which they are selected 
are simply the external symptoms or expressions of a particular 
quality or action of glands of internal secretion. These peculiar 
glands are the fundamental things that the breeders have uncon- 
sciously been selecting, since when they choose animals with the 
desired structural symptoms they also blindly choose the glandu- 
lar cause. Thus a certain gland type may be inherited and the 
dog breeders have produced the experimental proof of this over 
and over again. 


ACUTE STRUCTURAL REACTIONS 


Extremely abnormal or pathological cases could be cited in 
great numbers to show the acute action of the glands of internal 
secretion on growth and structural responses, but many of these 
are so well known as to be taken for granted in following the 
present discussion. However, before we pass to a brief consid- 
eration of the normal human being, one of the most striking 
experimental cases will be cited to illustrate the remarkable 
possibilities of these glands for determining personal types. 

In the breed of fowls known as the Golden Seabright Bantam 
the plumage of the cock is almost exactly like that of the hen in 
both coloring and feather form. The rooster’s head, however, 


274 CHARLES R. STOCKARD 


is decorated with the usual large fully developed comb and 
wattles of the male. Morgan, 719, has studied the conditions 
found in these chickens and has obtained most striking and 
valuable results. He finds that when both testicles are com- 
pletely removed from the hen-feathered rooster, a marked revo- 
lution in its appearance takes place at the next molt. The 
castrated male now develops the fine plumage of the rooster with 
plume-like hackles and saddle feathers and long sickle plumes in 
the tail. In plumage and color the capon no longer resembles 
the hen but is a perfectly feathered male. This is not all that 
has taken place. The large comb and wattles have now degen- 
erated in size until the head is much like that of the hen in 
appearance. The removal of the gonads has induced a sudden 
and extensive structural change. Morgan states, ‘Feathers 
that may have started their development at the time of the opera- 
tion show the old influence at the tip of the feathers and the 
new one in the rest of the feather. The change is abrupt, 
although the transition is perfect.” We could have no more 
striking evidence of the important influence of the gonads on 
bodily structure and appearance, nor of the changed action of 
something else in the body which calls forth the fine plumage 
after the gonads have been removed. We might suppose that 
since the comb and wattles were large before castration, their 
condition was due to some action of the testicles, but the sub- 
sequent feather development following castration is certainly 
not the result of genadal secretion; it is very probably due to a 
change in hypophyseal action after removal of the gonads since 
this gland seems to modify the growth of hair and feathers in 
many individuals after the gonads begin degeneration. 

Morgan has shown that the hen-feathered condition of the 
male is inherited in a perfectly definite way and is due to two 
dominant Mendelian genes. In other words this means that 
the strange gland condition which underlies the peculiar plumage 
of these birds is definitely inherited. The character of the 
plumage is merely the symptom indicating the presence of the 
unusual gland action. 


HUMAN TYPES AND GROWTH REACTIONS 275 


This experiment is of further interest to us in connection with 
certain general changes that occur during the life of human 
individuals. It will be found on observing a group of men that 
at about the age of thirty-five or a little later a coarse growth of 
hair begins on parts which in youth were not so hairy or on 
which the hair was fine. Strange coarse hairs grow in the eye- 
brows, on the pinna of the ears and at the entrance to the external 
meatus. The beard becomes coarser and heavier, and coarser 
hairs develop on the trunk and extremities. The man now 
possesses a more pronounced male hairiness than he did when 
under thirty. On first thought one might consider him to have 
fully arrived at the completely developed male state. This is 
not correct, however, since the gonads of such an individual 
have actually begun to decrease in their sexual power. The 
coarse hair growth is a plumage expression resulting from a 
decline in the male gonadal activity rather than the attainment 
of its zenith. The change is gradual but of exactly the same 
nature as that suddenly produced in the Seabright bantam by 
castration. In man this is truly to be,recognized as an early 
indication of senility. It is, however, peculiar that in man the 
hair growth reaction is not called forth by early castration but 
only follows a gradual degeneration of the gonads after middle 
life. 

It is also known that castration generally favors an extra 
accumulation of fat in mammals. The ox is more readily 
fattened than the bull. Here again in man at about the same 
time the coarse hair growth appears an accumulation of fat 
also takes place. The anterior abdominal wall often becomes 
prominent and a decided increase in waist measure occurs. These 
mild symptoms seem to accompany the physiological castration 
which is gradually taking place. The structural changes occur- 
ring at the menopause in women are similar in character but more 
pronounced and rapid in their development than the above 
changes in men, and very probably because the physiological 
castration in women is much more complete at this time. 

Thus we see that all normal human beings are experiencing 
developmental and growth changes which are noticeably due to 


276 CHARLES R. STOCKARD 


the usual fluctuations in function of the organs of internal 
secretion. The existence of these very evident changes serve to 
illustrate the fact that if we study closer the entire developmental 
history of the individual it will be found that growth and expres- 
sion are constantly being influenced and molded by the amount 
and quality of the internal secretions that have been inherited 
in the breed to which the individual belongs. 


NORMAL HUMAN TYPES 


When the reader reviews his personal acquaintances or any 
group of human beings it is evident that many of them show 
slight degrees of one or another of the peculiar structural condi- 
tions which we have surveyed above. There are those with 
short arms and flattened faces, others with heavy features and 
large wide hands, others with long narrow faces and delicate 
hands, some with rough hairy skin and others with smooth, 
until we may simply admit, that each is sufficiently peculiarto 
be distinguished from all the rest and addressed by name. We 
also recognize certain family and racial resemblances which aid 
us in classing or grouping our acquaintances. Such resemblances 
have been more or less taken as a matter of course and simply 
explained as being inherited, but actually to what are they due? 
Anthropologists have never explained why some heads are long 
and others wide, although they lay great stress on the fact that 
such is the case. Would it be possible for any baby to grow either 
a long or a wide head? 

Does a great heterogeneous population lend itself to a system 
of classing or grouping from the structural standpoint? If such 
is possible to what cause or causes is this division into structural 
groups due? The first question is very old and almost endless 
fanciful groups and classes have been arranged by various 
students of the subject. As a rule each has begun with a few 
classes, but later these have been divided until in the end a return 
to confusion resulted. 

One premise we may depend upon, namely, that all structural 
form in animals results from processes of unequal growth. Equal 
growth in all directions from the original spherical egg would 


HUMAN TYPES AND GROWTH REACTIONS 277 


result in a sphere. Spheres may differ in size but in form all 
are alike. Should the growth processes be exactly the same 
in two specimens their final structures will also be exactly alike. 
Whenever the growth processes of the two differ, the resemblance 
is modified. Thus the problem of human types is a problem of 
growth and all individuals that may be grouped together under 
one type are individuals with closely similar growth histories. 
In previous articles the writer has pointed out that in the embryo 
and the fetus the type of structure largely depends upon the rate 
of growth. A rapid growth and development gives one result 
and a slow growth produces, even in a twin individual, an entirely 
different result. The peculiar human adult and animal forms 
that have been described were also interpreted as due to modifica- 
tion of the usual growth processes by the actions of substances 
which affect metabolism and, therefore, growth rates. Certainly 
from the time of birth numerous growth affecting substances 
are being produced in the body and the action of these stuffs 
regulate and modify the rate and type of growth. Their usual 
effects are simply to increase or decrease the rate of metabolism 
and to cause the individual to grow faster or slower than another, 
thus giving rise to the variations above and below the mean. 

It is necessary to recall at this point several propositions dis- 
cussed in connection with embryonic development (Stockard ’21) 
in introducing the present conception of human types. In the 
first place initial growth and rapid growth tend to produce linear 

‘structure. In all plants and animals there is this primary tend- 
ency to form an axis or line of growth. Following this a 
lateral growth in width takes place. Crudely stated there is a 
tendency first to attain length and later width. Secondly, there 
is a certain degree of competition between these two tendencies 
so that as a rule the growth in width only expresses itself after the 
length growth has worn itself down and become slower. 

It follows that any organ capable of affecting the rate of 
metabolism or oxidation would necessarily affect the growth rate 
and must likewise affect the form and structure of the individual. 
The one organ in the vertebrate body which seems above all 
others to affect the rate of metabolism is the thyroid gland and 


278 CHARLES R. STOCKARD 


we actually know from convincing experimental proof that this 
gland also greatly affects the rate of growth and structural devel- 
opment. It is a fact that the cretin without a thyroid gland is 
incapable of growth and development. The tadpole without 
thyroid does not metamorphose into a frog. The thyroid is 
essential for growth from babyhood to maturity. Very probably 
the thyroid is not alone in its action, it may be affected by many 
other things and so may growth rate, but the point of primary 
importance is that the thyroid is the central body tending to 
control the rate of oxidation, and therefore growth rate in the 
individual. An active thyroid gives fast growing rapidly differ- 
entiating structures and linear rather than large lateral type 
individuals. 

It is impossible here to go into questions of the interactions 
among the internal glands and it is only necessary for our present 
* purpose to state that if a substance such as the secretion of the 
thyroid does regulate growth rate it must also tend to determine 
structural types. And since the rate of growth is the important 
thing in structural type, we should expect not more than two 
extreme types which will grade regularly into each other. The 
thyroid is so delicate in its response and is so certainly different 
in its action in different environments that the two types may be 
quite well separated, the one due to highly active thyroid and the 
other due to a low acting thyroid. The intermediate ideally 
balanced or indifferent condition would be the most difficult to 
attain on the part of the sensitive gland. 

The two groups into which almost all ordinary persons fall 
more or less exactly may, therefore, be termed the Linear Type 
and the Lateral Type. The linear type is the faster growing 
high metabolizing thin but not necessarily tall group, while the 
lateral type is slower in maturing and is stocky and rounder in 
form. 

Taking the tip of the nose as the extreme anterior point of 
the body and viewing the figure laterally, as seen in figure 1, we 
may draw a line which would indicate the morphological lateral 
line. This line on each side of the body separates the truly 
dorsal from the truly ventral surface regions. When these lines 


HUMAN TYPES AND GROWTH REACTIONS 279 


Fig.1 A side view of the human figure to indicate the superior tip of the 
body, the tip of the nose, and the general direction of the lateral line. 


280. CHARLES R. STOCKARD 


on the two lateral surfaces of the head and body are thought 
of in space we may imagine that the nearer they come together 
the more linear is the individual, and the wider apart they diverge 
the less linear and more lateral the individual type will be. 
Figure 2 illustrates this in the growth and development of the 
two types from the infant condition. 

Examining figure 2B it is seen that when the lateral lines are 
near together the head is of course narrow or dolichocephalic. 
The interpupillary distance is short and the eyes are close 
together, the nose bridge is narrow and therefore generally high, 
the mouth arch is narrow and for the same reason generally high, 
the lower jaw is small and narrow and usually not strongly de- 
veloped. The teeth are usually crowded and somewhat. ill-set. 
The neck is long and small in circumference, the shoulders are 
square, high and angular, the extremities are long and slender 
with long slender muscles and slender bones, the trunk is short 
and narrow tapering to the waist. The intercostal angle is 
quite acute. The stomach in such a person is long and narrow 
and rather vertical in position, extending to low in the abdomen, 
and the liver is generally small. 

The shape of the eye in this type is such that it is usually 
physiologically far-sighted though not pathologically so. They 
need no glasses on the street unless for astigmatism or some patho- 
logical condition. They are under weight for height according 
to the crude average tables now in use, and are often so as 
children. They arrive at puberty rather early than late and 
differentiate rapidly so that the males develop a large strong 
larynx and a low pitched bass or baritone voice. Their skin 
is thin and sensitive as is also the epithelial lining of their ali- 
mentary tracts. They are as a rule active, energetic and nervous, 
quite self-conscious and thus constantly exerting considerable 
nervous control. When in normal health they rarely laugh 
aloud and when suddenly shocked they resist the reflex jump and 
never scream. In this way they pass for cool, calm individuals 
with steady nerve, but as a matter of fact the body is almost 
constantly held under nerve control and they are actually 
nervous, usually suffering more after a shock than on the occasion. 


HUMAN TYPES AND GROWTH REACTIONS 281 


The lateral type when fully expressed is the antithesis of the 
linear type in all of the respects mentioned. The lateral lines 
are far apart and the head grows wide and not long (Brachy- 
cephalic), the interpupillary distance is wide and the eyes are 


Fig. 2 The ventral aspects of three figures to indicate the degree of divergence 
of the lateral lines in A, the infant; B, the ‘Linear Type’; and C, the ‘Lateral 
Type.’ 


far apart, the nose bridge is wide and often, though not neces- 
sarily, low. The mouth arch is wide and low the teeth are not 
crowded and are usually smoothly set. The lower jaw is large 
and strongly developed. The neck is short and large in circum- 
ference. The shoulders are round and sloping. The extremities 
are not long and are stocky with large bones and thick short 


282 CHARLES R. STOCKARD 


muscles. The trunk is inclined to be long and full, not constricted 
but bulging at the waist. The intercostal angle is quite obtuse. 
The stomach in such a person is large and tends to be transverse 
and high in position, the liver is generally large. 

The eye in the lateral type is so shaped as to be anatomically 
near-sighted instead of far and such persons frequently wear 
glasses on the street. This type is well rounded and over weight 
for height and also shows great fluctuations in weight, often 
gaining or losing as much as 15 or 20 pounds in a short space 
of time. Those of the linear type on the contrary do not experi- 
ence rapid weight changes but maintain a very constant weight, 
and may during the twenty years from about nineteen to thirty- 
nine vary a small number of pounds. The lateral type arrives at 
puberty a little late and is slow differentiating, the larynx of the 
male does not develop so suddenly as in the linear type and does 
not usually grow so large. The voice is thus high or tenor instead 
of bass. When men are under thirty years old the heaviest bass 
voices are almost always found among the thin linear individuals 
and these are very rarely tenors. The finest tenor voices are 
those of the round lateral type. Everyone recalls that the fine 
tenor is a fat man while the heaviest bass is a tall thin man. 

The two types are more clearly expressed in men than in 
women since the growth and glandular reactions are more decided 
in the male than in the female and are also freer from physio- 
logical disturbances. Many more physical points of difference 
and contrast could be cited for the groups but the above list is 
sufficient to make the differences clear. 

The actual recognition of the two types is not new. They 
have long been recognized just as we should expect, since if the 
contrasting features are really of any significant value anyone 
studying the physical characters of men should have discovered 
the types. Numerous workers have realized the existence of 
the two types and they have been designated by many names. 
The most recent, and what seem to me about the best physical 
distinctions have been recorded by Bean and his data bring out 
in a statistical way a number of physical differences which may 
be added to those mentioned above. My linear group Bean 


HUMAN TYPES AND GROWTH REACTIONS 283 


formerly termed hyperontomorph, meaning high developed 
structure, but he has since changed the term to hyperphylo- 
morph indicating a high phyletic origin. This change in terms 
means that Bean considers these types largely as fixed phyletic 
or hereditary entities rather than as ontogenetic or develop- 
mental results as his former terms indicated. The lateral type 
is close to his mesophylomorph. He arranges five types in all 
in considering the world races. It is a usual procedure to think 
of the races as grouped into separate types and this accounts 
for the change of term from onto- to phylomorph. It is the 
method of grouping end products to which I object from the devel- 
opmental and growth standpoint. I do not question the value 
of thoroughly analyzing the physical race differences but it 
must be more clearly emphasized that there are frequently 
extreme type differences within the race groups. hese differ- 
ences are often very probably of a genetic origin but they are 
complicated in other ways, and in all cases they are directly the 
result of definite growth and developmental reactions. The 
hereditary type is transmitted but the expression or develop- 
ment of this type depends upon numerous environmental influ- 
ences, and although a feature may be definitely inherited it may 
never be developed or expressed, just as the Seabright bantam 
cock inherits the male plumage but is unable to develop such 
plumage until his testicles are removed. Without this operation 
a casual observer might conclude that the rooster plumage had 
failed to be inherited when it has actually failed to be expressed. 

In the same way the human types are modified and changed 
by environment and the problem of expression or development 
is equally as important in understanding them as are the involved 
questions of heredity. The two types above outlined actually 
occur among all races and nations of men, but some races or 
groups show a great majority of one type and only a few of the 
other. So that, in general, it may be said that one race is of the 
linear type and another of the lateral, whereas the actual ancestry 
or stock of the races may have been closely similar. The upper 
classes of people in England and Germany illustrate the point. 
Almost all of the Englishmen of this cast are linear in type, thin 


284 CHARLES R. STOCKARD 


and dolichocephalic, while almost all of the Germans are of the 
lateral type, stocky and brachycephalic. Yet some of these 
Englishmen are decidedly lateral and some of the Germans are 
decidedly linear in type. All in all, however, the English may 
be called a linear type race, and the Germans a lateral type race. 

We may go still further and claim that these types among the 
English and Germans are more largely the result of the effects on 
growth of the environments in which they live rather than of 
hereditary differences in the stock. That is, the differences are 
more ontogenetic than phylogenetic in origin. This position 
will be borne out if we consider the types in conjunction with the 
geographical distribution of the peoples of the world. The linear 
types are usually found along the costal planes, in maritime 
climates where there is a good supply of iodine in the environ- 
ment and where the thyroid gland is normally active or even 
hyperactive. The lateral types are largely central continentals 
living in an inland environment away from the iodine supply 
of the sea. The thyroid gland functions poorly in these central 
continental regions, colloidal goitre is common and cretinism 
occurs in the extreme situations. This is a brief general state- 
ment of type distribution, all that space permits, and at once a 
number of exceptional cases occur to the reader but these have 
also occurred to the writer and when they are fully considered 
and analyzed they may be fitted into the general scheme. The 
high voleanic islands often have in certain of their central valleys 
lateral type people but the environment may not be actually 
maritime though it is insular. 

It is a well recognized fact that when the distribution of an 
animal species becomes very broad the species is frequently 
broken up into a number of varieties, each typical for a given 
geographic region. There are also certain similar characters 
developed in the varieties of different species in a given geographic 
region. The blue-jay has several geographic varieties as has also 
the bob-white and other birds in different parts of the United 
States. The northern varieties of these birds are as a rule larger 
than the southern, and the Florida types are often the smallest 
and frequently have longer feathers. The small mammals, 


HUMAN TYPES AND GROWTH REACTIONS 285 


squirrels and others, also show numerous geographic varieties. 
There are reasons to believe that food and other environmental 
conditions are the primary factors that have brought about 
these varieties. 

When we consider wider geographic ranges, it becomes rare 
indeed to find the same variety or even species of bird or mammal 
living on two different continents. The greater the degree 
and time of isolation for a given region the more the species 
differ from those of all other world regions, as is illustrated by 
Australia, New Zealand and other such regions of long isolation. 

A species of such wide distribution as the primitive man of 
several thousand years ago with only limited means of world 
migration must necessarily have broken up into the geographic 
varieties which we now find. But the species has in all of its 
ranges either a tendency to produce a majority of the linear 
type, long narrow individuals or a majority of the lateral type, 
stocky round persons. Whichever the tendency may be, we 
attribute the result to the action of the environment on thefune- 
tion of the thyroid gland and the type is the result of either a fast 
or slow rate of development. It is a growth reaction. 

In diagnosing these types it is very essential to take into con- 
sideration the age factor. The well expressed typical condition 
may only be depended upon during the twenties, at times younger 
than this the type may not be fully expressed and at periods 
above thirty it is becoming modified by age changes. It is of 
course possible to diagnose the type at any age but it is far more 
difficult and must be done in detail on the young and over mature, 
while during the twenties it may with practice be done very 
readily. 

During the twenties the linear type is always rather thin, but 
after thirty-five many of this type become fat and rounder and 
may at first sight be mistaken for a lateral individual, a change 
in the pituitary condition having probably occurred, but on close 
examination the head shape, interpupillary distance, tone of 
voice and other characters that have not changed make the 
linear type recognizable in this fat individual. 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 31, No. 3 


286 CHARLES R. STOCKARD 


The study made by Boas a number of years ago on the children 
of immigrants in New York City and later confirmed in Bos- 
ton by Guthe, ’18, furnishes most important data of the en- 
vironmental modification of types. Central continentals from 
Europe with brachycephalic heads and lateral type char- 
acters when bred in a maritime environment for several 
generations tend to become dolichocephalic and of the linear 
type. The gland quality that produces the type is certainly 
inherited but the action of the gland itself is actually modified 
by the environment, and the race stock in the new environment 
is changed in spite of heredity, the possibilities of which Morgan’s 
experiments on the chickens so beautifully illustrate. 

Again there are persons who do not properly fall into either 
type, nor are they typical intermediates, or blends of the two 
types. These individuals may possess well marked fully ex- 
pressed features of the linear type along with typically developed 
lateral features. They may be dolichocephalic with near-sighted 
eyes, wide palate arches, and tenor voices. These combinations 
are at once out of harmony. Such individuals are almost 
invariably found to be derived from parents of opposite types, 
and they are very common among the offspring of race mixtures. 
The fact that the purest type individuals are derived from two 
similarly pure type parents emphasizes the hereditary elements 
concerned. In spite of this there is much evidence to indicate 
that environment may modify the growth regulating mechanism 
and so tend to change the brachycephalic into the dolichocephalic 
head. 

If two distinct types actually exist in a wide population we 
should fail to obtain a usually proportioned figure by averaging 
a large number of physical measurements. A striking illustration 
of this fact is now at hand. Accurate physical measurements 
were collected from a great number of men in the recent army 


Fig. 3. A statue by Miss Davenport accurately constructed on the basis of 
average physical measurements obtained from the American army draft. The 
figure presents unusual proportions and probably indicates that the elements of 
the draft were not all simple variations about one common mean or single ana- 
tomical type. 

lam indebted to the American Museum of Natural History for this photograph. 


HUMAN 


TYPES 


AND 


GROWTH 


REACTIONS 


288 CHARLES R. STOCKARD 
draft by Professor C. B. Davenport of the Carnegie Institution. 
From these data averages were obtained for each of the several 
measurements collected. A statue of the human figure was 
then built on the basis of these averages by Miss Davenport. 

- This might be called a statue of the average young American 
male. Yet any anatomist examining the illustration shown by 
figure 3 will at once recognize a number of abnormal proportions. 
The abnormally long trunk renders the arms disproportionately 
short and throws the mid point of the figure entirely out of 
position. Other unusual details render the figure that of a 
person one would rarely ever see. In melting together the 
accumulated measurements an unusual or almost deformed figure 
is produced instead of a combination of commonly seen propor- 
tions. This is just such a result as would be expected when 
measurements from two or more distinct types are averaged. 
Failure to obtain a commonly seen figure from such measurements 
is very good evidence for the existence in the population of at 
least two distinct anatomical types. These are, I believe, those 
termed above the Linear and the Lateral growth types. 


— 


LITERATURE CITED 


Gutue, C. E. 1918 Notes on the cephalic index of Russian Jews in Boston. 
Amer. Jour. Phys. Anthrop., v. 1, pp. 213-223. 

Keirn, Str ArtHur 1922 The evolution of human races in the light of the 
harmone theory. Johns Hopkins Hosp. Bull. 33, pp. 155 and 195, 
May and June. 

Morean, T.H. 1919 The genetic and operative evidence relating to secondary 
sexual characters. Pub. Carnegie Inst. 

Srockarp, C. R. 1921 Developmental rate and structural expression: An 
experimental study of twins, double monsters and single deformities, 
and the interaction among embryonic organs during their origin and 
development. Am. Jour. Anat., vol. 28, pp. 115-278. 


Reprinted from the Proceedings of the Society for Experimental Biology and Medicine, 
1922, Xix, pp. 401-402. 


199 (1946) 
Morphology of cystic growths in the ovary and uterus of the 
guinea pig. 
By G. N. PAPANICOLAOU and C. R. STOCKARD. 


[From the Department of Anatomy, Cornell University Medical Col- 
lege, New York City.] 


The ovary in the guinea pig, as in most other mammals, fre- 
quently presents a cystic condition. The cysts are the result 
of a proliferation of the cuboidal epithelium which lines the epi- 
didymal portions of the embryonic Wolffian duct. The epididy- 
mal tubules are located towards one pole of the ovary and are 
connected with similar tubules lying outside the body of the ovary 
between it and the oviduct. 

Under certain conditions the walls of these blind tubules begin 
to proliferate, apparently forming a number of new tubules. A 
fluid accumulates in the interior of the tubules and distends them 
into spheroidal shapes. They become greatly distended and break 
into one another or fuse, thus forming large ‘‘ovarian cysts” in 
the case of those tubules lying within the ovary or ‘“‘parovarian 
cysts”’ in the tubules lying outside. 

Thus the ovarian and parovarian cysts are similar in struc- 
ture and their formation is of the nature of a tumor-like growth 
of the cuboidal epithelium which lines them. The accumulation 
of fluid which is essential to the formation of typical cysts is not 
to be considered their primary cause. 

In studying a great many ovaries for cystic conditions during 
several years we have never observed a follicular cyst. Large 
atretic follicles may be confused at times with small cysts, but 
such follicles always begin to disappear or atrophy before attain- 
ing significant dimensions. 

The uterine glands occasionally become cystic. Such cysts 
usually break into the lumen of the uterus when their epithelial 
lining becomes greatly distended. These are similar to the ovarian 
cysts in that both occur under identical conditions in tubules lined 
by epithelium. The fact that the uterine glands open directly into 
the lumen of the uterus makes the occurrence of such cysts excep- 
tional. 


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Reprinted from the Proceedings of the Society for Experimental Biology and Medicine, 
1922, xix, pp. 402~403. 


200 (1947) 
Experimental results bearing on the etiology of cystic growths in 
the ovary and uterus of the guinea pig. 
By G. N. PAPANICOLAOU and C. R. STOCKARD. 
[From the Department of Anatomy, Cornell University Medical Col- 
lege, New York City.] 

In experiments on underfeeding it was found that malnutri- 
tion readily gave rise to marked cystic conditions in the ovaries 
of healthy young guinea pigs. Such cystic conditions are, of 
course, frequently found in normal stock but here especially in 
old or unhealthy specimens. 

The changed nutritive conditions in the reproductive organs of 
underfed animals cause circulatory congestion, and as was pointed 
out in a previous communication! such conditions suppress the 
cestrous changes and prevent ovulation in these animals. The 
congestion and the high pressure resulting therefrom seem to favor 
the proliferation of the epithelial lining of the epididymal tubules 
located near one pole of the ovary, and the accumulation of fluid 
within the lumen of the blind tubules. 

The malnutrition expresses itself first within the ovary by a 
wholesale degeneration of developing follicles which seem to respond 
most delicately to changes in nutritive conditions. The conges- 
tion and follicular degeneration seem then to favor an over- 
growth of the more resistant epididymal tubules which become 
distended and crowd out the parenchymatous portion of the ovary. 

Uterine cysts seem to develop in the same way as those above 
as a response to the congestion resulting from malnutrition. 
The open mouths of the uterine glands make their cystic condi- 
tion rare so that among hundreds of ovarian cysts of all sizes we 
have observed only one perfectly typical case of uterine cyst. 

These experiments seem to indicate that ovarian and _paro- 
varian cysts represent growths of persistent embryonic tissue, 
and that an accompanying congestion and high pressure are nec- 
essary to the formation of typical cysts, and that these condi- 
tions may result from disturbed nutrition as is demonstrated by 
underfeeding the guinea pigs. 


1G.N. Papanicolaou and C. R. Stockard, Proc. Soc. Exp. BIOL. AND MEp., 
1920, xvii, 143. 


AUTHOR’S ABSTRACT OF THIS PAPER ISSUED Reprinted from Tum AMERICAN JOURNAL OF 
BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 2 Anatomy, Vol. 31, No. 2, November, 1922 


STUDIES ON THE GONADS OF THE FOWL 


III, THE ORIGIN OF THE SO-CALLED LUTEAL CELLS IN THE TESTIS 
OF HEN-FEATHERED COCKS 


JOSE F. NONIDEZ 
Department of Anatomy, Cornell University Medical College 
SEVEN FIGURES 


The discovery by Boring and Morgan (’18) of cells identical 
with the interstitial cells of the ovary in the testes of cocks of the 
Sebright bantam breed, in which all the males are hen-feathered, 
has promised to clear up one of the most interesting problems 
in the field of endocrinology. Since castration of these hen- 
feathered cocks is followed by the appearance of cock-feathering— 
a result similar to that obtained after ovariotomy in the female 
of other breeds—it seemed logical to suppose that the condition of 
the plumage in such males depended upon the presence of inter- 
stitial cells of ovarian type. The secretion from these cells was 
thought to inhibit the development of the plumage character- 
istic of the normal cock (Morgan, 719, 20). In this way a close 
relation between a specific tissue and the development of one 
of the most highly expressed secondary sexual characters would 
be established. This would also confirm the view, so often ex- 
pressed, that the interstitial tissue found in the gonads must be 
regarded as an endocrine gland. 

In a recent paper (22) I described the origin of the interstitial 
cells of the ovary, which have been regarded as luteal cells 
by Pearl and Boring. As previously shown by Firket (14), 
these cells arise from the degenerating sexual cords of the first 
proliferation and their epithelial origin seems well established. 
The clusters of interstitial cells I regarded as the remnants of 
the sexual cords infiltrated with fat. It was thought that if the 
cells found in the testis of the Sebrights are homologous with those 

109 


110 JOSE F. NONIDEZ 


in the normal ovary they must also arise from epithelium, either 
in the sexual cords of the early gonad or in the seminiferous 
tubules which represent their direct continuation. In the follow- 
ing pages I shall show the results of a study of developing testes 
which has amply confirmed this view. 

In the present paper the misleading term ‘luteal cells’ will be 
avoided, since a true corpus luteum or luteal cells in the ovary of 
the hen probably does not exist or is at least extremely doubt- 
ful. Indeed, the mere presence of these cells in the testis is 
sufficient proof that whatever their function may be they are not 
primarily concerned in the formation of a corpus luteum. On the 
other hand, their secretory function has not yet been established 
beyond a reasonable doubt. The term ‘interstitial’ has been 
so often associated with endocrine activity that it might convey 
the idea that the cells under question are glandular elements. 
Until their true function can be demonstrated, it seems more 
convenient to speak of the clusters as ‘remnants of the sexual 
cords,’ since in both sexes they are derived from these structures, 
and we may refer to the cells themselves as the ‘fat-laden cells’ 
of the clusters. 


MATERIAL 


The material used in the embryological part of the work con- 
sisted of a brood of eight Sebright eggs, generously put at my 
disposal by Prof. T. H. Morgan. Five of the embryos were males 
and two were females; one of the eggs failed to develop. The 
gonads from the male embryos while still attached to the sur- 
rounding organs were preserved in Bouin’s fluid at the tenth, 
seventeenth, eighteenth, twentieth, and twenty-first days of 
incubation. In addition to this material,-I was able through the 
kindness of Prof. H. D. Goodale, of Massachusetts Agricultural 
College, to study testes of four- and eight-day-old Sebright 
chicks. 

In order to ascertain whether remnants of the sexual cords 
also occur in chicks of breeds other than the Sebrights, the testes 
of four- and eight-day-old Rhode Island Red chicks were studied 
as well as a slide from a young Leghorn prepared by Professor 
Goodale. 


STUDIES ON THE GONADS OF FOWL Ua 


THE REMNANTS OF THE SEXUAL CORDS IN THE SEBRIGHT TESTIS 
a. Degeneration of the seminiferous tubules 


As in the case of the ovary, the clusters of the so-called luteal 
cells found in the testis arise as a result of the degeneration 
of the sexual cords. The sexual cords are represented in late 
stages of development of the testis by the young seminiferous 
tubules, which are their direct continuation. Degenerative 
processes in sonie of the tubules were only found in the embryos 
about the time of hatching (twenty and twenty-one days) and 
in young chicks. They were absent in the younger embryos 
the testes of which appeared normal. 

The seminal epithelium at the end of incubation and during 
the first weeks after hatching is made up of columnar cells with 
oval nuclei (fig. 1, 7). Seattered among these cells, either toward 
the periphery of the tubule or near the center there are large, 
round cells, the spermatogonia. Transitional stages represented 
by columnar cells with round nucleus of large size are not un- 
common. As reported by Firket (20), secondary spermatogonia 
are produced by the growth and rounding up of some of the epi- 
thelial cells. This columnar epithelium is also the source of the 
Sertoli cells, which do not appear until spermatogenesis begins. 

