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Marine Biological Laboratory

Re,„,eH July 19, 1041

Accession No. ooojI

Given Bv Tl^f^ 3la}:iston Co.

Philadelphia, Pa,
Place.

BIOLOGY

OF THE

LABORATORY MOUSE

BY THE STAFF OF
THE ROSCOE B. JACKSON MEMORIAL LABORATORY

BIOLOGY

OF THE

LABORATORY MOUSE

hy
THE STAFF

THE ROSCOE B. JACKSON MEMORLAL LABORATORY

CLARENCE C. LITTLE, Director

GEORGE D. SNELL, Editor
J. J. BITTNER W. E. HESTON

A. M. CLOUDMAN W. L. RUSSELL

E. FEKETE G. W. WOOLLEY

With a Chapter on
INFECTIOUS DISEASES OF :\IICE

J. H. Dingle
Harvard Medical School

THE BLAKISTON COMPANY
Philadelphia

FEINTED IN U. S. A.
BY THE MAPLE PRESS COMPANY, YORK, PA.

To the Trustees of
The John and Mary R. Markle Foundation

Whose generous grant

Made its preparation possible

This hook is dedicated

PREFACE

Of_all the laboratory mammals, probably none has contributed more
to the'*advancement of knowledge than the common mouse. Certainly
among all the mammals it is the most widely used, for not less than one
million mice are raised each year in this country for research in bac-
teriology, cancer and genetics.

A result of this extensive use of the mouse is that a large body of
information has grown up concerning it. This, however, is so widely
scattered through the literature that it is often a major undertaking for
the research worker who wishes to use it to locate and gather the par-
ticular facts that he needs. Much of this information is assembled in this
book. In a number of cases, where there are important gaps in the
literature, these have been filled in by special research projects. In
general, controversial material has been avoided or given only brief
mention. The emphasis is placed on established facts useful to the
research worker.

Certain fields, for example anatomy and endocrinology, have of neces-
sity been largely omitted. In most cases material omitted is adequately
covered in other recent books.

Because it deals with the mouse alone, this book presents a vertical
cross-section of biological knowledge rather than the more usual horizontal
cross-section. It contains information about one animal drawn from
various branches of zoology, rather than information about one branch of
zoology drawn from observation of a variety of animals. There is, I
believe, one notable virtue in this vertical method of presentation,
namely, that it makes the synthesis of biological knowledge somewhat
easier. There is a widespread feeling among biologists that progress will
depend increasingly on the synthesis of the specialized techniques which
have been developed within the individual cubby-holes into which science
is somewhat arbitrarily divided. The departmentalization of biology is a
convenience not to say an absolute necessity, but within the organism the
tissues, the genes, the endocrines, the diseases and the processes of develop-
ment are all intimately related, and the biologist frequently finds that
research in his own specialty is leading him straight into another field of

viii PREFACE

knowledge. At the present time there are, for example, increasingly well
beaten paths between genetics and embryology, between endocrinology
and cancer research, between cancer research and bacteriology, between
bacteriology and genetics. It is a major purpose of this book, by gather-
ing together the fundamental knowledge about the mouse from several
fields of study, to make it easier for the research worker using mice as his
experimental material to traverse these interconnecting paths of science.

The preparation of the book has been financed by a grant from the
John and Mary R. Markle Foundation. This generous support has made
possible the conduct of several pertinent research projects and the
preparation of many original photographs and drawings. The embryo-
logical studies described in Chapter i have also been aided by a grant from
the Alexander Dallas Bache Fund of the National Academy of Sciences.
In the preparation of their material the authors have been ably assisted
by the following persons: Miss Olive Bartholomew, preparation of
embryological and histological sections; Miss Bernette Bohen, drawings;
Mr. Joshua Burnett, tabulation of linkage data; Dr. Elizabeth Chase,
histological sections; Dr. Katrina P. Hummel, photography; Mr. Arthur
Lieberman, bibliography; Mr. John Mowat, photography and construc-
tion of apparatus; Mr. William Payne, photography; Miss Ella Rowe,
preparation of sections; Miss Elizabeth Keucher, assistance in preparation
of the index. Prof. C. H. Danforth has made valuable suggestions in
regard to several parts of the text.

In conclusion, the editor would like to express his appreciation to the
other members of the Laboratory Staff for their continued cooperation and
for many valuable suggestions, and to Dr. C. C. Little for his hearty
support and, in a broader sense, for the wise direction in a large measure
responsible for the friendly atmosphere so essential for successful
collaboration.

George D. Snell, Editor

Roscoe B. Jackson Memorial Laboratory
Bar Harbor Maine

^^\ *^ /^/

CONTENTS

Page

Preface vii

Chapter

1. The Early Embryology of the Mouse — George D. Snell . . i

2. Reproduction — George D. Snell 55

3. Histology — Elizabeth Fekete 89

4. Spontaneous Neoplasms IN Mice — Arthur M. Cloudman. . . 168

5. Gene and Chromosomes Mutations — George D. Snell .... 234

6. The Genetics of Spontaneous Tumor Formation — C. C. Little 248

7. The Genetics of Tumor Transplantation — C. C. Little . . 279

8. Endocrine Secretion and Tumor Formation — George W.

Woolley 310

9. The Milk Influence in Tumor Formation — John J. Bittner 321

10. In-bred and Hybrid Animals and Their Value in Research —

W. Lawson Russell 325

11. Parasites — Walter E. Heston 349

12. Infectious Diseases of Mice — John H. Dingle 380

13. Care and Recording — John J. Bittner 475

^oa9i

Cnapter 1

THE EARLY EMBRYOLOGY OF THE MOUSE

By George D. Snell, Roscoe B. Jackson Memorial Laboratory.

Fertilization, 2. Cleavage, 4. The blastula, 5. Implantation and early growth, 5
The formation of the entoderm, 7. Embryonic and extra-embryonic ectoderm, 8
The ectoplacental cone, 10. The inversion of the germ layers, 10. The primitive
streak and mesoderm formation, 15. The orientation of the embryo in the uterus, 15
Amnion, chorion and exocoelom, 16. The head process, 20. The neural groove, 23
The notochord, 24. The archenteron, 25. The allantois, 25. Fore-gut and hind-gut
26. The head fold, 28. The somites, 28. The primitive streak as a growth center
31. The coelom, ^2. Reichert's membrane, ;^,i. The amnion, 36. The yolk-sac, 36
The blood islands, 37. Changes in the uterus. 37. The nourishment of the embryo
3q. The giant cells, 40. The seven somite embryo, 41. The tail fold, 42. The
turning of the embryo, 44. The mid-gut, 44. The heart, 45. Blood vessels, 50
Change in shape of the yolk-sac, 51. Bibliography, 51.

The early embryology of the mouse and rat has been the subject of
numerous studies during the past 50 or 60 years. Because the results of
these studies are published in several languages and in many different
journals, some of them not accessible in most libraries, because errors were
inevitably present in the earlier articles, and because many of the published
figures are not adequate for conveying a quick and clear understanding of the
subject, the author has undertaken, and here presents the results of, a com-
plete reinvestigation of nearly the whole field. The material used in the
study consists of sections of embryos spaced at six hour intervals from 4
days to 9 days. In some cases ten or more embryos of a single stage have
been sectioned. The sections were prepared by Olive Bartholomew,
Elizabeth Fekete and the author. The technique used has been described
elsewhere (14). To this description need only be added that, because in
most cases the females used as mothers were hybrids between two strains,
and because the fathers were from a third strain, thus giving both embryos
and mothers a maximum of hybrid vigor, the stages as here described are
usually earlier, often by as much as a day or more, than comparable stages
described by other authors. While this procedure gave embryos which
developed rapidly and were normal in a high proportion of all cases, it did
not eliminate variability. No attempt has been made to describe the varia-

2 BIOLOGY OF THE LABORATORY MOUSE

tions that have been noted in the rate of development of embryos or in the
rate of development or form of separate parts. It should be emphasized,
however, that the range of variation in these respects is considerable.

Wherever it is applicable to the mouse we have in general followed the
terminology employed by Patten in the "Embryology of the Pig."

Contentious material is described in footnotes rather than in the text.
Some readers will wish to skip these altogether. A complete bibliography is
given at the end of the chapter, including a number of articles not referred
to anywhere in the text.

Fertilization. — By fertilization is meant the entrance of a sperm into the
egg. Fertilization in the mouse occurs in the upper end of each oviduct
where the eggs are found, usually gathered into clumps, after their discharge
from the ovaries. The sperm thus have to traverse the length of the uterus
and oviduct to reach the eggs, a process accomplished partly through their
own motility but for the most part through a churning action of the female
duct. Since the beginning of heat in the female commonly occurs about two
hours before ovulation, sperm may already be present in the oviduct when
ovulation occurs.

The egg consists of a sphere of living protoplasm, the vitellus, surrounded
by a transparent, non-living membrane, the zona pellucida (Fig. lA). The
zona pellucida in turn is surrounded by follicular cells which, however, are
dispersed soon after fertilization. Within the vitellus is the egg nucleus, not
clearly visible in living eggs such as the one shown in Fig. lA, but easily seen
in fixed and stained material.

Mature eggs within the ovary average about 95 jjl in diameter (outside
diameter of the zona). Following fertilization the zona pellucida expands
until its outer diameter becomes about 1 13 /x (= .0044 inches). This is just
within the Hmits of visibiHty for the unaided eye (35).

Usually only one sperm enters each egg. Almost immediately after
entry, which may occur through any part of the egg's surface, the vitellus
shrinks slightly in size and the zona pellucida expands, so that a space forms
between them (35, 50). This is the peri vitelline space. At this time only
the first polar body has been formed. Within the next few hours the second
maturation division occurs and the second polar body is budded off from the
surface of the vitellus (Fig. iB).

Not only the sperm head but also the middle piece and sometimes the
whole tail enters the vitellus. The sperm head carries in one complete set of
chromosomes from the male parent, while the middle piece contributes
mitochondria from this parent. These latter are soon distributed through-

THE EARLY EMBRYOLOGY OF THE MOUSE

out the vitellus, and at the hrst cleavage division are divided more or less
equally, alono; with the mitochondria already present in the egg, to the two

Follicular cells

Vitellus

Zona pellucida

Polar body

Perivitelline space

Fig. I. — rhotugraphs of mouse eggs (X6oo). A. Egg removed from ovary.
B. Fertilized egg from oviduct 20 hours after copulation. Two polar bodies and
sperm in perivitelline space. {From Lewis and Wright.)

daughter cells. There is some evidence that Golgi material is also carried by
the sperm into the egg (19, 22, 34).

The sperm and egg nuclei, now both within the vitellus, are known as the
male and female pronuclei. They move towards each other until they lie

BIOLOGY OF THE LABORATORY MOUSE

side by side, each appearing at this stage as a typical resting nucleus, though
the male element is a little the smaller of the two. At the first cleavage
division the nuclear walls break down, the chromosomes split longitudinally,
and one-half of each split chromosome is carried to each daughter cell.
Hence at this division, as at all future somatic divisions, each cell receives a
full complement of chromosomes from each parent.

Cleavage. — Cleavage in the mouse occurs while the eggs are still in the
oviduct. The first cleavage occurs about 24 hours after copulation and

Fig. 2. — Photographs of mouse eggs (X600). .\. Two-cell egg from oviduct 24
hours after copulation. Large second polar body and disintegrating first (on opposite
side). B. Seven-cell egg from oviduct 48 hours after copulation. Note one cell
on left larger than the rest. Division of this cell would give the eight-cell stage.
(From Lewis and Wright.)

results in two cells not quite equal in size (Fig. 2 A). Following divisions
occur somewhat more rapidly, giving rise to 4-cell, 8-cell stages, etc., and
are usually nearly synchronous in the different cells. Occasionally, however,
eggs are found with some divisions completed, others still incomplete, and
hence showing an odd number of cells (Fig. 2B). The actual act of division
requires only 5 or 10 minutes; the interval between divisions lasts about 12
hours. Eggs of 16 cells or more, but in which no cavity has appeared, are
called morulae. Eggs usually reach this stage about 60 hours after fertiliza-
tion, and pass from the oviduct, through which they have been gradually
moving, into the uterus, some 6 to 12 hours later (35). This is subject to

THE EARLY EMBRYOLOGY OF THE MOUSE

considerable variation, however, and in one study passage into the uterus at
4 days was found to be the rule (7).

The blastula. — Shortly after entering the uterus, and usually sometime
after the egg has reached the ,:;2-cell stage, an eccentrically located, fluid
tilled cavity appears among the cells of the morula. This enlarges rapidly

t^ ^

uterine lumen

^^(2) Q^ '*^^ G^- - --- ■

Inner cell mass

Blastocoele

Uterine epithelium

Decidua

B. Behen

Fig. 3. — Blaatula in uterine crypt 4 days after copulation. Projection drawing

(X600).

to produce the segmentation cavity or blastocoele (Fig. 3). The cavity is
bounded by only a single layer of cells except on one side where most of the
cells are grouped to form a structure called the inner cell mass. Eggs in
this stage are known as blastulae.

Implantation and early growth. — The uterus in the mouse is duplex, con-
sisting of two horns which unite just anterior to their junction with the
vagina, and each of which is attached to the dorsal body wall by a mesentery,
the mesometrium (Fig. 4). There are two layers of muscle in each horn, an
outer longitudinal layer and an inner circular layer. The uterine lumen is
lined with epithelium. Between the epithelium and the muscle layers is the
mucosa, a tissue which forms the bulk of the uterine wall. The epithelium
is indented by numerous small crypts.

BIOLOGY OF THE LABORATORY MOUSE

Very shortly after entering the uterus the eggs become spaced more or
less evenly throughout its length, and each egg finds its way into a uterine

Mesometrium

Blood vessel

Uterine lumen

" ^ ''^'' Circular muscle

Longitudinal muscles

Ovum

Uterine
lumen

Embryo

Decidua

Uterine gland

Fig 4 -Diagrams showing implantation (X45)- A. Longitudinal section
through horn of uterus about 5 days after mating. An ovum has recently become
implanted in one of the uterine crypts. B. Longitudinal section through implantation
site about 7 days after mating. {After Burckhard)

crypt on the ventral or antimesometrial side of the lumen, thereby coming
into close contact with the uterine epithehum (Fig. 3) • The presence of the

THE EARLY EMBRYOLOGY OF THE MOUSE 7

blastula quickly sets up changes at the implantation site. Within a few
hours the epithelium begins to loosen, and its nuclei to show degenerative
changes (Fig. 5). Within 15 hours it is sloughed off entirely (Fig. 6). At
the same time active growth commences in the mucosa, so that by i day after
implantation (5 days after mating) there is an appreciable swelling in the
uterus at the implantation site. The swollen mucosa at the implantation
site is known as decidua.

Meanwhile the zona pellucida has been lost from around the egg, perhaps
through a process of digestion by means of an enzyme secreted by the uterine
mucosa (11), though neither the exact time nor mechanism is thoroughly
known.

Up to the time of implantation there has been no growth in size in the
egg. Cleavage has resulted in a division of the egg, originally one large cell,
into numerous smaller cells, but little if any new protoplasm has been formed
in the process. Beginning with implantation, however, rapid growth com-
mences. At first the blastocoele enlarges, while the inner cell mass assumes
a flattened cup-shape with the concave face towards the cavity (Fig. 5).
In the living condition the blastocoele is probably distended with fluid, and
its walls tightly pressed against the uterine epithelium, but in fixed material
at this stage there is always some collapse. This initial expansion of the
blastocoele requires only a few hours and is quickly followed by a growth of
the inner cell mass down into the enlarged cavity (Fig. 6). Blastocoele and
inner cell mass both are known thereafter by new names; namely, yolk
cavity for the former and egg cylinder for the latter. A comparison of
Figs. 7, 8, 10 and 12 will show the rapid growth of the egg cylinder that
occurs during the next two' and one-half or three days.

The formation of the entoderm. — At the same time that the blastocoele
begins to enlarge, the inner cell mass can be seen to be composed of two types
of cells (Fig. 5). Adjacent to the blastocoele is a single layer of darkly
staining cells. This is the entoderm, one of the three primary germ layers.
The rest of the blastocyst is composed of ectoderm, divided into the ecto-
derm of the inner cell mass, and the trophectoderm, a single celled layer
bounding the blastocoele ventrally and laterally. The trophectoderm
(troph from the Greek word for nourishment) derives its name from the fact
that it probably plays a role in the nourishment of the young embryo. The
mesoderm has not yet appeared.

Very shortly after the first appearance of the entoderm, single cells or
strands of cells grow out from its margin down along the inner surface of the
trophectoderm. At first these cells are few and widely separated (Figs. 7

BIOLOGY OF THE LABORATORY MOUSE

and 8), but by 63^^ days they lie evenly spaced and quite close together over
the trophectoderm's entire inner surface (Fig. lo). The layer of cells thus
formed is known as the distal entoderm. Meantime the inner cell mass has
grown down into the yolk cavity to form the egg cylinder. This is composed
of an inner mass of ectoderm cells and an outer layer of entoderm cells
(Fig. 8). This layer of entoderm cells bounding the egg cylinder is known as
the proximal entoderm. The entoderm is thus divided into two distinct
parts, distal and proximal, lining the distal and proximal walls of the yolk
cavitv.

uterine lumen

Inner cell mass

Entoderm

Trophectoderm

Blastocoele

Uterine epithelium

S. Bohtn

Fig. 5.— Section of implanting blastula 4 days 5 hours after mating. Projection

drawing (X400).

Embryonic and extra -embryonic ectoderm. — At about 4}^ days, when
the egg cylinder first begins to form, it can be seen that the egg cylinder ecto-
derm is divided into two parts, a dorsal,* more darkly stainingf region with

* Most authors have used the terms mesometrial and antimesometrial to dis-
tinguish the two poles of the egg, the former being toward, and the latter away from,
the mesometrium or supporting mesentery of the uterus. However, as the dorso-
ventral axis of the embryo coincides with the dorso-ventral axis of the mother for at
least the first 8 days of development, the usage dorsal and ventral would seem to be
perfectly clear in most cases besides having the advantage of simplicity. The dorsal
side is up in the drawings.

I When counterstained with congo red.

THE EARLY EMBRYOLOGY OF THE MOUSE g

elongated nuclei, and a ventral, more lightly staining portion with round
nuclei* (Fig. 6). The former gives rise to various extra-embryonic struc-

fa ^.

t% r-3 uterine lumen

Droplets of
secretion

Extra-embryonic
ectoderm

Embryonic
ectoderm

Proximal
entoderm

Distal entoderm
Trophectoderm
Yolk cavity
— Giant cell

Degenerating
uterine
epithelium

(^W ^^'

^. Soh*.n

Fig. 6. — ^Longitudinal section of early egg cylinder stage at 4 days 15 hours after
mating. Projection drawing (X400).

tures and is, therefore, called the extra-embryonic ectoderm; the latter
gives rise to the ectoderm of the embryo proper and is, therefore, called
the embryonic ectoderm. While the difference in staining reaction and in the
shape of the nuclei has disappeared by 5 3-^ days, the division between the

* It is possible that the division between embryonic and extra-embryonic ectoderm
can be traced back to stages earlier than \V2 days. One author (41) contends that
the ectoderm of the inner cell mass at a stage corresponding to that shown in Fig. 5 is
divided into two regions, a lighter staining outer layer continuous with the trophecto-
derm and a darker staining area between this and the entoderm, but the existence of
such a division has also been denied {22, 61). In our preparations at the 4)^4 day
stage we find occasional llattened, dark-staining nuclei on the outer surface of the
inner cell mass and in some cases these appear to form a layer continuous with the
trophectoderm. It seems probable that these represent an early stage of the extra-
embryonic ectoderm. Phylogenetically the extra-embryonic ectoderm is probably
derived from the trophectoderm, so that a similarity of structure is not surprising.

BIOLOGY OF THE LABORATORY MOUSE

two regions is still quite distinct (Fig. 8). Strictly speaking the trophecto-
derm is also extra-embryonic ectoderm, but as a matter of convenience the
term will be used only for the extra-embryonic ectoderm of the egg cylinder.

At about 5 days a cleft or cavity, the proamniotic cavity, appears in the
embryonic ectoderm (Fig. 7). This is followed very shortly by the appear-
ance of a similar cleft in the extra-embryonic ectoderm, and by the fusion of
these two, so that by 5}^ days the egg cylinder contains a narrow lumen
(Fig. 8).

The ectoplacental cone. — Beginning at 5 or 5^^ days, active growth at
the dorsal end of the extra-embryonic ectoderm gives rise to a new structure.

Secondary giant cell
-Extra-embryonic ectoderm
Distal entoderm
Proximal entoderm
Proamniotic cavity
Embryonic ectoderm
Primary giant cell
Yolk cavity

£. 3ohKn

Fig. 7. — ^Longitudinal section of early egg cylinder. Age unknown, but probably
about 5 or 6 days. Projection drawing (X400).

the ectoplacental cone, which joins the egg cylinder ventrally, and extends
dorsally towards the lumen of the uterus (Fig . 8) . This develops rapidly , its
cells showing numerous mitoses, and by 6I2 days it composes almost one-
half of the total length of the embryo. Its structure, particularly at the
upper extremity, is porous, and the interstices between the strands of cells
that compose it soon become infiltrated with maternal blood (Fig. 10). In
later stages it becomes part of the placenta.

The inversion of the germ layers.— At sH days (Fig. 8) the egg cylinder
is a structure consisting of a double wall enclosing a narrow lumen. The
inner layer of the double wall is composed of ectoderm, the outer of ento-
derm. This relation of ectoderm and entoderm, found in the mouse, rat,

THE EARI.Y EMBRYOLOGY OF THE MOUSE

rabbit, guinea pig and their close relatives, proved very puzzling to early
embryo legists, for the reason that it is the reverse of the condition found in

IvV.^o"

Maternal blood

Ectoplacental cone

Distal entoderm

Proximal entoderm

Extra -embryonic ectoderm

Embryonic ectoderm

Proamniotic cavity

Yolk cavity

Trophectoderm

V ^l£±i

Giant cell

Degenerating uterine epithelium

Fig. 8. — ^Longitudinal section at 5 days 12 hours after mating. Projection drawing

(X300).

all other chordates. It has been called the inversion of the germ layers.
While at first sight it seems to indicate a drastic alteration in early develop-

BIOLOGY OF THE LABORATORY MOUSE

.Embryonic ectoderm

B. Bohe.n

Fig. 9. — Diagram comparing the eariy stages of development in a primitive rodent,
the thirteen-striped ground squirrel, and in the mouse. A, B and C. The thirteen-
striped ground squirrel. {After Mossman and Weisfeldt.) D, E and F. The mouse
{Mus muscidus). (A and B, X24; C, X8; D and E, Xioo; F, X50.)

THE EARLY EMBRYOLOGY OF THE MOUSE 13

ment, actually there is no very fundamental change in the relations of the
important structures. Those changes that are involved are easily under-
stood from a comparison of the early development of the mouse with that of a
primitive rodent, the thirteen-striped ground squirrel. Three comparable
stages for each species are shown diagrammatically in Fig. 9. Beginning
students of embryology will want to refer again to this figure after complet-
ing the study of later stages in the mouse. >

In primitive rodents, as represented by the thirteen-striped ground
squirrel, the embryonic area (embryonic ectoderm and underlying entoderm)
forms a disc that overlies an almost spherical yolk cavity . In the mouse , the
embryonic area forms a deep cup pushed far down into the yolk cavity,
which thereby is greatly reduced in size. The obvious explanation of this
condition is that during some period in the evolution of the mouse there
developed an invagination of the embryonic area into the yolk cavity, the
curvature of the embryonic area thereby being reversed and the relation of
ectoderm and entoderm inverted. The change is comparable to that
produced when a rubber ball has one side pushed in, being altered thereby
from a sphere to a cup.

In Fig. 9D the lumen of the egg cylinder is shown extending through the
ectoplacental cone to the outside. This condition is probably the exception
rather than the rule, but it has been described by Sobotta (61) and Melissinos
(41), and we have found it in a few cases in our material. It is significant
evidence for the theory that the inversion of the germ layers is due to an
invagination of the embryonic area.

Further evidence is provided by the later development of the thirteen-
striped ground squirrel (Fig. 9C). In this species the whole embryo sinks
down into the yolk cavity, carrying the splanchnopleure with it. The
splanchnopleure is thereby inverted, but no inversion of embryonic ectoderm
and entoderm occurs because of the advanced development of the embryo at
the time. However, if the sinking or invagination of the embryonic area
were pushed back to an earlier period of development, the condition found
in the mouse would result.

One interesting consequence of the inversion of the germ layers is the
production of a very compact form of early development. Much seemingly
waste space in the yolk cavity is eliminated. The reader should note in this
connection that the drawings of the mouse embryos in Fig. 9 are at a higher
scale of magnification than those of the ground squirrel embr}'os. Actually,
at comparable stages of early development, the total volume of a mouse
embryo is, in round figures, perhaps one-fiftieth that of the total volume of

Distal entoderm ■

Proximal entoderm

Extra-embryonic
ectoderm

Embryonic ectoderm

Maternal blood

Secondary giant cell

^3- ^®'

(5s/5.^

8^1 ^=2:^^0^ Ectoplacental cone

Posterior amniotic fold

Proamniotic cavity

-Reichert's membrane

Fig. io. — Scagittal section of mouse embryo of 6 days 13 hours, showing early
stage of mesoderm formation. Projection drawing (X300) except that Reichert's
membrane is drawn in part from adjacent section.

THE EARLY EMBRYOLOGY OF THE MOUSE 15

an embryo of the thirtecn-striped ground squirrel. This reduction in total
volume involves little if any reduction in the volume of the embrvonic area
proper.

The primitive streak and mesoderm formation. — At 6} 2 days the middle
germ layer or mesoderm makes its appearance (Fig. 10). The first meso-
derm cells are budded off from a narrow strip of embryonic ectoderm which

r . . . '

extends dorso-ventrally from the line of junction of the embryonic and extra-
embryonic ectoderm about half way to the tip of the egg cylinder. This
J stfip of ectoderm is known as the primitive streak. Since the primitive
streak lies at the posterior end of the embryo, an anterior-posterior axis is at
once established with its appearance.*

The mesoderm cells form a loose tissue of very characteristic appearance.
They multiply rapidly, wedging their way laterally between ectoderm and
entoderm toward the anterior margin of the egg cylinder (Fig. 14A). The
forward growth is particularly rapid along the line which marks the junction
between embryonic and extra-embryonic ectoderm, and in this line meso-
derm may be found at the anterior margin of the egg cylinder about 12 hours
after the first mesoderm cells appeared (Fig. 12) . Elsewhere the two lateral
wings of mesoderm do not penetrate to the mid-sagittal region until much
later. Some mesoderm cells also push dorsally between the extra-embryonic
ectoderm and the adjacent entoderm, thus leaving the region of the embryo
proper. These mesoderm cells, for the most part, are destined to take part
in the formation of the yolk-sac, an extra-embryonic membrane, which later
envelops the embryo and which is discarded at birth.

The orientation of the embryo in the uterus. — Since the primitive streak
is at the posterior margin of the egg cylinder, its formation, heralded by the
appearance of the mesoderm, establishes an anterior-posterior axis in the
embryo. It is appropriate at this point to consider how this axis and
the other axes of the embryo are oriented in relation to the uterus.

At the time of implantation the embryo settles to the ventral or anti-
mesometrial side of the uterus. When it first implants, the inner cell mass
is up or towards the mesometrium, the blastocoele is down or away from the

* In our material we have noted that from 5 to $14 days, the egg cylinder and more
particularly the proamniotic cavity instead of being round in cross section, are slightly
flattened along an axis perpendicular to the mesometrium. This is the same as the
future anterior-posterior axis. However, it cannot be determined until the appearance
of the mesoderm which end of the axis is anterior and which end posterior. With the
appearance of the mesoderm the flattening of the egg cylinder, if any, is along the
opposite axis.

BIOLOGY OF THE LABORATORY MOUSE

mesometrium (Fig. 4A). In terms of an older embryo, the ectoplaceiital
cone is up and the embryonic portion of the egg cylinder is down (Fig. 4B).
The dorso-ventral axis of the embryo is thus parallel to the mesometrium
and perpendicular to the long axis of the uterus (Fig. 11). The anterior-
posterior axis of the embryo likewise has a definite orientation with respect
to the uterus, being, as a rule, perpendicular to the mesometrium. Depar-

Mesometriuiti

Uterus

Embryo

Fig. II. — Diagram showing the onentation of an 8 day embryo in the uterus,
and of the planes in which sections are cut. Plane A: transverse to uterus, sagittal
to embryo. In the early egg cylinder stages this may be referred to also as a longi-
tudinal section of the egg cylinder. The orientation of the embryo is not always
consistent and may sometimes depart by as much as 45° from this plane. Plane B:
transverse section of embryo. Note, however, that in embryos past the egg cylinder
stage this plane though transverse to head and tail regions is frontal with respect to
the mid-trunk region. Plane C: frontal section of embryo. Note, however, that in
embryos past the egg cylinder stage this plane though frontal to head and tail regions
is transverse with respect to the mid-trunk region. In early egg cylinder stages this
may be referred to also as a longitudinal section of the egg cylinder.

tures from this orientation by as much as 45° may, however, occur. This
orientation persists until about 8 or 8^ 2 days when the embryo begins to shift
its position in the uterus.

Amnion, chorion and exocoelom. — When mesoderm cells first appear
between the ectoderm and entoderm at the posterior margin of the egg
cylinder, they cause the ectoderm at the line of junction between its embry-
onic and extra-embryonic portions to bulge into the proamniotic cavity.

Maternal blood

Extra-embryonic
ectoderm

Posterior amniotic
fold

Ectoplacental cone

Yolk cavity

Distal entoderm

Proximal entoderm

Exocoelom

Mesoderm

Anterior amniotic
fold

Primitive streak

Squamous entoderm

Head process

Fig. 12. — Sagittal section of embryo of 7 days i hour showing the amniotic folds and
the head process. Reichert's membrane omitted. Projection drawing (X300).

1 8 . r BIOLOGY OF THE LABORATORY MOUSE

This bulge is the beginning of the posterior amniotic fold* (Fig. lo) . In like
manner the lateral wings of mesoderm, progressing around the egg cylinder
toward its anterior margin, give rise to folds along the sides of the cylinder.
These are the lateral amniotic folds. Finally, when the mesoderm reaches
the anterior margin of the egg cylinder, a small anterior fold is produced
(Fig. 12). The posterior, lateral and anterior folds should be thought of,
not as separate structures, but as a continuous constriction about the middle
of the egg cylinder which is drawn tighter and tighter as the folds develop.
Because of the very precocious development of the posterior amniotic
fold as compared with the anterior one, the constriction is eccentric, the
point of final closure being far towards the anterior margin of the egg
cylinder, t

Before the anterior fold forms, small cavities J begin to appear in the
mesoderm of the posterior and lateral folds. These soon coalesce to form a
single large cavity, the extra-embryonic coelom, or, more concisely, the
exocoelom (Fig. 12). The exocoelom at this stage, and at all future stages,
is lined by mesoderm. For a short time a second cavity is present in the
posterior amniotic fold between the mesoderm and ectoderm (Fig. 12), but
this is a transitory structure of no particular significance.

In less than a day after the first appearance of the amniotic folds, the
girdle which they form has closed. For a time a vertical strand of cells
adjacent and usually attached to the inner anterior wall of the exocoelom
marks the point of closure, but this soon disappears and the separation is
complete. The resulting condition is shown in the sagittal section repro-
duced in Fig. 13.

Three cavities § are now present in the egg cylinder in place of the single
proamniotic cavity which it formerly contained. The most ventral of these

* In Fig. 10 there may be seen a second fold pushing into the proamniotic cavity
just dorsal to the posterior amniotic fold. Sobotta (62) shows this in his Fig. 5, but
interprets it as an artifact. Our material would indicate that it is regularly though
briefly present. Its significance is unknown, but it is perhaps indicative of the very
rapid growth that occurs in the whole posterior wall of the egg cylinder at the time of
mesoderm formation.

t In the rat, the anterior amniotic fold is much better developed than in the mouse,
and the constriction, therefore, less eccentric (26).

I None of the embryos in our collection show this early stage in the formation of
the exocoelom. This description is based on the observations of Jolly and Ferester-
Tadie (26).

§ In some cases, also, a transitory fourth cavity, the cavity between ectoderm and
mesoderm mentioned on page 18, second paragraph, is present.

THE EARLY EMBRYOLOGY OF THE MOUSE

Maternal blood

Ectoplacental cavity

Chorion

Exocoelom -

Amnion

Fore-gut

Amniotic cavity

Embryonic ectoderm

Head process

Squamous entoderm

Secondary giant cell

'" — Ectoplacental cone

Distal entoderm

Proximal entoderm

Extra-embryonic
ectoderm

03 Mesoderm

AUantois

Primitive streak

Fig. 13.— Sagittal section of embryo of 7 days 6 hours showing completion of amnion
formation. Reichert's membrane omitted. Projection drawing (X200).

20 BIOLOGY OF THE LABORATORY MOUSE

is the amniotic cavity, lined with embryonic ectoderm.* At this stage it is
cup-shaped, as can be seen from Figs. 13 and 14A, which show it in sagittal
and cross section respectively.

In the middle is the exocoelom, lined with mesoderm.

At the dorsal extremity of the egg cylinder is the ectoplacental cavity,
lined with extra-embryonic ectoderm. The amniotic cavity and the exo-
coelom, though neither one, as will be seen later, is included in the actual
body of the embryo, are important in its future development. The ecto-
placental cavity, on the other hand, already the smallest of the three,
gradually becomes narrower and finally disappears.

The membrane separating the amniotic cavity from the exocoelom is
called the amnion. It is composed of two thin, cellular layers, one of
ectoderm, the other of mesoderm. Separating the exocoelom from the
ectoplacental cavity is another membrane, the chorion, likewise composed of
ectoderm and mesoderm.

The head process, f — It will be remembered that mesoderm is first
proliferated by the primitive streak in embryos about 6^^ days old. The
growth is entirely from the lateral and caudal margins of the primitive
streak; no mesoderm is proliferated from its cephalad extremity. Beginning
at about 7 days, however, growth does occur in this region, but the structure
formed shows greater affinity to the entoderm than to the mesoderm. It is
known as the head process. In sagittal sections it first appears as a wedge
shaped group of cells between the ectoderm and entoderm at the ventral
extremity of the egg cylinder (Fig. 12). The base of the wedge is attached
to the ventral end of the primitive streak from which it takes its origin; the
tip of the wedge points forward towards the anterior margin of the egg
cylinder. Cells grow out rapidly from the margins of the wedge, forming a
thin, spreading sheet between ectoderm and entoderm. J

* We interpret the ectoderm of the amnion as embryonic ectoderm, the ectoderm
of the chorion as extra-embryonic ectoderm. The evidence on this point is not neces-
sarily conclusive, however, for concurrent with the appearance of the amniotic folds,
the division between the two types of ectoderm loses much of its distinctness.

t Sobotta (62) studied embryos representing the stages during which the head
process develops, but his drawings indicate that his otherwise admirable sections were
not close enough to the exact sagittal plane to reveal this structure clearly. Conse-
quently, it remained for Jolly and Ferester-Tadie (26) to first describe it correctly
for the mouse and rat. Our observations are entirely in accord with theirs.

t The entoderm and the margins of the head process are so thin and close together
at this stage that favorable conditions are necessary to distinguish them. In the

THE EARLY EMBRYOLOGY OF THE MOUSE 21

If the reader now will refer back to Fig. 10, he will see that the entoderm
over the ventral extremity of the egg cylinder is stretched and the cells
flattened, but that near the upper margin of the embr>'onic portion of the
cylinder there is a sudden change to a higher type of cell. The transition is
particularly abrupt at the anterior margin of the cylinder. The thin or
flattened entoderm we shall refer to as squamous entoderm, the thick ento-
derm as columnar entoderm, the line of junction between the two as the
transition line. The reader should take time at this point to note, in Figs. 10
and 12, the precise location of the transition Hne.

The limits of the head process are as follows. Caudad, it begins at the
anterior extremity of the primitive streak, that is to say just a little above
and caudad to the ventral tip of the egg cylinder. Cephalad, it extends to
the transition line. Laterad, at its broadest point it may extend almost
around the anterior half of the circumference of the egg cylinder (Fig. 14A),
but mostly it is narrower than this, fllling perhaps the anterior fifth of the
egg cylinder's circumference.

When its forward growth brings it to the transition line, the head process
fuses with the columnar entoderm with which it has thus newly come in
contact (Fig. 13). The fusion is so complete that in later stages the line of
junction is completely lost. Laterally, its outer margins fuse with the
squamous entoderm. Meantime the squamous entoderm underlying the
head process, already ven.- thin, becomes increasingly attenuated, its nuclei
become widely separated and ver}- flat, and the cytoplasm largely disappears
(Figs. 14A and B). At yf^ days no further trace of it remains.

In the course of the upward and laterad growth of the head process and
the forward growth of the mesoderm the two cefl layers come in contact and
overlap (Fig. 14A). In the regions of overlapping, the head process stays
adjacent to and advances over the surface of the entoderm, while the meso-
derm remains next to the ectoderm. At 7^2 days the development of the
mesoderm has brought it between ectoderm and head process ever}-w^here
except for a strip along the mid-sagittal plane of the embryo. As we shall
see later, the head process of this mid-sagittal strip gives rise to notochord,
while the remainder of the head process contributes to the lining of the gut.

section shown in Fig. 12 there are several cells at the anterior limit of head process
growth that cannot be classified definitely as either head process or entoderm. The
division in the drawing in this region is partly arbitrary. When head process and
mesoderm come into contact there is also possibility for confusion. However, in well
fixed preparations cut at a favorable angle, the division in this case can almost always
be precisely determined.

BIOLOGY OF THE LABORATORY MOUSE

Primitive streak -

Entoderm

Mesoderm

Ectoderm
Head process
Squamous entoderm

Mesoderm

Head process

Squamous
entoderm

3. Aohcn
Notochord

Fig. 14.— For descriptive legend see opposite page.

THE EARLY EMBRYOLOGY OF THE MOUSE

The neural groove. — It can be seen from Fig. 14B, which is a cross section
of the anterior part of an egg cylinder of a 7 14 day embryo, that the ectoderm
in the mid-sagittal plane forms a definite trough or truncated V. This

B. Boht
Archenteron Base of notochord

Ectoderm

Mesoderm

Head process
entoderm

C D

Fig. 14. — Transverse sections of head process. The location of sections is indi-
cated on the small key diagrams. A. 7 day i hour embryo. B. 7 day 6 hour embryo.
C and D. 7 day 10 hour embryo. Projection drawings (X400).

trough extends forward in the mid-sagittal plane from the cephalad end of
the primitive streak well towards the cephalad limit of the embryonic
ectoderm. Developed between the 7 and the 734 day stages (Figs. 14A and
B), it is the beginning of the neural groove which later gives rise to the
central nervous system. The further development of the neural groove will

24 BIOLOGY OF THE LABORATORY MOUSE

be discussed later, but the reader will do well at this stage to look ahead to
Figs. 19 and 20 which show the way in which it deepens and narrows and
ftnally closes at the top to form the neural tube. The point to be empha-
sized here is that the appearance of the neural groove establishes a perfectly
clear caudal-cephalic axis throughout the length of the embryo. The neural
groove anteriorly and the primitive streak posteriorly lie in the precise
mid-plane and together separate the right and left halves of the embryo.

The notochord. — At the same time that the neural groove is differentiat-
ing in the mid-sagittal area of the ectoderm, changes are also going on in the
mid-sagittal region of the head process which immediately underlies it
(Fig. 14). In this region the head process thickens, and the oval nuclei
become oriented in general perpendicular to the ectoderm. Elsewhere it
forms a thin membrane with the nuclei oriented parallel to the plane of the
membrane. The structure thus differentiated ventral to and in contact
with the ectoderm of the neural groove is the notochord. It is the axis about
which the vertebral column is later laid down. The remainder of the head
process, together with a part of the entoderm to which it is fused, becomes
the lining of the gut.* This part of the head process will hereafter be
referred to as gut entoderm. For a considerable period notochord and gut
entoderm remain joined. Eventually, however, the two halves of the gut
entoderm grow across the ventral surface of the notochord and unite in the
mid-ventral line, leaving the notochord as an axial, rod-like structure
between ectoderm and entoderm.

Huber (23) describes the head process in the guinea pig as giving rise to
notochord only. Our material, however, confirms completely the conten-
tion of Jolly and Ferester-Tadie (26) that in the mouse at least some gut
entoderm is also derived from the head process. The critical stage is that
shown in Fig. 14B in which it can be seen that the head process extends
laterally considerably beyond the limits of the differentiating notochord.

A much mooted question is whether the notochord should be classed as
ectoderm, entoderm or mesoderm (31). Since it is formed from the head
process and since the ver>' complete fusion of the margins of the head process
with the entoderm indicate a close affinity between the two tissues, classi-
fication as entoderm would seem logical. If, however, head process is
classed as entoderm, it must be remembered that its origin in time is quite
different from that of all the other entoderm, and two separate stages of

* It seems likely that most or all of the mid-gut is lined by head process. Whether
or not any of it enters into the formation of the fore- and hind-guts is not clear.

THE EARI.Y EMBRYOLOGY OF THE MOUSE 25

entodermal proliferation must be recognized. As to the place of origin,
there is a certain similarity between the two tissues, one forming at the
ventral margin of the inner cell mass, the other near the ventral tip of the
egg cylinder, which is, so to speak, simply the inner cell mass grown up.
Cell lineage studies might reveal a closer similarity in origin than is super-
ficially apparent.

The archenteron. — At 7^^ days there is a broad depression in the rather
thick base of the notochord adjacent to its junction with the primitive
streak (Figs. 14D and 15). The depression is a conspicuous landmark at
this stage, but it is a transitory structure, the first signs of it appearing at
7I4 days and disappearance being complete about twelve hours later. It
plays no part in later development and probably is best interpreted as a
vestigial structure corresponding to a similar structure that occurs in more
marked form in the development of reptiles,* and which in turn can probably
be traced back to the archenteron of the lower chordates. On the basis of
this probable homology it may be called the archenteron.

The allantois. — Soon after the exocoelomic cavity becomes well estab-
lished, a process begins to grow out into this cavity from the mesoderm at
the caudal end of the primitive streak. This is the allantois (Fig. 13), an
extra-embryonic, mesodermal structure whose function is to convey blood
vessels from the embryo to the placenta where they establish contact with
the maternal circulation. In many vertebrates the allantois contains a
cavity lined with entoderm and connected with the gut. There is no ento-
derm-lined cavity in mice; on the other hand there are numerous small
cavities in the mesoderm giving the organ a porous structure.

After its first appearance at 7I4 days the allantois grows rapidly across
the exocoelom in the direction of the ectoplacental cone (Figs. 15 and 16).
Meantime the chorion becomes flattened against the base of the cone,
constricting the ectoplacental cavity and finally eliminating it altogether.
When the allantois makes contact with the chorion at about 8 days, a con-

* See for example Figs. 21 and 22 of Prentiss and Arey (51). We have found no
trace of a neurenteric canal in the mouse, in the sense of a canal passing through the
ectoderm and the base of the notochord and connecting amniotic cavity and yolk
cavity. However, we have seen in a 7I4 day embryo a very short canal confined to
the base of the notochord. The ventral wall was thin, and it may be presumed that
it would shortly disappear, giving rise to the depression described above. Sobotta's
Fig. 14 (62) shows a canal somewhat similar to the one we have noted, except that our
material does not suggest, as his drawing does, that the canal is formed by invagination
of the entoderm. Jolly and Ferester-Tadie (26) have figured a section almost identical
with ours.

BIOLOGY OF THE LABORATORY MOUSE

tinuous structure is established connecting the posterior end of the primitive
streak with the ectoplacental cone. In due course embryonic blood vessels

ectoplacental cone

ectoplacental cavity
chorion

yolk sac

entoderm

archenteron

Fig. 15. — Sagittal section of mouse embryo of 7 days 15 hours. Reichert's membrane
omitted. Projection drawing (XiSo).

will find their way along this pathway to make contact with the maternal
blood supply.

Fore -gut and hind -gut. — In the early stages of its formation the digestive
tract consists of three quite distinct parts, the fore-gut, the hind-gut and the
mid-gut. These appear in the order named. The fore-gut can be traced

THE EARLY EMBRYOLOGY OF THE MOUSE

back to the 7 day stage when it consists merely of a deep notch in the thick
or columnar entoderm at the anterior margin of the egg cylinder (Fig. 12).
Six hours later there is Httle change (Fig. 13), but by 'jYi days (Fig. 15) the
notch has been replaced by a definite pocket in the entoderm, and the
entoderm surrounding the pocket together with the overlying ectoderm form
a bulge which projects into the amniotic cavity. From this stage on,

^^^ -Ectoplacenta

Reichert's
membrane

Ectoplacental
cavity

Exocoelom
Amnion

Somatic mesoderm

Pericardial coelom
Epi-myocardium

Endocardium
Fore-gut

Amniotic cavity

Chorion

.\llantois

Yolk-sac

Blood island

Omphalomesen-
teric artery

Hind-gut

Primitive streak

Ectoderm

&*'" ^Notochord

Fig. 16.— Partly diagrammatic sagittal section of embryo of 7 days 18 hours (Xioo).

Reichert's membrane omitted.

growth of the fore-gut is exceedingly rapid, the pocket changing in a few
hours into a deep pouch (Fig. 16). The process is due to a progressive
drawing together in the mid- ventral line of the folds of entoderm that bound
the anterior intestinal portal (Figs. 25C and 30), the portal thus being shifted
farther and farther towards the cudal extremity of the embryo. The process
has been aptly described as a "zipper action."

It should be noted that the fore-gut is lined by entoderm, and that this is
surrounded by mesoderm and ectoderm. Thus in this region the process of

28 BIOLOGY OF THE LABORATORY MOUSE

invagination has reversed the inversion of the' getfrn layers found in the early
egg cylinder. This is the first of the steps by which the germ layers in mice
are brought into the relation characteristic of the adult, i.e., entoderm on the
inside, ectoderm on the outside, mesoderm in between.

The hind-gut, less precocious than the fore-gut, appears at about 7^^
days as an invagination in the entoderm and overlying layers at the posterior
end of the primitive streak (Fig. 16).

The open ends of the fore- and hind-guts are eventually joined by the
mid-gut whose formation will be described in a later section. It is not these
open ends, but the blind ends of the two guts which, by breaking through to
the outside, give rise to mouth and anus. An early stage in the development
of the mouth may be seen in 8 day embryos (Fig. 22). In the ectoderm of
the head there is an invagination directed towards the anterior extremity of
the fore-gut. This is the stomodaeum. The wall between the stomodaeum
and the fore-gut is the oral plate. In course of time this ruptures and the
mouth opening is thereby estabhshed. The anus develops in a similar
manner at a somewhat later stage.

The head fold. — The invagination of the fore-gut involves a pushing or
folding of the adjacent tissues into the amniotic cavity. The structure thus
produced is the head fold (Fig. 15). First appearing at about 73^ days, it
becomes a large and conspicuous structure within less than twenty-four
hours (Figs. 22 and 26A). The growth of the neural folds in this region is
more rapid than elsewhere, presaging the formation of the brain, and the
heart, just ventral to the head fold, is also conspicuous by its rapid growth.
In 8 or S}^ day embryos the difference in size between the head region and
the middle of the trunk is striking. The head fold is thus the center of a
region of particularly rapid growth (24).

The somites. — Since the somites are mesodermal structures, it will be
useful before discussing their development to review the distribution of the
mesoderm at the 7^2 dav, stage when they make their first appearance. In
the extra-embryonic regidri,the entire exocoelom is lined with mesoderm.
The exocoelom contains also the allantois, a wholly mesodermal structure.*
In the embryo proper there is little mesoderm in the mid-sagittal region.
One small mass which will later contribute to the formation of the heart

* Not in the exocoelom, but also not part of the embryonic mesoderm, is a small
mass of mesoderm at the anterior extremity of the amnion (Fig. 1 5) . This is character-
istically in the form of two hollow, thin-walled vesicles, one on each side of the mid-
hne, though the range of variation is considerable. That the vesicles are paired is
probably due to the fact that the mesoderm in this region grew in from the two sides.
Between the two vesicles, and hence approximately in the mid-Une (it may be a little
to one side or the other), is a very small area where ectoderm and entoderm are unsepa-

THE EARLY EMBRYOLOGY OF THE MOUSE

occurs anterior to the fore-gut (Fig. 15). The primitive streak in the mid-
sagittal plane consists of a tissue which joins, and in structure is intermediate
between, ectoderm and mesoderm. Whether or not this should be called
mesoderm is a matter of definition. At the caudal extremity of the embryo
its structure is essentially that of true mesoderm, and it may accurately be
said that there is mesoderm in the mid-sagittal plane in this region.

.eVo*^

Fig. 17. — Section transverse to mid-trunk region of 8 day, 4 somite embryo. Through
2nd somite. Projection drawing (X150).

While the notochord blocks the entrance of mesoderm into most of the
mid-sagittal area, there are well developed sheets of mesoderm on each side
of this area. These lie between ectoderm and entoderm and are continuous
laterally with the extra-embryonic mesoderm (Fig. 17). Anterior to the
primitive streak, it is convenient to recognize two distinct areas in these
mesodermal sheets, an area of paraxial mesoderm adjacent to the notochord,
and an area of lateral mesoderm adjacent to the extra-embryonic coelom.
The former gives rise to the somites, the latter to the mesoderm of the
embryonic coelom. At 7I2 days there is no visible division between
the two areas (Fig. 19B), but beginning at about 7^4 days, coincident with
the development of the somites, they are separated by a longitudinal cleft
that becomes increasingly pronounced as the differentiation of the somites
progresses (Fig. 17).

The somites are paired, segmental structures arising in the paraxial
mesoderm (Figs. 18 and 25D). They are the first indication of metamerism
in the developing embryo. The first pair forms a little anterior to the

rated by any mesoderm,
the rabbit.

This area is probably homologous with the proamnion of

Auditory vesicle

Fig. i8. — For descriptive legend see opposite page.
30

THE EARLY EMBRYOLOGY OF THE MOUSE 31

anterior end of the primitive streak. Each member of the pair appears as a
localized denser area which grades off anteriorly into loose mesoderm, and f^^cU'^^'n^
which, posteriorly, is separated by a cleft from the undifferentiated caudal -'^^^<2
mesoderm. The second pair forms posterior to the first, and is Hkewise
separated by a cleft from the undifferentiated caudal mesoderm. Additional
pairs of somites form at more or less regular intervals, each new pair differ-
entiating just posterior to the pair last formed, until a total of 65 pairs*
has appeared (9). Continued and rapid proHferation of the mesoderm on
each side of the primitive streak maintains a supply of undifferentiated cells.
These push forward to about the level of the base of the notochord where the
new somites are segmented off in regular succession. As a consequence of
this manner of formation, the anterior somites are the oldest and, at any
given stage, the most highly differentiated while the posterior somites are
the youngest and the least differentiated.

Figure 18 shows in interesting fashion the way in which the "zipper
action" by which the fore-gut is formed moves the location of the anterior
intestinal portal steadily^ caudad. At the two somite stage the opening of
the shallow fore-gut lies well ahead of the first somite. At the seven somite
stage the anterior portal has moved backwards until it is just about at the
level of the first somite. At the eleven somite stage it has reached approxi-
mately the level of the sixth somite. (The portion of the gut shown in
Fig. 18C is mid-gut just caudad to the anterior intestinal portal.)

Because of the regularity with which successive somites develop, the
number of somites in an embryo is a convenient means of stating its stage
of development. V

The primitive streak as a growth center. — The primitive streak is ^-^
remarkable as being a region in which the three germ layers meet (Fig. 15). iQi

It is continuous dorsally with the ectoderm, laterally and posteriorly with

— —

* This figure is for the rat; the characteristic number may be slightly different for
the mouse. There is some individual variation. Butcher states in his excellent paper
on the somites of the rat that the first pair dedifferentiates and disappears at about
the seven somite stage. We have found no evidence of such a dedifferentiation in our
material. As may be seen from Figs. 18A, B, and C, the first somite can be traced
clearly at least to the eleven somite stage.

Fig. 18. — Sagittal sections through somites. A. Embr>'0 of 7 days 18 hours, with
2 somites formed. B. Embryo of 8 days i hour, 7 somites. C. Embryo of 8 days 11
hours, II somites. Because the embryo begins turning at about the 7 somite stage,
the plane at which this 11 somite embryo is cue, although sagittal to the first 6 somites,
is transverse to the mid-trunk region. Projection drawings (X150).

32 BIOLCGY OF THE LABORjiTORY MOUSE

the mesoderm, and anteriorly with the head process (which is entodermal in
nature and indistinguishably fused with the original proliferation of ento-
derm). Of these three, it gave rise to two, the mesoderm and the head
process. It may be added that the somewhat distinct proliferation of
mesoderm cells that produced the allantois occurred at its posterior end.
Its own cells are undifferentiated in nature and cannot be classified as either
ectoderm, entoderm or mesoderm. The only structure in primitive chor-
dates possessing these characteristics is the dorsal lip of the blastopore, and
it is probable that the primitive streak and the dorsal lip of the blastopore
are homologous. Besides being a point of origin for new tissues, it is the
center of a region of rapid growth. In sections it may be observed that the
adjacent mesoderm is full of dividing cells, and as we have seen, cells from
this region are continually pushed forward to give rise to somites anterior
to the primitive streak, so that much of the increase in length of the embryo
is due to growth in this region. We have already mentioned the head fold as
a growth center. There are thus two regions of particularly active growth
in the developing embryo, the primitive streak and the head fold (24). It is
interesting to note one point of contrast between these two; namely, that the
tissues in the head region are well advanced in difTerentiation while the
tissues of the primitive streak region remain relatively undifferentiated.

The coelom. — Coincident with the formation of the somites in the
paraxial mesoderm, the coelom or body cavity develops in the lateral meso-
derm. It is formed by a division of this mesoderm into two layers, a dorsal
or somatic layer adjacent to the ectoderm, and a ventral or splanchnic layer
adjacent to the entoderm. The coelom is the space between the two
(Fig. 17). Because the somatic mesoderm and the ectoderm are closely
associated and undergo many foldings in common, it is convenient to desig-
nate the two layers together by the term somatopleure. For the same
reasons splanchnic mesoderm and entoderm together are designated as
splanchnopleure. It should be noted that the mesoderm and ectoderm of
the somatopleure dorsal to the coelom are continuous with the similar layers
in the amnion. The amnion, therefore, may also be classed as somatopleure.
In like manner the mesoderm and entoderm of the splanchnopleure ventral
to the coelom are continuous with the similar layers in a tissue which bounds
the extra-embryonic coelom laterally. This tissue, therefore, may also be
classed as splanchnopleure.

It has been previously stated that there is a mass of mesoderm in the
mid-sagittal plane anterior to the fore-gut. This extends to right and left,
across the front of the fore-gut and is continuous laterally with the lateral

THE EARLY EMBRYOLOGY OF THE MOUSE 2>2>

sheets of mesoderm. It thus forms the base of a U of which the lateral
mesoderm forms the sides. By about the four somite stage or slightly later,
the coelom extends not only throughout the lateral mesoderm but also as a
passage through this anterior mesoderm (Figs. i6 and 29). The coelom,
also, is thus U-shaped. The whole posterior portion of the coelom opens
laterally into the extra-embryonic coelom (Figs. 17 and 28), The anterior
part of the coelom, on the other hand, forming the base of the U and extend-
ing as far posteriorly as the second somite, is separated from the extra-
embryonic coelom by a partition of mesoderm. Much of this anterior
portion of the coelom becomes the pericardial coelom, enclosing the heart.
The connection between the anterior and the lateral parts of the coelom is
called the pericardial-peritoneal canal (Fig. 28).

The relations of coelom and extra-embryonic coelom can be studied from
the series of sections of a seven somite embryo shown in Figs. 23, A to G.
The reader should note, however, that owing to the rapid development of the
heart between the four and the seven somite stage, the pericardial coelom
is already at this latter stage a more complicated cavity than when it first
appeared.

Reichert's membrane. — The mouse embryo is protected during its
development by three extra-embryonic membranes; namely, Reichert's
membrane, the amnion and the yolk-sac. There is no essential difference
between the amnion of rodents and that of other mammals. Reichert's
membrane, on the other hand, is found only in the Rodentia, while the yolk-
sac in this order has come to have rather unusual relations to other struc-
tures. The chorion, an important fetal membrane in most mammals is
present in the mouse but remains small and, as a protective structure,
unimportant.

To follow the development of Reichert's membrane we must go back to
the 5!^ day stage (Fig. 8). Except in the region of the ectoplacental cone,
the embryo is bounded by the trophcctoderm. This is continuous with the
margins of the cone and is separated from the egg cylinder by the yolk
cavity. Laterally its cells are in close contact with the maternal decidua, a
contact so intimate in fact that in some cases it is impossible to tell whether a
given cell is of embr>'onic or maternal origin. Ventrally it stretches across
the remains of the uterine lumen, now filled with a degenerating mass of
uterine epithelium. On the inner surface of the trophcctoderm are a few
widely separated entoderm cells.

A day later (Fig. 10) these entoderm cells have increased in number and
form a uniform though not quite continuous layer over the inner surface of

BIOLOGY OF THE LABORATORY MOUSE

— Ectoplacental cone
Reichert's membrane

Ectoplacental cavity

Extra-embryonic
coelom
Yolk-sac

Yolk cavity

Amniotic cavity

Embryonic ectoderm
Primitive streak

Allantois

Notochord

Ectoplacenta

Chorion

Yolk-sac
Amnion
Amniotic cavity
Extra-embryonic'coelom

Neural groove Intra-embryonic coelom

Fig. 19. — Frontal sections (X75) showing development of the yolk-sac. A.
7 days 6 hours. B. 7 days 10 hours. C. 8 days 10 hours, 9 somites, through 8th
somite. Reichert's membrane omitted except in \.

THE EARLY EMBRYOLOGY OF THE MOUSE

the trophectoderm. Between the two cell layers there soon begins to appear
a thin, non-cellular, pink-staining membrane called, after the man who first

'Ectoplacenta

Reichert's
membrane

Yolk-sac

Amnion

Exocoelom

Mid -gut

Blood island

Ectoplacenta

Neural tube

Allantois

Exocoelom

Yolk-sac

Reichert's
membrane

Mid-gut Dorsal aorta

Fig. 20. — Frontal sections showing development of extra-embryonic membranes
and formation of mid-gut. The sections are viewed from the head towards the tail,
so that the right side of the embryo is on the left side of the drawing, and vice versa.
.\. 8 days 11 hours, ii somites, through oth somite. Reichert's membrane omitted.
(X60.) B. S days 18 hours, 16 somites, through oth somite. (X45.)

described it, Reichert's membrane. The first signs of it are often visible at
the ventral extremity of the egg where there is apt to be a cluster of entoderm

36 BIOLOGY OF THE LABORATORY MOUSE

cells. Perhaps this indicates that the entoderm cells produce it. In any
case it soon becomes continuous over the entire inner surface of the trophec-
toderm. The fully developed membrane is of uniform thickness and, as can
be demonstrated by dissection, surprisingly tough for so delicate a structure.
Though non-living, it possesses the surprising property of being able to
increase its area to keep pace with the growth of the embryo. Presumably
this capacity for growth is dependent on the entoderm cells which are
distributed at quite regular intervals over its entire inner surface.

The amnion. — The early stages of the development of the amnion have
been described. Owing to the inversion of the germ layers, the amniotic
folds have only a short distance to grow, and amnion formation is conse-
quently precocious in the mouse as compared with most other mammals
(Figs. qB and E). For the same reason, the area of the amnion at first is
small. It expands rapidly, however, to accommodate the growing embryo
and by 8 days it forms a large sac over the embryo's entire dorsal surface
(Figs. 2 2 and 25C). In the later stages of development the embryo floats
free in the amniotic cavity attached only by the umbilical cord.

The yolk-sac. — The mammalian ovum contains virtually no yolk. The
mammals are, however, descended from reptilian ancestors in whose eggs
yolk was abundant, and this long period in their evolutionary history has
left an indelible, impress on mammalian development. Most striking, per-
haps, is the development of a yolk-sac so similar in many details to the
reptilian yolk-sac as to be unmistakably homologous. As is often the case
with vestigial structures, this has been modified in different ways in the
different groups of animals that have inherited it. In the rodents it gives
rise to a membrane enveloping the embryo and possessing the dual function
of protection and, during the middle stages of development, of absorbing
nourishment from the mother.

The yolk cavity of the mouse may be defined as the cavity derived from
the original segmentation cavity or blastocoele and lying between the egg
cylinder and Reichert's membrane (Fig. 19A). The yolk-sac is only a part
of the boundary of this cavity; namely, that middle portion of the egg cylin-
der wall which is composed of mesoderm and entoderm, or in other words,
of extra-embryonic splanchnopleure.*

* It should be noted that in many mammals, e.g., the pig, the allantois as well as
the yolk sac are derived from splanchnopleure. This is not the case in the mouse.
In this species the yolk-sac, as we are using the term, and the extra-embryonic
splanchnopleure are identical.

THE EARLY EMBRYOLOGY OF THE MOUSE 37

At 73<4 days the extra-embryonic splanchnopleure or yolk-sac is a struc-
ture of limited area forming the central or ectoderm free portion of the egg
cylinder wall (Fig. 19A). While small at first, it is an area of rapid growth
and by 8 or 8 V2 days forms an extensive membrane enveloping the amnion
and a greatly enlarged exocoelomic cavity (Figs. 19B and C). The whole
embryo changes its shape in the process, the egg cylinder becoming an ovoid
and the ovoid a sphere. At 8 days the yolk-sac is still attached to the
embryo along a band that runs anterior to the opening of the fore-gut and
posterior to the opening of the hind-gut, so that most of the ventral surface
of the embryo is outside it (Fig. 22). After the mid-gut has formed, how-
ever, this portion of the embryo, too, is enveloped by the yolk-sac (Fig. 20).
The details of this process will be described later.

The blood islands. — Associated with the yolk-sac splanchnopleure in all
species in which it occurs are structures known as the blood islands. These
appear in the mouse at 7)^ days as thickenings in the inner or mesodermal
layer of the yolk-sac about which they form an irregular girdle (Fig. 16).
As the name implies, the blood islands give rise to part of the circulatory
system. The peripheral cells differentiate to form the endothelium of a
system of blood vessels encircling the yolk-sac while the inner cells become
primitive blood corpuscles that circulate in the embryonic blood stream.

Changes in the uterus. — Implantation is accompanied by a rapid growth
of the uterine mucosa adjacent to the implantation site to produce a definite
swelling, the decidual swelling. For a while the uterine crypt containing
the embryo maintains its connection with the uterine cavity, but by about
7}^ days the growth of the decidua has blocked this off so that the cavity
containing the embryo is separated from the main lumen (Fig. 4B). The
bridge of tissue thus formed dorsal to the ectoplacental cone will later become
part of the placenta. Further growth of the decidua constricts and finally,
by about 8 days, completely closes the uterine lumen dorsal to the embryo
except for one or more small isolated chambers (Fig. 21). On each side of
the decidual swelling the uterine lumen remains open, but at this period in
development there is no continuous passage throughout the length of the
uterus. A little later a continuous lumen is reestablished, but the new
kimen is on the opposite side of the decidual swelling from the old, passing
ventral instead of dorsal to the embryo. An early stage in this reestablish-
ment of the lumen, may be seen at about 8 days (Fig. 21). The epithelium
lining the lumen on each side of the decidual swelling has grown in between
the muscle layers and the decidua ventral to the embryo. The extreme
limits of this growth consist of a double but unsplit layer of epithelium. In

BIOLOGY OF THE LABORATORY MOUSE

the slightly older epithelium nearer the lumen the two layers have split so
that two wedgeshaped spaces extend from the lumen between decidua and
muscles on each side of the decidual swelling. In course of time the wedges
penetrating from the two sides meet ventral to the embryo, thus completing
the formation of the new lumen.

Old uterine lumen

Decidua basalis

Mesometrium

Vascular zone

Blood vessel

New uterine
lumen

Giant cell / \ Uterine epithelium

Maternal blood cells Decidua capsularis

Fig. 21. — Longitudinal section (partly diagrammatic) of uterus at site of implantation
of 8 day 6 hour, 5 somite embryo. Cut parallel to mesometrium.