Since degeneration does not start simultaneously in all the tu- 
bules affected by this process, it was possible to establish a closely 
graded series of stages, some of which have been represented 
in figures 1 to 4. In the degeneration of the seminal epithelium 
not only the spermatogonia, but also a large number of the colum- 
nar epithelial cells are involved. While many of the latter shrink 
considerably, their cytoplasm becoming homogeneous and the 
nuclei pyknotic until no trace of structure can be recognized in 
them (figs. 1, 2 and 3, d), other epithelial cells are slowly in- 
filtrated with fat as shown by the vacuoles contained in their 
cytoplasm. These fat-laden cells persist as the so-called luteal 
cells. The whole process is very similar to the changes observed 
in the degenerating tubules of the adult under certain conditions 
(ligature of the vas deferens, partial castration, etc.) which 
result in the disappearance of all but the Sertoli cells. 


I 2 
Fig. 1 Embryo of twenty days. Early stage in the degeneration of a semi- 
niferous tubule of the right testis. d, degenerating cells; 7, fat-laden cells; n, 
normal portion of the seminal epithelium. 
Fig. 2 Embryo of twenty days. A more advanced stage in the degeneration 
of a seminiferous tubule of the right testis. d, degenerating cells; g, spermato- 
gonia; 7, fat-laden cells; i’, epithelial cells in early stages of fatty infiltration. 


112 


Fig. 3 Four-day-old chick. Clusters of fat-laden cells (7) still showing 
degenerated cells (d) and a normal spermatogonium (g). 

Fig. 4 Four-day-old chick. Degenerating tubule in the periphery of the 
testis between two normal tubules. a, albuginea; d, degenerating cells; i, fat- 
laden cells; t, portion of the tubule undergoing degeneration. 


113 


114 JOSE F. NONIDEZ 


In the upper portion of the tubule drawn in figure 1 the seminal 
epithelium (mn) appears normal as in the other tubules of the 
testis. The lower portion shows the beginning of degenerative 
processes In some of the epithelial cells, whereas others (7) are 
already infiltrated with fat and resemble very closely the fat- 
laden cells of the clusters found in later stages. This infiltration 
takes place following the same steps described by Firket (714) 
and more recently by the writer (22) in the ovary. It begins 
with the appearance of vacuoles, which cause the cell to become 
round or polygonal; at the same time the nucleus undergoes a 
slight decrease in size. Instead of a conspicuous chromatin 
network, it exhibits scattered chromatin granules and one or 
two small nucleoli; the nuclear sap stains more deeply than in 
the undifferentiated cells. Owing to these peculiarities, it is 
easy to identify the fat-laden cells in the midst of degenerating 
cells; they appear as polygonal or round elements with very 
clear cytoplasm and rather deeply stained nucleus. In the figure 
a normal spermatogonium is still seen in the lower part of the 
tubule, and directly above it there is another with a shrunken 
nucleus, the beginning of degeneration. A few pyknotic nuclei 
(d) are also seen. 

In figure 2 I have represented a seminiferous tubule in a state 
of more advanced degeneration. In this case a still larger 
number of pyknotie nuclei occur. The region corresponding to 
the lumen is filled with degenerating cells. In this tubule there 
are also more fat-laden cells (7) with highly vacuolar cytoplasm. 
Degenerating spermatogonia can also be detected; finally, two 
apparently normal spermatogonia (g) are seen in the figure. 

A still more advanced stage in the degeneration of the tubules 
has been represented in figure 3. The seminal epithelium is 
reduced to fat-laden cells and in some parts of the tubules a few 
degenerated cells and a normal spermatogonium still occur. 

As the tubules degenerate they become irregular in shape 
and shrink considerably, their epithelium disappearing as such 
a structure, while the fat-laden cells tend to crowd together. At 
the end of this process massive clusters are formed. That these 
clusters are the direct continuation of the seminiferous tubules 


STUDIES ON THE GONADS OF FOWL 115 


is best seen in figure 4, drawn from a section of the testis of a 
young chick (four days after hatching). In this figure a degen- 
erating tubule has been represented between two normal tubules. 
The portion nearer to the albuginea (a) contains only fat-laden 
cells (7), whereas the opposite portion (¢) situated towards the 
center of the testis still shows some slightly modified epithelial 
cells and two cells (d) undergoing degeneration. At a later 
stage, when all the epithelial cells which do not undergo fatty 
infiltration disappear, the tubule will appear as an elongated 
cluster of fat-laden cells, the remnant of a young seminiferous 
tubule. 

The possibility of mesenchyme cells entering the tubules was 
not overlooked. Indeed, it might be possible that such cells 
could work their way through the basement membrane and after 
entering the tubule become infiltrated with fat released by the 
degenerating cells. From my own abservations on testes in 
which some tubules are undergoing degeneration as a result of 
partial castration, it seems clear that the basement membrane is 
impervious and that neither leucocytes nor lymphocytes ever 
enter the tubules, although in some cases they may be very abun- 
dant in the vicinity. In the degenerating tubules of the embryo 
and young bird the basement membrane stains much deeper than 
in those which are normal, and owing to this fact it was possible 
to be certain that there was no break in its continuity, thus pre- 
venting the immigration of elements from the surrounding mesen- 


chyme. 


b. Developmental disturbances in the testis of embryos and 
young birds 


Aside from the degenerative changes just described, some of 
the embryos showed in their testes certain features which I 
regard as abnormal. Since they might have some bearing on the 
abnormal shape and color of the testis often found in adult birds 
(Morgan, 719) and on the sterility of some of the individuals, I 
will describe them briefly. 

In the embryo of twenty days circulatory disturbances in the 
right testis were very conspicuous. ‘The left testis appeared on 


Fig.5 Embryo of twenty days. Circulatory disturbances in the right testis. 
A, portion of the albuginea; B, genital artery of the right testis; C, genital artery 
of the left (normal) testis. 6, blood vessels showing blood stasis and hemorrhage ; 
e, degenerating erythrocytes; f, a portion of a degenerating tubule. 

Fig. 6 Embryo of twenty-one days. A ‘pit’ in the albuginea of the left 
testis. a, albuginea; e, epithelium of the serous layer. 
116 


STUDIES ON THE GONADS OF FOWL 117 


the whole entirely normal, but a slight degeneration in the periph- 
eral tubules could be noticed. In the right testis degeneration 
was widespread in some areas, a third or even a half of the tubules 
in a given section showing regressive changes in their epithelium. 
This degeneration was accompanied by diffuse hemorrhagic 
processes in certain areas of the albuginea. 

The study of the mesenchyme under the albuginea showed what 
at first sight might be regarded as a marked hyperemic condition 
accompanied with hemorrhage. In the normal albuginea the 
blood vessels are very thin and usually contain a few normal 
erythrocytes whose cytoplasm stains readily with eosin. In the 
portion of the albuginea near the degenerating tubules the 
capillaries were distended with blood and the erythrocytes were 
undergoing degenerative changes, their nucleus becoming round 
and spiny in appearance, while the cytoplasm no longer takes 
the eosin (fig. 5, A, b). The marked increase in the size of the 
capillaries and the degeneration of large numbers of erythrocytes 
within the vessels suggest that the condition just described is 
one of blood stasis. The walls of the vessels are not distinct, for 
the erythrocytes have passed into the surrounding mesenchyme, 
thus causing a hemorrhage. Indeed, it seems likely that the 
endothelial wall of the distended capillaries no longer exists, at 
least as a continuous structure; the presence of deeply stained 
nuclei in their vicinity suggests that there has been a prolifera- 
tion of the endothelium—a feature not uncommon in blood stasis 
under pathological conditions in man. 

The study of the right mesonephros showed that hemorrhage 
was also widespread in this organ, perhaps to a larger extent than 
in the testis, since the blood supply is more abundant. In some 
areas the mesonephric tubules appeared embedded in a continuous 
mass of degenerating erythrocytes. Hemorrhage was also noticed 
in the root of the mesentery. 

A comparison of the arteries supplying the testis of each side 
showed that the right genital artery (fig. 5, B) was much larger 
and had thicker walls than the left genital artery (C). This 
points to degenerative changes in the former, and perhaps to this 
condition are due the circulatory disturbances described above. 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 31, No. 2 


118 JOSE F. NONIDEZ 


In the embryo of twenty-one days degenerating tubules oc- 
curred only in the right testis, but they were less abundant than 
in the twenty-day embryo and were restricted to the periphery. 
In both testes the primitive relation of the sexual cords and the 
germinal epithelium (now the epithelium of the serous layer) 
had been retained. Under normal conditions the sexual cords 
lose their connections with the germinal epithelium when the 
albuginea begins to develop. In the embryo we are considering 
the albuginea developed normally, but mesenchyme cells failed 
to penetrate between the epithelium and the cords in the areas of 
proliferation. As a result of the thickening of the albuginea in 
certain places contact between the epithelium and the cords 
takes place at the bottom of depressions or pits, one of which has 
been represented in figure 6. The portion of the cords in im- 
mediate contact with the serous epithelium (e) showed slight 
degeneration in some of its cells. 

These ‘pits,’ of variable depth, were very abundant in both 
testes; over seventy were found in the left only. The arrange- 
ment of the peripheral tubules, which almost invariably converge 
into the pits, I regard as the result of their formation by prolifera- 
tion of the germinal epithelium in the restricted areas forming the 
bottom of the pits. 

Inasmuch as similar conditions were not found in the twenty- 
day embryo and in young chicks, I believe that the condition 
described is abnormal. It represents the persistence of an early 
stage in the development of the gonad in an embryo ready to 
hatch. Whether the albuginea becomes of uniform thickness 
in later stages of development or whether the condition described 
persists in the adult is not known. I might state, however, that 
in both cases the chances of degeneration of the peripheral tubules 
are great, thus contributing to a further increase in the number of 
clusters of fat-laden cells. 

In the four-day-old chick there were neither circulatory dis- 
turbances nor ‘pits’ in the albuginea, but an enormous amount of 
pigment appears within giant-cells in the intertubular spaces 
(figs. 3 and 4). The round shape of the pigment granules and 
their deep brown or black color suggest that this substance is 


STUDIES ON THE GONADS OF FOWL 119 


melanin. It appeared in both gonads, but was absent in adjacent 
organs, such as the adrenals and developing epididymis. The 
amount of melanin within the giant-cells was so large that their 
nuclei were almost invisible. 

The presence of variable amounts of pigment in the testes 
of the Sebrights has been reported by Morgan (719); in some cases 
both testes appear entirely black. The most striking feature of 
the pigmentary cells is their large size when compared with the 
ordinary cells of the mesenchyme. Pigmentary cells were not 
found in the late embryos, but in the embryo of ten days cells 
were observed in which pigment granules were beginning to de- 
velop. The similarity of these cells with the wandering hemo- 
blasts is so striking that one might believe that they are modified 
hemoblasts. They were most abundant in the loose mesenchyme 
separating the testis from the mesonephros and also in the mesen- 
chyme surrounding the adrenals. It seems likely that these 
cells ave the forerunners of the giant pigmentary cells found 
in later stages and in the adult cocks. In some regions of 
the testis of the seventeen- and eighteen-day embryos pigmentary 
cells were also found; some were round and others showed well- 
developed branches. 

The absence of pigmentary cells in the testes of some birds, 
their variable amount when present, and the unequal extent of 
their distribution suggest that the formation of pigment is an 
abnormal feature. The formation of melanin and related pig- 
ment in man is common in the melanoma, but in the testis 
it has never been found. On the other hand, in the Se- 
brights there is nothing suggesting neoplastic growth, unless 
the pigmentary cells themselves are regarded as a diffuse tumor 
which had invaded the gonad. 

Pigment was also observed in the upper half of the right 
testis of an eight-day-old Sebright chick; the other testis appeared 
entirely normal. 


120 JOSE F. NONIDEZ 


THE REMNANTS OF THE SEXUAL CORDS IN THE TESTIS OF YOUNG 
CHICKS OF OTHER BREEDS 

Clusters of fat-laden cells surrounded by a distinct basement 
membrane and identical in all respects with those described in 
the Sebrights were also observed in the testes of birds of other 
breeds in which they occur at least during the first days after 
hatching. Among my slides two series, one belonging to a four- 
day-old and another to an eight-day-old chick of the Rhode 
Island Reds breed showed typical clusters; they were absent in 
the adult cock of the same race. In the four-day-old chick 
degenerating tubules could be noticed in one of the poles of the 
testis and a few fat-laden cells in early stages of infiltration were 
seen in the midst of cells undergoing regression. 

In a slide of a young Leghorn chick, prepared by Prof. H. D. 
Goodale, the testes showed an enormous amount of fat-laden 
cells forming large clusters surrounded with a conspicuous base- 
ment membrane (fig. 7). Judging from the appearance of these 
testes and the abundance of remnants of the sexual cords, a 
widespread degeneration of seminiferous tubules must have 
taken place at some stage in development. The seminal 
epithelium appeared normal and showed the structure character- 
istic of the stage preceding spermatogenesis. 

From these facts it may be gathered that the formation of 
clusters of fat-laden cells persisting as the remnants of the sexual 
cords is by no means a characteristic feature of the Sebrights, 
but may also take place in the testis of birds of other races. That 
a few tubules may degenerate at early stages of their develop- 
ment prior to the full differentiation of the seminal epithelium 
is not surprising, and must be regarded as a result of the readjust- 
ment of the organ to functional conditions. In all probability, 
some portions of the sexual cords fail to become incorporated into 
the system of the seminiferous tubules, and under such con- 
ditions slowly degenerate, whereas normal tubules undergo the 
series of changes preceding spermatogenesis. 


STUDIES ON THE GONADS OF FOWL 121 


DISCUSSION 


The most significant feature in connection with the presence 
of clusters of fat-laden cells in the testis of the Sebrights is their 
origin from the epithelium of the sexual cords as represented in 
late stages by the seminiferous tubules. The clusters found in 


Fig. 7 A cluster of fat-laden cells in the periphery of the testis of a young 
Leghorn chick. a, albuginea; 7, fat-laden cells; ¢, normal tubules. 


the ovary also arise from these structures at an earlier stage of 
development. There can be little doubt about the homology of 
the cells in both organs. They represent the same kind of ele- 
ment, coming from identical origin and showing similar charac- 
teristics. If they perform a definite function in the gonads, it 
must be very similar in both sexes. The question naturally 


122 JOSE FP, NONIDEZ 


arises whether, as has been suggested, they are secretory ele- 
ments playing the role of an endocrine gland or whether they 
represent the result of fatty infiltration of degenerating cells. 
Unfortunately, from a study of the slides, no definite conclusion 
can be reached with regard to this important question. From 
a purely histological standpoint, the deep regressive changes 
undergone by these seminiferous tubules in which fat-laden cells 
arise strongly suggest that the formation of the clusters in the 
testis is an abnormal feature due to regressive differentiation of 
immature elements rather than to progressive changes leading to 
the formation of new morphological and physiological units. 

As shown in the figures, the spermatogonia without exception 
and many of the columnar cells which at the end of development 
constitute the bwk of the seminal epithelium are readily affected 
by degeneration, shrinking and finally disappearing without 
leaving any trace. But other epithelial cells undergo fatty in- 
filtration and persist, at least during the first weeks after hatch- 
ing. The cause of this different behavior is unknown. It may 
be due to the fact that they are elements of higher vitality or 
else that they do not require the same optimum of environmental 
conditions to survive as do the germ-cells. 

Under certain conditions (partial castration, ligature of the 
vas deferens, etc.), the seminal epithelium of adult tubules also 
shows similar phenomena, the only difference being that the 
elements which persist are fully differentiated and can be easily 
identified. As is well known, in these cases the germ-cells in 
the various stages of spermatogenesis degenerate and finally 
disappear, but the Sertoli cells are left behind and persist un- 
changed during a considerable time, if not throughout the life 
of the individual. As yet there is no evidence that the elements 
which in the embryo and young chick become fat-laden cells are 
the forerunners of the Sertoli cells, but it is possible that, although 
uniform in appearance, the epithelium of these early tubules 
already contains two different kinds of cells which cannot be 
distinguished from each other by any morphological characteris- 
tic. If some of the cells in this epithelium are already potential 
Sertoli cells, it is not surprising that their behavior may be differ- 


STUDIES ON THE GONADS OF FOWL [a5 


ent, for this is precisely what happens in the adult in which full 
morphological differentiation has already been attained. 

With regard to the factors which cause degeneration in some of 
the seminiferous tubules, very little can be said. As already 
mentioned, young chicks of breeds other than the Sebrights may 
show remnants of the sexual cords in the form of clusters of fat- 
laden cells. It is easy to conceive this degeneration as the result 
of the readjustment of the young gonad to functional conditions, 
since at the late stages in development and first days after hatch- 
ing there occur changes in the testis leading to the formation of 
the system of seminiferous tubules. If portions of the sexual 
cords have been cut off from this system by the increase in the 
mesenchyme which precedes the formation of connective tissue, 
their degeneration is only a question of time; they may disappear 
as such structures before spermatogenesis begins, leaving clusters 
of fat-laden cells, or they may persist until they become fully 
differentiated tubules, undergoing degeneration while distended 
by the spermatozoa, which are unable to leave the tubule on 
account of its lack of an outlet. 

In the late stages of the Sebright embryos degenerative changes 
in the tubules were accompanied by other conditions which can 
scarcely be regarded as normal. The existence of deep circulatory 
disturbances, the persistence of connections between the sexual 
cords and the germinal epithelium in one of the embryos, and the 
abundance of giant pigmentary cells in the young chicks are 
probably due to the influence of disturbing factors. Whether 
these abnormal features are always found in the late stages of 
embryos of this peculiar breed or whether normal development 
is the rule rather than the exception is a point to be established by 
further researches. I will, however, mention the fact that Mor- 
gan (719) has reported abnormalities in the gonads of adult cocks; 
according to this investigator, the testis “was often more flattened 
than is the testis of the typical male bird, that it was often some- 
what pear-shaped, and that frequently it was in part or entirely 
black” (p. 5). Sebright cocks are said to be often sterile. That 
these conditions may be the outcome of disturbances during 
development is extremely likely. Yet the factors causing the 


124 JOSE F. NONIDEZ 


abnormal development can only be surmised. Equally puzzling 
is the fact that degeneration of the tubules in the embryos studied 
was more conspicuous in the right testis than in the left, and that 
in the case of the eight-day-old chick pigment had only de- 
veloped in the upper half of the right gonad. This is important 
in connection with the constant degeneration of the right ovary in 
birds. 
CONCLUSIONS 


1. The clusters of the so-called luteal cells found in the 
testis of Sebright cocks arise during degeneration of the sexual 
cords and early seminiferous tubules as the result of fatty infil- 
tration of certain elements of the seminal epithelium. The 
spermatogonia and many of the columnar epithelial cells disap- 
pear without leaving any trace of their former existence. 

2. Degeneration of the young seminiferous tubules was only 
found in late embryos and young chicks; in the former it was 
more marked in the right testis. 

3. Aside from degeneration in some of the tubules, develop- 
mental disturbances, such as blood stasis, abnormal persistence 
of connections between the germinal epithelium and the seminif- 
erous tubules, and excessive formation of pigment, were observed. 

4, Remnants of the sexual cords in the form of clusters of 
luteal cells were also found in the testes of young birds of breeds 
other than the Sebright. 


LITERATURE CITED 


Borine, A. M., anp Morean, T. H. 1918 Lutear cells and hen-feathering. 
Journ. Gen. Physiol., vol. 1. 

Firxet, J. 1914 Recherches sur lorganogénése des glandes sexuelles chez les 
oiseaux. Arch. de Biol., T. 29. 
1920 Recherches, ete. 2eme Part., Ibid., T. 30. 

Morgan, T.H. 1919 The genetic and the operative evidence relating to secon- 
dary sexual characters. Carnegie Inst. Wash., Publ. 285. 
1920 The endocrine secretion of hen-feathered fowls. Endocrinol., 
vol. 4. 

Nonwez, J. F. 1922 Estudios sobre las gonadas de la gallina. IJ. El tejido 
intersticial del ovario. Volumen Jubilar de S. Ramon y Cajal, 
Madrid. 


[Reprinted from Tur JouRNAL or GENERAL PuysioLocy, September 20, 1921, 
Vol. iv, No. 1, pp. 33-39.] 


THE FORMATION OF THE ASTER IN ARTIFICIAL 
PARTHENOGENESIS.* 


By ROBERT CHAMBERS. 
(From the Cornell University Medical College, New York.) 


(Received for publication, May 24, 1921.) 


In normally fertilized eggs the development of the aster is attrib- 
uted to a substance carried into the egg by the spermatozoon. The 
aster first makes its appearance in the form of diminutive radiations 
surrounding the neck-piece of the spermatozoon within a few minutes 
after it has entered the egg. The writer! has shown that the formation 
of the radiations is accompanied by a jellying of the cytoplasm of 
the egg. The jellying process extends more and more as the aster 
increases in size and the entire egg becomes involved when the center 
of the aster comes to occupy the center of the egg. 

The formation of the aster is accompanied by an increase in size 
of a hyaline area in its center. This is Wilson’s hyaloplasm-sphere? 
also called centrosphere and astrosphere by other investigators. The 
microdissection method has demonstrated that this sphere area is 
liquid in contrast to the surrounding jellied cytoplasm. The pio- 
neer observers of mitotic division, such as Auerbach, Hertwig, 
Biitschli and Fol, described the accumulation of a hyaline plasma 
at the astral centers and suggested that the astral radiations are a 
result of protoplasmic currents. Later investigators, such as Morgan, 
Wilson and Conklin, considered this view as the most probable one. 


*The experiments, upon which this paper is based, were conducted in the 
Research Division of Eli Lilly and Company, at the Marine Biological Labora- 
tory, Woods Hole. The experiments constitute a part of a joint research project 
in which Dr. G. H. A. Clowes and the writer are engaged. 

1Chambers, R. Microdissection studies. II. The cell aster: A reversible 
gelation phenomenon, J. Exp. Zool., 1917, xxiii, 483. 

? Wilson, E. B., Experimental studies in cytology. I. A cytological study of 
artificial parthenogenesis in sea-urchin eggs, Arch. EntwckIngsmechn., 1901, xii, 529. 


33 


34 THE ASTER IN ARTIFICIAL PARTHENOGENESIS 


The movement of the egg nucleus is possibly also a case in point. 
As long as the egg nucleus is beyond the confines of the aster it is sta- 
tionary. As soon, however, as the extending aster reaches it, the 
nucleus begins travelling toward the sphere in which it finally lies 
close beside the sperm nucleus. The existence of a centripetal cur- 
rent may be inferred also from the following experiment. In an 
egg one may occasionally see one or more oil-like droplets 2 to 4 
microns in diameter. If one of these droplets be pushed by the 
needle from the liquid cytoplasm into the periphery of the aster the 
droplet will move along the rays toward the center. 

In view of the above observations it is highly probable that the 
liquid which accumulates in the center of the aster streams into it 
from all sides during the jellying of the cytoplasm. It is this stream- 
ing which probably occasions the innumerable radiations characteris- 
tic of the aster. After the aster has attained its full size the radia- 
tions begin to fade from view as the jelly state reverts to a more 
fluid one.. The liquid of the central sphere does not mix with the 
fluid cytoplasm but separates into two areas, one at each pole of 
the mitotic figure of the dividing nucleus. Astral radiations now 
appear about the two areas as the egg cytoplasm jellies again with the 
formation of two jellied masses instead of one, as heretofore. These 
grow at the expense of the fluid cytoplasm until all of the cytoplasm 
of the egg is taken up into two bodies, the two blastomeres of the 
segmenting egg. 

During the rapidly succeeding cleavages of the egg there is always 
a cap of liquid on the nucleus of each blastomere. With each mitosis 
this liquid flows around the nucleus to accumulate in two areas at 
the poles of the mitotic figure. These areas are periodically aug- 
mented during the formation of an aster and the ensuing jellying 
process. 

There is every evidence® that the mechanism of cell division de- 
pends upon a readiness of the cytoplasm to pass from a liquid to a 


3 Heilbrunn, L. V., Studies in artificial parthenogenesis. II. Physical changes 
in the egg of Arbacia, Biol. Bull., 1915, xxix, 149; An experimental study of cell 
division. I. The physical changes which determine the appearance of the spindle 
in sea-urchin eggs. J. Exp. Zool., 1920, xxx, 211; Chambers, R., Changes in proto- 
plasmic consistency and their relation to cell division, J. Gen. Physiol., 1919, 
ii, 49. 


pe ee ee 


ROBERT CHAMBERS 35 


jellied state and vice versa. The protoplasm must have its phase 
relations in a delicately balanced state in order that this may occur. 
In the egg we have seen that the reversal to a jellied state is probably 
accompanied by a separating out of a liquid. Something in this 
liquid may possibly control, in periodic rhythms, the physical state 
of the protoplasm surrounding it. We may assume that as long as 
there is a quantity of this substance localized in the egg it can induce 
aster formation. The idea suggests itself that one purpose of the 
spermatozoon is to accumulate this substance. In the mature unfer- 
tilized egg there is no localized area from which the jellying process 
may spread. The entrance of a sperm furnishes a focus as it were. 
Around this focus an aster develops with a steady accumulation of the 
liquid in its center. This liquid area surrounds the nucleus and puts 
the egg in a condition similar to that of a blastomere. The process of 
cleavage then becomes the same in both. 

An interpretation disconsonant with previous ones concerning the 
mode of aster formation in artificially parthenogenetic eggs has been 
recently put forward by Herlant.t Wilson? in Toxopneustes, had 
long ago shown that eggs treated insufficiently with a parthenogenetic 
agent may form monasters which disappear and reappear in several 
successive rhythms. Hindle® found this to be true also for the sea- 
urchin egg, if treated with butyric acid alone. A sufficient treatment, 
however, of a parthenogenetic agent results in the disappearance of 
the monaster followed by the appearance of an amphiaster. This 
results in cleavage of the egg. In the sea-urchin egg, the butyric 
acid treatment has to be followed by a bath of hypertonic sea water 
in order that this may occur. The hypertonic treatment often results 
in the formation of several cytasters in the egg. The cytasters pro- 
duced by the hypertonic treatment Herlant claimed to be due to 
dehydrative effects producing spots within the egg cytoplasm about 
which the asters appear. Herlant assumed that one of these cytas- 


* Herlant, M., Comment agit la solution hypertonique dans la parthénogenése 
éxperimentale (méthod de Loeb). I. Origine et signification des asters accessoires. 
Arch. Zool. exp. et gén., 1918, lvii, 511;.I1. Le mecanisme de la segmentation. 
Arch. Zool. exp. et gén., 1919, lviii, 291. 

5 Hindle, E., A cytological study of artificial parthenogenesis in Strongylo- 
centrotus purpuratus, Arch. Entwcklngsmechn., 1910-11, xxxi, 145. 


36 THE ASTER IN ARTIFICIAL PARTHENOGENESIS 


ters connects in some way with the monaster, thus forming the amphi- 
aster which initiates segmentation. The weakness in this interpre- 
tation is the lack of conclusive evidence for the union of the originally 
independent asters. Neither Wilson nor Hindle ever observed such 
a phenomenon. All my observations also indicate that the amphi- 
aster in parthenogenetic eggs arises from a previous single aster just 
as it does in normally fertilized eggs. 

My studies were mainly confined to the egg of the sand-dollar. 
In its behavior to parthenogenetic agents® the egg is almost identical 
with that of the sea-urchin which Herlant studied. The absence of 
pigment and the highly translucent nature of its protoplasm makes 
the sand-dollar egg an ideal object for observational study. 

The mature eggs, normally shed by the female, are placed in buty- 
ric acid (2 cc. 1/10 N in 50 cc. of sea water) for 35 seconds. During 
this treatment the eggs distinctly round up. They are then returned 
to sea water where, within a few minutes, the fertilization membrane 
lifts off. After 20 minutes the eggs are placed in hypertonic sea 
water (5 cc. 2.5 m NaCl in 50 cc. sea water). The eggs shrink slightly 
in this solution. After 20 minutes the eggs are transfered to a large 
quantity of normal sea water and the sea water is changed several 
times to free the eggs from any further action of the hypertonic 
solution. 

Up to this time no change whatever is to be seen in the cytoplasm 
or in the nucleus. While in the hypertonic solution thecytoplasm 
appears more granular and opaque than that of an untreated mature 
egg. However, on the return of the treated eggs to sea water the 
cytoplasm reverts to its former appearance and to the eye the eggs 
differ in no respect whatever from unfertilized eggs except for the 
presence of a fertilization membrane. 

It is not until the treated eggs have stood in sea water for several 
minutes that any cytoplasmic change is to be observed. ‘The first 
sign of a change consists in the appearance of faintly defined vacuoles 
about the center of the egg. Within a few minutes they coalesce 
to form a central clear area of about one-tenth the diameter of the 


6 Just, E. E., The fertilization reaction in Echinarachnius parma. II. The 
nature of the activation of the egg by butyric acid. Biol. Bull., 1919, xxxvi, 39. 


ROBERT CHAMBERS 37 


egg. The egg nucleus lies close to or within this area. Gradually 
rays begin to appear in the jellying cytoplasm about the area. These 
rays become more numerous and more pronounced until the entire 
ege is occupied by a large monaster which corresponds exactly with 
the fully developed sperm aster of a normally inseminated egg 
From now on the process is entirely analogous to that of a sperm ferti- 
lized egg. During the development of the aster the hyaline central 
area increases in size and the microdissection needle shows it to be 
a liquid area characteristic of that of the sperm aster. When the 
monaster disappears the liquid central area flows around the nucleus 
now undergoing mitosis and accumulates at the two poles of the nu- 
cleus into two polar areas. A jellying process now sets in with these 
two areas as centers and results in the amphiaster preparatory to 
the first cleavage of the egg. 

In the mode of aster formation the only difference between the 
sperm fertilized and the parthenogenetic egg consists in the manner 
in which a liquid separates out of the jellying protoplasm in connec- 
tion with the formation of the preliminary single aster. In the 
fertilized egg radiations appear immediately about the sperm-head 
and the accumulation of the liquid substance is from the beginning 
through the agency of the ray-like channels of the growing aster. 
In the parthenogenetic egg several vacuoles first appear in the cyto- 
plasm. These vacuoles collect in the center of the egg after which 
an aster appears. 

The frequent irregularities which obtain in ear Se eggs 
are apparently due to an incomplete fusing of the vacuoles and to 
a lack of polarity in the preliminary stages of the aster formation. 
In undertreatment, or when butyric acid alone is used, a monaster 
developes as usual. Upon the disappearance of the monaster, the 
persisting liquid centrosphere, instead of flowing to the two polar 
regions of the nucleus, remains a single body. With the return of 
the jellying period a single aster again forms and more fluid accumu- 
lates in the centrosphere which increases in size. This process 
repeats itself several times and segmentation of the egg never occurs. 

Eggs treated with butyric followed by a prolonged treatment of 
the hypertonic solution become abnormal. In cases of this kind 
the eggs, when returned to sea water from the hypertonic solution, 


38 THE ASTER IN ARTIFICIAL PARTHENOGENESIS 


exhibit vacuoles which, instead of being collected in the center of the 
egg, are scattered throughout the cytoplasm. Radiations appear 
about these vacuoles with the result that the egg becomes filled with 
many small asters. The longer the eggs have been left in the hyper- 
tonic solution the more numerous will be the asters, and most if not 
all of these asters develop independently of one another. Irregulari- 
ties may occur, even when the vacuoles collect in the center of the 
egg. In such cases an apparently normal single aster first results. 
Upon its disappearance, the central liquid area, instead of flowing 
away from the center into two polar bodies, produces three or four 
irregular lobes. About each of these lobes radiations appear in the 
egg cytoplasm producing a multipolar aster. In one instance one 
such lobe separated itself from the main body and a complete aster 
formed about it while a multipolar aster formed about the rest of the 
hyaline area. When the periphery of a multipolar aster reaches the 
surface of the egg cleavage furrows form between each lobe of the aster 
so that such eggs may segment simultaneously into three or four or 
more blastomeres. Asters which form independently of the central 
area never seem to be large enough to bring about segmentation of the 
egg into considerable masses. When such asters lie close to the periph- 
ery of the egg, furrows often grow in from the surface of the egg enclos- 
ing the asters. In this way a superficial type of segmentation results 
with the pinching off of small masses of the egg. The development 
of cytasters resulting in a spurious segmentation has already been 
described by Wilson.? 

The first aster appears at about the same time after the acid treat- 
ment, irrespective of whether the eggs have been subsequently treated 
with the hypertonic solution or not. However, with subsequent 
hypertonic treatment, the reappearance of the radiations following 
the fading away of the first aster occurs about more than one center. 
This results in segmentation of the egg. The reaction, therefore, 
which is peculiar to hypertonic treatment shows up only after the 
disappearance of the first aster. At that time the persisting central 
liquid area of the aster, instead of remaining as a single centralized 
mass, separates into two or more bodies with the result that the 
following reappearance of rays in the cytoplasm occurs as radiations 
about these bodies. This produces multiple asters. If there be 


ROBERT CHAMBERS 39 


only two focal points the liquid collects into two bodies, a typical 
amphiaster then develops, and the egg cleaves into two normal 
blastomeres. 

Aster formation not only consists in a jellying process but also in 
the separating out of a liquid. The optically visible phenomenon 
peculiar to the parthenogenetic egg consists in the manner in which 
this liquid begins to separate out of the egg cytoplasm preparatory to 
the formation of the preliminary single aster. In the sperm fertilized 
egg both processes are rapid and occur together, radiations appear 
immediately about the sperm-head, and the accumulation of the 
liquid substance is from the very start through the agency of the 
ray-like channels of the growing aster. In the parthenogenetic egg 
the jellying process is apparently very slow, and the separating out 
of a liquid takes place before the cytoplasm is stiff enough to exhibit 
channels through which the liquid flows to the center. The liquid 
first collects into several vacuoles and an optimum treatment is nec- 
essary to cause these vacuoles to fuse into one body with the subse- 
quent formation of a single aster. Overtreatment causes the appear- 
ance of many vacuoles scattered throughout the egg resulting in 
multiple asters. Undertreatment may result in the formation of a 
single aster which, however, periodically disappears and reappears as 
a single aster. 

The parthenogenetic treatment, in order to be successful, must 
not only bring about the separating out of a liquid from the egg 
cytoplasm, but must also induce polarity within the resulting hyaline 
area in order to enable it to form two centers about which an amphi- 
aster may develop. 


[Reprinted from THE JouRNAL or GENERAL PHysIoLocy, September 20, 1921, 
Vol. iv, No. 1, pp. 41-44.] 


STUDIES ON THE ORGANIZATION OF THE STARFISH 
EGG.* 


By ROBERT CHAMBERS. 


(From the Research Division of Eli Lilly and Company, Marine Biological Labora- 
tory, Woods Hole.) 


(Received for publication, July 19, 1921.) 


The following is a preliminary record of operative work on the 
starfish egg which throws some light on the nature of the fertilization 
membrane, the interaction between nucleus and cytoplasm, and the 
relation of the cortex to the interior of the egg. 

By means of the microdissection needle it has been possible to 
show that a morphologically definite membrane closely invests the 
unfertilized egg, and that it is this membrane which lifts off upon 
fertilization as the so called fertilization membrane. The description 
of two methods will suffice to demonstrate this. By carefully pressing 
an unfertilized mature egg between the surface of a cover-slip and the 
side of a slender glass needle the egg may be cut in two without tearing 
the investing membrane. This membrane now becomes apparent, 
bridging the gap between the two egg fragments and holding them 
together. Upon the addition of sperm this membrane lifts off as the 
fertilization membrane, in such a way that the two egg fragments 
come to lie within a single cavity. 

The unfertilized egg can also be slipped entirely out of its investing 
membrane. Such an egg will undergo normal fertilization and cleave 
into blastomeres having no investing membrane whatever. 

These two experiments definitely show that the normal unfertilized 
starfish egg is already surrounded by a membrane which, upon fertili- 
zation, becomes the fertilization membrane. 

The difference in behavior towards sperm of an egg, which has 
been denuded not only of its jelly but also of its membrane, and one 
which has not is very striking. In an egg enclosed in its membrane 


*The experiments reported in this paper constitute a part of the joint investi- 
gation of the mechanism of fertilization in which Dr. G. H. A. Clowes and the 
writer are engaged. 

41 


42 ORGANIZATION OF THE STARFISH EGG 


the spermatozoa quickly crowd about the egg as they are trapped in 
the jelly surrounding the membrane. In a membraneless egg no 
crowding of spermatozoa is noticeable and heavy insemination is 
necessary to bring about fertilization. With such eggs, when a 
cloud of sperm has been blown upon them, one may frequently observe 
a spermatozoon swim toward an egg, wander over its surface and 
then swim away. On the other hand the empty membrane with its 
investing jelly immediately becomes covered with a halo of active 
spermatozoa. 

The nucleus of the egg cell is a liquid drop surrounded by a mor- 
phologically definite membrane. The nucleus may be moved about 
within the egg with the needle, and can be considerably deformed by 
pressure. On removal of the needle the nucleus quickly resumes its 
spherical shape. Tearing the nucleus slightly causes the nucleus 
to shrink and the nucleolus to disappear; this is followed by a remark- 
able spread of a disintegrative process which involves the cytoplasm 
surrounding the nuclear area. In the immature egg, where the nucleus 
is large, the disintegrative process may extend throughout the entire 
egg. Inthe mature egg with a relatively small nucleus the destruction 
is restricted to a limited area. 

The disappearance of the nucleus or germinal vesicle during mat- 
uration has been described by several investigators. The nuclear 
membrane breaks down spontaneously and the nuclear sap spreads 
slowly throughout the cytoplasm. So long as the nuclear area, 
aside from the definitive egg nucleus, has not yet mixed with the 
cytoplasm, I find that a puncture of the area starts up the disintegra- 
tive process. When the nuclear sap has entirely mixed with the 
cytoplasm, any part of the egg, with the exception of the minute egg 
nucleus, may be torn with impunity. The mere presence of the glass 
needle in the nuclear sap is not sufficient to start up the disintegrative 
process. This process occurs only when the nuclear sap is agitated 
by the needle while the sap is in direct contact with the cytoplasm. 

Wilson! found in the Nemertine egg that any non-nucleated frag- 
ment, prior to the dissolution of the germinal vesicle, isnon-fertilizable 
whereas, any fragment from a mature egg is capable of being fertilized 
and undergoing cleavage. This I have found to be true also for the 


! Wilson, E. B., Experiments on cleavage and localization in the Nemertine 
egg, Arch. Entwcklingsmechn., 1903, xvi, 411. 


ROBERT CHAMBERS 43 


starfish egg. It is also of interest to note that the fertilizability of 
the egg fragments is directly connected with the extent of the mixing 
of the nuclear sap with the cytoplasm in the maturing egg. A non- 
nucleated fragment, taken from an egg in the early stages of the 
dissolution of the germinal vesicle, will admit sperm which will 
undergo several nuclear divisions with, at most, an abortive attempt 
on the part of the fragment to cleave. When the sap of the germinal 
vesicle has completely mixed with the cytoplasm, any fragment 
larger than a certain size limit is capable of being fertilized and 
undergoing cleavage. 

It is well known that immature eggs can be kept in sea water at 
room temperature for 24 hours or more without disintegrating and 
that unfertilized mature eggs go to pieces under the same conditions 
within a much shorter time.? The writer has found that nucleated 
fragments of the two kinds of eggs behave similarly, while non-nucleated 
fragments act quite differently indicating that the substance which 
prevents the disintegration is distributed differently in the two eggs. 
Non-nucleated fragments of immature eggs last for about 4 hours 
only. Similar fragments of mature eggs last from 8 to 10 hours, or 
about as long as the mature, nucleated fragments. The substance 
which prevents the destruction of the egg is apparently in the nuclear 
sap which, in the immature egg, is confined within the large nucleus 
or germinal vesicle, while in the mature egg this sap has escaped 
from the nucleus and spread throughout the entire egg. 

The following experiments indicate that the part of the starfish 
egg which is capable of development is chiefly confined to the cortex 
of the egg. It was long ago shown by Driesch,? Loeb‘ and others 
that starfish and sea-urchin eggs are highly fluid in that fragments 
quickly round up into spheres. That the cortex of the mature un- 
fertilized eggs is firmer in consistency than their interior has been 


? Loeb, J., and Lewis, W. H., On the prolongation of the life of the unfertilized 
eggs of the sea-urchins by potassium cyanide, Am. J. Physiol., 1902, vi, 305. 
Loeb, J., Maturation, natural death and the prolongation of the life of the unfer- 
tilized starfish eggs (Asterias forbesii) and their significance for the theory of 
fertilization, Biol. Bull., 1902, iii, 295. 

8 Driesch, H., Entwicklungsmechanische Studien. Der Werth der beiden 
ersten I peaneeaiee der Echinodermentwicklung, Z. wiss. Zool., 1891, liii, 60. 

4 Loeb, J., Ueber die Grenzen der Theilbarkeit der Eisubstanz, Arch. Physiol., 
1895, lix, 379. 


44 ORGANIZATION OF THE STARFISH EGG 


described by the writer. If the surface of the mature starfish egg 
be torn with a needle, and the egg then caught at the opposite side 
and pulled to the edge of the hanging drop, the compression on the 
egg produced by the shallow water at the edge of the drop will 
cause the fluid interior to ooze out through the tear, forming a perfect 
sphere. One may so manipulate the process as to cause the egg 
nucleus either to remain behind in the cortex (the cortical remnant) 
or to pass into the extruded sphere. 

The cortical remnant is relatively solid and remains more or less 
enclosed within the egg membrane and its jelly. If left long enough 
it will eventually round up so as to present the appearance of a dimin- 
utive egg surrounded by a collapsed and wrinkled egg membrane. 

The material which has escaped from the egg into the sea water 
is fluid and tends immediately to round up. On tearing with a needle 
its surface behaves like that of a highly viscous oil drop. These spheres 
adhere tenaciously to glass and, in the effort to remove them by 
blowing a current of water against them, they sometimes leave a torn 
of piece behind. The cortical remnant is readily fertilizable and 
undergoes normal segmentation. On the other hand, the material 
which has escaped from the interior of the egg whether nucleated or 
not, is non-fertilizable. It remains inert until it finally undergoes 
disintegration. As long as it possesses an intact surface it appears 
exactly like an egg fragment and will undergo disintegrative changes 
similar to those of entire eggs, on being torn with the needle. If even 
a small part of the original cortex is allowed to remain continuous 
with the sphere it is fertilizable and the more cortical material present 
the more will the sphere approach normal cleavage. 

It is significant that the fluid spheres which escape from the interior 
of the mature unfertilized egg, whether nucleated or not, withstand 
disintegration for a much longer period than do fragments, containing 
cortical material, which have been produced simply by cutting an 
egg into two or more pieces. 

It follows from these facts that the part of the starfish egg chiefly 
concerned in development lies in its periphery. The interior when 
separated from the cortex is incapable of developing. On the other 
hand, an egg containing cortical material alone is able to carry on its 
usual life activities. 


5 Chambers, R., Microdissectivn studies. I. The visible structure of cell proto- 
plasm and death changes, Am. J. Physiol., 1917, xliii, 1. 


Reprinted from the Proceedings of the Society for Experimental Biology and Medicine, 
1921, xix, pp. 87-88. 


46 (1793) 
The effect of experimentally induced changes in consistency 
on protoplasmic movement. 


By ROBERT CHAMBERS. 


[From the Department of Anatomy, Cornell University Medical 
College, New York City.] 


Agitation by means of a micro-dissection needle tends to cause 
the protoplasm of a living cell to pass from a more solid to a less 
solid phase. 

In marine ova, where one can closely follow the solidifying of 
the protoplasm just prior to cell division, mechanical agitation will 
cause the protoplasm to revert to its original liquid state so that 
the egg reverts to the shape of a sphere. If the egg so treated be 
subsequently left undisturbed the solidifying process starts up 
again with the result that the egg undergoes normal cleavage. 

In a previous communication! the writer has described the 
structural relations of changes in protoplasmic consistency of the 
Ameba to the formation of pseudopodia. The maintenance of 
pseudopodia depends upon a relatively solid state of certain parts 
of the Ameba. 

A resting Ameba, with numerous slender pseudopodia all over 
its surface, is relatively solid. Upon mechanical agitation the 
pseudopodia are retracted as the Ameba becomes more liquid. 
Fresh pseudopodia in an agitated Ameba tend to be broad lobate 
and, if the agitation be continued, all of the Ameba liquefies. 
The entire body then becomes, as it were, a single pseudopodium 
with a peripheral current of granules flowing away from its 
anterior end and a central current flowing forward. An Ameba 
in this extreme state does not change in position as the back flow 
tends to equal the forward flow. Amebe which are experimentally 
brought into this state have, so far, not been observed to return 
to their previous condition. The rate of flow of the currents 
gradually slows down until the animal dies. 


1 Chambers, Robert, Proc. Soc. Exp. Biot. AND MED., 1920, xviii, 66. 


2 SCIENTIFIC PROCEEDINGS (118). 


The protoplasm of an Ameba exists in a certain normal state 
of consistency from which it may deviate so as to solidify on the 
one hand or liquefy on the other. This normal state may be 
shifted not only by agitating the Ameba but also by injecting 
certain solutions. This I have been able to do with hydrochloric 
acid and with sodium hydrate. 

A trace of acid throws the normal state to the more solid side, 
while the alkali throws it to the more liquid side. An acidified 
Ameba forms long slender pseudopodia because the peripheral 
back flow in the developing pseudopodium is quickly arrested by 
a setting of the protoplasm. The area of the base of the pseudo- 
podium is, therefore, quickly limited and the extending pseudo- 
podium conforms to this narrow base. In an alkalinized Ameba, 
on the other hand, the peripheral back flow of a developing 
pseudopodium tends to be arrested much more slowly. Asa 
result of this the base of the pseudopodium spreads considerably 
before the protoplasm sets. The extending pseudopodium, having 
a larger base upon which to build, then becomes broadly lobate. ° 

These observations harmonize with my experiments on inject- 
ing ‘‘acid” and “basic” organic dyes. The basic dyes, which 
contain a relatively strong acid radicle, jelly the protoplasm, 
whereas acid dyes, with a strong basic radicle, liquefy it. 

It is interesting to note that these changes can be brought 
about in protoplasm while it is yet alive and that one can thereby 
change the character of the pseudopodia produced. 


MICRODISSECTION STUDIES, III. SOME PROBLEMS 
IN THE MATURATION AND FERTILIZATION 
OF THE ECHINODERM EGG 


{Reprinted from BroLocicaL BULLETIN, Vol. XLI., No. 6, December, 1921.7 


MICRODISSECTION STUDIES, III. SOME PROBLEMS 
IN THE MATURATION AND FERTILIZATION 
OF THE ECHINODERM EGG. 


ROBERT CHAMBERS. 
CorNELL Univ. Mepicat CoLitece, New Yorxk City. 


(From the Research Division of Eli Lilly and Company, 
Marine Biological Laboratory, Woods Hole, Mass.) 


This paper is a record of operative work on the starfish, sea- 
urchin and sand-dollar eggs to ascertain the morphological nature 
of changes which take place in the egg during its maturation and 
fertilization. Results were obtained on the effect of nuclear mate- 
rial on cytoplasm, the nature of cortical changes in the maturing 
and fertilized egg and the difference between cortex and medulla 
of the egg with respect to fertilizability and to other life activities. 
The dissection and injection of the living eggs were carried out at 
first by means of Barber’s (’14) apparatus and later with an 
improved micromanipulator of my own design (’21"). A de- 
scription of the technique as applied to microdissection has al- 
ready been published (Chambers, ’18"). A detailed description 
of the new micromanipulator will appear both in the Journal of 
Bacteriology and in the Anatomical Record. 


I. THE GERMINAL VESICLE IN THE MATURING STARFISH EGG. 


Starfish eggs, on being shed naturally, have already begun 
maturing. In order, however, to secure large quantities of eggs, 
it has been the general custom to remove the ovaries bodily from 
a ripe female and to cut them up in a bowl of sea water. This 
procedure brings the eggs into the sea water in the immature con- 
dition with germinal vesicles intact. The germinal vesicle begins 
to disappear anywhere from thirty to fifty minutes after the eggs 
come into contact with the sea water and maturation usually pro- 
ceeds in a normal manner (Wilson and Mathews, ’95). 

The undisturbed germinal vesicle or nucleus of a fully grown 

318 


MICRODISSECTION STUDIES. 319 


immature egg is a hyaline sphere containing a sharply differentiated 
nucleolus and occupying about one fifth the volume of the egg. 
With the microdissection needle the vesicle may be moved 
about in the fluid cytoplasm without injury to the egg. With 
the needle one may considerably indent the surface of the 
vesicle. On removal of the needle the vesicle reverts again to the 
spherical shape (Fig. 1). The vesicle possesses a morphologically 
definite surface membrane inclosing an optically homogeneous 
liquid (cf. Chambers, ’18°). Within this liquid lies a visible body, 
the nucleolus. By agitating the vesicle the nucleolus may be made 
to occupy any position within the nuclear fluid. The nuclear mem- 
brane is very easily injured. If, however, a microneedle be care- 
fully inserted into the nucleus, the membrane about the puncture 
adheres to the body of the needle and the tip of the needle may 
push the nucleolus about with no apparent injury. The existence 
of considerable tension in the nuclear membrane is shown in the 
following experiment. An egg was cut into three fragments in 
such a way that the surface film forming over the cut surfaces of 
the middle fragment pressed upon the nucleus, deforming it con- 
siderably (Fig. 2). The attempt of the nucleus to return to a 
spherical shape bulged out one end of the egg fragment until it 
was constricted off from the remainder of the fragment (Fig. 
2b-f). 

Tearing the nuclear membrane in most cases results in a de- 
struction of the nucleus. Ina few cases it was possible to produce 
a slight rupture with no noticeable injurious effects. Such a case 
is recorded in Fig. 3. At 10:44 A.M. undue pressure on the 
germinal vesicle when cutting an immature egg in two resulted in 
its rupture followed by a lobular extrusion bounded by a very 
delicate film. During the following ten minutes the vesicle began 
slowly to revert to its original shape (Fig. 3b andc). Before that 
was attained the maturation process began and, at 10:55, the out- 
line of the vesicle had disappeared. The nucleated egg fragment 
maturated normally and five hours and a half after insemination it 
had segmented in two. At 8:40 P.M. it had developed into a 
swimming blastula. 

The cytoplasm of the egg allows of considerable tearing without 


320 ROBERT CHAMBERS. 


apparent injury (Chambers, ’17-a). If, however, the nuclear 
membrane be torn, a very striking phenomenon occurs. The cyto- 
plasm immediately surrounding the nucleus disintegrates and 


Qa Fi 1g. 2 b G d e f &. 
310 
3.00 
245 


Fic. 1. Figures showing the extent to which the nucleus (germinal 
vesicle) of an immature starfish egg may be indented on one or both sides 
without rupture. On removing the needle the nucleus reverts to its original 


spherical shape. 

Fic. 2. a, immature starfish egg cut at 2:45 P.M. into three parts; the 
nucleus has remained intact but is laterally compressed in the middle frag- 
ment. b, c, d, e and f, successive steps in attempt of nucleus to round up; 
b, 2:50 P.M.; d, 3:00 P.M.; f, 3:10 P.M. 

Fic. 3. a, partial rupture of nucleus followed by a repair of its membrane. 
b and c, successive changes in the shape of the nucleus within the following 
ten minutes after which time it disappeared. 


liquefies. If the rupture of the nucleus be violent, the disintegra- 
tion of the cytoplasm spreads rapidly until the entire egg is in- 
volved. If the rupture be slight, the disintegrative process is 
quickly limited by a surface film which forms on the boundary 
between the disintegrating and the surrounding healthy cytoplasm 
(Fig. 4). This film tends to prevent any further spread of the 
destructive process. The destruction of the cytoplasm is evidently 
due to something which emanates from the injured nucleus. The 
injury to the cytoplasm does not start where the nuclear membrane 
is first torn, but from the entire surface of the injured nucleus. 


MICRODISSECTION STUDIES. 321 


This is analogous to results obtained by injuring red blood cor- 
puscles with a needle upon which hemoglobin escapes immediately 
from the entire surface (Chambers, ’15). 


4 

Fi Z Bae 

Fic. 4. Disintegration of cytoplasm surrounding the nucleus on tearing 
.the nucleus with a needle. (a) Faint hyaline sphere, a remnant of the 


destroyed nucleus. (b) Disintegrated .cytoplasm. (c) Cytoplasmic surface 
film separating disintegrated from healthy cytoplasm. 


Within the nucleus itself the immediate effect of the injury is a 
dissolution of the nucleolus. A nuclear remnant tends to persist 
after the injury as a hyaline sphere lying within the disintegra- 
tion products of the cytoplasm. On being touched with the 
needle it fades from view. 

In permanently immature eggs, such as eggs which have been 
standing in sea water for an hour or more without maturing, the 
disintegrative effect on the cytoplasm by injuring the nucleus tends 
to be much more restricted, and the nuclear sphere which persists 
after the injury can be shown to possess a morphologically definite 
membrane. Sucha sphere is easily dissected out of the egg. Fre- 
quently, when the germinal vesicle lies close to the periphery of the 
egg, the disintegration of the cytoplasm quickly reaches the surface. 
With the formation of a surface film over the healthy cytoplasm 
the disintegrative area lies in a deep bay on one side of the egg. 
This hollow is slowly obliterated as the semi-fluid substance of the 
egg strives to assume a spherical shape. In this way the disinte- 
grated material is forced out of the egg together with the persisting 
nuclear sphere. This nuclear sphere persists for some time in the 
sea water. It can be deformed by means of the needle and, on 


322 ROBERT CHAMBERS. 


tearing its surface, the fluid contents escape, leaving behind a col- 
lapsed membrane which disappears within 10 to 15 seconds. 

Fig. 5 shows the effect of cutting the mature egg nucleus of the 
starfish egg. By pushing the nucleus against the inner surface of 


pre Oe Cae 
Fig. 5 
Fic. 5. Effect of cutting mature nucleus of a starfish (Asterias) or sea- 
urchin (Arbacia) egg. a, intact egg nucleus; 6b, nucleus in process of being 
cut in two. The nucleus was pushed against the periphery of the egg as it 


was being cut by a vertical needle; c, the separated fragments of the nucleus; 
d, reunion of the fragments; e, reconstituted nucleus. 


the egg it is possible to pinch it into two pieces. Each piece 
rounds up but, if the two are allowed to come into contact, they~ 
will fuse into a single nucleus again. The same result obtains in 
the sand-dollar and sea-urchin eggs. If, however, the nuclear 
membrane be torn, a disintegration of the cytoplasm results 
analogous to that produced on rupturing the germinal vesicle. 
The extent of disintegration is much more limited, owing doubtless 
to the much smaller amount of nuclear material present. Similar 
results were obtained on tearing the nucleus of the Arbacia egg. 

It was found possible to destroy the cytoplasm of one egg by 
injecting into it nuclear material obtained from another egg. This 
experiment has to be performed very rapidly, for if the nuclear 
material be allowed to remain longer than ten seconds within the 
pipette it has no effect whatever when injected into the cytoplasm 
of an egg. If it be injected within that time the destructive effect 
is very pronounced. 

If an egg be allowed to undergo normal maturation, the ger- 
minal vesicle disappears except for a small remnant which be- 
comes the definite egg nucleus. This egg nucleus moves to the 
surface of the egg, where it gives off the two polar bodies. It then 
constitutes the female pronucleus, which remains quiescent until 
fertilization occurs. The disappearance of the germinal vesicle is 
a well-known phenomenon. In order, however, to locate definite 
stages selected for my operations I introduce the following sum- 


MICRODISSECTION STUDIES. 323 


mary. The germinal vesicle with an intact membrane is shown in 
Fig. 6. Within thirty to forty-five minutes after standing in sea 
water the nuclear membrane exhibits wrinkles and its outline be- 
gins to fade from view. Within a few minutes no membrane is 
visible and cytoplasmic granules can be seen moving into the region 
hitherto occupied by the nucleus, while the nuclear sap appears to 
be diffusing out (Fig. 6-c). As the nuclear membrane disappears 
the nucleolus fades from view. The invasion of the nuclear area 
by cytoplasmic granules continues until all of the area except a 
small portion is rendered indistinguishable from the general cyto- 
plasm of the egg. This small portion persists as the egg nucleus 
(Fig 6e and f). In Fig. 6-g two consecutive positions of the 
nucleus are shown. At 1:13 P.M. it lay deep in the substance of 
the egg. In twenty minutes it had moved to the periphery of the 
egg preparatory to the formation of the polar bodies. 


a eae 


ws “fk 
\ 


12.00 


ar 

Fic. 6. Camera lucida drawings of the successive steps in the normal 
dissolution of the germinal vesicle in the maturing starfish egg. The proc- 
ess was somewhat slowed down owing possibly to the compressed condition 
of the egg necessary for detailed observation. 

Fic. 7. a, intact germinal vesicle within the egg. b, nucleus after having 
been torn out of the egg and brought into sea water. c, d, e and f, successive 
changes undergone by the nucleus lying in sea water. 


324 ROBERT CHAMBERS. 


By means of the microdissection needle it is possible to show, at 
the stage shown in Fig. 6-d, that the membrane of the germinal 
vesicle no longer exists. By careful manipulation it was possible 
to push the cytoplasmic granules into the nuclear area. A slight 
rapid movement of the needle, however, was sufficient to give rise 
to disintegrative processes similar to those on tearing an intact 
germinal vesicle. In the normal maturation process the mingling 
of the nuclear sap with the cytoplasm is very gradual, being com- 
pleted in the case recorded not under ten minutes. It is this grad- 
ual mixing which apparently prevents disintegration. 

Morgan (’93) and Mathews (Wilson and Mathews, 95) found 
that maturation was accelerated by shaking starfish eggs shortly 
after they were placed in sea water. They concluded that the 
shaking ruptured the membrane of the germinal vesicle and so 
allowed the nuclear material to mix more quickly with the cyto- 
plasm. I have repeatedly tried to intermix cytoplasm and nuclear 
material by rupturing the nuclear membrane of the starfish egg 
with the needle, but in every case I get an explosive disintegration 
of the cytoplasm. The ruptured nuclear membrane which Mathews 
(W. and M., ’95) and Marcus (’07) describe in fixed and stained 
immature eggs which had been violently shaken is possibly the 
membrane of the sphere which I found to persist after injury to 
the germinal vesicle (see page 321). It is more likely that the 
shaking which accelerates processes within the egg leads to the 
normal gradual dissolution of the nuclear membrane and the subse- 
quent diffusion of the nuclear material throughout the egg. I have 
been able to do this occasionally with the needle. An intact ger- 
minal vesicle which to all appearances should take fifteen to 
twenty minutes to go into dissolution will often immediately ex- 
hibit a wrinkled outline on being gently agitated with the needle. 
Then follows the gradual fading from view of its outline with the 
subsequent changes as shown in Fig. 6. 

The intact germinal vesicle may be brought into the sea water by 
tearing away the surrounding cytoplasm. During the process the 
nucleolus fades from view. The slightest tearing of the nuclear 
surface then causes the entire liquid vesicle to disappear in the 
water. If, however, the nucleus be left alone, it shrinks for a 


MICRODISSECTION STUDIES. 325 


time and then swells. The changes appreciable to the eye are 
shown in Fig. 7. During the swelling of the nucleus a substance 
apparently separates out which collects into a small mass and 
persists as a gelatinous body. It is possible that this abnormal 
separating out is analogous to the formation of the definitive egg 
nucleus in the normal process of maturation. This separating out 
of a gelatinous material from a liquid nucleus upon injury may be 
similar to the method of precociously inducing chromosomes in 
spermatocytes of the grasshopper (Chambers, ’14). 


2. THE EXISTENCE OF AN EXTRANEOUS MEMBRANE ABOUT THE 
UNFERTILIZED Ecc. 


The existence. of a membrane about the unfertilized egg rising 
off as the fertilization membrane upon insemination was first sug- 
gested by the earlier investigators (e.g., Hertwig, "76; Herbst, 
93). Kite (712) and Glaser (’13) agreed with them whereas 
McClendon (’14), Harvey (714) and Elder (’13) claimed that the 
fertilization membrane is anew formation consequent to fertiliza- 
tion. Heilbrunn (’13) also identifies it with the actual proto- 
plasmic surface of the egg, which he considers to be in a state 
of a gel and which lifts off as the fertilization membrane, a new 
surface film forming over the egg underneath it. 

My experiments indicate that the unfertilized eggs of the 
starfish, sea-urchin and sand-dollar all possess a membrane ex- 
traneous to their true protoplasmic surface, and that it is this 
membrane which, upon insemination, is lifted off as the well-known 
fertilization membrane. 

In the unfertilized egg the membrane is more or less tightly 
glued to the surface of the egg just as Kite (’12) described it. In 
the sea-urchin egg it is extremely delicate and can be demonstrated 
only as follows (Fig. 8) : The needle is inserted as nearly as possi- 
ble through the periphery of the egg and left there. Within a few 
seconds the protoplasm, lying immediately under the egg mem- 
brane and distal to the needle, flow away from the needle until 
the needle lies in a small protuberance which is formed by a 
very slightly lifted portion of the egg membrane. 

The existence of the egg membrane is easily demonstrated in the 


326 ROBERT CHAMBERS. 


starfish egg. In Fig. 9 the disintegration of the cytoplasm follow- 
ing injury to the germinal vesicle has reached the surface of the 
egg. The disintegrated area is quickly localized by a surface 
film bounding a cup-shaped depression on the surface of the egg. 
Roofing over the depression is the egg membrane. The egg 
membrane can also be shown by cutting an egg in two by press- 
ing the egg against the coverslip with the side of a needle. The 
pressure of the needle cuts the egg in two without rupturing 
the membrane, which, on releasing the egg, bridges the gap 
between the pieces and holds them together (cf. Figs. 11 and 
12, page 329). 

The difference between the consistency of the egg membrane in 
the starfish and the sea-urchin egg is strikingly shown in the fol- 


e ? 
Fig. 9 

Fic. 8. Needle inserted at 11:36 A.M. through periphery of a sea-urchin 
egg and left there. At 11:38 the cytoplasmic granules have been flowing 
away from the needle. A new surface film begins to appear with the needle 
left outside. At 11:45 the original egg membrane appears as a delicate 
membrane partially lifted off the surface of the egg by the needle. 

Fic. 9. Lifting of a membrane from the surface of an immature starfish 
egg following injury to the egg. a, local disintegration of cytoplasm following 
destruction of the germinal vesicle (cf. Fig. 4). An egg membrane becomes 
apparent as the cytoplasm retreats from it. b and c, gradual separation of 
the membrane all over the surface of the egg. 


lowing experiments. With the eggs in a hanging drop the egg is 
pressed against the coverslip with the side of a glass needle until 


MICRODISSECTION STUDIES. 327 


the pressure divides the egg into two pieces. In the sea-urchin 
egg the two pieces immediately round up and roll away from one 
another. In the starfish egg the tougher membrane is not rup- 
tured, but holds the two pieces together. 

The membrane of the sea-urchin egg is so delicate that it is also 
possible to cut the egg in two in the following manner: In a hang- 
ing drop the horizontal end of the needle is brought over the egg 
(Fig. 10). The needle is now lowered. This brings the needle 


=; 


Fio. 10 
fe) 


Fic. 10. Side view of moist chamber to show one method of cutting an 
egg in two with the microdissection needle. 


against the upper surface of the egg and presses the egg down 
against the surface film of the hanging drop. On lowering the 
needle still further it passes through the egg and out of the drop, 
cutting the egg cleanly intwo. In the case of the starfish egg this 
procedure would drag the egg out of the drop along with the 
needle. The membrane of the sand-dollar egg is weaker than that 
of the starfish and stronger than that of the sea-urchin egg. 