Besides the changes in the uterine lumen there are interesting changes in
the histology of the decidua. Starting as a relatively homogeneous tissue,
dififerent zones differentiate within it, each with its characteristic structure.
As many as six zones have been distinguished (7,7,), but for our purposes it
will suffice to note no more than three (Fig. 21). Ventrally there is an
antimesometrial zone or decidua capsularis charactferized by large bi-, tri- or
tetra-nucleate cells.* The individual nuclei in this zone as well as the cells

I * In the rat this region is characterized by bi-nucleate cells. Krehbiel (33) states
that more than two nuclei do not occur.

THE EARLY EMBRYOLOGY OF THE MOUSE 39

are larger than elsewhere in the decidua, and this together with the grouping
of the nuclei gives the zone a ver>^ characteristic appearance. It will be
noted that it lies between the embryo and the new uterine lumen. With the
growth of the embryo it becomes stretched until, in the later part of the
gestation period, it is hardly more than a thin membrane separating embryo
and lumen. Dorsally there is a mesometrial zone, or decidua basalis. whose
cells at 8 days still closely resemble those of the unaltered mucosa. It later
contributes to the formation of the placenta. Between the antimesometrial
and mesometrial zones is an intermediate or vascular zone characterized
by the presence of numerous irregular endothelial-lined blood spaces or
sinusoids. Its cells tend to be multi-nucleate like those of the decidua
capsularis.

The nourishment of the embryo. — The source from which the embryo
derives its nourishment during its earliest growth period is somewhat uncer-
tain, but it is not unlikely that the degenerating cells of the uterine epithe-
lium that originally lined the implantation chamber serve as a source of food.
The epithelium is sloughed ofif and begins to undergo degenerative changes at
just about the same time that the first real increase in size of the embryo is to
be noted. At the mesometrial pole of the embryo at 4^2 days may be seen
droplets of secretion that contain perhaps an enzyme concerned with the
digestion of the epithelial cells (Fig. 6). This stage in the nourishment
of the embryo is brief; by 5I2 days only a remnant of the epithelial cells
remains (Fig. 8).

At the same time a new source of nourishment makes its appearance. It
has already been stated that the intermediate zone of the decidua contains
numerous blood-filled sinusoids. At 5^2 days these begin to rupture, pour-
ing their contents into the lumen surrounding the embryo. In a very short
time the embryo is completely bathed in maternal blood. It has recently
been shown that this blood is not stagnant as was once supposed, but that it
remains a part of the maternal circulation. In certain experiments with the
rat it was found that there is a complete replacement every twenty minutes

(13).

The maternal blood is separated from the embryo proper by Reichert's
membrane, the yolk cavity and, in later stages, by the yolk-sac. Reichert's
membrane probably plays an entirely passive role in the transportation of
nutrient substances from the maternal blood to the embryo, acting simply as
a semi-permeable membrane. The yolk-sac, on the other hand, probably
actively absorbs the food material. This is particularly true after the blood
islands which girdle the yolk-sac have developed into a capillary network and

40 BIOLOGY OF THE LABORATORY MOUSE

after the embryonic circulation is established. By the time this occurs the
yolk-sac has become pressed against and partly fused with Reichert's mem-
brane, obHterating the yolk cavity (Fig. 20B). The embryonic yolk-sac
circulation is thus brought very close to the maternal circulation, and the
yolk-sac is established as "an organ of exchange whose importance is not
secondary to that of the allantoic placenta" (13). It is interesting to
observe that in the rodents the yolk-sac has thus recovered in full measure
the role as an organ of absorption which it possessed in the reptiles, with,
however, the important difference that the material absorbed comes from the
maternal blood instead of from yolk deposited within the egg.

The sinusoids in the intermediate zone of the decidua extend from the
decidual cavity containing the embryo clear to the periphery of the decidual
swelling where this borders on the uterine lumen. Beginning at about 7 3^^
or 8 days there is bleeding into the uterus from these peripheral sinusoids
(64, 66). At about 10 days some of this blood finds its way into the vagina,
persisting there for 3 or 4 days (57). It is a convenient early sign of
pregnancy.

In the later stages of development the decidua basalis, the ectoplacental
cone, the chorion, and parts of the allantois fuse to give rise to a true
placenta which thereupon assumes a major role in transferring nutritive
material to the embryo.

The giant cells. — A conspicuous feature in sections of mouse embryos of
6 to 14 days is the presence of certain remarkably large cells lying between
Reichert's membrane and the decidua. These are the so-called giant cells
(Fig. 21). Because of the early and close fusion of embryonic and maternal
tissues in the region which the giant cells later occupy, their origin is difficult
to determine and has been the subject of extended debate (3, 22, 48 and
others). Some authors believe that they are derived from the decidua,
others that they come from the trophectoderm. Their function likewise has
been the subject of much discussion. Our own observations, briefly pre-
sented below, seem to us to be fairly conclusive on a few points, but to leave
others still in doubt.

It is convenient to distinguish three types of giant cells. The first large
and unmistakable giant cells to appear are at the ventral extremity of the
embryo (Figs. 6 and 8). They are quite evidently derived from the trophec-
toderm. Already quite large at 51^ days, they become, relatively speaking,
enormous by 7 days at which time they have penetrated for some distance
into the remains of the implantation cavity ventral to the embryo. These
are primary giant cells. The trophectoderm cells lateral to the egg cylinder

THE EARLY EMBRYOLOGY OF THE MOUSE 41

probably also give rise to similar though somewhat smaller primary giant
cells.

A second and much more numerous group of giant cells is quite probably
derived from the ectoplacental cone. Already at 5 days cells may be seen
growing down outside the trophectoderm from the region of the future cone
(Fig. 7). Later, when the embryo is surrounded by maternal blood, these
become long strands of cells extending down, within the blood or along the
inner surface of the decidua, from the cone towards the ventral extremity
of the egg cylinder." At first small, these cells increase in size and at 8 days
form a loose meshwork of large cells whose long protoplasmic processes
extend across the blood filled space between Reichert's membrane and the
decidua (Fig. 21). Other similar cells may be seen adjacent to the ecto-
placental cone. These are the secondary giant cells. At 8 days their
continuity with the cells of the ectoplacental cone is still quite obvious.
While this is the probable origin of the majority of the giant cells, the
possibility that at least some of them are derived from the decidua is not
ruled out. It should be pointed out that the division between primary and
secondary giant cells is partly arbitrary; the trophectoderm and the ecto-
placental cone are continuous structures, and cells from near the line of junc-
tion might be said to give rise to either type. One obvious function of the
giant cells is to anchor Reichert's membrane to the decidua. They quite
probably have other functions also, but what they are is uncertain.

The third class of giant cells consists of the so-called symplasia. These
cells, individually conspicuous but never very numerous, are multinucleate
cells first appearing in the decidua adjacent to the embryo at 7 or 73^^ days.
The number of nuclei per cell is extraordinary, mounting into the dozens by
8 days. The nuclei are dark staining and closely packed. The origin and
function of the symplasia is uncertain.

The seven somite embryo. — In embryos from genetically vigorous stock,
the seven somite stage is reached at about 8 days. As thereafter the embryo
begins a series of important changes, it will be useful to review here the
development attained at this point (Figs. 22 and 23). In sagittal section
the embryo is seen to form a letter S (facing to the left in Fig. 22) with the
head region convex, the trunk region concave towards the dorsal surface.
In transverse section, whereas the embryo was formerly conspicuously
cup-shaped with the ectoderm on the inside, it has now flattened out, in fact
in the regions of the fore- and hind-gut the entoderm has become the inner
layer. The neural groove, deep and well developed, is still open dorsally
though in the mid-trunk region the walls are quite close together. Cephalad,

BIOLOGY OF THE LABORATORY MOUSE

precocious growth of certain parts of the neural groove ectoderm indicates
early differentiation of the brain. The hind-gut is small, but there is a deep
fore-gut, and the heart, just anterior to the fore-gut, is a conspicuous struc-
ture. No mid-gut has formed. The allantois has almost reached the
chorion, in fact in some embryos at this stage has already reached and fused

Blood islands

Exocoelom

Allantois

Pericardial
coelom

Epi-myocardium

Fore-gut

Neural fold

Omphalomesen-
teric artery

Hind -gut

Amniotic cavity

Splanchnopleure

Somatopleure

Undivided
mesoderm

7th Somite

Fig. 22. — Drawing of reconstruction of 8 day i hour, 7 somite embryo. The
reconstruction is cut in the mid-sagittal plane and only the right half shown except
at the ventral extremity where the last 4 somites and part of the undivided mesoderm
of the left side are included. Cut areas are shown by horizontal shading. ( X 75.)

with it. The amnion, and the yolk-sac plus the chorion, form a double,
arched roof over the whole dorsal surface of the embryo. The blood islands
appear as a conspicuous hummocky band around the inner surface of the
yolk-sac. Within the embryo blood vessels have begun to form.

The tail fold. — The hind-gut, though much later to appear than the
foregut, soon overtakes it in development. In ten somite embryos the two
are of approximately equal size (Fig. 31). A necessary concomitant of
hind-gut growth is the appearance of a tail fold; the gut entoderm pushes the
overlying ectoderm and mesoderm ahead of it away from the yolk-sac wall.

THE EARLY EMBRYOLOGY OF THE MOUSE 43

Endocardium

Exocoelom Amnion
\

Dorsal aorta

Sinus venosus

Coelom'

Fore -gut

Endocardium

Pericardial
coelom

Epi-myocardium

Neural groove

Primitive streak
Amnion

Hind-gut

Exocoelom

Omphalomesenteric artery
Epi-myocardium -^ u

Fig ^, -Sections transverse to neural groove of 8 day i hour. 7 somite embryo.
.^1 except E are from the embryo shown in figure 22. The location of each section
is indicated on the key diagram. (X90.)

44 BIOLOGY OF THE LABORATORY MOUSE

A beginning of this process can be seen in six somite embryos (Fig. i5), and
in ten somite embryos the tail fold is well developed (Figs. 26 A and 27).
The process is strictly comparable to the formation of the head fold except
for one interesting difference; whereas the head fold lies entirely within the
amniotic cavity, the tail fold lies only partly within it. The ventral surface
of the tail fold is in the exocoelom. This is because in its growth away from
the yolk-sac it pushes the base of the allantois and the adjacent margin of the
amnion ahead of it. The amnion remains attached to its caudal and lateral
walls, and only its dorsal surface is within the amniotic cavity (Fig. 24).

The turning of the embryo. — Almost immediately after the seven somite
stage the embryo begins a process of turning which results in a reversal of
the curvature of the whole trunk region. Thus instead of being S-shaped
the embryo becomes C-shaped with the ventral surface everywhere on the
inside of the C. The turning begins in the head and tail folds, and consists
of a rotation of each along its long axis, or in other words, on axes parallel to
the fore- and hind-guts (Figs. 24-28). Viewing each fold from its cephahc
toward its caudal end, the direction of rotation is clockwise in each case.
Of course, both folds cannot be viewed in this direction from any one point,
because of the curvature of the embryo. Viewed from the mesometrial
pole, in sections the turning of the head fold appears to be clockwise, of the
tail fold counter-clockwise (Fig. 24).

At first the turning is confined to the head and tail folds; the mid-trunk
region, still firmly attached to the yolk-sac, remains in its original position.
At about 8^^ days, and at about the eleven or twelve somite stage, the mid-
trunk region turns also. The process is sudden. Transverse sections of the
trunk region at about this period show it to be either turned or not turned
(Figs. 19C and 20A). It is quite possible that after the growth of the head
and tail folds reduces sufficiently the attachment of the trunk region to the
yolk-sac, this region snaps over like a spring whose tension has come to
exceed the forces holding it. Some time elapses after the turning of the mid-
trunk before the head and tail regions complete their rotation, which even-
tually amounts to a full 180°. Essentially, however, by about 9 days the
embryo has become concave towards the ventral surface (Figs. 26B and C).

The mid -gut. — The turning of the mid-trunk region automatically results
in the formation of the mid-gut. Prior to turning, the two sheets of embry-
onic splanchnopleure in the mid-trunk region extend straight out from the
sides of the embryo, forming a virtually plain surface (Fig. 19C). There is
thus no indication of a mid-gut. When the mid-trunk region turns suddenly
towards its left side, the two sheets are pulled after it, forming between them

THE EARLY EMBRYOLOGY OF THE MOUSE

a groove which is continuous anteriorly and posteriorly with the fore- and
hind-guts. This groove is the mid-gut (Fig. 20A). The two sheets of
splanchnopleure rapidly draw closer together (Fig. 20B), and at the nineteen
somite stage, which may be reached as early as 8^4 days, have fused distally
to form a closed tube.

The heart. — It will be remembered that in 7)2 day embryos there is a
small region of mesoderm anterior to the fore-gut (Fig. 15). This forms the
base of a U of which the two lateral sheets of mesoderm form the sides.
Within this U the coelom develops and is, therefore, itself U-shaped. The
base of the U, and the two sides approximately as far caudad as the second

Fig. 24. — Transverse sections showing the turning of the embryo. A. 8 days i hour,
7 somites. B. 8 days 10 hours, 10 somites. (X50.)

pair of somites, contain that portion of the coelom which ultimately encloses
the heart and which, therefore, is known as the pericardial coelom (Fig. 22).
The curved shape of the pericardial coelom in cross section in Fig. 22
should not be confused with the U-shape of the pericardial coelom as a whole.
The heart is derived from the splanchnic mesoderm which forms the
ventral wall of the pericardial coelom (Fig. 29). In five somite embryos this
mesoderm has differentiated into two layers. Adjacent to the pericardial
coelom is a thick, continuous layer, known as the epi-myocardium because
it will give rise both to the heavy muscular layer of the heart wall (myo-
cardium) and to its outer covering (epicardium). Between the epi-myo-
cardium and the underlying entoderm are a number of irregular cavities
which later fuse to form the cavity of the heart. The lining of these cavities
is the endocardium.

BIOLOGY OF THE LABORATORY MOUSE

Placenta

Allantois

Neural groove

Yolk-sac

Anterior intestinal
portal

Neural groove
(anterior, open)

Somites

Fig. 25. — Photographs (X25) of mouse embryos. A. Lateral view of 7 day 18
hour, 6 somite embryo, with decidua and most of yolk-sac dissected. B. Dorsal view
of same embryo, amnion also dissected. C. 10 somite embryo, age Q-9^2 days.
Slightly retouched. Embryo from inbred stock. D. Same embryo as C, dorsal view,
amnion removed.

riJE EARLY EMBRYOLOGY OE THE MOUSE

AUantois

Neural groove of
tail fold

Yolk-sac

Placenta

Neural groove
(posterior, open)

Yolk-sac

Heart

Coelom

Mid-gut

Fig. 26. — Photographs (X25) of mouse embryos. A. 13 somite embryo, age
QJ-i^Q^i days, from inbred stock. B and C. 14 somite embryo, age 8 days 22 hours.
Note the greater degree of turning of this embryo, particularly in the mid-trunk
region, as compared with the one in A.

BIOLOGY OF THE LABORATORY MOUSE

Because of its relation to the U-shaped pericardial coelom, the heart is
itself a U-shaped structure at this stage* with the base of the U lying just
cephalad to the anterior intestinal portal (Fig. 30). As the intestinal portal

^^■•9':

Fig. 27. — Stereoscopic photograph (Xio) of rat embryo, age 10^:1 days. {From

Long and Burl inflame.)

105 .ujv 235 x20 i..id„v, 23a x20

Fig. 28. — Stereoscopic photograph (X20) of rat embryo, age lo^:^ days. Same

embryo as Fig. 27, more completely dissected. (From Long and Biirlingamc.)

moves caudad due to the ''zipper action'' which causes the progressive fold-
ing together and fusion in the mid- ventral line of the entoderm which bounds

* In many vertebrates the heart originates as two entirely distinct primordia which
later fuse. As has been clearly shown by Goss (16) and by Burlingame and Long (8),
this is not the case in the rat. Our observations indicate that the condition in the
mouse corresponds closely to that in the rat.

THE EARLY EMBRYOLOGY OF THE MOUSE

Somatic mesoderm Neural groove

Amnion

Exocoelom

Epi-myocardium
(Splanchnic mesoderm)

Endocardium

Entoderm

Coelom

Fig. 29. — Slightly diagonal transverse section through median endocardial pri-
mordium (see Fig. 30), just cephalad to fore-gut. Embryo of 8 days 6 hours, 5 somites.
Projection drawing (X150).

Forebrain

Cut edge of entoderm

Median endocardial
primordium

Aortic arch I

Atrium

Anterior intestinal portal

Fig. 30. — Diagram of the fore-gut region viewed from the ventral surface, showing
distribution of the endocardium. Endocardial tissue is represented by horizontal
lines. Rat embryo of 9 days 16 hours, 3 somites. {Modified after Goss.)

BIOLOGY OF THE LABORATORY MOUSE

it, the sides of the U are Ukewise brought into approximation and fused
together in the mid-ventral Une. The endocardium is thus transformed
from a U-shaped structure into a single tube. At the three somite stage (in
the rat) the different regions of the heart are not clearly set apart, though a
slight constriction serves to mark the boundary between the atrium and the
ventricle. As a result of subsequent foldings of the endocardial tube the
different regions of the heart are clearly differentiated (Fig. 31).

Internal carotid artery Aortic arch I Allantois Omphalomesenteric artery

Atrium
Omphalomesenteric vein

Vitelline
arteries

Yolk-sac

Fore-gut

Dorsal aorta Hind-gut

Fig. 31. — Diagram of the circulatory system in an 8 day 10 hour, 10 somite
embryo. The head and tail folds of this embryo have begun to turn but there is as
yet no turning in the mid-trunk region. Traces of the allantoic veins are present
but are not shown as they do not yet form a continuous channel. (X64.)

Blood vessels. — In ten somite embryos, still in the process of turning, a
number of blood vessels have become established (Fig. 31). The dorsal
aorta at this stage is a paired vessel running the length of the trunk. It
connects anteriorly with the heart by way of the aortic arches and the ventral
aorta. Posteriorly its two halves fuse at the caudal extremity of the hind-
gut to form the single, median, omphalomesenteric artery. This runs
cephalad for a short distance ventral to the hind-gut and then turns away
from the embryo towards the inner surface of the yolk-sac on which it
spreads out into a network of capillaries. These capillaries are derived from

THE EARLY EMBRYOLOGY OF THE MOUSE 51

the blood islands. At this stage actual blood channels have not appeared in
most of the blood islands, but when these are established, a capillary network
is formed encircling the yolk-sac. Blood is collected from this network
anteriorly by the paired, omphalomesenteric veins which convey it back to
the heart. When the heart starts beating, this system of blood vessels
provides a generous circulation through the yolk-sac which serves at this
time as the principal organ for the procurement of food from the mother.

Change in shape of the yolk-sac. — When the embryo starts turning, the
yolk-sac and ectoplacental cone form a slightly flattened sphere (Fig. 19C).
When turning has been completed, these bounding structures of the embryo
shortly assume the form of a slightly saucered-out hemisphere (Fig. 20B).
The ectoplacental cone becomes flattened and then dorsally concave, and the
yolk-sac adjacent to the cone pushes outward into the porous, blood-filled
vascular zone of the decidua. The embryo meantime, still attached to the
yolk-sac by the walls of the mid-gut, tips over so that it lies with its left side
adjacent to the yolk-sac, its right side facing the placenta.

BIBLIOGRAPHY

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10. Danforth, C. H., and S. B. dk Aberle. 1927. Distribution of foetuses in the
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52 BIOLOGY OF THE LABORATORY MOUSE

11. Defrise, a. 1933. Some observations on living eggs and blastulae of the
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12. Enzmann, E. V. 1935. Intrauterine growth of albino mice in normal and in
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13. Everett, J. W. 1935. Morphological and physiological studies of the placenta
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14. Fekete, E., O. Bartholomew and G. D. Snell. 1940. A technique for the
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15. Gerard, P. 1925. Recherches morphologiques at experimentales sur la vesicule
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17. Goss, C. M. 1940. First contractions of the heart without cytological differen-
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18. Grosser, O. 1927. Fruhentwicklung, Eihautbildung und I'lacentation des
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19. Gresson, R. a. R. 1940. Presence of the sperm middle-piece in the fertilized
egg of the mouse (Mus musculus). Nature 145: 425.

20. Hall, B. V. 1935. The reactions of rat and mouse eggs to hydrogen ions. Proc.
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21. Henneberg, B. 1937. Normentafel zur Entwicklungsgeschichte der Wander-
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22. Huber, G. C. 1915. The development of the albino rat, Mus Norvegicus
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the first to the end of the ninth day. J. Morph. 26: 1-114.

23. Hl^er, G. C. 1918. On the anlage and morphogenesis of the chorda dorsahs in
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24. Hyman, L. H. 1927. The metabolic gradients of vertebrate embryos. III.
The chick. Biol. Bull. 32: 1-38.

25. Johnson, M. L. 1933. The time and order of appearance of ossification centers
in the albino mouse. Am. J. Anat. 52: 241-272.

26. Jolly, J., and M. Ferester-Tadie. 1936. Recherches sur I'oeuf du rat et de la
souris. Arch. anat. micr. ^,2: 323-390.

27. Jolly, J., and C. Lieure. 1938. Le bourgeon caudal et la chorde dans les
cultures de I'oeuf du rat. Compt. rend. Soc. biol. 127: 595-598.

28. Jolly, J., and C. Lieure. 1938. La formation des premieres ebauches vascu-
laires dans I'embryon des mammiferes. Compt. rend. Soc. biol. 128: 252-255.

29. Jolly, J., and C. Lieure. 1938. La culture de I'allantoide des mammiferes et
la formation des ebauches vasculaires independantes. Compt. rend. Soc. biol.
128: 342-345-

30. Jolly, J., and C. Lieure. 1938. Sur les membranes qui enveloppent I'oeuf du
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31. Kingsbury, B. F. ig2o. The developmental origin of the notochord. Science
51: 190-193.

32. Kingsbury, B. F. 1924. The significance of the so-called law of cephalo-caudal
differential growth. Anat. Rec. 27: 305-321.

THE EARLY EMBRYOLOGY OF THE MOUSE 53

33. Krehbiel, R. H. 1937. Cytological studies of the decidual reaction in the rat
during early pregnancy and the production of deciduomata. Physiol. Zool. 10:
212-234.

34. Lams, H., and J. Doorme. 1907. Nouvelles recherches sur la maturation et la
fecondation de I'oeuf des Mammiferes. Arch. biol. 23: 259-365.

^5. Lewis, W. H., AND E. S. Wright. 1935. On the early development of the mouse
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36. LiTWER, G. 1928. tjber die Sekretion und Resorption in den Dotterentoderm-
zellen bei graviden weissen Miiusen. Z. Zellforsch. u. mikr. Anat. 8: 135-152

37. Long, J. A. 191 2. The living eggs of rats and mice with a description of appa-
ratus for obtaining and observing them. Univ. Cal. Pub. Zool. 9: 105-133.

38. Long, J. A., and P. L. Burlingame. 1938. The development of the external
form of the rat, with observations on the origin of the extraembryonic coelom and
fetal membranes. Univ. Cal. Press, Berkeley.

39. Long, J. A. A stereoscopic atlas of the rat embryo. Cal. Lab. Supply Co., Los
Angeles.

40. MacDowell, E. C, E. Allen and C. G. MacDowell. 1927. The prenatal
growth of the mouse. J. Gen. Physiol, i: 57-70.

41. Melissinos, K. 1907. Die Entwicklung des Eies der Mause von den ersten
Furchungs-Phanomenen bis zur Festsetzung der AUantois an der Ectoplacentar-
platte. Arch. mikr. Anat. 70: 577-628.

42. MossMAN, H. W. 1926. Comparative morphogenesis of the fetal membranes
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43. jNIossman, H. W., and F. L. Hisaw. 1940. The fetal membranes of the pocket
gopher, illustrating an intermediate type of rodent membrane formation. I.
From the unfertilized tubal egg to the beginning of the allantois. Am. J. Anat.
66: 367-391.

44. MossMAN, H. W., AND L. A. Weisfeldt. 1939. The fetal membranes of a primi-
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45. Nicholas, J. S. 1935. Thenormalstagesin the development of the rat. Photo-
static volume prepared at Osborn Zool. Lab., New Haven.

46. Nicholas, J. S., and D. Rudnick. 1938. Development of rat embryos of egg-
cylinder to head-fold stages in plasma cultures. J. Exp. Zool. 78: 205-232.

47. Nielson, p. E. 1940. The fetal membranes of the kangaroo rat, Dipodomys,
with a consideration of the phylogeny of the Geomyoidea. Anat. Rec. 77: 103-

-127.

48. Parodi, S. E., ANT) V. Wldakowich. 1920. El problema de la gastrulacion en
los mamiferos que presentan la inversion de las hojas blastodermicas. Contri-
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Hist. Nat. Buenos Aires 30: 445-500.

49. Patten, B. ^L 1931. The embryology of the pig. The Blakiston Company,
Philadelphia.

50. PiNCus, G. 1936. The eggs of mammals. The jNIacmillan Co., N.Y.

51. Prentiss, C. W., and L. B. Arev. 1918. A laboratory manual and text-book
of embryology. W. B. Saunders Co., Philadelphia.

54 BIOLOGY OF THE LABORATORY MOUSE

52. Ravn, E. 1894. Ueber die Arteria omphalo-mesenterica der Ratten und Mause.
Anat. Anz. 9: 420-424.

53. Ravn, E. 1894. Zur Entwickelung des Nabelstranges der weissen Maus.
Arch, Anat. u. Physiol., Anat. Abt. 293-312.

54. Ravn, E. 1895. tjber das Proamnion, besonders bei der Maus. Arch. Anat. u.
Physiol., Anat. Abt. 189-224.

55. Rietschel, p. E. 1929. Zur Morphologic der Genitalausfuhrungsgange im
Individualcyclus der weissen Maus. Z. wissensch. Zool. 135: 428-494.

56. Rogers, W. M. 1929. The development of the pharynx and the pharyngeal
derivatives in the white rat (Mus norvegicus albinus). Am. J. Anat. 44: 283-329.

57. Sato, K. 1936. Uber die Entwicklungsgeschichte des Mauseeies (I. Mitteilung).
Die intratubare Entwicklung deselben. Okayama-Igakkai-Zasshi 48: 423-441.

58. Sato, K. 1936. Uber die Entwicklungsgeschichte des Mauseeies (II. Mit-
teilung). Die intrauterine Entwicklung derselben, besonders der Entstehungs-
mechanismus des Amnions. Okayama-Igakkai-Zasshi 48: 792-832.

59. SiMKiNS, C. S. 1923. Origin and migration of the so-called primordial germ
cells in the mouse and rat. Acta Zool. 4: 241-278.

60. SoBOTTA, J. 1895. Die Befruchtung und Furchung des Eies der Maus. Arch,
mikr. Anat. 45: 15-93.

61. SoBOTTA, J. 1903. Die Entwicklung des Eies der Maus vom Schlusse der
Furchungsperiode bis zum x\uftreten der Amniosfalten. Arch. mikr. Anat.
61: 274-330.

62. SoBOTTA, J. 191 1. Die Entwicklung des Eies der Maus vom ersten Auftreten
des Mesoderms an bis zen Ausbildung der Embryonalanlage und dem Auftreten
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-63. SoBOTTA, J., AND G. BuRCKHARD. 1910. Reifung und Befruchtung des Eies der
weissen Ratte. Anat. Kefte 42: 433-497.

64. Stafford, E. S. 1930. The origin of the blood of the "placental sign." Anat.
Rec. 47: 43-57-

65. Thomas, L. J. 1926. Ossification centers in the petrosal bone of the mouse.
Anat. Rec. ^s: 59-68.

66. Venable, J. W. 1939. Intra-uterine bleeding in the pregnant albino rat. The
"placental sign." Anat. Rec. 74: 273-293.

67. Weiss, A. 1901. Die Entwicklung der Wirbelsaule der weissen Ratte, besonders
der vordersten Halswirbel. Z. wissensch. Zool. 69: 492-532.

68. WiDAKOWiCH, V. 1909. tJber die erste Bildung der Korperform bei Entypie des
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94: 240-298.

69.)ZuCKERKANDL, E. 1903. Die Entwicklung der Schilddriise und der Thymus bei
der Ratte. Anat. Hefte 21: 1-28.

Cnapter 2

REPRODUCTION

Bv George D. Snell, Roscoe B. Jackson Memorial Laboratory.

The vaginal plug, 55. Gestation, 55. Litter size, 56. Sex ratio, 57. Postnatal
development, 58. Ovogenesis, 59. Ovarian regeneration, 64. The estrous cycle, 65.
Ovulation, 76. Maturation and fertilization, 77. The transport of sperm and eggs,
78. Pseudopregnancy, 78. Corpora lutea, 80. Lactation, 81. Bibliography, 82.

Since the processes of reproduction are very similar in both mouse and
rat, the following discussion includes data from both species. Where no
mention is made of the species, it may be assumed that the mouse is the
animal referred to. Certain phases of reproduction in the mouse and rat are
dealt with much more thoroughly than others. The references listed below
contain important material not covered in this chapter.

Anatomy of the male and female reproductive systems : Chapter 3 of this
book.

Endocrines and reproduction: Parkes (102), Allen (6), Young (137).

Viabilitv and transport of spermatozoa: Chapter by Hartman, in Allen

Spermatogenesis : Hays (67) ; see also references in Chapter 3 of this book.

The vaginal plug. — Copulation in the mouse and rat is accompanied by
the formation of the vaginal plug, the presence of which is thus a convenient
sign that mating has occurred. The plug is formed by a mixture of the secre-
tions of the vesicular glands and the coagulating glands of the male (134, see
also p. 137), and in the mouse usually fills the vagina from the cervical canal
to the vulva, from which it may even protrude. Occasionally smaller, less
conspicuous plugs are formed, a condition particularly common in the case of
matings at the first pok partum estrus. Plugs in the mouse usually persist
for 18 to 24 hours, occasionally for as long as 48 hours, after which they are
sufficiently loosened, probably as the result of leukocytic action, to fall out
almost entire (100).

Gestation. — The gestation period in the non-suckling mouse is usually
iQ or 20 days,. (36, 73,. 97, lOoV The frequency distribution of gestation
periods of dift"erent lengths for t^yo inbred strains of mice is given in Table i.

BIOLOGY OF THE LABORATORY MOUSE

ijybrid stocks tend to have shorter gestation periods than inbred stocks.
The normal gestation period in the rat is shghtly longer than in the mouse,
ranging from 21 to 23 days (69).

Table i
Length of Gestation Period in the C57 Black and dba Strains (Fekete,

Unpublished Data)

Days

Number of Litters

C57 Black

dba

Birth of litters most commonly occurs at night (92). There is a decided
maximum in the number of births between midnight and 4 A.M., but
parturition. between 4 P.M. and midnight is not uncommon. Altogether, of
164 timedlDirths, two thirds occurred between 4 P.M. and 4 A.M.

An estrus occurs about 20 hours after parturition, and while fertile
matings at this time are not common in some stocks of mice (35) unless the
newly arrived Htter is killed at birth, they occur quite regularly in other
stocks. Thus lactation and gestation may proceed simultaneously. Under
these circumstances the gestation period is lengthened, the extent of the
lengthening being slightly correlated with the number of suckHng young
With only one or two young suckling, the prolongation does not exceed 7
days, with three or more young suckling prolongations up to 12 or 13 days
are not uncommon. The maximum recorded is 16 days (20, 47, 51, 136).
Kirkham (73) has shown that the prolongation is due to a delay in implanta-
tion, which, instead of occurring during the fifth day post coitus as normally,
occurs on some later day, the embryos meantime lying free in the uterus.
Mating may occur during pregnancy (^s)^ but that such matings are accom-
panied by ovulation is open to doubt.

Litter size. — ^Litter size differs greatly with the strain, with the age and
condition of the mother, and with order of the litter. Bittner (10) gives the
data reproduced in Table 2 which shows the relation between order of litter
and litter size for the highly inbred A strain.

REPRODUCTION

Many hybrid animals produce litters considerably larger than those
produced by the A strain. Griineberg (6i) reports taking 19 healthy
embryos just short of term from one hybrid female. Gates (56) reports an
average size of 7.4 with a range of 2 to 12 for 106 litters in a random bred
strain. This is fairly typical for many strains.

Table 2
Order of Litter and Litter Size in A Strain Mice (From Bittner)

No. of Litter

Mean Litter Size

I St

5. 13 + 0.08

2nd

6.35 ± 0.09

3rd

6.46 + 0.09

4th

6.21 + 0. 10

5th

5-53 ± on

6th

4.62 + 0. 13

7th

4.01 ± 0. 14

8th

3.50 ± 0-34

Total

5.68 ± 0.04

The number of corpora lutea formed at the time of the last mating is,
with possible rare exceptions, identical with the number of eggs ovulated.
This number is quite highly correlated with parity (order of litter) and with
weight of the mother, but only slightly correlated with age (88). It may be
used as an index of pre-natal mortahty. MacDowell (86) finds that 33.9 per
cent of the ova that come to maturity are not represented by living young at
birth. This is an average figure based on results from several strains; there
are considerable strain differences. Thus the dba strain shows a higher pre-
natal mortality than the C57 black strain (Fekete, unpublished data).

There is evidence that mouse ova may split to produce uniovular twins,
and that these may come to term, but the phenomenon is certainly rare
(15, 27, 59, 109, 129).

Sex ratio. — According to genetic theory, males produce equal numbers of
male-producing and female-producing sperm, so that, except for a possible
dift'erence in functional capacity of the two types of sperm, or a possible
selective effect of prenatal mortahty, the sex ratio at birth should be 1:1.
MacDowell and Lord (89) have recorded the sex of 106 litters of mice in
which the number born was no less than the number of corpora lutea, and
hence in which prenatal mortahty is ruled out. Their count showed

BIOLOGY OF THE LABORATORY MOUSE

261 males and 262 females, an almost exact 1:1 ratio. MacDowell and
Lord (90) also present evidence that there is no continuous selective elimina-
tion of one sex or the other before birth.

An alteration in the sex ratio through excessive breeding of the fathers
and through treatment of the fathers with alcohol has been both claimed and
denied (28, 57, 87, 104 and others). An effect through injection of the uterus
with sodium bicarbonate before breeding has also been claimed (14), and
there is evidence that diet may effect the ratio (11).

Postnatal development. — Mice are born hairless, except for short
vibrissae, and with eyes and ears shut. Sex can be distinguished at birth:
males have the larger genital papilla, and there is a greater distance between
this and the anus in males than in females. At nine days females show five
pairs of conspicuous nipples, though these tend to be obscured in a few days
by the lengthening fur. The external ears have opened by three days. A
well developed coat is present at two weeks. At twelve to fourteen days a
number of interesting changes occur. There is a break in the growth curve,
the eyes open, the external ears commence a rapid growth, the first jnoult_
begins, the larger follicles in the ovary develop an antrum, there is an
increase in muscular activity. At about the same time the young mice eat
their first solid food.

Table 3
Data Indicating the Age at Which Maturity Is Reached by Fem.4les in

Two Different Stocks

Stock

Mean Age
at First
Estrus

Per Cent of Cases
in Which First

Mating Occurred
at First Estrus

Per Cent of Preg-
nancies Resulting

when First Mating

Occurred at First

Estrus

Per Cent of Preg-
nancies Resulting
from Matings in
Mature Mice

Albinos
Colored

39 days
52 days

75%
85%

48%

47%

80-90%
80-90%

In young mice the vagina is closed by a membrane. The age at opening
varies considerably both within and between stocks. In one series of 100
mice the age at opening ranged from 28 to 49 days with the median at 35
days (45). The first estrus as indicated by cornification of the vagina occurs
soon after vaginal introitus. In one set of observations the interval was 24
to 120 hours (96). However, estrus, in the sense of willingness to mate.

REPRODUCTION 59

probably does not always occur at this time. Data on the occurrence of the
first estrus and the first mating have been pubHshed by Mirskaia and Crew
(95, 96) and are summarized in Table 3.

As this table shows, the time of the first fertile mating varies greatly.
Commonly it occurs_at sevento ten weeks. Thirty-nine days is exception-
ally early. Maturity in males occurs at about the same time as in females,
or perhaps somewhat later./

The useful breeding period of most inbred females terminates when they
reach ten or twelve months of age, for though litters may continue to be
produced after this, breeding is apt to be irregular and the litters small.
Hybrid females usually give fair sized litters and breed regularly until
sixteen or eighteen months of age. Males will usually breed several months
longer than females of the same stocky

Occasionally mice live to^hree years of age or even a few months past
this. ^^

Ovogenesis. — The problem of the origin of the female germ cells has been
the subject of extensive study. The following description is based on the
most important recent papers deaHng with ovogenesis in mice and rats and
does not present all the conflicting viewpoints found in much of the older and
some of the more recent literature. All statements are based on work with
the mouse unless otherwise specified. Investigations in this field have been
ably reviewed by Heys (67) and Pincus (108).

Beginning at about nine (18) to eleven (72) days post coitus, the gonads
of mouse embryos contain so-called primordial or primitive germ cells, char-
acterized by their large size and by the clear appearance of the cytoplasm.
These are present in both male and female gonads which at this early stage
are indistinguishable. At this same time or slightly later, cells closely
similar in appearance may be seen in tissues adjoining the gonads (18), a fact
that has led to extensive speculation as to their place of origin and possible
migrations. The view, at one time commonly held, that they migrate into
the ovary and there give rise to the germ cells is not supported by recent
evidence. The young primordial ova show numerous mitoses, though these
soon cease. Proliferation of ova from the germinal epithelium, however,
continues. By the twelfth to fourteenth day post coitus the nuclei of the
oldest ova enter on the complex series of stages characteristic of the meiotic
prophase, all of them reaching at least the pachytene stage by the time of
birth (18, 25). SHghtly before birth some of the primordial ova have begun
to be surrounded by follicle cells, and by three (55, 70) to six (18) days post
partum all the oocytes in the ovary proper have a follicular epithelium. The

BIOLOGY OF THE LABORATORY MOUSE

number of these primordial ova is enormous. Arai (8) estimates that there
is a total of approximately 35,100 in the two ovaries of a new born rat.

By birth, or shortly thereafter, another process has made its appearance;
namely, the degeneration of ova. This is very evident in the ovaries of rats
sixteen hours old and apparently reaches its height during the second and
third day post partum (25). Some follicles continue to grow, but degenera-
tion also continues, so that despite the production of new ova described
below, the total number of ova in both ovaries of twenty-three day old rats
is reduced to an average figure of about 11,000 (8).

The proliferation of ova by the germinal epithelium continues after birth
(Fig. 32). There is some evidence that the process temporarily ceases or at

Ovum in germinal
epithelium

Terminal epithelium

Tunica albuginea

Ovum with follicle
cells

Fig. 32. — The formation of ova from the germinal epithelium in a 45 day old rat.

{Af/er Hargitt.)

least is somewhat retarded from birth until several days thereafter (25, 70),
but this has also been denied (130). In any case, active proHferation is in
progress at six or seven days post partum. The young ova are distinguish-
able from the other cells in the germinal epithelium by their larger size, clear
cytoplasm, spherical and intensely staining nuclei, and by the fact that they
often occur in pairs. At eight days post partum ova may be seen separated
from the epithelium and in the process of passing through the thin tunica
albuginea toward the underlying stroma (25). At twelve to fifteen days
some of the follicles first acquire a small antrum (18, 44, 70). Accompany-
ing this (fifteenth day) the diameters of the larger folhcles show a sudden and
pronounced rise to a size almost equal to that of the follicle at the occurrence
of the first estrus (44). In rat ovaries, according to Lane (77), the percent-

REPRODUCTION 6i

age of follicles containing an antrum, as compared with the total number of
follicles having at least two layers of follicular cells, is ii% at fifteen days.
This figure rises to 39% at thirty-seven days, falls to 29% at fifty days, and
then ascends sharply until it reaches 50% at sixty-six days when ovulation
occurs. Hargitt (62) likewise has noted a decrease in the number of large
follicles in rat ovaries two to three weeks before the first ovulation, and finds
this to be due to an increased rate of degeneration of such follicles at this
time. Ovulation in his animals occurred at about 45 days, and the ovaries
at 29-32 days showed a drop in the number of large follicles. In the case
of mice, also, a reduction in the number of large follicles in the ovaries of
animals 28 days old, as compared with the number at 21 days, has been
noted (18).

According to a recent study with rats (124), ovogenesis between birth
and maturity is cyclic, with maxima occurring approximately every ten
days. In this investigation, as in others described above, the first maximum
wasTound to occur at six or seven days post partum. Other maxima
occurred at approximately ten day intervals until the onset of the normal
estrous rhythm. Follicular atresia during this period was found also to be
cyclic with about ten days between peaks. How this prepuberal rhythm of
ovogenesis and atresia is related to the prepuberal fluctuations in the pro-
portions of large follicles described by other authors is not yet clear.

The process of ovogenesis continues, though somewhat more slowly,
until fecundity is lost in old age. During maturity it shows fluctuations
that coincide with the estrous cycle (see p. 74). The process is less con-
spicuous in older mice because the newly formed ova do not attain such large
size while still in the germinal epithelium and hence are more easily confused
with epithelial cells. Some authors have disputed the continued production
of ova by the germinal epithelium during maturity, but recent work quite
definitely confirms its occurrence (4, 25).

Coincident with the occurrence of ovogenesis, continued ovular degenera-
tion is likewise going on. As a result there is a more or less steady reduction
in the number of ova present in the ovaries. Counts by Arai (8) in the rat
show a total of approximately 35,100 ova in both ovaries at birth, 11-10,000
at 2 1, days and 63 days, 6,600 at 70 days, 2,000 at 31 months. Except for
the period from 23 to 63 days, ovogenesis is not sufficiently rapid to replace
the ova lost through ovular degeneration and normal ovulation (For the
details of the degenerative changes in atretic ova and follicles see p. 154-)

In addition to abnormal ova due to degenerative changes, polyovular
follicles and polynuclear ova, probably not due to degeneration, have been

62 BIOLOGY OF THE LABORATORY MOUSE

described as occurring occasionally in ovaries of both mouse and rat (43, 78).

The earlier workers in this field were puzzled by the fact that while ova
formed before birth showed all the stages typical of meiotic prophase in the
male, these stages were not found in ova formed after birth. This problem
has been at least partly resolved by Swezy (130) in a study of the ovaries of
female rats from before birth to maturity. At five days post partum the
typical miotic prophase stages are, in fact, present. Deutobranch, lepto-
tene, synaptene, pachytene and diplotene nuclei can all be distinguished.
From then on the process is steadily modified and probably shortened. At
twenty days deutobranch nuclei and nuclei showing modified pachynema
stages may be seen. In the adult most of the different maturation phases
are lost altogether, or at least are not cytologically distinguishable. Crew
and Roller (32), however, have figured clear chiasmata in diplotene chromo-
somes in ova of mature female mice. This is excellent evidence that
synapsis (and crossing-over) has occurred, even though the stage at which it
occurs is difficult to see. Hence, however much the maturation stages may
be modified and telescoped in the developing ova of adult mice and rats,
there is little reason to doubt that they include the steps necessary for
accompHshing the pairing and crossing-over of the chromosomes required
by genetic evidence.

While the concomitant occurrence of ovogenesis and ovular degeneration
at all ages until senihty is reached seems well-estabUshed, the rate of turn-
over, and the consequent length of fife of the individual ovum, remains some-
what uncertain. The view of early investigators that ova formed in the
embryo are functional in the adult has been largely abandoned, and some
writers have gone to the other extreme, maintaining that "individual
folUcles have a functional life span of only a day or two, in all cases less than
the length of the estrous cycle" (49). Lane and Davis (79), as a result of
studies of mitotic activity and volume changes in rat follicles, take an inter-
mediate position. They write as follows : " Folhcles less than 200 fx in diam-
eter are inactive mitotically and are thought to be physiologically quiescent."
In the adult, folhcles of this size or smaller "represent a reserve from which
are drawn succeeding crops of foUicles for maturation at succeeding estrous
periods. This follicle reserve will develop or be maintained without the
assistance of the pituitary, but for the production of follicles larger than
200 to 300 IX, pituitary assistance is required. . . . Between 200 and 300 /x
diameter, the follicle in any stage of the cycle shows mitotic activity in the
granulosa and theca which is shghtly augmented. These folhcles are
thought to be on the way to maturation or atretic degeneration. ... It

REPRODUCTION 63

seems significant that the follicles in the size range of 401 to 500 /i should
exhibit the maximum activity in the granulosa. It is possible that these
constitute the group which will ovulate at the succeeding estrous period.
Numerically there are 6 of these follicles in the average metestrus ovary.
Allowing for atresia this number could easily produce the 3 to 5 ovulating
follicles which are present in each ovary at estrous."

A rough calculation of the length of life of the average follicle is possible
from pubhshed data. In two experiments (98, 125) female mice were
irradiated with x-rays and the condition of the ovaries determined by breed-
ing tests or by histological study. These experiments show that fertile
matings may be obtained from females irradiated with a dose of 260 r for a
period not exceeding 28 days following treatment. After this they become
completely sterile, presumably because no new ova are prohferated by the
germinal epithelium (125). That irradiation causes early cessation of
ovogenesis is shown by the fact that small or primary follicles are absent in
ovaries of irradiated mice (150 r) killed 2 days after treatment. At 21 days
only a few normal follicles of the older types are present, the gland being
mainly composed of degenerating remnants. At 43 days there is a total
absence of all follicular structure (98). These two experiments show that, at
least in irradiated ovaries, ova can survive for only about 28 days following
their proliferation by the germinal epithelium.

Further evidence as to the rate of development of ova is provided by
experiments in which rat ovaries were ligated, so that degeneration resulted
from loss of blood supply, followed by regeneration when the circulation was
re-established (26). At 8 or 10 days degeneration is usually complete
except for small peripheral regions. At 12 days small ova may be seen
recently differentiated from the germinal epithelium. At 21 days some
medium sized foUicles are present. At 30-34 days recovery is practically
complete, and the ovary contains fully developed Graafian follicles. The
interval from the first appearance of new ova to their final full development
is thus 18 to 22 days. This may be taken as the minimum time required for
the complete development of ovum and follicle. It is, of course, possible
that ova and follicles in normal ovaries develop at different rates and survive
for shorter or longer periods than do ova and follicles in ovaries under the
experimental conditions described above. The available evidence, however,
points to 18 to 28 days as a reasonable estimate of the time taken for the
mouse or rat ovum to mature following its separation from the germinal
epithelium. A quite difTerent line of evidence is provided by experiments
with ovaries of embryonic or new born rats and mice grown in vitro. Under

BIOLOGY OF THE LABORATORY MOUSE

these conditions, survival of primitive germ cells for at least 115 days has
been recorded (91).

The high mortality among ova under normal conditions can be appreci-
ated from figures presented by Allen (4). An average of 800 to 1000 ova
are differentiated in the two ovaries of a female mouse at each estrous period,
while only about 9 ova of an earlier generation mature. The percentage of
survival is thus about 1%.

Brambell (19) has made an interesting study of the growth of oocyte and
follicle, finding the relation shown in Fig. 33. It will be seen that at first
both oocyte and follicle increase in size, the relation between the two being

so-

600

200 300 400 500

DIAMETER OF FOLLICLE
Fig. ^^. — Graph showing the relation between oocyte size and follicle size. {From

Brambell 1928.)

linear. When the oocyte attains a diameter of approximately 70^1, and the
follicle a diameter of 125 yu, the former stops growing while the growth
of the follicle continues, so that the average follicle diameter at ovulation is
550 /z. The antrum first appears as an irregular fluid-filled cleft in the
middle of the follicular cells on one side of the oocyte in follicles about 200 /jl
in diameter.

Ovarian regeneration. — As a result of reports in the medical literature of
conception following complete double ovariectomy, there have been a
number of experiments with mice and rats to determine whether or not there
is any ovarian regeneration following removal of both ovaries. While the
results have been conflicting, the most recent experiments seem to indicate
that regeneration does not occur if the removal is complete (66, 107). If
regeneration does occur it is probably the result of a small piece of ovarian
tissue having been left. Regenerated ovaries contain follicles and may be
fully functional (37, 105).

REPRODUCTION 65

Butcher (26) has described regeneration of rat ovaries following ligation.
At eight or ten days the ligatcd ovaries consisted almost entirely of degener-
ate tissue. In the periphery of the ovary, particularly in the region where a
cavity had persisted between the gonad and capsule, small or primary
follicles were found, but in no case did the number encountered in one ovary
exceed twenty. Recovery was rapid, the ovary being practically normal at
thirty to thirty-four days. These cases of regeneration in the adult rat and
mouse are excellent evidence that ovogenesis can occur in sexually mature
animals.

In view of the extent of ovarian regeneration following partial removal it
is noteworthy that the ovaries of mice sterilized with x-rays, either at birth
or later in life, never regenerate any germ cells although they remain
functional in regulating the estrous cycle (22, 98).

Robertson (in) has described the successful transplantation of ovaries
between mice of the same inbred strain.

The estrous cycle. — Our present knowledge of the estrous cycle in rodents
dates from the discovery of Stockard and Papanicolaou that the cellular
contents of the vagina undergo cyclical changes and that by observing these
changes in vaginal smears the successive stages of the estrous cycle can be
accurately followed and the time of heat determined. The estrous cycle of
the mouse has been studied by /Allen 1(3), Rietschel (no), Clauberg (29), and
others. Long and Evans (83) have pubHshed a very thorough study of the
cycle in the rat. The following description is based on Allen's studies except
as otherwise noted.

Divisions of the estrous cycle. — ^The estrous cycle of the mouse and rat
is conveniently divided into 5 stages, namely, 'proestrus, estrus or heat,
metestrus-i, metestrus-2, diestrus. The first two are anabolic stages during
which active growth is in progress in various parts of the genital tract.
They culminate in ovulation and, where mating occurs, in fertilization. The
second two, metestrus-i and metestrus-2, are catabolic stages characterized
by degenerative changes in the genital tract. The last, diestrus, is a period
of quiescence or slow growth. The characteristics of each stage are sum-
marized in Table 4.

External signs of estrus. — There is a tendency at proestrus and estrus

for the vulva to show swelling and congestion, and for the vaginal orifice to

_gap£, but these appearances are so variable as to be unreliable signs of heat.

The onset of heat in the rat can be accurately determined by the '' copulaton,-

response" (65, 137).

BIOLOGY OF THE LABORATORY MOUSE

w
i-i
o

H
in

w
W

H
O

1:3

M u
t>

Q
O
PS
a,
W

Pi!
w

W
O

o
w

1-1

w
w

C/2

(S3
>

Follicles large and distended with
considerable liquor folliculi. Few
mitoses in germinal epithelium
and in follicular cells.

Ovulation occurs followed by dis-
tension of the upper end of ovi-
duct. Active mitoses in germinal
epithelium and in follicular cells.

Early corpora lutea present. Eggs
in oviduct. Many follicles under-
going atresia.

Growing corpora lutea. Eggs in
oviduct. Few mitoses in germ-
inal epithelium and in follicular
cells.

bO
Cu 0

^■£

bO 0
"^ -rt
-°^
m ^
OJ c«

c/l
3

Increasing hyperemia and disten-
sion. Active mitoses in epithe-
lium, few leukocytes.

Distension and mitotic activity
reach maximum during estrus, and
then decrease. No leukocytes.

JDistension decreased. Leukocytes
begin to penetrate epithelium.

Walls collapsed. Epithelium shows
degeneration. Mitoses rare. Leu-
kocytes numerous.

Anaemic, walls collapsed. Epithe-
lium healthy but contains many
leukocj'tes. Some secretion by
uterine glands.

'S-

'3d

>
0)

XI
'0
bC

cfi

Many cell layers (10-13). Outer
4-5 nucleated, stain lightly with
eosin. Under these, granulosa
layer showing increasing corni-
fication. Active mitoses. Few-
leukocytes.

Superficial nucleated layer lost.
Cornified layer now superficial.
About 12 layers of nucleated cells
under this. Mitoses decreasing.
Leukocvtes absent.

Cornified layer delaminated. Leu-
kocytes begin to appear under
epithelium.

4-7 layers of epithelial cells, with
very many leukocytes in outer
layers.

XI X

'^ 0

CJ cn

^^ OJ
cd >.

:g cd

<U — 1

XI ^

'a 3

0) 0
•^ G
0 •'-'
cn
cn a>
»-. .*-*
0 >.

>> CJ

rt 0

-5

-o

cn
<u
0

i s

8 i:

0 y

-1-
■+
CJ

+
+

w
+
+
0

g 3

OJ u
^ G

^ ...

REPRODUCTION 67

Vaginal smears. — Three methods are in common use for taking vaginal
smears, i. Pipette or lavage method. A pipette drawn to a rather fine
point and containing a few drops of water is inserted into the vagina, the
water ejected and immediately sucked in again. The water with its cellular
contents can then be transferred to a slide for examination. 2. Spatula or
curette method. Some of the cell contents of the vagina can be removed by

^ €^

# ^ •

i^****- '^'9 •♦ •

•• ' , i^

♦ ♦ ♦,

•o

Hi^

Fig. 34. — Photographs of vaginal smears stained with haematoxylin-eosin. A.
Diestrus. B. Late estrus. (X300.)

means of a spatula or, preferably, a fine curette. The cells are transferred
to a drop of water on a slide by tapping the curette on the slide. 5. Cotton
swab method. Cells can be removed with a fine, moist, cotton swab on the
end of a toothpick. It has been shown that frequent smearing with cotton
swabs will produce cornification of the vagina in spayed rats and mice, hence
leading to a smear that indicates estrus though in animals in which true
estrus cannot occur (133). The cycle in normal animals may likewise be
disturbed by this method of smearing (40) which is, therefore, not to be
recommended. The lavage method is less upsetting (40, 41). The curette
method is probably also satisfactory though it has been noted that frequent

BIOLOGY OF THE LABORATORY MOUSE

smearing with a spatula tends to disturb the regularity of the cycle (113,
133). The addition of a small amount of methylene blue to the water used
gives a very satisfactory stain. With this stain smears can be examined at
once without waiting for the water to dry.

Three types of cells are found in vaginal smears, i. Leukocytes (Fig.
34A). In unstained preparations these appear at first as small, round,
highly refractive cells, but they swell rapidly in water with resulting rupture
of the cell wall. In preparations stained with methylene blue the poly-
morphic nucleus takes a strong stain. 2. Cornified cells {¥\g. 2,A^)- These

IOO%i

50%

o%J

I I I I

0 DAYS 10 19
Fig. 35. — Graph showing the percentages of each of the three types of cells in the
vaginal smear of a mouse during the normal estrous cycle. Smears taken daily.
cornified cells, nucleated epithelial cells, leukocytes. {Voss 1930.)

are the largest cells in the smear. They are flattened, angular in outline,
quite regular in size, and lack nuclei. 3. Nucleated epithelial cells (Fig. 34A).
The typical epithelial cell is round, oval or polygonal, with clear cytoplasm
and a centrally placed nucleus that takes a strong methylene blue stain. A
number of variations occur. As estrus approaches the smear may contain
epithelial cells with dark staining cytoplasm and karyolytic nuclei. The
cytoplasm may contain droplets (of mucus?). A highly modified mucus-
secreting type also occurs (no). This characteristically is goblet-shaped
with the nucleus at the apex. The presence of mucus can be proved by the
use of appropriate stains. Cells intermediate between cornified cells and
nucleated epithelial cells occasionally occur.

More or less mucus may occur in the smear. Different accounts dift"er
greatly as to the amount normally present. It is possible that smearing
or other forms of irritation increase the amount. In adult ovariectomized
rats, mucification is produced by the combined administration of oestrone
and progesterone (115). During the latter two thirds of pregnancy in the
rat the vaginal mucosa actively secretes mucus (54, 75).

REPRODUCTION

The cyclical changes in the cell contents of the smear are shown in Figs.
35 and 36, taken from Voss (132). UnpubHshed data obtained at the
Jackson Laboratory indicate striking strain differences. However, the
cycles shown in Voss's figures may be taken as fairly typical. The smears
in terms of which the various stages of the cycle are defined are indicated
in Table 4.

Of particular interest is the smear characteristic of estrus or heat. The

complete or almost complete absence of leukocytes from the smear and

the presence of cornified cells, in moderate numbers and not clumped, are the

(usual criteria of estrus. In one study (126) with MacDowell-Bagg albino

mice it was found that the smear at the onset of heat, as determined by

PREG.

LACTATION

100%

50%-

DAYS

-I— I — I— r

20»
C

A BCD
Fig. 36. — Graph showing the percentages of the three types of cells in the vaginal
smear of a mouse during a postpartum estrus, lactation, and the normal estrus follow-
ing lactation. Smears taken daily. cornified cells, nucleated epithelial

cells leukocytes. (A) litter of four born and the beginning of lactation, (B)

two young weaned, (C) one more young weaned, (D) last young weaned. Note the
incomplete cornification at the post partum estrus and the occurrence of a normal
estrus while one young is still nursing. {Voss 1930.)

willingness to mate, still contained 5 to 75% of epithelial cells. The smear
marking the termination of estrus has not been so accurately determined,
but the presence of clumps or sheets of cornified cells is usually regarded as
marking the onset of metestrus. A typical late estrus or early metestrus
smear is shown in Fig. 34B.

The vagina. — No part of the genital tract undergoes more striking
histological changes during the estrous cycle than the epithelium of the
vagina. The successive stages are shown in Figs. 37 and 38 and sum-
marized in Table 4.

In proestrus the epithelium consists of three layers (Fig. 37A). The
outer layer is composed of epithelial cells sometimes more or less filled with

70 BIOLOGY OF THE LABORATORY MOUSE

"Epithelial cell layer

— Stratum granulosum

-Stratum germinativum
polygonal cells

. i— — — Stratum germinativum

basal cells

r - **. — Lamina propria

■ a — Degenerating

epithelial cell layer

Stratum corneum

-Stratum germinativum
polygonal cells

''^b'[*aAI^^^''* ^^■^''^"'"b^-Tciir''^"'"^

Fig. 37. — Photographs of vaginal epithelium of a mouse in different stages of the
estrous cycle. A. Proestrus. B. Estrus. {From Clauherg.)

REPRODUCTION 71

mucus and with nuclei showing signs of pycnosis. Below this is the stratum
granulosum which, with the approach of estrus, becomes the stratum
corneum. Third is the stratum germinativum, some seven cell layers in

%'•- '

• r«.*T^^K ' Leukocytes

l&X t^^X -^^-j'^ ih ,

stratum germinativum
polygonal cells

■Stratum germinativum
basal cells

-Lamina propria

_J| Stratum germinativum

polygonal cells

Stratum germinativum
basal cells

^ ' v-_ '.» 'T Lamina propria

'^^^..df-m

Fig. 38.— Photographs of vaginal epitheUum of a mouse in different stages of the
estrous cycle. A. Metestrus-2. B. Diestrus. {Frovi Clauherg.)

thickness. During proestrus and early estrus the cells of the outer layer
are delaminated into the vagina, producing the characteristic nucleated
cell smear. The'degree of delamination is not uniform in all parts of the
vagina, so that prior to the onset of estrus the cornified layer may be fully

\\.

72 BIOLOGY OF THE LABORATORY MOUSE

exposed in some regions, not at all in others. During late proestrus and
throughout estrus cells are delaminated from the corniiied layer (Fig. 37B).
The onset of metestrus-i is characterized by the peeling off of the whole
layer, with an accompanying rise in the cornified cell count in the smear.
During metestrus-2 there is a rise in the nucleated cell count (Fig. 35),
indicating that in the last stages of the delamination process some of the
superficial layers of the stratum germinativum are included. The super-
ficial layers of the stratum germinativum, meantime, have become heavily
infiltrated with leukocytes (Fig. 38A) which also appear abundantly in the
smear at this time. As a result of the delamination of the superlicial
layers, the vaginal epithelium at diestrus contains only one layer, the
stratum germinativum, some three to seven cell layers in thickness (Fig.
38B). Late in diestrus active growth begins in the stratum germinativum,
and by proestrus a stratum granulosum has formed several cell layers below
the surface, thus completing the cycle.

The uterus. — The uterus, like the vagina, undergoes a series of anabolic
and catabolic changes during the estrous cycle, but they are relatively
much less striking (Table 4 and Fig. 39). In proestrus and early estrus the
uterus shows marked hyperemia and is distended with fluid secreted by the
uterine glands. The distension starts to diminish in late estrus and in
diestrus the uterine wall is collapsed and anaemic. It has been reported
that in the rat the loss of some of this fluid is due to discharge into the
vagina (83). The uterine epithelium has been described as low columnar
in proestrus, with a distinct basement membrane, as high columnar in
estrus (3, 29). The increase in height is not marked, however, and in an
experiment with the rat a reverse change was noted accompanying the
increasing distension of the uterus (7). In metestrus-i degenerative proc-
esses become apparent. The basement membrane fades into a pink-
staining band which includes the basal sides of the epithelial cells and the
superficial stroma. The epithelium loses its definite organization and
shows vacuolar degeneration. Leukocytes appear in the region of the
basement membrane. In metestrum-2 the degeneration of the epithelium
is further advanced, so that almost all the epithelial cells are lost (no).
Cell walls at this stage are no longer recognizable and leukocytes are
numerous. The uterine glands show minimum activity. The onset of
diestrus is marked by the beginning of regenerative processes.

The oviducts. — In the case of most mammals the oviducts show hypere-
mia at estrus (6, p. 668) and the same is probably true of mice, though the
condition seems not to have been specifically noted. There is none of the

REPRODUCTION

periodic leukocytosis so marked in the rest of the genital tract. Cyclical
changes in certain non-ciliated cells in the epithelium of the ampulla have
been both described and denied (2, 3, 48, no). These cells protrude into
the lumen of the tube in an unusual fashion, and there is some evidence that
the protrusion shows cyclic changes. For some hours following ovul-
ation the upper part of the oviduct is distended with fluid.

D P EMIM2 D P EMIM2

Fig. 3q. — Graph showing various cyclic phenomena which accompany the estrous
cycle. The curves have been adjusted to make corresponding points of the cycle cor-
respond as nearly as possible. D = diestrus, P = proestrus, E = estrus, Mi = metes-
trus-i, M2 = metestrus-2.

The ovary. — Cyclic changes are pronounced in the ovary (Fig. 39 and
Tables 4 and 5). A conspicuous feature is the rapid growth of a few of the
follicles prior to ovulation. Lane and Davis (79) sectioned the ovaries of
twenty rats, five in each of the four major divisions of the estrous cycle, and
classified the follicles according to diameter. The results are shown in
Table 5. It will be seen that by proestrus the follicles that will ovulate at
estrus are fairly definitely set apart by their larger size. Brambell and

BIOLOGY OF THE LABORATORY MOUSE

Parks (21) have made a study of the rate of growth of maturing folUcles in
unmated mice. They find that the folHcles which will ovulate at the
following estrus are, on the average, only 380^1 in diameter at the beginning
of the estrous cycle. The follicles reach a maximum size of, on an average,
550 ^i in diameter immediately before rupturing. Most of this growth,
according to their study, occurs in the last 48 hours, during which period
the follicles increase 45% in diameter. Secretion of secondary liquor
folliculi, which at this time begins to replace the less fluid primary liquor
folliculi, may play a considerable role in the increase in size (6, p. 458; 131).
Just prior to ovulation the follicles bulge conspicuously from the surface of
the ovary.

Table 5

Average Distribution of Follicles According to Size throughout the

EsTROus Cycle in the Rat (From Lane and Davis)

Follicle Diameter
(in Micra)

Diestrus

Proestrus

Estrus

Metestrus

No.

35-100

130

61.3

53-3

50.0

56-4

101-200

26.0

31-8

32.5

30 -4

201-300

5-7

8.1

8.7

6.3

301-400

3-3

1-5

3-9

3-8

401-500

1.9

0.7

0.8

501-600

1-4

2. 2

1.6

601-700

0.4

2: 2

2-5

Average total

212

126

158

* The percentage of the total follicle content which falls in a given size range.

Several studies (4, 5, 79) have shown that the mitotic activity in the
ovary is cyclic, reaching a peak at estrus or metestrus- 1 (Fig. 39). Since
mitosis in the vaginal epithelium reaches a peak in proestrus or early estrus
(3, 83), it appears that the ovary responds to the estrus stimulus more
slowly than the vagina. Follicular atresia, like follicular growth, is cyclic,
reaching a peak in metestrus-2 (Fig. 39).

In metestrus- 1 newly formed corpora lutea are present. Since corpora
lutea in unmated mice persist for. two, three or four cycles before disappear-

REPRODUCTIOX 75

ing, numerous old corpora lutea are also present in females which have been
unmated for several previous cycles.

The mammary glands. — The mammary glands show cyclic growth and
regression, though the changes are slight compared to those occurring during
pregnancy (30, 82). In proestrus buds appear on the ducts particularly
around the periphery of each gland, and large blunt projections appear on
the main ducts near the nipples. In estrus the mammary ducts become
dilated, and the buds formed during proestrus prolongate. Metestrus-i
introduces regressive changes and by the end of metestrus-2 the ducts are
decreased in width and the duct endings collapsed. In diestrus the mam-
mary gland consists of a very open network of narrow, thread-like ducts
with comparatively few branches, the branches themselves being simple.

Other concomitants of estrus. — It has been noted that in the rat bodily
activity, as measured by the number of hourly revolutions of a rotating drum
placed in the cage, increases during estrus (65, 121, 135). A loss in weight
at estrus has been described in mice (i), but the weight cycle was not
regular except in mice with a very long estrous cycle (13-14 days), and it
does not seem to occur in rats (122). A cyclic change in the electrical
potential between the vagina and the symphysis pubis has been described
in rats (112). There is a marked peak in potential in late estrus, with an
abrupt fall when estrus terminates. A minor peak occurs about two days
before estrus.

The postpartum estrus. — An estrus occurs in mice and rats within about
20 hours of parturition. The range for mice in the interval between
parturition and the following ovulation has been found to be about 14 to
28 hours (84). The cornification of the vagina is not complete at this
estrus, and the cornified cell content of the smear never reaches 100%
(Fig. 36). Fertile matings are less often obtained during this period than
during the course of the normal cycle. There is less fluid in the uterus than
during a normal estrus (93).

The time relations of the cycle. — In the mouse the onset of heat usually
occurs in the night, most commonly between 10 P.M. and i A.M. Occa-
sionally it occurs between i and 7 A.M., in only rare instances during the
day (81, 126). Similar results have been obtained with the rat except that
the modal hour for the onset of estrus is several hours earlier, heat usually
beginning between 4 and 10 P.M. (12, 31, 121). The onset of heat may be
made to occur in the daytime in either mice or rats by keeping them in a
room that is dark in the daytime, lighted at night (23, 52, 65, 126).

76 BIOLOGY OF THE LABORATORY MOUSE

Observation of 608 heat periods in the rat showed an average duration
of 13.7 hours, with a range of i to 28 hours (12). Periods that start early
in the evening tend to run somewhat longer than ones that start later (31).
The duration has not been so accurately determined in mice, but is probably
much the same as in the rat. One estimate has placed it at about 12 hours
(126).

In some cases what is commonly regarded as the estrous smear may last
for long periods. Allen (3) found that "as diagnosed by the smear method,"
estrus usually lasts i or 2 days, but that unbroken estrous smears may con-
tinue for 9 days, and that 4 days of "heat" are not uncommon. These
cases of long continued estrous smear may be the result of the irritation due
to smearing (133), or they may occur normally in certain strains. The ce
strain commonly shows long intervals of cornification (Ossen, unpublished
data, 50). In any case they cannot be taken to indicate a long duration
of actual heat without further evidence.

The modal length of the complete cycle is commonly 4 days in rats
(12), 5 days in mice. Parkes (loi) found the following distribution for
1000 cycles in unmated mice: 2 days, .4%; 3 days, 2.9%; 4 days, 15.8%;
5 days, 29.3%; 6 days, 21.8%; 7 days, 12.2%; 8 days, 6%; 9 days, 3.1%;
10 days or more up to 28 days, 8.5%. There seems to be a tendency for
the length of the cycle to increase with the age of the female (120). There
are marked strain differences in the length of the cycle (1,4, 24). Strain
differences are even more pronounced in the matter of the individual stages
of the cycle as indicated by the smear. The diestrus interval is commonly
the longest interval, and also the most variable. The daily changes in the
smear throughout three typical cycles are shown in Fig. 35. In these
particular cycles the approximate lengths of the different stages are:
proestrus, i day; estrus, }'2 day; metestrus-i, i day; metestrus-2, i day;
diestrus, 23^^ days.

In the rat, low temperature has been shown to lengthen the cycle (16, 80).

Ovulation. — Ovulation occurs spontaneously during estrus in both
mice and rats, whether mated or unmated. Different accounts differ
considerably as to the time of ovulation in relation to the onset of estrus,
a fact perhaps due in part to the existence of significant strain differences.
Ovulation in mice has been said to occur both at or near the beginning of
estrus (21, 81, 126), and at or near the end of estrus (3, 131). In a recent
study (126) with MacDowell-Bagg albino mice it was found that ovulation
usually occurred between 12 M. and 2 or 3 A.M., but in one case at least as
early as 11:30 P.M. and in another at least as late as 4:40 A.M. Since

REPRODUCTION 77

mating most commonly occurred between 10 P.M. and i A.M., the average
interval between the onset of estrus and ovulation for the MacDowell-Bagg
albino strain at least is probably about 2 hours. The interval was found
to be quite variable, however, being certainly less than i hour in one case
and certainly more than 3 hours and 45 minutes in another. Ovulation
within 15 minutes of mating has been noted (81). Extensive data for the
rat (17) indicate that ovulation in the Wistar strain commonly occurs some
g hours after the onset of estrus, but may occur at least as early as 73^2 hours
and at least as late as 123^^ hours after the onset of estrus. It should be
remembered that the onset of estrus occurs much earlier in the evening in
this species than it does in the mouse.

The rupture of all the mature follicles in an ovary seems usually to be
approximately synchronous (83), but there is evidence that an appreciable
interval may separate the individual ovulations in some cases (3, 81, 126).
Ovulation may not occur at every estrus, particularly in young virgin
females (3, 131). Conversely, estrus may not always accompany ovulation
(137). The mechanism of ovulation is not entirely understood, but there
is evidence that a thinning of the wall at the outer surface of the follicle and
an increase in internal fluid pressure both play a part (6, 131).

Immediately after ovulation the eggs are found in the upper part of the
oviduct, presumably carried there by an outrush of follicular fluid at the
time of follicle rupture. The beating of the cilia of the infundibulum may
also help to carry them from the capsule into the oviduct. At the same
time the upper part of the oviduct becomes distended with fluid, a con-
dition easily seen in dissected animals under the microscope (83, 126, 127).
As the distension is not present prior to ovulation, it is a reliable sign that
ovulation has occurred (126). It has been stated that most, at least, of the
fluid is not derived from the follicles, but rather is secreted by the tubes
themselves (21, 83).

Maturation and fertilization. — Maturation and fertilization of the egg
in the mouse and rat have been described by several authors (71, 74, 76, 84,
127). The following description is based on the work of Long and Mark
(84) except as otherwise noted.

The whole maturation process requires not less than 4 nor more than 15
hours. At the onset of estrus the first maturation division is usually in
progress (126). Usually this division is completed, the first polar body
present, and the second maturation spindle already formed by the time
ovulation occurs. Occasionally, however, the egg is in the stage of the
first spindle or the first telophase at the time of ovulation, in which case

78 BIOLOGY OF THE LABORATORY MOUSE

first polar body formation is completed very shortly thereafter. The polar
body is quite large. Its future history is variable; it may degenerate while
the egg is in the one cell stage, or persist as late as the morula stage. Occa-
sionally it divides in two (8i). Where mating occurs at the onset of estrus,
sperm are probably usually present in the upper end of the tube at the time
of ovulation (126). Each egg is surrounded by a zona pellucida and, out-
side this, a covering of cumulus cells. The cumulus cells are sticky, and
all the eggs in one tube usually are massed into a clump. The sperm
penetrate these coverings quite rapidly, partly dispersing the cumulus cells
in the process, perhaps by enzyme action (108), and reach the vitellus in less
than 2 hours (81). The penetration of the vitellus may be regarded, by
definition, as the actual moment of fertilization. At the time it occurs the
second maturation spindle is invariably present. In the absence of fertili-
zation, the second polar body does not form; where fertilization occurs,
second polar body formation ensues rapidly (71, 81, 84), and the processes
of normal development are initiated.

Since estrus lasts for some 12 hours in mice and rats, mating may
occur several hours after ovulation, the eggs meantime lying unfertilized
in the oviducts. For several hours they retain their capacity for normal
fertilization and development, but in a relatively short time degenerative
processes make their appearance (13, 108).

The transport of sperm and eggs. — Sperm reach the upper end of the
uterus in the rat almost at once after mating (53, 58, 64, 1 14). Throughout
heat the uterus is distended with fluid, and transport of sperm to the mouth
of the oviduct is accomplished not by the sperm's own motility but as a
result of a churning action of the uterine wall acting on this fluid. Trans-
port of sperm through the oviduct is somewhat slower, but Lewis and
Wright (81) find that they may reach the ovarian end of the oviduct, where
fertilization occurs, within 15 minutes of mating. The mechanism involved
in this transport of the sperm toward the ovary, as also in the abovarian
transport of the fertilized eggs, is somewhat obscure, though a churning
action may again be involved in the sperm transport. The subject has
been thoroughly discussed by Parker (106) and Hartman (in Allen, 6), and
the interested reader is referred to these authorities.

The spermatozoa of the mouse retain their fertilizing ability in the ovi-
duct for about 6 hours; their motility ceases only after 133^2 hours. Their
period of survival in the uterus is shorter than in the oviduct (93).

Pseudopregnancy. — Sterile matings in the mouse and rat induce a
condition called pseudopregnancy, characterized by a delay of the next

REPRODUCTION 79

estrous period. In mice the average interval between a sterile mating
and the next estrus is 11 days {t^^, 100); in rats the average interval is 14.5
days and the range 7 to 19 days (122). It has been shown that pseudo-
pregnancy can be induced in the rat by several forms of artificial stimu-
lation. These include the brief insertion into the uterine cervix of a fine
glass rod (83), electrical stimulation of the vagina (60, 119), and intense elec-
trical stimulation through the head (63). Rats stimulated by the probe
method while under ether anaesthesia show only ten per cent pseudo-
pregnancies as against sixty-nine per cent for the controls (94). Spinal
anesthesia completely prevents the induction of pseudopregnancy. In the
rat, copulation without plug formation is a much less effective stimulus than
copulation with plug formation, and the chance that pseudopregnancy will
be induced seems to be still further increased if several completed matings
each with plug formation are permitted (9).

Pseudopregnancy is accompanied by important changes in the uterus
paralleling those that occur during the corresponding stages of pregnancy
and serving to prepare the uterus for the implantation of embryos. His-
tologically, the changes in the rat and mouse uterus are not as striking as
those occurring in the rabbit, but definite progressive changes in the
epithelium and stroma have been noted (7). More significant than the
histological changes is the capacity of the uterus during the early part of
pseudopregnancy to respond to appropriate stimuli by local growth of the
decidua, giving rise to swellings called deciduomata. Any slight local
injury to the uterus will incite their formation; a common practice is to use
a silk thread inserted through the uterine wall (83). In the pseudopregnant
mouse, the maximum capacity for deciduomata formation following local
injury of the uterus occurs about three days post coitum; by five days post
coitum the sensitivity is almost lost (103). The sensitive period thus corre-
sponds to the period of normal implantation.

The mammary gland undergoes development during pseudopregnancy.
The changes parallel those of pregnancy for the first nine days following
copulation. At the end of this period the pseudopregnant development of
the mammary gland reaches its peak, and regression sets in (30).

The available evidence, though not conclusive, seems to indicate that
the remarkable causal chain by which a stimulus applied to the uterine
cervix prepares the uterus to receive the young embryo involves a nervous
impulse from the cervix to the pituitary, an endocrine effect of the pituitary
on the corpora lutea, and a second endocrine effect of the corpora on the
uterus and mammary glands.

8o BIOLOGY OF THE LABORATORY MOUSE

Corpora lutea. — Following ovulation, the ruptured follicles, and occa-
sionally also large unruptured follicles (83) undergo changes which trans-
form them into corpora lutea. During the first few days of its development
each young corpus passes through characteristic stages from which an
approximate though probably not very accurate estimate of its age is
possible (38, 39, 128. See also p. 151). The subsequent history of the
corpus depends on the sexual history of the animal. On the basis of this
history, four types of corpora may be distinguished. The following descrip-
tion of these, except as otherwise noted, is based on the observations of
Long and Evans (83) on corpora lutea in the rat.

1. Corpora lutea of ovulation are corpora formed during an ordinary
estrous cycle where mating does not occur, or at a postpartum estrus if
mating or lactation do not occur. Such corpora may persist with little
obvious degeneration through two, three, or four cycles in the mouse (3),
possibly longer in the rat, so that an ovary from a mouse which has run
several uninterrupted cycles often contains as many as sixteen large, well
defined corpora. The youngest set is distinguished not only by the mor-
phological characteristics which set it apart for the first one or two days
but also by the fact that it stains blue with hematoxylin (3). Older sets
have a greater affinity for eosin. Perhaps a more critical test is a change
in certain lipoid droplets which can be detected in the luteal cells following
appropriate fixation. These are small and regular in size in young corpora,
become larger and less regular in size with the onset of the next estrus.
Long and Evans {d>T^) believe that the functional life of the corpus has
terminated by the time the changes in the droplets appear. Another test
of age is provided by the fact that the lutein cells of old corpora stain more
readily than those of young ones with the vital dye Dianil Blue 2R injected
intraperitoneally.

2. Corpora lutea of pseudo pregnancy are corpora lutea formed following
a sterile mating. Such a mating induces a diestrous interval of some eight
or ten days, and throughout this interval the lipoid droplets in the newly
formed corpora retain the small, uniform size characteristic of young cor-
pora of ovulation. Corpora of pseudopregnancy become more highly
vascularized (39) and attain a slightly larger size than do corpora of ovu-
lation. There is evidence that the prolonged diestrus following sterile
mating is caused by a lengthened functional life in the corpora of
pseudopregnancy .

J. Corpora lutea of pregnancy are corpora formed following a fertile
mating. For the first few days these cannot be distinguished from corpora

REPRODUCTION 8i

of ovulation or pseudopregnancy; for the next few days their development
parallels that of corpora of pseudopregnancy. On the eighth day, however
(in the mouse, 38), they begin a period of rapid growth largely accomplished
by an increase in cell size, and by the sixteenth day they are almost twice
the diameter of corpora of ovulation. They are made more conspicuous
by the fact that, during pregnancy, ovulation is suspended and no new
corpora formed, while meantime older sets of corpora rapidly regress.
With appropriate vital stains traces of the corpora of pregnancy may be
detected for three or four months post partum in rat ovaries.

4. Corpora lutea of lactation are the corpora that develop in non-pregnant
nursing mice from the follicles that ovulate at the first post partum estrus.
These corpora are distinguished from all others by the particularly small
size of the lipoid granules. Within twenty-four hours of the removal of a
nursing litter, the granules show the characteristic increase in size indicative
of cessation of function of the corpora. Corpora lutea of lactation attain a
size somewhat larger than that of corpora lutea of ovulation or pseudo-
pregnancy, but not equal to that of corpora lutea of pregnancy.

Lactation. — The normal duration of lactation in mice is about four
weeks. Milk production rises for the first ten days and then gradually
declines (46). In lactating mice a long period of diestrus occurs following
the first post partum estrus. If the nursing litter is of normal size, this
diestrous period, called the lactation interval, lasts from about 20 to 25 days
(34, T,'^). It may be terminated by removal of the litter, this inducing
estrus in two to four days. If the stimulus of suckling is maintained by
replacing the growing litters from time to time by very young litters, an
estrus occurs as usual at about three weeks, but future estrous periods are
delayed, the intervening diestrous intervals being some twelve to seventeen
days long (116). The stimulus of nursing can produce a marked effect on
females who have not recently lactated. Thus when normally cyclic
adult mice or rats are given on alternate days new litters of actively nursing
young, marked development of the mammary glands occurs. Milk secre-
tion may even be induced. The condition is accompanied by a lengthening
of the diestrous interval to two or three weeks, and by the appearance of the
capacity for deciduomata formation in the uterus. When the nursing
stimulus is removed, normal estrous cycles commence within a few days
(116, 117, 118). It may be noted in this connection that deciduomata
formation may also be induced during normal lactation (83, 85). Mammary
involution following removal of a litter may be retarded in mice by irritation
of the nipples with turpentine applied twice daily (68).

82 BIOLOGY OF THE LABORATORY MOUSE

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REPRODUCTION 83

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REPRODUCTION 85

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86 BIOLOGY OF THE LABORATORY MOUSE

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low environmental temperatures. Am. J. Physiol. 78: 246-253.

81. Lewis, W. H., and E. S. Wright. 1935. On the early development of the
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82. LoEB, L. 1923. The mechanism of the sexual cycle with special reference to
the corpus luteum. Am. J. Anat. 32: 305-343.

83. Long, J. A., and H. M. Evans. 1922. The oestrous cycle in the rat and its
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84. Long, J. A., and E. L. Mark. 191 i. The maturation of the egg of the mouse.
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85. Lyon, R. A. 1939. Survival of deciduomata during lactation in the rat.
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86. MacDowell, E. C. 1924. A method of determining the prenatal mortality
in a given pregnancy of a mouse without affecting its subsequent reproduction.
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87. MacDowell, E. C. 1928. Alcohol and sex ratios in mice. Am. Nat. 62:

48-54-

88. MacDowell, E. C, E. Allen and C. G. MacDowell. 1929. The relation of
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8g. MacDowell, E. C, and E. M. Lord. 1925. Data on the primary sex ratio
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90. MacDowell, E. C, and E. M. Lord. 1926. The relative viabihty of male
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91. Martinovitch, p. N. 1939. The effect of subnormal temperature on the
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92. Merton, H. 1938. Studies on reproduction in the albino mouse. I. The
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80-96.

93. Merton, H. 1939. Studies on reproduction in the albino mouse. III. The
duration of life of spermatozoa in the female reproductive tract. Proc. Roy. Soc.
Edinburgh 59: 145-152-

94. Meyer, R. K., S. L. Leonard and F. L. Hisaw. 1929. Effect of anaesthesia
on artificial production of pseudopregnancy in the rat. Proc. Soc. Exp. Biol,
and Med. 27: 340-342.

95. Mirskaia, L., and F. a. E. Crew. 1930. On the genetic nature of the time of
attainment of puberty in the female mouse. Quart. J. Exp. Physiol. 20: 299-304.

96. Mirskaia, L., and F. A. E. Crew. 1930. Maturity in the female mouse.
Proc. Roy. Soc. Edinburgh 50: 179-186.

97. Mirskaia, L., and F. A. E. Crew. 1931. On the pregnancy rate in the lactat-
ing mouse and the effect of suckling on the duration of pregnancy. Proc. Roy.
Soc. Edinburgh 51: 1-7.

98. Murray, J. M. 193 1. A study of the histological structure of mouse ovaries
following exposure to Roentgen irradiation. Am. J. Roent. and Rad. Therapy
25: I-4S-

REPRODUCTION 87

og. Parkes, a. S. 1926. Studies on the sex-ratio and related phenomena: (9)

observations on fertilit}^ and sex-ratio in mice, 1922-5. Brit. J. Exp. Biol. 4:

03-104.
100. F'arkes, a. S. 1926. Observations on the oestrous cycle of the albino mouse.

Proc. Roy. Soc. B 100: 1 51-170.
loi. Parkes, A. S. 1928. The length of the oestrous cycle in the unmated normal

mouse: records of 1000 cycles. Brit. J. Exp. Biol. 5: 371-377.

102. Parkes, A. S. 1929. The internal secretions of the ovary. Longmans, Green
and Co., London.

103. Parkes, A. S. 1929. The functions of the corpus luteum. II. The experi-
mental production of placentomata in the mouse. Proc. Roy. Soc. B 104: 183-
188.

104. Parkes, A. S., axd C. W. Bellerby. 1926. The mammalian sex-ratio. Biol.
Re\^ 2: 1-5 1.

105. Parkes, A. S., U. Fielding AND F. W. R. Brambell. 1927. Ovarian regenera-
tion in the mouse after complete double ovariotomy. Proc. Roy. Soc. B loi:
328-354.

106. Parker, G. H. 1931. The passage of sperms and of eggs through the oviducts
in terrestrial vertebrates. Phil. Tr. Roy. Soc. London B 219: 381-419.

107. Penchez, R. I. 1929. Experiments concerning ovarian regeneration in the
white rat and white mouse. J. Exp. Zool. 54: 319-339.

108. PiNCUs, G. 1936. The eggs of mammals. The Macmillan Co., N.Y.

109. Reed, S. C. 1938. Uniovular twins in mice. Science 88: 13.

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Individualcyclus der weissen Maus. Z. wissensch. Zool. 135:428-494.

111. Robertson, G. G. 1940. Ovarian transplantations in the house mouse.
Proc. Soc. Exp. Biol, and Med. 44: 302-304.

112. Rogers, P. V. 1938. Changes in electrical potential in normal, castrated, and
theelin-treated rats. Am. J. Physiol. 121: 565-573.

113. Rogers, P. V. and E. Allen. 1937. Epithelial growth caused by stimulation
with various smear methods as demonstrated by mitotic stasis with colchicine.
Endocrinology 21: 629-632.

114. RosSiMAN, I. 1937. Uterine contractions and the transport of sperm in the rat.
Anat. Rec. 69: 133-149.

115. Selye, H., J. S.L. Browne AND J. B. CoLLip. 1936. Effect of combined admin
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116. Selye, H., and T. McKeown. 1034. Further studies on intkience of suckling.
Anat. Rec. 60: 323-332.

117. Selye, H., and T. McKeown. 1934. The eftect of mechanical stimulation
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S86-890.

iiS. Selye,. H., and T. McKeown. 1934. Production of pseudopregnancy b}-
mechanical stimulation of the nipples. Proc. Soc. Exp. Biol, and Med. 31:
683-687.

88 BIOLOGY OF THE LABORATORY MOUSE

iig. Shelesnyak, M. C. 1931. The induction of pseudopregnancy in the rat by-
means of electrical stimulation. Anat. Rec. 49: 179-184.

120. Slonaker,-J. R. 1924. The effect of pubescence, oestruation and menopause
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121. Slonaker, J. R. 1925. Analysis of daily activity of the albino rat. Am. J.
Physiol. 73: 485-503.

122. Slonaker, J. R. 1929. Pseudopregnancy in the albino rat. Am. J. Physiol.
89: 406-416.

123. Slonaker, J. R. 1934. Superfetation in the albino rat. Am. J. Physiol. 108:
322-323.

124. Sneider, M. E. 1940. Rhythms of ovogenesis before sexual maturity in the
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y 125. Snell, G. D., and F. B. Ames. 1939. Hereditary changes in the descendants
of female mice exposed to Roentgen rays. Am. J. Roent. and Rad. Therapy 61:
248-255.

126. Snell, G. D., E. Fekete, K. P. Hummel and L. W. Law. 1940. The relation
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129. Stevens, W. L. 1937. Significance of grouping and a test for uniovular twins
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133. Wade, N. J., and E. A. Doisy. 1935. Cornification of vaginal epithehum of
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134. Walker, G. 1910. A special function discovered in a glandular structure
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135. Wang, G. H. 1923. The relation between spontaneous activity and the
oestrous cycle in the rat. Comp. Psychol. Mon. 2, serial No. 6:1.

136. Weichert, C. K. 1940. The experimental shortening of delayed pregnancy
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137. Young, W. C. 1940. Observations and experiments on mating behavior in
female mammals. Quart. Rev. Biol, (in press).

Cnapter 3

HISTOLOGY

By Elizabeth Fekete, Roscoe B. Jacksofi Memorial Laboratory.

Introduction, 89. Circulatory system, 90. The blood vessels, 90. The heart,
90. Lymphatic capillaries and vessels, 91. Blood, 92. Blood forming and blood
destroying organs, 94. Bone marrow, 94. Lymph nodes, 95. The spleen, 96.
Endocrine glands, 98. Hypophysis, 98. Thyroid gland, 100. Parathyroid
glands, 100. Adrenal glands, loi. Thymus, 103. Pineal body, 104. Skin and
hair, 105. The skin, 105. The hair and the vibrissa, 106. Hibernating, lacrimal
and Harderian glands, 108. Hibernating glands, 108. Lacrimal glands, 108.
Harderian glands, 109. Oral cavity and associated structures, no. The oral cavity,
no. The tongue, no. The pharyn.x, 112. Subma.xillary glands, 112. Major
subungual glands, 115. Parotid glands, 115. Digestive tube, 116. Esophagus, 116.
Stomach, 117. The small intestine, 120. The large intestine, 122. Mesenteries, 124.
Liver and pancreas, 124. The liver, 1 24. The gall bladder, 1 26. The pancreas, 1 26.
Respiratory system, 127. The larynx, 127. The trachea and the main bronchi, 128,
The lungs. 128. Urinary system, 130. The kidney, 130. Bladder, 132. Female
urethra, 132. Male genital system, 133. The testis and its excretory ducts, 133.
The accessory glands, 137. The urethra, bulbo-urethral glands, penis and preputial
glands, 143. Female genital system, 146. The ovary, 148. The oviduct, 155.
Uterus, 155. Vagina, clitoris and clitoral glands, 157. Mammary glands, 158.
Bibliography, 162.

Introduction

This chapter gives the histology of the organs rather than the tissues,
presupposing a general knowledge of the latter subject. The nervous sys-
tem and the special sense organs are omitted, and for these subjects the
reader is referred to the excellent work of C. W. Ariens Kappers, G. Carl
Huber and E. C. Crosby (58), "The Comparative Anatomy of the Nervous
System of Vertebrates Including Man."

The sections which serve as illustrations for this and for Chapter 4 were
fixed in a mixture of alcohol, formalin and acetic acid (70% alcohol 100 cc,
formalin 16 cc, acetic acid 5 cc.) for 4-24 hours, changed into 80% alcohol,
dehydrated in the usual way and imbedded in paraffin. This technique
gives satisfactory results with mouse tissue and is used in our laboratory

90 BIOLOGY OF THE LABORATORY MOUSE

routinely. Hematoxylin and eosin (H & E) stains were used, unless other-
wise stated.

Circulatory System

The blood vessels. — The walls of the blood vessels are formed of three
parts: the innermost part, the interna or intima; the middle part, the media;
and the outer part, the adventitia or externa.

The interna of the arteries consists of the endothelial lining, composed of
elongated flat cells with prominent oval nuclei, beneath which is a network
of elastic fibers forming the internal elastic membrane. The media is wide
and in the large arteries consists of alternating layers of circular smooth
muscle fibers and elastic membranes. According to Lowenthal (in Jaffe, 56) ,
in the aorta six to ten such layers are present, intermingled with fine col-
lagenous fibers. In smaller arteries the media contains less elastic and more
muscular elements. In the arterioles it consists of only a few individual
muscle fibers. The adventitia is composed of loose connective tissue fibers
and serves as a transition zone between the arteries and the surrounding
tissue.

The capillaries are formed of elongated endothelial cells usually separated
from the surrounding elements by a thin sheath of connective tissue. They
connect the terminal arteries with veins.

In some organs the connection between arteries and veins is accomplished
through irregular spaces, the sinusoids. The walls of the sinusoids unlike
the capillaries do not have a continuous endothelial cell lining, but are lined
by scattered phagocytic and non-phagocytic cells.

The intima of the veins consists of polygonal-shaped endothelial cells and
connective tissue fibers intermingled with fine elastic fibers. The media is
formed of smooth muscle fibers and is poorly delimited from the next layer.
The media of the wall of pulmonary veins contains cardiac muscle. The
adventitia is well developed and contains connective tissue fibers inter-
mingled with some longitudinal smooth muscle fibers. The valves of veins
are formed of a connective tissue membrane containing a network of elastic
fibers. Both surfaces of the valve are covered by endothelium.

The walls of all of the larger blood vessels are supplied with blood b>-
small vessels, the vasa vasorum. In general the walls of veins in relation to
the diameter of the lumen are thinner than the walls of the arteries.

The heart. — The heart consists of four chambers, the left and right atria
and ventricles. These chambers are lined by endothelial cells which rest on
a very thin layer of connective tissue. These together form the endo-

HISTOLOGY 91

cardium. The myocardium is composed of cardiac muscle fibers which are
arranged in spiral sheets. The myocardium of the left ventricle is consider-
ably thicker than the right. Both atria have very thin muscle walls. The
outermost layer is the epicardium. It is composed of a thin layer of connec-
tive tissue, covered by a single layer of mesothelial cells. A transparent,
serous membrane, the pericardium, encloses the heart and the proximal
portions of the great vessels. The heart lies in the pericardial cavity.

The right atrium communicates with the right ventricle by the tricuspid
valve; the left atrium with the left ventricle by the mitral or bicuspid valve.
The valves are composed of folds of the endocardium. They are covered
on both sides by endotheHum and contain some connective tissue. They are
attached by thin strands, the chordae tendineae, to the projections of the
papillary muscles of the ventricular walls.

According to Ohmori (74), the atrio-ventricular conducting system as
described by Tawara exists in the heart of the mouse.

Arterial blood leaves the left ventricle through the aorta. The opening
is guarded by the semilunar aortic valve. From the right ventricle the
pulmonary artery originates, guarded by the pulmonary valve, and carries
blood to the lungs. From the lungs oxygenated blood is transported to the
left atrium through the pulmonary veins. The superior and inferior venae
cavae bring venous blood into the right atrium.

The coronary arteries which branch off from the root of the aorta supply
the tissues of the heart with blood. Capillaries are numerous among the
heart muscle fibers.

Lymphatic capillaries and vessels. — The walls of lymphatic capillaries
are formed of a single layer of large, flat, polygonal, endothelial cells. The
lumina are irregular; dilations and constrictions occur frequently. The
capillaries form many branches, some of which end blindly while others
anastomose. The lymphatic vessels have thicker walls consisting of, in
addition to the endotheHum, collagenous bundles, elastic fibers, and smooth
muscle fibers. In the larger lymph vessels an intima, media and adventitia
can be distinguished. The intima is formed of endothelium, and a thin layer
of elastic fibers, the media of circularly arranged smooth muscle fibers, while
the well developed adventitia is composed of collagenous and elastic fibers
and smooth muscle bundles. The paired valves are similar to those of veins
and consist of folds of the intima. In the largest lymphatic vessel, the
thoracic duct, the division of the three parts of the wall is very indistinct.
Below the endothelium the collagenous and elastic elements form an inner
elastic membrane from which fibers project and mingle with the smooth

92 BIOLOGY OF THE LABORATORY MOUSE

muscle fibers of the media. The components of the adventitia are similar to
those of the media and they merge gradually into the surrounding tissues.

Blood. — The red blood corpuscles or erythrocytes of the mouse are similar
in shape to those of other mammals. They are very flexible, circular,
biconcave discs, capable of becoming cup-shaped when passing through fine
capillaries. The corpuscles contain hemoglobin and have lost their nuclei.
They have a diameter of 5.7 ju according to Kerti and Stengel (in Jaffe, 56).
Stained with Wright's stain, some of the erythrocytes show marked poly-
chromatophilia (about 10% according to Simonds, 87). Nucleated red
blood cells are seldom present in the circulating blood.

Haam (in Jaffe, 56) states that the hemoglobin content of the mouse
blood (based on the average of the observations of nine investigators) is
97% (Sahli).

The white blood corpuscles or leukocytes are true cells with a nucleus
and cytoplasm. Among them the lymphocytes are the most numerous.
They are somewhat larger than erythrocytes and have large, spherical,
slightly indented, eccentric nuclei which almost completely fill the cells. In
stained preparations the nucleus is very dark; the cytoplasm is homogeneous
and slightly basophilic.

The monocytes are the largest cells in the circulating blood. They have
eccentric, ovoid, bean-shaped, occasionally deeply indented nuclei which
stain Hghtly. The cytoplasm is abundant, non-granular and slightly
basophilic.

The granular leukocytes are somewhat larger than the lymphocytes.
Great variations exist in the shape of their nuclei, which may be ring-shaped
or show irregular constrictions and lobulations. According to the staining
reaction of the granules present in the cytoplasm of these cells, they are
divided in three groups: neutrophiUc, eosinophiUc, and basophilic poly-
morphonuclear leukocytes. The eosinophihc cytoplasmic granules stain
readily, while the neutrophilic granules stain faintly. The basophilic cells
are very rare; some investigators consider them absent. Simonds (87) gives
their number as less than 1%.

The blood platelets are very small, blue staining, granular bodies similar
to those present in the human blood. According to Klieneberger and Carl
(in Jaffe, 56) their number varies between 157,000 to 620,000 (mean 284,810)
per cu. mm.

To obtain blood for counts the tail vein and the ventricles of the heart are
most often used. Table i gives the total erythrocyte and leukocyte count
and the differential leukocyte count of several strains of mice maintained

HISTOLOGY

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BIOLOGY OF THE LABORATORY MOUSE

in the R. B. Jackson Memorial Laboratory. (The counts are based on
unpublished data of Dr. L. W. Law and Dr. W. E. Heston.) The peripheral
blood obtained from the tail vein contains a greater number of white blood
cells than the heart blood (Table 2).

Table 2
Comparison of Ventricle and Peripheral Blood in MacDowell-Bagg

Albino Mice

Sex

Total Leukocyte Count, Number per Cu. Mm.

Ventricle

Peripheral

9
9
9
9

9
<f

3 , 080
4,100
4,280
3,840
3,480
3,220
4,420
3,320

21,100
16,980
21,750
1 7 , 000
30,050
32,460
16,400
16,350

Mean

3.717

21,510

Blood Forming and Blood Destroying Organs

Bone marrow. — Within the cavities of the bones reticular stroma forms a
framework, the meshes of which are filled with marrow cells. The stroma
consists of reticular cells, fixed macrophages and reticular fibers. The
marrow cells give rise to the erythrocytes, the granular leukocytes and
perhaps blood platelets of the circulating blood.

The erythroblasts are immature red blood cells. The youngest of these
have basophilic cytoplasm and large, round, vesicular nuclei. As the hemo-
globin content of these cells increases, the cytoplasm becomes polychro-
matophilic. They divide by mitosis and some of the cells originating from
the division undergo further changes. The hemoglobin content increases
still more and the cytoplasm becomes acidophilic. At the same time the
vesicular nucleus becomes compact and dark staining. Such cells are called
normoblasts. After losing their pyknotic nuclei they are ready to enter the
circulation as erythrocytes.

HISTOLOGY 95

The myeloblasts are large cells with large, round, vesicular nuclei, contain-
ing coarse chromatin granules, surrounded by a small amount of non-granu-
lar cytoplasm. They undergo proliferation and give rise to myelocytes
which have indented bean-shapped nuclei and slightly granular cytoplasm.
The myelocytes divide by mitosis and give rise to metamyelocytes or pro-
leukocytes which have ring-shaped nuclei and cytoplasm containing some-
what coarser granules. These cells do not proliferate, but their nuclei
change into irregular lobulated shapes, typical of mature polymorphonuclear
leukocytes (Fig. ri8). The cytoplasmic granules of the myelocytes and
metamyelocytes may be eosinophilic or neutrophilic. BasophiHc myelo-
cytes are not found in mouse bone marrow (Haam, in Jaffe, 56). The
neutrophilic granules are fine and stain faintly, while the eosinophihc
granules are larger and stain intensely. The megakaryocyte is a giant,
irregular shaped cell which has a single lobulated nucleus. It undergoes
degeneration within the marrow. The theory that small processes of the
cytoplasm of megakaryocytes are pinched ofT and enter the circulation as
platelets is still under discussion. In addition to the cells described, the
presence of fat cells, large mononuclear cells and lymphocytes is constant in
the marrow.

Petri (77) gives the following average differential count based on the cells
of the femoral marrow of 14 white mice: nucleated red blood cells 23%,
myeloblasts 4.7%, myelocytes 9.2%, proleukocytes 6.5%, leukocytes 34.3%,
non-identified 18.7%, large mononuclear cells 0.1%, reticulo-endothelial
elements ^.2)%, megakaryocytes 0.2%.

The mature myeloid cells enter the circulation by passing through the
thin wall of the venous sinusoids. Arteries and veins are numerous in the
bone marrow.

The femur or sternum are suitable for obtaining marrow smears for
histological examination. According to Jaffe (56) the marrow of the long
bones is functional throughout the life of the mouse, and is not replaced by
fatty, yellow marrow.

Lymph nodes. — ^Lymph nodes are small, bean-shaped organs composed of
lymphatic tissue and located in the course of lymph vessels. At the
indented area, which forms the hilus, blood vessels enter and leave the node.
Intercommunicating large lymph spaces, the lymph sinuses, are present
throughout the organ. Each node is surrounded by a thin connective tissue
capsule which is especially well developed at the hilus, where it may project
for a distance into the medullary area. Trabeculae which are continuous
with the capsule divide the cortical part into alveolar areas and the medul-

96 BIOLOGY OF THE LABORATORY MOUSE

lary part into irregular spaces. In both areas reticular libers and primitive
and phagocytic reticular cells form the finer network.

In the cortex the lymphocytes may form rounded nodules which, how-
ever, are not constant structures and may vary in size and position (17), or
may be entirely absent, in which case the lymphocytes are arranged diffusely
without any definite structure. When a nodule is very active in producing
lymphocytes, its central area is lighter staining, and among its cell com-
ponents are many medium sized lymphocytes. Such areas contain mitotic
figures and have been called secondary nodules or germinal centers.

In the medulla the lymphatic tissue is arranged in cords, surrounded by
wide meshes of the medullary sinuses. In the cords among the lymphocytes
free macrophages, eosinophils, plasma cells and occasionally mast cells are
also present. The free macrophages originate from the phagocytic reticular
cells (fixed macrophages) and are capable of ameboid movement. They are
elongated, irregular shaped cells with oval nuclei containing coarse chromatin
granules. The plasma cells have eccentric round or oval nuclei, with large,
darkly staining chromatin granules distributed in a fairly regular pattern.
The cytoplasm is homogeneous and slightly basophilic. The mast cells are
large, oval or polyhedral cells, with small round nuclei and cytoplasm contain-
ing large granules which stain intensely with hematoxylin and often obscure
the nuclei.

Arteries enter at the hilus. They usually follow the course of the
trabeculae and branch repeatedly. The endothelial cells lining the capil-
laries are unusually tall, resembling in cross section cuboidal epithelial cells.
Several afferent lymph vessels enter through the capsule at the surface of the
organ. The lymph circulates throughout the sinuses, and lymphocytes
produced here are carried away by this constant flow. At the hilus the
lymph is collected into efferent lymph vessels.

Great variation exists in the size and shape of the lymph nodes, as well
as in the relative size and position of the medulla and the cortex. Often the
trabecular system is poorly developed, and the nodes contain a diffuse mass
of lymphatic tissue (57).

The spleen. — The spleen is a slightly curved, finger-shaped organ covered
with a capsule composed of dense connective tissue containing some smooth
muscle fibers. The trabeculae project in from the capsule, and with a net-
work of reticular cells and fibers form a framework which is filled by the
splenic pulp (Fig. 40). The splenic artery divides into two branches which
enter the spleen on its concave dorsal side, thus dividing the organ into three
approximately equal parts. Variations exist, and one or both of the second-

HISTOLOGY

ary arteries may divide forming more than two points of entrance. After
entering the organ the arteries divide repeatedly, decreasing gradually in
size. During their courses they give rise to numerous capillaries which
supply the lymphatic tissue, the white pulp, with blood. Lymphatic tissue
is arranged around the arteries in the form of a continuous sheath which
thickens at points where branching occurs. Around the small arteries the
tunica adventitia is replaced by lymphatic tissue. Lymphatic nodules, or

Central artery Splenic nodule Red pulp

White pulp

Megakaryocyte

Red pulp

Capsule

Trabecula

Fig. 40. — Spleen. (X50.)

splenic nodules (Malpighian bodies), are found arranged around central
arteries. The outline of these nodules is usually very indistinct. Their
central areas sometimes contain many medium sized lymphocytes forming
germinal centers, the secondary nodules. As in lymph nodes, these are
transitory structures.

The small arteries of the white pulp, after repeated branching, enter the
surrounding tissue, the red pulp, where they divide into many straight
arteries, the penicilli. A short distance before their termination, the walls
of these vessels are thickened by closely applied fibers of reticular tissue,
while the lumina remain narrow. These are the sheathed arteries of the
pulp, which after further branching give rise to arterial capillaries. The
question of "open" or "closed" circulation, depending on whether
the arterial capillaries open into the spaces between the reticular cells or into
the venous sinuses, is still under discussion.

98 BIOLOGY OF THE LABORATORY MOUSE

According to Knisley (63) who observed the circulation of the Kving
mouse spleen by transillumination, the branches of the penicilli divide into
arterial capillaries. Some of these capillaries after a somewhat curved,,
unbranched course connect directly with venules. Others after a short
course connect with the afferent ends of venous sinuses. Some of the
sinuses intercommunicate, forming multiple sinus routes; others form a single
sinus route. Both routes finally open into venules. The tissue between the
sinuses forms the splenic cords. According to the same author, in the
unstimulated spleen few erythrocytes are present in the splenic cords.
These leave the closed vascular system by individual penetration of the
walls. In traumatized and dying spleens rapid changes occur which result
in the passage of large numbers of erythrocytes into the pulp tissue.

The red pulp fills the spaces between the terminal venous sinuses, forming
the splenic cords. The framework is formed by reticular fibers, primitive
reticular cells and fixed macrophages. In addition to the lymphatfc ele-
ments and elements of the circulating blood, free macrophages, small groups
of myelocytes, erythroblasts and plasma cells are present. Megakaryocytes
are constant constituents although their number varies considerably. As
the red pulp of the mouse contains many lymphocytes and few erythrocytes,
it is not well delimited from the white pulp. The reticular cells of the red
pulp almost always contain varying amounts of pigment.

There is great variation in the size of the spleen. The distal end of the
organ occasionally shows bifurcation. Accessory splenic tissue in the pan-
creas or in fat lobules of the mesentery is often found.

Endocrine Glands

Hypophysis. — The hypophysis rests on a slight depression of the sphenoid
bone. It is attached to the floor of the third ventricle by a short stalk (96).
It consists of two main parts which are separated from each other by a
narrow cleft, the residual lumen of Rathke's pouch. The part which is
directly above the sphenoid bone and below the cleft is the anterior lobe or
pars distalis, while the parts above the cleft are the pars intermedia and the
pars nervosa (Fig. 41).

The anterior lobe is formed of epithelial cells arranged in cords or alveolar
groups which are separated from each other by delicate connective tissue
septa. Small cysts lined by ciliated cells have been found occasionally.
The epithelial cells can be classified as chromophobe and chromophil cells.
The chromophobe cells (also called chief cells) have large, light staining

HISTOLOGY

99
The

nuclei, surrounded by small amounts of non-granular cytoplasm
nucleus contains one or two intensely staining nucleoli.

There are two kinds of chromophil cells. Stained with hematoxylin and
eosin, the cytoplasmic granules of some cells take the hematoxylin stain —
these are the basophil cells; others take the eosin stain — these are the

-• •

^ ' *•

-Pars nervosa

Pars intermedia

Residual lumen

Pars distalis

Fig. 41. — Hypophysis. Fixed in io',~c formalin. (X^oo.)

acidophil cells. The basophils are large polyhedral cells with eccentric
nuclei and a large amount of cytoplasm. Variations exist in the amount and
size of the basophilic granules. The cytoplasm often contains vacuoles of
different size.

The acidophils are small, round or oval cells with centrally located nuclei.
The nucleus contains a large acidophilic and a smaller basophilic nucleolus.

\'ariations exist in the relative number of the three types of cells. The
number of degranulated basophil cells increases during pregnancy (61).
After castration the basophil cells increase in number and in size, and in some
of them cytoplasmic vacuoles are present (castration cells).

loo BIOLOGY OF THE LABORATORY MOUSE

According to Severinghaus (86) there is strong evidence that the chro-
mophobic cells are progenitors of distinct and divergent chromophilic cell
types, and that no transition between the basophils and acidophils is possi-
ble. The study of castrate pituitaries indicates that the chromophils may
revert to their chromophobic form.

The pars intermedia is above the cleft and is in close contact with the
pars nervosa. It is very well developed in the mouse. The epithelial cells
are arranged in small irregular groups. Most numerous are the polygonal
cells with oval nuclei and non-granular cytoplasm. These are similar to the
chromophobe cells of the pars distalis. Spindle-shaped cells often as long as
the width of the pars intermedia are also present. They have dark staining
oval nuclei. These cells are considered by Benda (in Jaffe, 56) as endo-
thelial cells lining very minute capillaries. More general opinion maintains
that the intermedia has poor blood supply.

The pars nervosa contains ependymal and glia cells and fibers. Elements
from the pars intermedia may project into this zone. Gersh (38) describes
specific parenchymatous cells which are distinguished from the neuroglia
cells elsewhere in the central nervous system by their characteristic cyto-
plasmic inclusions. In the mouse these cells have an oval nucleus and a
large prominent nucleolus. The cytoplasm contains osmophilic granules.
In other cells osmophilic granules are not present, but the cytoplasm is filled
instead by delicate basophilic granules which may be arranged in short
chains or clumped masses. Some cells are intermediate between these two
types. Gersh states that the parenchymatous glandular elements of the
neuro-hypophysis produce and secrete the antidiuritic substance.

Thyroid gland. — The lateral lobes of the thyroid are situated on the
sides of the trachea, just below the larynx. They are connected by a very
narrow transverse lobe, the isthmus. The lobes are surrounded by a fibrous
capsule. The organ is composed of follicles of varying size which are
filled with colloid (Fig. 42). In section this material is homogeneous and
stains well with acid dyes. The follicles are lined by simple cuboidal epi-
thelial cells, having large, spherical, central nuclei and clear cytoplasm.
Occasionally round droplets of colloid or clear vacuoles may be present in
the cytoplasm. The cell outHnes are distinct. It is considered that glands
composed of tall cells are more active than those composed of low cells.
The follicular epithelium is surrounded by the interfollicular reticular con-
nective tissue, which is very rich in blood and lymph supply.

Parathyroid glands. — Each parathyroid gland is surrounded by and
separated from the thyroid by a connective tissue capsule (Fig. 42). The

HISTOLOGY

lOI

positions as well as the number of lobes is variable. Usually they are sit-
uated at the caudal end of each of the two lateral lobes of the thyroid. The
gland consists of densely arranged groups or cords of polygonal cells. In
some of these cells the nuclei are round and contain fine chromatin granules;

Thyroid fDllicle
with colloid

Parathyroid

.^_ -.-»,»_ Blood vessel

Fig. 42. — Thyroid and parathyroid glands. (X200.)

in others the nuclei are elongated and contain a single large nucleolus.
According to Larionow (67), in young mice the former predominate, and in
old mice the latter, while mice of middle age occupy an intermediate posi-
tion. The supporting framework consists of reticular fibers and a network
of capillaries.

The adrenal glands. — The adrenal glands are situated immediately
anterior to the kidneys. Each gland has a thin connective tissue capsule
which projects into the parenchyma and forms supporting trabeculae. A
cross section shows a central medulla and a peripheral cortex. Dependent

I02

BIOLOGY OF THE LABORATORY MOUSE

upon the size and arrangement of the cells, the cortex may be divided into
three zones. Immediately beneath the capsule is the narrow zona glomeru-
losa formed by small cells arranged in arch-like groups. The cells have
relatively large nuclei and slightly basophilic cytoplasm. In the next zone,
the wide zona fasciculata, the cells are larger and are arranged in more or
less definite radial columns separated from each other by small blood

Capsule

omerulosa

fasciculata

Zona reticularis

^^f

■:, -©

y Medulla

Fig. 4j. — Adrenal gland. P'ixed in Bouin's fluid. (X200.)

vessels. The cells have vesicular nuclei, and the cytoplasm appears foamy,
due to the presence of finely distributed lipoid droplets. This zone mor-
phologically resembles the corpus luteum. The third zone, the zona
reticularis, is composed of strands of small cells which form a network

(Fig. 43)-

According to Howard-Miller (51), in the adult male mouse the fas-
cicular and reticular layers are not definitely limited, and their separation
into two zones is not justified. The same author observed that in the young
adult nulliparous female the zona glomerulosa and fasciculata are similar to
those of the male, but that in addition to these zones there exists a wide
third zone composed of cells which differ from the cells of the zona fas-
ciculata by being smaller and staining more intensely. She named this

HISTOLOGY 103

highly developed reticular zone, which has specific variations under certain
conditions, the X zone. The X zone is present in the male until about the
time sexual maturity is reached. It persists in castrated males at least four
months longer. In the female it continues to develop until at 4-5 weeks it
occupies a much larger area than in the male of the same age. The zone
disappears during first pregnancy, but in virgin females it persists for a
longer period, gradually degenerating between the third and seventh month.
The gradual degeneration leads to hyperemia and widespread vacuoliza-
tion and the separation of the medulla and cortex by connective tissue.
In a later article (52) the same author states that: "Mice of different strains
show considerable variation in the amount of adrenal X zone tissue which
they normally develop."

The cells of the medulla are arranged in rounded groups and wide
reticular cords, separated from each other by sinusoidal blood spaces. The
cells and consequently the reticular cords are considerably larger than those
in the zona reticularis of the cortex. The nuclei are large and central!}'
located and the cytoplasm is pale staining. If the gland is treated with
potassium dichromate, small brown granules are visible in the cells. Chro-
mic acid stains the cells evenly brown, giving the so-called chromaffin
reaction.

Arteries enter the gland from the capsule. They form the capillaries
of the cortex. The sinusoidal blood spaces of the medulla drain into the
tributaries of a large central vein and leave the gland at the hilus.

The presence of small accessory adrenals in the vicinity of the gland is
not infrequent.

Thymus. — The thymus is situated in the thorax ventral to the aortic
arch. It consists of two lobes of unequal size lying close together. The
lobes are covered by fibrous connective tissue from which septa project in
and produce lobulation without dividing the gland into distinctly separate
lobules. In a cross section of the thymus a light staining medulla and a
dark staining cortex can be differentiated (Fig. 44). In both parts reticular
cells form a supporting framework. In the cortex densely packed, small,
round cells are present. These cells are considered identical morphologically
with small lymphocytes by some authors, while others consider them of
epithelial origin and call them small thymocytes. They have dark staining,
slightly eccentric, round nuclei with dark chromatin granules and
prominent nucleoli, surrounded by a very small amount of cytoplasm.'
Because of the dense arrangement of these cells, the reticular cells are
difficult to see.

I04 BIOLOGY OF THE LABORATORY MOUSE

Toward the medulla the density of the thymocytes changes rather
suddenly and the light staining reticular cells become much more promi-
nent. These cells are entodermal in origin and their epithelial character is
evident during embryonic life. The reticular cells have pale spherical
nuclei with fine chromatin granules and indefinite cell outlines. Thymo-
cytes are present, although not densely arranged. Eosinophils and plasma
cells are usually found here. Small groups of large, pale staining epithelioid

Capsule

Cortex

v.. •.-.

Fic. 44-

-Thymus.

(X200.)

cells with large oval nuclei occur in the medulla. These, however, are not
flattened and concentrically arranged like the typical Hassall's corpuscles.
Cysts of varying sizes are frequently present. The smaller cysts are often
lined entirely by cuboidal, ciliated cells; in the larger ones the lining con-
sists partially of fiat, partially of ciliated cuboidal cells.

The gland is relatively large during embryonic life and in postnatal life up
to the time of puberty but undergoes involution after sexual maturity is
reached. During involution the thymic cells of the cortex gradually become
less dense and the division into cortical and medullary part is less prominent.
Adipose tissue is not deposited in the involuted thymus of the mouse.

Arteries enter the capsule, are distributed first to the cortex, then to the
medulla. Veins arise in the medulla and leave the organ at the hilus.

Pineal body. — The pineal body (epiphysis cerebri) is a small, cone-shaped
body which is situated above the roof of the third ventricle and is attached
to its posterior part by a stalk. The gland has a fibrous connective tissue

HISTOLOGY

105

capsule, from which septa project into the parenchyma and divide it into
irregular areas. The cells are arranged in cords and spherical groups

(Fig. 45)-

Neuroglia cells with long stellate processes form a reticular framework
for the cell cords. Some of the cells of the cords have compact, small,
round, dark staining nuclei and homogeneous cytoplasm. Others have
indistinct cell outline and possess large, pale staining, oval nuclei with finely

Acervulus

Capsule

Fig. 45.

-Pineal body. (X200.)

distributed chromatin granules. Sand granules (acervuli) are present in
old animals, situated beneath the capsule or in the center of cell groups.
The blood supply is rich and small capillaries form a dense network around
the glandular cells.

Skin and Hair

The skin. — The skin is composed of two parts, the epidermis which is a
stratified squamous epithelium, and the corium or dermis, the underlying
connective tissue. The epidermis of the mouse is very thin at all surfaces
where hair covers and protects the animal. At areas where the hair is
thin or absent (as at the anus, around the nipples, tail, feet, etc.) the
epidermis is considerably thicker.

Around the nipple the epidermis consists of about ten to twelve layers of
epithelial cells. The cells of the basal layer which are next to the corium
are columnar in shape and are placed perpendicularly to the skin surface.
They have large oval nuclei and frequently show mitotic figures. The next
few layers of cells are polyhedral or flattened squamous cells. These cells

io6 BIOLOGY OF THE LABORATORY MOUSE

and the basal cells together form the stratum germinativum (also called
stratum Malpighii or stratum spinosum). The cells of this layer are
separated from each other by intercellular spaces. Spines protruding from
the cell surface form bridges connecting the cells with each other, and the
spines of the basal cells penetrate the connective tissue of the dermis.

The next three to four layers of cells in the epidermis of the nipple are
flattened, have light staining oval nuclei, and possess cytoplasm containing
coarse keratohyalin granules. These cells form the stratum granulosum.
The intercellular spaces between these cells are considerably narrower,
gradually becoming indistinct.

The next layer consists of about four to five layers of flattened, dead,
cornified cells, the peripheral layers of which are constantly in the process of
desquamation. This layer is the stratum corneum.

The surface of the dermis forms projecting elevations, the dermal
papillae, which project into the epidermis. The dermis is composed of
fibrous connective tissue and adipose cells. It is rich in blood supply.

Where the epidermis is thin some of the strata described above are not
present (Fig. 8i). The cells of the stratum germinativum are cuboidal or
polyhedral in shape and form only about one to two cell layers. The stra-
tum granulosum is absent. The stratum corneum consists of one to two
layers of cornified cells. Dermal papillae are absent.

Below the dermis loose connective tissue and adipose cells form the sub-
cutaneous layer. Where the panniculus carnosus is present, the striated
muscle fibers of this layer separate the cutaneous layer from the sub-
cutaneous connective tissue. At other places the subcutaneous tissue is a
loose continuation of the dermis.

Pigment is present in the basal cells of the epidermis and in the cells of
the dermis of the tail and ear in many difi'erent strains.

The hair and the vibrissa. — The part of the hair which projects above the
skin surface is the hair shaft ; the part within the skin is the root. The root is
enclosed in a tubular sac, the hair follicle, which is composed of both the
epidermal and the dermal layers of the skin. The dermal part of the
folhcle is continuous with the papilla, which projects into the basal part of
the hair root. The epithelial cells around the papilla form the hair matrix.
These cells multiply, move upward and are responsible for the growth of the
hair. The cytoplasm of these cells, in animals which have colored fur,
contains pigment.

As the histological details are essentially similar to those in human hair,
for a description of the cell layers of the hair the reader is referred to

HISTOLOGY

T07

Maximow and Bloom (73). Dry (30) describes the develoi)ment and suc-
cession of the various types of overhairs, underfurs (zigzags), vibrissae, hair-
lets, and hairs of the arioles of the mouse.

Vibrissae, tactile hairs or sinus hairs are long coarse hairs with deeply
imbedded hair follicles (22, 23). Between the connective tissue sheath and

Hair shaft
Medulla
Cortex
Hair cuticle
Skin

Sebaceous gland
Ring sinus
Hyaline membrane

Superior swelling of
the root sheath

Pulvinus

Nerve fibers

Inferior swelling o
the root sheath

Cavernous sinus

Connective tissue
sheath

Nerve fibers

Hair bulb
Papilla

Fig. 46. — Diagram of a vibrissa. (X125.)

the hyaline membrane of the hair follicle a cavity is present, the lower
part of which is divided into reticular spaces by a network of fibers originat-
ing from the inner surface of the fibrous sheath, while the upper part con-
tains one circular space (Fig. 46). These cavities are filled with blood and
form the cavernous and circular (or ring) sinuses respectively. The cells
forming the reticular network often contain pigment. Extending into the
circular sinus is an oval-shaped projection, the pulvinus. According to

io8 BIOLOGY OF THE LABORATORY MOUSE

Vincent (94) this is much shrunken in prepared sections, but in expanded
state surrounds the foUicle entirely and fills in the space between the walls.
The root sheath shows two slight enlargements forming the superior and
inferior swellings. Vincent stated that many nerve fibers have their
terminations here.

Striated muscle fibers surround the connective tissue sheath. The con-
traction of the muscles around the opening of the follicle permits the hair to
vibrate freely. Muscle fibers connect the walls of adjacent foUicles and
are responsible for the uniform, almost continuous movement of these hairs.

Hibernating, Lacrimal and Harderian Glands

Hibernating glands. — Some of the adipose tissue differs from the usual
white fat and has a characteristic light brown color which is due to the
presence of pigment. It is divided into lobules by loose fibrous connective
tissue and was considered to be a gland of internal secretion by the early
investigators. Many different names were suggested for it, among them
hibernating gland, interscapular gland, multilocular adipose tissue, oil
gland, etc. It is present as a large bilobed mass between the scapulae, in
the superior mediastinum about the thymus, in the cervical region, in each
axillary fossa, and in the abdominal cavity as perirenal lobes (80).

The fat cells have large, round, centrally located nuclei with fine chroma-
tin granules. The distribution of fat is multilocular, being present in the
form of numerous small droplets. The abundant granular cytoplasm con-
tains small spherical vacuoles (dissolved fat droplets), the outline of which
stains intensely with eosin. Groups of cells are supported by reticular fibers
which also surround the capillaries. The blood supply is rich.

According to Rasmussen (80), "The structural differences such as the
more granular character of the cytoplasm of the cells . . . are not sufficient
evidence to warrant the conclusion that the structure under consideration is
of any endocrine significance."

Lacrimal glands. — The exorbital lacrimal glands are situated slightly
below and in front of the ears. The gland is tubulo-alveolar and is composed
of small lobes, which are enveloped and divided into lobules by fibrous con-
nective tissue (Fig. 47). Structurally the gland resembles the parotid
gland. It differs from it in that its intra-lobular ducts are lined by low
cuboidal epithelial cells, which lack basal striations. The alveoli are
slightly larger and more loosely arranged than the similar structures of the
parotid gland.

HISTOLOGY

109

The main duct leads toward the posterior corner of the eye. Here the
duct joins the intra-orbital lacrimal gland which consists of a small lobe, and
is identical structurally with the exorbital gland.

'^^9^^

-Alveolus

■Blood vesse

Duct

Fig. 47. — Exorbital lacrimal gland. (X200.)

Harderian glands. — The Harderian gland lies behind the eyeball and
partially encircles the optic nerve. It is tubulo-alveolar in structure
(Fig. 48). A thin connective tissue membrane surrounds and divides the
gland into lobes and lobules. The tubules and alveoli are lined by tall

Fig. 48. — Harderian gland. (X200.)

columnar epithelial cells in which the pale staining round nuclei are at the
bases of the cells. The cytoplasm contains minute fat droplets which are
seen in sections as small vacuoles, separated from each other by acidophilic
granules. In many tubules the cells are broken down and the lumina con-
tain a fatty secretion which is the product of cell degeneration.

The gland cells rest on a delicate lamina propria, the cells of which
occasionally contain pigment granules. The pigment may color the secre-
tion present in the lumina and is visible grossly as small dark granules and

no BIOLOGY OF THE LABORATORY MOUSE

ill section as homogeneous brown drops. The short excretory ducts are
lined by cuboidal epitheHal cells and open at the base of the nictitating
membrane.

Oral Cavity and Associated Structures

The oral cavity. — The lips are covered on the outside b}' skin containing
deeply imbedded hair follicles. At the zone of transition from the skin to
the mucous membrane covering the inner surface of the lips, the hairs dis-
appear and the stratified squamous epithelium becomes much thicker, its
outer layers being cornified. Similar epithelium lines the entire oral
cavity. Below the epithelium the fibrous lamina propria forms low,
broad papillae. At the corners of the mouth there are large sebaceous
glands which open directly through short ducts to the surface of the lips.

The dental formula of the mouse is: incisor i/i, cuspid o/o, premolar
o/o, molar ^/_^. The incisors in both jaws are bow-shaped with the root
projecting far back below the root of the third molars. According to Weber
(in Jaffe, 56) the crown on the outer convex side is covered by enamel, while
on the inner concave side the enamel is absent and the dentine is covered by
cementum. As the incisors are growing continuously, their apical foramina
stay open. The molars are similar structurally to the human molars.

The anterior part of the roof of the mouth, the hard palate, bears rows of
membranous ridges. The three anterior ridges are transverse; the five
pairs of posterior ridges are V-shaped. They are covered by stratified
squamous epithelium showing keratinization, and are supported by the
dense fibrous lamina propria which takes part in their formation. The
mucous membrane is firmly attached to the surface of the bones.

The posterior part of the roof of the mouth, the soft palate, is composed
of striated muscle fibers and fibrous connective tissue covered by mucous
membrane. On the oral surface and at the posterior margin of the soft
palate the epithelium is cornified stratified squamous, while toward the nasal
surface, a short distance from the margin, this changes into columnar
pseudostratified, and still farther into pseudostratified ciliated columnar
respiratory epithelium. Mucous glands are present on the oral surface
beneath the mucous membrane. They are surrounded by loose vascular
connective tissue and open through short ducts into the oral cavity.

The tongue. — About one third of the distal part of the tongue lies free
in the oral cavity. Farther caudad it is attached to the floor of the mouth
and the wide proximal part is attached also on the sides, here forming the
floor of the mouth cavity. The tongue is covered by stratified squamous

HISTOLOGY

III

epithelium, the superficial layers of which are cornitied. Except for a small
proximal part, the dorsal surface is covered by small elevations, the papillae
(Fig. 49). Morphologically three kinds of papillae can be distinguished.
The filiform papillae are the most numerous, the fungiform papillae being

Fungiform papilla

Filiform papilla

Taste bud

Stratified souamous
epithelium

Lamina propria

Striated muscle

Fig. 4q. — Tongue with papillae and taste bud. (X400.)

present in much smaller numbers. A single circumvallate papilla is situated
on the midline close to the base of the tongue. The cone-shaped filiform
papillae are formed entirely of epithelial cells. The parts projecting above
the surface are composed of overlapping cornified cells. The fungiform
papillae are elevated only slightly above the surface (Fig. 49). The epithe-
lium as well as the underlying lamina propria takes part in their formation.
The free surface is covered by three to four rows of epithelial cells. At
its center, each papilla has a single taste bud (75). The circumvallate
papilla is surrounded by a deep circular groove and does not project above

112 BIOLOGY OF THE LABORATORY MOUSE

the surface. The stratified squamous epithehum fining both waUs of the
circular groove lacks the superficial cornified cell layers and contains
numerous taste buds. These are barrel-shaped structures occupying the
thickness of the stratified epithelium. Two kinds of cells take part in their
formation : the tall peripheral supporting cells which have pale staining oval
nuclei, and the slender neuroepithelial cells which have dark staining, spindle-
shaped nuclei and end in hair-like processes. The former cells enclose a
small central opening, the taste pore, into which the hair-like processes of the
neuroepithelial cells project. The taste buds of the fungiform papiUae are
similar in structure, but dift'er in that they project below the epithelium into
the lamina propria (Fig. 49).