The consistency of the membrane varies with the age of the 
egg. The full-grown immature egg of the starfish has a relatively 
tough membrane. On the other hand, young ovarian eggs possess 
very delicate membranes and they can be cut in two with the same 
ease as mature sea-urchin eggs. 

The strongest argument regarding the existence of a mem- 
brane about the unfertilized ege is that a membrane may be 
stripped off the egg whereupon the egg, which was previously 
non-adherent, now sticks to everything it touches. The fer- 
tilizability of such naked eggs is discussed under the next head- 
ing. 

The existence of egg membranes is a fairly universal feature and 
it is, therefore, not surprising that we should find them in the 


328 ROBERT CHAMBERS. 


echinoderm eggs which have generally been considered as naked. 
The unfertilized Cumingia egg has an extremely tough mem- 
brane, so tough that it is difficult to rupture it without com- 
pletely destroying the egg contents. The vitelline membranes in 
the frog and in the chick are undoubtedly analogous structures. 


3. THE Ecc MremBRANE AND THE FERTILIZATION MEMBRANE 
ARE IDENTICAL. 


Prior to fertilization no membrane enveloping the egg is vis- 
ible. Upon fertilization a membrane lifts off which can easily 
be cut away from the egg. Figs. 11 and 12 indicate the iden- 
tity of a preexisting membrane with the fertilization membrane. 
Fig. 11-a shows an egg cut in two with an investing membrane 
holding the pieces together. Upon fertilization the membrane 
lifts off, enclosing the two pieces in a single cavity (Fig. 11-b). 
One only of the pieces happened to ségment, and the fact that 
the two pieces lie in one cavity is shown in Fig. 11-c, where the 
blastomeres of the segmented portion have encroached on the 
area around the nonsegmented piece. In Fig. 12 an egg was 
cut into three pieces, the egg nucleus lying in one of the pieces. 
Upon fertilization the membrane lifted off the pieces, each of 
which received sperm and developed into swimming larve. Fig. 
12-c shows the empty fertilization membrane after the three larve 
had escaped. In Fig. 13 is shown an egg which, on being cut in two, 
was rolled about in an attempt to separate the pieces. The egg 
membrane between the two pieces was twisted into a thread 
joining the two. Upon fertilization each piece exhibited a com- 
plete fertilization membrane, but the fact that the two investing 
membranes are portions of one common membrane is shown by 
the connecting thread. . 

A conclusive test for the starfish and sand-dollar egg is the 
removal of the egg membrane prior to insemination. Occa- 
sionally, pricking the egg is sufficient to elevate the membrane. 
No subsequent development takes place. It is possible, however, 
to remove this membrane by tearing it and the egg then be made 
to slip out. This is more easily done on eggs which have been 
standing for some time in seawater. On catching at the sur- 


MICRODISSECTION STUDIES. ‘329 


face of such eggs with the needle, the membrane is often torn 
in such way that the egg slips out leaving the membrane stuck 
to the needle. Such an egg, when inseminated, is fertilized and 
subsequently segments with no investing membrane whatever. 


Fic. 11. a, starfish egg cut in two without destroying the investing mem- 
brane. 6, after insemination the investing membrane lifts off both fragments 
- as the fertilization membrane. c, one of the fragments segmented, the other 
did not. That both fragments lie in a common cavity is shown by the en- 
croaching of blastomeres of one fragment into the region of the unsegmented 
fragment. 

Fic. 12. a, starfish egg cut into three pieces. One piece was squashed and 
produced an exovate. b, on being fertilized the exovate was pinched off 
as an endoplasmic sphere (cf. Fig. 25). The rest of the fragments produced 
a common fertilization membrane. Each of the three enclosed fragments 
developed into a swimming larva. 

Fic. 13. a, sand-dollar egg rolled as it was cut in two. The egg membrane 
between the two pieces: was twisted into a thread joining the two. b, egg 
shortly after fertilization showing fertilization membrane about each con- 
nected by a filament. c, the two pieces in an early segmentation stage. 


The difference in reaction of sperm to an egg which has been 
denuded of its membrane as well as of its jelly, and to one which 
has not is very striking. An egg within its membrane is quickly 
surrounded by spermatozoa as they are trapped in the jelly sur- 
rounding the membrane. In a membraneless egg no crowding 
of spermatozoa is noticeable and heavy insemination is necessary 


330 ROBERT CHAMBERS. 


to bring about fertilization. When a cloud of sperm has been 
blown upon a naked egg, one may frequently observe a sperma- 
tozoon swim toward it, wander over its surface, and then swim 
away. On the other hand, the empty membrane with its in- 
vesting jelly immediately becomes covered with a halo of sper- 
matozoa. This observation accords with the interpretation of 
Buller (’02), that the investing jelly determines the direction of 
the sperm which are captured by it, and that there is no apparent 
chemotactic substance excreted by the egg to attract the sperm. 

The difference in position of the polar bodies in the starfish egg 
with respect to the fertilization membrane as shown by Gemmill 
(12) (see also Chambers and Mossop, ’18, and Garrey, ’19) may 
be explained as follows: When the polar bodies form prior to fer- 
tilization they rise off the surface of the egg, carrying with them 
the closely adherent membrane. When they are pinched off the 
egg membrane remains continuous about the egg and subsequent 
insemination results in the formation of a fertilization membrane 
with the polar bodies lying outside. If, however, the eggs are in- 
seminated before extrusion of the polar bodies, the egg membrane 
lifts off as the fertilization membrane and, when the polar bodies 
are formed, they lie within the membrane. 

In the sea-urchin egg the identity of the egg membrane with the 
fertilization membrane is more difficult to demonstrate. In Fig. 
14 is shown the effect of locally injuring the surface of the sea- 
urchin egg. In a is a disintegrated mass produced by tearing a 
spot on the surface with a needle. In b this area is shown as a 
bulge which may be explained as being produced by the interior 
pressure of the egg on a surface weakened by the loss of an invest- 
ing membrane. In c the egg has been fertilized. The fertiliza- 
tion membrane is formed over all the surface except at the in- 
jured place. In d segmentation has occurred and a blastomere 
protrudes through the gap in the fertilization membrane. 

A better demonstration is the case shown in Fig. 15. At 4:26 
the tip of a needle was punched through the cortex. Within a few 
seconds the cytoplasm distal to the needle flowed away, leaving the 
needle lying under a delicate membrane (Fig. 15-a). At 4:27 
the egg was inseminated with the needle still in place. At 4:29 


MICRODISSECTION STUDIES. 331 


the fertilization membrane was formed, showing its continuity 
with the delicate membrane previously noticeable (Fig. 15-b). 


€ d 


12.40 


115 


Feril. oe 


ST 


Tig. 16 


Fic. 14. Sea-urchin egg with surface torn producing local cytolysis. a, a 
new surface film has formed under the cytolyzed area which is being ex- 
truded. b, a bulge appears in the region of the new surface showing this 
region to be weaker than elsewhere on the egg surface. c, egg after fertiliza- 
tion exhibiting a fertilization membrane over the egg except at the place 
previously torn. d, the same egg 35 minutes later with a blastomere pro- 
truding through the tear. 

Fic. 15. a, needle piercing sea-urchin egg near its periphery. The cyto- 
plasmic granules are flowing in the direction of the arrows. One minute 
later the egg was inseminated. c, an intact fertilization membrane forms, 
inclosing both egg and needle tip. 

Fic. 16. a, protrusion on surface of egg produced by pulling at cortex 
with needle. b, three minutes later the investing membrane lifted off surface 
of protrusion. c, one minute after fertilization. The protrusion has been 
pinched off from the egg and its investing membrane can be seen to be con- 
tinuous with the fertilization membrane. d, empty and collapsed fertiliza- 
tion membrane. 


In the sea-urchin egg the membrane often rises off a protrusion 
caused by pulling at the cortex with the needle. Such a case 
is shown in Fig. 16. The protrusion was formed at 12:43. At 
12:46 a membrane had lifted off the protrusion. At 12:51 the 
egg was inseminated, and one minute later the membrane was 


332 ROBERT CHAMBERS, 


found continuous with the fertilization membrane. The protrusion 
subsequently pinched itself off and persisted in a sac-like protuber- 
ance of the fertilization membrane (Fig. 16-d-e). 

In all of the various eggs studied a change in the consistency 
of the membrane takes place very soon after it has been elevated. 
The membrane, at first very soft and delicate, progressively 
toughens until it becomes almost parchment-like during the later 
segmentation stages. It is of interest to note that Harvey (’10) 
found a difference between the unfertilized and the fertilized 
sea-urchin egg when subjected to sulfuric acid. The acid dis- 
solves the unfertilized egg completely, whereas it dissolves all 
of the fertilized egg except the fertilization membrane. Some 
chemical change apparently takes place as the membrane lifts 
off the egg. 

Outside the membrane is a considerable zone of a structureless 
jelly. In the sand-dollar egg the jelly very loosely adheres to the 
membrane. On cutting into the jelly the egg with its membrane 
easily slips out. This is to a somewhat lesser degree true for 
the starfish egg. In the starfish egg one often sees the under sur- 
face of the jelly pushed away from the surface of the unfertilized 
egg by the protruding polar body. 

The question as to whether the membrane lifts off the surface of 
the egg or whether the egg shrinks leaving the membrane behind 
has been raised by Glaser (714) in spite of McClendon’s (’10) 
statement to the contrary. Glaser, by making a large series of 
measurements, claims that the egg shrinks upon fertilization, and 
that the initial diameter of the completed fertilization membrane is 
equal to that of the unfertilized egg. Glaser’s measurements were 
made on the assumption that the eggs always maintain a spherical 
shape. This is not true. The mature unfertilized egg is very soft 
_ and if allowed to lie on the bottom of a glass dish tends to flatten 
into the shape of a disc. Upon fertilization the egg rounds up as 
the fertilization membrane leaves its surface. One can readily see 
if the observations are taken of eggs in one plane only that erro- 
neous conclusions may be arrived at. 

I used two methods to ascertain the diameter of starfish eggs 
before and after fertilization. One method was to place a drop 


MICRODISSECTION STUDIES. 333 


containing a few eggs on a gelatin-coated slide. The eggs were 
rolled over by means of a micro-needle and only those which main- 
tained their spherical shape were measured. With a micro-pipette 
sperm were introduced into the drop without disturbing the rela- 
tive positions of the eggs. A second method was to place several 
eggs in a hanging drop in a Barber moist chamber. By piercing 
the surrounding jelly with a needle the egg to be measured could 
be held suspended in the middle of the drop. Numerous measure- 
ments of the starfish egg were made at different times through 
several summers and in every case the egg maintained its original 
size as the fertilization membrane rose off its surface. Not only 
does the egg not decrease in volume, but it slightly imcreases in 
size until segmentation occurs. The accompanying table is one 
sample of the measurements made: 


Minutes after Fertilization. 


Un- | 
| fertil. el Bete he! ie De |) - 70" 
Egg diameter......... | 340 3-4: laa) | 3:4 [3:5%3-55 | 3-5 X3.6 | 3.53.6 
Fertilization membrane | | | 
GIAMIPLED yoy. 5 5 i. = | Bey [hse gona 73:05 3.7 3:75 3:75 3-9X3.9 


The conclusions from this table apply both to starfish and sea- 
urchin eggs. They may not necessarily be true for other species. 

Fig. 17 shows successive steps in pulling a starfish egg out of 
its fertilization membrane. No second membrane is ever formed 
even with superimposed insemination. Occasionally the hyaline 
plasma layer in such an extruded egg swells up and simulates a 
second membrane, and it is probably this that has been described 
by certain investigators as a second fertilization membrane. The 
hyaline plasma layer will be discussed under heading 5. 

An unfertilized mature sea-urchin egg may be rolled about and 
its contents churned to the extent of producing “ fountain cur- 
rents’ within the egg (Chambers, ’17-b). This is done by push- 
ing an egg in a drop shallow enough to compress the egg. Cur- 
rents are produced which flow backward immediately under the 
surface of the egg and forward along its central axis (Fig. 18). 
By careful manipulation it is possible to do this without rupturing 


334 ROBERT CHAMBERS. 


the investing membrane. Such an egg is capable of forming a 
normal fertilization membrane when inseminated. If the pushing 
process be carried too far, a distinctive quiver can be recognized, 
as of something giving way. On subsequent insemination such 


Fig.17 


Fic. 17. a@ and b, successive steps in pulling a starfish egg out of its 
fertilization membrane. c, empty membrane at 4:00 P.M. d, ditto four hours 
later at 8:00 P.M. The membrane persists as a collapsed remnant for a 
long time. 


eggs produce a collapsed fertilization membrane. The quiver 
undoubtedly was due to a rupture of the egg membrane. On 
account of this rupture the fluid, which presumably collects under 
the membrane, leaks out and the membrane is not lifted uni- 
formly. 


4. THE CorTEX AND INTERIOR OF THE UNFERTILIZED Ecc. 


The cytoplasm of the immature starfish egg is uniformly semi- 
solid. A gash made in it with a needle is maintained for some 
minutes before closing up. When the germinal vesicle breaks down 
naturally, the egg protoplasm becomes more fluid so that a gash 


MICRODISSECTION STUDIES, 335 


through such an egg quickly closes up. The cortex—i.e., the sur- 
face of the egg immediately beneath the egg membrane—tends 
always to remain more solid (Chambers, ’17-a). Because of this 
difference in consistency the cortex and medulla of the egg can be 
separated from one another as follows (’21*): If the surface of 
the mature starfish egg be torn with a needle and the egg then 
be caught at the opposite side and pulled to the edge of the 


Rots 


Fig-19 


Fic. 18. Currents produced within a sea-urchin egg by pushing a sea- 
urchin egg held against a coverslip by a shallow film of water. The direction 
of the currents is shown by the arrows. The nucleus, after being carried 
about with the current, tends to come to rest in the location shown in the 
figure. 

Fic. 19. Part of the cortex of a fertilized ege after the appearance of the 
hyaline plasma layer. The cortex was ruptured in one place and cytoplasmic 
granules can be seen issuing through the rupture in the hyaline plasma layer 
and the investing fertilization membrane. 


hanging drop, the compression on the egg produced by the shal- 
low water at the edge of the drop will cause the fluid interior 
to ooze out through the tear to form a spherical exovate (see 
Fig. 25, page 344). One may so manipulate the process as to 
cause the egg nucleus either to remain behind in the cortex (the 
cortical remnant) or to pass into the extruded sphere of endo- 
plasmic material. 

The cortical remnant is relatively solid and remains more or less 
inclosed within the egg membrane and its jelly. If left long 
enough it will eventually round up so as to present the appearance 
of a diminutive egg surrounded by a collapsed and wrinkled egg 
membrane. 


336 / ROBERT CHAMBERS. 


The endoplasmic material which has escaped from the egg 
into the sea water is fluid and tends immediately to round up. 
On tearing with a needle its surface behaves like that of a highly 
viscous oil drop, adheres tenaciously to glass. As long as it 
possesses an intact surface it looks exactly like an egg frag- 
ment and will undergo disintegrative changes similar to those 
of entire eggs on being torn with the needle (cf. Chambers, 
"17-a). 

The ability to produce endoplasmic spheres is possibly due to 
the relatively tough egg membrane in the starfish egg which helps 
to keep back the adherent cortex. In the sea-urchin egg, with an 
extremely delicate egg membrane, it has been impossible to cause 
the interior to flow out, as the cortex tends to flow with it. 

The sand-dollar egg behaves very much like the starfish egg. 
The egg membrane is appreciable in the unfertilized egg and endo- 
plasmic spheres are readily produced. 

A difference in the functional activities of the cortex and inte- 
rior of the starfish egg is discussed under the headings 6 and 7. 


5. THE HyaLtnE PLaAsMA LAYER. 


Prior to fertilization the cytoplasmic granules in the sea-urchin 
and sand-dollar egg lie close to the surface. Within ten minutes 
after fertilization the granules have undergone a centripetal migra- 
tion, leaving an appreciable peripheral zone of a hyaline appearance 
which has been called the hyaline plasma layer (Loeb’s gelatinous 
film, ’13, p. 19). 

The microdissection needle indicates that this layer is relatively 
firm and gelatinous. The very fluid internal cytoplasm may be 
made to flow out through a rupture in this layer if the egg be torn. 
This is shown in Fig. 19. The cytoplasmic granules lie against the 
inner boundary of this layer and may be seen oozing out through 
the small tear in this layer and through a tear in the fertilization 
membrane to the exterior. 

The hyaline plasma layer adheres very tenaciously to the needle 
and when an egg has been deprived of its fertilization membrane 
the egg sticks to everything it touches. 

Loeb has called attention to the fact that the hyaline plasma 


MICRODISSECTION STUDIES. 


Ww 
Ww 
“I 


layer in a segmented egg bridges the segmentation furrow. When 
the furrow is first formed, however, the hyaline plasma layer does 
not bridge the furrow, but is carried in on the walls of the cleavage 
furrow (Fig. 20-a, b,c). The layer is thicker in the floor of the 


7 
Fig-20 

Fic. 20. Contour of a sand-dollar egg at various stages of its cleavage 
into two blastomeres. In a and b the hyaline plasma layer is seen carried in 
on the walls of the deepening furrow. In c the egg has segmented in two 
with the hyaline plasma layer on opposite sides of the furrow tending to 
merge into each other. In d this process is carried further. In e the two 
blastomeres are tending to assume the shape of hemispheres with the hyaline 
plasma layer bridging the furrow. 


furrow, but it is only later when the furrow has cut through the 
egg that the hyaline plasma layers on the opposite surfaces of 
the furrow run together. Each half of the segmenting egg tends 
to assume the shape of a sphere owing to the separation of the 
two asters of the amphiaster (Chambers, ’17-b, ’19). If there 
were no other forces at play, the two blastomeres,-when formed, 
should be spheres. In the sea-urchin egg the adhesiveness of the 
hyaline plasma layer tends to draw the two blastomeres together; 
also the fertilization membrane, not rising to any great extent off 
the surface of the egg, must exert some pressure on the two blas- 
tomeres. In the sand-dollar the fertilization membrane is well 


338 ROBERT CHAMBERS. 


lifted, so that there is plenty of room within the membrane, per- 
mitting the two blastomeres to assume almost spherical shapes 
(Fig. 20-c). When the cleavage furrow is completed the two 
blastomeres are contiguous only where the two spheres touch. 
At this place the hyaline plasma layers of the two blastomeres 
merge. We have here, apparently, two opposing forces; first, 
the jellied aster holding each blastomere to a spherical shape, 
and, second, the affinity of the plasma layer substance surround- 
ing the two blastomeres. As soon as the asters disappear and 
the cytoplasm of the blastomeres reverts to a more fluid state 
the plasma layers of the two blastomeres merge more and more 
and the blastomeres are pulled together till they assume shapes 
approaching those of hemispheres (Fig. 20-e). The outlines in 
Fig. 20 are camera lucida drawings taken during the successive 
stages of one sand-dollar egg. - 

In the starfish, where there is no appreciable hyaline layer, and 
where the fertilization membrane is lifted far beyond the surface 


1It has recently been intimated that the microdissection method is unre- 
liable as a means of ascertaining changes in viscosity in the dividing egg 
because of supposed discrepancies in the results obtained by Seifriz (’20) 
and myself (’17” and ’19). As a matter of fact the results of Seifriz har- 
monize perfectly with mine. Seifriz states “there is a pronounced decrease 
in viscosity of the central region of the cell with the first appearance of 
the amphiasters.” This statement has been interpreted as running counter 
{to mine. This is not true for although my results indicate that the astral 
portion of the amphiaster is jellied, I definitely state (p. 494, ’17) that the 
central region and the zone between the two halves of the egg are fluid 
where “a distinct flow of granules medianward can be observed.” 

Again, on completion of cleavage Seifriz notes that the two blastomeres 
become liquid. This statement also fits in with my results. I state (p. 51, 
’r9) that, immediately after cleavage and while the two blastomeres are 
still spherical, the firmness of the cytoplasm persists. Later, when the asters 
disappear the cytoplasm liquefies and the two blastomeres crowd up against 
one another. Seifriz noted this last liquid state of the two blastomeres 
without considering the state prior to it. ° 

I may mention here a possible criticism of the centrifuge method in ascer- 
taining viscosity variations. There are critical stages in the developing asters 
during which agitation causes their disappearance. This was noted long ago 
by Wilson. On bringing the eggs to rest the asters reappear and develop- 
ment proceeds normally. I have already discussed this matter fully (19). 
The centrifuge and miscrodissection methods of studying the physical state 
of protoplasm should serve as valuable checks on one another, if only the 
investigators in these fields would agree on cooperation. 


MICRODISSECTION STUDIES. 339 


of the egg, the blastomeres are practically non-adhesive, and they 
maintain more or less spherical shapes till well on into the later 
segmentation stages. 


6. THe LocarizaATION oF A MaTertAL WuicH AFFECTS THE 
LIFE OF THE UNFERTILIZED STARFISH Ecc. 


It is well known that immature starfish eggs can be kept in sea 
water at room temperature for 36 hours or more without disinte- 
grating. That the germinal vesicle or nucleus is responsible for this 
length of life can be demonstrated by cutting an immature egg in 
two. The nucleated fragment lasts fully as long as the entire egg. 
The non-nucleated portion, on the other hand, disintegrates within 
three to four hours. In mature unfertilized eggs the conditions 
are quite different. In the mature egg the germinal vesicle has 
broken down and the nuclear sap has diffused throughout the egg. 
Loeb (’o2) and Mathews (’07) showed that such eggs have a 
higher rate of oxidation than immature eggs and if left unfer- 
tilized disintegrate within 8 to 10 hours whereas the immature 
eggs last for days. 

The non-nucleated fragment of the mature egg lasts as long as 
the whole egg, evidently owing to the dispersed nuclear sap of the 
dissolved germinal vesicle. What is significant is that the nucleated 
fragment lives no longer than the non-nucleated fragment. Both 
contain the dispersed nuclear sap, while the nucleated fragment 
possesses also the definitive mature egg nucleus which is ultimately 
to become the female pronucleus. Apparently it is the dispersed 
nuclear sap and not the definitive mature egg nucleus which is 
chiefly concerned. In the formation of the nucleus of the mature 
egg we have possibly something analogous to the state of affairs in 
many Protozoa where the nuclear apparatus consists of a tropho- 
or macro-nucleus concerned chiefly in the metabolic activities of the 
cell, and the kineto- or micro-nucleus which has only to do with the 
reproductive activities. In the starfish egg we may consider the 
germinal vesicle as a combined tropho- and kineto-nucleus. On the 
approach of maturation the tropho-nuclear material (nuclear sap) 
diffuses throughout the egg, leaving behind the kineto-nuclear part, 
the mature egg nucleus, which gives off the polar bodies to become 
ultimately the female pronucleus. 


340 ROBERT CHAMBERS. 


The fluid interior of the mature unfertilized egg, if isolated by 
being made to escape through a tear or the cortex, withstands dis- 
integration for 24 to 36 hours. The presence of even a small part 
of the original cortex in organic continuity with it causes it to 
disintegrate in about the same time as an entire mature egg. This 
would indicate that the reactions which make for disintegration 
reside chiefly in the cortex. This, together with the fact that the 
cortex of the egg is necessary for fertilization, would indicate that 
the cortex is the seat of the initial activation processes of the egg. 
The relatively inactive central material of the starfish and sand- 
dollar egg somewhat resembles that of the Linerges, the Scy- 
phomedusan, which Conklin (’08) has described. Conklin speaks 
of “the large cavity in the line of the first cleavage furrow filled 
with gelatinous or fluid substance, which forms the ground sub- 
stance of the central area of the unsegmented egg.” He found 
that most of the ground substance escapes into the cleavage cav- 
ity and suggested that it is the fluid yoke which is gradually used 
up in the nourishment of the embryo. The central substance of 
the Linerges egg is probably not strictly analogous with that of 
the starfish or sand-dollar egg. In Linerges cleavage is of a type 
peculiar to yolk-laden eggs and the central substance escapes 
during the first cleavage. On the other hand, in the echinoderm 
egg the nucleus lies well within the central substance of the egg 
and, upon fertilization, all of the endoplasm is used up in the 
formation of the cleavage asters and nothing apparently escapes 
into the early cleavage cavity. We can not, therefore, con- 
clude that the interior of the Echinoderm egg consists of entirely 
inert material. It lacks certain essential features, but when co- 
existent with the cortex it plays a full part in the cleavage of 
the egg. 


7. THE LocaALizATION OF A SUBSTANCE WHICH RENDERS A STAR- 
FISH EGG FERTILIZABLE. 


Wilson (’03°) in Cerebratulus and Renilla and Yatsu (’04 
and ’o8) in Cerebratulus have shown that non-nucleated frag- 
ments of the egg are capable of fertilization only after the ger- 
minal vesicle has broken down. With more delicate methods 


MICRODISSECTION STUDIES. 341 


rendered possible by the microdissection instrument it has been 
possible to work out this problem in detail and to ascertain to 
some extent the distribution of the material which renders fer- 
tilization possible. 

A number of fully grown immature starfish eggs were enucleated 
by carefully dissecting out their germinal vesicles. None became 
fertilized when inseminated. In another lot of immature eggs the 
germinal vesicle was torn while in the egg (Fig. 21). Immediate 


Je 


Fig. 21 
Fic. 21. A starfish egg whose germinal vesicle is eliminated by puncturing 


it (cf. Fig. 9). The cytoplasm surrounding this nucleus was also destroyed. 
This enucleated remnant is nonfertilizable. 


dissolution of the nuclear membrane took place with a disintegra- 
tion of the cytoplasm around the nuclear area. Those eggs which 
succeeded in forming a protective surface film to prevent spread 
of the disintegration process subsequently rounded up. Upon in- 
semination none of the eggs showed any sign of being fertilized. 

Eggs were then taken with the germinal vesicle in various stages 
of normal dissolution and cut into nucleated and non-nucleated 
portions. The eggs may be grouped into stages b, ¢ and d, accord- 
ing to the stage of dissolution of their germinal vesicles, as shown 
in Fig. 6 (page 323). Whenever the cut passed through the nu- 
clear area during the nuclear stages b, c and d, disintegration al- 
ways took place, involving all of the nucleated portion and a small 
part of the non-nucleated piece (Fig. 23 a, b and c). When the 
cut did not pass through the nuclear area all persisting nucleated 
portions matured normally and upon insemination formed fer- 
tilization membranes and segmented. Of the non-nucleated por- 
tions those from eggs in stage b are non-fertilizable (Fig. 22). 
Those from eggs in stage c form fertilization membranes upon 
insemination. Nuclear division also takes place, so that the egg 


342 ROBERT CHAMBERS. 


fragment becomes multi-nucleated but remains unsegmented (Fig. 
23-c). Non-nucleated fragments of eggs in a later stage (stage 
d) proceed somewhat farther (Fig. 24). The multi-nucleated 
masses arising from them make several periodic attempts at seg- 
mentation (Fig. 24-c). Small furrows appear over the surface 
of the egg, cutting in between the peripherally arranged nuclei. 


Fic. 22. Starfish egg in stage corresponding to b in Fig. 6 cut into two 
fragments, The non-nucleated fragment contains no material from the 
germinal vesicle and is nonfertilizable. 


Fic. 23. Starfish egg in a later stage corresponding to c in Fig. 6 cut 
through the nuclear area. The cytoplasm in the injured nuclear area disin- 
tegrated leaving a non-nucleated fragment, b. That the fragment is fertiliza- 
ble is shown in c by the formation of a fertilization membrane and the re- 
peated division of the sperm nucleus. The fragment, however, is unable to 
segment. 

Fic. 24. a, starfish egg in stage d of Fig. 7 cut into a nucleated and non- 
nucleated fragment. b, both fragments fertilized. The nucleated fragment 
segmented in the normal way with a number of blastomeres. The non- 
nucleated fragment became multinucleated and furrows appeared over its 
surface in an attempt at segmentation. 


MICRODISSECTION STUDIES. 343 


These furrows then disappear, to reappear again after a short 
interval. This may occur several times until the egg finally re- 
verts to a spherical shape and remains so. In stage f the ger- 
minal vesicle has disappeared except for the definitive egg nu- 
cleus. Of such eggs any non-nucleated portion down to a cer- 
tain size is capable of being fertilized and undergoing cleavage. 

The above experiments lead one to infer the existence of a sub- 
stance in the germinal vesicle which, on dissolution of the nuclear 
membrane, diffuses throughout the cytoplasm. The fertilizability 
of any egg fragment apparently depends upon the extent of dif- 
fusion of this substance. An egg fragment taken when a minimum 
amount of this substance has diffused into it will allow the sperm 
nucleus which has entered into it to divide. The presence of a 
little more of this substance will allow the fragment to undergo 
abortive segmentation. It is not until a sufficient amount is dis- 
tributed throughout the egg that any fragment can develop 
properly. 

Mature eggs were now studied, and it was found that any egg 
fragment in order to be capable of fertilization must contain a 
portion of the original cortex. The cortex and interior of mature 
unfertilized eggs were separated according to the method described 
under heading 4 (Fig. 25 aand b). The endoplasmic sphere and 
the cortical remnant were then inseminated. The fragment con- 
sisting of the cortical remnant is readily fertilizable and undergoes 
segmentation (Fig. 25 b and c). The endoplasmic sphere is non- 
fertilizable, no matter whether it contains the egg nucleus or not. 

That the protoplasm of the endoplasmic spheres has not been 
irreparably injured in the process of flowing through a small tear 
in the cortex is shown in the following experiment. Eggs were 
squashed until the endoplasm protruded as lobate processes, where- 
upon the pressure on the eggs was lifted and the extrusion allowed 
to flow back into the egg. Such eggs are fertilizable and are capa- 
ble of undergoing cleavage. One such case is illustrated in Fig. 26 
where the cortex was torn in two places on squashing the egg and 
two exovates were formed. The nucleated exovate was allowed to 
pinch itself off. The other exovate flowed back into the remainder 
of the egg upon insemination (Fig. 26 b and c). A fairly com- 


344 ROBERT CHAMBERS. 


plete fertilization membrane formed around the egg except at 
the two torn spots and cleavage followed. 

Endoplasmic exovates were also produced which remain con- 
nected by a bridge of protoplasm to the collapsed cortical portion 


Qa 


Fic. 25. a, nucleated exovate of internal cytoplasm produced by squashing 
a starfish egg. b, fragments inseminated after the endoplasmic sphere was 
pinched off. Only the ectoplasmic remnant forms a fertilization membrane. 
c, the endoplasmic sphere remains inert and nonfertilizable (cf. Fig. 12). 

Fic. 26. a, starfish egg squashed producing two endoplasmic exovates. 
b, the nucleated exovate was pinched off. Upon insemination the other ex- 
ovate drew back into the ectoplasmic remnant which formed a fertilization 
membrane. c, d and e, the ectoplasmic remnant underwent segmentation 
showing that the disturbance due to the squashing does not prevent segmen- 
tation. The endoplasmic sphere remains inert (d). 


of the egg. On being inseminated the exovate either is drawn back 
into the cortical portion as the latter rounds up with the formation 
of a fertilization membrane or is pinched off, after which it remains 
as an inert body. 

The possibility suggested itself that the substance which renders 
an egg fertilizable has a tendency to collect in the surface film of 
an egg and that, if an exovate remained in organic continuity with 
the egg, this substance might spread to the surface film of the 
exovate, thus rendering it fertilizable. Endoplasmic exovates were, 
therefore, produced which remained connected for varying lengths 
of time with the cortical portion of the egg. Some of the exovates 
remained connected for as long as fifteen minutes. Before insemi- 


MICRODISSECTION STUDIES. 345 


nation they were pinched off from the cortical portion of the eggs. 
None developed of those which were separated in such a way that 
there was no question as to their lacking any of the original cortex 
of the egg. 