Loose connective tissue forms the lamina propria, which is thin except
where it projects into and takes part in the formation of the fungiform and
vallate papillae. Below the propria is the musculature of the tongue. This
consists of vertical, longitudinal and transverse striated muscle bundles.
Blood vessels branch between the muscle layers, and in the lamina propria
capillaries are numerous. Only near the base of the tongue are glands pres-
ent, surrounded by and separating the muscle bundles. Those near the
vallate papilla are serous glands (the glands of Ebner) which have short ducts
opening at the base of the groove of the papilla. There are small lobules of
mucous glands farther laterally and also dorsally which open with short
ducts directly on the surface of the tongue.

The pharynx. — The oral cavity opens caudally into the pharynx, which
connects it with the esophagus. The pharynx also serves as a connection
between the oral and nasal passages and the larynx. Except for a small
area, where the respiratory epithelium of the posterior nares persist, the
entire surface of the pharynx is lined by stratified squamous epithelium
with cornified superficial layers. The lamina propria is composed of dense
connective tissue and is directly continuous with the muscular wall, which
is composed of striated muscle fibers. Between the muscle fibers groups of
mucous glands are present which open to the surface through short ducts,
lined by stratified squamous epithelium. Lymphatic tissue is not
present (75).

Submaxillary glands. — The submaxillary glands are two large lobes
which slightly overlap on the midventral line of the neck. They are com-
pound, branched tubulo-alveolar glands. Each lobe is divided into several
lobules which are surrounded by and separated from each other by con-
nective tissue membranes. The glands have an extensive duct system.
The main duct of each lobe opens on the floor of the mouth. At its orifice

HISTOLOGY 113

the duct is lined by stratified squamous epithelium which toward the gland
changes into pseudostratified columnar type. The interlobular ducts are
lined by columnar epithelial cells. The intralobular ducts are the so-called
striated tubules, and are lined by rodded epithelial cells. These cells have
centrally located, large, round nuclei and characteristic basal striations in
their cytoplasm. The central intralobular ducts divide into terminal
tubules which in turn connect with the alveoli. The alveoli are composed
of "special serous cells" (18). They are pyramidal in shape and have
large, oval, darkly .staining nuclei near the bases of the cells. The granular
cytoplasm is basophilic and unlike the serous cells of the parotid gland it
does not contain chromophil substance (18). The cells rest on a basement
membrane. Scattered between this membrane and the epithelial cells,
stellate basal cells or "basket" cells are present.

According to Oppel (Vol. Ill, 57) the submaxillary glands of the rat and
mouse are serous glands and do not contain any mucous cells. He states
that even those cells which resemble them are not true mucous cells.
Stormont (in Cowdry, 18) considers all the cells of the submaxillary gland of
rabbit, rat, mouse and muskrat as "special serous cells" and gives the
definition of this type of cells as "those non-mucous cells which differ in
important respects from the serozymogenic type but which, notwithstanding
a vast amount of research, remain, as yet, functionally and cytologically ill
defined." According to the same author the special serous cells forming the
gland of the mouse are of two types: "The gland tubules are composed of
tropochrome cells, but the terminal segments of the intralobular ducts con-
tain in their cells large coarse, highly refractive granules similar to those
present in the homeochrome cells of the rabbit's submaxillary gland." He
describes the tropochrome cells of the rabbit's submaxillary as clear,
palely stained cells which show coarse reticular structure. The nucleus is
usually shrunken and basal in position in fixed material. In preparations
fixed in sublimate these cells present an appearance very similar to mucous
cells, from which they differ by the fact that the contents of the cell spaces
do not stain with any of the ordinary staining reagents for mucin. The
homeochrome cells of the rabbit, according to him, have large oval nuclei
situated at the bases. Unlike the serozymogenic cells, they do not contain
chromophil material. The cytoplasm is abundant and filled with darkly
staining large granules.

Our own observations which include the examination of the submaxillary
glands of more than 200 animals from the dba and C57 black strains showed
differences in the structure of the adult normal male and female animals.

114

BIOLOGY OF THE LABORATORY MOUSE

The central intralobular ducts of both sexes are Hned by rodded epithelium.
In the adult female this type of epithelium also lines the terminal tubules
into which the central intralobular ducts divide (Fig. 50A). In the adult
male the lining of the terminal tubules and some of the alveoli opening into
them consists of tall columnar epithelial cells with the nuclei near and often
flattened against the bases (Fig. 50B). These cells resemble mucous cells

Terminal tubule

Central intralobular
duct

Fig. 50. — Subm;i.\illary gland. A

Terminal tubule

Alveolus

Central intralobular
duct

]''emale mouse. B. Male mouse. (X200.)

and are perhaps identical with the tropochrome cells described by Stormont.
They do not stain red with Mayer's mucicarmine stain. In young animals
of both sexes up to about live to seven weeks the structural differences do
not exist, and the tall, light staining cells are not present.*

* The above is based on observations by the author and Paul Ossen. Since these
observations were recorded a recent paper by Lacassagne (65) which describes the same
dimorphic structural sex differences has come to our attention.

HISTOLOGY 115

Blood vessels ramify in the interlobar connective tissue. They follow
the course of the ducts, and a capillary network provides the tubules and
alveoli with a rich blood supply.

Major sublingual glands. — The major sublingual glands are in close
proximit}' to the lateral surface of the submaxillary glands.

-Intralobular
duct

Fig. 51. — -Sublingual gland. (X200.)

Loewenthal (69) referred to this gland as the retrolingual gland and con-
sidered it to be an accessory submaxillary gland. It is composed usually of
one large lobe divided into smaller lobules by connective tissue septa.
The main excretory duct is lined by pseudostratified columnar epithelium
and has a parallel course with the duct of the submaxillary gland. It opens
through a separate orifice in close proximity to the submaxillary duct. The
intralobular ducts are striated tubules and are lined by rodded epithelium.
The short and narrow intercalated ducts are lined by very low cuboidal
epithelial cells. In the mucous cells which constitute the alveoli the
nuclei are flattened to the bases and the cytoplasm appears clear and
slightly basophilic. Stained with thionin the cells contain a purple-red, with
Mayer's mucicarmine a red network of precipitated mucigen. The delicate
basement membrane and stellate basal cells are like those described in the
submaxillary gland (Fig. 51).

Parotid glands. — The paired parotid glands are composed of several
small elongated lobules. Extending from the ventro-lateral surface of the
neck, the posterior lobes reach the shoulders. The main duct is formed by
several branches and opens in the oral cavity opposite the molar teeth (43).
The intralobular ducts are striated tubules, lined by rodded epithelial cells.
The intercalated ducts are lined by low cuboidal epithelial cells. The serous
cells of the secretory alveoli are pyramidal in shape. Around and below

ii6

BIOLOGY OF THE LABORATORY MOUSE

the nucleus the cytoplasm contains chromophil substance, staining intensely
with basic stains and causing vertical striations. The nucleus is relatively
large and round or oval in shape. Above the nucleus coarse zymogen
granules are present and can be demonstrated by special technique. Basal
cells are present between the epithelial cells and the basement membrane
(Fig- 52).

Rodded epithelium

Alveolus

Intralobular duct

-Parotid gland. (X200.)

Digestive Tube

The wall of the digestive tube is composed of several layers. The inner-
most layer is the mucous membrane or tunica mucosa, which consists of a
surface layer of epithelium and the underlying connective tissue, the
lamina propria (also called stratum proprium or tunica propria). In some
parts of the tube a layer of smooth muscle fibers, the muscularis mucosae,
forms the limit of the mucous membrane and separates it from the sub-
niucosa, which is composed of loose connective tissue. Where the mus-
cularis mucosae is not present the lamina propria changes gradually into
the submucosa. The muscularis externa, also called tunica muscularis,
consists of layers of muscle fibers. In the stomach and intestines this layer
is surrounded by the serosa, composed of a thin connective tissue membrane
and covered by mesothelium. The esophagus and the rectum are attached
to the adjacent tissue by a layer of loose connective tissue, the tunica
adventitia.

Esophagus. — The esophagus is a tube which connects the pharynx with
the stomach. The stratified squamous epithelium lining the lumen con-
sists of a thin stratum germinativum, a somewhat heavier stratum gran-
ulosum and a stratum corneum which forms about one half of the total

HISTOLOGY 117

thickness. The lamina propria is composed of fibrous connective tissue
and does not form papillae. The mucous membrane forms longitudinal
folds. The muscularis mucosae is developed only in the lower, caudal third
while in the upper two thirds the lamina propria is continuous with the loose
connective tissue of the submucosa. The muscularis externa is composed
of striated muscle fibers throughout the entire length of the tube to the

Cardiac antrum

Region of pyloric glands

Region of gastric glands

Esophagus

Right Jf-H-v-^rr "f V^^r''/ A ^*'*

Non-glandular part

Ridge

Fig. 53. — Stomach, outline drawing.

cardiac opening of the stomach. The outer surface of the esophagus is
attached by a layer of loose connective tissue, the tunica adventitia. No
glands are present (39).

Stomach. — The esophagus enters the stomach in about the middle of the
lesser curvature. Grossly the stomach shows two parts, the thin-walled,
slightly transparent, grayish part on the left, and the thick-walled, white
part on the right. The mucous membrane of the former is devoid of glands,
while the latter contains the digestive glands (Fig. 53). Since the wall of
the stomach distends and stretches easily, the size relationship of the two
parts is not always the same but depends on the amount of food present
in each. The lining of the glandless part is a stratified cornified squamous
epithelium similar to the lining of the esophagus. The lamina propria
forms numerous papillae. At the boundary of the non-glandular and
glandular part the mucous membrane of the former forms a ridge. This
ridge is particular!}' prominent dextral to the entrance of the esophagus,
there forming a channel-like extension of the non-glandular part, the cardiac

ii8 BIOLOGY OF THE LABORATORY MOUSE

antrum. It probably has the function of directing the swallowed food
toward the non-glandular part for storage.

The glands of the stomach are compactly arranged, simple, branched,
tubular glands lying parallel to one another, perpendicular to the surface
and occupying the thickness of the mucous membrane. On the surface of
the stomach a multitude of small depressions form the gastric pits, foveolae

Ridge

Gastric pit

Gastric gland

Muscularis mucosae
Submucosa

Muscularis externa
Serosa

Lamina propria

Non-glandular part

^^^:<::

Fig. 54. — Stomach, region of ridge between non-glandular and glandular part. ( X 100.)

gastricae. Into the bottom of each pit small groups of gland tubules open
through slight constrictions.

The inner surface of the glandular stomach is covered, and its pits are
lined, by tall simple columnar epithelial cells containing mucigen. They
have large oval nuclei, located in the lower halves of the cells. In sections
the cytoplasm above the nucleus shows faint granulation.

The glandular area of the stomach may be divided into two main parts,
the larger containing the gastric glands, also called fundic glands, and the
smaller containing the pyloric glands. At the junction of the glandless and
glandular areas the stratified squamous epithelium covering the ridge is
replaced by simple tall columnar cells (Fig. 54). Here a very short tran-
sitional zone exists where two to three rows of gland tubules are present,

HISTOLOGY

119

lined by simple columnar cells which do not show special secretory granules.
Bensley (7, 8) considered these as cardiac glands.

The gastric glands have a fairly straight course and open into short
gastric pits. The cells which are most numerous in the lower third of the
tubules are the serous chief cells or zymogenic cells. They are columnar
cells having large oval nuclei, situated at the center, and granular cytoplasm.

•-A

'Epithelium

-Villus

Lamina propria

glands

Circular muscle
layer

Longitudinal muscle
layer

■ Serosa

Fig. 55. — -Duodenum, longitudinal section. (X200.)

In stained preparations the cells show basophilic granules above and basal
striated chromophil substance below the nuclei. Mitotic division cannot be
found among these cells. In the upper part of the tubules and in the neck
there are cells which are similar in structure but contain lighter staining
cytoplasmic granules and slightly compressed nuclei. These are the mucous
neck cells. Mitoses occur occasionally among these cells. Scattered among
the serous chief and mucous neck cells but occurring in particularly large
numbers in the upper part of the tubules are the parietal cells which are
large, round or wedge-shaped cells with clear acidophilic cytoplasm. The

I20 BIOLOGY OF THE LABORATORY MOUSE

nuclei are large and round and may be found two in a cell. Mitoses are not
found among them.

The gastric pits of the pyloric region are deep and the gland tubules
are short. The glands are lined by columnar cells in which the nuclei are
near the base and the cytoplasm contains fine granulations. These cells
resemble the mucous neck cells of the gastric glands and are mucous cells.
The transition between the fundic and pyloric glands is gradual.

The lamina propria of the glandular part of the stomach separates the
gland tubules from each other, forms the walls of the foveolae and fills
the spaces between the glands and the muscularis mucosae. It consists of
connective tissue containing fibroblasts, lymphocytes, some eosinophil
leukocytes and plasma cells. The muscularis mucosae in both the glan-
dular and non-glandular parts consists mainly of longitudinally arranged,
smooth muscle fibers. In the glandular part thin strands of smooth muscle
project between the glands. The submucosa is composed of loose connec-
tive tissue and contains blood and lymph vessels. The muscularis externa
is thin in the non-glandular and better developed in the glandular part. It
consists of an irregular inner oblique, a well developed middle circular and
a thin outer, longitudinal, smooth muscle layer. The circular layer is par-
ticularly well developed at the pylorus. About the organ is a serous mem-
brane consisting of loose connective tissue containing adipose cells and
covered by mesothelium. Frequently a solitary lymph node is present in
the serosa at the lesser curvature.

The small intestine. — The small intestine extends from the pyloric
valve to the caecum (about i8 inches). It may be divided into three parts:
the duodenum, the jejunum and the ileum.

The inner surface of the small intestine is covered with villi, delicate
finger-like projections of the mucous membrane. Phcae circulares are not
present. The villi of the duodenum are tall and leaf-shaped, being wide at
the base and narrow at the tip (Fig. 55). In the jejunum and ileum they
are cyUndric in shape, tall in the former and short in the latter (Fig. 56).
Between the villi are the openings of the simple tubular intestinal glands
(crypts of Lieberkiihn). The surface of the villi and the areas between them
are covered by simple, very tall, columnar epitheHal cells, having oval
nuclei situated in their lower thirds and striated cuticular borders at their
free surfaces. These epithelial cells continue into the glands, becoming
somewhat shorter near and at the base. The cells near the base of the
glands show numerous mitoses. Oval-shaped goblet cells are present,
scattered among the columnar cells. The nuclei are pushed to the base,

HISTOLOGY

121

and the mucigcn-coiitaining upper parts of the cells are distended. They are
especially numerous in the ileum. Some of the cells lining the bases of the
tubules contain acidophilic cytoplasmic granules above their nuclei. These
are the cells of Paneth, which become more conspicuous after several hours
of fasting and are most frequent in the jejunum.

^ Villi

Epithelium

Lamina propria

''^.';

••• • • • , - •

Fig. :;6.

Intestinal gland

Muscularis mucosae

Submucosa

• Circular muscle
layer

Longitudinal muscle
layer

Serosa

-Ileum, longitudinal section. (X200.)

The lamina propria forms the center of each villus and tills the spaces
between the glands. It is composed of reticular tissue containing many
lymphocytes, some granular leukocytes (especially eosinophils), and plasma
cells.

The muscularis mucosae is very delicate. The submucosa consists of
loose connective tissue. In the submucosa of the pyloric valve and extend-
ing for a few millimeters along the wall of the duodenum, coiled, tubulo-
alveolar mucous glands are present. These are the duodenal glands (of
Brunner) (Fig. 55). Although they are located in the submucosa, some
parts of the glands may be present in the mucosa. The glands are lined

122 BIOLOGY OF THE LABORATORY MOUSE

by cuboidal epithelium with spherical nuclei and pale staining cytoplasm.
Their excretory ducts open at the bases of the intestinal glands. In the
submucosa near the entrance of the pancreatic duct small groups of pan-
creatic acini are often present. An inner well-developed circular and a
thin outer longitudinal smooth muscle coat covered by the serosa complete
the wall.

Solitary lymph nodules occur in the lamina propria of the small intestine.
Aggregations of lymph nodules known as Peyer's patches also occur.
Where these are present the villi are absent or shortened. These aggre-
gated nodules extend into the submucosa and are covered only by the thin
muscle coat and serosa. They cause a bulging of the outer surface which is
visible grossly. Our observations are in accord with those of Hummel (54)
in rats, who found that variations exist in the number and location of the
patches, although in position the nodules are usually opposite the attach-
ment of the mesentery.

Each villus usually contains a central, endothelial-lined, lymphatic
vessel, the lacteal, which drains the absorbed fat or the white lymph (chyle).
The lacteals anastomose with lymph vessels of the muscularis mucosae.

The large intestine. — The large intestine consists of the caecum, the
colon and the rectum.

The caecum. — The caecum is a curved, blindly ending sac which com-
municates with the ileum and the colon. At its inner curvature it has
several transverse folds, while most of its surface is smooth. Villi are not
present. The epithelial cells of the lumen and the glands are like those in
other parts of the large intestine. The lamina propria of the proximal part
contains few lymphocytes. The muscularis mucosae is well developed and
takes part in the formation of the transverse folds. The distal, blind end
contains an aggregation of lymphatic tissue between the surface epithelium
and muscularis externa. The inner circular, smooth muscle layer of the
muscularis externa is well developed, while the outer longitudinal layer is
thin. The serosa is like that of the small intestine. The caecum does not
end in a typical vermiform process.

The colon and the rectum. — The colon and the rectum are devoid of
villi. Except that the tubules are straighter and slightly longer, the glands
of the large intestine are similar in structure to those of the small intestine.
Goblet cells are present in large numbers, while the cells of Paneth are
absent. The free surface between glands is covered by simple columnar
epithelial cells with striated borders. The lamina propria contains the
same elements described in the small intestine. The muscularis mucosae is

HISTOLOGY

123

poorly developed in the colon and well developed in the rectum. The mus-
cularis externa and the serous membrane are like those of the small intes-
tine. Taeniae are absent (56).

Epithelium

Goblet cell

Intestinal gland

Lamina propria

Muscularis mucosae

Solitary follicle

ibmucosa

-Muscularis externa

Fig. 57. — Colon, longiludinal section. (X200.)

Solitary lymph follicles are present in varying numbers. They extend
into the submucous layer. Peyer's patches are also found. The ascending
part of the colon contains several (about fourteen) parallel transverse folds,
which are made up of the mucous membrane (Fig. 57). In the descending
colon and rectum the fecal material is pellet-shaped. Where pellets are
found the lumen is slightly distended and smooth, while between them
longitudinal folds (colic ridges) are present (55). The first part of the

124 BIOLOGY OF THE LABORATORY MOUSE

rectum is similar structurally to the colon. There is a gradual increase in
the thickness of the inner circular smooth muscle layer of the muscularis
externa. The serous covering is replaced by the loose connective tissue of
the adventitia, which attaches the rectum to the surrounding tissue.
Toward the anal opening the glands become shorter and disappear as the
lining of the lumen changes into thick, stratified squamous epithelium which
is continuous with the thin, stratified squamous epithelium of the skin.
The musculature at the anus is composed of striated muscle fibers. Sur-
rounding the anus are the anal glands. They are similar structurally to
sebaceous glands and are arranged in small lobules separated by con-
nective tissue.

Mesenteries. — The stomach and the intestines are attached to the
abdominal wall by the mesogastrium (omentum) and mesenteries respec-
tively. These are thin, transparent membranes composed of loose connec-
tive tissue containing many adipose cells, lymphocytes and granular
leukocytes, and covered on their free surfaces by mesothelium. They con-
tain many blood and lymph vessels.

Arteries enter and leave the intestinal walls through the mesentery. In
the submucosa they form a network which in the small intestine gives off
two kinds of branches, both of which enter the muscularis mucosae. Some
of the arterial branches supply the intestinal glands with dense capillary
networks, others supply the viUi. The latter enter the base of each villus
and form a capillary network which is in close proximity to the epithelium.
At the tip of the villus the capillaries collect into veins which have a parallel
course with the arteries.

Liver and Pancreas

The liver. — The liver consists of four main lobes: a large median, a right
and left lateral and a left caudal. All the lobes except the left lateral lobe
are partially divided by deep bifurcations. The gall bladder is attached to
the caudal surface of the median lobe.

From the very thin connective tissue capsule covering the lobes, strands
of connective tissue project into the gland and form the supporting frame-
work, or capsule of Glisson, for the interlobular vessels and bile ducts.
This framework is very poorly developed and divides the gland into indis-
tinct polygonal areas, the lobules. In the center of each lobule is the cen-
tral vein, surrounded by anastomosing, radially arranged cords of liver
cells (Fig. 58). The central veins are intralobular tributaries of the hepatic
veins. The interlobular vessels which are surrounded by the capsules of

HISTOLOGY

125

Glisson are branches of the hepatic artery and of the portal vein. Accord-
ijig to Higgins (47), "Two rather large hepatic ducts accompany each main
branch of the portal vein, through the lobes of the liver, while usually but a
single one follows the smaller distal branches of the vein. Branches of the
hepatic vein are not associated with bile ducts."

The cords of liver cells radiating from the central veins are separated
from each other by the hepatic sinusoids, which connect the branches of the
portal veins with the central intralobular veins. They also receive blood

Intralobular
vein

Capsule

Gall bladder

Liver and gall bladder. (X75.)

from branches of the hepatic artery. The network of reticular fibers which
surrounds the sinusoids is a continuation of the fibers of the capsule of
Glisson.

The sinusoids are lined by two kinds of cells: the undifferentiated
reticular cells, possessing small, dark staining, elongated nuclei, and the
stellate cells of Kupffer, containing large oval nuclei. The cells of Kupffer
are phagocytic and often contain pigment granules.

The liver cells are large polygonal cells with large round nuclei. The
presence of two nuclei in a cell is frequent. Mitosis is rarely seen in the
normal liver of an adult animal. The cytoplasm is extremely variable in
appearance, depending on the functional state and the amount of glycogen
or fat in the cell. The cell outline is often indistinct.

The bile canaliculi are present between adjacent liver cells and require
special methods for demonstration. The canaUculi of one liver cell cord

126 BIOLOGY OF THE LABORATORY MOUSE

receive short lateral branches from adjoining cords. These collect into the
interlobular bile ducts, which are lined by cuboidal epithelial cells and are
in proximity to the branches of the portal vein. As the tributaries of each
lobe come together they form the hepatic duct. The common bile duct,
formed by the hepatic and cystic ducts, also receives a branch of the
pancreatic duct before entering the duodenum. The common bile duct is
lined by tall columnar epithelial cells.

The gall bladder. — The gall bladder is attached to the posterior surface
of the median lobe of the liver. It is lined by simple columnar epitheHal
cells which have cuticular borders. The thin lamina propria is composed
of connective tissue. The mucous membrane forms many folds when the
wall is not distended. The irregularly arranged smooth muscle fibers form
a very thin layer. A delicate loose connective tissue sheet, part of the
peritoneum, covers the free surface of the organ. The cystic duct connects
the gall bladder with the common bile duct.

The pancreas. — The pancreas is a compound acinous gland, composed
of many irregularly shaped lobes of varying size, distributed in the mesentery
of the duodenum with its distal end in close proximity with the spleen.
The lobes are covered by a thin connective tissue membrane which also
divides them into lobules. The small secretory acini, which resemble those
of the parotid gland, are composed of polyhedral shaped cells with dark
staining round nuclei near the bases. Below and around the nucleus the
cytoplasm stains deeply with basic stains, while above it the cytoplasm is
light and contains granulations (zymogen). Mitotic figures are rare
although they occur occasionally. Cytological variations exist due to the
difference in the functional phase of the gland, and perhaps also due to
the fact that the gland undergoes post mortem changes very quickly.

The secretion is collected in minute intercalated ducts lined by flat
epitheHal cells. As these ducts enter the acini they appear to be continuous
with the centro-acinous cells. These cells are flattened, have relatively
large, dark staining nuclei and a small amount of cytoplasm. The intra-
lobar ducts are lined by cuboidal epithelial cells. One duct of the gland
enters the common bile duct, while another enters the duodenum close
to the entrance of the bile duct. At the entrance of the pancreatic duct into
the duodenum small groups of pancreatic acini are usually present in the
submucosa of the duodenum. In obese animals adipose cells are found
in the interstitial tissue of the gland.

Irregularly distributed among the acini or the interstitial tissue are
the pancreatic islands (of Langerhans), which function as glands of internal

HISTOLOGY

127

secretion (Fig. 59). They are separated from the surrounding tissue by thin
membranes. The cells of the islands are round, cuboidal or polyhedral in
shape and form irregular cords. The round nuclei stain faintly. By special
staining methods the presence of cytoplasmic granules can be demonstrated
in the cells, showing differences in their staining reaction. Between the
cords of cells, capillaries provide an intimate blood supply.

Pancreatic acinus

Blood vessel

Duct

Pancreatic island

Fig. 59. — Pancreas with pancreatic island.

Respiratory System

(X200.)

The larynx. — The larynx connects the pharynx with the trachea. Its
walls contain cartilages covered by mucous membrane. The cartilages of
the larynx are mostly hyaline; only in the epiglottis and in the vocal process
of the arythenoid are elastic fibers present in the hyaHne ground substance.
The entrance of the larynx is guarded by the epiglottis which is attached
by a stalk antero-ventrally to the thyroid cartilage. The lingual and the
upper part of the laryngeal surface of the epiglottis and the aryo-epiglottic
folds of the larynx are covered by stratified squamous epithelium. At the
base of the epiglottis the epithelium changes from a stratified squamous type
into a pseudostratified ciliated columnar type which extends over the entire
surface of the larv'nx, except the true vocal cords.

The lamina propria of the epiglottis is continuous with and firmly
attached to the perichondrium. The mucous membrane is rich in glands.

128 BIOLOGY OF THE LABORATORY MOUSE

Small accumulations of lymphatic tissue are occasionally present near the
base of the epiglottis. Projecting into the glottis are the false vocal cords,
paired folds of the mucous membrane. Below these the true vocal cords
arise. Between the false and the true vocal cords are lateral pouches, the
ventricles of the larynx. The surface of the true vocal cords is covered by
stratified squamous epithelium. The lamina propria consists of dense
fibrous elastic tissue below which groups of striated muscle fibers are present.
There are no glands in the mucous membrane of the true vocal cords. With
the exception of these areas, glands are present throughout the entire length
of the larynx. They consist of small groups of branched alveolar glands
containing serous or mucous secreting acini and opening through short ducts
into the lumen.

The cartilages of the larynx in old animals often show calcification.

The trachea and the main bronchi. — The trachea is continuous with the
larynx. Its lumen is lined by pseudostratified ciliated columnar epithelium.
The fibrous lamina propria is rich in blood vessels. The rigidity of the wall
is due to the presence of hyaline cartilage rings. The rings are incomplete
and one end of each ring is connected by smooth muscle fibers to the opposite
end, forming the dorsal membranous wall of the trachea. The attachment
of the musculature is on the outer (dorsal) side of the cartilage. The mucous
membrane of the membranous wall is thrown into longitudinal folds. The
perichondrium which surrounds each cartilage is continuous with the lamina
propria. Only at the cephalic end of the trachea are there glands present
in the lamina propria between the cartilage rings. Loose connective tissue
forms the adventitia and attaches the trachea to the surrounding tissues.

The trachea divides into two main bronchi which are similar to it in
structure. Here the cartilages form small irregular plates that completely
surround the tube. Circular smooth muscle fibers complete the wall.
Cartilage is not present in the walls of the bronchi beyond the point where
they enter the lungs.

The lungs. — The thoracic cavity is lined by and divided into right
and left pleural sacs by a very thin membrane, the pleura. The membranes
of the two sacs meet in the median plane and form the mediastinal septum.
The lungs are covered by the visceral pleura, a thin serous membrane
composed of connective tissue containing collagenous and elastic fibers and
covered by mesothehum. The left lung has one, the right four lobes. The
inferior median lobe of the right lung is separated from the inferior lateral
lobe by the inferior vena cava, and is enclosed in a separate pleural sac
(Lauche in Jaffe, 56).

HISTOLOGY

129

Each lobe receives, at its hilus, a branch from one of the two main
bronchi. These branch repeatedly, gradually diminishing in size. The
large bronchi of the lung are Uned by pseudostratified ciliated columnar
epithelial cells, among which goblet cells are present in varying number.
The lamina propria consists of fibrous connective tissue containing elastic
fibers. The mucous membrane forms longitudinal folds. Beneath this

Branch of pulmonary
vein

Bronchium

Artery

membrane delicate smooth muscle fibers complete the wall. The bronchial
tubes of the lung do not contain any cartilage. In the smaller bronchi the
epithelial lining is simple columnar and ciliated. Short terminal bronchioles
are formed by the division of the smaller bronchi and are lined by low
columnar cells, cilia and goblet cells being absent.

The terminal bronchioles give rise to respirator}- bronchioles, each of
which in turn forms several alveolar ducts. Alveolar sacs, containing
several alveoli, open from the alveolar ducts and form intercommunicating
spaces separated from each other by thin walled septa in which capillaries
anastomose. The respiratory bronchioles are lined by cuboidal epithelial
cells, which are surrounded by connective tissue containing elastic and

I30 BIOLOGY OF THE LABORATORY MOUSE

collagenous fibers. Elastic and reticular fibers are present in the inter-
alveolar septa. The cells which line the alveolar walls are the so-called
"septal cells." They are large flat cells with oval nuclei and are closely
attached to the walls of the capillary network. The entodermal or mesen-
chymal origin of these cells is still uncertain. The alveolar wall of the mouse
contains a varying number of lymphocytes and occasionally granular
leukocytes.

The lungs receive blood from the branches of the pulmonary arteries
which follow the course of the bronchi. From these an arteriole passes to
each alveolar duct and forms the network of capillaries in the walls of the
alveoli. The pulmonary veins are formed by capillaries of the alveolar
septa and of the pleura, and follow the course of the bronchi. The smaller
bronchial arteries supply arterial blood to the wall of the bronchi and collect
into the bronchial veins. The media of the walls of the veins in the lung
are composed of cardiac muscle fibers (Fig. 60).

Urinary System

The kidney. — The kidney is a compound tubular gland composed of
uriniferous tubules enclosed within a thin connective tissue capsule. A
rrfedian section through the middle of the kidney shows a division into a
cortical part containing mostly convoluted tubules, and a medullary part
containing radially arranged straight tubules. The medulla is pyramidal
in shape with the broad surface outward, and the apex ending in a single
nipple-shaped dorsoventrally flattened papilla (Fig. 61). Columns of
straight medullary tubules project part way into the cortex where they form
the medullary rays.

The uriniferous tubules of the mouse are similar in structure to those of
man, and for their detailed description the reader is referred to Maximow
and Bloom (73).

It has been reported that in some mice the parietal or capsular epithelium
of the capsule of Bowman consists partially or entirely of cuboidal epithelial
cells (20, 42). Such capsules appear in greater number in the male than in
the female animals (20).

The circulation of the mouse kidney is similar in general to the circulation
in the human kidney (56). In the media of the glomerular arteries, in
addition to the ordinary smooth muscle cells, larger, more afibrillar cells are
present. These are similar to the cells described by Goormaghtigh (40)
who states that they tend to accumulate in groups at the vascular poles of
the glomeruli and form the ''juxtaglomerular apparatus" (41).

HISTOLOGY

131

The renal pelvis and ureter. — The funnel shaped pelvis surrounds the
renal papilla. In its wide part the epithelial lining consists of a single
layer of squamous cells which change gradually toward the narrow part first
to polyhedral, then to two or three layered, still farther to four or five
layered transitional type. At the wide part the lamina propria is very
delicate and becomes better developed at the narrow part. An inner circu-

Glomerulus

Renal vein

— Renal artery

Renal pelvis

Capsule

Fig. 61. — Kidney, region of pelvis and papilla. (X30.)

lar smooth muscle layer appears first about at the level where the epithelium
becomes stratified, while still lower an outer longitudinal smooth muscle
layer is also distinguishable. Outside of this, loose connective tissue con-
taining many adipose cells surrounds the narrow end of the pelvis and the
ureter which arises here (Fig. 61).

The ureter is a narrow tube which conducts the urine from the kidney
to the bladder. Its wall is composed of transitional epithelium, a fibrous
lamina propria, an inner circular and an outer longitudinal smooth muscle
coat and the adventitia consisting of loose connective tissue and many

132

BIOLOGY OF THE LABORATORY MOUSE

adipose cells. The mucous membrane forms low longitudinal folds. The
ureters enter the dorsal wall of the neck of the bladder close to one another.
Bladder. — The bladder is lined by transitional epithelium consisting,
when the organ is empty, of about four to five layers of cells. The fibrous
lamina propria is rich in blood vessels. The mucous membrane is thrown
into wide irregular folds and occasionally contains an aggregation of lympho-
cytes. When the bladder is in a distended condition the folds are absent
and the epithelial lining is very thin. The smooth muscle coat consists of

Epithelium

Blood vessel

Lamina propria

Fig. 62. — Bladder. (X200.)

irregular muscle bundles of varying size, separated from each other by
considerable amounts of connective tissue (Fig. 62). At the neck of the
bladder the direction of the muscle bundles is circular.

Female urethra. — The female urethra is a dorsoventrally slightly flat-
tened tube which originates at the neck of the bladder and opens into the
clitoral fossa. Near its origin the tube is lined by transitional epithelium
which soon changes into stratified squamous type. The lamina propria is
formed by loose connective tissue. The mucous membrane forms longitu-
dinal folds. The epithelium forms invaginations which are continuous with
gland tubules of the urethral glands. These glands are similar in structure
to the urethral glands (of Littre) in the male. The circularly arranged
smooth muscle fibers forming the outer wall are well developed. Near the

HISTOLOGY 133

clitoris striated muscle fibers are also present. Loose connective tissue
attaches the urethra to the ventral wall of the vagina.

Male Genital System

Figure 63 shows the dissected male genital system which includes the
testes, a system of excretory ducts, the accessory glands, the urethra and
the penis.

The testis and its excretory ducts. — The testis is a compound tubular
gland in which the male sex cells, the spermatozoa, develop. The organ is
covered by a fibrous connective tissue capsule, the tunica albuginea, from
which, at the hilus, thin septa project into the gland and divide it into
lobules. The lobules contain the convoluted seminiferous tubules. Arteries
enter at the hilus, form a network on the inner surface of the tunica albu-
ginea, penetrate with the septa, form a capillary network among the semi-
niferous tubules and collect into veins, the courses of which correspond with
the courses of the arteries.

The tubules are lined by seminiferous epithelium resting on a basement
membrane which, in turn, is surrounded by a thin layer of fibrous connective
tissue. The interstitial stroma is rich in blood and lymph vessels and con-
tains small groups of interstitial cells (of Leydig). The seminiferous epi-
thelium is composed of two kinds of cells, the sustentacular Sertoli cells and
the spermatogenic cells.

Under normal conditions the Sertoli cells lie near the basement mem-
brane and are spaced at fairly regular intervals. The cells have large, oval,
often indented nuclei and contain a compound nucleolus consisting of one
central acidophil and two peripheral basophil bodies. When the cell is
resting the nucleus is parallel with the wall of the tubule and the cell is
polygonal in shape. When it is fulfilling its function of supporting the
developing spermatogenic cells, the nucleus is perpendicular to the wall and
the cell is pyramidal in shape. Under abnormal conditions, resulting in the
degeneration of the seminiferous cells, the highly resistant Sertoli cells
alone line the tubules and their cytoplasm forms a shapeless syncytium.

The primary spermatogenic cells, the spermatogonia, initiate spermato-
genesis by repeated cell division. As the spermato- and spermiogenesis of
the mouse do not differ in essentials from other mammals, for a detailed
description the reader is referred to Maximow and Bloom's Textbook of
Histology (73). Certain phases of spermatogenesis of the mouse are dis-
cussed by Cutright (21), Cox (19) Regaud (82) and Yocum (97).

134

BIOLOGY OF THE LABORATORY MOUSE

Ampullary glands

Ductus deferens^ \

Membranous
urethra

Vesicular gland

Coagulating gland

Bladder

Ventral prostate

IschiocavernosuE
muscle

Bulbo-urettiral
gland

Bulbocavernosus
muscle

Bulbo-urethral
gland

Urethral

diverticulum

Corp. cav. urethra

Corp. cav. penis
Preputial gland

Vesicular gland

Ampullary gland

Membranous urethra-

Coagulating gland
Dorsal prostate
Ventral prostate

Fig. 63. — Drawings of the male genital system. A. Ventral view. The urethra
is completely straightened. The left corpus cavernosum penis and bulbocavernosus
muscle (right side of the drawing) are dissected, to show the urethral diverticulum, and
the bulbo-urethral gland. On the right side (left side of the drawing) these structures
are in normal position. The preputial sack is cut open. (X2M.) B. Dorsal view
of the cephalic end of the male urethra. {X2%.)

HISTOLOGY

135

The spermatozoon is composed of the head, the middle piece and the
tail or fiagellum. The head is flattened and hook-shaped, and ranges from
.0068-.0102 mm. in length with a mean length of .0081 mm. The total
length ranges from .1190-.1265 mm. with a mean length of .1227 mm.
(Figures are based on 30 measurements made by Margaret Nickson.)

Fatbody^

'Caput epididymidis

Ductuli efferentes-

x -\

-•5,

Corpus epididymidis

J — Ductus deferens

Testis

"Cauda epididymidis

Fig. 64. — Drawing of the testis, efferent ducts, epididymis and ductus deferens. ( X6.)

It was mentioned previously that the intertubular tissue contains small
groups or cords of interstitial cells of Ley dig. These cells have large round
nuclei which contain one, or more often two, nucleoli and rather coarse
chromatin granules. The cytoplasm stains intensely with eosin.

The excretory ducts of the testis include the rete testis, the efferent
ducts, the epididymis w^hich has three parts, the caput, corpus and cauda,
and the ductus deferens (Fig. 64).

At the hilus the seminiferous tubules are collected into the network of an
anastomosing system of canals, the rete testis, which is lined by simple, low
cuboidal or at some places flattened epithelium. The network opens into a
single lacuna which, outside the tunica albuginea, branches into as many
parts as the number of efferent ducts. According to Benoit (6) this number

136

BIOLOGY OF THE LABORATORY MOUSE

varies from three to seven. The number of efferent ducts in about 10
animals examined by us was three to five. The efferent ducts have two
parts: beginning at the testis, in the first part the ducts have a short,
straight, then convoluted course and are surrounded directly by the fat
body of the testis; in the second part the ducts are highly convoluted and

Ductus epididymidis

Capsule Efferent duct joining the ductus epididymidis Efferent duct

Fig. 65. — Efferent duct joining the ductus epididymidis. (X200.)

are surrounded by a connective tissue capsule which becomes continuous
with the capsule of the epididymis. The efferent ducts enter and form
the first small segment in the caput of the epididymis. Our own observa-
tions are in agreement with those of Benoit (6) and Young (98) who found
that the efferent ducts unite into a single duct which is in continuity with
the duct of the epididymis (Fig. 65).

The lining of the efferent ducts is composed of alternating groups of
tall and low columnar epithelial cells, which give the lumen a characteristic
scalloped outline. The epithelial cells rest on a basement membrane.

HISTOLOGY 137

below which a few circularly arranged smooth muscle fibers complete the
wall.

The epididymis is covered throughout its entire length by a continuous
sheath of fibrous connective tissue, which in the caput projects in and divides
the convoluted tubules into seven to eight segments or lobules. As stated
previously the first one of these lobules contains efferent ducts. In the
second lobule the lumen of the ductus epididymidis is lined by very tall
columnar epithelial cells. In most cells the oval nucleus is located in the
lower third of the cell, while in some the nucleus is in a higher position.
The cells possess non-motile stereocilia. Beginning at the third segment the
epithelium lining the duct becomes considerably lower; the nuclei are at an
even height and the duct is narrower. Approaching the cauda the duct
becomes wider. On the inner surface of the basement membrane small
round basal cells are present. The cross section of any part of the duct
shows a very regular circular outline. A few circularly arranged smooth
muscle fibers complete the wall.

As the ductus epididymidis leaves the cauda it becomes the ductus
deferens. This duct is lined by tall columnar epithelium which at some
places seems to be pseudostratified. The lamina propria is formed by fibrous
connective tissue and the mucosa forms several prominent longitudinal
folds. An inner circular and an outer longitudinal smooth muscle coat
form a rather thick wall. Loose connective tissue, the adventitia, covers
the duct (Fig. 66). Before entering the urethra the duct opens into the
ampulla through a papuliferous projection. The epithelium changes sud-
denly, and the ampulla and its narrow neck, which connects it with the
urethra, are lined by low columnar cells which have large, oval, deeply
staining nuclei and small amounts of cytoplasm. The deHcate lamina
propria is surrounded by smooth muscle fibers. The mucous membrane
forms many deep folds (Fig. 67).

The accessory glands. — Before giving the histological details of the
accessory glands, a few general remarks are needed. The seminal vesicles
are correctly referred to in the more recent literature as vesicular glands,
because they do not contain or store spermatozoa but produce a secretion.
The naming of the lobes of the prostate may cause confusion. There are
three pairs of prostate glands, one pair of which is attached to the lesser
curvatures of the vesicular glands. Because the secretion of this gland,
according to Walker (95), produces coagulation when mixed with the
secretion of the vesicular gland, it is often referred to as the coagulating
gland, and this name will be adopted in this text. Rauther, (81) in a draw-

138

BIOLOGY OF THE LABORATORY MOUSE

ing which has been frequently reproduced, illustrated the male urogenital
system of the mouse and labelled this gland prostate I. The other two
prostates are dorsally and ventrally located and will be designated in the
text as dorsal and ventral prostates. Rauther referred to these as prostate
II and III respectively. Occasionally the two lobes of the dorsal prostate
are connected by a very small median lobe, but more often this lobe is

— ^Epithelium
Lamina propria

Circular mus-
cle layer

Longitudinal
muscle layer

Fig. 66. — Ductus deferens. Fixed in Bouin's fluid. (X66i2-)

absent. Around the base of the ductus deferens is a small group of glands
whose ducts open into the ampulla. These are the ampullary glands (Fig.

63)-

Figure 67 is a composite drawing of three adjacent sagittal sections of
the urethra, slightly lateral to the midline, showing the entrance of some of
the structures joining it. A short description of the more lateral sections
is needed. The lateral wall of the cephalic end of the urethra is surrounded
by the coagulating gland and the ventral and dorsal prostates. The dorsal
prostate has many ducts, some of which are lateral to all the other ducts
entering the urethra. Each coagulating gland has two ducts; they enter
the dorsal wall of the neck of the bladder. The ventral prostate has several
ducts which have a curved course caudad to the neck of the bladder, and
enter the ventral wall of the urethra. The ductus deferens opens into a
vestibule, the ampulla, which narrows down considerably before entering
the urethra. Each vesicular gland has a rather wide duct and enters in

HISTOLOGY

139

close proximity and dorsal to the neck of the ampulla. According to
Dissclhorst (28), the ducts of the vesicular gland and ductus deferens (neck
of ampulla) join to form the ejaculatory duct before entering the urethra.
In six animals examined by us the neck of the ampulla and the duct of the
vesicular gland entered separately. However, variations exist and in a
seventh animal the two ducts joined on the right side but entered separately

AmpuUary gland Ductus deferens Ampulla

Vesicular gland

Dorsal prostate

Duct of dorsal prostate

Striated muscle

Duct of vesicular gland

Duct of coagulating gland

Neck of bladder

CoUiculus seminalis

Urethral glands

Urethra

Ventral prostate Bladder

Fig. 67. — Cephalic end of the male urethra. A composite drawing of three adjacent

sagittal sections. (X13.)

on the left side. The entrance of the neck of the ampulla and vesicular
gland form a caudally diminishing elevation on the dorsal wall of the urethra,
the colliculus seminalis.

AmpuUary glands. — The ampullary glands are groups of branched
tubular glands which open into the vestibular part of the ampulla. They
are lined by low columnar cells having large oval nuclei. The lamina
propria is very thin and the mucous membrane is thrown into many delicate,
deep, longitudinal folds. The tubules are surrounded by a very thin laver

140

BIOLOGY OF THE LABORATORY MOUSE

of circular smooth muscle fibers and held together in groups by a thin con-
nective tissue membrane (Fig. 68). The color and apparent consistency of
the secretions of the accessory glands in preparations stained with hema-
toxylin and eosin is characteristic and helpful in identifying them. The
tubules of the ampullary glands contain an intensely red staining, dense,

Secretion

Fig. 68. — Ampullary gland. (X200.)

homogeneous secretion which has a tendency to shrink away from the
epithehal lining and lie free in the lumina.

Vesicular glands. — The vesicular glands are long, narrow and curved at
their tips. Internally there is a large, elongated cavity with medial alveolar
outpocketings. The epithelial lining consists of a single layer of tall
columnar cells having distinct cell outlines. The large oval nuclei are near
the bases of the cells. The slightly basophilic cytoplasm contains dark
secretion granules which are surrounded by lighter staining areas (halos)
(Fig. 69). When the lumen is distended by secretion the epithelial cells
are lower and do not contain secretion granules. The mucosa, especially on
the side containing the alveolar outpocketings, is thrown into many fine,
intricate folds. The gland is surrounded by smooth muscle fibers and

HISTOLOGY

141

covered by a connective tissue sheath. The secretion, in sections stained
with hematoxyUn and eosin, is intensely red and has a tendency to crack
and form parallel fissures.

Epithelium

Secretion

Fig. 60. — \'esicular gland.

(XI75-)

Coagulating glands. — The branched, tubular coagulating glands
are lined by simple columnar epithelial cells having round, centrally
located nuclei and eosinophilic cytoplasm. The mucous membrane forms
curved longitudinal folds, some of which project far into the lumen (Fig.
70). Even in the distended tubules some mucous folds are almost always

Epitbelium

■Secretion

Fi(,. 70. ('o;i<;ulating gland. (X200.)

present. Each coagulating gland usually has two ducts which are lined by
low columnar epithelial cells having deeply staining nuclei and slightly
basophilic cytoplasm. Due to the folds present in the mucosa the ducts
of these glands have a wavy lumen on section. The gland tubules are
surrounded by a delicate layer of circular smooth muscle fibers and have a
common connective tissue covering which attaches the gland to the lesser

142

BIOLOGY OF THE LABOILiTORY MOUSE

curvature of the vesicular gland. The secretion is a faintly pink (H. E.
stain) homogenous substance which forms fissures in sections of the
larger tubules.

^^i^

^WKF^

■«. •

-Epithelium

Secretion

Fig. 71. — Dorsal prostate.

Dorsal prostates. — The tubules of the dorsal prostates (Fig. 71) although
considerably narrower, are very similar structurally to those of the coagulat-
ing glands. The color and apparent consistency of the secretion in section is

-Epithelium

J ^ 'uBi^t •'l^— Secretion

Fig. 72. — \'entral prostate. (X200.)

also similar; perhaps because the narrower tubules contain less secretion,
the formation of fissures is rare. The gland has several ducts in which the
mucous membrane, in contrast to the ducts of the coagulating gland, is free
from folds.

HISTOLOGY 143

Ventral prostates. — In the ventral prostates (Fig. 72) the gland tubules
are lined by low columnar epithelium, having deeply staining spherical nuclei
and slightly basophilic cytoplasm. The distended tubules do not contain
mucous folds; in the smaller tubules folds are present. The secretion in a
stained preparation shows a tendency to form round, pink staining globules
of varying size. The gland tubules have a thin circular smooth muscle coat
and are held together by a common connective tissue membrane.

The urethra, bulbo -urethral glands, penis and preputial glands. — The
neck of the bladder is lined by diminishing rows of transitional epithelial
cells. This changes into stratified squamous epithelium (two to three layers)
lining the ventral wall of the urethra. The colliculus seminalis is covered
by a continuation of the simple low columnar type of epithelium which
Hnes the ducts entering on this projection. Similar epithelium lines the
dorsal wall of the urethra. At a sHghtly lower level this also changes into
stratified squamous epitheHum which lines the membranous urethra
throughout its entire length. Loose connective tissue forms the lamina
propria, which is very rich in blood vessels and forms a framework for the
urethral glands present in the mucosa (Fig. 67). These glands, as well
as the thick layer of striated muscle fibers which surround them, appear on
the dorsal wall near to its cephalic extremity and spread gradually caudally
toward the ventral wall to form a complete sheath around the tube below the
neck of the bladder. The urethral glands (of Littre) are composed of small
groups of alveoli, the cells of which have oval nuclei near the base and
cytoplasm containing basophilic secretion granules. Their short ducts,
lined by cuboidal epithelial cells, open separately into the urethral lumen
at different levels.

The root of the penis is attached to the pubic bone by the crura which
are the terminal extensions of the corpora cavernosa penis. From the
enlarged base of the crus penis, the ischeum, the ischio cavernosus muscle,
arises and passes forward. The corpus cavernosum urethra proximally
expands into the urethral bulb, over which extend the bulbo cavernosus
muscle. The urethra forms paired lateral diverticula at the region of the
bulb (Figs. 63 and 73). The lumen of each diverticulum shows variations
in size and shape, depending on the amount of secretion present. It is
lined by transitional epithelium, the apparent thickness depending on the
dilated or relaxed condition. Below the epithelium, glands similar in
structure to those of the membranous urethral wall are present. The diver-
ticulum is surrounded b}- a fibrous membrane with circularly arranged,
smooth muscle fibers as an inner layer. From here trabeculae composed of

144

BIOLOGY OF THE LABORATORY MOUSE

fibrous connective tissue intermingled with smooth muscle fibers, project
among the glands and form endothelial lined cavernous spaces. When
these are distended, small groups of glands are widely separated from each
other. When they are collapsed the glandular tissue appears compact. A
heavy outer muscle sheath composed of striated fibers (m. bulbocavernosus)
involves the diverticulum.

Penis

Membranous urethra

Tail and duct of bulbo-
urethral gland

Urethral diverticulum

Bulbocavernosus
muscle

Fig. 73. — Sagittal section of the urethra showing the region of the bulb. Decalcified
section of the pelvic region. (X30.)

Bulbo-urethral glands. — The paired bulbo-urethral glands (glands of
Cowper) are retort-shaped organs composed of tubulo-alveolar glands. The
body is partially covered by the musculus bulbocavernosus, while the tail
runs throughout the muscles and its duct enters into the cephalic wall of
the urethral diverticulum (Fig. 73). The tail is an aggregation of small
gland lobules covered by a connective tissue membrane.

The body is surrounded by striated muscle fibers; inside this a very thin
connective tissue membrane involves the gland and, projecting inward,
forms the delicate inter-alveolar tissue. The tubules and alveoli are lined
by tall columnar epithelial cells which have small, dark staining nuclei
flattened against the bases of the cells. The cytoplasm stains pale blue with

HISTOLOGY

145

hematoxylin-eosin stains. The cells rest on a well developed basement
membrane. Occasionally the gland may be distended and contain large
central cavities into which the tubules and acini open directly. In the lower
part of the body a duct lined by cuboidal epithelium is present which anasto-
moses with the central ducts of the gland lobules of the tail. The gland
lobules of the tail are composed of small alveoli lined by low columnar cells
which have dark, round nuclei near the base and dark staining granular
basophilic cytoplasm. Small groups of light staining cells similar to those
present in the body are intermingled with these cells but in the part of the
tail near the urethra these disappear and only the dark staining cells are
present (Fig. 74). The long central duct of the lobule nearest to the urethra
opens into the urethral diverticulum between the aggregation of the glands

Tail of bulbo-urethral gland

Capsule

Head of bulbo-
urethral gland

Bulbocavernosus
muscle

Bulbo-urethral gland.

present in its lateral wall and the glands encircling the lumen of the muscular
urethra (Fig. 73).

According to Hall (44) the secretion of Cowper's glands gives a positive
reaction when stained specifically for mucin, while the glands of the urethra
and the sinus give a negative reaction.

The penis. — The body of the penis consists of the thin corpus cavernosum
urethrae and the two thick corpora cavernosa penis. The corpus caverno-
sum urethrae begins at the distal part of the diverticulum of the bulb, where
it lies between the crura of the corpora cavernosa penis. It is composed of
cavernous spaces surrounded by a layer of dense fibrous connective tissue,
the tunica albuginea, the inner surface of which contains a layer of circular
smooth muscle fibers. The cavernous spaces are formed by trabeculae con-

146 BIOLOGY OF THE LABORATORY MOUSE

sisting of fibrous tissue, containing elastic fibers and a few smooth muscle
fibers. The cavernous spaces are lined by endothelium. The urethral
lumen, which occupies the center of the body, is lined by stratified columnar
epithelium, which near the external orifice changes into stratified squamous.
The fibrous lamina propria becomes continuous with the surrounding cavern-
ous tissue. Glands are absent in the penial urethra. The proximal part of
each corpus cavernosum penis is surrounded by its own tunica albuginea.
Toward the distal part this becomes a narrow septum which finally dis-
appears, and the cavernous spaces intercommunicate. The cavernous spaces
are smaller near the periphery and larger toward the center. A small bone,
the OS penis, is found within the fibrous septum of the two corpora cavernosa
penis and projects somewhat beyond the orifice of the penis.

The terminal end of the penis, the glans, lies within a protective chamber,
the prepuce (or foreskin). The stratified squamous epithelium covering the
glans forms low filiform papillae which make the surface slightly rough.
Hair follicles are not present. The dense subcutaneous tissue contains
some smooth muscle fibers. The mucous membrane which lines the pre-
putial sack is a continuation of the covering of the glans (Fig. 63).

Preputial glands. — The large, flat, leaf-shaped preputial glands are
homologous with the clitoral glands of the female (Fig. 63). They are large
sebaceous glands surrounded by connective tissue capsule and consisting of
rounded areas made up of large, flat, polyhedral epithelial cells with pale
staining nuclei. The nuclei gradually disappear, and the cells degenerate
forming a fatty secretion. Each gland has a long duct lined by stratified
squamous epithelium which opens on the side of the tip of the prepuce.
Near the orifice the epithelial cells of the duct and the subcutaneous tissue
around it usually contain some pigment in non-albino animals.

Female Genital System

Figure 75 shows the dissected female genital organs which include the
ovaries, the oviducts, the uterine horns, the corpus uteri, the cervix and
the vagina. The following description of the attachment of the female
genital system is based on the observations of Drahn (29) (Fig. 76). The
ligamentum suspensorium ovarii which originates at the ovarian hilus
extends anteriorly to the lateral surface of the kidney and attaches to the
dorsal abdominal wall. This ligament contains some smooth muscles from
which fibers project for a short distance into the ovarian capsule, increasing
its elasticity and serving as constrictor muscles. The ligamentum ovarii
proprium connects the hilus of the ovary to the cephalic end of the uterine

HISTOLOGY

147

horn. This ligament is also rich in smooth muscle fibers which project into
the mesotubarium and to the infundibular muscle. The infundibular mus-
cle besides having connections with the above mentioned ligament has fibers
projecting to the ovarian hilus. A narrow connection exists composed

Ovary

Oviduct

Uterine horn

Uterine lumen

Corpus uteri

Vaginal fornix

Cervix

Vagina

Clitoral eland

Clitoris

Fig. 75. — Drawing of the ventral aspect of the female genital system. The
uterine horns as well as the corpus uteri, cervix and the vagina are cut open on the mid-
ventral line. (X3.)

partly of connective tissue, partly of smooth muscle fibers between the tube
and the ovarian capsule, and also with the ovary itself. Each uterine horn
is attached to the dorsal wall by the mesometrium (broad ligament) which
contains varying amounts of fat. Near the uterine horn the mesometrium
contains longitudinal, smooth muscle fibers which are continuous with the

148

BIOLOGY OF THE LABORATORY MOUSE

uterine musculature. Where the horns unite externally the mesometria
join and end on the dorsal wall of the corpus.

The Ovary. — The ovaries are paired glands in which the female sex
cells, the ova, develop. The free surface of the ovary is envolved in a thin
transparent membrane, the ovarian capsule, which encloses the periovarian

per. sp.

col. tub.

lig.o.

BUS

m. of cap.

inf. of m. inf. ist. lig. o. pr.

Fig. 76. — Reconstruction of the ovary, oviduct and part of the uterine horn.
{After Drawn.) o. v., ovarian vein; o. art., ovarian artery; per. sp., periovarian space;
cap., capsule; amp., ampulla; u. v., uterine vein; u. art., uterine artery; col. tub.,
colliculus tubarius; u. h., uterine horn; lig. o. pr., ligamentum ovarii proprium; ist.,
isthmus; inf., infundibulum; inf. of m., infundibulum-ovarial fold of muscular meso-
tubarium; m. of cap., musculature of ovarian capsule; h., hilus of ovary; Hg. o. sus.,
ligamentum ovarii suspensory.

space. The ovarian capsule consists of a thin membrane of connective
tissue covered on both surfaces by mesothelium. Small, blindly ending
vestigial tubules of the Wolffian body, the epoophoron, may be present in or
near the mesovarium. These are lined by cuboidal, often ciliated, epithe-
lium and are surrounded by a thin circular smooth muscle wall.

A cross section of the ovary of an adult mouse (Fig. 76) shows an inner
zone, the medulla (or zona vasculosa), and a surrounding outer zone, the
cortex. Blood vessels enter and leave the organ at the hilus. They con-

HISTOLOGY 149

tinue their course in the medulla which contains many large blood vessels
separated from each other by a rather dense fibrous stroma. The free
surface of the cortex is covered by a layer of cuboidal epithelial cells, the
germinal epithelium, beneath which a thin layer of dense fibrous connective
tissue forms the tunica albuginea. The primary folUcles are immediately
beneath the tunica albuginea, while those which are further developed are
more deeply located.

A primary follicle consists of a large spherical cell, the primary oocyte,
surrounded by a layer of squamous follicular cells. The nucleus of the
oocyte is vesicular, contains small chromatin granules and a prominent
nucleolus. Follicles which are somewhat further developed are lined by two
or more layers of cuboidal follicular cells. Each such follicle contains a
larger oocyte which is separated from the follicular cells by a transparent cell
membrane, the zona pellucida. The connective tissue cells of the stroma
are arranged concentrically around the follicle and form the theca folliculi.
Around the larger follicles this layer has an inner part, the theca interna,
which is rich in capillaries and contains large, loosely arranged cells, and an
outer part, the theca externa, which contains concentrically arranged dense
fibers.

In those folHcles in which the development is still further progressed,
small irregular spaces filled with a clear fluid, the primary liquor folliculi,
appear among the follicular cells. These spaces gradually open into each
other and form a single large fluid-filled cavity, the antrum. The antrum is
lined by a stratified layer of follicular cells which in this position sometimes
are called granulosa cells and which form the membrana granulosa. This
membrane is thicker in the region where the oocyte, encircled by a group of
follicular cells to form the cumulus oophorus, is attached. The follicular
cells which immediately surround the zona pellucida are elongated and
radially arranged. They are attached to the ovum by delicate cytoplasmic
processes and form the corona radiata. The formation of the antrum and
the increase in the amount of the liquor foUiculi enlarges the follicle. Due to
this expansion the follicle extends to the surface of the ovary and finally
bulges out into the periovarian space. Such a follicle is called a mature
vesicular or Graafian follicle. According to Brambell (11) the mean
diameter of a ripe follicle in a section is about 530 ju. A single follicle may
occasionally contain two or more ova.

Changes preceding and following ovulation.— Tht primary liquor
folliculi becomes more viscid as estrus approaches. Preceding ovulation
the secondary liquor folliculi is formed which is more fluid in character (85).

15°

BIOLOGY OF THE LABORATORY MOUSE

Small liquid filled cavities appear among the cells of the cumulus oophorus
and the granulosa cells which line the antrum. These gradually detach the
cumulus oophorus from the surrounding cells so that it floats free in the
antrum (Fig. 77). In the meantime, in the nucleus of the ovum which lies
near the surface of the cell, the nuclear membrane becomes faint, irregular
and gradually disappears. The nucleolus also disappears, and the chroma-

mm-

•i ■

Germinal epithelium

Tunica albuginea

Cumulus oophorus

Membrana granulosa

-Theca interna

Liquor foUiculi

Theca externa

Blood vessel

tin granules form small dense chromosomes (20 tetrads). Delicate achroma-
tic fibers form a rather narrow spindle and the chromosomes collect at the
equatorial plane. There are no centrosomes or astral radiations. As the
chromosomes begin to move toward the poles, 20 diads and a small amount
of the cytoplasm are separated from the ovum by constriction and the first
polar body is formed. It lies within the zona pellucida causing a bulging
of its surface. The nuclear material of the ovum does not return to a resting
condition. Instead preparations begin at once for the next division.

HISTOLOGY 151

At this stage the ovum is free in the cavity of the mature follicle, sur-
rounded by the cells of the cumulus oophorus. Ovulation occurs soon after
this condition is established.

Parallel with the changes taking place in the ovum, changes also occur
in the surrounding tissues. The large blood vessels of the medulla are
engorged and the capillaries around the Graafian follicles show congestion.
The thin wall of each follicle protruding into the periovarian space consists
of flattened germinal epithelium, stretched tunica albuginea, the cells of
which seem to be loosened by the congested capillaries, and one or two rows
of granulosa cells. The opposite wall is considerably thicker, consisting of
many layers of granulosa cells, and the theca interna which in section
appears to project in waves into the follicle. When the thin wall ruptures,
the ovum with the first polar body and the second polar spindle in the
process of formation, surrounded by the cells of the cumulus oophorus,
imbedded in liquor folliculi, are expelled into the periovarian capsule which,
consequently, becomes distended. Several ova escape in a relatively short
time interval, and due to the viscosity of the liquor folliculi they have a
tendency to clump. They remain in the periovarian capsule only for a very
short time, passing almost at once into the ampulla of the oviduct, which
becomes distended. Fertilization takes place here and if spermatozoa enter
the ova, the second divisions are completed. If fertilization does not take
place, further development does not occur and the ova fragment and
degenerate.

The ruptured follicle and the formation of the corpus leuteum. — After
the bulging wall of the follicle has ruptured the tension is relieved and
only a relatively small gap and cavity remain. The rupture does not
cause bleeding, and normal ovulation is seldom followed by the formation
of a hemorrhagic follicle. The free surface of the ruptured area contains
enlarged capillaries, and the rich blood supply probably facilitates the
rapid healing (Fig. 78). About 2 hours after ovulation the germinal epi-
thelium and the tunica albuginea are united and the rupture is closed.
From this time on the follicle is called the corpus luteum. In the young
corpus luteum the theca externa keeps its circular outline, w^hile the cells
of the theca interna, which were beginning to project into the follicle even
before ovulation, now penetrate still farther, carrying with them a network
of developing capillaries, and are thus converted into vascular, radially
arranged trabeculae providing support and blood supply to the granulosa
cells. The trabeculae extend to the small inner cavity in which they form a
loose network (Fig. 79). Later when the lutein cells are fully hypertrophied

152

BIOLOGY OF THE LABORATORY MOUSE

this cavity disappears. At the beginning of this process mitotic figures are
seen among the theca interna cells as well as among the granulosa cells.

Ruptured surface
Blood vessel

Germinal epithelium

Granulosa cells

, — i_«;.,»^ ^- Theca interna

Fig. 78. — Ruptured follicle. (X62.)

Theca externa

Germinal epithelium

Central cavity

Lutein cells

Trabecula

Theca externa

Fig. yq. — -Young corpus luteum.

Later the granulosa cells rapidly hypertrophy with resulting increase in size
of the corpus luteum as a whole. While the small granulosa cells have oval

HISTOLOGY

153

shaped nuclei with dark staining, coarse chromatin granules, surrounded by
very little, slightly basophilic cytoplasm, the fully developed lutein cells are
large and polyhedral with clear, slightly vacuolated eosinophilic cytoplasm
and large round vesicular nuclei. The change from one type of cells to the
other is gradual. The lutein cells are arranged in radial strands, surrounded
by a network of sinusoidal capillaries. By the time they become estab-
lished the central cavity has been obliterated.

Corpora lutea. — As ovulation usually occurs spontaneously in rr.ice and
the presence of one set of corpora does not inhibit ovulation, the ovaries
may contain many sets of corpora lutea. According to Allen (2), ''The most
recent corpora lutea are easily distinguishable from the older ones by their
blue color, the latter staining more heavily with eosin."