An endoplasmic sphere, in order to develop at all, apparently 
must incorporate in its substance at least a part of the original 
cortex of the egg. This is shown in Fig. 27. An exovate was 


a Fertil. 1.25 
; @) 
cS x 
a BAIS 
WAS 
Fis 27 


Fic. 27. a, an exovate is produced by squashing and most of the ecto- 
plasmic part is cut away along line of arrow. b, the endoplasmic sphere formed 
itself incorporating a small part of the cortex. Upon fertilization the small 
cortical region formed a partial fertilization membrane. c, many furrows 
form simultaneously over the surface of the egg showing that it has been 
fertilized. (Note that the small cortical piece to one side of the egg has 
segmented in two.) d, the egg has reverted into a multinucleated nonseg- 
mented mass except for three blastomere-like bodies which were pinched off. 
e, the fragment is again attempting to segment. 


produced by crushing an egg (Fig. 27-2). However, before the 
exovate was set free most of the cortical remnant was cut away, 
leaving a very small piece which was drawn into the circumference 
of the endoplasmic sphere. On being inseminated a small shred 
of the egg membrane lifted off from this remnant, and this was 
all that constituted the fertilization membrane (Fig. 27-b). A 
sperm on entering this sphere underwent nuclear division several 
times. This was followed by cleavage furrows which formed on 
the surface of the egg between the peripheral nuclei and gave to 
the egg the appearance of a mulberry (Fig. 27-c). Some of the 
furrows deepened sufficiently to pinch off nucleated bodies. A 
few minutes later the furrows became obliterated and the main 
body of the egg appeared again as a non-segmented but multi- 
nucleated mass (Fig. 27-d). This process may occur several 
times (Fig. 27-e). The ability of an exovate to approximate 
normal segmentation is a function of the amount of the original 
egg cortex which it incorporates. 


346 ROBERT CHAMBERS. 


The inability of the endoplasmic sphere to develop is not due 
to the lack of successful sperm entry. Sections show that the 
sperm enter with ease but they remain unchanged and no asters 
form about them. In this regard the sperm react exactly as 
they do when they have entered immature eggs. 

There must be something localized in the cortex which is nec- 
essary for successful fertilization and development (cf. Lillie, 
*I4, °18). On the evidence presented here we may assume that 
this substance, originally within the germinal vesicle, diffuses 
out upon its dissolution and accumulates in the cortex of the egg. 
It is held in the cortex of the egg and is not carried out in the 
endoplasmic spheres on crushing the egg. The spheres are, 
therefore, incapable of being fertilized. Finally, the variation 
in the ability to segment among exovates containing varying 
amounts of cortical material indicates that there must also be 
a definite minimum amount of this substance present in order 
that an egg fragment may develop. 


CONCLUSIONS. 


1. The nucleus possesses a morphologically definite membrane. 

2. Tearing the nucleus results in an immediate change of the 
nuclear membrane, followed by a disintegration of the cytoplasm 
surrounding it. This is most striking in the feeb large 
nucleus (germinal vesicle) of the starfish egg. 

3. Injection of the germinal vesicle sap of one egg into the 
cytoplasm of another egg starts up disintegration processes in the 
injected area. 

4. The mature egg nucleus can be pinched into two fragments. 
The fragments behave like fluid droplets and will run together 
when contiguous. Eggs whose nuclei have been operated upon 
in this manner are capable of normal segmentation. 

5. A membrane can be demonstrated adhering to the surface of 
the unfertilized starfish, sea-urchin and sand-dollar eggs. This 
egg membrane is most pronounced in the starfish and least of all 
in the sea-urchin. In the starfish and sand-dollar the membrane 
can be stripped off without injuring the egg. In the starfish a very 
delicate egg membrane can be demonstrated investing half-sized 


MICRODISSECTION STUDIES. 347 


immature eggs. This membrane becomes more pronounced as the 
eggs reach their full growth and still more so as the egg matures. 
In the sea-urchin the immature eggs exhibit no trace of a mem- 
brane until the eggs begin maturation. In the mature unfertilized 
sea-urchin egg the membrane has reached a development com- 
parable to that of the half-grown immature egg of the starfish. 

6. The egg membrane rises off the surface of the egg upon fer- 
tilization and constitutes the fertilization membrane. No appreci- 
able diminution in volume of the egg occurs during this process. 

7. An egg, whose membrane has been removed, is fertilizable 
and segments without a fertilization membrane. 

8. The hyaline plasma layer, which forms on the surface of the 
sea-urchin and sand-dollar egg within ten minutes after fertiliza- 
tion, binds the blastomeres together. In the starfish egg no such 
layer is formed, and, if the fertilization membrane be removed, the 
blastomeres tend to fall apart. 

g. The fertilizability and approach to normal development of 
an egg fragment is directly proportional to the amount of a 
substance which emanates from the germinal vesicle during 
maturation. 

10. The unfertilized mature egg possesses a more solid cortex 
of appreciable thickness inclosing a highly fluid interior. The 
fluid interior of the starfish and sand-dollar eggs can be made to 
ooze out through a tear in the cortex, whereupon it forms a sur- 
face film on coming into contact with sea water. In this way the 
internal and cortical material of the egg can be isolated from one 
another. Both round up, the internal material immediately and 
the cortical after some time. 

11. Endoplasmic material, possessing a small part of the original 
cortex, is fertilizable and the approach to normal development is 
in direct proportion to the amount of cortical material present. 
The presence of even a small amount of cortical material causes 
disintegrative changes to set in at about the same time as in a 
whole egg. 

12. The following table gives, for the various kinds of frag- 
ments of immature and mature starfish eggs, the length of time 
that they withstand disintegration when left standing in seawater 
and also whether they are or are not capable of being fertilized: 


348 


ROBERT CHAMBERS, 


Mature 


Immature 
i Nucl. or Non-nucl. 
nc sears Nonsnncls ecg Maem. - - 
enticcters fragm. entirelers fragm. SeIOPESE Se 
Longevity | — a + = = 74 
in 
hours... 24-36 2-3 8-10 8-10 8-10 24-36 
Fertiliza- 
bility... = oF + = 


(when mature 


As regards longevity it will be seen that the immature egg de- 
pends upon its nucleus (germinal vesicle) to prevent disintegra- 
tion, for a fragment lacking the nucleus disintegrates very quickly. 
On the other hand, the mature egg, which has become permeated 
with the nuclear sap of the germinal vesicle, behaves quite ditter- 
ently. The non-nucleated fragment of a mature egg lasts longer 
than that of an immature egg and it is significant that the pres- 
ence of the nucleus of the mature egg, which consists of not 
much more than the chromosomal constituents, has no effect in 
preventing disintegration. 

The long period that the endoplasmic sphere withstands disin- 
tegration indicates that the factors which make for disintegra- 
tion reside chiefly in the original cortex of the mature egg. 

In regard to fertilizability it is evident that the substance which 
renders cytoplasm fertilizable emanates from the germinal ves- 
icle and finally becomes localized in the cortex of the mature egg. 

We can, therefore, distinguish three factors in the starfish egg ; 
one affecting longevity, the second affecting disintegration and 
the third affeeting fertilizability. The first and third have been 
traced to the germinal vesicle of the immature egg. The second 
is a function of the egg cortex. 


BIBLIOGRAPHY. 
Barber, M. A. 
’r4 The Pipette Method in the Isolation of Single Micro-organisms and 
in the Inoculation of Substances into Living Cells. Philipp. Jour. Sc., 
IX., Sec. B, 307. 
Buller, A. H. R. 
%o92 Is Chemotaxis a Factor in the Fertilization of Animals? 
Micr. Sc., XIV., 145. 
Chambers, Robert 
15 Microdissection Studies on the Physical Properties of Protoplasm. 
Lancet-Clinic, Mar. 27. 


Quar. Jour. 


MICRODISSECTION STUDIES. 349 


17° Microdissection Studies I. The Visible Structure of Cell Protoplasm 

and Death Changes. Amer. Jour. Physiol., XLIII., 1. 

17” Microdissection Studies II. The Cell Aster: a Reversible Gelation 

Phenomenon. Jour. Exp. Zo6l., XXIII., 483. 

738" The Microvivisection Method. Biol. Bull., XXXIV., 121. 

’78” A Report on Results Obtained from the Microdissection of Certain 
Cells. Trans. Roy. Soc., Sec. IV., 41. 

719 Changes in Protoplasmic Consistency and their Relation to Cell Divi- 
sion. Jour. Gen. Physiol., II., 40. 

’21° Studies on the Organization of the Starfish Egg. Jour. Gen. Physiol., 
IV., 41. 

’21° A Simple Apparatus for Accurate Micromanipulation under the Highest 
Magnification of the Microscope. Sc., N.S., LIV., 411. 

Chambers, R. and Mossop, Bessie. 

7718 A Report on Cross-fertilization Experiments (Asterias X Solaster). 

Trans. Roy. Soc., Gan., Sec. IV., 145. 
Conklin, E. G. 

708 The Habits and Early Development of Linerges mercurius. Carn. 

Inst. Publ., 103, 153. 
Elder, J. C. 

73. The Relation of the Zona Pellucida to the Formation of the Fertiliza- 
tion Membrane in the Egg of the Sea-urchin (Strongylocentrotus 
lividus). Arch. Entwicklungsmech., XXXV., 145. 

Garrey, W. E. 

’t9 The Nature of the Fertilization Membrane of Asterias and Arbacia 

Eggs. Brot. Butyt., XXXVII., 287. 
Gemmill, J. F. 

%12 The Development of the Starfish, Solaster endeca, Forbes. Trans. Zodl. 

Soc., Lon., XX., 1. 
Glaser, 0. 

713, On Inducing Development in the Sea-urchin (Arbacia punctulata) 
etc. Sc., N.S., XXXVIII., 446. 

’14. The Change in Volume of Arbacia and Asterias Eggs at Fertilization. 
Brot. Bury., XXVI., 84. 

Harvey, E. N. 

"190 The Mechanism of Membrane Formation and Other Early Changes in 
Developing Sea-urchins’ eggs, ete. Jour. Exp. Zodl., VIII, 355. 

’r4 Is the Fertilization Membrane of Arbacia Eggs a Precipitation Mem- 
brane? Brox. BuLt., XXVII., 237. 

Heilbrunn, L. V. 

"13. Studies in Artificial Parthenogenesis. I. Membrane Elevation in the 
: Sea-Urchin Egg. Brox. Burz., XXIV., 343. 
Herbst, Curt. 

793, ~Ueber die kiinstliche Hervorrufung von Dottermembranen an unbe- 
fruchteten Seeigeleiern nebst einigen Bemerkungen iiber die Dotter- 
hautbildung tiberhaupt. Biol Centrabl., XIII., 14. 

Hertwig, 0. : 
76 Betrage zur Kenntnis der Bildung, Befruchtung und Theilung des 
thierischen Eies. Morph. Jahrb., 1, 347. 


350 ROBERT CHAMBERS. 


Kite, G. L. 
‘12 The Nature of the Fertilization Membrane of the Egg of the Sea- 
Urchin (Arbacia punctulata). Se., N.S.. XXXVI., 562. 
Lillie, F. R. 
’r4 Studies of Fertilization: VI. The Mechanism of Fertilization in 
Arbacia. Jour. Exp. Zool., XIV. 523. 
718 Problems of Fertilization. Univ. of Chicago Press. 
Loeb, J. 
¥o92 Maturation, Natural Death and the Prolongation of the Life of the 
Unfertilized Starfish eggs (Asterias forbesii) and their Significance 
for the Theory of Fertilization. Brot. Butt., III., 295 
13° Artificial Parthenogenesis and Fertilization. Univ. of Chicago Press, 
p29: : 
Marcus, H. 
’07 Uber den Agegregatzustand der Kernmembran. Sitzungsber. Ges. 
Morph, Physiol., Miinch, XXIII, 61. 
Mathews, A. P. 
’o7_ A Contribution to the Chemistry of Cell Division, Maturation and 
Fertilization. Amer. Jour. Physiol., XVIII., 89. 
McClendon, J. F. 
’r9 Increase in Permeability of the Sea-urchin’s Egg to Electrolytes at 
the Beginning of Development. Sc., N.S., XXXIL., 317. 
’r4 On the Nature and Formation of the Fertilization Membrane of the 
Echinoderm Egg. Intern. Zeits. physik. chem. Biol., I., 163. 
Morgan, T. H. 
93 «Experimental Studies on Echinoderm Eggs. Anat. Anz., TX., 141. 
Seifriz, W. 
220 Viscosity Values of Protoplasm as Determined by Microdissection. 
Bot. Gaz., LXX., 360. 
Wilson, E. B. 
293" Experiments on Cleavage and Localization in the Nemertine Egg. 
Arch. Entwickelungsm., XVI., 411. 
93” Notes on Merogony and Regeneration in Renilla. Brot. Butt., IV., 
215. 
Wilson, E. B. and Mathews, A. P. 
’95 Maturation, Fertilization and Polarity in the Echinoderm Egg. Jour. 
Morph., X., 319. 
Yatsu, N. 
’04 Experiments in the Development of Egg Fragments in Cerebratulus. 
Brot. Butt., VI. 
%98 Some Experiments on Cell Division in the Egg of Cerebratulus lacteus. 
Annot. Zool. Japon., VI., 267. 


Reprinted from the Proceedings of the Society for Experimenta] Biology and Medicine, 
1922, xix, pp. 320-321. 


147 (1894) 
Merogony experiments on sea-urchin eggs. 
By ROBERT CHAMBERS and HIROSHE OHSHIMA. 


[From the Marine Biological Laboratory, Woods Hole, Mass.] 


By merogony in the broader sense is meant the fertilization 
and development of egg fragments whether nucleated or not. 

By means of the more accurate method of using a mechanical 
apparatus for microdissection an attempt was made to repeat the 
work of earlier investigators (O. and R. Hertwig, Boveri, Driesch, 
Morgan, Loeb, Wilson and others) especially for the purpose of 
cross-fertilizing egg fragments of the sea-urchin and sand dollar. 
Owing probably to the lateness of the season the cross-fertiliza- 
tion experiments were unsuccessful. 

However, the following results were obtained in the self- 
fertilization of sea-urchin egg fragments which indicate that the 
size of the nucleus in the swimming larve depends directly upon 
the initial size of the nucleus in the fertilized egg fragment whereas 
the size of the larva bears no direct relation either to the size of 
the nucleus or to the initial amount of cytoplasm in the fertilized 
egg. Mature eggs were deprived of their nuclei by cutting them 
out together with a minimum amount of cytoplasm. The non- 
nucleated fragments were about 4/5 the size of the entire eggs. 
These, when fertilized, developed into dwarf larve of about half the 
size of the control and with abnormally small nuclei. Other eggs 
were deprived of more than half of their cytoplasm. These, upon 
fertilization, developed into dwarf larve of about half the size of 
the control but possessed nuclei equal in size to that of the control. 


Reprinted from the Proceedings of the Society for Experimental Biology and Med- 
cine, 1921, xix, pp. 85-87. 


45 (1792) 
Apparatus for micro-manipulation and micro-injection. 


By ROBERT CHAMBERS. 


[From the Department of Anatomy, Cornell University Medical 
College, New York City.] 


This apparatus is designed for the purpose of dissecting living 
cells or injecting substances into them, and for isolating micro- 
organisms. Its advantage over that which Barber described in 
the Philippine Journal of Science in 1914 is its simplicity of con- 
struction, and the accuracy with which it can be manipulated. 

The apparatus consists of two instruments, the micro-manipu- 
lator for producing movements in the microscopic field in any of 
three dimensions and, second, the micro-injection instrument for 
securing the necessary pressure to drive or suck substances through 
a micro-pipette. The method of making glass micro needles and 
pipettes is given in full in Barber’s paper and in mine in the 
Biological Bulletin of 1918. 

The micro-manipulator is small and compact and can be 
attached to the stage of any microscope. It consists of a system 
of rigid metal bars connected together with spring hinges. By 
turning certain screws the bars are forced apart. On reversing 
the screws the springs return the bars to their original positions. 
The instrument moves the tip of a needle or a pipette in three 
arcs at right angles to one another. The arcs are small enough 
so that, in the microscopic field, the needle moves practically in 
straight lines. The movements are fine and steady enough to be 
under perfect control when viewed under the highest power of the 
microscope. The instrument can be used singly for one needle 
only or with a companion when two needles, or a pipette and a 
needle, are to be used simultaneously. 

In the micro-injection instrument mercury or an inert oil 
(Nujol) is used to procure the necessary pressure. The instrument 
consists of a thin-walled steel tube about six inches long and half 


SCIENTIFIC PROCEEDINGS (118). 2 


an inch in diameter, one end of which is provided with a stopcock. 
The other end leads into a small steel tube fine enough to be 
flexible and long enough and so bent that, while the large tube lies 
on the table beside the microscope, the tip of the fine tube can 
be held in the pipette carrier of the micro-manipulator. Into 
this tip a glass Barber pipette is sealed. Mercury or oil is intro- 
duced through the stopcock of the large tube and is forced on into 
the micro-pipette. The stopcock is then shut off. By means of 
leverage clamps on the thin-walled tube the mercury or oil can 
be driven through a pipette having an aperture of only one micron 
in diameter. By turning the screws of the micro-manipulator 
the tip of the pipette can be brought into a hanging drop in a 
Barber’s moist chamber. Release of pressure on the steel tube 
draws substances into the pipette. Injection and suction in micro- 
scopic quantities is accurately controllable as the meniscus of the 
mercury or oil in the pipette responds instantly to the pressure 
of the leverage clamps. 


Reprinted from Tar ANATOMICAL RecorD 


AUTHOR'S ABSTRACT OF THIS PAPER ISSUED 
Vol. 24, No. 1, August, 1922 


BY THE BIBLIOGRAPHIC SERVICE, JULY 3 


NEW APPARATUS AND METHODS FOR THE DISSEC- 
TION AND INJECTION OF LIVING CELLS 


ROBERT CHAMBERS 
Cornell University Medical College, New York City 


FIVE FIGURES 


INTRODUCTION 


Operative work on the living cell has long been the aim of 
investigators in cytology and in experimental embryology. It 
was, however, not till Barber developed his method that any 
serious attempt could be made to dissect cells under magnifica- 
tions high enough to enable one to observe in detail the various 
steps of the operation. The big feature of his method, aside: 
from the making of needles and pipettes stiff and yet fine enough 
to puncture red blood corpuscles, consists in his moist chamber, 
which allows the needle tips to be operated in a drop hanging 
from a cover-slip in the chamber. This method eliminates all 
obstacles between the objective and the cover-slip, thereby per- 
mitting the use of the highest-powered objectives. Unfortu- 
nately, his instrument for manipulating the needles, unless very 
skillfully made, has too much lost motion, and wear and tear 
soon render the movements jerky and undependable. 

Barber uses his apparatus principally for the isolation of 
bacteria. In 1912 Kite (Kite and Chambers, ’12) applied Bar- 
ber’s method to cytological investigation. The difficulty of 
handling Barber’s apparatus limited the number of investigators 
in this field and as the work in microdissection progressed the 
need of a more accurate and simple instrument became 
imperative. 

The instrument described in this paper, a preliminary account 
of which has been published (’21), has the following advantages 
over any instrument hitherto made: a) simplicity of construction, 

1 


» ROBERT CHAMBERS 


b) no lost motion through wear and tear, ¢) accurate and con- 
tinuous control of the movements of the needle or pipette tip in 
any direction under the highest magnifications of the microscope, 
d) maintenance of the needle tip in one plane while it is being 
moved back and forth in any of the three directions, and e) exist- 
ence of preliminary adjusting devices which facilitate placing the 
needle or pipette quickly into position. 

The basic principle of the instrument consists in rigid bars 
which are screwed apart against springs. The movements im- 
parted are in ares of a circle having a radius of about two and a 
half inches. As the extreme range of movement of the fine ad- 
justments is only 2 mm. (of which only one is necessary) the 
curvature of the are is unnoticeable. 

The movements performed by the instrument are so accurately 
controlled that one can readily carry out such delicate operations 
as puncturing mammalian blood corpuscles, tearing off the 
sarcolemma of a muscle fiber, drawing out nuclear chromatin 
strands and even cutting up the chromosomes of insect germ 
cells. The glass needles used for these operations taper rapidly 
to a point invisible under the oil immersion objective. With the 
micropipette, the bore of which need be no larger than one micron 
in diameter, one can either inject substances into or withdraw 
material from a cell. 

For the isolation of bacteria the instrument is not only steadier 
than Barber’s apparatus but has new features which facilitate 
greatly the method of procedure. Its application to bacterio- 
logical purposes is more specifically dealt with in the Journal of 
Infectious Diseases. 

I take this opportunity of expressing my deep obligation to 
Mr. W. H. Farnham, mechanician in the department of Chemical 
Engineering in Columbia University, to whose skill and faithful 
workmanship the practical evolution of the instrument is due. 
I wish especially to acknowledge assistance from Dr. Milton 
J. Greenman of The Wistar Institute and Dr. C. V. Taylor of 
the University of California. I wish also to express my apprecia- 
tion to many friends for valuable suggestions. The principle 
involved in the construction of the micromanipulation instru- 
ment is patented. 


DISSECTION AND INJECTION OF LIVING CELLS 3 


A MECHANICAL MICROMANIPULATOR FOR CONTROLLING THE 
MOVEMENTS OF A MICRONEEDLE OR MICROPIPETTE IN 
THE FIELD OF A COMPOUND MICROSCOPE 

The principle of this device is demonstrated on considering the 
mechanism for the movements in one plane only (fig. 1, b). This 
consists of three bars of rigid metal connected at their ends to 
form a Z-like figure by resilient metal acting as a spring hinge. 


Fig. 1 Diagram showing the working principle of the micromanipulator. In 
l,a, where the instrument is viewed from the side, screw, J, moves needle tip 
through vertical are, y-z. In 1,b, where the instrument is viewed from above 
serews, Gand H, move the needle tip through the horizontal ares m—n and o-p. 


By the action of certain screws the bars can be forced apart; 
on reversing the screws the bars return to their original position 
owing to the spring action at the end of the bars. By these 
means arc movements may be imparted to the tip of a needle 
when placed in the proper position. 

The needle or any instrument, the tip of which is to be manip- 
ulated, is held in a carrier fastened to the free end of a bar, 
A at X. The needle is made to extend so that its tip is at the 
apex of an imaginary triangle at D. In order to obtain two 
movements at right angles to one another and in the horizontal 
plane the tip of the needle must be at the apex, D, of a right- 


4 ROBERT CHAMBERS 


angled isosceles triangle the base of which is a straight. line 
joining the centers, # and F’, of the two springs holding the three 
bars, A, B, and C, together. The shank of screw, G, passes 
through a large hole in bar, C, and is screw-threaded in bar, B. 
Turning it spreads apart bars, A and B, and imparts an are 
movement to the needle tip at D at right angles to that procured 
by turning screw, H. 

The movement in the vertical plane at right angles to the 
aforementioned movements is produced by screw, J (fig. 1, a), 
which is screw-threaded in a rigid vertical bar, J, and abuts 
against a vertical extension, K, of bar, C. The extension, K, is 
parallel to the bar, J, and is connected to it at its top by means 
of a solid spring hinge. Turning screw, J, spreads apart bars, 
J and K, and lifts the whole combination (A, B, and C) and 
imparts an are movement in the vertical plane to the tip of the 
needle at D. To procure a vertical movement, the tip of the 
needle at D must lie in the same horizontal plane, L-D, with 
the spring fastening K and J together. When screw, /, is turned, 
the needle tip will then move in an arc, Y to Z, more nearly 
vertical than any other are on the same circumference of which 
the point, D, is the center. 

There are two models of the micromanipulator. One is fitted 
with a clamping device with which it can be fastened directly to 
the front of the microscope stage (fig. 2; ef. fig. 3, e).! The other 
is fastened to a rigid pillar rising from a large metal base on 
which the microscope is clamped (fig. 3, a). The horizontal bars 
of the instrument extend diagonally across the corner below the 
level of the stage. They do not interfere with the substage acces- 
sories of the microscope nor with any of the known types of 
mechanical stages. 

The necessity of having one or two instruments is, of course, 
conditioned by the type of work to be done. For picking up 
bacteria one is sufficient. For microdissection in experimental 
embryology a great deal can be done with one instrument, but 
for cell injection in general and for tissue cell dissection two 


1 Steadiness may be assured by a brace, one end being screwed to the rigid 
vertical part of the instrument and the other end to the foot of the microscope. 


DISSECTION AND INJECTION OF LIVING CELLS 0 


instruments are indispensable so that two needles or a needle 
and a pipette may be manipulated simultaneously. When two 
instruments are to be used both must be placed at the front of 
the microscope so that the needles may extend, side by side, into 
the moist chamber from the front. As the horizontal bars of 
each instrument extend diagonally under the microscope stage 


f 


Fig. 2 Left-handed micromanipulator to be clamped to microscope stage. 
a, needle carrier with clamping screw; b, screw to clamp post of needle carrier; c, 
serew for up-and-down movement; d and e, screws for lateral movements; /, 
dise guide for the horizontal bars; g, stationary or rigid part of instrument with 
lugs by means of which instrument is clamped to microscope stage. Screw, ), 
clamps the coarse adjustments. 


one must be a mirror image of the other. According to their 
position with respect to the microscope these two models have 
been designated as left-handed and right-handed. For bacterio- 
logical work, where it is more convenient to work from the left, 
the right-handed model is to be preferred as it can be swung 
around and fastened to the left side so that the pipette may 


6 ROBERT CHAMBERS 


extend into the moist chamber from the left. For cytological 
work, if one desires to have only one instrument, it is advisable 
to secure the left-handed form and to use it as shown in figure 3, e. 
The mechanical stage may then be operated with the right hand 
and the instrument with the left. Eventually this instrument 
may be supplemented with a right-handed form to be clamped 
to the stage or attached to a pillar. When a pair of instruments 
is to be used the best combination is a left-handed one clamped to 
the microscope and a right-handed one attached to a pillar (fig. 
3). This allows one to hold the tissue on which one is operating 
with one instrument while the microscope is being temporarily 
removed for renewing the pipette of the other (see page 14). 


THE SETTING UP AND THE WORKING OF THE INSTRUMENT 


Figure 3 shows two instruments in place ready for work. 
They should be as close together as possible so that the open 
end of the moist chamber need not be too wide to accommodate 
the needles. This leaves ample room on either side for the attach- 
ment of a mechanical stage on the microscope.’ 

The instrument is provided with means for a preliminary 
adjustment of the needle in any direction. By these means the 
needle tip can be quickly centered in the field of a low-powered 
objective and raised close to the hanging drop in which it is to 
operate. Before centering the tip the bars which control the 
fine adjustments must be put into a state of tension by giving 
a few turns to the milled heads of each of the three screws. 
The instrument is now ready for action. 

The milled heads of the screws which control the lateral 
movements are provided with holes for rods to be used as levers. 
A most useful accessory is a wire-wound flexible shaft about 2 
feet 6 inches long (fig. 3, c) with a milled head at one end (fig. 3, d) 
and the other end attached to the screw controlling the up-and- 
down movement. Curving the shaft around one side of the micro- 
scope brings the control of this screw, which is the one most 


2 In the case of the Bausch & Lomb and Spencer stages, it may be necessary 
to replace the screw clamping the front end of the stage by one with a smaller 
head, 


Ve —_—_—? TTT 


<= a ~ . 
8 a 4 2 


Fig. 3 Microscope with two micromanipulators and the microinjection 
apparatus in place. a, right-handed manipulator on pillar set in collar, b, fastened 
to base on which microscope is clamped; c, flexible shaft attached to screw for 
up-and-down movement with its milled head at d. (Note that screws for lateral 
movements are controlled by levers.) e, left-handed manipulator clamped to 
left front of microscope stage. In its needle carrier is clamped brass collar, f, 
within which shaft of needle slides. (See detail in fig. 3!.) The coarse adjust- 
ment for raising and lowering needle carrier is done by screw, g; that for the 
lateral movements is done by turning the post on its axis. 

Injection apparatus. h, Luer syringe set in its butt, 7, cemented to curved 
brass tube, 7. This is clamped to base at k. Its other end is cemented into glass 
tube, 1 (see detail in fig. 32), clamped in needle carrier of the right-handed manip- 
ulator, a. 

Most of the holes in the base are unnecessary. Foot of microscope is held by 
two screw clamps. The adjustable guide, m, keeps microscope in proper alignment. 

Fig. 3! Detail of brass collar (f in fig. 3) which facilitates in-and-out movement 
of needle or pipette; m, screw which presses on a spring to clamp the needle in 
the collar. 

Fig. 32. Detail of glass tube of injection apparatus (l in fig. 3) cemented on 
brass tube, j; 0, shank of micropipette cemented into end of glass tube. The 
pipette is readily changed by softening the sealing wax which holds it. 


7 


S ROBERT CHAMBERS 


frequently used, close to that of the fine adjustment of the micro- 
scope. The shaft also facilitates the use of both hands for the 
various movements of the one instrument. 

Another useful accessory is a brass collar 13 inch long (fig. 3) 
with a spring which projects into its lumen through a slot. The 
shaft of the needle is slipped through the collar and the screw, 
clamping the spring, tightened sufficiently to enable one to slide 
the shaft evenly. The collar is then clamped into the needle 
carrier of the instrument. This arrangement facilitates sliding 
the needle into or out of the moist chamber without danger to 
the tip of the needle. 

The micromanipulator is intended to be used with the mechani- 
cal stage of the microscope. The mechanical stage moves the 
moist chamber (fig. 3). As the cell or tissue to be dissected lies 
in a drop hanging from the roof of the chamber, the motion im- 
parted by the mechanical stage moves the cells against the micro- 
needle. Indeed, most of the dissection, where a single needle is 
used, is done by first bringing the needle tip into the cell and then 
dragging the cell away by means of the mechanical stage. 

The horizontal movements of the micromanipulator are used 
mostly for the purpose of bringing the tip of the needle accurately 
into a desired spot in the field of the microscope preparatory to 
the actual operative work. In order to insure the greatest 
possible steadiness to the vertical movement, the part of the 
instrument which imparts this movement adjoins and is mani- 
pulated from the stationary and rigid part of the instrument. 
To make this possible the present design incorporates a theoreti- 
eal error which can be understood from figurel,a. Turning screw, 
TI, to produce the vertical movement throws the combination of 
bars A, B, and C, out of the horizontal, and it is these bars upon 
which the lateral movements of the needle depend. However, 
the angle at which these bars are placed minimizes the error so 
that it is unnoticeable. 

Guides exist in the instrument to insure a true travel of the 
bars as they spread apart or come together. The guide for the 
bar which produces the vertical movement consists of a depres- 
sion in the stationary part of the instrument into which the verti- 


DISSECTION AND INJECTION OF LIVING CELLS 9 


cal bar fits. The guides of the lateral movements are two metal 
dises which can be tightened or loosened by screws. The upper 
one is seen in figure 2, f. They correct two possible errors which 
may occur on reversing the direction of movement, viz., a drop- 
ping of the needle or pipette out of focus and a shifting to one side. 

The first error can be corrected by tightening one or both of 
the guides; the second, by loosening them. The guides, there- 
fore, must be neither too tight nor too loose. The first error is 
the more serious of the two. It is due to an unequal tension in 
the springs which throws the tip of the moving screw to a dif- 
ferent spot on the bar against which it abuts. If this be not 
corrected, the screw will in time wear a depression in the brass 
bar that is out of center thus accentuating the error. The second 
error is due to the guides being too tight so that they bind and 
prevent the bars from making a true return. If not corrected, 
this error will be gradually eliminated with the wear of the 
frictional surfaces. 

By an accidental knock the horizontal bars of the instrument 
may be jarred out of place or the fine adjustment screws injured. 
If the upper and lower surfaces of the horizontal bars are not 
flush loosen the guide dises (fig. 2, f) also the screws of the springs 
on the ends of the bars and, with a wooden mallet, gently hammer 
the bars till they are flush. Then tighten the guide dises to keep 
the bars flush and carefully tighten the screws of the spring. 
If the screws have been bent by the accident they must be 
changed otherwise tightening them will again pull the bars out of 
place. If the guide dises are bent they also must be changed. 
A more serious accident is when the fine adjustment screws are 
injured. The steel shafts of the screws may be bent or they may 
have cut into the brass so as to loosen the threads. This tends 
to throw the shaft of the screw out of center. In such a case 
somewhat larger screws must be made and accurately centered 
opposite the bar against which it abuts. 