Deansley (26) conducted a detailed study of the fat accumulation in the
corpora lutea of the mouse during the estrous cycle. (The fat granules
were blackened by osmic acid preparation.) She found that in the corpus
luteum of ovulation the fat and lipoid contents of the lutein cells gradually
increase and that the granules become coarser as the next estrus approaches.
At the time of the next ovulation the granules are less distinct, and 2 days
after metestrus the cells contain hardly any fats or lipoids. Simultaneously
the cells become smaller and cell outlines are less distinct. The corpora
lutea of pseudo-pregnancy accumulate fat at a slower rate. Their cells are
slightly larger, and the nuclei are smaller. In the corpus luteum of preg-
nancy, fats and lipoids are absent until about the 8th day after copulation.
After that it contains finely distributed granules. The corpus enlarges
until about the 13th day (mean diameter 976 fj., nearly i mm.). After this,
little change takes place until about the i8th day when the corpus accumu-
lates fat and a gradual shrinking starts. At parturition the outline of the
corpus becomes indistinct and the fat and lipoid granules are coarser than
at any time before, but not as irregular as at the end of estrus or pseudo-
pregnancy. After parturition, although degeneration occurs, the body
persists for a considerable time.

At the time of 10-12 days pregnancy, all the corpora lutea present in the
ovary (except those of pregnancy) rapidly degenerate, forming fibrous
masses containing large fat globules.

During lactation the corpora of pregnancy show a gradual shrinkage.
During the first week the fats and lipoids show some increase, but this is
followed by a decrease and a loss of the regular distribution. At the end of
lactation diestrus (38 days after parturition) the corpora are fat free and
have a mean diameter of 480 ju.

154 BIOLOGY OF THE LABORATORY MOUSE

The corpora lutea of lactation are formed from follicles which ovulate
post partum. They remain small and free from fat. The size of the cells
is equal to those of the corpora of ovulation, but the nuclei are very small.

Atresia. — All of the follicles present in the ovaries do not mature and
ovulate. Many of them undergo involution and gradual degeneration.
This involution which is called atresia is a normal occurrence in the ovaries.
Atresia may take place at any phase of the developing foUicle. In the
process of atresia of a primary follicle the ovum shrinks; it becomes wrinkled,
the follicular cells become pyknotic and fragment, following which the sur-
rounding stroma soon refills the space. In a larger follicle, after the degen-
eration of the ovum, the collapsed zona pellucida forms a hyaline clump
which may persist for a considerable time. Occasionally the ovum shows
pseudomaturation spindles or polar body formation. Atypical cell division
of the ovum may lead to the formation of several cells of varying sizes
enclosed in the zona pellucida. Such so-called parthenogenetic development
is followed by degeneration. In some cases the partial degeneration of the
follicular cells preceeds the degeneration of the ovum, and the latter is
found "naked" in the middle of the follicle where it soon shows signs of
karyorrhexis and cytolysis. Connective tissue cells and capillaries invade
the follicle and replace the degenerated cells. The cells of the theca interna
hypertrophy and form large polyhedral epithelioid cells, called theca lutein
cells, which form the corpus luteum of atresia. Structurally such a corpus
is similar to the normal corpus luteum, but usually contains some remains
of the degenerated ovum or granulosa cells. These gradually shrink and
are replaced by connective tissue. Strands of theca lutein cells may per-
sist for a considerable time.

Occasionally (in virgin females quite often) a peculiar atresia takes place
in a ripe follicle which fails to rupture. The granulosa cells do not degen-
erate, but hyertrophy and form a corpus luteum at the center of which the
ovum is present. Sometimes the antrum of a follicle in the process of this
type of atresia contains blood, and later is not filled in entirely by luteal
cells but contains a loose connective tissue core. Gradually the ovum
degenerates and hyalinization, progressing from the central area toward
the periphery, sets in. The hyalinized corpus may persist as a round bod}-
for a considerable time, but finally shrinks and is gradually imbedded in
the stroma.

In an Aschheim-Zondek test, after the injection into an immature
female mouse of the urine of a pregnant woman, a similar type of atresia
takes place. The follicles of the immature mouse ripen, pseudomaturation

HISTOLOGY 155

spindles form and atypical, parthenogenetic development occurs. The
antra fill with blood and later the granulosa cells hypertrophy to form
corpora lutea atretica around the degenerating ova.

According to Engle (34), "A count of atretic follicles at four stages of the
estrous cycle shows that there is a cyclic variation, both in the number of
pseudomaturation spindles and in the total number of atretic follicles. The
destruction is at its highest point during the first day of the diestrum, and at
its lowest on the second day."

The oviduct. — The oviduct is often called the uterine tube or Fallopian
tube. It is a narrow, coiled tube which connects the periovarian space
with the uterus. The part nearest to the ovary, called the ampulla, ends
in a funnel-shaped opening, the infundibulum (Fig. 76). The fringe-like
edges of the ampulla, the fimbriae, extend into the periovarian space. The
ampulla is continuous with the narrow isthmus, while the distal end of the
oviduct, the intramural part, runs for a short distance within the wall of
the uterus entering the lumen slightly eccentrically (pars interstitialis).
Simple columnar epithelium lines the entire length of the lumen of the
oviduct. In the ampulla these cells are tall, possess centrally located
oval-shaped nuclei, strongly acidophilic cytoplasm and long motile cilia.
Scattered among these cells are some non-ciliated club-shaped cells, which
at certain phases of the estrous cycle protrude into the lumen (2). There
is a short transitional zone between the ampulla and the isthmus, where
ciliated and non-ciliated cells intermingle. The latter gradually prevail,
and the rest of the oviduct is lined by low columnar cells without cilia.

The lamina propria consists of fibrous connective tissue. The mucous
membrane of the ampulla forms narrow, high longitudinal folds. In the
isthmus a few broad, low folds are present, while in the intramural part the
folds are again somewhat higher. The muscular coat, which is formed by
circularly arranged smooth muscle fibers, surrounds the mucous membrane.
It is progressively better developed toward the intramural part. The tube
is surrounded by a serous membrane which attaches it to the mesotubarium.
As the intramural part of the oviduct enters eccentrically into the lumen of
the uterus, it forms a papillary projection, the colliculus tubarius (Fig. 76).
The projecting colliculus tubarius and encirchng sulcus make difiicult the
injection of fluid into the oviduct from the uterus.

Uterus. — The uterus is composed of two horns which join to form an
undivided caudal part, the corpus uteri (Fig. 75). The lumen of the uterine
horn is lined by simple, columnar cells. Projecting down from the lumen
are simple branched tubular uterine glands which are lined by low columnar

156

BIOLOGY OF THE LABORATORY MOUSE

epithelium and have a spiral course deep in the mucosa. Occasionally they
may penetrate into the muscular layer. The lamina propria consists of
reticular tissue and contains many small polyhedral cells with relatively
large, round nuclei. Lymphocytes are present and are especially numerous
near the muscle wall. The mucosa (the epithelium, uterine glands and the

Lumen of uterine horn

Lumen of corpus uteri

4i> Vaginal fornix

Cervix

^^^„„„ Vagina

Fig. 8o. — Transverse section of the uterine horns, corpus uteri, cervix and vagina.

(X30.)

lamina propria) of the non-pregnant uterus is called the endometrium. It
is well supplied with blood vessels. The endometrium is elevated into
circular folds. The small polyhedral cells of the endometrium change
during pregnancy into large epithelioid decidual cells.

The myometrium surrounding the mucous membrane consists of a
compact ring of circularly arranged smooth muscle fibers, outside of which a
layer of loose connective tissue containing large blood and lymph vessels

HISTOLOGY

157

forms the stratum vasculosum. This in turn is surrounded by longitudinal,
smooth muscle fibers. A serous membrane covers the horns and connects
them with the broad ligaments.

As the horns come together the structure of the fused walls changes,
losing first the longitudinal muscle layers and later the strata vasculosum.
The circular muscle layers persist farther but disappear gradually and the
two lumina are separated only by a wedge-shaped septum composed of
longitudinal smooth muscle and connective tissue. Finally, the two lumina

— Epidermis

Dermis

~ Clitoral gland

■* — Hair follicle

Capsule

Fig. 81. — Clitoral gland of a two weeks old mouse. (X25.)

join to form the single lumen of the corpus uteri. Laterally on each side
of the corpus uteri the lumen of the vagina forms a deep fornix, while the
mid-dorsal and mid-ventral walls of the corpus are fused with the wall of
the vagina (Fig. 80). The corpus uteri opens into the vagina at the cervix,
which is dorso-ventrally flattened. The epithelium of the corpus uteri
consists of low cuboidal cells changing to stratified squamous at the cervix.
A few shallow glands are present in the mucous membrane. The lamina
propria is more fibrous and not as cellular as it is in the uterine horns.

Vagina, clitoris and clitoral glands. — The wide, dorso-ventrally flattened
lumen of the vagina is lined by stratified squamous epitheHum which
undergoes cyclic changes during estrus. The lamina propria is formed of

158 BIOLOGY OF THE LABORATORY MOUSE

vascular fibrous connective tissue. The mucous membrane which is devoid
of glands forms longitudinal folds. The thin muscular coat contains some
inner circular and outer longitudinal smooth muscle fibers, which are inter-
mingled with considerable amounts of connective tissue. The wall is
covered by loose connective tissue.

The vagina opens at the vulva. Immediately cephalad to the vaginal
orifice the clitoris forms a small elevation. The subcutaneous tissue of the
clitoris is rich in blood vessels, but does not contain any erectile tissue.
The clitoris contains a small pouch, the clitoral fossa, which is lined by
cornilied stratified squamous epithelium. The urethra opens on the dorsal
wall of this pouch, while ventro-laterally on each side open the ducts of the
two clitoral glands (Fig. 75). These glands are considerably smaller, but
similar in structure and position to the preputial glands of the male. The
excretory ducts are lined by stratified squamous epithelium. The sac-like
secretory alveoli are surrounded by a thin, connective tissue capsule, and
consist of large pale staining, often vacuolated cells which like all sebaceous
glands produce an oily secretion by cell degeneration. Each gland contains
a single hair folhcle with the hair shaft projecting into the duct (Fig. 81).

Mammary Glands

The mammary gland is a compound tubulo-alveolar gland. Mice have
five glands on each side, three in the thoracic and two in the abdomino-
inguinal region (Fig. 89). The gland undergoes several progressive and
regressive changes during the lifetime of a breeding female. In the male a
very small rudimentary duct system is present.

In the formation of the nipples all three layers of the epidermis take part
(germinativum, granulosum and corneum). The skin covering the nipples
is thickened and forms circular folds which allow for the stretching of the
nipples at the time of nursing. In the formation of the duct system the
stratum germinativum takes part. One main duct leads from each nipple
into the subcutaneous fat pads and forms the collateral and terminal
branches. The fat pads form a framework and seem to limit the growth of
the fully developed glands. Each nipple with its main duct, collateral and
terminal branches is a separate unit and is not in communication with
the others.

The ducts are lined by cuboidal epithelial cells which have dark staining,
oval nuclei and small amounts of cytoplasm. They are surrounded by
circularly arranged connective tissue fibers. The connective tissue coat is

HISTOLOGY

159

thicker around the main and primary ducts and gradually becomes thinner
around the terminal ducts.

Before puberty the gland consists of long ducts which have few side
branches. Shortly before puberty (between four to six weeks) more side
branches develop and the distal terminal branches end in enlarged end-bulbs

Fig. 82. — Mammary gland of an eight weeks old mouse showing rapidly growing end-
bulbs. (X65.)

lined by several layers of cuboidal epithelial cells which contain mitotic
figures (16, 35, 93) (Fig. 82). Increased mitotic activity and formation of
the end-bulbs of the distal ducts at the approach of each estrus has been
noted by several investigators.

Fig. 83. — Developing mammary gland on the ii-th day of pregnancy. (X20C.)

A gradual increase in the epithelial elements of the gland by cell division
is evident during the first part of pregnancy (Fig. 83). This increase reaches
its peak at about the iith-i2th day, and results in the formation of alveoli.
By the 14th to 15th day of pregnancy the alveolar system is well developed
and mitosis is infrequent. Further development consists of an increase in
size of the epithelial cells and an enlargement of the lumina of the ducts and
alveoli. Secretor}' activity is established gradually, starting first in the
alveoli proximal to the nipple and progressing distally. In the cytoplasm

i6o

BIOLOGY OF THE LABORATORY MOUSE

small droplets of secretion appear which gradually fuse into large drops.
The nuclei are pushed toward the base away from the lumina. At 17 to
19 days of pregnancy secretory activity is generally well established (Fig.

Fig. 84. — Mammary gland showing secretory activity near the end of pregnancy.

(X200.)

84). (Parturition at 20 days.) Parallel with the glandular development
there is an intensive development of blood vessels with which developing
ducts and alveoli come in intimate contact. As the glandular parenchyma

Fig. 85. — Mammary gland on the 7-th day of lactation. (X 100.)

occupies more and more space, the adipose cells of the fat pads rapidly
disappear.

During lactation the ducts and alveoli are dilated and contain milk
(Fig. 85). The original lobes and lobules of the fat pad supply the frame-
work of the lactating gland, and adipose cells serve only to fill in the space

HISTOLOGY

i6i

between the parenchymatous elements. The appearance of the epithehal
cells of the alveoli is not uniform, indicating different phases of secretory
activity. In some cells the nucleus is in the middle of the cell and the cyto-
plasm is homogeneous. In others the cytoplasm appears foamy or contains
large protruding fat droplets.

If the litter is small and all the nipples are not suckled, some of the
glands may undergo partial regression while others are still functioning
(16,35). The young mice usually suckle for about 21-23 days. Thelength
of the suckling period depends somewhat on the size of the litter, large

^jjg. --.s;^l-g^'»'"'««^'^

Fig. 86. — Mammary gland 24 hours after lactation stopped.

for 22 days. (X 100.)

Lactation had continued

litters usually suckling longer than small litters. About three weeks after
parturition the glands begin to show signs of regression.

Twenty-four hours after suckling ceases, milk has accumulated in the
ducts and alveoli, which are distended. Epithelial cells have become
detached and are lying loose in the lumen. These degenerate, the cytoplasm
becoming swollen and the pyknotic nuclei fragmenting (Fig. 86). In some
epithelial cells the swollen cytoplasm forms globules which are discharged
into the lumen, but the nuclei with small amounts of cytoplasm remain
intact. The shrunken alveoli lose their close contact with the capillaries.
The lack, of blood supply hastens the process of regression. During this
process the space between the shrinking alveoli is being filled by adipose
cells. Some of these cells seem to develop from fibroblasts which are in

1 62 BIOLOGY OF THE LABORATORY MOUSE

dose proximity to capillaries. The nuclei of these fibroblasts become
rounded and the cytoplasm increases in amount. Gradually fat accumu-
lates in the cytoplasm and the nucleus is pushed to the periphery. The cells
increase immensely in size during this change and adipose cells rapidly
rebuild the fat pads. The collapsed alveoli form irregular clumps of cells
which gradually undergo further degeneration. In the completely regressed,
resting gland the lumina of the ducts are narrow, the epithelial cells lining
them are small and darkly staining. The connective tissue sheath surround-
ing the ducts is increased in thickness. The glands remain in resting condi-

FiG. 87. — Mammary gland at resting stage. (Xioo.)

tion until the following pregnancy, when the described changes are repeated
(Fig. 87).

In old females the glands undergo gradual involution. Part of each duct
system atrophies and only the main ducts and a few secondary branches
remain. The connective tissue surrounding the ducts becomes less cellular
and more homogeneous.

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1 66 BIOLOGY OF THE LABORATORY MOUSE

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HISTOLOGY 167

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Cnapter 4

SPONTANEOUS NEOPLASMS IN MICE

By Arthur M. Cloudman, Roscoe B. Jackson Memorial Laboratory .

Introduction, i68. Definition and characteristics of tumors, 169. Classification of
tumors, 171. Histological classification of mouse tumors, 172. Tumors of the
mammary region, 174. Classification of tumors in or near the mammary glands, 175.
Adenomas of the mammary glands, 176. Adenocarcinomas of the mammary glands,
182. Carcinoma simplex of the mammary glands, 190. Carcinosarcoma of the
mammary glands, 192. Fibrosarcoma of the mammary gland stroma, 192. Tumors
in or near the mammary line and its branches but not originating from the mammary
gland proper, 193. Tumors of the skin, subcutaneous and body wall tissues, 195.
Tumors of the epidermis, 195. Tumors of the dermis, subcutaneous and body wall
tissues, 199. Tumors of the lung, 208. Tmnors of the blood forming and blood
destroying tissues, 212. Lymphocyte tumors, 215. Myeloid cell tumors, 216.
Monocyte tumors, 217. Tumors of the digestive system and associated glands, 219.
Digestive tube and submaxillary gland tumors, 219. Hepatic and gall bladder
tumors, 219. Pancreatic tumors, 221. Tumors of the uro -genital system, 221.
Kidney and urinary bladder tumors, 221. Ovarian tumors, 222. Uterine tumors,
225. Tumors of the testes, 227. Tumors of the central nervous system, 228. Other
rare sites of tumors, 229. BibUography, 230.

Introduction

In our laboratory there have been over 20,000 mice that have developed
spontaneous tumors. Many of these mice have developed multiple tumors
involving different regions of the body. All of these cases have been care-
fully recorded and permanent preparations have been made of the vast
majority of these tissues. Some have been studied as frozen sections.

As in the case of human neoplasms, practically all body regions have
given rise to spontaneous new grov^ths. Also, as with tumors in humans, the
malignant nature of mouse neoplasms has been definitely established (23,
71). This has been a natural result of the enormous amount of research
involving the investigation of all phases of mouse tumors since their value
for this purpose was pointed out over forty years ago (42).

In this section spontaneous tumors will be treated as fully as the space
permits. Most of the tumors described have occurred in the Roscoe B.
Jackson Memorial Laboratory mice.

168

SPONTANEOUS NEOPLASMS IN MICE 169

During the past ten years the staff of the laboratory has kept detailed
records on large colonies of mice representing a considerable number of
inbred stocks. One phase of the record-keeping included the collecting of
detailed data on the incidence of spontaneous tumors. Complete autopsies
were routinely performed and tissues were saved from all body regions which
offered any suggestion of abnormal growth. Furthermore, no tumor
experiments have been considered ready for publication until these tissues
have been studied as to their histopathology. Our collection of tissues from
mice which spontaneously developed tumors represents data from stocks
that vary widely in their tumor incidences. Data on the high tumor strains
show that in some stocks over 90 per cent of all breeding females living into
the tumor age develop some form of neoplasm, e.g., the A and C3H strains,
while in the low tumor strains abnormal growths are rarely found, even in
mice which have attained extreme senility, e.g., Mus bactrianus.

Naturally, the number of recorded mice with spontaneous tumors is no
indication of the vast numbers of mice which have been employed by the
staff, for the stocks vary so markedly from one to another in their population
tumor incidence. However, inbreeding has been carried on to such a degree
that, on the basis of previous observations within a stock, one can predict
with considerable accuracy what types of tumors will probably occur, at
what average age they will be found and in the case of certain types of
growths, in what per cent of another large population these new growths will
develop when one employs the same stock.

Definition and Characteristics of Tumors

A tumor is an autonomous new growth of tissue (Fig. 88). Also, tumors
are atypical growths with atypical structure, apparently of independent
origin. They exhibit no useful function, are without limit to growth and, if
uninterrupted, can result in the destruction of the host. These growths arise
either from embryonic cell rests or from the body cells of the host. They
start as a localized disease involving a few cells and progressively increase in
size by cell division.

Some masses of cells may grow by expansion. This will result in the
formation of a connective tissue capsule due to the pressure of the growth on
the surrounding supporting cells. Others may grow by infiltration, spread
along tissue spaces and lymphatics and may be found at some distance from
the spontaneous tumor. This is a more malignant type than the former.
Another form of growth combining the two above types is called interlocking.
The second and third types of growth are the most difficult to remove by

I70 BIOLOGY OF THE LABORATORY MOUSE

operation and unless complete removal is effected recurrence takes place,
often accompanied by an increase in rate of growth.

As long as neoplasms are in contact with serum they acquire an inde-
pendence of growth. Spread, or metastasis, through serum is the greatest
danger to the life of the host and one of the chief factors of malignancy.
Within the host metastases may be lymphogenous,* hemogenous, implanta-
tion or transplantation. New colonies of similar tumor cells are established
at a distance from the primary tumor, and these in turn may spread to other
locations until a generalized involvement of the entire host organism results.

Tumor cells are parenchymatous neoplastic cells of connective tissue or
epithelial descent. The connective tissue tumors form their own stroma

Fig. 88. — A spontaneous mammary gland carcinoma (X}^)-

and blood vessels, while in the epithelial tumors the stroma and blood vessels
are from the surrounding tissues, with the result that in the latter tumors the
vascular supply is abnormal, atypical and more sinusoidal than in normal
tissue. There are no new-formed lymphatics in tumors, and it is generally
believed that there are no nervous system attachments (21, 31, 38).

A mouse frequently appears in excellent health when a tumor is small but
grossly detectable. As the tumor increases in size, it places increasing
nutritional demands upon the host and at the same time there frequently are
metabolic changes due to infiltration of, or pressure upon, vital organs.
This is accompanied by degenerative changes in the tumor resulting from the
faulty blood supply, so that parts of the tumor become necrotic and their
waste products find their way into the blood stream. The mouse will
develop progressive signs of general ill health with loss of weight and evi-
dence of malnutrition, until in the late stages there is a rufiling of the hair,
weakness, lowered body temperature and a tendency to kyphosis which gives

* Rare in mice for lymphatics are so small and delicate that they are easily occluded

(44).

SPONTANEOUS NEOPLASMS IN MICE 171

tlic animal a shrunken appt-arancc. With certain ncnjphisia there also
develop respiratory dilTiculties and in some cases marked edema. There is a
great variation in the speed at which different tumors grow, so that in some
cases the mouse may die from other causes without having been greatly
inconvenienced by a slow growing neoplasm.

A vast amount of work has been done on the etiology but the exact cause
of cancer remains unknown (9, 11, 12, 37, 71). It is known that chronic
inflammation of either intrinsic or extrinsic origin may accompany the onset,
but chronic inflammation of itself is not enough to change normal body cells
into outlaw cells the chief function of which is unrestrained growth and
which exhibit a total disregard for normal tissue boundaries. Neoplasms
may develop from any cell, organ or tissue of the body which is capable of
growth.

To summarize the characteristics of a neoplasm, we can say that it is an
autonomous new growth of undetermined origin which starts locally, serves
no useful function, may invade the adjacent tissues and even be transferred
to distant body regions of the host, grows progressively and, if uninterrupted,
eventually results in the death of the host.

Tumors may be either fnalignant or benign. The benign forms are
homeotypic in structure, relatively slow growing, grow by expansion and are
encapsulated so that they do not infiltrate and do not metastasize. This
makes complete removal possible, in which case they will not recur. On the
other hand, the malignant forms are heterotypic in structure and possess no
capsules, so that infiltration and metastases are the most important charac-
teristics of this group. A benign tumor may develop into a mahgnant form
showing infiltration and metastasis. This is fairly frequently seen in
carcinoma of the breast in mice, where a small, partially encapsulated
adenoma may be continuous with an adenocarcinoma.

Classification of Tumors

There are several methods of classification of tumors (11, 12), but the
histological structure offers the simplest means, especially with small experi-
mental mammals such as the mouse. A tumor receives a name according to
the tissue which it most resembles. However, this applies best to only the
simple and benign tumors since many malignant forms do not resemble any
normal tissue. The terms sarcoma and carcinoma, therefore, have been
employed to designate the two main groups of the malignant neoplasms (11).
A sarcoma is a malignant tumor composed of cells of the connective tissue
type. It is formed on the connective tissue plan, developing its own stroma

172 BIOLOGY OF THE LABORATORY MOUSE

and blood vessels so that the stroma and blood vessels are in intimate contact
with the tumor cells. The main object in this classification is to separate a
large group of malignant tumors from the carcinomas; however, this method
ignores certain embryological considerations. A carcinoma is a malignant
tumor originating from the epithelial cells of the skin, the mucosa or their
derivatives. In general, sarcomas present a smooth, rounded contour, while
the carcinomas appear less uniform in consistency and frequently give a
nodular appearance.

Histological Classification of Mouse Tumors

I. Connective tissue (types i through 7 are benign).

1. Fibroma — connective tissue origin.

2. Myxoma* — mucous connective tissue origin.

3. Lipoma — fat tissue origin.

4. Chordoma* — Chorda dorsalis tissue origin.

5. Chondroma — cartilage tissue origin.

6. Osteoma — bone tissue origin.

7. Angioma

a. Hemangiona — blood vessel origin.

b. Lymphangioma — lymph vessel origin.

8. Sarcoma — a mahgnant cellular tumor composed of anaplastic
tissue of any of the above types i through 7.

a. Fibrosarcoma.

b. Neurogenic sarcoma.

c. Myxosarcoma.

d. Liposarcoma.

e. Chondrosarcoma.

/. Osteogenic sarcoma.

g. Angio-endothelioma.

h. Round cell sarcoma.

(i) Lymphocytoma.

(2) Myelocytoma.

(3) Monocytoma.
II. Muscle tissue.

I. Myoma (benign).

a. Leiomyoma — smooth muscle tissue origin.

b. Rhabdomyoma — striated muscle tissue origin.

* As yet not reported in mice.

SPONTANEOUS NEOPLASMS IN MICE 173

2. Myosarcoma (niali^naiil).

a. Leiomyosarcoma.

b. Rhabdomyosarcoma.

III, Elements of the nervous system.

1. Neuroma — nerve tiber origin.

2. Neuroganglioma — nerve fiber and ganglion cell origin.

3. Glioma and medulloblastoma — neuroglia tissue origin.

4. Neuro-epithelioma -from neuro-epithelium.

IV. Tumors of pigment cells.

1. Melanoma.

2. Malignant melanoma.
V. Endothelium.

I. Endothelioma — blood and lymph vessel endothelium origin.
VI. Epithelial tissue (pavement and glandular).

1. Papilloma — a benign tumor of pavement epithelium with support-
ing tissue in a normal arrangement.

2. Adenoma — a benign tumor of glandular epithelium with supporting
tissue in normal arrangement.

3. Epithelioma (epidermoid carcinoma, squamous cell carcinoma,
acanthoma) — a malignant tumor of pavement epithelium in atypi-
cal arrangement.

4. Carcinoma — a malignant tumor of glandular epithelium in atypical
arrangement.

VII. Complex tissue tumors.

1 . Simple mixed tumor — composed of more than one type of neoplas-
tic tissue and named according to composition — carcinosarcoma,
adenofibroma, fibro-adenoma, etc. The predominating type is
named last.

2. Teratoma — composed of tissues and organs of one, two or three
germinal layers, such as monodermal, bidermal or tridermal types.

3. Embryoma — composed of tissues from three germinal layers in a
more or less orderly imitation of the fetus.

VIII. Cysts, not neoplasms, but related to them and in mice often mistaken
for them by gross observation.
Some types of tumors have been reported in the literature as rare. How-
ever, our experience has been that the frequency of spontaneous tumors of
any particular type is dependent, to a certain extent, upon the lines of inbred
mice under observation. Had our observations been limited to the C57
black stock and other lines low in epithelial tumors of the mammary glands

174

BIOLOGY OF THE LABORATORY MOUSE

such as C57 leaden, black hairless and Mus htutrianus, our impression would
have been that mammary tumors are rare. The reverse would have been
true had we been using only such stocks as the A albino, the C3H, the dba
and similar lines with a high incidence of spontaneous breast cancer. To
give more concrete examples, the A albino line is high in the incidence of
both lung and mammary cancer, while lung cancer is uncommon in most of
our other stocks. On the other hand, the ce (extreme dilution) stock has an
abnormally high number of tumors of the ovary, while most of the melanomas

Fig. 89. — A diagrammatic drawing of the maximum extent of the mammary system
of the mouse shown in the ventral and lateral aspects. The large black dots represent
the nipples and the stippled areas the mammary glands.

have been observed in the dba (dilute brown) stock, and tumors of all types
are rare in Mus hactrianus. Certain tumors will probably always be con-
sidered uncommon, but as an increasing number of inbred stocks are devel-
oped and studied, we shall be able to find certain lines of mice which will be
of exceptional value in advancing our present knowledge of the more rare
forms of spontaneous neoplasms.

Tumors or the Mammary Region

Since the majority of investigators, especially the pioneers, working with
spontaneous tumors in mice confined their studies mainly to the most avail-
able forms of neoplasms, they investigated chiefly those outside the body
cavities and thus much of the work has been done with tumors of the mam-

SPONTANEOUS NEOPLASMS IN MICE 175

niary region. 'J'hc normal mammary glands have been full}- discussecJ
under the section on histology, but for convenience the distribution of the
mammae can be briefly reported here. There are five pairs of glands
arranged symmetrically along the ventral surface of the mouse (Fig. 89).
This rather extensive distribution of the mammary glands is referred to here
as the mammary line and its branches.

Because of the accessibility to observation the subcutaneous tumors are
probably better known than those of other body parts, and since the major-
ity of tumors observed are in or near the mammary region, they must be
studied histologically to determine their true nature.

Mammary region tumors have been divided into two main groups in an
attempt to include all the types of tumors which occur in the region of the
mammary line and its branches. First are those tumors which originate
from the mammary gland proper while the second group comprises all other
tumors in this same location, but not arising from the mammary gland or its
supporting stroma. This grouping is intended to cover the masses
which, by gross observation, might be mistaken for tumors of mammary
gland origin as well as those which arise from the gland itself.

Classification of Tumors in or Near the Mammary Glands

I. Tumors originating from the mammary gland proper.

A. Benign tumors.

1. Simple adenoma.

2. Polylocular cyst adenoma.

3. Papillary cyst adenoma.

4. Fibro-adenoma (adenofibroma).

B. Malignant tumors.

1. Adenocarcinoma — definite evidence of mammary- gland origin
predominates the histological picture.

a. Simple adenocarcinoma.

h. Adenocarcinoma (variable type).

c. Papillary cyst adenocarcinoma.

d. Intracanalicular adenocarcinoma.

e. Macroglandular adenocarcinoma.

2. Carcinoma simplex — little evidence of definite gland formation.

a. Round cell or medullary.

b. Spindle cell.

3. Carcinosarcoma — originating from both the mammary gland
epithelium and the stromal connective tissue.

4. Fibrosarcoma — originating from the mammary gland stroma.

176 BIOLOGY OF THE LABORATORY MOUSE

II. Tumors originating in or near the mammary line and its branches but not
arising from mammary glands or their stroma.

A. Benign tumors.

1. Fibroma.

2. Chondroma.

3. Osteoma.

4. Lipoma.

5. Angioma.

a. Lymphangioma.
h. Hemangioma.

B. Malignant tumors.

1. Fibrosarcoma.

2. Melanoma.

3. Osteogenic sarcoma.

4. Rhabdomyosarcoma.

5. Carcinomas of skin appendage.

6. Round cell sarcoma — axillary and inguinal lymph nodes.

7. Endothelioma.

a. Hemangio-endothelioma.

b. Lymphangio-endothelioma.

There are also non-neoplastic masses that grossly resemble true neoplasms
and these will be merely listed.

1. Cysts.

a. Mammary duct cysts.

b. Skin cysts.

c. Hygromas — thin-walled, endothelial-lined, cysts filled with
lymph. Seen in C57 black stock.

2. Chronic inflammation.

a. Subcutaneous in general.

b. Chronic mastitis — fibrosis and lymphoid infiltration of the
mammary gland, usually not accompanied by cyst formation in
the mice.

3. Lymphoid hyperplasia.

Adenomas or the Mammary Glands

The tumors within this benign group have certain characteristics in
common. Grossly they are comparatively small, frequently indistinguish-
able from soft non-hemorrhagic carcinomas, and sometimes they appear to
be soft, cystic, translucent masses. As seen under the microscope they have

SPONTANEOUS NEOPLASMS IN MICE

177

a connective tissue capsule which may be thick in some places and difficult to
follow in others. The capsule is not invaded by the tumor cells. The
epithelial cells are arranged as gland-Hke structures which are easily recog-
nized as mammary gland in origin. These structures may vary considerably
in size and arrangement, but they are always lined by a single layer of fairly
uniform, usually small, and relatively inactive epithelial cells. Under these

Cystic space

Epithelium

Stroma

Blood vessel

Fig. 90. — Simple adenoma of the mammary gland (X200').

cells the basement membrane remains intact and around this there are seen
a few small, thread-like, wavy, spindle-shaped connective tissue cells.

Simple adenoma of the mammary gland. — This is not commonly seen as
such, but it is not rare to observe the remains of this type in direct continuity
with carcinoma of the mammary gland. These adenomas contain gland-like
arrangements of the mammar\^ epithelial cells (Fig. 90). They range from
small abortive structures which appear to be attempts at gland formation to
large, round or oval, dilated cyst-like spaces containing more or less eosino-
philic amorphous material. The lining cells are arranged in a single, smooth,
orderly layer. Generally they are cuboidal and uniform in size, shape and

178

BIOLOGY OF THE LABORATORY MOUSE

staining properties. The basement membrane is well preserved. These
cells do not differ strikingly from inactive, normal mammary gland epithelial
cells. In some cases the lining epithelial cells of the cysts range from the
small cuboidal type with moderately deeply staining nuclei and scant cyto-
plasm to fairly large, oval cells which bulge into the cyst cavity. These
latter cells have large, pale, centrally placed oval nuclei which contain

Fig. 91. — Polylocular cystadenoma of the mammary gland showing intercommuni-
cating cysts (X200).

scattered chromatin granules and multiple nucleoli. The cytoplasm is
eosinophilic, uniformly staining and fairly abundant. In simple adenomas
the stroma does not bulge into the epithelial-lined cavities. These gland-
like arrangements may have foci that are uniformly large or small but are
usually distributed so that the whole range can easily be found in a single
low power field. Mitoses are seen but are infrequent.

The stroma is rather loose in the foci where the gland-like structures are
most widely separated and contains scattered strands of connective tissue.
Beneath the basement membrane of each of the gland structures the connec-

SPONTANEOUS NEOPLASMS IN MICE

179

tive tissue is more compact and may consist of one to several layers. These
connective tissue cells are wavy and thread-like in appearance, have
centrally placed spindle-shaped nuclei and possess pale eosinophilic cyto-
plasm. The nuclei are moderately pale, with somewhat evenly distributed
small chromatin granules. It is beneath this compact layer of connective
tissue that the loose stroma is found when present. Where the adenomatous

Cyst

Epithelium

Edematous stroma

Tip of papillary structure Cyst Blood vessel

Fig. 92. — Papillary cystadenoma of the mammary gland (X200).

structures are most compact the adjacent epithelial layers of different glands
may be in very close relationship with only narrow septa of stroma between
them. Large and small, irregularly shaped, thin-walled, endothelial lined
blood spaces are scattered throughout the stoma, most prominently in the
looser foci. The capsule is composed of a dense connective tissue layer
which may vary somewhat in thickness so that in some foci it is difficult to
distinguish.

Polylocular cyst adenoma of the mammary gland. This type shows
many large and some small irregularly shaped, frequently intercommunicat-

i8o

BIOLOGY OF THE LABORATORY MOUSE

ing, epithelial lined cysts (Fig. 91). These lining cells form a single orderly
layer, are low cuboidal, closely packed and uniform in size, shape and stain-
ing properties. They have scant cytoplasm and somewhat rounded, deeply
staining nuclei. Mitoses are infrequent. The walls of the cysts are not
smooth as in simple adenoma, but have an irregular wavy appearance with-
out the formation of papillary ingrowths.

The stroma is composed of coarse and fine, wav>% eosinophihc non-
nucleated fibrils, throughout which are scattered thread-like connective

Large spindle cell

Adenomatous foci

Fig. 93. — Fibro-adenoma of the mammary gland (X200).

tissue cells. It varies from the thin compact foci which barely separate the
adjacent walls of many of the larger cysts to the dense compact foci around
the smaller cysts. The stroma is quite cellular in the region of the capsule
and blends with it. Numerous thin-walled blood vessels are scattered
throughout the entire stroma.

Papillary cyst adenoma of the mammary gland. — This type is so named
because of the characteristic architecture which shows large and small,
branching and anastomosing irregular growths extending into cystic epithe-
lial lined cavities (Fig. 92). These papillary structures may have one or
more broad or narrow points of attachment with the remainder of the tumor.
The stroma extends into these structures and thus makes up a considerable
part of the papillary formations. In the tumor in general some of the

SPONTANEOUS NEOPLASMS IN MICE

i8i

glands are nearly the same size as normal mammary gland. ]\Iost of them
form irregularly shaped cysts, which vary greatly in extent and which derive
their outlines from the size, shape and number of papillary growths which
extend into them.

Fibro-adenoma. — This tumor of the mammary gland usually has the
same type of gland-like arrangement seen in the simple adenoma. Histo-

Large spindle cells

Adenomatous foci

Fig. q4. — Adenofibroma of the mammary gland (X200).

logically, the stroma has the same type of wayy, thread-like connective
tissue cells with spindle-shaped nuclei around the adenomatous foci. The
difference between these adenomas lies for the most part in the two chief
characteristics of the hbro-adenoma ( Fig. 93) . First, that the stroma is more
abundant and makes up nearly as much of the bulk of the tumor as does the
adenomatous parts. Second, that there are strands and bundles of large,
closely packed connective tissue cells running in all directions throughout the
stroma. These large cells are narrow, long, tapering at the ends and have
centrally placed nuclei. The cytoplasm is abundant and uniform, taking a

I82

BIOLOGY OF THE LABORATORY MOUSE

fairly deep eosinophilic stain. Nuclei are elongated, narrow and blunt at
their ends with moderately pale, coarse and fine chromatin granules.

These interlacing strands of large connective tissue cells vary in amount
in different fibro-adenomas, but they are always present. When they
definitely predominate over glandular parts, the tumor would then be called
an adenofibroma (Fig. 94).

The capsule varies in thickness but not in direct relationship to the extent
of their fibrous parts. Mitoses are rare throughout the entire tumor.

Adenocarcinomas of the Mammary Glands

These tumors form a group of malignant neoplasms in which stromal
variations play a somewhat miner role in regard to diagnosis. However, the
mammary gland epithelium gives rise to epithelial tumor cells which may
assume a wide range of variations in arrangement and distribution within the
stroma without becoming so undifferentiated as to lose all trace of gland
origin.

A high percentage of all the spontaneous tumors which have occurred in
the mice raised in our laboratory have been of mammary gland origin.
Most of these mammary gland tumors have been some form of adenocarci-
noma. The histological examination of these adenocarcinomas has shown
that some arose from pre-existing adenomas, and some appeared to have
developed directly from the mammary glands in the absence of adenomas.
When a large series of these mammary tumors is examined, a few characteris-
tic types emerge, each of which shows some variations and together they
cover the various forms of adenocarcinomas observed. The tumors are
classified according to the most outstanding cell arrangement. For example,
a papillary cyst adenocarcinoma may have a small focus of tumor cells
arranged as in intracanalicular adenocarcinoma or as in macroglandular
adenocarcinoma.

Simple Adenocarcinoma. — This growth is composed of small, narrow
coiled ducts which are generally evenly distributed throughout the stroma.
These ducts are uniform in diameter, are about the size of the ducts of the
resting mammary gland and are lined by one to two layers of cuboidal
epithelial cells (Fig. 95). These cells are small, closely packed, possess a
scant amount of eosinophilic cytoplasm and oval, rather hyperchromatic,
nuclei.

The duct-Hke structures are usually so closely packed that there is little
stroma between them, yet they may be spread through foci of loose stroma.
The tubules are generally so coiled that the majority of them are cut in cross

SPONTANEOUS NEOPLASMS IN MICE

i»3

section or near this angle. Mitoses are frequent and infiltration around and
into normal adjacent structures, such as muscle and nerves, can be seen.
Small central islands or scattered peripheral foci of other forms of mammary
carcinoma are often seen in this type of tumor. The outstanding character-
istics are the uniformity in size and distribution of these small duct-like,
coiled structures lined by one or two layers of small, cuboidal epithelial
tumor cells.

Adenocarcinoma (variable type). — This shows gland-like formations
which may exhibit a wide range of size, arrangement and degree of similarity

Wui m

'f-'H ■

Tubules of carcinoma

Stroma

Sinusoidal blood space

Fig. 95. — Simple adenocarcinoma of the mammary gland showing small duct-
like cancer tubules (X200). The area illustrated shows more stroma than is usually
observed.

as compared to the normal mammary gland. However, their origin from
mammary glands is always evident, since some degree of attempted gland
formation is a characteristic feature (Figs. 96 and 97). There is a varying
degree of definite lumen formation, and around this the epithelial tumor cell
lining ranges from one to several layers in thickness. These cells show fre-
quent mitoses, may be large or small, cuboidal shaped and exhibit consider-
able loss of normal orientation. They often grade over from glands with
definite lumen formations to disorganized nests of epithelial tumor cells.

Different tumors of this type may show a variety of arrangements. One
may show broad, ramifying and branching strands of closely packed abortive

i84

BIOLOGY OF THE LABORATORY MOUSE

Abortive gland

^4/

Normal connective
tissue

Fig. 96. — Adenocarcinoma (variable type) of the mammary gland ( X ^00)

r36t^?r^,-^-i?!*w^-« -•*^v- '^^^v ^'^S"
-.. -'3*' v^ • *- ^ ^^\ ^^^ i^-T. v**3

Fig. 97. — -Adenocarcinoma (variable type) of the mammary gland (X200). This
shows a greater gland forming tendency than is seen in Fig. 96. There is also more
abundant stroma and greater mitotic activity, g.f., gland-like formations; lu., lumen;
m.f., mitotic figure; str., stroma.

SPONTANEOUS NEOPLASMS IN MICE 185

gland-like formations separated by thin septa of stroma with fairly large
blood spaces. Another may contain large irregularly shaped nests of closely
packed, poorly formed glands varying in size and lined by large and small
cuboidal epithelial tumor cells with numerous, small thin-walled blood
vessels and little stroma within the tumor nests but with dense stroma
separating them. A third type may exhibit pseudoglandular arrangements
of large and small, or fairly uniform size, imperfectly formed glands about a
focus or stroma which consists largely of a thin walled blood vessel, or about
a necrotic focus of tumor cells. Some are composed of clusters of large and
small, blood-filled endothelial lined spaces, surrounded by poorly formed
glands which may be markedly compressed by the blood spaces. Still other
types are seen where there may be metaplasia producing true epithelial pearl
formations with the stroma varying in amount and density. This descrip-
tion does not cover completely all the varieties which might be observed for
this tumor type.

The distinguishing characteristics are some degree of attempted gland
formation by the majority of the epithelial tumor cells. The glands may
vary in size and arrangement and are lined by large or small cuboidal
epithelial cells. Gland walls vary from one to several layers in thickness and
show frequent loss of normal orientation of the cells where the walls have
become thickened. Mitoses are abundant. Infiltration of the surrounding
tissues and metastases to the lungs are often observed.

Papillary cyst adenocarcinoma. — At least a large proportion of this group
arises in pre-existing papillary cyst adenomas. For this reason there is a
striking similarity in the general arrangement of the stroma in both the
benign and malignant tumors. However, in the latter the stroma is fre-
quently less abundant, except at the base of and within the central portion
of the papillary structure. The epithelial tumor cells cover the surfaces of
the poorly defined cysts and the branching papillae. On the latter they
often form irregular finger-like projections which contain a small amount of
connective tissue extending from the central stromal core (Fig. 98). In the
larger papillae the distal portions are composed chiefly of epithelial tumor
cells. These cells may be arranged in groups of gland-like formation,
nodules, sheets of cells or a combination of these with or without imperfectly
formed glands of different sizes. Even in the larger tumor masses thin
strands of stroma can be found in the form of scattered groups of small
connective tissue cells and small, thin-walled blood vessels.

The epithelial tumor cells are medium sized, cuboidal or low columnar,
with oval, moderately hyperchromatic nuclei containing scattered chromatin

i86

BIOLOGY OF THE LABOILiTORY MOUSE

granules. The cytoplasm is fairly abundant and eosinophilic. These cells
vary somewhat in size. Mitoses are fairly frequent. On the surfaces of the
papillae and within the gland-hke formations the epithelial cells vary from
one to several cell layers in thickness and normal orientation is frequently
lost. Invasion of surrounding normal structures and metastases to the
lungs occur.

stroma Cyst Papillae

Fig. g8. — Papillary cyst adenocarcinoma of the mammary gland (X200).

The distinguishing characteristics are large and small branching and
anastomosing papillary tumor growths within cyst-like cavities which are
often so filled with these papillary structures that the cysts are poorly
defined. The walls of the cysts are fined by medium sized cuboidal or low
columnar epithelial cells which also extend over the surfaces of the branching
papillary formations. Here they form a cover of one to several cell layers in
thickness. The stroma forms a definite core of connective tissue containing
thin-walled blood vessels in the papifiary structures, and the stroma may not
be clearly defined in the distal portions of their branches. However, stromal

SPONTANEOUS NEOPLASMS IN MICE

187

connective tissue and blood vessels infiltrate and can be found, by careful
observation, even within the finer branches of the papillae which arc com-
posed largely of disorganized nests and partial gland-forming foci of epithe-
lial tumor cells.

stroma

Disorganized tumor cells

Palisading layer

Fig. qq. — Intracanalicular adenocarcinoma of the mammary gland. A, shows typical
architecture (X200); B, shows the cellular detail (X400).

Intracanalicular adenocarcinoma. — This tumor grows as finger-like
branching and anastomosing strands of epithelial tumor cells extending into
a loose stroma (Fig. 99A). The edges of these strands are smooth due to an
orderly paHsade arrangement of a single outer layer of epithelial cells.
Within this palisade layer the entire remainder of these finger-like processes
is composed of epithelial tumor cells of the same type and size but with a
disorderly arrangement due to loss of normal orientation (Fig. 99B).

These tumor cells are closely packed and cuboidal in shape, somewhat
larger than normal resting mammar}^ gland epithelium, with moderately

1 88 BIOLOGY OF THE LABORATORY MOUSE

hyperchromatic, oval nuclei and scant cytoplasm. The stroma consists of
loosely scattered, threadlike connective tissue cells and thin-walled blood
vessels.

This type of tumor derives its name from its manner of growth, which is
intraductal, filling the lumen with wildly growing, epithelial tumor cells, but
having an outer layer of orderly arranged cells. This arrangement of cells,
together with the manner of infiltrating the stroma in branching finger-like

Lumen

Epithelial tumor
cells

Fig. ioo. — Macroglandular adenocarcinoma of the mammary gland (X200).

processes, constitutes the distinguishing characteristics for intracanalicular
adenocarcinoma.

Mitoses are frequent. Infiltration of normal adjacent structures occurs
and metastases to the lungs are fairly frequent.

Macroglandular adenocarcinoma. — This type of growth is probably what
Apolant (i) called fissure forming carcinoma of the mammary gland. Here
occurs what appears to be enormous gland-like structures with long, branch-
ing, irregular lumena (Fig. 100). The walls are thrown into folds and are
composed of four or five to many cell layers in thickness. The epithelial
tumor cells forming the walls are medium sized, oval and closely packed,
often growing in wild disorder with frequent mitotic figures in evidence.

SPONTANEOUS NEOPLASMS IN MICE 189

They have a small amount of eosinophilic cytoplasm and oval, somewhat
hyperchromatic nuclei.

The lumena of these glands are very prominent. Between the glands the
supporting stroma may be reduced to narrow but conspicuous septa of dense
fibrous connective tissue containing some thin-walled blood vessels.

The chief characteristic of this type of tumor is the enormous, irregularly
branching, duct-like structures whose lumena may extend for considerable
distances. The walls, which are composed of compact tumor cells, are from
four or five to twenty or more cells in thickness and follow fairly closely the
contours of the lumena. This gives the appearance of giant thick-walled
ducts. Invasion of adjacent normal tissue is commonly seen and metastases
in the lungs are often found.

In quite a number of breast tumors there can be seen large and small,
blood-filled, cyst-like spaces which are often clustered closely together.
These are always surrounded by small epithelial tumor cells, which may form
compact strands varying from three or four to many cell layers in thickness.
These cells may or may not show some flattening where they come in close
contact with the blood-filled cyst-like spaces. These spaces have an acellu-
lar, membrane-like, eosinophilic zone between the epithelial cells and the
blood. In some instances there are scattered, flattened cells present which
suggest an endothelial lining within these spaces.

Some investigators have considered these cystic tumors as belonging to a
type called hemorrhagic cyst adenocarcinoma (Fig. loiB). One can find
simple adenocarcinoma with foci where clusters of blood filled spaces are
separated by thin, compressed strands of epithelial tumor cells. Similar
spaces are also frequently found in cases of adenocarcinoma, variable type, as
well as in nests of tumor cells which are in the midst of and continuous with
intracanalicular adenocarcinoma and even papillary cyst adenocarcinoma.
In the latter type papillary growths may extend into the cyst-like blood
filled spaces. When this is taken into consideration, it may be advisable to
consider these hemorrhagic cysts not as a separate type but more as a com-
mon characteristic of adenocarcinomas in general.

In gross observation these tumors show many bulging, blood filled cysts.
The tumor is turgid and when cut open will collapse into a soft hemorrhagic
mass.

There is a somewhat similar situation in the case of the frequent appear-
ance of epithelial pearls, composed of cornified, squamous epithelial cells
grouped in concentrically arranged foci (23). These pearls can be found in
all types of adenocarcinoma. This may even progress to the stage where the

I go

BIOLOGY OF THE LABORATORY MOUSE

tumor shows a central amorphous mass which grades over into desquamated
stratified squamous epithehum. This in turn grades over into definite
adenocarcinoma. This can be considered as adeno-acanthoma, but a more
probable explanation is that metaplasia has occurred changing glandular
epithelial cells into stratified squamous epithelium (Fig. loiA).

These two characteristics in their most extreme manifestations could be
considered as special types of breast cancer. However, an interpretation of

Fig. loi. — Two features frequently observed in adenocarcinoma of the mammary
gland. A, metaplasia of the glandular epithelium in a nest of tumor cells (center of
figure) to keratinized squamous epithelium (X200); B, cystic blood-filled spaces sur-
rounded by the tumor cells of adenocarcinoma (X200).

the histological picture presented by the various forms assumed by
adenocarcinoma of the mammary gland probably does not require these
subdivisions.

Carcinoma Simplex of the Mammary Glands

Histologically this tumor is so undifferentiated that its appearance is
frequently difficult to associate with that of the mammary gland from which
it originated (Fig. 102). However, one can find small foci and traces of
adenocarcinoma that blend with the carcinoma simplex cells forming the
bulk of the tumor mass.

The architecture of the tumor shows compact masses of epithelial tumor
cells growing in long, broad, branching and anastomosing bands or in a com-

SPONTANEOUS NEOPLASMS IN MICE

191

pact mass without any definite arrangement and with rather inconspicuous
stroma. In the first type there is often considerable debris resembHng
necrotic material between the bands of tumor cells, and pseudoglandular
arrangement around this debris and surrounding the thin-walled blood
vessels is not uncommon. Clusters of large and small, blood filled, cystic
spaces similar to those observed in adenocarcinoma are sometimes found in
this type of tumor.

The epithelial tumor cells are usually quite large and vary in size. They
are compact 1\- arranged with rather indistinct cell boundaries. In outline

Tumor cell

■Mitotic figure

-Normal duct

^9 -'

<ir-

-i<u

■i'

-Stroma

'X:-

»L

Fig. 102. — Carcinoma simplex of the mammary gland (X400).

the cells vary from rounded and polyhedral to somewhat spindle-shaped.
Their nuclei are hypochromatic, have one to many nucleoli, are round to
nearly spindle-shaped, and vary in size. Mitoses are frequent. Some
mononuclear tumor giant cells are present. The cytoplasm is pale, eosino-
philic, and varies from a scant amount in the rounded cells to abundant in
the spindle-shaped epithelial tumor cells and in the polyhedral cells. The
stroma is usually represented by numerous, large and small, thin-walled
blood vessels with a small amount of connective tissue, except between large
nests of tumor cells where well defined septa of stromal connective tissue are
present.

The rounded and spindle-shaped epithelial tumor cells can often be found
in the same high power fields. The latter can be seen arising from the

192 BIOLOGY OF THE LABORATORY MOUSE

rounded epithelial tumor cells and represent a more undififerentiated form of
carcinoma simplex. These spindle cells often grow in nests and strands
with dense irregular strips of connective tissue cells scattered between them.
In some respects they may be confused with fibrosarcoma. However,
fibrosarcomas have more distinct cell boundaries and the cells are more
definitely tapering and spindle shaped. Also, these cells usually have less
cytoplasm and possess smaller, more hyperchromatic, nuclei which are more
pointed at the poles. The spindle-shaped carcinoma cells are greatly
elongated epithelial cells and grade into polyhedral and rounded epithelial
tumor cells at the periphery of the tumor cell nests.

The polyhedral cells are the most uncommon carcinoma simplex cell in
our stocks. The cases we have show pale, rather large, many sided cells
which in some respects resemble squamous cells of the epidermis, but are
without keratinization. They grow in closely packed, irregular masses with
dense stroma between them. Their origin can be traced to mammary gland
epithelium. Mitoses are frequent and the tumor freely invades surrounding
tissues.

Carcinoma simplex as a whole grows rapidly, shows extensive infiltration
into adjacent tissues and metastasizes to the lungs. Epithelial pearls may
be found, especially in the branching and anastomosing forms.

Carcinosarcoma of the Mammary Glands

This type originates from a pre-existing fibroadenoma. In this tumor
the adenomatous elements become malignant as shown by invasion of the
basement membrane, followed by the spreading of the epithelial tumor cells
into the stroma in dense, irregularly arranged nests. There is also a malig-
nant change involving the large connective tissue cells found in the stroma.
These multiply and spread as interlacing strands of connective tissue tumor
cells. Thus the resultant histological picture is that of a fibrosarcoma grow-
ing around nests of adenocarcinoma (Fig. 103). Both types of tumor show
frequent mitoses. Should the sarcoma outgrow the carcinoma, the picture
is predominantly that of fibrosarcoma.

Fibrosarcoma of the Mammary Gland Stroma

This may originate from a carcinosarcoma, as above, or from an adeno-
fibroma in which the fibromatous elements alone have become active. This
tumor can also originate from the stroma about the mammar}^ glands in the
absence of an adenoma. The resultant fibrosarcoma forms a dense tumor
mass composed of closely packed spindle-shaped connective tissue tumor

SPONTANEOUS NEOPLASMS IN MICE

193

cells. The architecture exhibits the same characteristics as are described
under fibrosarcomas of the subcutaneous tissue in general. Mammary
glands are present around this tumor and usually are invaded by the
infiltrating tumor mass.

Tumors in or Near the Mammary Line and Its Branches but Not
Originating from the Mammary Gland Proper

Tumors in or near the mammary line and its branches and not originating
from mammary glands or their stroma may be either benign or malignant.
While such tumors are in no essential way different from tumors in general

Carcinomatous foci

Sarcomatous focus

Fig. 103. — Carcinosarcoma of the mammary gland (X200).

which are found subcutaneously, it is convenient to list and briefly describe
them here because of the fact that confusion with true mammar}- tumors is
possible if diagnosis is not carefully made. Normal appearing mammar\-
glands are found either adjacent to or invaded by these tumors. For the
details of their histological appearance the reader should turn to the section
on Tumors of the Dermis, Subcutaneous and Body Wall Tissues (p. 199).
Only the names and a brief description are given below.

Fibromas, chondromas and osteomas.— These originate from fibrous
connective tissue, cartilage and bone respectively and are uncommon in this

194 BIOLOGY OF THE LABORATORY MOUSE

region. At least this is true for our mice and according to the pubHshed
literature from other sources.

Angiomas. — These tumors are found especially in the C57 black stock
mice. This is true for both hemangiomas and lymphangiomas. The former
has been mistaken for primary carcinoma of the mammary gland on super-
ficial inspection of the living mouse. Even on gross section it may resemble
somewhat the mammary gland tumors with dilated, blood-filled cysts.

Hemangiomas may be formed anywhere in the mammary gland region.
They are composed of the elements of blood vessel walls and develop as a
benign tumor with a poorly formed capsule. Lymphangiomas, on the other
hand, are found in the axillarv^ or the inguinal regions. They are benign
tumors formed from the elements of the lymph vascular system.

Lipomas. — These are tumors of adipose tissue. They are benign tumors
with cells larger than normal and without the vascular arrangement of
normal fat tissue.

Fibrosarcoma. — This growth may occur near the mammar\' glands and
may invade and destroy them. Here the epithelium is not a part of the
tumor. The tumor has spread around the normal tissue as is the case in the
infiltration of other normal tissues. Fibrosarcoma here is the same as that
which will be described later under subcutaneous fibrosarcoma (Fig. 112).

Melanoma. — This is a pigmented tumor sometimes seen at the base of
the tail in the dba females which are of a high mammary tumor stock. How-
ever, the tumor is usually black and not confused grossly with a mammary-
carcinoma, even when the latter has blood filled cysts.

Rhabdomyosarcoma. — This is a sarcoma of the striated muscles and has
been found in the mammary gland region of dba stock female mice. The
same is true of hybrids between the dba and C57 black stocks.

Osteogenic sarcomas and chondrosarcomas. — Sarcomas of bone origin in
the mammary region are uncommon in our stocks. One chondrosarcoma
originating from rib cartilage has been noted. As stated under the sub-
cutaneous tumors, osteogenic sarcoma has occurred several times. J. A.
Murray (43) reported on a chondro-osteosarcoma in the left groin of a female
mouse. Pybus and Miller (45) developed a branch of the Simpson strain
with a fairly high incidence of bone sarcomas, several of which were in the
mammary region.

Carcinoma of skin appendage. — These arise from specialized sebaceous
glands. The preputial (23) and clitoral glands also fall into this group.
This carcinoma can be traced to the skin appendage glands as its site of
origin (Fig. 109).

SPONTANEOUS NEOPLASMS IN MICE 195

Tumors of the Skin, Subcutaneous and Body Wall Tissues

Tumors of the dermis, subcutaneous tissues and body wall may be con-
sidered together. A tumor of the epidermis is fairly easy to determine
grossly, but tumors of the dermis might be confused with many of the new
growths occurring in the subcutaneous and body wall tissues. In the
mammary line and its branches the tumors not of mammary gland origin
would be the same as those of the subcutaneous tissues in general, except for
those of the axillary and inguinal lymph nodes. The majority of these
lymph node tumors belong in the groups to be discussed under lympho-
cytomas, myelocytomas and monocytomas.

Tumors or the Epidermis

Tumors of the epidermis are not common in any of our stocks. Papil-
lomas and epidermoid carcinomas have been found in small numbers in
many of the stocks, chiefly in the C57 black, X, W, ce, dba and their hybrids.
Papillomas occur most frequently on the external genitalia of the female,
around the anus, on the eyelids, ears, lower lip and occasionally on the skin
of other parts of the body. Epithelial horns are rare but have been found
about the head and shoulders in the C57 black and the dba mice.
Epidermoid carcinomas have been seen arising from the skin of the
dorsal and ventral surfaces (Fig. 106), the shoulders (Fig. 107), the lower
lip (Fig. 105), the eyelid and the skin around the anus and external genitalia
of the female. Frequently the epidermoid carcinoma occurs within a pre-
existing papilloma.

The Papillomas. — These are benign epithelial tumors which are elevated
above the skin surface, often pedunculated, and contain varying amounts of
stroma. The epithelium is the active part of these tumors and shows thick-
ening and overgrowth. This results in the formation of the blunt, elevated
papillae and in the varying degrees of epithelial downgrowth into the dermis
(Fig. 104). Within the thickened epithelial layer the normal orientation of
epidermis is not lost and the basement membrane is intact, but marked
keratinization and cornification are usually present. The elevated mass
may consist almost entirely of epithelium with only thin finger-like processes
of stroma extending between the irregular epitheUal downgrowths and form-
ing a central core within the papillae.

Epithelial Jwrns are really papillomas which show a marked degree of the
piling up of the cornified epithelium until grossly a horn-like growth about
two centimeters long may develop. This structure tapers from the base to

igS

BIOLOGY OF THE LABORATORY MOUSE

the tip. At the base the epithehal downgrowths may penetrate below the
level of the epidermis and contain only thin, loose strands of stroma (Fig.
104). The basement membrane remains intact and the arrangement of the
epithelium at the base is that of a papilloma.

Epidermoid carcinomas of the skin.— These are malignant tumors of the
epidermal epithelium. The\- vary from early forms beginning in papillomas,
such as on the lip (Fig. 105A) or external genitalia, to wildly growing types

Sebaceous glands

Stroma of dermis

Stroma of tumor

Base of papilloma

Fig. 104. — Subcutaneous focus from the base of an epithelial horn (papilloma) of the

skin (X200).

with marked anaplasia. This is accompanied by extensive invasion and
occasional metastases to lymph nodes (Fig. 108A) and lungs. The low
grade forms show loss of orientation, extension through the basement mem-
brane and invasion of the adjacent normal structures. Except in the most
rapidly growing forms, marked keratinization and extensive formation of
large and small epithelial pearls are common (Fig. 106). The epithehal
tumor cells grow in nearly solid masses without much stroma. Epithelial
pearls are usually scattered throughout and show concentrically arranged,
flattened, cornified epithelial cell debris that takes an eosinophilic stain.
Around these pearls are irregular clumps of large polyhedral cells with large.

SPONTANEOUS NEOPLASMS IN MICE

197

pale, oval frequently pyknotic, nuclei. The cytoplasm is abundant,
acidophilic and often contains coarse keratohyalin granules. These cells
grade over into smaller, closely packed, disorderly, polyhedral to somewhat
spindle-shaped cells. They possess a relatively small amount of eosinophilic
cytoplasm and contain oval, moderately hypochromatic nuclei with scattered
coarse chromatin granules. Scattered foci of brown pigment resembling
melanin are often seen. Mitoses are frequent in these smaller cells.

Epithelial pearl

YiG. 105. — Epidermoid carcinoma from the lip of a female mouse. A, tumor invad-
ing dermis around vibrissa (X200); B, cellular detail of this same tumor (X400).

Occasionally a very malignant form shows little epithelial pearl formation
(Fig. 107). It may consist of wildly growing spindle-shaped epithelial
tumor cells that blend with narrow strands and small nests of the smaller
types of polyhedral epithelial tumor cells (Fig. 108B). Unless the origin can
be traced to the epidermis in this type of tumor, the architecture is so mis-
leading that it could confuse one in interpreting the histopathology . ]\Iitoses
are abundant.

Carcinomas of skin appendages. — These all give the same general picture.
They originate in the specialized sebaceous glands of the head region of

1 98 BIOLOGY OF THE LABORATORY MOUSE

males and females, most commonly in the A stock. They are also seen
arising from the preputial glands of the male (23) and the clitoral glands of
the female mouse. The chief characteristic is the resemblance of the cells
to the normal cells of the sebaceous glands. They are large round cells with
pale cytoplasm which appears to be filled with fine droplets (Fig. 109). The
nuclei are relatively small, pale, oval and centrally located. These tumor
cells grow in irregular masses as well as in broad branching strands. The

-Epithelial pearl

-Polyhedral cells

Undifferentiated tumor
cells

^ , _. . ,_.- « . , Pearl with keratohyalin
i^*"*jkf^ .♦ T> j granules

*♦'•■'- ,.■^ *

Fig. 106. — Epidermoid carcinoma of the skin on ilie ventral abdominal surface (X200).

most rapidly growing parts may contain small round, rather deeply staining,
cells which have a small amount of cytoplasm and oval nuclei. These cells
resemble the small undifferentiated cells of epidermoid carcinomas. Some-
times stratified squamous cells are found in some of the tumors of the clitoral
glands. Since the smaller tumor cells may also rather closely resemble small
undifferentiated cells of carcinoma of the mammary gland, it is not always
easy to determine whether one is looking at a carcinoma of the clitoris or a
carcinoma of the mammar\^ gland invading the clitoral glands. Both of
these conditions do occur. Usually the clitoral glands show active growth
with dedifferentiation when they are the primary site of the neoplasm.

SPONTANEOUS NEOPLASMS IN MICE

199

Tumors of the Dermis, Subcutaneous and Body Wall Tissues

Many tumors of the dermis and subcutaneous tissues are not easily
separated, and for the purposes of this section no attempt to separate them
will be made. Benign and malignant forms are found here. These are
representative of the type of tissues normally found in the subcutaneous and
body wall region.

Fig. 107. — Rapidly growing epidermoid carcinoma of the skin ( X::oo).
little epithelial pearl formation in contrast to Fig. io6.