THE SUBSTAGE CONDENSER AND THE METHOD OF MAKING 
BARBER’S MOIST CHAMBER AND GLASS NEEDLES 


For critical illumination the height of the moist chamber must 
be equal to the working focal distance of the substage condenser. 


10 ROBERT CHAMBERS 


The Abbe condenser can be used by removing the top lens. The 
foeal distance of the remaining lens is almost one inch. In the 
Bausch and Lomb microscope the substage can easily be arranged 
to raise this lens sufficiently to have at least half its focal dis- 
tance above the surface of the stage. This is ample, for one 
seldom requires a moist chamber as high as half an inch. The 
focal distance of this lens can be reduced and its illuminating 
power correspondingly increased by placing the lens of a 10X 
dissecting lens on top of it. This comhination has a focal dis- 
tance of about 2 of an inch and, if the substage can be raised to 
bring the top lens flush with the upper surface of the stage, all 
of this distance may be used for the height of the moist chamber. 
Better results are secured with a triple lens condenser with its 
top lens removed. Such a condenser from Leitz which I am using 
has a working focal distance of 3 of an inch. One may also use 
condensers which are made with a specially long working dis- 
tance for projection apparatus, in which a cooling trough is 
placed between the condenser and the slide. 

If the working focal distance of the condenser be less than 2 of 
an inch, it is well to have two moist chambers, one for critical 
work and the other, from 2 to 3 an inch high, for ordinary work. 
This is advisable, because it is easier to make needles for the 
higher chamber. 

The moist chamber is of glass (fig. 4). The base is a thin 
glass slide about 23 x 2 inches in size. The sides consist of strips 
of plate glass about 17 inches long and } inch wide, and of a 
height determined upon by the available condenser. One end 
of the chamber is closed with a strip of glass of the same height 
as the sides and backed by another strip a fraction higher, in 
order to prevent a cover-slip from sliding beyond it. The 
trough of the chamber should be from ? to 7 of an inch wide. 
The strips are cemented with any ordinary glass cement. Heated 
Canada balsam serves well. Near the closed end of the trough a 
small strip of glass should be cemented across the trough to pro- 
vide a well for water. When cementing the long strips to the 
base, care must be taken to have the top surface of the strips 
horizontal. This may be done while the cement is still soft by 


DISSECTION AND INJECTION OF LIVING CELLS 11 


focusing on the upper surface of the strips and by manipulating 
the strips until all parts of their surfaces lie in one focal plane. 
The well in the chamber is to be filled with water and, in order 
to distribute the moisture throughout the chamber, strips of 
blotting-paper should be placed along the sides of the trough 
with the inner end in the water well. One may substitute for 
the well strips of blotting-paper laid across the trough. This 
moist chamber is designed for cover-slips of a size 24 x 40 mm. 
The cover-slip is sealed on the chamber with vaseline. Square 


mar, 


Fig.4 Moist chamber and cardboard trough for closing open end of chamber. 
When the needles are in place (ef. fig. 3), the trough is placed over shanks of 
needles (dotted lines at open end of chamber) and filled with vaseline. 


cover-slips may also be used, if the rest of the chamber be 
roofed with other strips of cover-glass. 

The moist chamber is open at one end to permit the entrance 
of the microneedles or pipettes. To prevent undue evaporation, 
especially when a preparation is to be left over night, the open 
end may be temporarily closed by means of a paraffined thin card- 
board trough of a shape shown in figure 4. The trough is placed 
over the shafts of the needles and filled with soft vaseline con- 
taining a few threads of cotton to give substance to the vaseline. 
The vaseline closes around the shafts of the needle and seals the 
opening of the chamber without interfering with the movement 


12 ROBERT CHAMBERS 


of the needles. To prevent the vaseline from spreading on the 
floor of the moist chamber, it is well to have a shallow pan of 
cardboard set under the shafts of the needles for the trough to 
rest upon. 

The hanging-drop containing the cells or tissue to be operated 
upon is placed on the cover-slip which is then inverted over the 
moist chamber.’ To prevent the vaseline from spreading on the 
cover-glass and from contaminating the hanging-drop, a thin 
film of melted paraffin may be spread and cooled on the cover- 
glass bounding the area to be occupied by the hanging-drop. 

The needles are made from either soft or hard glass tubing. 
If a brass collar is used to serve as a guide for the in-and-out 
movement (fig. 3), the glass tubing should be selected to fit the 
collar. What I use is a fraction less than % inch in outside di- 
ameter. The thicker the wall of the tubing the firmer tends to 
be the tip of the needle made from it. The method of making 
the needle is given in a paper of Barber’s (14) and in one of 
mine (’18). A brief account will suffice here. Acetylene or 
ordinary illuminating gas may be used. For a microburner use 
a piece of hard glass tubing bent at right angles and with the 
burner end closed except for the smallest aperture that will retain 
a flame. This may be done by heating the end and pinching it 
with forceps. The size of the flame may be regulated by a screw 
pinch-cock on the rubber tube, figure 5, h. 

To make the needles, proceed as follows: 1) In an ordinary 
burner draw out one end of a glass tube with a capillary of 
about 0.3 to 0.5 mm. in diameter (fig. 5, a). 2) Lower the flame 
of the microburner to the smallest flame possible. Now hold 
the shank of the tube in the left hand and grasp the capillary 
at its end either with the thumb and finger of the right hand 
or with forceps having flat tips coated with Canada balsam. 
Bring the capillary over the flame and pull gently till the capil- 
lary parts. The hands should remain on the table during the 

3 For placing a hanging-drop after the moist chamber has been covered, a 
convenient pipette is one with its end drawn out into a curved capillary and the 
tip bent at an angle so that, on insertion into the moist chamber, the tip will 
touch the undersurface of the cover-slip. With a rubber tube to reach one’s 
mouth, a small drop is readily deposited. 


DISSECTION AND INJECTION OF LIVING CELLS 13 


process and, as the capillary parts, lift the glass away from the 
flame by turning the hands slightly outward. The capillary will 
separate with a slight tug. The tip should be like that in 
figure 5, c. If too little heat be used and the pull made too sud- 
denly, the capillary may part with a snap with a broken tip. 


Fig. 5 Method of making the needles. h, position of hands when making 
needles over microburner; a, glass tube with capillary; b, needle with tip bent 
up; c, a good needle tip; d, needle tip serviceable for converting into a pipette; 
€, unserviceable tip drawn out into a hair; f, needle with stout shank; g, needle 
with tip bent back for cutting purposes. 


Tf too much heat be used, the tip is drawn out into a long hair, 
figure 5,e. 3) Bend the capillary at right angles by heating it 
just back of the point and pushing up with a dissecting needle, 5, b. 
The length of the needle beyond the bend is conditioned by the 
height of the moist chamber to be used. The type of needle 
shown in 5, g, is used for cutting by bringing the upper limb of 
the needle below and up into the cell. 


14 ROBERT CHAMBERS 


APPARATUS FOR INJECTION AND FOR THE WITHDRAWAL 
OF MATERIAL FROM A LIVING CELL 

Barber’s mercury pipette method, which depends upon the 
expansion and contraction of mercury by heat and cold, 
although excellent, is troublesome to make and easily broken. 
Taylor ('20) devised an instrument which depends upon a 
plunger to exert pressure on an enclosed mercury column. With 
mercury, however, it is difficult to maintain a plunger for any 
length of time without leakage. I described an apparatus (’21) 
in which mercury or Nujol oil is enclosed in a thin-walled steel 
cylinder. Pressure on the wall of the cylinder exerts the driving 
force necessary for injection. This works very well, but it re- 
quires special apparatus and the difficulty of securing a cylinder 
the walls of which are sufficiently resilient renders the apparatus 
somewhat unserviceable. 

The apparatus shown in figure 3, does all the work of any device 
hitherto described and has the advantage of being extremely 
simple to make. All that is required is a carefully selected glass 
Luer syringe of about 2 ec. capacity, a piece of fine brass tubing 
of about 2 mm. outside diameter and two feet long (small, extra 
soft brass tubing used for lighting purposes is also serviceable), 
a metal rod ji inch long with a hole through it large enough to 
receive the brass tubing, a piece of §-inch glass tubing, some de 
Khotinsky cement or ordinary sealing wax and an ordinary small 
horseshoe clamp. 

First seal the metal butt of the Luer syringe to one end of the 
brass tubing. Slip the metal rod over the tubing and cement it 
an inch or two away from the syringe attachment. At the other 
end of the brass tubing seal a short piece of }-inch glass tubing, 
the free end of which has previously been drawn out into a 
capillary an inch or so long and about 1 mm. in inside diameter 
(fig. 37). 

When cementing the brass tube to the syringe attachment and 
to the glass tube, have a wire inserted far into the brass tube 
before applying the cement. The tip of the brass tube, from 
which the wire projects, is then coated with cement and the part 
to be cemented pulled over it. While the tube is still warm, 


DISSECTION AND INJECTION OF LIVING CELLS 15 


withdraw the wire with a gentle twirling motion. This draws the 
cement out around the ends of the brass tube on the inner sur- 
face of the projecting glass tube and prevents the formation of 
pockets in which air may be trapped. In the make-up of the 
entire system one must exercise care to prevent air from being 
trapped, for the presence of the air-bubbles vitiates the accurate 
control of pressure in the apparatus. 

The brass tube where the metal rod encloses it is to be clamped 
to the foot of the microscope or to a base which is rigidly at- 
tached to the microscope, figure 3. The short end of the tube, 
projecting from the rod, is bent so that the syringe, when set into 
its butt stands more or less upright. The long end of the tube 
is carefully curved and bent, so that the glass tube which is sealed 
on the end will rest in the needle carrier of the micromanipulator 
and its capillary project over the stage of the microscope with 
its end about 14 inches from the field of the microscope objective. 

The Luer syringe must now be charged with distilled water 
which has been boiled and the apparatus filled to within § of an 
inch from the tip of the glass capillary. Before stopping, how- 
ever, it is well to run water through the apparatus for some time 
to drive out all the air. Before charging the syringe for the last 
time the plunger should be coated with heavy stop-cock grease. 
This much of the apparatus can be kept permanently ready foruse. 

The micropipettes are made from microneedles drawn out of 
thin-walled capillary glass tubing. When finished, the shaft 
of the needle should be at least 14 inches long and large enough 
to fit snugly into the glass capillary of the apparatus. This can 
be readily done by drawing out a supply of thin-walled glass 
capillaries and preserving those which fit a sample the size of the 
capillary of the apparatus. The needle end of the shaft should 
be bent at an angle, the length from the knee of the bend to the 
tip depending upon the height of the moist chamber. The shaft 
of the needle near its end is now thinly coated with de Khotinsky 
cement or sealing wax and, while the cement is still soft, inserted 
into the glass tube of the apparatus. An extra coat of cement 
should be added over the joint to insure the seal. The apparatus 
is now ready for use. The tip of the needle is brought into a 


16 ROBERT CHAMBERS 


hanging-drop of water or a solution to be injected and converted 
into a pipette by jamming the tip against the under surface of 
the cover-slip until it breaks off. During the process continual 
pressure should be exerted on the plunger of the syringe in order 
to prevent pieces of glass from being sucked into the pipette. 
Occasionally, while attempting to make the needle in the flame, 
a serviceable pipette results instead. When the pipette is finally 
in place, all or most of the air in it should be driven out. 

One can readily see that the sealing of the micropipette into 
the apparatus must be done away from the microscope. It is 
in this operation that the type of micromanipulator fastened on a 
pillar is of advantage. The pipette has to be frequently changed, 
and it is very convenient to be able to release the microscope 
from its base by loosening its clamps and to slip it out of the way. 
As soon as a fresh needle has been inserted, the microscope is 
readily slid back into place. For this purpose the base on which 
the microscope rests is provided with guides to insure its true 
return. When exchanging a pipette, care must be taken not to 
clog the lumen. This can be done by using a minimum amount 
of cement and by having the lumen of the tube into which the 
shaft of the pipette is to be inserted as clean as possible. 

The use of thin-walled tubing for making the micropipette is 
to insure having the largest bore possible at the tip of the pipette. 
The thickness of the wall and the size of the lumen of the glass 
tube tend to maintain their original proportions when drawn 
out in a flame. Often, however, it is more convenient to have 
pipettes with stouter walls. Such pipettes are less readily broken 
but, owing to the smaller-sized lumen, run the risk of quickly 
clogging. The best pipettes are made from hollow needles with 
a rapidly tapering tip (fig. 5, d), for needles with a long taper are 
apt to break anywhere. 

A necessary precaution is to have the capillary from which the 
needle is to be made perfectly dry. The presence of the least 
moisture may result in alternating columns of water and air in 
the pipette tip which no amount of pressure will expel. 

Water seems to be the best medium for transmitting pressure 
in the apparatus. Mercury is apt to break and allow air or 


DISSECTION AND INJECTION OF LIVING CELLS iN? 


water to leak past it when it reaches the tip of the pipette. 
When this occurs, the separated droplet of mercury clogs the 
aperture. Mercury also tends to leak past the best plunger 
made.‘ The disadvantage of using water is the risk of its dif- 
fusion into the solution to be injected. If a considerable amount 
of the solution be drawn into the pipette, this risk is minimized. 
A good method is to color the water (e.g., with Nile-blue chlorhy- 
drate or with neutral red). The solution drawn into the pipette 
from a hanging-drop is then visible by contrast. For ordinary 
purposes a cushion of air between the water and the injection 
fluid serves well. 

Oil is unsuitable because, in spite of all precautions, it occa- 
sionally comes into contact with the hanging-drop containing 
the tissue to be operated upon; it then spreads over the surface 
of the drop and injures the preparation. It also dissolves de 
Khotinsky cement and sealing wax which are so convenient for 
cementing the pipette to the apparatus. 

Manipulation of the syringe-is facilitated by fastening it in 
a frame and by using a milled screw to press the plunger. I usea 
microscope for this purpose with the objective, substage and 
mirror removed. The syringe is passed through the center of 
the microscope stage where it is held firmly with a tight-fitting 
collar of cork. The lower end of the microscope tube rests on the 
top of the plunger so that pressure can be brought to bear on it 
by either the coarse or fine adjustments. There is no need of 
fastening the plunger to the microscope tube, because the resil- 
iency of the water in the apparatus is sufficient to cause suction 
in the micropipette when the plunger is released from pressure. 


APPENDIX 


Barber’s instrument is based on the principle of a carrier pushed 
along a groove by a screw at one end. By having a series of 
three carriers built up on one another, each traveling in a dif- 
ferent direction, movements in any one of three dimensions may 


4 Leakage in the syringe can be avoided by placing a cushion of oil between the 
plunger and the mercury. This may also be done when water is used. 


18 ROBERT CHAMBERS 


be imparted to a needle clamped to the top carrier. Hecker (’16) 
improved Barber’s instrument, but added materially to the in- 
tricacy of its make up. 

Other investigators that I know of who have devised instru- 
ments for micro-operative work are Schmidt (’69, ’70), Birge 
(82), Chabry (’87), Schouten (’05, ’11), Tchahotine (712, ’21), 
McClendon (’07), Malone (18), Bishop and Tharaldsen (’21),. 

Schmidt’s instrument is one of historic interest only. I have 
already described it (’18). Chabry used a delicate spring device 
with which he could shoot the tip of a glass needle into an ovum 
to any desired depth. Schouten uses his for the isolation of 
bacteria. It consists of a pillar carrying a needle which may be 
mechanically raised and lowered. For the horizontal movements 
Schouten depends upon pushing the microscope on a base. 
McClendon attached an up-and-down movement to a Spencer 
mechanical stage. ‘Tchahotine uses a mechanism attached to the 
tube of his microscope from which extends a glass needle curved 
in such a way as to bring its tip into the field of a low-power 
objective where it is brought into focus. Dissection of cells is 
carried out by moving the microscope tube and by pushing the 
cells against the needle tip by means of the mechanical stage of the 
microscope. Malone uses Schouten’s method, but, instead of 
having a special pillar with a raising device, he mounts his pipette 
earrier on the tube of a second microscope whose adjustments 
serve as a means for raising and lowering the pipette. Bishop 
and Tharaldsen have a simple instrument based on a principle 
somewhat resembling mine but lacking in proper control forone 
of the two lateral movements. Recently I have heard that 
Zeiss is manufacturing a micro-dissection instrument which 
is a modification of Barber’s apparatus with both coarse and 
fine adjustments. 

Tchahotine and Bovie have recently devised a method for 
producing localized injury in a cell by means of ultra violet rays. 
The method is very ingenious but, of course, is rather limited in 
its application to micro-dissection. 


DISSECTION AND INJECTION OF LIVING CELLS 19 


BIBLIOGRAPHY 


Barper, M. A. 1904 A new method of isolating micro-organisms. Jour. 
Kans. Med. Soc., vol. 4, p. 487. 
1911 A technic for the inoculation of bacteria and other substances 
and of micro-organisms into the cavity of the living cell. Jour. Inf. 
Dis., vol. 8, p. 348. 
1914 The pipette method in the isolation of single micro-organisms 
and in the inoculation of substances into living cells. The Philippine 
Jour. Se., See. B, Trop. Med., vol. 9, p. 307 (reviewed in Zeitschr. wiss. 
Mikr., Bd. 32, S. 82, 1915). 

BisHop AND THARALDSEN 1921 An apparatus for microdissection. Amer. 
Nat., vol. 55, p. 381. 

Cuasry, L. 1887 Contribution a l’embryologie normal et teratologique des 
Ascidies simples. Jour. del’Anat. et de Physiol., T. 25, p. 167. 

CuHamBers, R. 1918 The microvivisection method. Biol. Bull., vol. 34, p. 121. 
1921 a A simple apparatus for micromanipulation under the highest 
magnifications of the microscope. Sc., N.S., vol. 54, p.411. 
1921 b A simple micro-injection apparatus made of steel. Sc., N.S., 
vol. 54, p. 552. 

Hecker, F. 1916 A new model of a double pipet holder and the technic for the 
isolation of living organisms. Jour. Inf. Dis., vol. 19, p. 306. 

Kirr, G. L., anp CuamBers, R. 1912 Vital staining of chromosomes and the 
function and structure of thenucleus. Sc., N.8., vol. 36, p. 639. 

McCienpon, J. F. 1907 Experiments on the eggs of Chaetopterus and Asterias 
in which the chromatin was removed. Biol. Bull., vol. 12, p. 141. 

Matongz, R. H. 1918 A simple method for isolating small organisms. Jour. 
Path. and Bacter., vol. 22, p. 222. 

Scumipt, H.D. 1869 and 1870 The microscopical anatomy of the human liver. 
New Orleans Med. Jour., vol. 22, p. 627, and vol. 23, pp. 66 and 274. 

Scuoutgen, 8. L. 1905 and 1907 Reinkulturen aus einer unter dem Mikroskop 
isolierter Zelle. Zeitschr. wiss. Mikr., Bd. 22, S. 10; ibid., Bd. 24, 
S. 258. 
1911 Pure cultures from a single cell isolated under the microscope. 
K6nigl. Akad. Wetensch., Amsterd, Proc. Sect. Sc., vol. 18, pt. 2, p. 840. 

Taytor, C. V. 1920 An accurately controllable micropipette. Sc. N.S., vol. 51, 
iD Uv 

Tcuanotine, 8. 1912 Eine Mikrooperationsvorrichtung. Zeitschr. wiss. Mik- 
ros., Bd. 29, S. 188. 
1912 Die mikroscopische Strahlenstichmethode. Biol. Centralbl., 
Bd. 32, 8. 623. 
1920 La méthode de la radio-piqire microscopique, un moyen d’ana- 
lyse en cytologieexpérimentale. C.R.del’Acad.desSc., T.171, p. 1237. 
1921 Nouveau dispositif pour la méthode de la radio-puncture micro- 
scopique. C.R.delaSoc. de Biol., T. 85, p. 137. 


THE ANATOMICAL RECORD, VOL, 24, NO. 1 


Reprinted from JourNAL or Bacrnriotody 
VoIMII, No. 1, January, 1922 


A MICROMANIPULATOR FOR THE ISOLATION OF 
BACTERIA AND THE DISSECTION OF CELLS 


ROBERT CHAMBERS 
Cornell University Medical College, New York City 


Received for publication June 5, 1922 


I have recently described (Chambers, 1922, b) an apparatus 
for the manipulation of micro needles and micro pipettes under 
the highest magnifications of the microscope. This apparatus is 
an improvement on Barber’s Pipette Holder (Barber, 1914) 
because of its simpler construction and the greater accuracy 
with which one can control its movements. An additional 
advantage consists in the existence of certain devices for bringing 
the pipette or needle, quickly into position before starting actual 
operation. 

The working principle of the apparatus (which is being pat- 
ented) is illustrated in figure 1. It consists in the use of bars 
of rigid metal connected at their ends to form a Z like figure by 
resilient metal acting as spring hinges. The bars are forced 
apart by screws and return when the screws are reversed. By 
these means are movements are imparted to the tip of a pipette 
which is attached to one of the bars. As the radius of each are 
is about two and a half inches, the fine movements imparted 
to the tip of the pipette are practically in straight lines because 
the excursion never exceeds one millimeter. 

The instrument can be used by itself for one needle or pipette, 
or with a companion apparatus when two needles, or a needle 
and a pipette are to be used simultaneously. When a pair is 
used, one is a left handed and the other a right handed appara- 
tus, both being clamped to the front of the microscope stage. 
For the isolation of bacteria, one instrument is sufficient. It 
may be clamped on the left side of the microscope stage, figure 
2, so that the pipette projects into the moist chamber from the 

1 


JOURNAL OF BACTERIOLOGY, VOL. VIII, NO.1 


2 ROBERT CHAMBERS 


left. The tip of the needle or pipette is bent up so as to pro- 
ject from below into a drop suspended from the coverslip which 
roofs the chamber. The cells to be operated upon lie in the 
hanging drop. When a cell is to be dissected or injected it 
tends to retain its position on account of the shallowness of the 
drop and the inertia of the cell. However, it is more satisfac- 
tory to use two instruments, one with a needle for holding the 
cell or tissue, and the other with a needle or pipette for the 
actual operation. 


Fig. 1. DracraMm SHOWING WorKING PRINCIPLE OF MICROMANIPULATOR 


(a) Side view. Screw J in stationary pillar J pushes against K, and causes 
needle tip D to move through vertical are y-z. 

(b) Surface view. Screws G and H move the needle tip through horizontal 
ares m-n and o-p. 


For dissecting purposes, the glass needles may be curved or 
straight and with obtusely or gently tapering tips. They can 
be made fine enough to puncture red blood corpuscles and to 
tear up leucocytes. 

For injecting and for withdrawing materials from a living 
cell, the micro pipettes are made with apertures varying from 
two to less than half a micron in diameter. I have recently 
described (Chambers, 1922, a) an effective and easily madeappara- 
tus for exerting the necessary pressure to drive materials through 


ISOLATION OF BACTERIA AND DISSECTION OF CELLS 3 


such small pipettes, and at the same time to control, with con- 
siderable accuracy, the amount to be injected or withdrawn. 
For isolating bacteria, much coarser pipettes are used, which 
‘can be blown into by the mouth through a length of rubber 
tubing, figure 2. At my suggestion, Dr. Kahn has kindly pub- 


Via. 2. MickomManiputator Mountep on Lerr Sipe or MicroscoPr FOR 
IsoLaTInG BacTERIA 
Note Barber’s moist chamber with the coverslip marked with cross lines to 
aid in locating areas. The chamber shown here is higher than necessary. The 
screw producing the vertical movement is connected with a flexible shaft, which 
allows its control to be brought into close proximity with the fine adjustment 
of the microscope. 


lished an account (Kahn, 1922) of the procedure, together with 
a discussion of the application of the micromanipulator to the 
isolation of bacteria. In brief, the procedure is as follows: 
A sterile, hollow glass needle is first made. The bent up tip is 
then inserted into a test tube of a liquid culture of bacteria, 


4 ROBERT CHAMBERS 


and converted into a pipette by breaking the tip against the wall 
of the test tube. A small amount of the culture is sucked up, 
and the filled pipette placed in the micromanipulator attached 
to the microscope. The tip of the pipette is then brought into 
the microscopic field and brought close to the coverslip of the 


MM, | 


Fig. 3. Derain SHowinG Devices ror PRELIMINARY ADJUSTMENTS OF THE 
PIPETTE 
Carrier, a, for clamping brass collar, b, in which needle or pipette has been in- 
serted. The needle or pipette slides evenly within the collar for the in and out 
movement. Telescoping pillar, c, for lengthening vertical post of carrier. Post, 
d; rotates and serves to move needle tip laterally, Screw, e, raises and lowers 
post, d, to move needle tip vertically. 


moist chamber by means of the preliminary adjusting devices 
shown in detail in figure 3. The tip is now further raised by 
means of the fine adjustment screw until it reaches the under- 
surface of the coverslip. By alternately raising and lowering 
the pipette, and by moving the moist chamber with the mechani- 


ISOLATION OF BACTERIA AND DISSECTION OF CELLS 5 


cal stage, a series of hanging droplets! are placed on the coverslip. 
The pipette is then removed from the instrument and discarded. 
A search is now made for droplets containing only a single or- 
ganism. Each such droplet is drawn up into a fresh sterile 
pipette, which is then removed from the instrument and in- 
serted into a tube containing a suitable sterile medium. The 
contents of the pipette are now expelled by blowing. In this 
way, one can quickly obtain cultures known to have originated 
from a single organism. 

The micromanipulation technic is not very difficult. The 
making of the glass needles and pipettes, and the working of the 
instrument can be quickly mastered. 

For the bacteriologist, the isolation method as introduced by 
Barber, has long proved most successful. With the apparatus 
described here, it should soon be more widely used. 

For the cytologist and cell physiologist, the problem is to 
find the proper material with which to work. Through micro- 
operations on certain tissue cells and on such material as Protozoa 
and marine ova, considerable light has already been thrown 
upon the nature of living protoplasm. 


REFERENCES 


Barper, M.A. 1914 Philipp. Jour. Sci., Ser. B, Trop. Med., 9, 307. 
CuamsBers, R. 1922a Anat. Rec., 24, 1. 

CuambBers, R. 1922b Jour. Infect. Dis. now in print. 

Kaun, M.C. 1922 Jour. Infect. Dis., now in print. 


1 Barber uses coverslips smeared with petrolatum to aid in the maintenance of 
the droplets. The excess, having been washed off with soap and water, the 
slips are dried with a cloth, and then heated and wiped a second time while still 
warm. They are sterilized by flaming. 


{Reprinted from THE JourRNAL oF GENERAL PuystoLocy, November 20, 1922, 
Vol. v, No. 2, pp. 189-193.] 


A MICRO INJECTION STUDY ON THE PERMEABILITY 
OF THE STARFISH EGG. 


By ROBERT CHAMBERS. 
(From the Eli Lilly Research Division, Marine Biological Laboratory, Woods Hole.) 


(Received for publication, September 27, 1922.) 


It is well known that selective permeability, or semipermeability, 
is one of the essential characteristics of the living cell. So far, how- 
ever, there is no evidence as to whether the semipermeability of pro- 
toplasm is a property of its entire mass or of its surface only. 

Apparently the only means by which the action of substances on 
the interior alone of protoplasm may be studied is by injection. 
Animal cells can be injected by using the very fine glass pipettes and 
the mercury injection method which Barber devised for bacteriologi- 
cal work. I have used this method. The pipettes, both as regards 
their size and the ease of making, leave nothing to be desired. The 
method, however, is not only very difficult, but is unsatisfactory, 
owing to the fact that the pressure required for injection depends 
upon the expansion of mercury by heat, and this cannot be instantly 
controlled. Kite! tried it, but substituted for most purposes the far 
cruder method of blowing into his pipettes through a rubber tube. 
This operation necessitates larger pipettes than can be properly used 
for cell injection. The erroneous conclusions arrived at by Kite 
were due not only to the difficulty of the procedure, but mainly to 
the extraordinary ability of protoplasm to form films over torn sur- 
faces. Pushing a pipette, especially a comparatively large one, into 
an egg cell frequently causes the surface of the cell to become in- 
vaginated and thus forms a deep pocket. The tip of the pipette, 
even if it should finally break through the surface, is apt to be separ- 
ated from the protoplasm of the interior by the formation of a new 
surface film continuous with the original surface of the cell. Kite 
apparently did not guard against this contingency, and his experi- 


1 Kite, G. L., Biol. Bull., 1913, xxv, 1: 
189 


190 PERMEABILITY OF STARFISH EGG 


mental results indicate that his solutions never actually entered the 
protoplasm of the cell. The injected fluid simply seems to have filled 
the bottom of a pocket and then flowed down the side of the pipette 
to the exterior. When Kite says that he found no difference in 
permeability, whether a substance be applied to the surface of a cell 
or to any spot in its interior, he was perfectly correct. The spot in 
the interior was an infolding from the surface of the cell. At best, 
he was only comparing the permeability of the original surface of the 
cell with that of a newly formed surface film which surrounded the 
tip of his pipette. 

I have recently succeeded? in devising a simple but efficient piece 
of apparatus with which one can accurately and easily control the 
injection of fluids through a micro pipette having an aperture of 
less than 1 micron in diameter. The pipette, when properly made, 
tapers rapidly to a tip with a sharp cutting edge. The apparatus is 
so constructed that the pipette can be quickly changed. By keeping 
in mind the ease with which protoplasmic surface films are formed, 
one can, with this method, readily and accurately inject fluids directly 
into the interior of the protoplasm of a cell. 

This summer Jacobs kindly set at my disposal a manuscript which 
the reader will find in this number of this Journal, in which are de- 
scribed the interesting results that were obtained by immersing neu- 
tral red vitally stained starfish eggs in ammonium chloride, and in 
sodium bicarbonate solutions. Jacobs found that a 1/2 m NH,Cl 
solution, which is sufficiently acid to redden neutral red, will cause 
the neutral red within the eggs to turn yellow, indicating the entrance 
into the eggs of NH; and not of HCl. He also found that neutral 
red stained eggs will turn a deeper red when immersed in an alkaline 
solution of 1/2 m NaHCO; charged with COs, indicating the entrance 
into the eggs of COz and not of NaOH. 

Jacobs’ results confirm those of Loeb,’ Bethe,* Warburg, Harvey, 


2 Chambers, R., Anat. Rec., 1922, xxiv, 1. 

3 Loeb, J., Biochem. Z., 1909, xv, 254. 

‘ Bethe, A., Arch., ges. Physiol., 1909, cxxvii, 261. 

5 Warburg, O., Z. Physiol. Chem., 1910, lxvi, 305. 

6 Harvey, E. N., J. Exp. Zool., 1911, x, 507; Internat. Z. phystk. Chem. u. Biol., 
1914, i, 463. 


ROBERT CHAMBERS 191 


and Crozier’ that weak acids and bases freely penetrate living cells, 
whereas strong acids and bases do not. This is presumably because 
of their solubility in the organic solvents (lipoids) of the protoplasmic 
surface layer. 

I have injected the solutions which Jacobs used into starfish eggs 
vitally stained with neutral red, and obtained decided and consistent 
results, which show that HCl and NaOH will permeate protoplasm 
freely as long as there is no protoplasmic film to serve as a barrier. 
The semipermeability of protoplasm, in all probability, depends upon 
the surface film having properties different from those of the contin- 
uous internal protoplasm. 


EXPERIMENTS. 


Mature starfish ‘(Asterias forbesii) eggs were vitally stained with 
neutral red. They were then placed in a hanging drop in Barber’s 
moist chamber, and those eggs selected which showed a neutral tint 
of an orange-red hue. All the experimental work was done under a 
Leitz + oil immersion objective and ocular 15. This objective gives 
a remarkably long working distance together with a sharp definition 
that allows of the use of high powered oculars. 