This shows

Fibroma. — This is a benign tumor, not commonly observed, composed of
connective tissue cells. These cells are uniform in size and shape and are
distributed throughout the intercellular substance. The tumors are
encapsulated and invasion does not occur. Mitotic figures are rare.

Chondroma. — This is a benign tumor originating from cartilage. The
cartilage cells are atypical, larger than normal and arranged in irregular
islands. They show a tendency to mucoid degeneration or calcification.
Blood vessels may be fairly abundant. Mitoses are rare and a well formed
capsule is present. This type of tumor is not common among our stocks.

200

BIOLOGY OF THE LABORATORY MOUSE

Osteoma. — This growth originates from bone and is a benign encapsu-
lated tumor. It is composed mostly of dense compact bone, usually with
little marrow (Fig. no). This is another uncommon form of tumor and is
probably overlooked when small and inconspicuous.

Lipoma. — This is a benign tumor of fat or adipose tissue and is usually
composed of large fat cells. The tissue looks nearly normal but lacks

Fig. io8. — Epidermoid carcinoma of the skin. A, inguinal lymph node metastasis
of the carcinoma shown in Fig. io6 (X200); B. cellular detail of the tumor in Fig. 107
(X400).

trabeculae, normal vascularity, and the fat cells are larger than ordinary.
Due to the lack of normal vascularity retrograde changes may occur, such as
necrosis followed by calcification. This tumor has been seen in the yellow
stock, in which there is a tendency for the mice to become obese.

Angiomas. — These are benign and are composed of the elements of either
the blood vascular or the lymph circulatory systems. When they are
formed from blood vessels they are called hemangiomas, and lymphangiomas
when formed from lymph vessels.

Hemangiomas grow either as a diffuse mass or as closely clustered groups
of endothelial lined spaces supported by a dense stroma. The endothelial

SPONTANEOUS NEOPLASMS IN MICE

20I

Sebaceous-like
tumor cells

Undifferentiated
''3& epithelial

ji.-;;^ tumor cells

Fig. ioq. — Carcinoma of skin appendage origin from the head region (X200)

Marrow

Fig. no. — Osteoma of a tail vertebra (X75).

202

BIOLOGY OF THE LABORATORY MOUSE

cells are flattened and pavement like. The spaces are blood filled and irregu-
lar in size and shape (Fig. iiiA and B). Only narrow septa of fibrous con-
nective tissue stroma are present between the spaces. Epithelial elements do
not appear as a part of these tumors. Within the tumors foci of thrombosis
and considerable old blood pigment are often seen. The connective tissue
capsule is not wtU formed. Mitotic figures are rare. In mice this tumor

Fig. III. — Hemangioma

A, shows typical arcliiteclure (. X50)
endothelial lined spaces (X200).

tilled

frequently shows a mixture of capillary -like and cavernous blood filled
spaces.

Lymphangioma is most often found in the axillary and the inguinal
regions. Sometimes it occurs near these sites where it may have originated
from lymph nodes. Irregular, large or small lymph filled sinusoidal spaces
are seen lined by flattened endothelial cells. The connective tissue stroma
forms nodular septa containing small lymph vessels and normal appearing
lymphocytes in varying degrees of concentration. In its most benign form
this tumor exhibits broad bands of connective tissue stroma surrounding
long, narrow, irregular, endothelial-lined spaces filled with lymphocytes.
This may involve a large part of a lymph node.

SPONTANEOUS NEOPLASMS IN MICE

20.;

Fibrosarcoma. — Among the more common malignant tumors in the
subcutaneous region, the fibrosarcoma is the tumor most often observed.
However, no stock of mice shows a frequency of subcutaneous tumors which
would enable one to call it a high tumor stock in that respect. It is unusual
to find a stock showing over 15 per cent of the mice with tumors other than
mammary carcinoma in this region. Many fines of mice show considerably
less.

Fibrosarcoma originates from the fibrous connective tissue cells. The
basic architecture is the same whether it develops in the mammary line and

Fig. 112.

-Fibrosarcoma of the subcutaneous connective tissue showing the typical
interlacing pattern of the spindle cells (X200).

its branches or in any other subcutaneous focus. Grossly, the tumor is a
compact mass with a smooth, rounded, white surface. The cut surface is
uniform, bulging and varies from soft to firm. Microscopically it shows
closely packed spindle-shaped tumor cells (Fig. 112). Their architecture
presents a picture of solid masses of cells alternating with large and small
whorls and interlacing bands of fibrous connective tissue tumor cells. The
tumor cells exceed the stroma and the latter is difficult to identify, but
appears to be represented by inconspicuous stromal connective tissue cells
surrounding the individual tumor cells. Fibrosarcoma tumor cells often
appear slightly separated, as though shrunken away from the intercellular
stroma. Small, endothelial lined thin-walled blood vessels are abundant.
Invaded tissues such as striated muscle, nerves, large blood vessels and mam-
mary glands may be seen, for no capsule is present and infiltration occurs.

204 BIOLOGY OF THE LABORATORY MOUSE

The sarcoma cells vary in size from medium to large, while very large
tumor giant cells are sometimes present. In shape the tumor cells range
from blunt, to long, narrow spindle cells. The cytoplasm is pale, eosino-
philic and appears to have faint, longitudinal striations. Nuclei are elon-
gated, moderately hypochromatic, more or less irregular in outline, and have
one or more large nucleoli. The nuclei are centrally located and between the
nucleated cells are many smaller non-nucleated fragments. These frag-
ments represent the tapering ends of long cells cut at such an angle that the
nuclei are not included. Mitotic figures are abundant.

In some undifferentiated fibrosarcomas the spindle-shaped cells are often
in the minority. These tumors show many polyhedral cells that are large,
pale and closely packed. They grade into very large mononucleated and
multinucleated tumor giant cells. These have an irregular outline and
abundant, rather deeply eosinophilic, cytoplasm. Some of the largest cells
may have a stippled appearance due to the presence of tiny vacuoles. This
is a degenerative change which can advance into a signet ring type of cell
where the nucleus and cytoplasm are compressed into a small peripheral mass.
As a rule the more undifferentiated the cells, the less the amount of stroma
and the more rapid the growth of the tumor.

Liposarcomas. — These are malignant tumors originating from fat tissue
as in a lipoma. They are among the rare tumors in mice, but have been
observed in yellow stock animals.

Neurogenic fibrosarcoma. — This type is difhcult to separate from fibro-
sarcoma of connective tissue origin in the mouse. However, it can be
identified when the origin is definitely traced to nervous tissue. There is
also a more marked tendency to show a herring bone pattern type of arrange-
ment of the spindle-shaped tumor cells in neurogenic fibrosarcoma. It is
probable that the rapidly growing undifferentiated tumors of this group are
often classed with the fibrous connective tissue tumors, fibrosarcomas.

Melanomas. — These tumors have occurred for the most part in our dba
stock and the common site has been on or near the tail. There have been
cases of melanoma of the eye, ear and the skin in general. The tumor is
grossly brown to black and the color is often visible through the skin of the
living mouse. The tumor is smooth and rounded and may show tiny black
foci extending into the adjacent tissues. Lymph nodes when involved often
appear black to the naked eye, and lung metastases may be so extensive that
the lungs are sometimes solid and nearly black in color. The cut surface is
bulging, smooth and black or nearly black.

These tumors are usually malignant and the majority of them may
properly be called malignant melanomas. There is neither space nor neces-

SPONTANEOUS NEOPLASMS IN MICE

205

sity here to enter into the controversy over the exact tissue of origin and
whether they should be called melanocarcinomas or melanosarcomas. For
our purposes it is sufficient to designate them as either melanomas or
malignant melanomas.

The histopathology usually shows a tumor whose architecture and cellu-
lar detail is heavily masked by the intense pigmentation (Fig. 113). Around
the edge of the tumor the cellular detail is visible and shows large and oval or
smaller and spindle-shaped cells whose cytoplasm is filled with a closely

Epidermis

Dermis

Sebaceous gland

Melanotic tumor
cells

Fig. 113. — Melanoma of the skin (X200)

packed fine brown pigment, melanin. The most rapidly growing foci show
the least pigmentation and the cellular detail is easily seen under the micro-
scope. ^Mitotic figures are often abundant and invasion of the adjacent
tissues is extensive. This is one of the most widely metastasizing types of
tumors found in the mouse.

Rhabdomyosarcoma. — This is a malignant tumor originating from
striated muscle (Fig. 114). In the subcutaneous region it appears to occur
generally in mice of about the same age as animals bearing other types of
subcutaneous tumors. However, cases are sometimes observed in young
mice probably from embryonic rests in the striated muscle. The earliest

2o6

BIOLOGY OF THE LABORATORY MOUSE

case in our records occurred in a two and one half months old dba female.

This tumor is composed of cells which resemble embryonic muscle grow-
ing in wild confusion. These can be seen to originate from normal muscle
and may become spindle cells which resemble spindle cell sarcoma of fibrous
connective tissue origin.

The tumor cells are eosinophilic but paler than normal muscle. The
largest cells are most differentiated and possess finely granular cytoplasm.

Tumorous muscle
cell

Fig. 114. — Rhabdomj'osarcoma that originated in the striated muscle (X400).

There are usually some cells showing definite cross striations, as in normal
muscle, but most of these cells resemble embryonic muscle. Nuclei are
large, nearly round, fairly deeply staining and centrally located. The
stroma consists of an abundant blood supply and a small amount of
connective tissue. Where the cells are smaller and less differentiated, they
become more like fibrosarcoma in cellular characteristics and general
arrangement.

Infiltration of normal tissue occurs. Mitotic figures are not very abun-
dant in the foci which are most like normal muscle. This tumor can be
distinguished from fibrosarcoma of the connective tissue invading normal
muscle, for in the latter, muscle is being destroyed, while in the former there
are foci resembling embryonic and regenerating muscle cells.

SPONTAXEOUS NEOPLASMS IX MICE

207

Osteogenic sarcoma. — This is a malignant tumor of bone origin (Fig.
115). Primary tumors in the bone would include those originating from
osteogenic tissue and those arising from the bone marrow cells. In this
section we will consider only those which develop from osteogenic tissue and
which retain, more or less, the ability to form bone. The myelocytomas or
bone marrow tumors are considered in the section on the tumors of the blood
forming and blood destroying tissues.

We have not found a large number of osteogenic sarcomas in our labora-
tory. However, they have occurred in scattered body regions including the
skull, jaw, humerus, ribs, pelvis, femur and tail vertebrae. The stocks

■Osteogenic spindle-
shaped cells

\ H

fc ^^ >: I '^Tt^r^^'lrr il4 U.^g;*ajMrr-^Newly formed bone

Fig. 115. — Osteogenic sarcoma from a rib (X200).

showing most of these osteogenic sarcomas have been the yellow, the C57
black, dba, X, Danforth's posterior duplication and Zavadskaia's brachyury.
Hybrids of some of these stocks with other lines have also developed
osteogenic sarcoma.

Pybus and Miller (45), on the other hand, were able to derive sublines of
the Simpson strain of mice that develop a high incidence of spontaneous bone
tumors. These developed at about sixteen months of age and originated in
the skull, jaw, fore and hind limbs, ribs, sternum, pelvis and spine, most
often in the last named site. Among the early reports on bone tumors were
those by Ehrlich (10), Haaland (22), and J. A. Murray (43).

These tumors may develop as compact, rapidly growing, spindle-shaped
cells resembling tibrosarcoma. However, most of these tumors retain their
bone forming potentiality and show varying amounts of cartilage and bone
in all stages of development. Cartilage may or may not be found, and
frequently the tumors show foci of branching and anastomosing bony
trabeculae. Surrounding the bone and cartilage are interlacing strands of

2o8 BIOLOGY OF THE LABORATORY MOUSE

small, closely packed, spindle-shaped tumor cells and larger polyhedral
tumor cells.

Mitotic figures are usually quite abundant in the spindle cells. Invasion
into the surrounding normal tissues is quite extensive. Metastasis to the
lungs has been observed in several cases and definite bone formation is seen
in the pulmonary metastases.

When a sarcoma originating from bone shows definite cartilage formation
without newly formed bone, it is called a chondrosarcoma. The bone
sarcomas with some cartilage and considerable bone may be called chondro-
osteosarcomas. It is convenient, however, to call all malignant bone forming
tumors osteogenic sarcomas.

Angio-endotheliomas. — These are malignant tumors that appear to have
arisen within some of the pre-existing benign hemangiomas and lymphangi-
omas due to the malignant changes which involved the endothelial cells.
Malignant changes produce sarcomas which are called hemangio-endotheli-
omas and lymphangio-endotheliomas respectively.

In hemangio-cndothelioma the general architecture of the hemangioma is
evident. However, the endothelial cells are enlarged, rounded, vary in size
and have invaded much of the stroma. There are foci of solid masses of
cells in which the blood filled spaces have been obliterated. The cells have
rather deeply staining, finely granular, eosinophilic cytoplasm and moder-
ately hypochromatic oval nuclei. The nuclei contain many finely divided
chromatin granules. Mitoses are often abundant. Invasion of adjacent
normal tissues occurs and the simultaneous presence of this tumor in the leg
and spleen has been observed. Whether this is a case of multiple primaries
or metastasis is not easily determined.

The lymphangio-endothelioma shows the same type of malignant endo-
thelial cells invading the stroma. These tumor cells also grow into the
endothelial lined spaces. Infiltration of adjacent normal tissues is seen.
Mitoses may be frequent.

Tumors of the Lung

There have been several publications (4, 20, 50 and 67) on tumors of the
lung, but probably the first was by Livingood in 1896 (36).

The primary tumors of the lung are mainly those originating from the
lining cells of the bronchi and the alveoli. They may be classified as:

1. Adenoma.

2. Adenocarcinoma.

J. Papillary adenocarcinoma.

SPOXTANEOUS NEOPLASMS IN MICE 209

4. Carcinoma simplex.

5. Carcinosarcoma.

Workers have disagreed as to the degree of malignancy of lung tumors.
Among our tumor slides there are a large number of cases of spontaneous
tumors of the lung, many of which show characteristics definitely demon-
strating the malignant nature of lung tumors.

Adenoma. — Some small tumors are classed as adenomas because of their
cell arrangement and comparative inactivity, but small size alone is not a
true indication of the mass being benign. We have not observed a well
developed capsule, possibly because of the looseness of the lung architecture.
Adenomas appear fairly frequently as subserous, pearly white, slightly
elevated nodules, often one half to two millimeters in diameter. On section
they may be lenticulate to round. Their histopatholog>' shows rather
closely packed, poorly staining, polyhedral cells whose arrangement as
twisting, branching tubules with blunt ends is suggestive of immature,
uninflated air cells. Between these poorly defined structures is a network
of thin-walled, capillar}'-like blood vessels. The polyhedral cells are not
markedly different from many of the lining cells of normal pulmonary alveoli.
They have centrally placed, rounded or oval, somewhat hypochromatic
nuclei with one or more nucleoli, and abundant pale cytoplasm filled with
fine eosinophilic granules. The tumor cells differ from the normal in that
some are twice as large as the normal cells, some have lobulated nuclei and
others show two nuclei within a single cell. Not all of these tumors are at
the surface but are most likely to be observed there in gross dissection.

Some other tumors no larger than the above may differ from them chiefly
in that the parenchyma cells are more closely packed, have more eosinophilic
cytoplasmic granules, and exhibit a preponderance of large irregular cells.
These have hypochromatic nuclei which often appear as two distinct nuclei
within a single cell. It is not uncommon to see nuclei with multiple lobules,
and sometimes a dozen or more closely clustered, rounded nuclear masses are
seen within a single tumor cell. This indicates amitotic division. Mitotic
figures are also occasionally seen.

The outlines of these two types of tumors dift'er in that the latter may be
more irregular. In this type there are some foci of infiltration into normal
alveoli, while other foci show compressed, collapsed alveoli resulting from
the pressure of the tumor growth by expansion. Invasion of the smaller nor-
mal bronchi can sometimes be seen. We call this type an adenocarcinoma.

Adenocarcinoma. — It is a common belief that this tumor originates from
the lining cells of the alveoli but it may also originate from the bronchi.

2IO BIOLOGY OF THE LABORATORY MOUSE

Here the tumor consists of poorly formed, gland-like structures scattered
through nests of irregularly arranged tumor cells. These cells vary from
polyhedral to columnar, possess hypochromatic nuclei and abundant cyto-
plasm filled with fine eosinophilic granules which are similar to those of the
epithelium of the bronchi. The stroma consists principally of thin-walled
blood vessels. Mitoses are frequent. Metastases in the liver have been
observed.

Papillary adenocarcinoma of the lung. — This type may be seen as definite
masses within, or continuous with, the more benign adenomatous form dis-
cussed above, and apparently arises through malignant changes. This type
of carcinoma may also be in direct continuity with one of the smaller bronchi.
The papillary type of growth is by far the most commonly seen among our
carcinomas of the lungs of mice. Even tiny masses show it as definitely as
the large tumors, which may involve an entire lobe of the lung. The archi-
tecture shows closely packed, branching and anastomosing, thin, finger-
like strands with a stromal core of capillaries and a varying amount, usually
small, of connective tissue stromal cells (Fig. ii6). The papillary adeno-
carcinomas are darker staining than the benign type, have more cells, many
of which are larger and show piling up to form several cell layers at many
foci on the papillae. The tumor masses are chiefly composed of papillary
structures without cyst formation. Occasionally one can observe dense foci
of connective tissue stroma from which several of the papillary growths
extend to form the main tumor mass. The stroma in the bulk of the tumor
is often scant. The tumor cells of papillary adenocarcinomas vary in size
and shape. Eosinophilic cytoplasmic granules are prominent, nuclei are
hypochromatic and vary in size and shape, with some multilobulated,
bilobed and binucleated forms present. In some tumors, foci of columnar
tumor cells show well developed goblet cells. A few tumors may exhibit one
to several layers of epithelial tumor cells lining intercommunicating spaces
with only thin strands of stroma between them. Into these spaces project
short, branching papillae giving an irregular appearance to the lining. On
cross section these structures appear as large glands, not as cysts, presenting
frequent foci of ciliated columnar epithelial tumor cells. These are located
between the more piled up foci of tumor cells.

Papillary adenocarcinomas are often found close to the smaller bronchi,
and it is not uncommon for these tumors to extend into these bronchi, nearly
occluding them.

Carcinoma simplex. — This may originate in a papillary adenocarcinoma
and often appears as closely packed cells arranged in an irregular pattern.

SPONTANEOUS NEOPLASMS IN MICE

211

This type contains very little stroma except for numerous small, endothelial
lined, capillary-like blood vessels. The outlines of the tumor cells may be
vague but suggest round and polyhedral shapes. They have irregular, oval,
hypochromatic nuclei and the cytoplasm is filled with rather deeply staining

7iit^'^:^^

Fig. ii6. — Primary papillary adenocarcinoma of the lung (X200). This tumor
is sub-pleural and has been invaded by metastatic carcinoma of the mammary gland
(lower left on the illustration). 1., lung; m.m., metastatic mammary gland carcinoma;
p.l., primary lung carcinoma; str., stroma.

eosinophilic granules. Sometimes the cytoplasm is reduced in amount,
nuclei vary in size and have prominent nucleoli. These cells show no defi-
nite arrangement and are accompanied by more stroma than the above.
The undifferentiated tumor cells may blend with definite papillary adeno-
carcinoma. This tumor shows abundant mitotic figures and may develop
widespread metastases.

212 BIOLOGY OF THE LABORATORY MOUSE

Carcinosarcoma. — This type may occur when the stromal connective
tissue of a lung carcinoma becomes malignant. We have seen it most
commonly in papillary adenocarcinomas. The sarcomatous part is com-
posed of rather large spindle cells irregularly arranged in interlacing strands.
Nuclei are spindle-shaped, darker than in the carcinoma, and the eosino-
philic cytoplasm appears to have longitudinal striations but no granules.
Mitotic figures are abundant.

Primary tumors of the lung other than the above types are rare. How-
ever, the lung is a common site for metastases of carcinomas and sarcomas
from many other body regions (Fig. ii6). This is especially true of carci-
nomas in the mammary region which grossly may resemble carcinomas
primary in the lung. On histological examination a primary tumor of the
bronchi or alveoli can be identified as such by the characteristics of the
tumor cells. This includes their close resemblance to the normal lining cells
of the bronchi and alveoli in cell outline and staining properties. Primary
lung carcinoma cells are paler than mammary carcinoma cells and contain
fine eosinophilic cytoplasmic granules, as well as lobulated nuclei and multi-
nucleated cells which are not characteristic of the breast carcinomas. Other
types of pulmonary metastases that have been seen are from carcinoma of
the liver, malignant melanoma, osteogenic sarcoma, lymphocytoma, mono-
cytoma, etc.

Tumors of the Blood Forming and Blood Destroying Tissues
(Round Cell Sarcomas)

Enlargement of the mesenteric lymph nodes is not uncommon in old mice
from many of our stocks. Frequently this enlargement is benign and is
associated with some chronic infection. The usual finding in such cases is
lymph node hyperplasia. There is, however, a tendency for a small per-
centage of the mice from nearly all of the stocks to develop spontaneous
neoplasms of any of the lymph nodes, the spleen and sometimes the thymus.
Occasionally a tumor appears at a single focus, such as the mesenteric lymph
node. When the axillary and inguinal lymph nodes are involved, there is
usually a bilateral enlargement of these glands as well as of the cervical
lymph nodes. The spleen and internal lymph nodes may or may not
become enlarged when bilateral enlargement of the subcutaneous lymph
nodes occurs.

Mice with neoplasms involving the spleen and lymph nodes do not
usually live long after the symptoms become marked. The outstanding
gross characteristics are signs of ill health, such as dull, rough coat and

SPONTANEOUS NEOPLASMS IN MICE 213

general emaciation accompanied by weakness and kyphosis. The abdomen
becomes greatly distended by either enlargement of the spleen or ascites, or a
combination of these two conditions. In some advanced cases marked
subcutaneous edema obscures the emaciation. When the body cavity is
opened, the edematous subcutaneous tissue is found to contain a clear
serous-like fluid, and the intra-abdominal liquid may be serous or sero-
sanguineous. Hydrothorax is also a fairly common finding when ascites is
marked.

Lymph nodes are not uniformly enlarged. The mesenteric lymph node
is usually but not always involved, and may be enlarged to three centimeters
in length. Sometimes the spleen is enormous, light gray, friable and granu-
lar. Other lymph nodes may be enlarged in varying degrees. In a condi-
tion that is generalized the subcutaneous, mediastinal and intraperitoneal
lymph nodes are enlarged and tumor cells from them show infiltrations into
adjacent normal tissues and organs. The organs outside the lymphatic
system which most frequently show gross involvement and tumor nodules
are the liver, kidneys and the lungs.

The microscopic picture presented by these neoplasms is varied, since the
tumors may be made up of cells which are predominantly from the unre-
stricted proliferation of lymphocytes, of immature myelocytes or of mono-
cytes. These cells produce tumors with var\dng frequency in different
stocks of mice. Numerous workers have published on this group of tumors
in mice. Probably the earliest report was by Eberth (1878). However,
many early reports were on small numbers of animals and the terminology
employed has sometimes been confusing. More recently there have been
several reports employing large numbers of mice and a fairly clear classifica-
tion is in use. Tumors resulting from the unrestricted proliferation of
lymphocytes and immature myeloid cells are well understood (Table i).
The third group, however, is less understood. This is largely because the
origin and nature of monocytes are among the most debated problems of
morphologic hematolog>\ The interpretation used here is based upon the
classification of human tissues employed in the Lymphatic Tumor Division
of the American Registry of Pathology (6). From a review of the literature
and from experiments conducted by himself and his co-workers, J. Furth
(15) gives his conclusions on terminology and says: "that monocytes,
histiocytes, macrophages, clasmatocytes, polyblasts, Kupffer cells and
microglia cells are synonymous terms for one cell type, which is capable of
perpetuating itself by mitotic division. In this (Furth's) communication
we shall refer to the round forms of this type of cell seen in the circulating

214

BIOLOGY OF THE LABORATORY MOUSE

blood, as monocytes, and to all other forms as histiocytes. Tumors of
monocytes or histiocytes will be named histiocytomata (monocytoma) and
the systemic disease characterized by these cells histiocytoma tosis (mono-
cytomatosis). Monocytic leukemia is a synonymous term for leukemic

Table i

Tumors and Tumor-like Conditions or Blood Forming and Blood

Destroying Tissues in Mice

Cells

Lymphocytes

Myeloid Cells

Monocytes
(Histiocytes)

Granular
Leukocytes

Red Blood
Corpuscles

Nonneoplastic
increase in cells

1. Hyperplastic
lymph nodes
(Lymphoma)

2. Benign lymph-
oid infiltrations

1. ExtrameduUary
myelopoiesis

2. Leukocytosis

Poly-
cythemia*

Granuloma
(infections)

Neoplastic (inva-
sive with cells
fairly uniform)

Lymphocytoma

1. Leukemic

2. Aleukemic

Myelocytoma

1. Leukemic

2. Aleukemic

Erythro-
cytoma*

Monocytoma

1. Leukemic

2. Aleukemic

Neoplastic (inva-
sive and cells
pleomorphic)

Lymphosarcoma

1. Leukemic

2. Aleukemic

Myelosarcoma

1. Leukemic

2. Aleukemic

Erythro-
sarcoma*

Monocyte sar-
coma

1. Leukemic

2. Aleukemic

Early site of
neoplasm

Germinal centers
of spleen and
lymph nodes

Red marrow and
outside germinal
centers — red pulp
of spleen and med-
ullary tissue of
lymph nodes

Histiocytes of
liver, spleen and
lymph nodes

* No cases on record in mice.

histiocytomatosis (monocytomatosis)." In consideration of the above we
have employed the classification shown in Table i.

Each of these types of tumors may or may not show an abnormal blood
picture. When these tumors are accompanied by a marked increase of the
tumor cells in the circulating blood, outside of the lymphatic system, this
condition is called leukemia. The absence or paucity of these tumor cells in
the circulating blood of animals that have developed tumors of this group is

SPONTANEOUS NEOPLASMS IN MICE 215

called aleukemia (pseudo-leukemia). Without blood smears, it is not easy to
classify these tumors as to whether they are leukemic or aleukemic. Some
workers (48) have drawn their conclusions from a study of the large blood
vessels of the liver, lungs and kidneys. Tissue imprints with special stains
have been very valuable in determining the types of abnormal cells present in
the tissues {t,^). Generally, the greater the number of tumor cells in the
blood, the less the lymph nodes are enlarged and vice versa.

Most authors agree that there are no benign tumors produced bv either
lymphocytes, myeloid cells or monocytes. The non-neoplastic condition
which has caused the most confusion is probably non-malignant extramedul-
lar)' myelopoiesis. This condition is very common in the spleen of older
mice of some stocks (3). In extramedullary blood forming foci all the ele-
ments of the normal marrow are usually present. The granulopoietic
elements most often predominate over the er>'thropoietic and megakaryo-
cytic elements. This condition is found most frequently in the spleen and
liver. The sites usually involved by extramedullary myelopoiesis are
similar to those in cases of myeloid leukemia. In the former all stages of
development of myeloid cells are present, while in the latter most myeloid
cells are immature. Additional information on the dilTerences between
these two conditions can be found under myeloid tumors.

Lymphocyte Tumors (Lymphoblastoma)

The most commonly observed tumors of the lymphatic system are those
of the lymphocytes (Fig. 117). They appear first in the nodules of the
lymph nodes and in the Malpighian bodies of the spleen. The primar\' foci
increase in size, due to proliferation of the lymphocytes, and progress until
they obliterate the normal architecture of the lymph nodes and spleen,
leaving only uniform masses of lymphocytes. These tumors always invade
the lymph node capsules (48). Due to the extent of the lymphatic sys-
tem, infiltration of adjacent tissues is difticult to differentiate from true
metastases.

Lymphocytoma. — This shows fairly uniform cells of the lymphocyte
type; however, they are larger than normal cells. Usually they belong to
the large lymphocyte variety. Mitotic figures are often numerous (Fig.
117). The liver, lungs and kidneys are the organs most often invaded. In
the liver the periportal foci are first involved, in the lungs perivascular
infiltration is most marked and in the kidney the infiltration extends inward
from the hilus. This tumor mav be leukemic or aleukemic, focal or more or

2l6

BIOLOGY OF THE LABORATORY MOUSE

less generalized. In the generalized type there is usually a focus of greatest
lymph node enlargement.

Lymphosarcoma. — (Lymphoblastoma sarcoma type.) This type may be
leukemic or aleukemic. The cells are more pleomorphic than in lympho-
cytoma, showing atypical lymphocytes with irregular nuclei and little cyto-
plasm. The more atypical the cells the more malignant the tumor. Mitoses
are abundant.

Lymphocyte tumor cells Mitoses Normal lymphocytes

'^^^^^^^

Fig. 117. — Lymphocytoma in the mesenteric lymph node (X400).
Myeloid Cell Tumors

Only those tumors from myeloid cells which form the granular leukocytes
will be considered. These tumors are rare in most stocks of mice, but
Barnes and Sisman report that several cases have been seen in their stock Rf
and in stock S, and the same is true of Strong's F strain (3, t,^)- The sites
of early involvement are bone marrow, the red pulp of the spleen and the
medullary tissue of the lymph nodes. Lymph nodes are sometimes greenish
(chloroma). In the spleen and lymph nodes the immature myeloid cells
surround the germinal centers and may obliterate them. The following
criteria have been suggested as an aid in distinguishing between myeloid cell
tumors and non-malignant extramedullary myelopoiesis in mice (3).

SPONTANEOUS NEOPLASMS IN MICE

217

Myeloid Cell Tumors
Most myeloid cells are immature

Erythrogenic foci are absent among
myeloid cells

Megakaryocytes are few and present
only in the organs (the spleen, liver
and lymph nodes) where they are
found in non-leukemic conditions

Myeloid cells often invade muscle and
other non-hematopoietic tissues

Blood usually contains immature mye-
loid cells

Liver is usually enlarged and gray-
brown

Most of the lymph nodes are usually
enlarged

Hemorrhages are frequent in viscera
(lungs, lymph nodes, etc.)

Transmissible to other mice

Not shown to be produced by bacteria

Non-Malignant Extramedullary

Myelopoiesis

All stages of development of myeloid

cells are present
Erythrogenic foci are usually present

Megakaryocytes are usually numerous

Cells are non-invasive

Blood is normal or there is leukocy-
toses with numerous mature forms

Liver is usually not enlarged and is
brown-red

Most of the lymph nodes are usually
of normal size

Hemorrhagic manifestations are ab-
sent

Not yet shown to be transmissible

Can be produced by bacteria

Myelocytoma. — This is a tumor made up of immature myeloid cells.
The predominating tumor cells may be myeloblasts or different kinds of
immature granulocytes (Fig. 118). There may be considerable variation
between the cells of different tumors, but the cells have a tendency to be
rather uniform in individual cases. Variations in size and staining power of
these tumor cells indicate an approach to the sarcoma type. Myelocytoma
may be leukemic or aleukemic. Mitoses may be fairly frequent and inva-
sion into adjacent normal tissues occurs.

Myelosarcoma. — This is similar to myelocytoma, except that the cells
are more variable in size and assume bizarre shapes. Mitoses are frequent.
Extensive infiltrations into normal tissues may occur.

Monocyte Tumors (Monocytoma or Histiocytoma*)

These tumors are rare in many stocks of mice. However, Tyzzer (67)
and Slye (51) report cases, without diagnoses, which from their pictures and
descriptions appear to be similar to a type of tumor (probably monocytoma)

* (The neoplasms sometimes called reticulum cell tumors probably belong in this
group.)

2l8

BIOLOGY OF THE LABORATORY MOUSE

MYELOBLAST PROMYELOCYTE

seen in the C57 black, C57 brown and C57 leaden stocks. There are occa-
sional reports in the literature on reticulum cell tumors and references to
cases in mice resembhng Hodgkin's disease (15, 26). J. Furth states that
most human neoplasms of the histiocytes have been described as reticulum
cell sarcoma or reticulosis, leukemic and aleukemic, but since the relation of

histiocytes to reticulum fibers and to reticular
libroblast-like cells of blood forming organs is
obscure, this terminology is not desirable. He
suggests the use of the term histiocytoma or
monocytoma to cover these tumors in the blood
forming organs.

Monocytoma. — Monocytes may form tumors
in the liver and spleen and infiltrate these and other
tissues. The liver becomes enormously enlarged
and mottled with minute irregular gray -white
masses of tumor cells, hemorrhagic areas and
yellowish-gray necrotic foci. The spleen and
lymph nodes may or may not be enlarged grossly.
Microscopically the spleen usually shows small
foci of tumor cells, hemorrhage and necrosis.
Occasionally the lungs show large or small areas
of hemorrhage with yellowish-gray foci of tumor
cells. Death usually results from internal
hemorrhage starting from the lesions in the
liver.

There are leukemic and aleukemic forms of
this tumor. The cells are the large monocyte type
with oval, bean-shaped or irregularly lobed,
eccentric nuclei. Considerable non-granular
basophilic cytoplasm is present. Infiltration of
the liver is diffuse with frequent formation of small
tumor nodules. MaHgnant cells are seen within the blood vessels of the
liver, sometimes nearly occluding them. The spleen may show the same
type of tumor cells and blood vessel involvement. Lymph nodes may or
may not be involved. When involvement occurs, it is around the nodules.
Mitoses are somewhat numerous. The tumor cells have some phagocytic
ability. A condition exactly like Hodgkin's disease has not been found in
mice, but there are certain similarities between monocytoma and Hodgkin's
disease (15).

POLYMORPHONUCLEAR

LEUKOCYTE
Fig. 118. — Diagram
showing the development
of granular leucocytes
from the immature myelo-
blast to the fully formed
polymorphonuclear leuko-
cyte. Note the changes
in number and size of the
granules. {Redrawn from
Barnes and Sisman.)

SPONTANEOUS NEOPLASMS IN MICE 219

Monocyte sarcoma.— -This tumor is similar to the above but mitoses are
more frequent and the cells show more variation in size. Large tumor giant
cells and bizarre shaped cells are often seen.

The writer is well aware that there is considerable difference of opinion
concerning the monocyte tumors. Because of this the above discussion is
necessarily brief and somewhat incomplete. The work now being done at
several institutions should be of real value in clarifying their classification
and nomenclature.

Tumors of the Digestive System and Associated Glands
Digestive Tube and Submaxillary Gland Tumors

Tumors of this region are rare but they do occur. In the submaxillary
gland we have seen an adenoma in a yellow stock mouse and a carcinoma
simplex in an A stock albino female. Tumors of the oral cavity and the
esophagus have been extremely rare (68). However, we have had a papil-
loma develop in the esophagus of a mouse.

Marked hyperplasia of the epithelium in the glandular part of the
stomach has been observed in several of our mice. This has also been
recorded in other laboratories. Wells (68) reviews the literature and reports
three adenocarcinomas of the pylorus. Epidermoid carcinoma of the non-
glandular part of the stomach has been seen. Here the normal lining is
stratified squamous epithelium and the tumor shows definite epithelial pearl
formation. In a C57 black mouse this type of tumor has been observed to
infiltrate through the stomach wall and to begin the invasion of the pancreas.
Similar tumors have been recorded by Slye (54, 68) and others.

A few intestinal polyps have occurred. In one case beginning adeno-
carcinoma was observed in this type of polyp. Carcinomas are rarely
observed, even though the intestine is inspected routinely at autopsy. In
Slye's laboratory a few squamous cell carcinomas and adenocarcinomas of the
intestine have been found (68). Cavernous hemangioma and primary
fibrosarcoma have been found in our C57 black, the dba mice and their
hybrids. Other fibrosarcomas have been seen in the mesentery of the small
intestine. Lymphocyte tumors also occur in the lymph nodes of the intes-
tines in several stocks.

Hepatic and Gall Bladder Tumors

The liver is a relatively common site for primar>' tumors (hepatomas).
Regeneration adenomas are the result of rapid proliferation as an attempted
repair following injury. Such lesions have not been classified as tumors.

2 20 BIOLOGY OF THE LABORATORY MOUSE

True adenomas of the liver parenchyma cells occur most frequently in the
C57 black, yellow, and dba stocks but are not limited to them. These
tumors are circumscribed growths of atypical parenchyma cells with atypical
arrangement but without signs of infiltration or marked activity. Slye (51)
found a few similar tumors. Carcinoma of the liver is encountered quite
often among our primary liver tumors. The usual picture is of large and
small liver parenchyma cells growing in wild confusion with frequent tumor
giant cells and bizarre cell forms (Fig. 119). Normal architecture is lost and

b.v.

m.f.

Fig. 119. — Carciiuuna ui iht- liver parenchyma cells (xjoo). Ij.v., blood vessel;
h.s., hepatic sinusoid; m.f., mitotic figure; p.c, tumorous liver parenchyma cells.

invasion of adjacent normal liver occurs. Mitoses may be frequent and
metastasis to the lung occurs. The tumors of the liver parenchyma cells
appear grossly as elevated or pedunculated masses that are almost the same
color as normal liver. There is a rare form of carcinoma of the liver com-
posed of large, pale cells whose arrangement suggests attempted gland
formations. Mitoses are frequent. Papilloma of the gall bladder has been
observed.

Non-epithelial liver tumors are also seen somewhat frequently. Tumors
of the lymphoid, myeloid, and monocyte cells are taken up elsewhere.
Hemangiomas are a fairly common type of liver tumor, and hemangio-
endotheliomas are occasionally observed in some stocks. A lymphangio-

SPONTANEOUS NEOPLASMS IN MICE 221

endothelioma has been seen in the Hver of an A stock female. Fibrosarcomas
of the Uver have been observed in several stocks.

Pancreatic Tumors

Primary tumors of the pancreas are rare. Adenocarcinoma has been
observed in one Mus hactrianus female and in two hybrids of this stock
crossed with C57 black. Slye (61) reported two cases in 125,000 autopsies.

Fig. 120. — Adenoma of the renal tubules (X400). ad., adenoma; n.r.t., normal renal

tubule.

Carcinoma of the pancreatic islands has been seen twice in our stocks.
Hueper (27) reports another case. Fibrosarcoma also occurs in this gland.

Tumors of the Uro-genital System

Kidney and Urinary Bladder Tumors

The kidney is a fairly frequent site for secondary involvement of tumors
of the blood forming and blood destroying tissues. However, primary
tumors of this organ are not common. We have had one case of adetioma
of the renal tubules (Fig. 120) and one case of papillary cyst adenocarcinoma.
Several papillomas of the renal pelvis have occurred (Fig. 121A). A few of

222

BIOLOGY OF THE LABORATORY MOUSE

these papillomas showed beginning adenocarcinoma (Fig. 121B). Primary
fibrosarcoma also occurs in this organ. Slye (57) reported on kidney tumors
of several types.

Although tumors of the urinary bladder are extremely rare, papillomas
have been found. One extensively invading and rapidly growing carcinoma

P^iG. 121. — Tumors of the renal pelvis. A, papilloma of the renal pelvis (X200);
B, adenocarcinoma in a papilloma of the renal pelvis (X200).

of the transitional cell epithelium occurred in a Riga stock female. Hem-
angiomas are also seen (Fig. iiiA).

Ovarian Tumors

Among the first reports on tumors of the ovaries in mice were those of
Jobling (30), Tyzzer (67) and Haaland (23). Slye, Holmes and Wells (56)
stated that they had found 44 primary ovarian tumors in 22,000 autopsies.
Gardner, Strong and Smith (19) described a case of spontaneous bilateral
granulosa cell tumors in an old mouse. J. Furth and Butterworth discuss

SPONTANEOUS NEOPLASMS IN MICE 223

the types of ovarian tumors found in mice subjected to x-rays and state that
spontaneous tumors of the ovaries are very rare (16). This last paper is
of interest to us in that we have observed, in our spontaneous tumors of the
ovaries, types that compare with most tumors which developed following
irradiation.

Probably because of the complexity of the ovary it can be the primary
site of a fair number of tumors in some stocks of mice. This is true of the
ce (extreme dilution) stock in our laboratory. Scattered cases have been
found in several of the pure stocks. Also offspring of crosses between pure
stocks have developed several spontaneous tumors especially where the
C57 black mice have been crossed with the dba, A albinos and Mus bac-
trianus. Altogether we have found over 50 spontaneous ovarian tumors
in our mice. This does not include simple cysts which are common and are
not malignant. They are probably associated with abnormal physiology.
These cysts may be lined by a single layer of flattened or cuboidal epithelium
and occasionally are distended by hemorrhage into the cyst cavities. Those
lined by ciliated cuboidal epithelium arise from the vestigial tubules, the
epoophoron.

Ovarian tumors in mice show considerable variation in appearance. The
majority of these tumors already observed would probably tit into one of
the following classes. No doubt additional forms will be found.

A. Cystic tumors.
Papillary cyst adenoma.
Papillary cyst adenocarcinoma.

B. Solid tumors.
Granulosa cell tumor.
Hemangioma.
Hemangio-endothelioma.
Fibroma.
Fibrosarcoma.

C. Embryonal tumors.
Teratoma.

Embryonal adenoma.
Embryonal cell carcinoma.

Cystic tumors. — A cyst that grossly appears to be simple may contain
numerous small or large papillary ingrowths. These papillae may have
simple cuboidal or columnar epithelial cells covering their surfaces (Fig.
122). Such tumors are papillary cyst adenomas and are benign . A papillary
cyst adenocarcinoma may arise in the same manner.

224

BIOLOGY OF THE LABORATORY MOUSE

Solid tumors. — In the non-tumorous ovary the changes of the follicular
cells into granulosa cells and their further differentiation to form lutein
cells are not sharp but gradual. For this reason definite lines cannot be
drawn, and borderline cell types can be observed. Therefore, there is
justification for considering that the tumors made up of cells resembling
the above phases should be grouped together. The growing tendency is to
call all such tumors granulosa-ccll tumors. This type is the most common of

Fig. 122. — Papillary cyst adenoma of the ovary (X200).

the solid ovarian tumors observed here. Grossly they are usually rather
large.

This places in one group tumors that show a wide variation in the type
and arrangement of cells. The cells may be fairly uniform and rather
closely resemble foUicular, granulosa, theca or lutein cells. However, there
is often what appears to be a mixture of two or more of these cell types.
The cells may be arranged in a pattern that resembles closely packed, large
and small follicles, some distinct and some confluent, separated by thin
septa of stroma. Sometimes the cells grow in irregular cords which bear a
resemblance to the trabeculae seen in the early stages of corpus luteum
formation. There may also be seen more or less solid masses of cells with
some stroma and scattered, almost gland-like foci that resemble attempted
follicle formations. There are other tumors with large, pale, spindle shaped

SPONTANEOUS NEOPLASMS IN MICE 225

cells which show foci that appear almost sarcomatous. Probably at least
part of the latter are from theca interna cells. In the ce stock, at least,
large clusters of Sertoli-Hke cells are often encountered with the last men-
tioned form of tumor cells. The large, clear, lutein-like cells seen by J.
Furth and Butterworth (16) have not been found as the type cell of any
of our spontaneous ovarian tumors. However, MacDowell-Bagg stock
albinos treated with x-ray have produced several, and these have shown
occasional mitotic figures. This is mentioned because the potentiality for
the formation of lutein-like tumor cells is present and these tumors will
probably appear spontaneously in rare cases.

All the above variations of spontaneous granulosa-cell tumors are
probably benign. There are, however, mitotic figures in some cases, and
the tumor masses may be fairly large and nodular in outline. Some
sarcoma-like tumors have foci that suggest granulosa-cell tumors. These
are difficult to diagnose with certainty.

Cavernous hemangioma is occasionally seen in the ovaries of mice. Still
more uncommon is hemangio-endothelioma which has been observed a few
times. True fibrosarcoma of the ovary is also rare in our stocks. A few of
these tumors have been diagnosed as primary at this site. Fibromas have
not been observed in our mice.

Embryonal tumors of the ovary. — A rare, benign tumor of the ovary is
the teratoma. This usually shows a mixture of bone, cartilage, striated
muscle and gland structures as well as other tissues. There may be skin,
nerve or almost any tissue in this type of tumor (Fig. 123).

Occasionally there is a tumor composed of closely packed epithelial cells
arranged as in embryonic gland formation. This is called an embryonal
adenoma and is benign. The cells are uniform, small and deeply staining.
Mitoses are rare.

Embryonal cell carcinoma is composed of large, rounded, pale epithelial
cells varying in size. They have a fair amount of pale cytoplasm and
rounded, hypochromatic nuclei with coarse chromatin granules. These
cells are compactly arranged without much stroma; mitoses are abundant.

Uterine Tumors

Epithelial tumors at this site are rare in mice (58). Our records show
that adenomas have been observed twice in the dba stock. Carcinoma
simplex has also been observed in two mice, both hybrids, one from a cross
between dba and C57 black, the other from a cross of dba with yellow. The
former is shown in Figs. 124A and 125. Here the epithelium can be seen

226

BIOLOGY OF THE LABORATORY MOUSE

grading over into carcinoma simplex tumor cells which are invading the
uterine wall.

Of the non-epithelial tumors fibrosarcoma is the most common tumor of
the uterus (Fig. 124B). Quite a number of cases have been seen. This,
however, does not represent a high incidence in any of the pure stocks or

mu. bn.

Fig. 123. — Teratoma of the ovary (X200). bn., bone; cart., cartilage; gl., gland; mu.,

striated muscle.

their hybrids. Of the pure stocks it is probably most common in the dba.
It is, however, seen in the C57 black mice. Most of our cases have devel-
oped in crosses between these two stocks or in hybrids between C57 black
and A albino.

With this tumor the uterus is greatly enlarged, firm and friable. The
enlargement is usually bilateral and these tumors are not multiple as is the
case of the fibroid tumors in the human.

Histologically this tumor is composed of small, closely packed, short
spindle cells with little stroma. The cells are arranged in an irregular inter-
lacing pattern of whorls (Fig. 124B). Mitotic figures are not common but

SPONTANEOUS NEOPLASMS IN MICE

227
The

blood vessel invasion occurs and metastases in the liver are seen.
ovaries are sometimes involved by extension of this tumor.

Leiomvosarcoma is a tumor which is grossly like fibrosarcoma but micro-
scopically is composed of larger, longer spindle cells. These cells are
arranged in irregular interlacing bundles and arc of the smooth muscle type
characteristic of the uterine wall. Mitoses are not frequent.

Fig. 124. — Primary tumors of the uterus. A, carcinoma simplex that originated
in the uterine epithelium and invaded the uterine wall (X200); B, fibrosarcoma in the
wall of the uterus (X200). car., carcinoma simplex; ep., epithelium; n.u.w., normal
uterine wall; sar., fibrosarcoma.

Other tumor forms seen include adenofibrosarcoma, hemangioma and
hemangio-endothelioma. All these are rare. The first shows a few uterine
glands deep within a fibrosarcoma. The third type has been seen but once
and was in the oviduct.

Tumors of the Testes

Tumors are rare at this site. Slye (55) reported 28 primar}' tumors in
the testes of mice. The majority of her tumors appear to be similar to two
cases that we have called embryonal cell carcinoma. These tumors show-
many of the characteristics described by her. The tumor cells are large,
rounded and pale with abundant cytoplasm and hypochromatic nuclei

228

BIOLOGY OF THE LABORATORY MOUSE

containing coarse chromatin granules. Some nuclei are vesicular. Mitotic
figures are quite numerous. The architecture shows cords and dense
masses of epithelial cells without much stroma. One of our cases appeared
in the I stock and the other in the black-eved white (AMC) stock.

b.v.

'>;c^^

Fig. 125. — Carcinoma simplex of the uterus (X400). b.v., Ijlood vessel; car., car-
cinoma simplex; e.p., epithelium grading over into carcinoma; u.l., uterine lumen.

No other types of tumors of the male reproductive organs have been
found in our stocks. Slye reported sarcomas found in the testicle and in
the seminal vesicle.

Tumors or the Central Nervous System

Brain tumors. — These neoplasms are rarely found in mice. We have
observed a medulloblastoma (Fig. 126). Another tumor has been diagnosed
as a glioma. Both of these were in C57 black females. It is of interest that
in this same stock hydrocephaly has been observed in a number of young of

SPONTANEOUS NEOPLASMS IN MICE

229

both sexes. The only other cases in the Uterature are a papillary adenoma
of the ependyma cells of the lateral ventricle, an endothelioma of the cere-
brum and an adenoma of the hypophysis in 11,118 autopsies by Slye (60).
One other adenoma of the hypophysis has been reported (19). In our stocks
we have diagnosed two adenocarcinomas of the hypophysis composed chiefly

Normal brain

— Medulloblastoma

Fig. 126. — Medulloblastoma from the brain of a mouse (X200).

of eosinophile cells. These were in hybrids from a cross between the C57
black and C57 brown stocks.

Other Rare Sites of Tumors

Among the rare sites of tumors is the heart. We have observed a
rhabdomyosarcoma of this organ and Hertzog (25) reported a papillary
fibroma of the cardiac valve. Slye (59) reported tumors of the thyroid.
However, we have not observed neoplasms at this site. In the glands
around the eye we have found two papillary cystadenomas, a papillary
adenocarcinoma and an adenocarcinoma.

This chapter has been intended to emphasize, mainly, the types of
spontaneous tumors that are most commonly encountered. As data on

230 BIOLOGY OF THE LABORATORY MOUSE

spontaneous tumors are being steadily accumulated, there will be addi-
tional types of tumors found and more information will be available on the
tumors at other sites.

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44. Murray, J. A. 1921. Lymphatic dissemination in spontaneous and transplanted
tumors of mice. Imperial Cancer Res. Fund, Scient. Rep. 7: 63-66.

45. Py'bus, F. C. and E. W. Miller. 1938. Spontaneous bone tumors of mice.
Am. J. Cancer ^^: 98-1 11.

46. ScHABAD, L. 1928. Spontaneous tumors of lung in mice. Compt. rend. Soc.
biol. 99: 1383-1384.

47. ScHABAD, L. AND V. KoRKHOFF. 1935. Hematomas of the mouse ovary resem-
bling spontaneous tumors. Neoplasmes 14: 7-14-

48. SiMONDS, J. P. 1925. Leukemia, pseudoleukemia and related conditions in
the Slye stock of mice. Am. J. Cancer 9: 329-373.

49. Slye, M. 1927. Studies in incidence and inheritability of spontaneous tumors
in mice. Twenty-fifth report. J. Cancer Res. 11: 54-71.

50. Slye, M., H. F. Holmes and H. G. Wells. 1914. The primary spontaneous
tumors of the lungs in mice; studies on the incidence and inheritability of spon-
taneous tumors in mice. J. Med. Res. 30: 417-442.

51. Slye, M., H. F. Holmes AND H. G. Wells. 1915. Spontaneous primary tumors
of liver in mice. J. Med. Res. t,^'- 170-182.

52. Slye, M., H. F. Holmes and H. G. Wells. 1916. Spontaneous sarcoma in
the mouse. J. Cancer Res. i : 360.

53. Slye, M., H. F. Holmes and H. G. Wells. 191 7. Primary spontaneous
sarcoma in mice. J. Cancer Res. 2: 1-27.

54. Slye, M., H. F. Holmes and H. G. Wells. 191 7. Comparative pathology of
cancer of stomach with particular reference to the primary spontaneous malignant
tumors of the alimentary canal in mice. J. Cancer Res. 2: 401-424.

55. Slye, M., H. F. Holmes and H. G. Wells. 1919. Primary spontaneous
tumors of the testicle. J. Cancer Res. 4: 207-229.

56. Slye, M., H. F. Holmes AND H. G. Wells. 1920. Tumors of the ovary in mice.
J. Cancer Res. 5: 205-226.

57. Slye, M., H. F. Holmes AND H. G. Wells. 1921. Primary spontaneous tumors
in kidney and adrenal of mice. J. Cancer Res. 6: 305-337.

58. Slye, M., H. F. Holmes and H. G. Wells. 1924. Primary spontaneous
tumors of the uterus in mice. J. Cancer Res. 8: 96-117.

59. Slye, M., H. F. Holmes and H. G. Wells. 1926. The comparative pathology
of cancer of the thyroid with report of primary spontaneous tumors of thyroid.
J. Cancer Res. 10: 175-194.

60. Slye, M., H. F. Holmes and H. G. Wells. 193 i. Intracranial neoplasms in
lower animals. Studies in the incidence and inheritabiHty of spontaneous tumors
in mice. Am. J. Cancer 15: 1387-1400.

SPONTANEOUS NEOPLASMS IN MICE 233

61. Slye, M., H. F. Holmes and H. G. Wells. 1935. Comparative pathology of
carcinoma of pancreas with report of two cases in mice. Am. J. Cancer 23: 81-86.

62. Strong, L. C. 1936. Production of the CBA strain of inbred mice: Long life
associated with low tumor incidence. Brit. J. Exp. Path. 17: 60-63.

63. Strong, L. C. and G. M. Smith. 1936. Benign hepatomas in mice of the CBA
strain. Am. J. Cancer 27: 279-284.

64. Strong, L. C, G. M. Smith and W. U. Gardner. 1936. Epithehoma of the
anus in a mouse of pure strain (F). Bull. Assn. frang. etude cancer 25: 111-119.

65. Tyzzer, E. E. 1907. Spontaneous tumors in mice. Harvard Univ. Cancer
Com. 4: 29-65.

66. Tyzzer, E. E. 1907. A series of twenty spontaneous tumors in mice, with the
accompanying pathological changes and the results of the inoculation of certain
of these tumors into normal mice. J. Med. Res. 12: 155-197.

67. Tyzzer, E. E. 1909. Series of spontaneous tumors in mice with observations
on the influence of heredity on frequency of their occurrence. Harvard Univ.
Cancer Com. 5: 153-192.

68. Wells, H. G., M. Slye and H. Holmes. 1938. Comparative pathology of
cancer of the alimentary canal, with report of cases in mice. Am. J. Cancer
2>y. 223-238.

69. Whitehead, R. 1932. AbnormaHties of the mouse suprarenals. J. Path, and
Bact. 35: 415-418.

70. Williams, A. C, L. E. SiLCOx AND B. Halpert. 1935. Carcinoma of the mam-
mary gland in an inbred stock of albino mice. Am. J. Cancer 24: 823-830.

71. Woglom, W. H. 1913. The spontaneous tumor. George Crocker Special
Res. Fund i: 183-226.

72. Woglom, W. H. 1914. Carcinosarcoma in the mouse. Proc. New York Path.
Soc. 14: 195-201.

Cnapter 5

GENE AND CHROMOSOME MUTATIONS

Bv George D. Snell, Roscoe B. Jackson Memorial Laboratory.

Gene mutations, 234. Characters inherited in an irregular or undetermined manner,
240. Induced chromosome mutations, 242. Rules for assigning symbols to muta-
tions, 242. The chromosomes of the mouse, 243. Negative linkage data, 244.
Bibliography, 246.

Gene Mutations

In the following list of gene mutations are given (j) the symbol for the
mutation adopted by the International Committee on Mouse Genetics
Nomenclature; (2) the name of the mutation; (j) if a linkage is known, (a)
the number of the chromosome to which the linkage group has been assigned,
{h) the per cent of crossing-over between the known genes on this chromo-
some, (c) a reference to an article giving linkage data; {4) a brief description
of the mutation; (5) a statement as to the dominance shown by the mutation
(if no statement is made about dominance it may be assumed that any
mutation represented by a small letter is completely recessive to its normal
or type allele); {6) a reference to one or more important, and if possible
recent, articles describing the mutation.

A agouti. — Chromosome 5. Linked with pa (20% crossing-over).
Roberts and Quisenberry, 1935, Am. Nat. 69: 181-183. The normal or
type allele of the agouti locus.

a non-agouti. — aa mice are solid black, lacking the sub-apical yellow
band on each hair which gives wild-type mice their characteristic brownish
color. In most stocks a is completely recessive to +, but strains have been
reported in which +a mice have a dark or black back merging into nearly
typical agouti on the sides and belly. In these strains -f-f mice are only
slightly darkened. There is some evidence that one principal partly domi-
nant modifying gene, umbrous {U), is involved. Mather and North, 1940,
J. Genet. 40: 229-241.

a' black-and-tan. — Allele of a. Mice of the constitution a'a^ and a^a
have a light belly (dirty yellow to white) and a black back. The line of
demarcation between light and dark regions is quite sharp. Aa*^ mice have
a light belly and an agouti back. Dunn, 1916, Am. Nat. 50: 664-675.

234

GENE AND CHROMOSOME MUTATIONS 235

.!"' lighl-hiilicd agouH. — Allele of </. Like a' except that the back is
agouti instead of black. Morgan, 1915, Am. Nat. 49: 379 383.

.P lethal yellow. — Allele of a. Mice heterozygous for A" have a bright
yellow coat and show a tendency to put on fat. Homozygous A^A^ mice
are non-viable, dying in the early stages of development. Little, 1919,
Am. Nat. 53: 185 187. Kirkham, 1919, J. Exp. Zool. 28: 125-135.

ae absence of corpus callosum. — Brain lacks corpus callosum. Behavior
normal. Keeler, 1933, Proc. Nat. Acad. Sc. 19: 609-611.

b brown. — Coat color cinnamon brown in combination with agouti,
chocolate in combination with non-agouti. Completely recessive except
in the genotypes sisi and pp in which it is partially dominant. Little, 1913,
Carnegie Instn. Wash. Pub. No. 179: 11-102.

C color. — Chromosome i. Between sh-i (3.1% crossing-over) and p
(17.3% crossing-over). Griineberg, 1936, J. Genet. 2)2>'- 255-265. C is the
type allele of the albino series.

c albinism. — No pigment in coat or eyes. Recessive to C except
that Ccpp mice are lighter than CCpp mice. Dunn, 1936, J. Genet. ^^ : 443-

453-

c* extreme dilution. — Allele of c. Coat quite light, eyes black, yellow
pigment suppressed. Recessive to C, partly dominant to c, c''^, and c'.
Detlefsen, 1921, Am. Nat. 55: 469.

(■'''* chinchilla. — Allele of c. Coat lightened, but much less than by c^,
eyes black, yellow pigment suppressed, c"^^ has more effect on the coat of A
mice than of aa mice. Thus in aaB mice separation of c''V' from C is some-
times difficult, although with practice c'^V' may be distinguished by the
lesser saturation of black which tends to be dull and slate colored near the
base of the fur, and especially by the shade of the hairs lining the ears which
in C forms are yellowish, in c'^^c'''' creamy or nearly white. Again, in aabb
mice the genotypes C, c'^V' and c'^^c^ are indistinguishable, whereas if A is
present they can be separated. Recessive to C, partly dominant to c, c^
and c'. Dunn, 1936, J. Genet. ^7^: 443-453-

6"' intense chinchilla. — Allele of c. Similar to c'"'' but causes somewhat
less lightening of the coat. Feldman, 1935, J. Mammal. 16: 207-210.

Ca caracul. — Chromosome 6. Linked with A (1.8% crossing-over).
Cooper, 1939, J. Hered. 30: 212. Vibrissae curled and coat wa\T. Recog-
nizable at one day after birth by slight curling of vibrissae. Less pronounced
in old mice than in mice with the first pelage but easily recognized by the
curled vibrissae and the waviness of the guard hairs. Very similar to Re.
Completely dominant to type. Carnochan, 1937, J. Hered. 28: ^2>2>~i6A-

236 BIOLOGY OF THE LABORATORY MOUSE

d dilution. — Chromosome 2. Linked with sc (.06% crossing-over).
Snell, 193 1, Genetics 16: 42-74. The coat is diluted to a blue-gray or leaden
color. In aa mice the color is similar to that of a Maltese cat. The pig-
mentation of the eyes at birth, as seen through the unopened eyelids, is
slightly lighter than in D mice. Little, 1913, Carnegie Instn. Wash. Pub.
No. 179: 11-102.

dw dwarf. — Causes practical cessation of growth at 14 days. Prior to
this age there is some retardation of growth, so that by 7 days dwdw mice
can usually be recognized by their smaller size. Sterile in both sexes.
Due to pituitary deficiency. De Beer and Griineberg, 1940, J. Genet. 39:
297-300.

/ flexed tail. — Tail flexed due to fusion of vertebrae, newborn young
anaemic, often accompanied by white spot on belly. The anaemia is due
in part to a deficiency in the number of erythrocytes; more important
however is a deficiency in the total amount of haemoglobin. It largely
disappears at two weeks. The flexed tail condition is recessive with some
normal overlaps, perhaps sometimes dominant. Mixter and Hunt, 1933,
Genetics 18: 367-387.

gl grey-lethal. — Homozygous glgl mice, otherwise of wild phenotype, have
a pure grey coat without a trace of yellow. In aa or c'^c^ genotypes, the gl
gene produces little effect on coat color. There is a major effect on growth.
glgl mice are slightly smaller than normals from birth to 14 days, thereafter
they loose weight. The teeth do not erupt, their shape is abnormal and the
roots uncalcified. The long limb bones are abnormal. Death occurs
usually between the 22nd and 30th day. Griineberg, 1938, J. Genet. 36:

153-170-

hr hairless. — Chromosome 3. Linked with s (about 9% crossing-over).
Snell, 193 1, Genetics 16: 42-74. Homozygous hrhr mice develop normally
until about 14 days of age when, at just about the same time that the eyes
Open, they can be distinguished from normal sibs by loss of hair on the upper
eyelid. At about the same time shedding begins on the under jaw and
on all four feet just back of the toes, and slightly later at the base of the tail.
During the next week shedding spreads from these centers, especially that
around the eye, until the animal is naked except for a few scattered hairs.
The vibrissae usually remain. There is sometimes a very slight regeneration
of hair at about six weeks. Females are poor breeders or often completely
sterile. David, 1932, Z. Zellforsch. u. mikr. Anat. 14: 616-719.

hr'^'^ rhino. — Allele of hr. At 13 or 14 days hair begins to shed above
eyes just as in hrhr mice, but there is less definite anterior-posterior progres-

GENE AND CHROMOSOME MUTATIONS 237

sion of shedding, hair tending to thin out all over. Also hair persists on
feet as late as seventh week instead of falling out at 2 weeks as in hrhr mice.
At about 3 weeks hf^hr''^ mice begin to show a wrinkling of the skin which
becomes very pronounced in old animals, giving ''rhinoceros" appearance.
Recessive to hr. Howard, 1940, J. Hered. 31: 467-470.

hy-i hydrocephalus- 1. — The lateral and third ventricles and the foramina
of Monroe are distended with accumulated fluid. The aqueduct of Sylvius
is occluded. The swelling of the head may be detectable at birth but usually
does not become noticable until a week or two later. Affected mice become
grotesque in appearance, lack coordination and finally die during the third
or fourth week of postnatal life. Clark, 1934, Anat. Rec. 58: 225-233.
Clark, 1935, Proc. Nat. Acad. Sc. 21: 150-152.

hy-2 hydrocephalus-2. — The ventricles of the brain are distended with
fluid and the skull enlarged. These brain defects are detectable at least as
early as 4 days. Growth is much retarded and there is a high mortality
particularly during the first week. Adults are sterile and usually about one
half normal size, though the size varies considerably. Zimmermann, 1933,
Z. ind. Abst.-u. Vererb. 64: 176-180.

/;/ leaden. — Phenotypically almost indistinguishable from d. Murray,
1933, Am. Nat. 67: 278-283.

?ny myeleneephalic blebs. — ^Large blisters tilled with clear fluid appear
on the back of 7-8 mm. embryos and move toward the extremities where
they tend to cause bleeding and various foot and eye defects that persist
in the adult mouse. The expression of the gene in the adult mouse, at least,
appears to be subject to frequent normal overlaps. Bonnevie, 1934, J.
Exp. Zool. 67: 443-520.

A naked. — Chromosome 6. Linked with Ca (which see). NN mice are
almost completely hairless from birth; vibrissae absent. Sterile, except
that occasional males show low degree of fertility. In Nn mice the coat
appears almost normal up to 14 days, though usually a little short and dull;
thereafter the hairs break off before attaining normal length, so that parts
of the skin are naked, other parts covered by hair. Fertility of heterozygotes
is normal. David, 1932, Z. Zellforsch. u. mikr. Anat. 14: 616-719.

p pink-eye. — Chromosome i (see C). Eyes pink, coat much lightened,
tending towards brown or yellow. Recognizable at birth by lack of pigment
in the eye. Little, 1913, Carnegie Instn. Wash. Pub. No. 179: 11-102.

pa pallid (pink-eye-2). — Chromosome 5 (see A). Rather similar pheno-
typically to p but causes more extreme dilution of coat color. Eyes pink,
unpigmented. Roberts, 193 1, Science 74: 569.

238 BIOLOGY OF THE LABORATORY MOUSE

r rodless retina.— Chiomosome 4. Probably linked with silver (about
12% crossing-over). Keeler, 1930, Bull. Howe Lab. Ophthalmology 3:111.
Rods lacking or reduced in number. Blind. Keeler, 1925, Anat. Rec. 31: 341.

Re rex. — Phenotypically similar to or identical with Ca. Crew and
Auerbach, 1939, J. Genet. 38: 341-344.

5 piebald. — Chromosome 3 (see hr). White spotting. There is always
a white area on the belly, usually one on the back, often a white blaze on
the head, but the amount and location of the spotting is variable, being
affected by both modifying genes and environment. In one ''all-white"
strain 99% of the dorsal area, on the average, is white, but this has been
shown to be due to a group of "k" genes capable of causing 3 to 35% dorsal
white in the absence of s. Ss mice may show some white, particularly on
the belly. Dunn and Charles, 1937, Genetics 22: 14-42.

Sd short-Danforth. — In heterozygotes the tail is shortened, terminating
in a contorted filament, or lacking; sacral region frequently shortened due
to malformations of the sacral vertebrae; one or both kidneys may be
reduced or missing. Viability reduced. In homozygotes tail is lacking
and spinal column shortened, usually terminating at the second lumbar
vertebra; anus imperforate; kidneys absent; bladder and urethra sometimes
absent. Do not survive more than 24 hours after birth. Dunn, Gluecksohn-
Schoenheimer and Bryson, 1940, J. Hered. 31: 343-348.

se short-ear. — Chromosome 2 (see d). The ears do not grow after 14
days thus remaining quite short. Prior to 14 days cannot be distinguished
from normal sibs. The gene produces several other minor effects, in particu-
lar a muscular waviness of the tail that disappears in etherized animals.
Lynch, 1921, Am. Nat. 55: 421-426. Snell, 1935, Genetics 20: 545-567.

sh-i shaker-i. — Chromosome i (see C). Nervous, rapid, up and down
movements of the head. Internal ear histologically normal up to 12 days,
thereafter abnormalities appear which are later accompanied by deafness.
Recessive, except that Sh-ish-i Vv mice usually go deaf at from 3 to 6
months of age. Lord and Gates, 1929, Am. Nat. 63: 435 442. Griineberg,
Hallpike and Ledoux, 1940, Proc. Roy. Soc. B 129: 154-173.

sh-2 shaker-2. — Chromosome 7. Linked with u'a-2 (25% crossing-over).
Snell and Law, 1939, J. Hered. 30: 447. Nervous movements of the head
which are indistinguishable from those of sh-ish-i mice. Clark, 1935,
Proc. Nat. Acad. Sc. 21: i^'j-i^i.

si silver. — Chromosome 4 (see r). Some of the hairs in coat partly or
wholly unpigmented. Quite variable. The silvering is more pronounced
when one h gene or one W^ gene is present. Ordinarily recessive, but partly

GENE AND CHROMOSOME MUTATIONS 239

dominant in the presence of one W" j^ene. Dunn and Thi^pen, kj.^i, ).
Hered. 21 : 495 498.

si shaker short. — Recognizable at birth by absence or reduction of the
tail and by the presence of one or two small blood-blebs in the dorsal median
line of the head. Disturbances of equilibrium suggestive of shaker- 1 appear
at about 5 days. Semicircular canals and endolymphatic appendage are
lacking, and the cochlea and cortical organ are abnormal. Deaf. Sterile
in both sexes. Bonnevie, 1936, Genetica 18: 105-125.

/ type allele of T.

T brachyury. — Tt mice are short-tailed (brachyuric). TT gives abnor-
mal embryos which die about 11 days after fertilization. Dobrovolskaia-
Zavadskaia and KoboziefT, 1934, Arch. zool. exp. et gen. 76: 249 358.

/° lethal allele of t. — Tt^ mice are tailless; /"/" gives abnormal embryos
which stop developing at between 53^-^ and 7 days embryo age; tt^ mice are
normal. Males heterozygous for t^ and either T or / transmit t^ to more
than half their progeny, probably due to an efTect of t'^ on segregation.
Gluecksohn-Schoenheimer, 1940, Genetics 25: 391-400.

t^ lethal allele of t. — Tt^ mice are tailless; t^t^ mice die before implanta-
tion; t^t^ males are sterile, the females normal. Like t^ in its effect on ratios.
Dunn and Gluecksohn-Schoenheimer, 1939, Genetics 24: 587-609.

T^ fused. — Allele of /. TH mice usually have a kinked tail due to fusion
of vertebrae, but there are normal overlaps in some stocks. T^T^ mice
show a more marked expression of the same trait, the tail often being very
short. Reed, 1937, Genetics 22: 1-13.

V waltzing. — Shaking movements of the head and a tendency to run in
circles. Deaf. Probably due to a defect of the inner ear. Not always dis-
tuinguishable phenotypically from sh-i and sh-2. Gates, 1926, Carnegie
Instn. Wash. Pub. No. 337: 83-138.

w type allele of W.

W dominant spotting. — WW mice are anaemic, usually living for only
a few days after birth. Those surviving long enough to develop a coat are
all white with black eyes. In the presence of certain recessive modifying
genes, m{w), at least 3 in number, W is partly dominant, Ww mice showing
90-98% white. In the absence of the modifiers, Ww mice show no spotting;
with only some of the modifiers present, the degree of spotting is inter-
mediate. One dose of 5 increases the spotting in Ww mice provided some
or all of the modifiers are present. A" tends to reduce the amount of white
spotting. Dunn, 1937, Genetics 22: 43-64. Dunn, MacDowell and
Lebedeff, 1937, Genetics 22: 307-318.

240 BIOLOGY OF THE LABORATORY MOUSE

W viable dominant spotting. — Allele of W. W-'w mice are similar to Ww
mice. W^W^ mice usually live to maturity. They are all white with black
eyes, usually sterile, but occasionally with a limited fertility. The eryth-
rocyte count is about one half normal. The W^ gene lightens sisi and makes
si partly dominant to Si. Little and Cloudman, 1937, Proc. Nat. Acad.
Sc. 23: 535-537. Griineberg, 1939, Genetics 24: 777-810.

wa-i waved-i. — Hair wavy, vibrissae slightly curly. Recognizable in
mice at about 5 days of age because of curling of vibrissae. Quite pro-
nounced at 7 or 8 weeks, thereafter becomes less distinct and in older mice
remains only as a slight curling at the tip of the vibrissae and a tendency of
the hairs on the back to incline towards the mid-line of the body. Crew,
1933, J- Genet. 27: 95-96.

wa-2 waved-2. — Chromosome 7 (see sh-2). Like iva-i but more pro-
nounced. Keeler, 1935, J. Hered. 26: 189-191.

Characters Inherited in an Irregular
OR Undetermined Manner

There are a number of structural and physiological characters in mice
which genetic tests have shown to be inherited, but the exact manner of
whose inheritance is not yet adequately determined. Most of them give
imperfect ratios so that they cannot be classed as simple recessive or domi-
nant factors due to a single gene. These characters are listed and briefly
described below and a reference given.

Agglutinin absorption ability of blood corpuscles. — The blood corpuscles
of different strains of mice may be classified as strong or weak according to
their ability to absorb agglutinin. Strong ability may be inherited as a
simple dominant. Gorer, 1936, J. Genet. 32: 17-31.

Anophthalmia. — An anophthalmic strain gives 90% complete eyeless-
ness on both sides and 10% of various degrees of smallness of the eyes.
Chase and Chase (in press).

Cleft palate and harelip. — Usually recessive in Fi but occasionally domi-
nant. Ratios are imperfect, showing variable but usually large number of
normal overlaps. Reed, 1936, Genetics 21: 361-374. Steiniger, 1939, Z.
Menschliche Vereb. u. Konstitutionslehre 23: 425-462.

Diaphragm imperfectly formed. — Causes death in newborn mice due to
leakage of air from ruptures in lungs. Wang, 1938, Anat. Rec. 71: 469-
476.

GENE AND CHROMOSOME MUTATIONS 241

Edromelie. ^Ahstnce of tibia from hind legs. Perhaps recessive with
normal overlaps. Rabaud and Hovelacque, 1923, Bull. biol. France et
Belgiqiie 57: 401-468.

Eyelids open at birth. — Often not symmetrical on two sides. Perhaps
recessive with normal overlaps. Loeffler, 1932, Z. ind. Abst.- u. Vereb.
61 : 409-446.

Hcaddot. — White dot on head. Irregular recessive probably distinct
from piebald. Little, 1926, Anat. Rec. 34: 171. Keeler, 1935, Proc. Nat.
Acad. Sc. 21:379-383.

Hound-ear. — Varies from slight reduction of pinna to absence of outer
ear. Recessive with numerous normal overlaps. Feldman, 1932, Proc.
Sixth Int. Cong. Genet. 2: 51-52. McPheters and Little, 1933, J. Hered.
24: 157-158. Kobozieff and Pomriaskinsky-Kobozieff, 1940, Compt. rend.
Soc. biol. 133: 386-389.

Hyperglycaemia and hypoglycaemia. — Griineberg and Haldane, 1940,
Nature 145: 704-705.

Hypotrichosis juvenilis. — The first coat of hair is thin or almost lacking.
At 5 weeks the second coat begins to appear and grows in normally, start-
ing at the head and progressing tow^ards the tail. Perhaps due to recessive
gene, but the percentage of normal overlaps ranges from 3% to 66% in
males, higher in females. Loeffler, 1934, Z. ind. Abst.-u. Vererb. 67:
209-211.

Microphthalmia. — Eyes small or opaque, and show various histological
abnormalities. Quite variable. Koch and Gowen, 1939, Arch. Path.
28: 171-176.

Palatal ridges reduced. — One pair of palatal ridges missing. Woolley,
1937, Rec. Genet. Soc. Am. 6: 176-177.

Polydactylism. — Occurs in a small percentage of mice in certain inbred
strains. Murray, 1932, Science 75: 312. Fortuyn, 1939, Genetica 21:
97-106.

Posterior duplication. — Varying degrees of duplication of structures at
posterior end of body. Lethal in extreme forms. Recessive with normal
overlaps. Danforth, 1930, Am. J. Anat. 45. 275-288.

Pseudcncephalie. — Brain defect due to failure of the neural groove to
close. Perhaps recessive. Bonnevie, 1936, Norske Videnskaps-Akademi I
Oslo. I. Mat.-Naturv. Klasse 9: 1-38.

Tail tip pigmentation. — White tail tip, inherited perhaps as recessive
with normal overlaps. Griineberg, 1936, J. Genet. 2)3,'- 343^345-

242 BIOLOGY OF THE LABORATORY MOUSE

Vaginal occlusion. — Occurred in a number of individuals of silver strain.
Marx, 1936, Anat. Rec. 66: 449-454.

Induced Chromosome Mutations

X-rays and neutron rays when applied to mature ova and spermatozoa of
mice are a prolific source of translocations.* Other sorts of chromosome
mutations probably are induced also, but are not detectable by the genetic
methods at present available. The detection of translocations is relatively
easy, due to the fact that mice heterozygous for a translocation are semi-
sterile, consistently producing small litters even when mated to unrelated
and entirely normal mice.

T-F1146 translocation-F\i46. — Average size of litters from the mating
T-Fii46/-\- X +/+ is 4.6 as compared with the normal value for the stock
of 8.3. Reduction in litter size is due to death in utero of approximately
45% of the embryos. Most of these embryos die shortly after implantation ;
a few live to later stages, occasionally even to term, but show brain abnor-
malities due to failure of the neural groove to close at the anterior end.
Of the viable young, one half on the average are semi-sterile, one half
normal. The chromosomes involved are 5 and the chromosome carrying b
(0% crossing over between a and break, 20% between break and h). Snell,
Bodemann and Hollander, 1934, J. Exp. Zool. 67: 93-104. Snell, 1941,
Genetics 26: 169.

T-i translocation- 1. — Very little reduction in litter size, but almost
4.6% of the offspring from the cross T-i/-\- X +/+ show brain abnor-
malities due to failure of the neural groove to close at the anterior end.
These frequently come to term. The evidence that this is a translocation
is not complete. Snell and Picken, 1935, J. Genet. 31: 213-235.

Rules for Assigning Symbols to Mutations

The following rules for assigning symbols to mutations have been
adopted by the Committee on Mouse Genetics Nomenclature, j

1. The initial letter of the mutant symbol shall be the same as the
initial letter of the mutant gene, e.g., d for dilution.

2. Additional letters shall be added to the initial letter if necessary to
distinguish it from symbols already in use. These shall be, preferably,
those immediately following the initial letter, or suggestive letters, espe-

* Snell, 1935, Genetics 20: 545-567; Snell and Ames, 1939, Am. J. Roent. Rad.
Therapy 41: 248-255.

t Dunn, Gruneberg and Snell, 1940, J. Hered. 31: 505-506.

GENE AND CHROMOSOME MUTATIONS 243

cially consonants, from the rest of the name, e.g., dw for dwarf, ac for absence
of corpus callosum.

J. Recessive mutations shall be indicated by the use of a small initial
letter for the symbol of the mutant gene, the type allele being distinguished
by a capital letter, e.g., a for non-agouti, A for agouti.

4. Dominant mutations shall be indicated by the use of a capital initial
letter for the symbol of the mutant gene, the type allele being distinguished
by a small initial letter, e.g., Re for rex, re for the type allele of rex.

5. The icild type may also be represented by a + rather than by a letter
when this is more convenient, or by a small letter with a + superscript, e.g.,
+ or /^ for the type allele of T, -\- or a^ for the type allele of a.

6. Multiple alleles (except lethals) shall be indicated by the use of
superscripts (always small letters, never capitals) added to the symbol of
the original mutant type, e.g., c^ for extreme dilution. It is suggested that
the letter selected be the initial letter of the name of the mutation, e.g.,
7^^ for the fused allele of brachyury. The initial letter of the name of the
discoverer may also be used. Lethal alleles in a multiple series may be
indicated by the use of superscript numerals, e.g., f^ and /' for the lethal
alleles of brachyury.

7. Mimics, i.e., mutants of similar phenotype but different location,
shall be indicated either by entirely different names and symbols (e.g., In for
leaden and d for dilution) or by the same name and symbol with the addition
of distinguishing numbers (e.g., wa-i for waved- 1 and wa-2 for waved-2).
The latter procedure is not recommended.

S. In published articles in American journals in which symbols are used,
the symbols should be set in italics.

The Chromosomes of the Mouse

The mouse has 20 pairs of chromosomes. In males, one pair consists of
two chromosomes of unequal size. These are the sex chromosomes, the X
and the Y, the X being the larger.*

Presumably, in course of time, the number of linkage groups in the
mouse will come to equal the number of chromosome pairs. At the present
time seven linkage groups are known, p c sh-i, d se, hr s, r si, a pa, Ca N and
sli-2 u'a-2. In addition to these, nine genes, b, dw,f, hy-i, In, T, v, W and
wa-i have been tested against most of the other known genes without

* Painter, 1928, Genetics 13: 180-189.

244 BIOLOGY OF THE LABORATORY MOUSE

showing linkage, so that most or perhaps all of these mark additional
chromosomes, making perhaps sixteen chromosomes in all with known
marker genes.

No known mutant gene in the mouse, or in fact in any of the rodents,
sufficiently clear cut in its effects to serve as a "marker" gene, is sex-
linked. In view of the large size of the X chromosome and the ease with
which sex-linked mutations, if they occur, can be detected, this is a note-
worthy fact.

In all cases of linkage sufficiently well tested to give critical evidence,
the crossover percentage has been higher in the female than in the male.
In accord with this, the number of chiasmata observed during gameto-
genesis is higher in the female than in the male.*

Negative Linkage Data

Most of the gene mutations that have been found in mice have been
tested for linkage against other known gene mutations. Where these tests
have led to the discovery of a linkage, this is indicated in the section on Gene
Mutations (p. 234). In the majority of cases no linkage has been found.
These negative linkage data are summarized in the accompanying table. In
this table all the genes are listed in the first vertical and also in the first
horizontal line. Where several genes lie on the same chromosome they are
listed as a unit. The crossover data for any two genes are found in the
rectangle where the horizontal line from one gene and the vertical line from
the other gene intersect. In a number of cases several tests have been made
for a single pair of genes. In such cases the data from one test only, that
involving the most animals or for other reasons the most satisfactory, have
been use. The data given at each intersection consist of the following:

1. A number referring to a reference in the bibliography.

2. An abbreviation indicating the type of cross used. The abbreviations
are:

BC, a cross of the type AaBb X aabb
Fo, a cross of the type AaBb X AaBb
MC, a cross of the type AaBb X Aabb

3. The observed crossover per cent plus or minus its standard error, or
where this cannot be given, the data themselves. In the case of backcross
(BC) data, the standard error has been calculated from the tables given by

* Bryden, 1933, J. Genetics 27: 421-433.

GENE AND CHROMOSOME MUTATIONS
Table of Negative Linkage Data

245

d s,

hr s

r si

a pa

c p sh-i
d se
hr s
r si

9BC 50.0

± 2.7

SBC 47-8 + I
gBC 46.4 + [

•4
•3

12BC51.1 + 3.2

12BC51.3 ± 5-5
12BC 41.5 ± 9.3

SBC 49.3 ±1.6
13F2 48-9 ± 2.2

SBC 46.2 ±1.7
12BC52.8 ± 5.5

T.\BLE OF Negative Linkage

Data.-

-(Continued)

j A- Ca.

sh-2 wa-2

c p sh-i

I6BC53-6 ± 35

10F2 47. 1 ± 2.7

7BC 49.7 ± 0.8

i7F2> 57-3 + ?*

d sc

I6BC 49.3 + 4.6

loFo 54.6 ± 2.4

9BC45

5 + 1-3

i7F2>57.3 ± ?*

hr s

16BC 60.3 + 6.1

4F2 57. 1 ± 6.0

9BC 51

4 + 1-3

17MC 14: 17:6:.;

r si

16BC 61. 5 ± S.4

4F2 44-7 + 9-5

12BC 49

2 ± 6.5

a pa

16BC 54.3 + 2.6

4F2 56.3 ± 7-3

7BC50

3 ± 0.8

17MC 35:39:10:15

X Ca

4BC53.2 ± 4-8

16BC 53

5 ± 2.8

17MC 26:33:10: II

sh-2 u'a-2

4F2 50

5 + 6.9

4F2 4S.2 ± S.I

Crossover per cents greater than 57.3 are not given in Stevens' tables.
T.\BLE OF Negative Linkage Data. — (Continued)

hy-i

c p sh-i

3F2 514 + 5-2

5F2 54-3 ± 6.0

I4F2 182:61:9611

6BC51.8 ±5.5

d sc

3F2 45-3 + 4-3

5F2 >57-3 ± ?*

14BC51.7 ±3-5

6BC 50.0 + 6.4

3F2 44-9 + 7-6

5F., 46.6 ± 7.2

14BC 42.8 + 5.2

6BC32:22:/j:ii«

r si

3F2R:r:27:6t

5F,R:r::23:5t

a pa

3F2 55-5 ± 6.9

5MC 46:37:12:77

14BC 50.0 ± 4.4

6BC 47.1 ± 8.6

N Ca

3BC48.6 ± 5.9

5MC3i:42:<?:i3

14BC46.S ±3.9

sh-2 U'a-2

3F2>57.3 ± ?*

5F2 35-3 ± 5-5§

4F2 51-4 + 7-5

No linkage tests have been made

3F2 56.1 ±3-5

5MC 14:14:3:^

14BC 50.4 + 2.9

6BC 60.5 ± 7.1

d-u'

3F2 56.4 ± 71

-F, ^ -- 9 -1- ^*

3^2 ^3/3 - •

14F248.3 + 8.4

5F2 56.0 ± 7.4

3BC 50.0 ± 4.1

Xo linkage tests have been made

hy-i

5F2 53-9 ± 6.9

hv-2

Xo linkage tests have been made

6BC 22:32:9: 15

* Crossover per cents greater than 57.3 are not given in Stevens' tables,
t Only the ^^ fle.xed mice were classified as to whether they were tj'pe or rodless.
X Only the 28 hydrocephalus- 1 mice were classified as to whether they were type or rodless.
§ Tests of i4of the sh-2sh-2 Ily-i mice for the presence of hy-i showed more than the e.xpected
number of crossovers in this group.

]] The original cross was c X In: the expected F: ratio is 190:63:85.

% Waved-i as well as rex was involved in this backcross, giving an expected ratio of 3 : 3 : i : i .

246

BIOLOGY OF THE LABORATORY MOUSE

Table of Negative Linkage Data. — {Continued)

wa-i

c p sh-i

2BC 48.1 ± 5.7

qBC 50.3 + 2.6

18BC 52.4 + 2.9

iiBC 555 ± 3-7

d se

2BC 49.2 ± 4.5

9BC 470 ± 1.3

iiBC 52.1 ± 3-6

Iir s

2BC 43-7 ± 5-9

9BC48.4 ± 1.3

8BC 48.6 ± 2.2

1F2 55-4 ± 5-6

r si

2BC45-5 + 50

12BC 41 .9 + 7.6

1F2 43-7 ± 7-4

a pa

2BC 47.0 ± 5.0

9BC 55-8 + 4-7

18BC 51.3 ± 2.6

iiBC 511 + 2.5

N Ca

2BC 51.0 + 5.0

16BC 60.0 ± 9.1

16BC 47.0 + 7 .0

iMC 18:24:17:7

sh-2 wa-2

2BC53-4 + 5-3

4F2 94:54!

loBC 52.9 + 6.1

1BC52.8 ± 5.9

No linkage tests have been made

2BC4S-5 ± 4.8

9BC 49.9 ± 1.3

17BC 51.7 + 6.6

iiBC 48.1 + 2.5

2MC 15:23:3:7

17MC 26: 12:7:6

17MC 12:17:4:7

iF., >57.3 ± ?*

2BC 46.8 + 3.4

3F. >57.3 ± ?*

3MC 28:23:6:9

IF2 53-6 ± 5-6

No linkage tests have been made

hy-i

5MC48:48:/2:i8 5F242.o±q.4

5MC 21: 20:5:4

hy-2

No linkage tests have been made

2BC 50.5 ±4-8 I4F2 45-9 + 6.5

14BC48.7 ±3-3

iF. >57.3 ± ?*

No linkage tests have been made

1 6BC 34: 20:74: loj

No linkage tests have been made

2BC46.5 ± 5-4

2BC 49-3 + 5-9

iBC 47 0 ± 3.4

1F2 50.0 ±7.0

iBC 56.7 ± 6.4

* Crossover per cents greater than 57.3 are not given in Stevens' tables.
t The waltzing and shaker-2 animals were grouped into one class; the expected ratio is
83:65.

X Waved-i as well as rex was involved in this backcross, giving an expected ratio of

3:3:1:1-

Castle.* In the case of Fo data, the crossover per cent and the standard
error have been calculated from the tables given by Stevens, t In the case of
all data from mixed crosses (MC) the data are given in full. The data are
given in such order that the expected ratio is A B:Ab:aB:ab: ■.^:^: 1:1, and
of the last two numbers, the one that represents the crossover class is given
in italics.

BIBLIOGRAPHY

No attempt can be made in this chapter to cover completely the ver>' extensive
bibliography dealing with the genetics of the house mouse. Important references

* Castle, W. E., 1934. Outline for a laboratory course in genetics. Harvard Univ.
Press, Cambridge.

t Stevens, W. L., 1939. Tables of the recombination fraction estimated from the
product ratio. J. Genet. 39: 171-180.

GENE AND CHROMOSOME MUTATIONS 247

concerned with individual mutations are given in the body of the chapter, and certain
others are given in footnotes. Below, in addition to the bibliography of the linkage
table, are given a few general references each of which has an extensive bibliography.

Bibliography of linkage table. — (/) Burhoe, 1936, J. Hered. 27: 1 19-120. {2)
Clark, 1934, Proc. Nat. Acad. Sc. 20: 276-279. (j) Clark, 1934, Genetics 19: 365-393.
{4) Clark, 1935, Proc. Nat. Acad. Sc. 21: 247-251. (5) Clark, 1936, Proc. Nat. Acad.
Sc. 22: 474-478. {6) Crew and Auerbach, 1940, J. Genet. 39: 225-227. (7) Detlefsen
and Roberts, 1918, Genetics, 3: 573-598. {8) Dunn, 1920, Genetics 5: 325-343.
ig) Gates, 1926, Carnegie Instn. Wash. Pub. No. 337: 83-138. {id) Gates, 1934,
Am. Nat. 68: 173-174. (//) Griineberg, 1936, J. Genet. 32: 1-3. {12) Keeler, 1930,
Bull. Howe Lab. Ophthalmology 3: i-ii. {ij) Little and Phillips, 1913, .^m. Nat.
47: 760-762. {14) Murray, 1933, Am. Nat. 67: 278-283. (/j) Reed (unpublished
data). {16) Snell, 193 1, Genetics 16: 42-74. (/") Snell (unpublished data). iiS)
Wachter, 1921, Am. Nat. 55: 412-420.

General references. — The following are references to books and review articles
each of which contains an extensive bibliography.

1. Castle, W. E. 1940. Mammalian genetics. Harvard University Press. Cam-
bridge.

2. CuENOT, L. 1928. Genetique des Souris. Bibliographia Genetica 4.

3. Gates, W. H. 1926. The Japanese waltzing mouse: its origin, heredity and
relation to the gametic characters of other varieties of mice. Carnegie Instn. Wash.
Pub. 337:83-138.

4. Keeler, C. E. 1931. The laboratory mouse. Harvard Univ. Press, Cambridge.

5. Little, C. C. 1913. E.xperimental studies of the inheritance of color in mice.
Carnegie Instn. Wash. Pub. 179: 11-102.

6. Snell, G. D. 193 i. Inheritance in the house mouse, the linkage relations of
short-ear, hairless, and naked. Genetics 16: 42-74.

Cnapter 6

THE GENETICS OF SPONTANEOUS TUMOR
FORMATION

By Clarence C. Little, Roscoe B. Jackson Memorial Laboratory.

Introduction, 248. Mammary epithelial tumors, 251. Evidence that the tendency
to form such tumors is not due to the action of a single recessive gene, 251. Evidence
of an extra-chromosomal influence, 251. Evidence that at least part of the extra-
chromosomal influence can be transferred by foster nursing, 257. Evidence that
genetic factors also influence the incidence of breast tumors, 259. Evidence that
hormonal influences also affect the incidence of breast tumors, 259. Evidence that
coat color may play a part in influencing the incidence of mammary tumors, 260.
Summary, 261. Epithelial lung tumors, 261. Absence of extra-chromosomal influ-
ence, 262. "Dominant" nature, 262. Hormonal influences, 264. Coat color, 264.
Non -epithelial tumors, 264. Absence of extra-chromosomal influence, 266. Relation
of incidence to age, 268. Relation of incidence to sex, 268. Relation of incidence to
coat color, 269. Relation of incidence to hybridization, 269. Leukemias, 270.
Evidence of extra-chromosomal influence, 273. Comparison of the four general types
of neoplasms, 273. Bibliography, 273.

Introduction

The application of genetic methods in the analysis of the incidence of
spontaneous tumors in mice is a matter of considerable complexity. Various
factors and influences serve to modify the actual effects of genes. Yet in
spite of this fact there is compelling evidence that the genetic constitution
of an organism plays a part in determining whether or not it will develop a
tumor or tumors.

The earliest work which contributed to this conclusion was that of Tyzzer
(loi), J. A. Murray (73), Bashford (8), Haaland (43") and Loeb (48). By
191 2 these investigators had independently demonstrated that families and
strains of mice differed in their capacity for producing breast tumors.

A good deal of this earlier work lacked certain qualities which would
have greatly increased its accuracy. In some cases histological diagnosis of
the palpated nodules was absent. In others the number of individuals
studied was none too large. In some, knowledge of the more remote

248

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 249

ancestry of the material was fragmentary and insufficient. In all, the long
continued process of inbreeding so necessary to establish the genetic homogeneity
oj the strains before they were used was lacking.

This handicap weighed heavily against the later work of Slye (86-92) and
of Lathrop and Loeb (48-50). It has provided the most important point of
difference between the work completed before 1925 and that of the fifteen
years that have followed.

It is not at all surprising that the earlier work lacked this preliminary
process of genetic purification. There was more than one reason why this
was the case.

In the first place extra-peritoneal tumor nodules in mice are striking and
superficially obvious. To the early workers in experimental genetics they
gave a false sense of simplicity and a feeling that a tumor was something as
definite and predictable as coat color or any of the routine Mendelian
characters.

Then too, the great interest of all in the cancer problem was a constant
challenge to begin work on it without delay. The period of years necessary
in order to carry out the required preliminary inbreeding was not at all
appealing to the geneticists who had found abundant "surface gold" in the
shape of genetic differences capable of immediate analysis. Once an investi-
gator had embarked upon a program of rapid genetic methods, it was not
likely that he would scrap that work, retrace his steps and make a fresh start
with more uniform material.

Yet it is probable that more progress would have been made had this been
done. The experiments of Tyzzer, of J. A. Murray and of Haaland reached
the limit of their potentiality for detailed analysis by showing that female
mice with breast tumors had more female ancestors with similar tumors than
did tumor-free animals. The findings of Loeb and Lathrop indicated that,
in addition, there were "strain differences" in the age at which such tumors
developed, and that the actual incidence of the tumors might have a quanti-
tative basis on multiple factors. Not even the later painstaking statistical
analysis of their data by Bernstein (9) could add basic accuracy or further
knowledge of the nature of the genetic process.

The most extensive series of experiments between 1900 and 1930 were
those of Slye who raised and observed thousands of animals. These repre-
sented many pedigree lines of descent but had to rely by their very nature on
the ex post facto combination and summation of a large number of scattered
small-progeny matings in order to establish trends, groups or genetic
principles.

2 50 BIOLOGY OF THE LABORATORY MOUSE

In the meantime Wright (105, 106), who had started the genetic analysis
of a large number of strains of closely inbred guinea-pigs at the U.S. Depart-
ment of Agriculture, began to publish results which showed, (i) that the
incidence of certain morphological genetic abnormalities could differ in
different inbred lines, and (2) that non-genetic factors often influenced the
incidence of such characters, within a strain, more than did genes. The
foundation for a much more complex interpretation of the bio-genetics of
tumor formation was thus laid by evidence of a far from simple situation in
the genetics of other growth abnormalities.

The history of the development of our knowledge concerning the genetics
of spontaneous tumor formation in mice has followed the trend of recognizing
more and more complicating factors.

Slye's original theory (1913-1937) that all types of cancer in mice were
due to a single recessive Mendelian gene has been replaced by evidence that
there is a high degree of specificity as regards type and location of neoplastic
change. Various physiological factors such as age, sex and coat color have
some influence on the expression of the genetic constitution and its relation
to tumor formation.

Lynch (61) gave evidence suggestive of the possible partial dominance of
the tendency to form breast tumors. Little (52) showed that Slye's data
were not incompatible to some such interpretation. The discovery of an
extra-chromosomal maternal influence on the incidence of breast tumors in
mice was announced by the staft" of the Roscoe B. Jackson Memorial
Laboratory (44) and independently by Korteweg (46). This was further
investigated by Murray and Little (77). Bittner (15) made an important
discovery that an extra-chromosomal influence affecting breast tumor
incidence could be transmitted from parent to offspring apparently through
the milk.

In the meantime data were being gathered to show that lung tumors
(chiefly adenocarcinomas) and non-epithelial tumors, chiefly lymphosar-
comas, fibrosarcomas and endotheliomas, were two other categories of neo-
plasms quite largely distinct from mammary carcinomas and from one
another. A fourth group, strictly speaking a subdivision within the non-
epithelial tumor class, may well be made to include at least certain of the
leukemias. The excellent work of MacDowell, Richter and others (67-70)
supports such a subdivision. All of these steps were clear indicators of an
increasing complexity in the inherent nature of the genetic process.

We may very briefly review the more important data which have led to
the creation of at least four distinct biological groups of spontaneous tumors
in mice.

THE GEXETICS OE SPOXTANEOUS TUMOR EORMATION 251

Mammary Epithelial Tumors

Evidence that the tendency to form such tumors is not due to the action
of a single recessive gene. — Preliminaty evidence was provided by the work
of Lynch (61) who, in a series of crosses between various strains of mice,
showed that mammary tumors occurred in Fi animals. The strains used
were the best then available but left much to be desired as regards the extent
of genetic analysis previous to crossing.

Statistical analysis based on tabulations of Slye's extensive pedigree data
by Little (52) showed that her results could be as well explained on the basis
of dominance of the tendency to form breast tumors as on its recessive
nature.

Data published by the Staff of the Roscoe B. Jackson Memorial Labora-
tory (44) and confirmed independently by Korteweg (46) who published at
almost the same time showed,

1. That Fi hybrids between ''high" breast tumor and "low" breast
tumor strains formed large numbers of mammary tumors.

2. That this result was more in agreement with a theory of dominance
than of recessive nature of the tendency to form these tumors.

Evidence of an extra-chromosomal influence. — These same experiments
showed that a most interesting and unexpected difference exists between the
reciprocal crosses which produced such Fi generation mice.

Where the cross was made between "high tumor" strain female and "low
tumor" strain male, the rate of breast tumor incidence in Fi generation
females approached that of the "high tumor" parent strain. When, how-
ever, the cross was made between "low tumor" strain females and "high
tumor" strain males, the tumor rate in Fi females was little if any above that
of the "low tumor" parent strain. The F2 generations followed closely the
rate of mammary tumor incidence shown by the type of Fi generation from
which they were derived. These results have now been confirmed and
established by the work of a number of investigators.

Tables taken from ISIurray and Little (77) show the incidence and age
distribution of mammary' tumors in the Fi hybrids from reciprocal crosses
between the "high tumor" dba strain and the "low tumor" C57 black
strain. In Table i is the hybrid generation derived from the cross dba
9 X C57 black d" .

It will be noted that the rate of tumor incidence usually falls between
30 and 45 per cent. This may be contrasted with the reciprocal generation
(Table 2) produced by crossing C57 black females with dba males.

2s2

BIOLOGY OF THE LABORATORY MOUSE

Here the tumor incidence averages approximately 6 per cent. The
Difference is mathematically significant.

Table i
dB Fi Generation (dba 9 X C57 Black cf )

Age Group

eaths

No. Alive
at Start
of Period

No. Which

Later

Formed

Tumors

Percentage

Which
Formed
Tumors

Tumor

Non-tumor

151-180

113

39.82

181-210

107

41-

211-240

102

241-270

271-300

301-330

331-360

361-390

391-420

421-450

451-480

481-510

511-540

541-570

571-600

601-630

631-660

661-690

691-720

721-750

751-780

781-810

811-840

841-870

•57

871-900

.76

901-930

.00

931-960

.00

961-990

The tumor incidence in the reciprocal F2 generations shows that the
difference still persists. In Table 3 are included the F2 mice descended from

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 253

Table 2
Bd Fi Generation (C57 Black 9 X dba d^)

Age Group

Deaths

No. Alive
at Start
of Period

No. Which

Later

Formed

Tumors

Percentage
Which

Tumor

Non-tumor

Formed

Tumors

6.06

151- 180

379

181- 210

378

211- 240

378

241- 270

375

271- 300

374

301- 330

365

iT^-i-- 360

357

361- 390

343

391- 420

322

421- 450

291

451- 480

263

481- 510

234

511- 540

216

541- 570

206

571- 600

203

601- 630

193

631- 660

183

661- 690

167

691- 720

154

721- 750

142

751- 780

125

781- 810

114

8n- 840

102

841- 870

871- 900

901- 930

931- 960

961- 990

991-1020

1021-1050

1051-1080

1081-1110

1111-1140

1141-1170

1 1 71-1200

254

BIOLOGY OF THE LABORATORY MOUSE

inbred dBP^i animals (F2 from the cross dba 9 X C57 black cf )• This is
the dBF2 generation.

Table 3
dB F2 Generation

Age Group

Deaths

No. Alive
at Start
of Period

No. Which

Later

Formed

Tumors

Percentage
Which
Formed
Tumors

Tumor

Non-tumor

211-240

664

236

35-54

241-270

663

235

271-300

661

234

301-330

656

233

331-360

594

223

361-390

532

210

391-420

464

^95

421-450

413

178

451-480

381

162

481-510

362

153

511-540

341

138

541-570

317

121

571-600

296

108

601-630

268

631-660

234

661-690

207

691-720

182

721-750

163

751-780

141

781-810

112

811-840

841-870

871-900

901-930

931-960

961-990

991-1020

The incidence in all animals which lived as long as or beyond the age
of the animal in which a tumor first appeared is 35.54 per cent.

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 255

Table 4
Bd Yi Generation

Age Group

Deaths

No. Alive
at Start
of Period

No. Which

Later

Formed

Tumors

Percentage
Which
Formed
Tumors

Tumor

Non-tumor

211- 240

687

5 96

241- 270

685

598

271- 300

684

5-99

301- 330

679

6.03

331- 360

666

6.15

361- 390

635

598

391- 420

601

582

421- 450

563

5.68

451- 480

516

5-8i

481- 510

488

6. 14

511- 540

473

5-49

541- 570

458

5-24

571- 600

434

5 29

601- 630

418

5 50

631- 660

395

5.06

661- 690

365

5-47

691- 720

335

4-77

721- 750

312

5-12

751- 780

287

4-52

781- 810

271

4 05

811- 840

240

2.91

841- 870

203

2-95

871- 900

169

3-55

901- 930

136

2.94

931- 960

0.00

961- 990

0.00

991-1020

0,00

1021-1050

0.00

1051-1080

0.00

1081-1110

0.00

!56

BIOLOGY OF THE LABORATORY MOUSE

In sharp contrast to this is the low incidence (5.96 per cent) among the
BdF2 mice produced by inbreeding the BdFi animals (Table 4).

The eventual weakening and disappearance of the high tumor producing
tendency has been shown by Murray and Little (80) (Table 5) in a series of
backcross generations which were intended to provide a test of the relative
importance of genes and of other influences.

The data obtained from first generation animals backcrossed with parent
strains showed only a slight decrease in the incidence of mammary tumors in
those animals with extra-chromosomal influences (E) derived from "high"
tumor female ancestors. These backcross generations are shown in the

Table 5
Incidence of Mammary Cancer

Stock

dba

dBFi

dBF2

BdFi

BdF2

Number

297

113

664

379

687

250

252

250

244

Observed

tumors

iSi

236

Formula

CCCCE

CCccE

CCcce

CCCce

CCCcE

Cccce

CcccE

Per cent of

cancer

50.84

39.82

35-54

6.06

5 96

2.40

35-71

0.41

34.00

columns marked B and D in Table 5. The animals descended from "low"
cancer females with extra-chromosomal influences (e) showed a greater
proportional decrease in incidence of mammary tumors (columns A and C,
Table 5). The relative independence of all from chromosomal influences
(C = high tumor, c = low tumor) is also shown in this table for A and B had
three representatives of C while C and D had only one.

The system of matings used, in further studies of advanced backcross
generations, to concentrate the chromosomes from respective parent strains
is shown in Table 6.

Starting with individuals of the 8th backcross generations (Table 6),
which were virtually homozygous, a variety of crosses were made. Indi-
viduals from these crosses were identified as follows: S, T, U and V were
animals originally derived from maternal ancestors with the "high" tumor
extra-chromosomal influence (E). They were, however, eight generations
removed from the pure strain originally employed. Had the extra-chro-
mosomal influence remained unchanged, there should have been approxi-
mately 196 mammary tumors among the 372 mice recorded. Actually there
were 6. This is only 3 per cent of the former tumor rate. The extra-

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 257

chromosomal influence has, therefore, largely disappeared. When crosses
\V, X, Y and Z which lacked the extra-chromosomal high tumor influence are
compared with these, there are found to be 4 tumors when 122 were expected.
The percentage is similar to the previous crosses but the original extra-
chromosomal influence was different.

Having eliminated the extra-chromosomal influence, we may next com-
pare the various crosses as regards their chromosomal composition. Crosses
S and U should resemble the original low cancer strain. Actually this was
the case as no mammary tumors were recorded in them.

Table 6

Per Cent C57

Female Male

Per Cent dba

Black Chromatin

Chromatin

dBFi X Blk

BdFi X dba

ist BC X Blk

ist BC X dba

87-5

2nd BC X Blk

2nd BC X dba

87-5

3rd BC X Blk

3rd BC X dba

4th BC X Blk

4th BC X dba

5th BC X Blk

5th BC X dba

6th BC X Blk

6th BC X dba

7th BC X Blk

7th BC X dba

8th BC X Blk

8th BC X dba

Crosses T, \, W and Y had roughly the same formulae as the original
outcross BdFi and BdFo. The number of animals observed should have
given a total of 24 mammary tumors if the tendency to form mammar}-
tumors had been transmitted through the chromosomes. Actually 7 or 2^%
of that number were observed.

Crosses X and Z should be comparable to virgin females of the "high
tumor" strain. There should have been 144 mammary tumors. Actuallv
there were 3. Even if the extra-chromosomal influence was ruled out, there
should have been approximately 14 mammary' tumors formed.

There has, therefore, been a ver>^ clear decrease in cancer incidence which
requires further study.

Evidence that at least part of the extra-chromosomal influence can be
transferred by foster nursing. — There are two important wa>'s in which
evidence of the importance of foster nursing in determining the incidence of
mammarv tumors in mice can be obtained.

258

BIOLOGY OF THE LABORATORY MOUSE

The first is by the direct transfer of new-born young, reported and exten-
sively studied by Bittner (13, 18, 19, 22-28, see also Chapter 9). This
method brought to light the entirely unexpected and very interesting fact
that the new born young from "high" breast tumor stocks, when transferred
to nursing females of a "low" breast tumor stock, develop into animals
which in later life show an incidence of breast tumors very similar to that of
their foster mother. To a considerable degree the converse is also true.
The incidence of breast tumors among mice of "low" tumor strains can be
materially increased if they are fostered by "high" tumor females (Table 7).

Table 7

Stock

Incidence of
Breast Cancer

"High" stock females (unfostered)
"High" stock females (fostered)
"Low" stock females (unfostered)
"Low" stock females (fostered)

83-6%

7-9%

0.5%

approximately g.o%

Fi 9 's produced by H 9 X L cf (unfostered)
Similar mice fostered by H 9
Similar mice fostered by L 9

94-9%
95 0%

0.0%

Fi 9 's produced by L 9 X B. d^ (unfostered)
Similar mice fostered by H 9
Similar mice fostered by L 9

1.9%

93 0%

8.0%

The second method of testing the influence of foster mothers is by the
transfer of fertilized ova from the fallopian tubes of "high" breast tumor
mice to the uteri of pregnant "low " tumor females. This technique as used
by Fekete and Little has given rise to a considerable number of viable young
developed from transferred ova. These mice upon maturity have shown a
breast tumor incidence characteristic of the strain from which their foster
mother was derived. The exact quantitative relationship as regards breast
tumor incidence among the fostered and transferred mice obtained by the
two methods has not yet been determined. The possibility remains that the
intra-uterine influences may prove to be more extensive and stronger than
those of the milk alone. On the other hand no such difference may be
obtained. The matter is under investigation.

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 259

Evidence that genetic factors also influence the incidence of breast
tumors. — Perhaps the most interesting experiments to test this point are
those recently conducted by Bittner (25, 26). These are based upon the fact
that reciprocal crosses between "high" (A) and "low" (C57 black) breast
tumor strains produce very different degrees of tumor incidence in Fi.
These were compared with the pure "high" and "low" tumor stocks by
fostering young in both series.'

The results are summarized in Table 7.

According to Bittner 's theory all Fi hybrids whether produced from
H 9 X L cf or from L 9 X H d^ should carry one group of "high" tumor
genes from their high parent. When to this genetic tendency the extra-
chromosomal influence is added, the results are very different from the
parallel fostering between the pure stocks, one of which lacks the "high"
tumor genes. Thus pure "low" stock females fostered by "high" stock
produce only 9% breast tumors, while Fi females, themselves ''low," produce
93% breast tumors when fostered by high tumor females.

While further experiments are necessary, the evidence at present favors
Bittner's theory that some influence of genes is active.

Evidence that hormonal influences also affect the incidence of breast
tumors. — Primary evidence for this theory is to be found in the comparative
behavior of breeding and virgin females in three high tumor strains of mice.
These strains are designated respectively as C3H, dba and A. The approxi-
mate incidence of breast tumors is shown in Table 8.

Table 8

Stock

Virgin 9

Breeding 9

C3H

dba
A

95%^
51%

/ 93%

-^ 85%

84%

It is very evident that the absence of pregnancy and lactation has a
markedly different effect in the three strains.

Further evidence -of hormonalinfiuence has been derived from the experi-
ments of Bagg and others (5-7.) who have shown that forced breeding with-
out opportunity for nursing- increases the incidence of breast cancer in
animals where some genetic tendency to form such cancer exists.

There is also a series of experifnents involving the artificial prolongation
of lactation and nursing in mice reported by Fekete (40). In this case the

26o

BIOLOGY OF THE LABORATORY MOUSE

incidence of breast cancer was somewhat reduced as compared with normal
breeding females.

All of this suggests that the cyclic changes to which the breast tissues are
subjected in pregnancy and lactation are periods during which the risk of
setting up a neoplastic process is very definitely increased.

Evidence that coat color may play a part in influencing the incidence of
mammary tumors. — It will be well at the outset to make clear the fact that
various degrees of incidence of breast tumors exist in distinct inbred strains
of different coat colors. This, however, does not necessarily mean that coat
color per se affects the incidence directly or even that it represents a general
physiological type which is more or less susceptible. Selection which estab-
lishes any characteristic coat color for a given inbred strain may also fix the

Table 9

Generation

Total

Yellow

Total
Non-yellow

Per Cent of

Yellow Mice

with Breast

Tumors

Per Cent of

Non-yellow

Mice with

Breast Tumors

57
156

223

38.6
37-2

64.8
51-6

Total

213

277

37-5

54-2

tumor incidence coincident with but entirely independent of the question of
color.

The real test of the influence of coat color depends upon the comparison
of different colored animals within the same generation of mice, where other
genetic influences have been equalized as nearly as possible.

The opportunity to study this type of situation is offered by comparing
yellow and non-yellow mice among the animals of Fi and Fo generations
following an outcross.

Since all yellows so far observed are heterozygous,, being ^^a or A'-' A in
formula, the Fi generation of a cross with aa (non-yellow) mice consists of
yellows and non-yellows in approximately equal numbers.

The F2 generation gives yellows and non-yellows in proportions which
var>' according to the color of the Fi animals selected for breeding.

In a cross reported by Little (54) the figures shown in Table 9 were
obtained.

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 261

This is, in all probability, a significant difference and is, therefore, of
interest.

In analyzing the possible reason for the decreased incidence of mammar}^
tumors in yellow mice, the following suggestions were made by the writer:

'*A study of the physiology of reproduction of yellow and non-yellow
mice within the yellow stock suggests that the yellows pass through their
reproductive cycle earlier than do the non-yellows. The duration of the
cycle in the two forms is essentially equal. This fact would satisfactorily
explain the earlier incidence of mammary tumors in yellow mice.

"The lower incidence of mammary tumors in yellows as compared with
non-yellows may be at least in part due to the same phenomenon. This
would follow because the opportunity for mammary tissue in yellow mice of
cancer age to be continuously affected by ovarian secretion would be less
than in non-yellows. This would result in a higher percentage of vellows
reaching an age at which stimuli from the ovary ceased before the mammary
tissue had reached an age at which tumor formation is most frequent."

These data show that it is not accurate to lump together different colored
mice in calculating the incidence of mammar\' tumors. They also indicate
the need for further study of this general topic.

Summary. — To summarize the situation as regards epithelial breast
tumors in mice, one may state:

1. These are the commonest type of spontaneous neoplasm in unselected
material.

2. Strains have been produced by inbreeding and selection which mav
give as high as 93^0 or as low as 0.5*^ incidence of these tumors in breeding
females.

J. There is conclusive evidence that the incidence of these tumors is not
due to a single recessive gene.

4. There is a definite extra-chromosomal influence which is directlv
transferable from female parent to her progeny.

5. This influence is at least in part, if not entirely, transferable through
the milk of the mother.

6. It is probable that genetic factors also play a part in determining the
tendency to form tumors.

7. Hormonal influences also affect the incidence of breast tumors.

Epithelial Lung Tumors

Most of the observed lung tumors in mice are epithehal in origin, being
of the adenoma, adenocarcinoma or carcinoma simplex types. These

262

BIOLOGY OF THE LABORATORY MOUSE

tumors in their incidence and relationship to various genetic factors present
interesting contrasts with the mammary group. We may review briefly
certain of these differences.

Absence of extra-chromosomal influence. — It will be remembered that
reciprocal crosses between strains that were "high" and those that were
"low" in breast tumors gave very different results. Such is not the case in
similar crosses between "high" and "low" lung tumor strains.

Lynch (63, 64) gave the first data, describing in a preliminary way crosses
of this sort between two inbred strains. She mentioned no difference
between reciprocal crosses but also gave no figures to differentiate between
them. This was not surprising, for, at that period, no such distinction
between reciprocal crosses had been described for mammary tumors where
later it was found to exist. A later paper by the same writer (65) gave
further results of a similar nature.

The most conclusive data on this point, however, are those of Bittner (23)
who, having corrected his figures by the elimination of the disturbing factor
of "breast tumor" incidence, found the results shown in Table 10.

Table 10

Cross

Fi Generation

F2 Generation

No.
Mice

Per Cent
Lung Tumor

No.
Mice

Per Cent
Lung Tumor

High 9 X Low cf
Low 9 X High d'

203
202

76.4
76.7

204

222

59-3

54-1

There is no evidence, therefore, of "extra-chromosomal" influence.

"Dominant" nature. — Lynch (63) gave as one of her conclusions the
statement that the tendency to form lung tumors in mice appeared to be
"dominant" in heredity.

She, however, quite properly recognized the fact that dominance was far
from being regular or complete.

Again, her later work as well as that of Andervont and Bittner (i, 13)
provided further supporting evidence.

It remained for Bittner (26), however, to give the most complete data
on this question. Using, from his data, comparable groups of mice in differ-
ent generations, we find the results shown in Table 11.

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 263

Table 11

No.

Per Cent Lung

Parent stock C57 black virgin 9 9
Parent stock A virgin 9 9
Fi hybrids virgin 9 9
F2 hybrids virgin 9 9

133
221

367
376

0.0
90.0
87-5
673

The close similarity of the lung tumor incidence in the high tumor stock
and in the Fi hybrids is striking.

There is evidently a small percentage of potentially and genetically lung
tumor animals which fail to develop the neoplasms to a degree or at a
sufficiently early age to be recorded. This percentage may be taken as 11.3
which represents an average of lo.o and 12.5, the A stock and Fi percentage
respectively of normal overlaps.

Using 88.7% as the incidence of lung tumors in a stock in which all
animals carried the hypothetical dominant gene for these neoplasms, we may
calculate the expectation for F2 as 75% of that figure or 66.5. The actual
percentage observed in that generation was 67.3. The close degree of corre-
spondence between the calculated and observed figures is strong evidence in
support of the theory that a dominant Mendelian gene may, in certain
crosses, play the main role in determining the incidence of these tumors.

The situation is not, however, quite so simple. Certain crosses of other
stocks recently made by Heston (unfinished data) and reported at the 1940
meeting of the American Association for Cancer Research show that modify-
ing genes or other genetic agents influence the percentage of lung tumors
formed. Not all ''low" tumor stocks behave in a similar manner when
crossed with a single "high" tumor strain.

Furthermore, the percentage of mice showing multiple nodules in the
lungs was quite different when certain "low" tumor strains were employed
from what it was when others were used.

The age at which the nodules became visible also varied according to the
parent strains used.

We can thus conclude that the available evidence suggests that a domi-
nant gene is at times clearly indicated but that its influence is subject to
modification by secondary genes which affect actual incidence of any
lung nodule, the number of nodules, and the age at which they are usually
formed.

264

BIOLOGY OF THE LABORATORY MOUSE

Hormonal influences. — Although the distribution of lung tumors is not
exactly equal between the sexes, the investigations reported by various
workers give conflicting results.

Slye, Holmes and Wells (93) recorded 57.4% of their lung tumors in
female mice and 42.6% in males. Lynch, on the other hand, in a large group,
obtained among females an incidence of 16% and in males 22%.

Bittner's figures are again the most extensive available. The incidences
in the two sexes and in various generations are shown in Table 12.

Table 12
Lung Tumor Incidence Following Reciprocal Crosses between A and

Cs7 Black (B) Strains

Generation

No.

Per Cent Tumor

Generation

No.

Per Cent Tumor

ABFi cf cf
BAFi d'd^
ABF2 c^cT
BAF2 cf cT

91
99
90
98

92.3
80.8

75-6

571

ABFi 9 9
BAFi 9 9
ABF2 9 9
BAF2 9 9

94
83
90
98

89.4
88.0
65.6
714

All d^d"

378

Average 76.4

All 9 9

365

Average 78.6

The incidence in the two sexes is thus approximately the same, and no
evidence of hormonal influence exists.

Coat color. — Heston's work, referred to above, was planned to detect any
signs of linkage between certain of the common genes for coat color and the
tendency to form lung tumors if any such relationship existed. The genes
involved were the following pairs:

C = color
A = agouti
B - black

No evidence for linkage was found.

c = albinism
a = non-agouti
b = brown

Non-epithelial Tumors

Under this very broad heading are included a large number of different
types of neoplasms.

In spite of a large amount of pedigree data collected by Slye (86-92) and
others, there still is lacking a sulhcient number of animals with any one type
of tumor in any one inbred line of mice to give adequate and significant

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 265

ratios with which to test the exact genetic nature of the process of tumor
formation.

There are, however, certain general factors about non-epithehal tumors
that differentiate their time, place and rate of origin from that of epithelial,
mammary or lung tumors.

As, in the case of mammary or lung tumors, one must begin his investiga-
tion of the genetics of the incidence of non-epithelial tumors in inbred
material unless he wishes deliberately to handicap himself.

Even in types of tumors which histologically are relatively consistent, as
the adenocarcinomas of the mammary gland or lung, there are modifying
and complicating influences which afifect the genetic processes. This is even
more applicable to the non-epithelial tumors so that, in this chapter, empha-
sis will be placed upon a study of a few inbred strains and their hybrids.

In this connection a general statement contained in a recent paper by
Little, Murray and Cloudman (60) may be helpful. The authors, in describ-
ing the commoner types of non-epithelial tumors, state :

" Tumors of lymph cells may occur wherever lymphatic tissue is present.
They generally are primary in the spleen or in the various mesenteric or other
peritoneal nodes. When they are thus situated the clinical symptoms are an
abdominal swelling, with hardening, frequent turgidity, ascites or general-
ized edema, and occasional asymmetry. Often by careful palpation the
enlargement of the spleen or the presence of other peritoneal nodules can be
detected. In some animals the peritoneal cavity may be grossly distended
by fluid. This fluid is of three types. It may be hemorrhagic, of the deep
color of venous blood; in other cases it is milky with a pink tinge; while in
still others it is clear and watery. As yet no consistent correlation between
any of the three types of fluid and any particular character of the lymphatic
tumor has been detected.

"The next most frequent type of non-epithelial tumor is a reticulo-endo-
thelioma of the liver. Fluid within the peritoneal cavity is rare in associa-
tion with tumors of this type. Abdominal swelling occurs, however, due to
the enlargement of the liver. Naturally this swelling tends to be more
anterior in position than many of the masses in the lymphocyte tumor
group.

" Fibrosarcomas are apt to occur in scattered sites. Those on the jaw or
leg or in the dorsal or lateral subcutaneous tissue are readily discernible as
hard, fixed nodules of firm texture. Those in the uterus are usually recog-
nizable by posterior distention of the abdomen and by the presence of an
irregular palpable nodule.

266

BIOLOGY OF THE LABORATORY MOUSE

''Melanomas, which are rare, are usually conlined to the base of the tail
and are deeply pigmented.

"Osteogenic sarcomas, which have ordinarily appeared in the long bones
or jaw, are superficially very much like fibrosarcomas in those regions.

"Pathological diagnosis has been obtained for all tumors included in this
report."

The inbred stock on which there has been recorded the most extensive
observations is the J AX C57 black stock of the Jackson Memorial Labora-
tory (Tables 13, 14 and 15).

Table 13

Tumor Incidence in the C57 Black Stock among Animals in Which There

Are Data for the Full Life Cycle and for Non-epithelial Tumor

Incidence

Type of

Total

Non-

Non-epi-

Epithelial

Mean
Age at

Dp;ith

Mean
Age at

Animal

Mice

tumor

thelial Tumor

Tumor

Non-
tumor

Death,
Tumor

Breeding 9

570

499

64 (11.22%)

10 (1.75%)

608

706

Virgin 9

^^33

109

26 (19-54%)

I (0.75%)

814

711

Males

174

142

31 (17-81%)

5(2.87%)

720

741

It is doubtful whether any of these groups differs significantly from the
others except in the mean age at death of the virgin and of the breeding
female non-tumor mice. In the case of the breeding females there is a
distinct suggestion of the existence of a greater mortality risk. The virgin
females have definitely a greater life span than the other groups. This may
account at least in part for the high incidence of tumors since the opportunity
to have such tumors which occur at an average age of 711 days was well
afforded by the fact that mean age of survival of non-tumor mice was 100
days beyond that figure.

With this brief introductory statement we can next consider the relation
of non-epithelial tumor incidence to various factors such as extra-chromo-
somal influences, age, sex, etc.

Absence of extra-chromosomal influence. — In the various experiments
with non-epithelial tumors there are no cases of reciprocal crosses between

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 267

high and low tumor strains that are extensive enough in the first hybrid (Fi)
generations to give significant results.

Table 14
Types of Non-epithelial Tumors in the C57 Black Stock

Type of Tumor

Incidence

Lymphoblastoma

73 (60.3%)

Endothelioma

26 (21.5%)

Fibrosarcoma

8(6.6%)

Lymphangioma

5 (4.1%)

Osteogenic sarcoma

3 (2.5%)

Hemangioma

3 (2.5%)

Melanoma

I (0.8%)

Reticulum-cell sarcoma

I (0.8%)

Undifferentiated

I (0.8%)

Total

121

It is necessary, therefore, to compare the F2 hybrids formed from a
reciprocal cross between two inbred strains. While the two strains are not

Table 15
Distrlbution by Site of 121 Tumors in the C57 Black Stock

Location of Tumor

Incidence

Spleen and nodes

75 (62.0%)

Liver

31 (25.6%)

Uterus

4(3-3%)

Mammary line and branches

4(3

3%)

Subcutaneous other than mammary line

3(2

5%)

Tail

I (0

8%)

Jaw

I (0

8%)

Eye

I (0

8%)

Intestine

I (0

8%)

classifiable with complete accuracy as "high" and "low," there does seem to
be a difference between them so that we can tentatively classify the dilute
brown (dba) strain as "lower" in incidence of non-epithelial tumors and the

268

BIOLOGY OF THE LABORATORY MOUSE

JAX C57 black strain as "higher." The F2 generations give the results
shown in Table 16.

Table 16

Generation

Origin

No. of
Animals

No. of Non-
epithelial
Tumors

Per Cent

Incidence of

Non-epithelial

Tumors

F2 (C57 black 9 X dba d')
F2 (dba 9 X C57 black cf)

Lower 9 X higher cf
Higher 9 X lower cf

468
649

61
90

13.09
13.61

There is very evidently no sign of extra-chromosomal influence in the Fo
generations.

Relation of incidence to age. — One fact seems clear in all the experiments
thus far recorded. This is the distinctly later age at which non-epithelial
tumors usually appear as compared with epithelial mammary tumors.

A tabulation of the mean age at death of mammary tumor and non-
epithelial tumor mice in the same stocks and their hybrids can be made from
data derived from the work of Murray, Cloudman and the writer (Table 17).

Table 17

Stock and
Generation*

Mean Age in Days at Death

Excess in Age of
Non-epithelial
Tumor Group

Mammary

Non-epithelial

C57 black (B)

dba (d)

dBFi

BdFi

dBF.,

BdFo

433 .

575

711

566

623

706

t
806
808
706
704

+ 231
+ 97

+ 140
+ 81

* Female is listed first, male second, in the crosses.

t Numbers of tumor animals too small to provide a significant value for
the mean age of incidence.

It is very evident for the above data that there is involved a very different
set of physiological factors in the incidence of the two types of tumors.

Relation of incidence to sex. — Scattered data derived from various
workers over a period of several years indicates that non-epithelial tumors

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 269

located in tissues or organs not specific to or unequally developed in the
sexes are as frequent in one sex as in the other. In this respect they
resemble, as might be expected, more closely the epithelial lung tumors than
those of the mammary gland.

It may well be that further and more detailed observations will reveal
tendencies for certain types of non-epithelial tumors to occur more fre-
quently in one sex than in the other. Such differences will, however, in all
probability be minor and secondary and will occur as a reflection of the
influence of a physiological distinction between the sexes, less important
than those commonly recognized as secondary sexual characters.

Relation of incidence to coat color. — The difhculty of exact studies in this
field is clear. There are, however, certain indications of relationships

Table 18

Stock

Total Non-epithelial
Tumor

Lipoid
Tumors

Per Cent
Lipoid

Hybrids of yellow X non-yellow
Hybrids of non-yellows involving
same strain

44
199

20.4
0.0

between coat color of certain types and its accompanying physiology on one
hand and the incidence of non-epithelial tumors on the other.

One of the more interesting of these suggestive relationships is to be found
in yellow mice which have long been known to be addicted to adiposity.

In a cross between yellow and non-yellow strains of mice there were
among the hybrids nine cases of lipoma or liposarcoma. These are entirely
absent in other crosses involving the same non-yellow strain. The actual
figures are given in Table 18.

It seems likely, therefore, that yellow ancestry introducing physiological
tendencies towards excess formation of lipoid tissue provides an increased
opportunity for the origin of tumors in that tissue.

Another less clearly defined but potentially interesting relationship
between coat color and non-epithelial tumor formation is to be found in the
incidence of this type of tumor in "intense" pigmented mice with the gene D
as compared with "dilute'' mice homozygous for its allele d (Table 19).

Relation of incidence to hybridization. — The possible eftect on tumor
incidence of crosses between strains of mice that differ widely from one

270

BIOLOGY OF THE LABORATORY MOUSE

Table 19

The Incidence of Non-epithelial Tumors in Intense and in Dilute

Mice in Two Series of Crosses

Series

Intense

Dilute

Mice

Non-epithelial Tumor

Mice

Non-epithelial Tumor

I
II

732
264

105 (11.4%)
13(4-7%)

236
115

46(16.3%)
10(8.0%)

Total

996

118 (10.59%)

351

56(13.75%)

another in various physiological activities has been pointed out by Little

(58).

The parent strains used were :

(a) Mus bactrianus, a small, slowly maturing, relatively infertile species.

(6) JAX C57 black, a strain derived from Mus musculus, large, rapidly
maturing and fertile.

The tumor incidences in these strains and in their Fi hybrids are com-
pared in Table 20.

Table 20

Stock

Total
Mice

Mice with Non-
epithelial Tumors

Per Cent Non-
epithelial Tumors

Mus bactrianus
JAX C57 black
Fi hybrids

i59
877
121

0
1x6

0.0

13-2
39-7

The increase in the hybrids is striking. There was also a definite increase
in multiple tumors among the hybrids (Table 21).

Leukemias

By far the most extensive and important work in this field has been done
by MacDowell and his associates. It has been admirably summarized and
discussed by him (67) in a recent general paper.

He describes the origin of his material which is based upon carefully
controUed inbreeding over an extensive period. As he states, in one of his

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 271

inbred strains, C58, "A surprising number of animals were found at autopsy
to have enormous spleens, large livers and swollen lymph nodes. They
were dying with leukemia, in most cases of the lymphatic type."