Treatment with 1/2 Mm NH,Cl. 


Eggs were placed in a hanging drop of 1/2 m NH,Cl in which, as Jacobs has 
shown, their vitality remains unimpaired for a period of 10 to15 minutes. With 
a microdissection needle deep cuts were made in the eggs. The cut surfaces 
were immediately bounded by surface films continuous with the surface of the egg, 
and no injurious effect of the surrounding medium was noticeable. During this 
time, the neutral red within the egg, gradually turned yellow. 

This experiment indicates that the NH4Cl does not prevent the formation of 
films over the cut surfaces of the egg, and also that the solution will not, within the 
time limits of the experiment, penetrate those films. 

The interior of stained eggs was made to flow out in a drop of 1/2 m NH4Cl by 
the following means. The egg was torn at one spot on its surface and then caught 
on the other side and pulled to the edge of the drop. In every case the rapidly 
outflowing interior turned rose-red upon coming into contact with the surrounding 
solution and cytolyzed into a frothy semisolid mass. The change from an 
orange color to a red, with an accompanying cytolysis, extended from the out- 


7 Crozier, W. J., J. Biol. Chem., 1916, xxiv, 255. 


192 PERMEABILITY OF STARFISH EGG 


flowing area into the egg itself, and spread to the original cortex until the entire 
egg was cytolyzed. 

This experiment shows that if the egg be torn in such a way as to cause its 
interior to flow out rapidly, no surface film forms. ‘The NH4Cl at once penetrates 
the protoplasm which undergoes the characteristic color change and cytolysis. 

Stained eggs were placed in a hanging drop of sea water and 1/2 m NHC] in- 
jected under the egg membrane. This is fairly easy to do in the unfertilized egg 
but more so in the fertilized egg in which the membrane has already lifted off as 
the so called fertilization membrane. In the unfertilized egg, the injected solution 
at first bulges the membrane giving rise to a localized blister, and then usually 
spreads quickly over the egg, lifting the membrane from its entire surface. The 
permeability of the egg to the NH,Cl is not affected by this treatment. This 
demonstrates that it is not the egg membrane which protects the egg from the 
NH,Cl, but the actual surface film of the protoplasm lying under the membrane. 

Stained eggs in a hanging drop of sea water were punctured with a glass pipette 
having an aperture of 1 micron in diameter, and a minute quantity of 1/2 m NH4Cl 
injected directly into the interior of the egg. The injected area immediately 
changed from an orange to a rose-red color, and then underwent cytolysis. The 
color change and accompanying cytolysis spread from the injected area. In 
some cases this spread was arrested by the formation of a surface film which con- 
verted the injected and disintegrated area into a vacuole. In other cases the 
cytolysis spread till it reached the cortex which disintegrated from within outward. 

This experiment demonstrates that 1/2 m NH,Cl, which causes an alkaline 
color change within eggs immersed in it, will, when injected into the interior of the 
eggs, produce the acid color change and accompanying cytolysis which charac- 
terizes the presence of HCl. 


Treatment with 1/2mM NaHCO; + COs. 


Stained eggs were cut and torn in a hanging drop of the NaHCO; solution. In 
contrast to the reaction in the presence of NH4Cl there was no tendency for the 
formation of surface films over their cut surfaces. The protoplasm simply flowed 
out and was dispersed in the solution, the color changing meanwhile from red to 
yellow. 

Injection of NaHCO; beneath the egg membrane of eggs in sea water had no 
other effect than that produced upon eggs by immersing them in the solution; 
viz., deepening of the red color in the egg owing to the selective penetration of CO2. 

Stained eggs were injected with the NaHCO; solution. The injected area 
immediately turned yellow, and cytolysis with liquefaction took place. No surface 
film formed about the cytolyzing area, and the yellow color spread throughout. 


ROBERT CHAMBERS 193 


CONCLUSION. 


The experiments with the NH,ClI are similar to, and corroborate 
micro injection experiments performed in connection with some work 
on mustard gas in which the writer collaborated. Eggs immersed 
in sea water containing decomposed mustard gas, at a certain low 
concentration are not affected. If, however, the solution be in- 
jected, the egg quickly cytolyzes owing to the free HCl present. 

A similar impermeability of the protoplasmic surface film to cer- 
tain substances was also encountered in injection work on Ameba.® 
Amebe immersed in an aqueous solution of eosin will not take the 
stain till after death. On the other hand, the eosin, when injected 
into the Amaba, quickly permeates the protoplasm, to be arrested 
only at the surface. 

The semipermeability of a living cell appears primarily to be a 
function of its surface film. It is immaterial whether this film be 
that of the original cortex of the cell, a film newly formed over a cut 
surface, or a film that surrounds an artificially induced vacuole within 
the cell. As long as such a surface film exists neither the acid group 
of the NH,Cl nor the alkaline group of the NaHCO; can, within cer- 
tain concentration limits, penetrate the protoplasm. These solutions, 
if injected beneath the surface film, however, will produce their 
characteristic effects upon the protoplasm. 


8 Lillie, R. S., Clowes, G. H. A., and Chambers, R., J. Pharmacol. and Exp. 
Therap., 1919-20, xiv, 75. 
® Chambers, R., Proc. Soc. Exp. Biol. and Med., 1920-21, xviii, 66. 


Reprinted from the Procrepincs or Tw& Soctery ror Exrentmentat Brotocy anp Meprctne, 
1922, xx, pp. 137-138 


67 (2027) 
A note on the entrance of the spermatozoon into the starfish egg. 


By ROBERT CHAMBERS. 


[From the Department of Anatomy, Cornell University Medical 
College, New York City.] 


In 1876 Fol made the classic discovery that the spermatozoon 
actually enters the egg in fertilization. This fact he observed 
in the starfish egg. Fol’s treatise was apparently so exhaustive 
and so carefully worked out that no one has questioned the de- 
tails of his observations and his interpretation of the process is 
generally accepted to this day. Conical elevations were seen 
to form on the surface of the egg and the spermatozoa travelled 
in a straight line toward them. When a spermatozoon reached 
a cone its head penetrated it. Fol called the conical elevation the 
“attraction cone” and believed that it attracted the spermatozoon 
from a distance. 

The starfish egg is surrounded by a zone of glutinous jelly 
the thickness of which is about one fifth the diameter of the 
egg. When the eggs are placed in a sperm suspension all the 
spermatozoa that accidentally come into contact with the sur- 
face of the jelly stick and are unable to penetrate it to any ex- 
tent. 

My observations confirm those of Fol regarding the formation 
of the cones on the egg’s surface. The number of cones depends 
upon the age of the egg and upon the density of the sperm sus- 
pension surrounding it. An overripe egg forms these cones 


PAS Scientific ProcEEpincGs (127) 


quickly and in considerable numbers. A fresh mature egg forms 
only a few cones unless the sperm suspension is very dense. 

Fol, however, failed to observe the following: From the tip 
of each cone a slender filament grows outward piercing the 
jelly until it reaches the periphery where the trapped spermatozoa 
are lying. If there be no spermatozoa in the immediate vicinity 
nothing more happens. If, however, the tip of the filament 
comes into contact with a spermatozoon the cytoplasm of the 
tip and that of the sperm head immediately flow together so that 
the sperm nucleus now lies within the cytoplasm of the egg fila- 
ment. An extraordinary reaction then takes place. The fila- 
ment begins to draw back into the egg dragging the spermato- 
zoon along with it. Not only this but all the other filaments 
projecting from the egg are similarly withdrawn. Apparently, 
a wave of response is started when a filament fuses with a sper- 
matozoon. This wave must travel down the filament and over 
the egg. 

As the filament with a spermatozoon on its tip shortens, the 
spermatozoon is pulled deeper and deeper into the jelly and the 
lashing of its tail becomes more and more restricted. The sper- 
matozoon behaves like an unwilling victim and occasionally, 
frees itself, especially when- other filaments have been slightly 
ahead in activity and have also secured spermatozoa which they 
are now pulling in. With the microdissection needle one may 
free a spermatozoon by breaking the filament to which it is at- 
tached. Such a spermatozoon is generally unable to extricate 
itself from the jelly in which it lies embedded. After a few vi- 
brations of its tail it becomes permanently quiescent. 

By the time the filament has dragged the spermatozoon half 
way through the jelly the base of the cone changes in shape. 
The convexly rounded border, which gives it the appearance of 
a rounded nipple, draws in so as to become concave. In doing 
so it leaves the egg membrane behind and this now becomes 
plainly visible owing to the space intervening between it and the 
surface of the cone. By the time the filament is withdrawn so 
as to bring the sperm head to the summit of the cone, the lifting 
of the egg membrane has spread from the base of the cone over 
the egg and is recognized as the fertilization membrane. 

When the filament is completely withdrawn into the base of 
the cone the head of the spermatozoon is taken in with it. The 


ENTRANCE OF SPERMATOZOON INTO STARFISH EGG 3 


tail of the spermatozoon remains for a time outside the fertiliza- 
tion membrane. As long as the tail maintains organic continu- 
ity with its head it keeps up a feeble oscillatory movement. As 
the cone recedes into the egg, the strand extending from it to 
the tail outside the fertilization membrane breaks and the tail then 
lies motionless. The tail can be seen for several minutes mark- 
ing the site where the sperm head had gone in. 


DISTURBANCES IN MAMMALIAN 
DEVELOPMENT PRODUCED BY 
RADIUM EMANATION 


BY 


HALSEY J. BAGG, PH. D. 


Huntington Fund for Cancer Research, Memorial Hospital and the 
Department of Anatomy, Cornell University Medical College, 
New York City 


Reprint from ‘‘Radium,”’ Vol. 1, New Series, No. 2. 


July, 1922. 


DISTURBANCES IN MAMMALIAN DEVELOPMENT 
PRODUCED BY RADIUM EMANATION * 


By Hatsey J. Bacc, Ph.D. 


Prantaigion Fund for Cancer Research, Memorial Hospital and the 
Department of Anatomy, Cornell University Medical College, 
New York City 


The effect of radium on animal development has been the subject 
sf several researches since the early work of Bohn (1), in 1903, upon 
the ova and larve of the sea-urchin. [Experiments on developing nema- 
todes, molluscs, amphibians, fishes, and birds are associated with the 
names of Perthes (2), P .Hertwig (3), Schaper (4), O. Hertwig (5), 
and G. Hertwig (6). These investigators report developmental retarda- 
tions following radiation of the ova and developing embryos. They 
found a particular susceptibility of the nuclei of the cells and a gen- 
eral slowing up in the developmental processes, especially in the case 
of the central nervous system. The total disturbances, depending upon 
the period of development when the radiation was applied, resulted in 
the formation of monstrosities conforming more or less to a general 
type.t 

Similar experiments concerning the effects of x-rays on development 
have been conducted by many investigators. After exposure to x-radia- 
tion, Perthes (7) noted abnormal cell division and a retardation in the 
development of the ova of Ascaris megalocephala. Gilman and Baetjer 
(8), after radiating the ova of Amblystoma, and Baldwin (9), the 
fertilized ova of frogs, were able to produce a fairly constant type of 
development defect. Injurious results have followed in all cases where 
mammals have been exposed to x-radiation. It has been shown that 
when any particular part of a young animal is exposed to a sufficient 
amount of radiation, that part fails to reach its normal size and is 
unable to exercise a full degree of function. 

Arrests in development and the production of abnormal types may 
be induced not only by radio-activity, but by many physical or chemical 
agents. Abnormal temperature changes, treatment by many chemicals, 
lack of oxygen supply, or the overabundance of carbon dioxide, etc., 
have produced marked changes in the developing embryo. 

The present experiments are mainly concerned with disturbances in 
mammalian development, before and after birth, as a result of exposing 
the embryos of rats, at various times during the prenatal period, to 
irradiation from radium emanation. The effect on the embryos following 
radiation of the mother at varying intervals before mating was also 
determined. These experiments were designed not only to study the 
factors underlying the production of abnormal types, but through an 


*Reprinted by permission from the American Journal of Anatomy, xxx, 133- 
161, Jan. 1922. 


7In connection with the above statement, and applying to x-ray treatments as 
well, the question of dosage is an important one. A survey of the literature 
shows that there was a very wide range in the severity of the dose employed, and 
in several cases the experimental settings were inadequately described (Bohn used 
‘some centigrams’ of pure radium bromide for from twenty minutes to two hours). 
The amount of radium metal used in the investigations that have been men- 
tioned varied from 2 mg. to 35.1 mg., and the time from a few seconds to several 
hours. The deleterious changes in the animal tissues varied with the amount of 
radium and the time of exposure. 


3 


examination of the abnormal to gain a clearer insight into the nature of 
normal development and differentiation.§ 

I acknowledge with pleasure my indebtedness to Dr. James Ewing 
for his aid in the interpretation of the pathological results. 

METHODS AND APPARATUS 

Two methods were used for applying the radium emanation. In 
the first method an ‘active deposit’ was obtained by exposing a definite 
quantity of common salt to a comparatively large amount of radium 
emanation, about 500 millicuries were used, or the amount of radium 
emanation initially equivalent to one-half a gram of radium metal. To 
the radio-active salt thus produced sufficient water was added to make 
a physiological solution. The pregnant rats were injected subcutaneously 
in the shoulder region and intravenously through the caudal vein; 3 to 
4 minims constituted the usual dose. Because of the rapid loss of 
radio-activity of these solutions, the injections were made immediately 
after the preparation. The details involved in preparing and measuring 
the doses, as well as the methods for protecting the experimenter, are 
described elsewhere (10 and 11). The activated solution exhibited all 
the known phenomena of radium metal itself; alpha, beta, and gamma 
rays were present, but the greatest physiological effects were probably 
due to alpha-ray activity. After long experimentation, a dose of 5 milli- 
curies was found to be the maximum applicable to the aims of this 
experiment. In the second method gamma-ray radiation was applied 
through the ventral body wall of pregnant rats at nearly full term. A 
large amount of radium emanation was used, an amount equivalent to 
1% grams of radium metal, filtered by 2 mm. of lead and %4 mm. of 
silver. The source of emanation was 1 cm. away from the animal. The 
applicator, called a ‘lead tray’ in clinical usage, was 6 cm. in diameter 
and 1.5 cm. high. This was placed in the bottom of a small wire cage, 
10 by 13 cm. in diameter and 10 cm. high, and was covered by a thin 
sheet of cardboard. The animal was placed on this paper immediately 
above the applicator. 

Preliminary tests showed that a dose of about 1300 millicurie hours 
was sufficient to produce developmental arrests in the embryos without 
killing the pregnant animals. Doses as high as 2900 me. hrs., how- 
ever, were successfully used in some cases. ‘The embryos were killed 
by ether, and histological material procured at various periods after 
the treatment. The tissues were fixed in Bouin’s solution, cut in serial 
section, and stained with haematoxylin and eosin. 


EXPERIMENTAL RESULTS 
Series A. Injections of Radio-Active Solutions. 


1. Subcutaneous Injections After Mating. Sixty-five full-grown, 
normal, pregnant rats were treated in this series. They were divided 
into four groups, each treated at different periods after mating. Ten 
pregnant females were injected 7 days after mating; twenty-four, 10 
to 14 days after; twenty-one, 15 to 17 days after; and ten, 18 to 21 
days after mating. Many of the animals were killed at weekly inter- 
vals after treatment, although some were allowed to reach full term. 

Various degrees of developmental disturbances were noted, as 
shown in the following groups: 

§Dr. J. F. Gudernatsch was a co-investigator with the writer during the year 
1919. A preliminary report of the work done with him at that time is given in 


the Proceedings of the Society for Experimental Biology and Medicine, 1920, 
vol. 17, p. 183. 


4 


1. There was a large number of cases where no embryos devel- 
oped, in others many began development, but were absorbed or aborted 
at an early time. The females in which no embryos were found, 
although they were definitely considered pregnant before treatment, 
occurred among cases treated soon after mating and in those instances 
where females were autopsied a considerable time after treatment. Figure 
1 shows the remnants of maternal and embryonic structures; from the 
size of the placentae one can see that the foetuses had reached a fair 
degree of development before the radiation retarded the normal physio- 
logical processes. In one case (fig. 2) a small ovoid sac was found 
attached to the uterine wall by a thin stalk. This apparently repre- 
sented the remnants of a former embryo and placenta. [xtravasated 
blood and cell detritus were found in this sac and a great many large 
cells of an epithelioid nature that probably belonged to the former em- 
bryonic syncytium. The wall of this cyst was formed by fibrous con- 
nective tissue. 


2. Embryos were killed by the treatment, but were removed from 
the mother and preserved before they were absorbed. These showed 
various extravasations from the vessels of the subcutaneous connective 
issue, within the meningeal sinuses, and mainly along the dorsal mid- 
line of the body. Figure 3 shows a typical example of such a lesion 
which was situated in the mid-dorsal line. The mother of this embryo, 
No. 1167, was mated on April 22, 1919, injected with 4.9 millicuries on 
May 7th, and was killed two days later. When the embryo was cut 
in serial section, it showed that the haematoma in the dorsal subcu- 
taneous tissues had exerted sufficient pressure upon the spinal colimn 
to produce at one place a complete dislocation. Microscopical examina- 
tion of the viscera showed no pathological changes. Not all the foetuses 
of a litter were affected in the same degree. In one case seven foetuses 
were found, three showing haemorrhagic lesions, two beginning to 
macerate, and two in the progess of absorption. This variation in re- 
sistance was due either to the higher or lower vitality of the embryos 
themselves or to the amount of radioactivity which passed the placentae. 
In another case the foetuses, although injured, were carried to full term, 
and among a litter of six young, two were apparently normai and four 
showed haemorrhagic spots on the head, face, and along the dorsal 
midline of the body. 

3. Several young of a single litter showed areas of extravasation 
and were born alive. Their mother died, however, and foster moth>rs 
refused to nurse them. 

4.. Eight litters gave normal living young. This number is low, 
because, as previously stated, many pregnant rats were killed by the 
experimenter at various intervals after treatment. The average number 
of young per litter was 4.8, as compared with 6.5 per litter for the 
control rats, but the probable errors indicate that this difference is, very 
likely, not significant. Only one litter, containing four young, survived 
a treatment given seven days after mating. Several of the rats of this 
group, which had apparently escaped the full radium exposure during 
the uterine period or perhaps they were more resistant to it, when mated 
inter se produced litters of apparently normal young of normal fer- 
tility. The offspring of these animals, about twenty in number, were 
observed for two generations, but no abnormalities were noted. 

Il. Subcutaneous Injections Before Mating. Seventy-seven 
females were treated in this group, eleven died as a result of the injec- 


5 


Fig. 1. Two well-developed placentae are shown at the left attached to 
a uterus which has been partly opened. The remnants of embryonic tissue are 
superimposed on the placentae. At the right is a placenta which has been 
dissected from the uterus, and shows more clearly the remains of embryonic 
material, here represented as a lighter area in the upper portion of the draw- 
ing. Female mated April 22, 1919, injected May 7th, killed May 6th. Dose = 
4.6 mc. (subcutaneous). 

Fig. 2. A stalked sae partly dissected from the uterus, showing the rem- 
nants of a former embryo and placenta. Female mated April 22nd, injected 
May 7th, killed May 16th. Dose = 4.8 mc. (subcutaneous). 

Fig. 8. This is a dorsal view of a rat embryo, showing a characteristic 
area of extravasation due to the treatment of the mother during pregnancy. 
Female mated April 22nd, injected May 7th, killed May 9th, at which time 


6 


tion before they were mated, while several were killed at weekly intervals 
after mating, and some were allowed to continue to full term. Thirty- 
four animals were injected between 5 and 7 days before mating; seven- 
teen, 10 to 14 days before, and fifteen, 20 days before mating. 


Only three litters in this group showed abnormal young. The most 
interesting was a litter of seven, in which case the female was treated 
with 4.2 me., 22 days previous to fertilization, and the foetuses, approxi- 
mately 16 days old, showed very pronounced areas of extravasation, 
which in one case (fig. 4) covered a large area on one side of the head 
and a few small scattered areas on the other side. These areas were 
not only along the dorsal midline, but also on the lateral surfaces of 
the body as well (fig. 5). ‘The lesions were much more widely dis- 
tributed and more variable in size than in the cases recorded under 
Section I. Although the conditions that produced these results were 
repeated many times, the above is the only case where positive data were 
obtained. Usually the female had either been rendered sterile or the 
young were killed and absorbed during early stages. There were two 
other cases, however, where young were found with haemorrhagic areas, 
and these occurred in a group of females that were treated seven days 
before mating. Female 85 was given a dose of 6.6 mc. on November 7, 
1919. It was mated on November 14th, and as three young were born 
December 11th, fertilization took place about fourteen days after the 
treatment. Two of the young were apparently normal, but one showed 
a large haemorrhagic area, which involved most of the right side of the 
snout, the right eye, and a portion of the lower jaw on that side. This 
area disappeared after three days. Female 99, injected and mated et 
the same time with female No. 85, received a dose of 5.6 mc. Five 
young, three males and two females, were born on December 13th, 
making the date of fertilization about sixteen days after treatment. One 
male and one female showed definite haemorrhagic areas on the face. 
Consideration of these cases will be deferred until later. 


Seventeen females following treatment were killed at varying in- 
tervals after mating and showed markedly haemorrhagic or cystic ovaries 
and congested uteri. In these cases radium emanation apparently had 
either so altered the maternal tissues as to prevent fertilization or devel- 
opment when started was soon followed by the death of the embryo and 
its absorption. Many nodules were found in the uteri in which it was 
impossible to differentiate between embryonic and maternal structures. 

The remaining females (as previously stated, eleven died between 
the period of treatment and mating) produced either full-term normal 
young or young apparently normal at autopsy. Several of these living 


seven foetuses were found about fifteen days in development. Two of the 
litter were macerated and two absorbed. Dose = 4.9 me. (subcutaneous). 


Fig. 4. Areas of extravasation are shown in the two views of this 
embryo, similar to the condition shown in figure 3, but in this case resulting 
from treating the mother twenty-two days before fertilization. There are a 
few small scattered areas over the right side of the head and a large area of 
extravasation on the left side. Female injected April 22nd, mated May 12th, 
killed May 30th. Seven foetuses were found, fifteen to sixteen days old. Dose 
= 42 me. (subcutaneous). 


Fig. 5. These are three views of another foetus, a litter mate of the one 
shown in figure 4, showing the wide distribution of the extravasated areas over 
both sides and back of the animal. The experimental conditions are the same 
as for figure 4. 


young grew normally and were mated inter se, but produced no abnor- 
mal offspring, although observed for two generations. 

Ill. Jntravenous Injections After Mating. The intravenous injec- 
tions were primarily planned to act as a check on the series of subcu- 
taneous treatments. The object was to determine the immediate reac- 
tions that might occur in the embryo as a result of injecting a compara- 
tively large dose of radio-active solution into the circulation of the preg- 
nant female, and whether these reactions would be similar to those 
already recorded for the subcutaneous series. The toxic reactions were 
so prompt and fatal that it was not necessary to treat many animals 
to settle this point. A typical case in that of female No. 123. This 
animal, of about nineteen days’ pregnancy, was treated with 30 me. in- 
jected directly into the blood stream through the caudal vein. This was 
six times greater than the usual dose in the first two series. Three 
young were born dead twenty-four hours later. They showed very 
definite radium changes, typical of those already recorded for the sub- 
cutaneous series. Figure 6 shows a foetus still attached to an appar- 
ently normal placenta, but a characteristic area of extravasation was 
found over a considerable portion of the left side of the head. In figure 
7A a dorsal view shows another embryo with two comparatively small 
haemorrhagic areas along the dorsal midline, and the placenta in this 
case is also normal. The third foetus in this litter was apparently nor- 
mal, but the placenta (fig. 7B) had acted in the nature of a ‘shock ab- 
sorber’ in protecting the foetus from exposure to the radio-activity, and 
it was so swollen and completely filled with blood as a result of its 
injury, that it had the appearance of a large haemorrhagic sac. 


Series B. Results from Radiating Nearly Full-Term Pregnant Rats 
With Gamma-Ray Radiation. 


Ten rats were treated at the end of about nineteen days of preg- 
nancy. It was found that exposure to about 1350 me. hrs. of radium 
emanation was sufficient to produce very decided changes in the embryo 
and yet leave the pregnant females sufficiently uninjured to be able to 
nurse their young and care for them until after the weaning period. 
When the dose was increased to 3378 me. hrs., the young were severely 
injured, and were either killed outright or died two or three days after 
birth. 

The following are the conditions that resulted in the first genera- 
tion of animals treated in utero with a dose of about 1350 me. hrs.: 


1. The young of each litter were born two or three days after the 
treatment, alive and apparently normal. 


2. About ten days after treatment, about half of each litter be- 
came markedly anemic, showed symptoms of diffuse edema, and promptly 
died. There was an easily recognizable slow development of meningeal 
and spinal-cord haemorrhages, similar to those already described as a 
result of treatment by radio-active solutions. A series of these lesions 
is shown in figures 8, 9 and 10. Figure 8 shows a young rat with the 
dorsal integument partly dissected away, exposing a typical haemorrhagic 
area in the region of the frontal lobes. The slow development of this 
lesion could be easily noted through the thin, transparent scalp. This 
young was one of several treated in utero with 1350 me. hrs. of gamma- 
ray radiation on February 21, 1920. It was born two days later, and 
died on March 3rd. ‘The young rat shown in figure 9 was a litter mate 
of the previous animal. It shows the presence of three distinct haemor- 


8 


rhagic areas, a small frontal lesion, a fairly extensive one in the occipital 
region, and a small lesion in the subcutaneous tissues in the thoracic 
region, near the middorsal line on the left side of the body. This animal 
also died on March 3rd. A third animal belonging to the same litter 
is shown in figure 10. Here is seen a still more acute reaction, as shown 
by the fact that the animal died a day sooner than in the two cases 
above. There is an extensive area of meningeal haemorrhage which 
covers most of the dorsal portion of the brain, involving the frontal 
and occipital regions and the medial area between, as well as a consid- 
erable portion of the right temporal area. In addition, a distinct, 
rounded haemorrhagic lesion may be noted on the reflected skin on the 
left side of the body. This lesion occurred in the midshoulder region 
of the back. 

The heads of several of the young rats showed marked lateral com- 
pression. In one case a haemorrhage so affected the spinal cord as to 
produce complete paraplegia. The tissues of these animals were studied 
histologically. Save for the mechanical disturbances produced by the 
presence of the extravasated areas, the most marked pathological con- 
ditions were seen in the liver and intestines. In the first case there was 
a pronounced fatty degeneration of the hepatic cells, and in the second, 
a desquamation of the lining cells of the intestinal mucosa. 

3. It is interesting to note that the other half of each litter sur- 
vived the treatment, grew to a normal size, and some animals have 
lived for over eighteen months. They showed the effects of the late 
uterine treatment by the following arrests in development: 

a. The first pathological condition noted was that the eyes became 
smaller, the pupils opaque, and there finally was a complete, or nearly 
complete, closing of the lids and total blindness. This condition was 
first observed a short time after the eyes had opened. The photographs 
in figure 11 show three views of a female rat about one year old with 
typical eye deformities. The upper view shows the entire animal, which 
had grown to normal size and weight for its age. The left eye was 
nearly completely closed, as is shown more clearly in the lower right- 
hand view of the head at a higher magnification. Both pupils were 
opaque, but, as shown in the illustration, the right eyelids were slightly 
more opened than those of the other side. The animal was one of a 
litter treated in utero on March 8, 1920, was born six days later, and 
the photograph was taken on March 1, 1921. The dose in this case was 
2920 me. hrs. of gamma-ray radiation, which was a dose higher than 
that usually tolerated. 

b. Mating tests showed that both the males and females were com- 
vletely sterile in the first lots, but subsequently a first-generation female, 
that had been treated with 1350 mc. hrs., mated with a male similarly 
treated, gave birth to nine apparently normal young. 

c. Before these adult offspring of treated animals were killed for 
histological examination, their neurological reactions were very carefully 
studied. The animals, being blind, when startled assumed various de- 
fensive attitudes, but save for these reactions their behavior was re- 
markably normal. There was no ataxia in locomotion or in any of the 
feeding reactions, auditory acuity was normal, and there was no cutan- 
eous hypoesthesia or other sensory disturbances. Except for blindness, 
there was nothing to suggest abnormal sensory function. 

d. When these animals were autopsied, marked developmental dis- 
turbances were noted in the condition of the central nervous system. 


9 


Fig. 6. There is a large and a small area of extravasation on the head 
of this foetus. The mother was injected intravenously with 30 me. of radio- 
active solution and three young, about full term, were born twenty-four hours 
‘later. All were dead. In this case the attached placenta is apparently 
normal. 


lod 


Fig. 7. The lower figure (A) shows an embryo with two dorsal head 
lesions and an apparently normal placenta. This is a litter mate of the animal 
shown in figure 6. At B is indicated a large haemorrhagic placenta from the 
third young of this litter, which itself was apparently normal. 


Fig. 8. Dorsal view of a young rat with the skin dissected to either side. 
There is a prominent area of meningeal extravasation in the frontal region. 
This animal was treated in utero with 1350 me. hrs. of gamma-ray radiation 
on February 21st and was born apparently normal on February 23rd. It died 
on March 3rd. 


Fig. 9. Dorsal view of a young rat showing aieas of frontal and occi- 
pital extravasations which were within the meningeal sinuses. There is a 
smaller lesion in the left dorsal thoracic region. This is a litter mate of the 
animal shown in figure 8, and the experimental conditions were identical. Death 
occurred on March 3rd. : 


Fig. 10. There is an extensive meningeal extravasation over a consider- 
able portion of the hemispheres, and a haemorrhagic lesion is shown on the 
reflected skin from the dorsal interscapular region. This is a litter mate of 
the animals shown in the two preceding figures. Death occurred on March 2nd. 


10 


‘Lhe cerebral hemispheres were greatly reduced in size, and in several 
eases very little cortical material remained. ‘Those portions of the brain 
that were ontogenetically older (the archiostriatum and the cerebellum) 
were apparently normal. ‘The optic tracts were markedly atrophic. Cor- 
related with this disturbance in brain development, the skull was found 
to be asymmetrical, narrow, thicker than normal, and concave in the 
frontal region. 


Figure 12 shows a dorsal view of a normal, untreated brain of an 
adult rat, magnified five diameters. In figures 13 and 14 are dorsal and 
lateral views of a brain of one of the rats which belonged to the same 
litter as those of section 2 of this series. This animal was treated with 
1350 me. hrs. on February 21, 1920, was born on February 23rd, and 
vas killed December 31, 1920. ‘This was one of the animals which (ex- 
cept for blindness) showed no abnormal neurological reactions. The 
magnification in figures 13 and 14 is the same as that for the control 
brain in figure 12. The dorsal view in figure 13 shows an apparently 
normal cerebellum and normal olfactory lobes, but the part of the brain 
which represents the rudiments of the hemispheres shows a great lack 
of development of cortical substance. In a side view of the brain in 
figure 14, the cortex may be seen to be very thin; indeed, not completely 
covering what should normally be the frontal, occipital, and lateral 
aspects of the brain. The remains of the hemispheres do not suffi- 
ciently approach each other in the median line to cover the colliculi be- 
neath. In figure 13 the meninges on the left side of the brain have been 
removed, but on the other side they have been left in place. It was 
possible in this specimen to see the lateral ventricles through the trans- 
parent membranes. Several other brains have been studied which showed 
various degrees of developmental arrests resulting from radium treat- 
ment. In some cases the hemispheres were markedly reduced in size, 
were widely divergent in the median line, and yet the pallium was com- 
plete over the entire surface. In all these cases there was marked optic 
atrophy. These brains are now being sectioned, and a study of them in 
greater detail will be the subject of a separate communication. 

e. A histological study of the eye showed that the eyeball was re- 
duced to one-fourth the normal diameter. The retina was missing, but 
traces of the choroid remained as a few scattered pigment cells. The 
cornea was three times as thick as normal and covered with four or five 
layers of opaque squamous epithelium. The optic nerve was extremely 
small, not more than one-third the normal dimensions. 

f. The testes of the radiated animals were decidedly atrophic, and 
a comparison with the normal is shown in the photograph in figure 15. 
The diameters of the testicle alone (minus the epididymis) of the experi- 
mental animal was 14 mm. for the length and 7 mm. for the width, while 
ihe control measurements from normal animals of the same age and 
weight and with the same method of fixation were 21 mm. for the length 
and 11.5 mm. for the width. The epididymis of the radiated testis was 
practically missing. A small portion of the tail remained, but the head 
and body of the epididymis had failed to develop. Histological examina- 
tion shows that there is little evidence of spermatogenesis. Some tubules 
seem to contain imperfect spermatoblasts and forming spermatozoa, but 
the great majority of tubules show complete degeneration and loss of 
epithelial cells, and contain loose granular material, which in places is 
calcified. Some spermatic tubules are greatly dilated and filled with 
granular material. Very few interstitial cells are visible. 


11 


The ovary of the radiated animals was reduced to one-fourth or 
ene-fifth the normal size. The graffian follicles were entirely missing. 
Groups of lutein cells persisted in small numbers, but showed marked 
hydropic degeneration. Some of the large vessels about the ovary were 
sclerosed. 

g. The liver, kidney, lungs, spleen, and the other organs were 
examined, but showed no pathological disturbance. 


CONTROL GROUP 


Pregnant rats of the same stock, the same age and weight, were 
injected subcutaneously and intravenously with equal amounts of solu- 
tions that previously had been strongly radioactive, but were allowed to 
‘decay,’ until they had lost their radio-activity. These experiments gave 
absolutely negative results. As a control to the gamma-ray experiments, 
pregnant rats, sisters of the treated animals, were allowed to breed under 
exactly the same experimental conditions. No abnormal young were 
observed. 


DISCUSSION AND SUMMARY OF RESULTS 


It has been shown that when doses of radio-active solutions are 
injected into an animal marked physiological reactions take place. Large 
doses produce severe toxemia, resulting in pronounced pathological 
changes in the various viscera of the white rat (10). A study of meta- 
bolic changes in dogs, as determined by urine analysis, showed that, 
following intravenous injections of such solutions, there were very de- 
cided increases in the total nitrogen content of the urine, the urea, crea- 
linine, uric acid, and the total phosphates (12). A prompt reduction 
occurred in the number of white blood cells of the dog after intravenous 
injections of these solutions, associated with a marked decrease in the 
relative percentage of circulating lymphocytes (13). In order to reduce 
as much as possible the severity of the reaction, very small doses of 
radio-activity were used in the experiments recorded in this article. 
But even with comparatively small doses, certain rats treated in uterg 
showed very acute reactions. Many were killed by the treatment and 
and were absorbed or aborted. Others were found showing pronounced 
areas of subcutaneous extravasations, mainly situated along the mid- 
dorsal line of the body and within the meningeal sinuses. This condi- 
tion was probably due to the destructive action of radium on the endo- 
thelium of the blood vessels, as well as a possible increase in blood 
pressure, as was shown to occur in the dog by Burton-Opitz and Meyer 
(14) after intravenous injections of very small quantities of radium 
bromide. <A similar reaction of the blood vessels to radiation was pre- 
viously reported by Halkin (15) for the skin of pigs, and by Danysz 
(16) for radiated mice. This destructive action of radium on the blood 
vessels is in line with clinical observations on the usual prompt regres- 
sion of very vascular tumors (the angiomata, in particular) after expo- 
sure to irradiation. 


The changes in the rat embryos of this experiment are interesting 
in so far as they show that a sufficient amount of radio-activity was able 
to pass the placenta and subsequently affect the developing embryo. This 
occurred after subcutaneous as well as intravenous injections of the 
mother. By far the most interesting observation concerned the presence 
of lesions similar to those described above in rat embryos whose mother 
was treated with radio-active solutions a considerable time before mating. 


12 


Fig. 11. The upper photograph shows an adult rat with eye deformities due 
to gamma-ray irradiation during the prenatal period. The size and weight are 
normal for its age. The lower figures show the lateral views of the head at 


a higher magnification. Both pupils are opaque and the left eyelids are 
nearly completely closed. Mother treated on March 8, 1920, young were born 
on March 14th, and photograph was taken on March 1, 1921. Dose = 2920 
me. hrs. 


15 


Fig. 12. Dorsal view of a normal untreated brain of an adult rat. 
Fig. 13. Dorsal view of the brain of an adult rat showing the marked devel- 


14 


mental arrest in the formation of the neopallium. The meninges on the left 
side of the brain are removed. Mother treated with gamma-ray irradiation 
on February 21, 1920, young were born on February 23rd. Brain from radiated 
young removed on December 31, 1920. Dose 1350 me. hrs. (For further 
reference see text). The magnification is the same as for the drawing of the 
normal brain. 


) 


Fig. 14. A lateral view of the brain shown in Fig. 13. This shows the thin- 
ness of the remains of the cerebral cortex and its total absence in the frontal 
and occipital regions. (For further reference see text). 


Fig. 15. At the right is shown a normal untreated testicle of a white rat sur- 
mounted by a well-developed epididymis, which is partly obscured by fat. 
At the left is a radiated testis showing considerable atrophy. The single small 
lobe at the very bottom of the photograph represents the remains of the 
tail of the epididymis, the head and body of that part being completely missing. 
The animal was treated in utero on February 21, 1920, was born February 
23rd, and was killed December 31, 1920. Dose = 1350 me. hrs. of gamma-ray 
irradiation. 


The writer has no explanation to account for this phenomenon, It 
would appear that the treatment of the mother several days previous to 
conception has lessened the faculty of the later-developing embryo to 
form proper endothelium of the blood vessels, and the wide distribution 
ot these lesions over the body of the embryo (peculiar to this group of 
animals) would tend to substantiate this view. One female was injected 
twenty-two days before fertilization, and since the solutions lose their 
radio-activity very rapidly (there is about a 50 per cent reduction in 
the first hour after the preparation) the likelihood of any radio-activity 
remaining over during this period and affecting the egg at a later critical 
moment is remote ‘The amount of radio-activity remaining after twenty- 
two days, if present at all, should, as determined from physical compu- 
tation, be infinitesimally small. 


The series of intravenous injections again emphasize the specific 
action of radium emanation in the production of ty pical areas of subcu- 
taneous extravasations in the developing young, and in addition shows 
that the placenta may act in the nature of a ‘shock absorber’ and prevent 
the embryo from receiving the full effect of the radiation. 


We now come to a consideration of the cases wherein pregnant 
females were treated with external applications of comparatively large 
doses of gamma-ray radiation. At this time a report is given only for 
embryos treated towards the end of pregnancy. The writer plans to 
continue this line of investigation and treat at earlier prenatal periods. 


The results emphasize the well-known delayed reaction associated 
with gamma-ray radiation. There was approximately a ten-day interval 
following treatment during which no changes were noted in the embryo, 
and during this period the young animals were born in an apparently 
normal condition. Acute reactions promptly occurred at the completion 
of this time, killing half of each litter. The young rats died showing 
typical radium changes, such as anemia, diffuse edema, and meningeal, 
spinal-cord, and subcutaneous extravasations. These extravasations 
were markedly similar to those already described for the series of solu- 
tion treatments. The liver in these animals showed a fatty degeneration 
of the hepatic cells similar to the condition reported by Mills (17) after 
exposing a series of mice to gamma-ray radiation. The only other patho- 
logical change noted in these embryos was a desquamation of the lining 
cells of the intestinal mucosa. This observation is in line with the results 
emphasized by Hall and Whipple (18) in their experiments on Roentgen- 
tay intoxication in dogs. 

While the animals described above died after showing acute reac- 
tions certain of their litter mates (half of the litter) continued to de- 
velop apparently normally. This difference in reaction may possibly be 
due to individual variability or tolerance for the radiation, but 1t prob- 
ably can be explained by the fact that certain embryos were slightly 
farther away from the source of radiation than others, and as the in- 
tensity of radiation varies inversely as the square of the distance, even 
such slight differences in distances that did exist would be sufficient to 
subject the embryos to a considerable range in intensity of radiation. 
This is especially important in this case because the source of radiation 
was only I cm. from the body wall of the mother. The quality of radia- 
tion, however, remained the same for all the embryos. 

It was soon apparent that the animals that lived over the ten-day 
period had not completely escaped the effect of the radiation, as was 
shown by a suppression of the full development of the eyes. Eye defects 


16 


were noted, such as opaqueness of the pupil, atrophy of the lens, and 
closing of the lids, which resulted in complete blindness. These animals 
grew to a normal size, successfully competed with their cage mates for 
food, and showed absolutely no abnormal neurological condition, except 
those clearly incident to blindness. At autopsy, in some cases over a 
year after birth, very decided developmental arrests were noted in tne 
structure of the brain. All grades of such maldevelopment were noted 
in the condition of the neopallium, from merely a decrease in the size 
of the hemispheres, which permitted the corpora quadrigemina to be 
clearly visible from above, to a more marked absence of the cerebral 
cortex until only a very thin lamina of tissue remained to represent that 
structure, and there were large areas in the frontal, occipital, and tem- 
poral region where no cortex existed at all, so that when the meninges 
were removed the basal ganglia were clearly seen from without. 


This correlation between defects in the development of the eye and 
the brain has been emphasized by Stockard (19) in his recent paper 
on developmental rate and structural expression. He states as follows: 
“The periods of arrest necessary to induce the eye and the brain modifi- 
cations are so close together or so nearly the same, that one generally 
finds combinations and mixtures of the defects among the same experi- 
mental group of embryos.” Again in the same article Stockard has 
shown that the type of deformity that results from experimental dis- 
turbance depends upon the developmental moment at which the inter- 
ruption occurs. It is significant that the animals of this experiment 
showed arrests in the development of the neopallial portions of the brain 
and not in those regions which are ontogenetically older. Apparently 
the radium emanation, acting towards the end of pregnancy, had affected 
ihe development of the brain after the basal ganglia, the cerebellum, and 
medulla had become fairly well differentiated, and therefore those por- 
tions showed no gross changes. But the radium had slowed the develop- 
mental rate of the neopallium (which we know is one of the last por- 
sions of the brain to differentiate) during its period of active cell pro- 
liferation, and that portion of the brain was never able to re-establish 
its proper rate of development in relation to the other parts of the brain. 
If the period of treatment had occurred earlier in prenatal existence, 
other portions of the brain would probably have shown disturbances as 
well. The writer does not believe that the deformities in the brains of 
ihese animals were due to the early production of vascular disturbances 
later recovered from, but to an actual inhibitory effect of the radiation 
upon the developing nerve cells. If extravasations had occurred in this 
group of animals, and were so situated as to affect the development of 
the cerebral cortex in particular, they probably would have been de- 
tected as were even the comparatively small lesions which were asso- 
ciated with the acute reactions. Also, if the effect was largely due to 
vascular disturbances, from the nature of the radiation employed, one 
would expect more generalized changes throughout the entire brain. 
However, on the other hand, Craigie (20), in his recent paper on the 
relative vascularity of various parts of the central nervous system of 
the albino rat, suggests that “the vascularization of the more recently 
evolved centers (of the cortex cerebri) is more susceptible than the 
miore ancient regions to sexual, hereditary or environmental influences.” 


From a neurological point of view, it is interesting to consider that 
the animals with practically no cerebral cortex reacted so normally in 
their ordinary behavior. Except for blindness, there was no other 


17 


apparent sensory disturbance, and motor co-ordination appeared perfect. 
The physiological functions localized in the cerebral cortex of the rat 
were in these animals apparently transferred to the basal ganglia and 
other paleokinetic portions of the brain, showing the remarkable degree 
of compensation possible in the mammalian brain when the disturbing 
element acts at an early period in its development. In this connection 
it is worth mentioning that the radium emanation did not produce a 
sudden traumatic effect, as is normally the case with the experimental 
production of brain lesions, and in fact, the radium changes were prob- 
ably prolonged over a considerable period. This condition favored the 
establishment of compensatory reactions, and exists (as shown by the 
writer in a recent article (21) even in the case of radium lesions ex- 
perimentally produced in adult mammalian brains. 


The reproductive system completes its development a considerable 
time after birth, and so it is not at all surprising that these structures. 
should have shown a considerable amount of atrophy due to the develop- 
mental arrest during the prenatal period. ‘The ovaries and testes ap- 
peared to suffer with equal severity. 

An interesting correlative relation was shown by the fact that the 
<ther viscera (digestive, excretory, etc.) of the animals that showed 
marked developmental arrests of the nervous and reproductive systems 
were apparently normal and the animals grew to an average size. The 
lungs, liver, kidneys, etc., had differentiated before the physical agent 
was employed. Further studies with earlier prenatal treatments should 
throw some light on establishing the critical growth periods of the 
various embryonic structures. 

As a final point, the results of this investigation may be of interest 
to the clinicians and the laboratory workers who handle large quantities 
of radium and utilize x-rays. Although the results of this paper deal 
only with irradiation of the female, there is no reason to believe that 
the germ cells of the male are more resistant to these destructive agents 
than those of the female, and, in fact, there is very good experimental 
evidence to show that spermatozoa of some animals are especially likely 
to produce abnormal young after exposure to comparatively small quan- 
tities of irradiation. Physicians should guard against the possibility of 
producing developmental arrests such as shown in this article when 
treating pregnant women, as well as the possibility of altering the human 
germ cells by irradiation previous to conception. || 


CONCLUSIONS 


1. The marked selective action of radium emanation on fast-grow- 
ing embryonic structures was noted in these experiments. 


2. Very decided developmental arrests occurred in the differen- 
ulation of the nervous and reproductive systems of mammalian embryos: 
exposed to irradiation towards the end of pregnancy. 


3. Experimental animals with greatly reduced, or practically no. 


The writer does not mean to be understood as stating that present-day clinical 
‘rradiation treatments produce such effects in the developing young, but it is 
his personal opinion that such changes are biologically possible. It is not possible 
to obtain desired information by comparing the amount of exposure that a small 
mammal can stand with the corresponding dose that a man should tolerate, 
judging by comparative weights. The small mammal can tolerate very much 
more radiation in proportion to its weight than a man can. 


12 


neopallium, gave apparently normal neurological behavior, except for 
blindness. 


4. Radium emanation, used either in the form of a radio-active 
solution injected into the adult female, or employed as an external 
gamma-ray radiation, produced marked areas of extravasation in the 
subcutaneous connective tissue of the developing young. This suggests 
that the action of radium emanation might be selective upon the endothe- 
hum of blood vessels. 


5. xtravasations occurred in the developing young of females 
treated with radio-active solutions a considerable time before fertiliza- 
tion, and suggest that in some way the faculty of the later developing 
embryos to form proper blood vascular endothelium had been interfered 
with. 


6. The results so far obtained indicate that gamma-ray radia- 
tion is a physical agent admirably adapted to the study of experimen- 
tally produced developmental arrests in mammalian embryos. 


7. When women are subjected to therapeutic irradiation, especially 
during the early stages of pregnancy, the clinician should be forewarned 
concerning the possibility of producing very grave disturbances in the 
developing child. 


LITERATURE CITED 


1eBohns Bs 1903 Action des rayons du radium sur les teguments. 
Compt. rend. Soc. de biol, T. 40, p. 1442. 


2. Perthes, G. 1904 Versuche uber den Hinfluss des Roentgenstrahlen 
und Radiumstrahlen auf die Zellteilung. Deut. med. Woch., Bd. 30, S. 682. 


3. Hertwig, P. 1911 Durch Radiumbestrahlung hervorgerufene Veran- 
derungen in der Kernteilungsfiguren der Hier yon Ascaris megalocephala. 
Archiv. f. mikros. Anat., Bd. 77, No. 2, 8. 301. 


4. Schaper, A. 1904 Experimentelle Untersuchungen uber die Wirkung 
des Radiums auf embryonale und regenerative Entwicklungsvorgange. Deut. 
med. Woch., Bd. 30, S. 1434. 


5. Hertwig, O. 1911 Die Radiumkrankheit tierischer Keimzellen. 
Archiv. f. mikros. Anat., Bd. 77, No. 2, S. 1. 


6. Hertwig. G. 1911 Radiumbestrahlung unbefruchteter Froscheier und 
ihre Entwicklung nach Befruchtung mit normalem Samen. Archiv. f. mikros. 
Anat., Bd. 77, No. 2, S. 165. 


7. Perthes, G. 1904 Versuche uber den Einfluss der Rontgenstrahlen 
und Radiumstrahlen auf die Zellteilung. Deut. med. Woch., Bd. 30, S. 668. 


8. Gilman, P. K., and Baetjer, F. H 1904 Some effects of the Rontgen 
rays on the development of embryos. Amer. Jour. Physiol, vol. 10, p. 222. 


9. Baldwin, W. M. 1919. The artificial production of monsters con- 
forming to a definite type by means of x-rays. Anat. Ree., vol. 17, no. 3, 
p. 135. 


10. Bagg, H. J. 1920. Pathological changes accompanying injections 
of an active deposit of radium emanation. Jour. Cancer Res., vol. 5, No. 1, p. 1. 


11. Duane, W. 1917. Methods of preparing and using radioactive sub- 
stances in the treatment of malignant diseases and of estimating suitable doses. 
Boston Med. and Surg. Jour., vol. 177, no. 23, p. 787. 


19 


12. Theis, R. C., and Bagg, H. J. 1920. The effect of intravenous in- 
jections of active deposit of radium on metabolism in the dog. Jour. Biol. 
Chem., vol. 41, no. 4, p. 525. 


18. Bagg, H. J. 1920. The response of the animal organism to re- 
peated injections of an active deposit of radium emanation. Jour. Cancer. 
Res., vol. 5, no. 4, p. 301. 


14. Burton-Opitz, R., and Meyer, G. M. 1906. Effects of intravenous 
injections of radium bromide. Jour. Exp. Med., vol. 8, p. 245. 


15. Halkin, H. 1908. Uber den Kinfluss der Becquerelstrahlen auf die 
Haut. Archiv. f. Dermat. u. Syphilis, Bd. 65, S. 201. 


16. Danysz, J. 1903. De l’action pathogene des rayons et des emana- 
tions emis par le radium sur differentes tissus et differentes organismes. 
Compt. rend. Soe. de biol., T. 186, p. 461. 


17. Mills, G. P. 1910. The effect of radium on healthy tissue cells. 
Lancet, vol. 2, p. 462. 


18. Hall, C.C., and Whipple, G. H. 1919. Roentgen-ray intoxication: 
Disturbances in metabolism produced by deep massive doses of the hard 
Roentgen rays. Amer. Jour. of the Med. Sciences, vol. 157, no. 4, p. 453. 


19. Stockard, C. R. 1921. Developmental rate and structural expres- 
sion: An experimental study of twins, ‘double monsters’ and single deformi- 
ties, and the interaction among embryonic organs during their origin and 
development. Am. Jour. Anat., vol. 28, no. 2, p. 115. 


20. Craigie, E. H. 1920. On the relative vascularity of various parts 
of the central nervous system of the albino rat. Jour. Comp. Neur., vol. 31, 
p. 429. 


21. Bagg, H. J. 1921. The effect of radium emanation on the adult 
mammalisn brain. Amer. Jovy. Reent., vo!. 8, no. 9, p. 586. 


20) 


Reprinted from Tur Anaromicat Recorp, 
- Vol. 24, No 5, December, 1922. 


A SIMPLE APPARATUS FOR DELICATE 
INJECTIONS. 


ALICE L. BROWN 
Cornell University Medical College 


ONE FIGURE 


Methods and apparatus for dissecting and injecting cells have 
been worked upon for a number of years. With the micromani- 
pulator apparatus recently described by Chambers! is an in- 
jection device consisting of a syringe, a brass tube, and a glass 
cannula. This device, which is a very simple one, may be easily 
adapted to injecting the blood vessels of chick embryos or any 
delicate accurate free-hand injection work under a binocular 
dissecting microscope. The accompanying illustration shows 
this device held in a system of ball-and-socket joints which takes 
the place of the micromanipulator. In addition is a scheme 
providing for a warm injection mass. 

For descriptive purposes this apparatus is divided into three 
parts: 

First, the actual injecting part consists of a syringe? (A); a 
hypodermic needle with the tip sealed into one end of a coiled 
piece of fine extra soft brass tubing? (C’); and into the other end 
of which (n) is sealed a, piece of 14 inch glass tubing (D). The 
glass tube (D) is drawn out at one end (p) to about 1 mm. inside 

1 Chambers, R. New apparatus and methods for the dissection and injection 
of living cells. Anat. Rec., vol. 24, 1922. 

? A 2-c.c. Luer syringe is shown in the figure, but any convenient syringe may 
be used. 

* The soft brass tubing used here is one with 2 mm. outside diameter. This 
or others of somewhat larger sizes may be obtained from any wholesale brass 


dealer or automobile supply house. If the tubing is too stiff, it may be softened 
by annealing. 


295 


296 ALICE L. BROWN 


diameter, into which the cannula (Z) is sealed.t The cannula 
is made by drawing out a glass capillary which will fit into the 
drawn out end of Dat p. The tip of the cannula is made by 
momentarily heating the capillary over a microburner and pulling 
it out just as it softens. This procedure results in a tapering 
tip which may be broken off to form a pipette of the size which 
suits the operator. The cannula may be bent at any desired spot 
to facilitate insertion into the tissue to be injected. 

Second, the support for the injecting device in the figure is 
built out of two Leitz dissecting stands. One complete stand, 
and the ball-bearing arm, d’, of a second, connected together 
with wooden plugs, as shown in the figure, gives ample freedom 
for moving the glass tube (D) which bears the injecting cannula 
(Z). The coils of the brass wire allows the cannula to be moved 
about while the syringe is held rigid by the clamp, c’. 

Third, if a warm injection be desired, this is accomplished by” 
arranging a flow of warm water to heat the injection fluid in the 
glass tube (D). This is done by means of a 500-ce. flask (J) 
fastened on the support (b’) and a 14 inch rubber tube connected 
with it at 7. The glass tube (D) is thrust through the wall of 
the tubing in two places as indicated in the figure. The lower 
end of the rubber tube is shunted off to one side of the apparatus 
and the outflow of the warm water is regulated by the screw clamp 
(III). The water in the flask (J) can be heated by an alcohol 
lamp or a small Bunsen burner and its temperature regulated 
with the aid of a thermometer. Through the cork (m) a small 
funnel may be inserted for the addition of more water. If a - 
flask with an opening as shown in the figure is not available, a 
second glass tube must be thrust through the cork,of the flask (J) 
to allow the entrance of air. A third tube may be added for the 
addition of water. 

4 The sealing may be done with ordinary sealing-wax or de Knotinsky cement, 
which can be obtained from any dealer in chemical and physical apparatus. 
As the micropipette has to be frequently changed, it must be sealed with a 


cement which can be readily softened. The de Knotinsky cement is admirable 
for this purpose. 


APPARATUS FOR DELICATE INJECTIONS 297 


Once this apparatus is set up, the bases a and a’ may be screwed 
down to a board of convenient size, thus making a permanent 
apparatus of which only the cannula has to be renewed. 

To operate the apparatus the brass tube (C) must be filled 
with water through the syringe before the cannula (£) is sealed 
at p. The injection mass is drawn up through the cannula, an 
air space being left between the water and injection mass to 
prevent diffusion. The tissue or embryo to be injected is placed 
in position under a binocular dissecting microscope, and the 
injecting device brought into position with the cannula immedi- 
ately over the tissue. The operator’s fingers control the cannula 
by gripping D where the rubber tubing covers it.® 

5 For other types of apparatus for similar purposes, see Knower, H. McE., 
A new and sensitive method of injecting the vessels of small embryos, etc., 


under the microscope. Anat. Rec. vol. 2, no. 5. 


Chambers’ Micromanipulator for the Isolation 
of a Single Bacterium 


ME © FR OMNES Ca ease 


Reprinted from 
Tue JourNAL oF INnFecTIous DisEAses, Vol. 31, No. 4, Oct., 1922, pp. 344-348 


CHAMBERS’ MICROMANIPULATOR FOR THE ISOLA- 
TION OF A SINGLE BACTERIUM 


Morton C. KAHN 


From the Department of Hygiene, Cornell University Medical College, New York City 


As I have been working for several years on the isolation of bacteria 
with Barber’s apparatus and have recently been using Chambers’ instru- 
ment for the same purpose, Dr. Chambers suggested that I make some 
comments on the relative merits of the two instruments together with 
a short review of Barber’s isolation method. 

The mechanical principles involved in the construction of this 
instrument are entirely different from those of Barber’s pipet holder, 
while the basic methods of manipulation are essentially the same. There 
are several noteworthy features in the Chambers’ instrument which 
modify and improve the original Barber technic. These features may 
be best described under two headings: (1) advantages in construction, 
and (2) advantages from the use of various accessories to aid in 
manipulation. 


ADVANTAGES IN CONSTRUCTION 


The mechanical principles on which the instrument is based are 
fully described in the preceding article by Chambers.1_ The absence 
of parts which may loosen by wear and tear renders possible great pre- 
cision in the manipulations. While excellent work may be done with the 
Barber pipet holder, the parts wear somewhat after several months’ use, 
giving rise to a certain amount of false motion. For instance, when one 
desires to move the pipet laterally one may encounter an unexpected 
vertical motion. The Chambers apparatus used by me had been in use 
for two years, and in spite of this I was unable to detect any false 
motion. 

A second advantage is that the instrument clamps directly on the 
stage of the microscope giving much greater rigidity than is possible 
with the metal flange which has to be attached to the stage of the 
microscope when Barber’s instrument is used. Third, the smaller size 
of the instrument brings all manipulations closer to the microscope and 


Received for publication, June 9, 1922. 


1 Jour. Infect. Dis., 1922, 31, p. 334. 


4 M. C. KAHN 


eliminates accidental jostling of the pipet holder which may shift the 
needle out of focus. This compactness also makes it feasible to use a 
short pipet which is easier to focus and is in less danger of contamina- 
tion as less of the pipet is exposed. 


ADVANTAGES OF THE ACCESSORIES IN CHAMBERS’ APPARATUS 


Barber’s instrument possesses no accessories so that all the adjust- 
ments, both preliminary and operative, have to be made by means of the 
same finely threaded screws with a consequent waste of considerable 
time. 

In Chambers’ instrument the several accessories are as follows: 

1. There is a brass collar (fig. 2’) through which the shank of 
the pipet is inserted before clamping it in the pipet carrier of the 
instrument. Besides insuring rigidity to the pipet and thus greater 
accuracy for manipulation, the collar facilitates bringing the pipet into 
the field of the microscope. It also steadies the pipet as it is being 
withdrawn from the moist chamber, thus minimizing contamination or : 
injury to the delicate tip. 

2. For the vertical manipulation of the pipet there are three different 
adjusting devices: first, the telescoping pillar for roughly adjusting 
the pipet to the height of the moist chamber, after which it may be 

_ tightly clamped ; second, another coarse adjustment operated by a spring 
screw with which one may bring the pipet into focus, and, third, the 
fine adjustment of the knurl headed screw (fig. 1), which is used in 
the actual operation of isolation. The first two devices are for the 
coarse adjustment and enable one to use moist chambers of practically 
any height. They aid greatly in the technic of the vertical adjustment, 
which is the most important one from the bacteriologic point of view. 

3. The use of levers on the screws controlling the lateral move- 
ments insures great delicacy to the touch and is a decided aid in bring- 
ing the pipet directly under the droplet containing the organism, 
especially when working with the higher power or oil immersion lens 
where the slightest movement is greatly magnified. 


4. The flexible shaft attached to the vertical control screw brings 
the movement of that screw behind the microscope away from all work- 
ing parts and close to the fine adjustment of the microscope. 

For bacteriologic work it is more convenient to have the moist 
chamber as Barber originally devised it, viz., with its open end so 
placed that the pipet may project into it from the left side. This 


CHAMBERS’ MICROMANIPULATOR 5 


facilitates the frequent interchange of pipets so necessary in isolating 
bacteria. For this purpose it is necessary to have the pipet holder 
attached to the left side of the microscope. This can be done with the 
form which Chambers designates as right-handed by fastening it on 
the left side of the microscope stage near the outer corner (fig. 1). 

In the preceding article it has been recommended to have the height 
of the moist chamber equal to the working focal distance of the sub- 


a 
cal 


Micromanipulator mounted on the left side of the microscope for isolating bacteria. Note 


Barber's moist chamber with the coverslip marked with cross lines to aid in locating areas 
The chamber shown here is higher than necessary. 


stage condensor. This limits the height to one which may make the 
chamber too shallow to be convenient for frequent interchanges of the 
pipet. I, therefore, use a chamber °4 of an inch high, 1 inch wide and 
21% inches long. It will be noted that this moist chamber is not only 
deeper but also wider than the one Chambers uses for cytological 
work. 


6 M. C. Kann 


THE ISOLATION METHOD 


For the isolation of a single bacterium one must have the under sur- 
face of the coverslip so treated as to hold minute droplets without fear 
of their spreading or possibly running together. The droplets placed on 
the coverslip must be slightly hemispheroidal in order that their outlines 
may be distinct and all parts of it clearly visible. Also, in order to 
maintain these droplets throughout the operation, the moisture condi- 
tions within the chamber must be sufficient to prevent their evaporation 
and at the same time must not be too great for fear of flooding them. 

It is necessary, therefore, to have the surface of the coverslip 
specially prepared. Barber, after smearing the cleaned coverslips with 
petrolatum, washes them with soap and water to get rid of the excess 
of petrolatum. The coverslips are then carefully cleaned with a dry 
cloth, heated enough to soften the petrolatum and rubbed again while 
still warm. The aim is to remove as much petrolatum as_ possible 
without the use of excessive heat or any fat dissolving reagent other 
than soap. If an excess of petrolatum is left on the cover, small 
particles will appear in the droplets and may be mistaken for bacteria. 
If all petrolatum is removed, the droplets run together and make 
successful isolation impossible. Instead of soap and water one may use 
95% alcohol with equally good results. One must realize that success 
or failure in isolation work depends on a proper treatment of the 
coverglass. 


The method of procedure for the isolation of a bacterium may be 


summarized as follows: ? b 

1. Prepare a young liquid culture from a subculture not more than 18 
hours old. 

2. Insert the tip of a needle into a tube of the liquid culture and convert 
the needle into a pipet by gently rubbing it against the wall of the tube. Then 
with a rubber tube on its shank suck up a small amount of the culture. 

3. Insert the pipet in the brass collar, then clamp it in the pipet holder of 
the instrument and bring the tip into focus in the center of the microscopic field 
(see figure). Raise the pipet until its tip touches the undersurface of the cover- 
slip and expel an appreciable droplet. This may have to be diluted with sterile 
fluid if the culture is too dense. 

4. After securing a moderately dilute preparation fill the same or a new 
pipet to a little below its bend. Lower the pipet, and with the aid of the 
mechanical stage, bring another portion of the coverslip into view. By alter- 
nately raising and lowering the pipet a series of minute droplets will be pro- 
duced on the coverslip wherever the pipet touches it. The fluid runs out by 
capillary attraction and needs no blowing. Some of these droplets will be 
found to contain a single micro-organism. 


2 The method of making the pipets is described in the preceding article by Chambers. 


CHAMBERS’ MICROMANIPULATOR 7 


5. Replace this pipet with a new sterile one containing a small amount of 
sterile liquid medium which must not run below the elbow. This new pipet is 
now brought directly under a droplet containing a single micro-organism. The 
pipet is then slowly raised and as soon as it touches the surface the droplet with 
the contained organism will flow into it. This occurs by capillary attraction 
and no suction is required. 

6. This pipet, which is known to contain only one micro-organism, is care- 
fully removed from the apparatus and its tip inserted into a tube containing a 
suitable sterile medium. The entire contents of the pipet are now to be expelled 
by blowing. As an added precaution it is well to break off the tip of the pipet 
in the culture medium. The blowing may be done by mouth or by a rubber 
bulb operated either by the hand or the foot. 


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