Table 21

Per Cent

Stock

Mice with

of Tumor

Mice with

Multiple

Growths

I Tumor

2 Tumors

3 Tumors

4 Tumors

Mus bactrianus

0.0

J AX C57 black

116

7-9

Fi hybrids

23.6

* Both epithelial and non-epithelial tumors are included.

In this strain, among over 700 mice observed until death, the incidence of
spontaneous leukemia was 90%. Since the strain was presumably homo-
geneous genetically, the presence of a group of 10% which failed to develop

Table 22

Total

Incidence of

Difference -^ Probable

Mice

Leukemia

Error

High tumor stock C58

606

89.6%

Fi from high 9 X low cf

139

61.9%

Fi from low 9 X high cf

106

42.5%

4-5

BC derived from

46.5%

Fi 9 X low d"

159

BC derived from

low 9 X Fi d^

19.8%

7.0

leukemia showed that in these mice extrinsic influences of some sort were
deciding whether or not an animal became leukemic.

When males of this strain were crossed with females of a non-leukemic
strain, the incidence of leukemia in the resulting Fi hybrids was 45%.

MacDowell defines the term leukemia as a neoplastic growth of the white
blood cells, differing from cancer in the fact that these cells do not remain
localized but move throughout the body. He further points out that

272

BIOLOGY OF THE LABORATORY MOUSE

Coat Color
Influence

aj
aj ..

-a
-a
0

c
0

Lipomas may be re-
lated to yellow an-
cestry. Dilute coat
color may be related
to some types.

-0

0
<J
aj

c
0

1 §

1/1

OJ
Ph

•a
>

c
0

-a
>

C
0

T3
(U

-a

1)
C
0

Milk
Influence

PLh

T3
>

C
0

-a

OJ
tn

C
0

-T3

(U
TD
;.^
0
(J

ID
OJ

-S 0
P<

0
>. *^

= -a

0 C
CJ C

SS 8

T3
>

0
OJ
C

"3.

c
0

0
0

c
0

Nature of
Genetic
Influence

^§

=^ 'c

-Q S

0 0

(in

Probably
multiple
factors

Probably
incomplete
dominance

Extra-
chromosomal
Influence

C
0

cJ5

lU
in

0
0

Genetic

Influence

Chromosomal

Slight but
probably
present

fcC

c
£
en

'S .2

OJ C

■S .2
<u c

u- B
0 «

Epithelial
mammary
tumors

in
0

^ B

•- 2

-c c
.ti 3

a -^

X. ^

'2- 2
V E
c 3
0 -^

OJ
3

h-I

THE GENETICS OF SPONTANEOUS TUMOR FORMATION 273

leukemic cells are not changed blood cells but are "a special race of cells hav-
ing independent origin." He states that they arise from reticulum cells
by focal proliferation. Migration obscures all trace of the point of origin.
By careful observation the earliest stages of this proliferation have often
been detected. They are so numerous that very clearly all of the early sites
of proliferation do iwl become sources of origin of the disease. This appears
to be evidence against the proliferation of normal lymphoid cells or organiza-
tions as being a precursor to the occurrence of leukemia.

Evidence of extra-chromosomal influence. — It is interesting to note that
in somewhat the same manner as that reported for epithelial mammary
tumors, MacDowell (6g, 70) has recorded reciprocal cross differences in the
incidence of spontaneous leukemia in mice derived from a cross between a
"high" and a "low" line (Table 22).

There is no doubt, therefore, that some influence which is extra-chromo-
somal in nature is operative. Further studies of the genetic behavior of
spontaneous leukemia should be important.

Comparison of the Four General Types of Neoplasms

Table 23 may help to summarize some of the main points of resemblance
and difference in the four main groups of neoplasms considered.

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274 BIOLOGY OF THE LABORATORY MOUSE

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THE GENETICS OF SPONTANEOUS TUMOR FORMATION 275

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2 76 BIOLOGY OF THE LABORATORY MOUSE

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THE GENETICS OF SPONTANEOUS TUMOR FORMATION 277

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278 BIOLOGY OF THE LABORATORY MOUSE

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Cnapter 7

THE GENETICS OF TUMOR TRANSPLANTATION

By Clarence C. Little, Roscoe B. Jackson Memorial Laboratory.

Genetic studies on tumor transplantation, 279. The Mendelian nature of the genetic
influences determining susceptibility and non-susceptibiUty to transplanted tumors,
279. Evidence of mutations in transplanted tumors, 288. Transplantation of
leukemia, 2qo. Practical considerations, 292. Methods of transplantation. 292.
Sites of transplantation, 2Q:i,. Material used in transplantation, 294. Practical
suggestions, 296. Transplantation of genetically controlled tumors in relation to the
study of growth and individuality, 297. Factors influencing successful transplanta-
tion 297. Relation to individuaUty, 301. The relation of transplantable tumors to
spontaneous tumors, 301. Relation to transplantation of normal tissue, 304. Con-
clusion, 305. Bibliography, 305.

Genetic Studies on Tumor Transplantation

Under this heading will be considered (a) the Mendelian nature of the
genetic influences determining susceptibility and non-susceptibility to the
growth of transplanted tumors, (b) evidence of mutations in transplanted
tumors, (c) transplantation of leukemia.

The Mendelian Nature of the Genetic Influences Determining
Susceptibility and Non-susceptibility to Transplanted Tumors

The early work of LeoLoeb (52, 53) showed that tumors which originated
in a strain of Japanese waltzing mice would grow, upon transplantation, in
approximately ioo<^ of animals of that strain. The same tumors failed to
grow in an unrelated strain of non-waltzing mice. This provided material in
which there was a clear cut and uniform difference in susceptibility between
two strains.

Acting upon this suggestive result, Tyzzer (85) made certain carefully
controlled experiments on which he reported in 1909. His results are
summarized in Table i.

From these results Tyzzer concluded that susceptibility to the carcinoma
JwA was inherited but not according to Mendel's law or any other type of
inheritance then known. This conclusion seemed justifiable since what
looked like Mendelian dominance in Fi had completely disappeared in F-..

279

28o

BIOLOGY OF THE LABORATORY MOUSE

The subsequent occurrence of a susceptible animal among mixed hybrids of
F2 and more advanced generations reopened the question, however, and
suggested the need of further study (86).

Table i

Growth or an Adenocarcinoma or the Mammary Gland (JwA) of Japanese

Waltzing Mice, in Japanese Waltzing Mice, Common Mice and

Their Fi, F2 and F3 Hybrids

Stock

Japanese waltzing mice
Common mice
Fi hybrids
F2 hybrids
F3 hybrids

142

54
16

In 19 16 Little and Tyzzer (51) reported on a larger series of mice inocu-
lated with tumor JwA. A total of 629 mice were used. The results in the
more important generations are summarized in Table 2.

Table 2
Growth of Tumor JwA in Japanese Waltzing Mice, in Common Mice
and in Various Hybrids between These Two

Stock

Per Cent

Japanese waltzing mice

Common mice

Fi hybrids

F2 hybrids

Fi hybrids X Japanese waltzing mice

Fi hybrids X common

38
0

61
3

63
0

180

100. 0

0.0

98.4

1.6

100. 0
0.0

The incidence of susceptible animals in F2 required further analysis and
if possible a genetic explanation.

In 1 9 14 Little (44) had published a brief note on a type of inheritance
which might occur and which would give the appearance of being non-
Mendelian, although actually depending upon Mendelizing genes. It was
the continuation and development of simpler experiments already recorded

THE GENETICS OF TUMOR TRANSPLANTATION 281

and it gave somewhat striking and startling results. It depended upon the
hypothesis that certain characters of an organism depended upon the
simultaneous presence of more than one Mendelizing gene.

Certain characters of this type were already known. Since it was this
hypothesis which was applied successfully to the reaction of mice to trans-
planted tumors it may be discussed to advantage at this time.

PARENT I.

PARENT 2.

SUSCEPTIBILITY

NON-SUSCEPTIBILITY

SUSCEPTIBILITY GENE PRESENT

SUSCEPTIBLE

I
NON-SUSCEPTIBLE

PERCENT SUSCEPTIBLE 75.0

Fig. 127. — Diagram showing the inheritance of susceptibility to transplanted tumors

where susceptibility is due to the presence of a single dominant gene.

Characters dependent upon one pair of genes. — It is, of course, well
known that Mendelian inheritance when one pair of genes is involved gives a
3 : 1 ratio in F2, in this case 3 susceptible mice to i non-susceptible mouse
(Fig. 127).

Characters dependent upon two pairs of genes. — If now we suppose that
two genes, A and B, are needed simultaneously to produce susceptibility we
should have a ratio, not of 3:1, but of 9:7 or 1.3:1 (Fig. 128).

Characters dependent upon three pairs of genes. — If we continue this
principle to a character dependent upon the simultaneous presence of 3
genes, the ratio will change still further (Fig. 129).

2»2

BIOLOGY OF THE LABORATORY MOUSE

Genetic theory of transplantation. — It would be cumbersome to continue
to develop this theory further by diagrams. We may, however, give a table
which shows the percentages of susceptible mice to be expected when larger
numbers of genes are needed (Table 3). In this table the data already
shown in diagrams will be included.

PARENT I.

PARENT 2.

SUSCEPTIBILITY NON-SUSCEPTIBILITY

A a B BOTH PRESENT SUSCEPTIBILITY

/////

3
A

3
B

SUSCEPTIBLE

7
NON-SUSCEPTIBLE

PERCENT SUSCEPTIBLE 5625

Fig. 128. — Diagram showing the inheritance of susceptibility to transplanted tumors
where susceptibility is due to the simultaneous presence of two dominant genes.

It will be noted that as the number of genes needed increases the Fi and
backcross with the susceptible parent give constant figures. The behavior
of the F2 generation and of the backcross with the non-susceptible parent is
quite different. As the number of genes increases the percentage of suscepti-
ble animals in these generations decreases with great rapidity. The decrease
is more rapid in the backcross than in the F2 generation and after 11 or 12
genes are involved would result in practically negligible occurrence of
susceptible animals in the former generation.

If we now compare the results obtained by Little and Tyzzer with the
expectation for 14-15 genes we get the situation shown in Table 4.

THE GENETICS OF TUMOR TRANSPLANTATION 283

Table .^
Thk Relation between the Percentage of Mice Susceptible to a Trans-
planted Tumor and the Number of Genes Responsible for the

Susceptibility

Pairs of Genes,
the Simul-

Per Cent

Per Cent Susceptible in
Backcross of Fi

taneous Pres-
ence of Which
Is Needed

Susceptible
in Fi

Susceptible
in F2

X Susceptible
Parent

X Non-sus-
ceptible Parent

100. 0

75-0

100. 0

50.0

100. 0

56.2

100. 0

25.0

100. 0

42. 2

100. 0

12.5

100. 0

31.6

100. 0

6.2

100. 0

23-7

100. 0

17.8

100. 0

1.6

100. 0

133

100. 0

0.8

100. 0

10. 0

100. 0

0.4

100. 0

7-5

100. 0

0. 2

100. 0

5-6

100. 0

0. 1

lOO.O

4.2

100. 0

0.05

100. 0

0.02

100. 0

2-3

100. 0

O.OI

100. 0

1-7

100. 0

0.005

100. 0

1 .0

100. 0

0.002

Table 4
Comparison of Observed and Expected Results in Growth of Tumor JwA

Susceptible
Parent

Non-
susceptible
Parent

Backcross

with

Susceptible

Parent

Backcross
with Non-
susceptible
Parent

Observed
Expected

100. 0
100. 0

0.0
0.0

98.4
100. 0

1.6
I . 7-1 .0

lOO.O

100. 0

0.0
0.0

With this beginning as a working hypothesis, experiments were continued
and extended.

A sarcoma of the Japanese waltzing mouse JwB gave simpler results
indicating that from 4 to 5 genes were needed (46).

284

BIOLOGY OF THE LABORATORY MOUSE

Some years later (1924) Little and Strong (50) described, in some detail,
the behavior of two transplanted adenocarcinomas of the dilute brown (dba)
strain of mice, dBrA and dBrB. Strong and Little (81) had previously
shown that these two tumors, although apparently identical histologically.

PARENT I.

PARENT 2.

SUSCEPTIBILITY

NON-SUSCEPTIBILITY

A B a C ALL PRESENT SUSCEPTIBILITY

2 27

SUSCEPTIBLE

9
A B

B C

37
NON-SUSCEPTIBLE

RATIO I* TO 1.3- PERCENT SUSCEPTIBLE 42.2

Fig. 1 29. — Diagram showing the inheritance of susceptibility to transplanted tumors
where susceptibiHty is due to the simultaneous presence of three dominant genes.

gave distinctly different percentages of successful growth when inoculated
simultaneously on opposite sides of the same animals.

The results of inoculating these two tumors in a large number of dilute
brown mice, unrelated Bagg albino (A) mice and various hybrid generations
between them are shown in Table 5.

With the exception of the backcross Fi X Bagg albino inoculated with
dBrB, the two tumors give figures which coincide closely with the expecta-
tion for two genes in the case of dBrB and three for dBrA (Table 6).

THE GENETICS OF TUMOR TRANSPLANTATION 285

Table 5

The Growth of Tumors dBrB and dBrA in Two Strains of Mice and

IN THE Progeny of Crosses between These Strains

Stock or Generation

Tumor dBrB

Tumor dBrA

Per

Cent +

Per

Cent +

Dilute brown (dba)

All

100. 0

All

100. 0

Bagg albino

130

0.26

131

0.0

Fi hybrids

139

99.2

145

99-3

203

141

58.1

156

188

45-35

BC Fi X dba

100. 0

BC Fi X Bagg

131

16.0

131

17.6

Anah'sis of the simultaneous reaction of the animals to the two tumors
indicated that two of the three genes that were involved in the case of the
tumor dBrA are the same as those which are active in the case of dBrB.
The two thus bear the following relation to one another.

Table 6
Expected and Observed Percentage Takes of Tumors dBrB and dBrA

Per Cent

+ Bagg

Albino

Stock

Per Cent

Per Cent
+ Back-

+ dba

+ Fi

+ F2

cross

Stock

Hybrids

FiX
dba

Fi X
Bagg

Observed dBrB

100. 0

0. 26

99.2

58.1

lOO.O

16.0

Calculated 2 gene

ratio

100. 0

0.0

100. 0

56.25

100. 0

25.0

Observed dBrA

100. 0

0.0

99-3

45-35

100. 0

17.6

Calculated 3 gene

ratio

100. 0

0.0

100. 0

42.2

100. 0

1-^-5

Tumor dBrA needs genes A, B and C.
Tumor dBrB needs genes A, B.

These experiments helped to strengthen the probability that the working
hypothesis based on Little's earlier theor}^ was correct.

286

BIOLOGY OF THE LABORATORY MOUSE

F'nvdl confirmation, however, came from two series of experiments, one b\-
Strong (77, 78) and a still more importance piece of work by Bittner (9).

Strong's 1926 (77) paper established a "one gene" ratio in the case of a
transplanted adenocarcinoma dBrCsp. This climaxed the long series of
tested tumors which had begun ten years earlier.

His later paper (78) described an interesting tumor FiDb which showed a
four gene ratio in F... One of the genes which alTected the growth of the
tumor was, however, sex-linked. It thus provided important confirmatory
evidence of the Mendelian nature of the susceptibility to tumor transplants.

Bittner's work was with certain transplantable tumors which occurred
spontaneously in Fi hybrid mice. It was, in some ways, related to an earlier
piece of work reported by Little and Johnson (49) .

In this earlier experiment splenic tissue had been used instead of tumors.
Three groups of mice were used. These were (a) Japanese waltzers, (b)
Bagg albinos and (c) Fi hybrids between these two strains. Bits of spleen
from animals in each group were inoculated subcutaneously into animals of
the same group and into mice from the other two groups. In Table 7 are
shown the results obtained in animals where the physical condition remained
good throughout the experiment.

Table 7

The Results of Transplanting Spleen Tissue w^ithin and between Two

Inbred Strains of Mice and Their Hybrids

Spleen from

Spleen Inoculated into

Japanese Waltzers

Bagg Albinos

Fi Hybrids

Japanese waltzer
Bagg albino
Fi hybrid

81
0
0

0
16

17
0

0
0

The Fi spleens failed to grow in the Japanese waltzers, thus showing that
they were characteristic of hybrid animals. On the other hand they grew in
other Fi hybrids as did the Japanese waltzers' spleens.

The tumors with which Bittner worked originated in Fi hybrids between
the dilute brown (dba or *'D ") strain and an albino (A) strain derived from
Bagg albinos (Fig. 130).

THE GENETICS OF TUMOR TRANSPLANTATION

>87

The most important and critical scries of crosses were tliose in which
tumor 13714BX, originating in an Fi generation mouse, was used for
transplantation.

This tumor, inoculated in F2 generation mice gave 94+:25o— . This
indicated that either 4 or 5 genes were involved. The experimental results
lie between the expectation for this number of genes and afford no basis for
choice as to the exact number.

Fig. 130. — Diagram showing the relationship existing between the various stocks
and generations of mice employed by Bittner in studies of transplantable tumors
arising in hybrid mice. The two parent strains are called "A" (albino) and "D"
(dba or dilute brown). Two arrows pointing towards a square indicate that the
particular generation was produced by crossing the two stocks or generations from
which the arrows come. One arrow indicates that the matings were inter se to produce
the next generation. {From Bittner.)

The most interesting figures come, however, from the two backcross
generations, that of Fi X A (ZBC) and that of Fi X D (iBC). The actual
figures, compared with expectation for various numbers of genes which are
introduced by the respective parent stocks, are shown in Table 8.

It appears, therefore, that probably four or five pairs of genes, of which
one or two are introduced by the A stock and three or four are introduced by
the D stock, are involved.

The fact that the evidence provided by the backcross generations sup-
ports the probability that four or five genes are involved, is important.

288

BIOLOGY OF THE LABORATORY MOUSE

It agrees with the F2 generation results and thus goes far to establish the
correctness of the theory used to explain the observed figures.

In a review of the genetics of tumor transplantation by Haldane (33) the
theory herein advanced is accepted.

Table 8
Comparison of Observed and Expected Takes of Tumor 13714BX, Orig-
inating IN an Fi Mouse, in Animals Produced by Crossing Fi Mice
TO THE Parent Stocks

Generation

Observed

Expected i Gene

Expected 2 Genes

ZBC
iBC

34+ 434-
23+ 44-

29-75+ 438.75-
Expected 3 genes

16.75+ 50.25-

58.5+ 409.5-
Expected 4 genes

33-5+ 33.5-

More recent experiments by Gorer (29, 30) have provided evidence that
in the case of certain transplanted mouse tumors the genes involved have a
chemical basis in certain iso-agglutinogens which have been identified.

Evidence of Mutations in Transplanted Tumors

Once the principle of Mendelizing units underlying transplantation of
tumors was established, a means was provided for interpreting more accu-
rately the nature and significance of variations in the number or percentage
of successful "takes" in difference generations or experimental groups of
mice.

Utilizing very cleverly selected inbred lines of mice and their hybrids,
Bittner (10) was able to explain and to reproduce at will the complicated
curves on which the investigators at the Imperial Cancer Research Fund in
London had based a theory of fluctuating virulence of the tumor which was
supposed to be rhythmic.

Bittner showed that different proportions of various genetic types was
all that was required. There was no need of hypothesizing either fluctuating
virulence or rhythm in that fluctuation.

It was, however, desirable to set up a series of experiments designed to
show whether transplantable tumors did change and if so in what respects.

In order to provide the proper conditions for such a test it was necessary
to keep constant the genetic constitution of the various populations studied
so that when variation occurred it would be due to some change in the tumor
itself.

THE GEXETICS OF TUMOR TRAXSPLAXTATIOX 289

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290 BIOLOGY OF THE LABORATORY MOUSE

Strong (75), working with an adenocarcinoma dBrC which originated in
the dilute brown (dba) strain, found that it grew in all animals of that strain
which were inoculated. It also grew in 180 Fi mice produced by a cross
between strain dba and A albinos. The F2 generation gave a ratio (line i,
Table 9) which indicates that probably six genes were involved.

During routine inoculations of this tumor a very rapidly growing sub-
strain of it was observed. This was designated as tumor dBrCX (line 12 in
Table 9) . From this tumor two further sub-strains seeming to show a dififer-
ence in growth rate and specificity were isolated. These were called dBrCm
and dBrCsp. They were carefully tested with Fo animals and gave results
shown in lines 5 and 9 of Table 9. There had evidently been a genetic
change from a probable six factor tumor to two factors in the case of dBrCm
and to one factor in the case of dBrCsp.

Similar results with other tumors have later been described by Bittner
(7) and by Cloudman (22). In every case the change has been in the direc-
tion of decreased specificity and there have been ratios indicative of fewer
factors after the change than before it.

Since the changes appear to be sudden and since they are perpetuated
from one cell generation to another, they are properly definable as mutations.
It will, of course, be necessary' to discover a method of identifying the genes
borne within the tumor cells before the mutations can be considered as estab-
lished "gene" mutations. They are, however, abrupt genetic modifications
which are self-perpetuating.

Tr.\nspl.\ntation of Leukemia

One of the most extensively studied types of neoplasm in mice is the series
of leukemias reported by MacDowell and his associates.

An excellent discussion and review of this field has been given by
MacDowell (57). In all of his work he has employed significant numbers of
inbred genetic strains which provide authenticity and a sound foundation for
future investigation.

Having demonstrated that the estabhshment of a true leukemic condition
depends upon the multiplication of an invasion by inoculated leukemic cells,
the parallel between that situation and the growth of transplanted tumors is
established.

The elimination of extra-cellular agents including bacteria further
strengthens the similarity of the two processes.

There also exists a high degree of specificity within the inbred genetic
strain so that transplantation of leukemic cells within the strain is uniformly

THE GENETICS OF TUMOR TRANSPLANTATION 291

successful while transfer to unrelated strains is unsuccessful. This, of
course, also applies in the various transplanted tumors of mice which have
been studied.

It is perhaps in the field of immunization that many of the most interest-
ing and important contributions by MacDowell and his associates have been
made.

Although they recognize that much remains to be explained, and that
many complicating factors serve to obscure the true nature of the process,
they have made very definite progress to which some brief reference may be
made.

By introducing very small numbers of leukemic cells — as, for example,
1/4,000,000 of the standard dose — the mouse may survive. If it does, it
shows that it can become modified to tolerate increasingly larger doses of
cells until it is finally able to "overcome massive doses of leukemic cells given
repeatedly." It is, however, clear that this immunity has no effect upon any
tendency to form spontaneous leukemia in the same animals.

It is also interesting to note at this point that often the first transplants
of leukemic cells derived from a spontaneous growth, will not kill the host
before 20 to 90 days, while after a long series of transfers from mouse to
mouse, death may result in 3 or 4 days. This has also been the history of
some transplanted tumors, but not of all.

In such "immunized" mice there is no trace of any antibodies in the
serum. This is in accord with the results obtained with transplanted tumors.

Although there are no antibodies present "a susceptible host can be
immediately protected against a lethal dose of leukemic cells by treatment
with minced tissue from an actively immunized animal." Whatever causes
this protection is "intimately associated with living cells." By forcing the
minced tissue out of a syringe "held firmly against the bottom of the vessel"
all the cells are torn apart and the protective property is destroyed. Very
evidently these facts raise the possibility that some unknown mechanism of
resistance is involved.

The injection of entirely normal tissue from an unrelated mouse may
also confer immunity. Various organs, both embryonic and adult, may
be used. Genetic constitution has a role to play. MacDowell states, how-
ever, that "before making the test there is no means of knowing the effect
of normal tissue of a given genetic constitution, except that the tissue of
the same genetic constitution as the host is ineft'ective."

The resistance produced by a single treatment with normal tissue " difiers
from that induced by leukemic cells in that it cannot be passively transferred

292 BIOLOGY OF THE LABORATORY MOUSE

to another host, and, while regularly delaying the progress of leukemic
invasion, does not always give lasting resistance." There may be delayed
appearance of leukemia or "curious subcutaneous tumors may appear with
the histological characteristics of lymphosarcoma." Such tumors have not
been obtained elsewhere. When transplanted into normal hosts these
peculiar tumors give rise to leukemia of the same type peculiar to the line of
leukemic cells previously inoculated. In some cases, however, the inocu-
lated lymphosarcoma type of tumor reappears in one or more transplant
generations in untreated hosts. This suggests a different type of resistance
mechanism on the part of normal tissue to that of leukemic cells.

The importance of continued studies in this field is thus obvious and
should be generally recognized.

With the general conclusion that the genetics of tissue transplantation
has a Mendelian basis, and that the number of genes involved varies in
individual cases according to the degree of genetic similarity or difference
between donor and host, we may consider certain of the more practical
aspects of tumor transplantation.

Practical Considerations
Methods of Transplantation

The commonest method of transplantation is by use of a trocar. In this
and all other types, great care to maintain asceptic conditions should be
taken. An infected tumor or site of implantation results in the introduction
of factors which importantly influence the continued growth of the implant.
A tumor when removed under asceptic conditions and placed in a sterile dish
may be cut into a number of small bits. These may be loaded in a trocar one
at a time and by a blunt plunger be pushed out through the sharpened end
of the trocar after that has been inserted to the site at which the implant is
desired. This method can be used for subcutaneous or intraperitoneal
implantation. In the case of the former, the trocar can be withdrawn
through a constriction formed by grasping the skin with the forefinger and
thumb just above the tip of the trocar, thus preventing the implant from
being pulled out of place.

Another method closely allied to the above is the implantation of bits of
tissue by fine pointed forceps. This method may at times possess certain
advantages of greater accuracy in location of the implant. With the excep-
tion of the instrument used, it varies little, however, from the trocar method.

The use of a fairly coarse hypodermic needle is often helpful. In this
case the sterile tumor, after removal, is cut into bits which are then ground

THE GENETICS OF TUMOR TRANSPLANTATION 293

into a mush by mortar and pestle. If an emulsion which will pass through a
fine needle is desired the process of grinding must, of course, be more pro-
longed and careful. Either normal salt solution or Ringer's solution may be
used as a medium for thinning the emulsion. This method is naturally more
delicate than either of the foregoing and is valuable in reaching relatively
inaccessible or restricted sites.

Sites of Transpl.\ntation

The ear. — This provides a site easy of observation. There is a relatively
slight blood supply ; however, and the temperature is apt to be below that of
the peritoneal cavity or various subcutaneous sites. For this reason the ear
is a favorable site for testicular transplants.

The forehead. — This is a convenient site and one in which the oppor-
tunity for invasion of underlying tissue is definitely limited by the proximity
of the skull. The blood supply is relatively low.

Subcutaneous axillary and inguinal. — The paired sites thus provided are
very frequently used. The blood supply of both areas is good, that of the
axillary region being the better. In using these sites it is well to make the
incision through which the trocar or the forceps are inserted at some distance
at some lateral location.

Subcutaneous mid-dorsal and mid-ventral. — These are also frequently
used. Accurate location of the implant is more difificult than in either the
axillary or inguinal sites, but the blood supply is good.

Subcutaneous tail. — This region pro\ddes the lowest blood supply and
slowest growth of any yet studied. It is advantageous because of ease of
observation and because the tail can be wholly or partially removed, thus
providing a convenient aid in studies of induced immunity.

Intracranial. — This site can be approached after removal of a small
amount of bone which can later be replaced or through a fine hole drilled in
the skull. It can also be utilized by the careful insertion of a small hypo-
dermic needle if solutions are used for the implant. Its advantages are those
common to the site in other forms. There appears to be in this site an
unusual degree of non-specificity. There are several records of successful
transplants of mouse tumors in rats, guinea pigs or even pigeons. Quite
obviously extensive growth of any implant is accompanied by serious symp-
toms and disturbances.

Intraperitoneal. — Mice are particularly resistant to infection and to
operative shock so that any site in the peritoneal cavity is available with
comparatively little danger or difficulty. Suspension of tumor cells injected

294 BIOLOGY OF THE LABORATORY MOUSE

into the peritoneal cavity often gives rise to many small nodules of healthy
tissue convenient for reinoculation.

Intrathoracic. — This cavity can be reached either through the diaphragm
or the intercostal spaces. The postoperative results are usually satisfactory.

Intra -uterine. — Because of the small size of the os and the danger of tear-
ing it if inoculation through it is attempted, it has been found that exposure
of the uterus by abdominal operation, by either dorsal or ventral incision, is
preferable. The uterus provides an excellent site for transplantation.

Intratesticular. — By maintaining pressure which keeps the testis in the
scrotum it becomes easily available as a site for implantation. If a more
delicate technique of transplantation is desired, an abdominal operation is
simple and effective!

Intravenous.- The most accessible site is the tail vein in which inocula-
tions can easily be made. By proper care and experience this vein can be
used many times in a series of experiments without great difficulty.

The eye. — In rabbits a satisfactory technique for implanting bits of
tumor tissue in the anterior chamber of the eye has been developed. In this
site vascularization appears to be rapid and extensive. Possibly as a result
of this factor alone or in combination with decreased specificity of reaction to
foreign tissue in that area, successful growth of homologous and even of
heterologous tissue has been reported. As yet this technique has been little
used in mice although it offers real promise.

By application. — If desired a bit of tumor tissue may be applied to the
surface of an organ or tissue and be held in place there either by some adhe-
sive membrane such as that formed by collodion or by a single suture. This
method has the advantage of affording an opportunity to study surface reac-
tions between any two tissues.

Material Used in Transplantation

Since new tumors are constantly being discovered and are being used for
transplantation, it is impossible to make, at any one time, a complete and
permanent list of this material.

On the other hand, it may be helpful in giving a picture of the opportuni-
ties for research in this field if some of the more interesting and commonly
used tumors are mentioned.

Imperial Cancer Research Fund, London, England

No. or Symbol Type of Tumor

27 Adenocarcinoma of the mammary gland

37S Spindle cell sarcoma

THE GENETICS OF TUMOR TRANSPLANTATION 295

No. or Sxmbol

113

155

173

206

2146

Twort

Melanotic Harding-Passey

3187

Berlogh

B.P.

Oestrin

Type of Tumor
Alveolar* carcinoma of the mammary gland
Alveolar and adenocarcinoma of the mammarv

gland
Alveolar carcinoma of the mammary gland
Adenocarcinoma of the mammary gland
Tar carcinoma — undifferentiated squamous
Alveolar carcinoma of the mammary gland
Tar carcinoma — polymorph
Alveolar carcinoma of the mammary gland
Melanotic sarcoma — unpigmented strain
Mast-cell sarcoma
Anaplastic carcinoma (Originally from Silberstein

Vienna as Ehrlich mouse carcinoma)
Sarcoma — benzpyrene in subcutaneous tissue
Mammarv carcinoma

Columbia University, New York, N.Y.

Ehrlich Chondroma

Sarcoma 37

Mi 80 (Crocker 180)

M2163

Received direct from Frankfort 1924 — slow

growing
Originally 37S from London. Polymorphous cell
(1914) Polymorphous cell sarcoma
(1938) Left axilla — undifferentiated carcinoma

with some areas of adenocarcinoma

Huntington Hospital, Harvard Unrersity, Boston, Mass.
Ovarian embryo ma (1938) Ovarian embryoma originating in C3H mice

Yale Unrersity School of Medicine, New Haven, Conn.

Brain tumor

Brain tumor
Rhabdomyosarcoma

Hepatoma
Ovarv

Meningeal sarcoma (obtained from Drs. Seligman

and Shear)
Glioma (obtained from Drs. Seligman and Shear)
Obtained following injection of methylcholan-

threne, C3H stock
Originated in CBA stock
Carcinoma, CBA mice

Many types of tumors induced by hormones or synthetic chemical
carcinogens are usually available.

* The term "alveolar" is used in this connection to denote tumors having solid
masses of cells in contrast to the glandular or adenocarcinomatous type.

296 BIOLOGY OF THE LABORATORY MOUSE

Dr. Margaret Reed Lewis and Dr. Warren Lewis, Wistar Institute,

Philadelphia, Pa.

No. or Symbol Type of Tumor

6 sarcomata Transplantable in Bagg albino mice

3 sarcomata Transplantable in Little C3H stock

5 sarcomata Transplantable in Little C57 black stock

1 sarcoma Transplantable in Murray dba stock

4 white blood cell tumors Myeloid cells, 3 in dba and i in C57 black
4 white blood cell tumors Lymphoid cells, 3 in dba and i in C3H

2 white blood cell tumors Monocytic, i in A stock and i in C57 black

RoscoE B. Jackson Memorial Laboratory, Bar Harbor, Me.

15091a Spindle cell carcinoma of the mammary gland

(1928) originally diagnosed by Ewing and
Warthin as adenocarcinoma. Thirteenth gen-
eration became carcinoma simplex. Twentieth
generation gave evidence of transformation of
epithelial cells to spindle cells. A stock

L946A 11. Fibrosarcoma originating in osteogenic sarcoma of

the tail (1936). C57 black stock. No bony
elements

E060 Papillary adenocarcinoma of the mammary gland

(1936) C57 black

C617 Adenocarcinoma of the mammary gland (1938)

C57 brown stock

dbrB Adenocarcinoma of the mammary gland (1920)

dba stock

S91 Melanoma (1937) primary at base of tail dba stock

C252 Fibrosarcoma (1936) subcutaneous pelvic region

C57 leaden stock

C198 Reticulo-endothelioma liver — rare type (1936)

C57 leaden stock

P208 Melanoma (1937) on side of dba strain mouse

P764 Embryonal cell carcinoma of the testis (1939) dba

strain

From the above list the great diversity of available material will be
evident.

Practical Suggestions

The following suggestions are made to those who desire to utilize genetic
knowledge in the transplantation of tumor tissue in mice.

THE GENETICS OF TUMOR TRANSPLANTATION 297

1. For routine carrying on of tumors use one or more strains produced
and maintained by brother to sister or parent to offspring matings. Use
either the strain in which the tumor originated, which should give approxi-
mately 100% takes, or if this is impossible, any inbred strain that gives a
high proportion of positive animals.

2. For routine carrying on of tumors at rapid rate of growth, maintain
one or more pure strains as above indicated. Use animals from such strains
to cross with one or more unrelated inbred strains to produce first generation
(Fi) hybrids. Use these for inoculation. They usually grow the tumor
more rapidly than the inbred animals themselves.

3. To use an inoculated tumor as a means of measuring the degree of
physiological difference or similarity between strains several steps are neces-
sary: Maintain two or more distinct inbred strains, one of which is the strain
in which the tumor originated, the other being the strain or strains which are
to be compared with it.

4. The storage of tumor tissue in dry ice refrigerators (about — 7o°C.)
has also proven a satisfactory method of preserving this type of tissue. Best
results have been obtained when the tissue is frozen slowly, thawed rapidly.
Some investigators have found this method satisfactory for all types of
tumors, others have reported success with some tumors but unreliable results
with others.

Transplantation or Genetically Controlled Tumors

IN Relation to the Study of Growth

AND Individuality

Factors Influencing Successful Transplantation

In addition to the genetic constitution as an important factor in deter-
mining success or failure of transplanted tissue there are a number of other
things which may influence the final result.

Among these, several may be briefly discussed.

Diet. — Various experimental, unbalanced and defective diets have been
reported as influencing the number of "takes" and the rate of growth of
transplanted tumors. There is no doubt that diet may play a part in deter-
mining the reaction of the animal. On the other hand, the fact that the
investigators have not used inbred strains to reduce and control the genetic
variables, leaves it uncertain as to the cause and effect relationship between
diet and the changes in percentage of growth. This fact, coupled with an
almost complete disregard of criteria of mathematical significance between

298 BIOLOGY OF THE LABORATORY MOUSE

the groups that are being compared, seems to have left the problem of (Het in
a most unsatisfactory condition. For this reason no attempt is made in this
volume to cover the extensive but non-critical bibliography. The whole
problem will have to be approached ''from the ground up" by investigators
who understand and utilize genetics, biochemistry and mathematics.

Irritating agents. — There have been several types of experiments dealing
with the effects of irritants of various sorts in the response of an animal to
implants of tumor tissue.

Perhaps the simplest approach to this problem is through the introduc-
tion of a mechanical irritant which is not able to exert any evident chemical
reaction.

A series of experiments of this sort was reported by E. E. Jones (37) who
found that growth of an adenocarcinoma was obtained in a number of mice
belonging to stocks, otherwise negative, when a bit of sterile non-dyed
flannel was inoculated with a bit of the tumor.

This interesting result indicated that possibly local factors as well as
those affecting general lymphocytic reaction may be operative.

It would seem that further study of this general held would prove
fruitful.

It is also known that previous exposure of transplantation sites to physi-
cal agents such as heat, cold or radiation may affect the percentage of
successful implants and their rate of growth. As yet, however, data on these
effects are so fragmentary and diffuse as to prevent any general conclusions
being drawn. Biochemical irritants of some types have also been used.

Perhaps a typical and interesting result is that obtained by Koenigsfeld
who found that animals painted with carcinogenic tar and inoculated at the
same time with a transplantable tumor showed increased response to the
former and more rapid growth of the latter. This interesting mutual
activation remains unexplained and is in contrast with the experience of
investigators who have compared the interaction of centers of benign growth
with a center of malignant growth. In this case the usual experience has
been that pregnancy slows the rate of growth of transplanted cancer except
in the case of certain exceptional tumors. These are mammary adenomas
which in some instances have grown more rapidly when the host is pregnant
than at other times.

In all of these experiments, as in those dealing with dietary factors, the
present need is for a more accurate control of the too numerous variables
which, influencing the fate of the transplant, may serve to mask or to dis-
tort the relationship between any one experimental factor and the end result.

THE GENETICS OF TUMOR TRANSPLANTATION 299

Age, sex and other biological factors. — In 1920 Little (45) showed that
temporary growth of tumors destined to eventual regression and disappear-
ance was more readily obtained in very young animals than in young adults.
Strong later showed that the same is true of very old animals as compared
with those in the prime of physiological activity.

A difference in the rate at which the sexes acquired the ability to elimi-
nate transplants of tumors was also demonstrated by Little (Table 10).

Table 10

DlFFKRKNCE IN THE RaTE AT WhICH THE SeXES ACQUIRE THE ABILITY TO

Eliminate Transplanted Tumors

Males
Females

Age in

Obser-

Days at

vations

Inocu-

Showing

Nega-

lation

Mass

tive

2-10

212

12-20

209

2-10

231

12-20

150

Per Cent

Showing

Mass

14-51 ± I-5I
15-38 ± 1-55
12.12 + I . 76
25.74 ± 2.07

Diff. ± P.E.

13. 16 + 2.71

Diff.

p1^

0.87 + 2. i6| 0.4

S-o

In this case the mice used were those of a hybrid generation in which
some of the animals would presumably show progressive growth of the
implants and others (the majority) would show regression and eventual dis-
appearance. The female mice in the older age group gave a significantly
higher percentage of "takes" than did the males. This was in all prob-
ability due to the earlier assumption by some of these animals of the
biological make-up which reflects the presence of genetic factors for sus-
ceptibility. Female mice mature distinctly more rapidly than do males.
They would, therefore, begin earlier to express their characteristic genetic
constitution. This actually is the case.

As a contrast to the hybrid mice among which are to be found a number
of animals with a genetic constitution favoring susceptibility, may be cited
the results of inoculating females of a completely non-susceptible strain.
These results are shown in Table 11.

Here it will be noted that the non-susceptible genetic constitution is
expressing itself rapidly and definitely in a significant decrease in positive
observations.

300

BIOLOGY OF THE LABORATORY MOUSE

Complete or partial castration and ovariectomy have also been studied
in relation to growth of transplanted tumors. The results obtained by dif-
ferent investigators have varied as have the conclusions drawn from them.
This is probably due to the fact that various stocks, ages and tumors
have been used. An additional variable has been provided in the interval
between operation and implantation of the tumor.

One of the most complete and careful studies of this question has been
made by Strong (72). He concludes:

I. Removal of the gonads does not change the massed percentage reac-
tions for individuals of a non-susceptible race. This bears out the previous
conclusion that the number of percentage reactions in a given strain depends
upon the genetic constitution of the individuals.

Table 11

Age in Days
of Females
at Inocu-
lation

Observa-
tions
Showing
Masses

Observa-
tions
Negative

Per Cent

Showing
Masses

Diff. ± P.E.

Difif./P.E.

2-10

12-20

331
463

19.46 + I. 31
9-39 ± 0-87

10.7 ± I. 51

7.0

2. Gonadectomy produced, in the stock employed, a significant increase
in percentage reactions in mice attaining sexual maturity.

3. Gonadectomy causes an approach towards a "neutral" type (loss of
characteristic differences between sexes) in the percentage of reactions — just
as it does in the case of morphological characteristics.

4. By the removal of the gonads, the individuality of tissues and the
normal functioning of the age factor can be interfered with.

5. A severe shock caused by such an operation as gonadectomy produces,
in some cases at least, a resistant state to transplantable tumors, that is at
its maximum from five to ten days after the operation.

Other investigators have found similar shock effects following operative
removal of the spleen.

In all of these physiological studies a common criticism can be made.
It is roughly the same as that applied to investigations of diet ; namely, that
too little work has been reported on material in which the number of vari-
ables has been reduced to a minimum.

THE GEXETICS OF TUMOR TRAXSPLAXTATIOX 301

It will be necessary to wait until far more extensive and carefully con-
trolled studies have been made before any conclusions of general applica-
tion can be drawn.

Relation to Indriduality

Transplantation studies afford one of the most promising methods
of investigation of the process of acquisition of complete biological
individuality.

By the growth of heterologous adult tissue in embryonic culture media,
such as the allantois of the chick embryo, and by the opposite process of
growing embryonic tissue for a considerable period in heterologous adult
individuals, evidence is clearly provided that full expression of the bio-
chemical characteristics of the species, strain or individual is gradually
developed.

Tumors which represent a source of supply of rapidly growing tissue in
which the degree of biochemical specificity may. to some extent, be measured
by genetic tests are valuable aids in such research.

By holding the source of tumor material constant and by varying the
degree of biological differentiation of the host that receives the implant,
information concerning the process of differentiation both chemical and
morphological should be obtained.

Similarly by the inoculation of several types of tumors in a single host
the reaction of that host can be measured in terms of its response to different
biological stimuli.

The REL.A.TION OF Transplant.able Tumors to Spontaneous Tumors

The bearing of genetic work with transplanted tumors on the genetics
of spontaneous tumors in mice is one on w^hich a great deal of difference of
opinion exists. One of the commonest points of view is that a clear and
distinct line should be drawn between experimental work on (/) transplanted
tumors, (2) induced tumors and (j) spontaneous tumors. While there is
no doubt that characteristic differences exist between the three groups as
regards the t>^e of problem which each is best fitted to cover, it seems likely
that an extreme point of view^ such as that cited is incorrect. One of the
reasons why a point of view of that sort has developed is that there is proper
objection to applying, in toto, the results obtained with either transplanted
or induced tumors to the field of the spontaneous tumors. This does not
mean, however, that work with spontaneous and induced tumors may not

302 BIOLOGY OF THE LABORATORY MOUSE

contrilnitf (Iftinitt'l}- to (»ui uiKJcrstaiiding of the i)i(K:rssfs of foniialion and
growth of spontaneous neoplasms.

One principle may safely guide us in this discussion. It is the fact that
only those who have had direct and continuing, first-hand knowledge of
experimentation in all of the three lields are qualified to evaluate with any
degree of probable accuracy the relationship between them. This again
does not mean that the student of transplanted tumors alone may not con-
tribute greatly to our knowledge of the cancer process. The same, of course,
applies to investigators who use only induced tumors or who study only
spontaneous tumors. All that is meant is that relations between the three
types of experimentation are best understood by those who have engaged in
all of them.

With this preliminary discussion we may consider briefly three prin-
ciples established by abundant experiments with transplanted tumors which
have an important bearing on the problem of spontaneous tumors. These
have been considered in a paper by Little (48) . They are as follows :

1. Transplantation in known and controlled genetic material provides a
more delicate test of biological and physiological differences between certain
neoplasms than does any other test at present available.

2. Transplantation experiments in which somatic mutational changes in
the genetic constitution of a tumor have been demonstrated afford a most
helpful avenue of investigation on the nature and incidence of somatic
mutation as a process of importance in cancer research.

J. Transplantation experiments on the genetics of spontaneous tumors
arising in Fi and other hybrid mice, derived from a cross between two
inbred strains, give an unusually good opportunity for linkage studies
between tumor genes, derived from the parent races, and genes for other
characters of a more easily detectable nature. They also should enable us
to determine whether hybridization as a process has any influence on the
genetic complexity of tumors formed.

In each of these cases transplantation is being used as an experimental
method as an aid in analysis and not as a process which creates important
facts de novo.

Transplantation and the physiological individuality of tumors. — In
1920 Strong and the writer (81) published evidence which showed that
two mammary adenocarcinomas of the mouse, although histologically
indistinguishable, gave very different percentages of continuing growth when
inoculated into hybrid mice of known genetic origin. The rate at which
these two tumors were eliminated by a negative strain of mice also showed a

THE GENETICS OF TUMOR TRAXSPLAXTATIOX 303

clear and persistent difference. The amount of temporary growth which
each exhibited was also different. The use of stocks of mice in which
temporary growth of transplanted neoplasms is followed by regression gives
a very delicate physiological test of the nature and activity of that tumor.
A series of tumors compared in this way often reveals more subtle and minute
differences than are detectable by any other known test. Cloudman has
made an intensive comparative study of the transplantation of mammary
tumors arising spontaneously in a single mouse and has shown that in the
case of three adenocarcinomas of the breast very different genetic constitu-
tions were involved. These tumors appeared at essentially the same time.
It is clear that the transplantation test provides a method of determining
whether these three tumors were independent primary growths or metastases
of the same primary neoplasm.

It is also evident that by a comparison of the genetic factors in such
a series of tumors much information can be derived as to the factors which
all possess in common and those w^hich are specific to a single growth. It
is quite conceivable that if extensive studies of this type were made we
might, by plotting the relationships of the genetic factors, obtain a valuable
picture of the process of tumor formation as a whole from a biological point
of view.

Similarly during the lifetime of an individual successive neoplasms occur-
ring at intervals as the age of the animal increases may be maintained
through transplantation and studied in comparison with one another to
find out whether older animals give rise to tumors which are character-
istically different from those produced by younger ones. All this type of
work in its various implications should contribute very definitely to our
knowledge of the process of disintegrating individuality in ageing animals.

Transplantation experiments and somatic mutation. — The question
of somatic mutation is discussed further in Chapter 6. For the present it
will suffice to point out that the occurrence of mutations in transplanted
tumors which increase the percentage of takes of these tumors is a well-
established phenomenon supported by the work of Strong, Bittner and
Cloudman. Ordinarily in tumors involving a number of genes these changes
also aft"ect more than one gene, also there have been cases where apparently
a change in a single gene resulting in a change from a two factor to a one
factor ratio has been observed. In these tumors the mutational change is
clearly somatic since the tumor in which the changes occur is composed of
somatic and not germinal tissue. Tyzzer and many others subsequently
have suggested that the change from a normal to a tumor cell may be in

304 BIOLOGY OF THE LABORATORY MOUSE

the nature of a somatic mutation. The question is still undecided, but it is
certain that some of the most favorable material in which to study it is to
be found in the modification of transplantable neoplasms. Such tumor
tissue can be subjected in various amounts to chemical and physical condi-
tions which have been shown to be mutation producing agents. Changes
in the tumor can afterwards be studied and recorded. Controlled series
of normal tissue subjected to the same agents can be maintained.

Treatment of various clearly defined sites in animals of known tendency to
produce spontaneous tumors of different types with agents likely to produce
mutation should give interesting information as to whether these agents
increase the incidence of spontaneous cancer. If they do so the relation of
this increase to the higher mutation rate in germ cells affected by similar
agents should be interesting and important.

Genetic studies of tumors originating in hybrids. — Transplantation
studies of tumors of this type should add more knowledge to the genetic
analysis of spontaneous tumors by providing evidence for linkage between
genes which underly the growth of transplanted tumors and some other
known Mendelizing genes. As the number of known genes in mice increases
and linkage becomes more generally recorded, the chance of finding genes
related to the process of spontaneous tumor formation should similarly
increase. If there is no evidence of such linkage when it may fairly be
expected, the negative findings will themselves be important in determining
the relative roll of chromosomal inheritance and other etiological factors in
spontaneous tumor formation. Preliminary evidence of linkage between
genes determining the growth of certain spontaneous tumors and those for
certain types of coat color has already been obtained. The need of obtain-
ing rapidly the largest possible number of genes is evident and the field of
tissue transplantation (more particularly that of tumors) should give us
valuable new information.

At all events, the genetic analysis of transplanted and induced tumors
has a direct and permanent bearing on similar studies with spontaneous
neoplasms.

Relation to Transplantation of Normal Tissue

Little has been said in this chapter on the bearing of tumor transplanta-
tion to the genetics of normal tissue transplants.

This omission is not due to the fact that the subject lacks importance or
interest. It results from the somewhat extraordinary fact that so little

THE GENETICS OF TUMOR TRANSPLANTATION 305

work has been done in this field — with properly controlled material — that it
remains practically an open door for experimentation.

Up to the establishment of the Mendelian nature of the genetic factors
influencing growth of transplants, Loeb had presented the only comprehen-
sive theory to attempt to explain success or failure of implants of normal
tissues. In 1924 Little (47) reviewed and criticized Loeb's work up to that
point. Discrepancies between experimental results and Loeb's theory
were pointed out.

Later Loeb and Wright (56) and Loeb and King (55) investigated the
transplantation of normal tissues in inbred and hybrid strains of guinea
pigs and rats. The data obtained from these experiments were in agree-
ment with the genetic theory of transplantation of tumors as given earlier
in this chapter.

So also were the results of Bittner (17) working with mice.

We may, therefore, conclude that distinct advances in our knowledge
can be made when further studies along these lines have been conducted.

Conclusion

In conclusion we may point out the fact that few investigators as yet
recognize and utilize the great opportunities for new attacks on many basic
biological problems afforded by the recent advances in our knowledge of the
genetics of tissue transplantation.

With inbred strains of mice now available there is a whole new field of
attack, not only on the problems of experimental cancer, but on those of
the nature of individuality and of the fundamental processes of ontogeny.

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to transplantable tumors. J. Exp. Zool. 36: 67-134.

73. Strong, L. C. 1924. Indications of tissue specificity in a transplantable sar-
coma. J. Exp. Med. 39: 447-456.

THE GENETICS OF TUMOR TRANSPLANTATION 309

74. Strong, L. C. 1926. Changes in the reaction potential of a transplantable
tumor. J. Exp. Med. 43: 713-724.

75. Strong, L. C. 1926. On the occurrence of mutations within transplantable
neopilasms. Genetics 11: 294-303.

76. Strong, L. C. 1926. A genetic study of the growth of a transplantable tumor
(adenocarcinoma dBrB). J. Exp. Zool. 45: 231-253.

77. Strong, L. C. 1926. Genetic studies on the nature of cancer. Am. Nat. 60:
201-206.

78. Strong, L. C. 1929. Transplantation studies on tumors arising spontaneously
in heterozygous individuals. J. Cancer Res. 13: 103-115.

79. Strong, L. C. and. A. M. Cloudman. 1930. Inbreeding and fixation of genetic
factors underlying the transplantability of cancerous tissue. Papers Mich. Acad.
Sc, Arts and Letters 11: 481-483.

80. Strong, L. C. and R. T. Hill. 1937. A sex difference encountered in the trans-
plantation of a carcinoma of the ovary. Science 85: 119.

81. Strong, L. C. and C. C. Little. 1920. Tests for physiological differences in
transplantable tumors. Proc. Soc. Exp. Biol, and Med. 18: 45-48.

82. Strong, L. C. and G. M. Smith. 1936. Successful transplantation of a hepatoma
in mice. Am. J. Cancer 28: 11 2-1 14.

83. Tyzzer, E. E. 1907. The inoculable tumors of mice. Harvard Univ. Cancer
Com. Rep. 4: 9-23.

84. Tyzzer, E. E. 1907. The inoculable tumors of mice. J. Med. Res. 12: 137-151.

85. Tyzzer, E. E. 1909. A study of inheritance in mice with reference to their
susceptibility to transplantable tumors. J. Med. Res. 21: 519.

86. Tyzzer, E. E. 191 5. The tumors of the Japanese waltzing mouse and its
hybrids. J. Med. Res. ^,2: 331.

Cnapter 8

ENDOCRINE SECRETION AND TUMOR
FORMATION

By George W. Woolley, Roscoe B. Jackson Memorial Laboratory.

With no outside addition of hormones, 311. With unusual addition of hormones, 312.
Hormones and transplantable tumors, 314. Hormone production with tumors, 315.
Bibliography, 315.

The secretions of those glands which hberate their substances into the
blood and lymph have a marked and important relation to tumor formation
in mice. This has been well demonstrated but only a few of the probably
many details of these relationships are known at the present time. For
mammary gland tumor production the presence of a certain type of hormone
is an indispensable factor. In addition to furthering the problems arising
from the primary differences due to sex itself, there are many possibilities
for extension of knowledge in this field. Strains of mice which develop
different but comparatively uniform percentages of mammary, as well as
other, tumors furnish excellent material for following up the indications of
at least quantitative differences in the hormonal control mechanisms and
of their relation to tumor incidence. The endocrine differences which
limit generalization not only between species but within a species such as
Mus musculus are no doubt important stepping stones along the path to a
more complete analysis of the interrelationships. The isolation and
chemical determination of many of the sex hormones, together with the
synthesis of related compounds, is rapidly leading to more extensive and
specific modification of hormones within experimental animals. All of
these are greatly aiding the progress of our knowledge. To make clear the
relation of these secretions and of related substances to tumor formation is
the first step. To be able to use this knowledge to aid in the control of
tumor formation is the eventual goal.

Because of the incompleteness of the picture of the relation of internal
secretions to tumor formation at the present time an attempt is made only
to call attention to some of the studies now available rather than to try to
fit the picture together. The bibliography is not exhaustive but through its

310

ENDOCRINE SECRETION AND TUMOR FORMATION 311

use reference may be had to many of the original studies. The material is
restricted to the mouse. In developing the review, use has been made of the
divisions which the mechanics of experimentation have projected into
the field. For example, those studies where no hormones have been added
from outside of the body have been separated from those where an addition
in one form or another has been made. Transplanted tumor studies and
evidences of hormone production in tumors are in still separate sections.

With no outside addition of hormones. — Of all the types of tumors that
have been found in experimental mice none have been more extensively
studied or more closely related to the endocrine system than those of the
mammary glands. That an endocrine factor was involved in the develop-
ment was indicated by the experiments of Loeb (55, 56), Lathrop and Loeb
(52) and subsequently confirmed by many experimental workers. Three
facts stood out. First, mammary tumors developed spontaneously in
female but never or very rarely in male mice. Second, the incidence of
mammary tumors in female mice varied according to the breeding activity :
virgin females had a lower percentage than parous or multiparous females.
Third, the tumors of virgin females appeared later than those of breeding
females.

That an endocrine factor was ovarian was indicated by the removal of
ovaries at various ages, a technique which caused a decrease in mammary
tumor percentage in direct proportion to the time of the ovary removal
(55, 52). Experiments by Cori (15) showed that ovariectomy at 16 days of
age reduced the percentage to almost zero. Further experiments with
ovariectomized mice supported and added to the work of Loeb and Cori
(70, 71).

Many experiments have demonstrated that the ovarian factor is not the
sole factor leading to mammary tumor development. The genetic and milk
influences are discussed in other chapters of this book. That the endocrine
factor need not always be ovarian has recently been demonstrated (87).
Following ovariectomy at birth, Jackson Laboratory dilute brown mice
developed nodular hyperplasia of the adrenal cortex. This was followed
by stimulation of the vagina, uterus and mammary glands. Twenty-seven
per cent of the ovariectomized females developed mammary gland tumors.
These changes leading all the way to tumor production are not limited just
to the dilute brown strain of mice, though they do not occur to the same
extent in some of the low tumor strains of mice (86). Recently mammary
tumors have appeared in male mice which were castrated at birth. This
again followed development of nodular hyperplasia of the adrenal cortex

312 BIOLOGY OF THE LABORATORY MOUSE

and subsequent growth of the mammary rudiments into extensive duct
systems.

It has been found that lymphosarcoma in one Hne of mice was nearly
twice as frequent in females as in males (6i). In another study, daughters
from reciprocal matings showed the same difference in incidence of leukemias
as the sons (59).

A brown degeneration occurring in the adrenal glands of both sexes of
mice has been described. Efforts to correlate this degeneration with varia-
tion of estrogenic hormones and the incidence of mammary cancer have been
made (16, 19).

The reproductive physiology of strains of mice with various percentages
of mammary tumors has been investigated (57, 43, 44, 37, 10, 68, 7, 80).
Special characteristics of the estrous cycle such as unusual duration of
phases of the cycle have not been consistently correlated with tumor
incidence.

It has been observed that the frequency of breeding had in some cases
marked influence on the incidence of mammary tumors in mice (3, 4, 53, 25).
Whether the result was related to the rapidity of the pregnancies in them-
selves or to the irritation of stagnating products in the mammary ducts is
not certain.

In an extensive study of mice painted with tar it has been found that the
males showed a distinctly delayed tar tumor reaction as compared with the
females (40).

With unusual addition of hormones. — Experimental studies have shown
that sex hormones can awaken malignant changes at least on a substratum
that is usually thought of as hereditarily susceptible to cancer. Thus
Murray (71) found that mammary tumors appeared in fifteen male mice out
of 210 castrated at 3 to 4 months of age when ovaries from sisters were
implanted. An inbred Jackson Laboratory tumor strain of mice was used.
Feminization of the male mouse, in which the mammary rudiments undergo
little if any development throughout life (30) , induced growth of mammary
glands and also the development of mammary tumors. This was confirmed
by dejongh (21).

Following the injection of estrogen,* males from high tumor strains of
mice developed mammar\' tumors as frequently as multiparous females
(41, 42, 27, 32). Males from low tumor strains developed tumors with

* Estrogen: a generic name for female sex hormone. The term as here used is
intended to include synthetic as well as naturally occurring hormones.

ENDOCRINE SECRETION AND TUMOR FORMATION 313

greater frequency than multiparous females of the same strains. Mammary
gland tumors have not yet been obtained following estrogen injections in
male mice from some of the very low tumor strains even though prolonged
efforts have been made to produce them (8, 44, 27). It might be assumed
that estrogens act in conjunction with some intrinsic factor predisposing
to tumor formation. Mammary tumors were produced in males of one low
tumor strain following injection of an estrogen only when nursed on high
tumor mothers (84). The method of injection is of importance. Using a
high tumor strain it was found that 3000 rat units of estrogen over a three
day period at two weeks of age was not effective in producing mammary
tumors in males but 16 weekly doses of 100 rat units each produced a high
incidence of tumors (11). Synthetic estrogens which differ markedly in
molecular structure from naturally occurring forms will produce tumors in
male mice (48, 75). This makes it difficult to assume that there is car-
cinogenic action associated with hormone molecular structure (50).

Mammary gland carcinomas have developed in females of very low
tumor strains following estrogenic treatment, though only after long periods
of treatment (9). The incidence of mammary tumors in mixed stocks has
been increased as compared to the controls (74). The incidence of mam-
mary tumors was increased in female mice of both high and low tumor
strains following estrogen injections (81).

The activity of the corpora lutea may be a contributing factor to mam-
mary gland cancer production (58). However, progesterone alone or
in combination with estrogen did not alter mammary tumor percentages
(50, 28).

A considerable increase in tumor rate in non-breeding mice of
several strains was observed following subcutaneous transplants of three
or four anterior lobes of the hypophysis from male and female litter mates

(58).

^Modification of the incidence of mammary tumors in mice has been
attempted with male hormone preparations. It has been reported that
testosterone administered to female mice of a highly susceptible strain will
result in a marked fall in the incidence of mammary tumors (51, 72). The
mechanism of the inhibition is not understood although evidence has been
marshalled indicating that the action is through the pituitary (50). The
lowering of incidence when treatment is started with mature animals has
not been suitably confirmed (50).

The appearance of mammary cancer has been prevented by use of the
thyrotropic hormone of the pituitar>^ gland (20). In another study the

314 BIOLOGY OF THE LABORATORY MOUSE

same hormone failed to prevent the occurrence of mammary tumors in
females, or in males treated with estrogen (49).

Cancerous lesions in or near the cervix have been reported following the
injection of estrogens (45, 56, 82, 29). One of these tumors was grafted
into young male and female mice in which it continued to grow without
further hormone administration (29). Lesions of the cervix similar to
malignant tumors appeared in mice receiving estrogen and 1:2:5:6 dibenz-
anthracene (74, 73).

Hemorrhagic chromophobe adenomas of the pituitaries developed in
mice following long continued injections of estrogen or its cutaneous appli-
cation (17, 18, 12). Hypophyseal tumors did not appear in six inbred
strains following the injection of several estrogens for prolonged periods.
In another strain 15 of the 106 mice treated showed pituitary enlargement
with the largest (46 to 87 mg.) consisting largely of adenomas of non-
glandular chromophobe cells (33).

Two sarcomas were observed among 16 castrated male mice bearing
ovarian grafts (21). There have been many reports of sarcomas developing
in mice following the injection of estrogens, usually estrogens in oil (15, 32,
27, 81, 46). In some cases they developed in relation to the oil cysts.
Lymphoid leukemia and lymphosarcomas have been observed in a number
of strains following the injection of estrogens while none of the controls
have shown such tumors (27, 47, 31).

The effect of estrogens in conjunction with carcinogenic agents has been
investigated. Reviews of the early studies are available (28, 14).

Hormones and transplantable mouse tumors. — In 1932 Zondeck, Zondek
and Hartoch (88) reported an inhibition of growth of the Ehrhch mouse
carcinoma following the administration of an extract containing both Prolan
A and Prolan B. Using over 400 mice the authors found that the average
tumor growth in animals treated over a three week period was 0.2 grams
while the control tumors averaged 1.65 grams. Furthermore, it was found
that this reduction in growth persisted during later transplants. Many
workers have attempted to modify the growth of transplanted sarcomas and
carcinomas with pituitary hormones since that time. Some studies con-
firmed retardation of growth (13, 66, 6). Some reported stimulation (23, 24,
79) and others found no effect upon the growth (36, 39). One of the most
serious hazards in such experiments is the difficulty in differentiating the
direct and the indirect effects of Prolan on the tumor growth (5). The
transplant has been exposed directly to Prolan extract in vitro following
which it was inoculated into the host. No evidence of inhibition or accel-

ENDOCRINE SECRETION AND TUMOR FORMATION 315

cration was secured with mouse sarcoma 180 and slight inhibition of growth
was observed with mouse sarcoma S3 7 (83). Tumor grafts grew more
slowly in hypophysectomized animals than in controls of the same age but
the relation of final tumor weight to body weight of operated and control
mice of equal age was the same (38).

Gonadectomy and sex itself has been considered as a factor effecting
the growth of transplantable tumors. Both have been reported of some
influence with particular tumors (76, 77, 69, 85).

The extracts of many internal secreting glands in addition to those
already mentioned have been used in attempts to alter the growth of
spontaneous and transplantable mouse tumors (64, 65, 2, 26, 67, 22, 62, 63).
The subject offers interesting possibilities.

Hormone production with tumors. — An adenocarcinoma arising pre-
sumably from follicle tissue of the ovary has been tested and found to secrete
estrogenic hormone (78). Evidence of estrogenic activity was also seen in
one mouse with bilateral granulosal cell tumors (34).

A lengthening and finally cessation of estrous cycles has been noted
following transplantation with tumors (60). With the growth of spon-
taneous mammar}' gland tumors estrous cycles became infrequent with long
continued periods of diestrus, and finally disappeared. Sections of the
genital organs showed them to be in extremely atrophic condition approach-
ing that of ovariectomized animals. Cycles were obtained by injection of
estrogen. The acyclic condition in tumor mice probably involves primarily
the gonadotropic function of the pituitary (i).

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between the incidence of mammary cancer and nature of the sexual cycle in
various strains of mice. Am. J. Cancer 26: 761.

81. Suxtzeff, V., E. L. Burns, M. Moskop and L. Loeb. 1936. The eft"ect of
injections of estrin on the incidence of mammary cancer in various strains of mice.
Am. J. Cancer 27: 229.

82. Suxtzeff, V., E. L. Burxs, M. Moskop axd L. Loeb. 1938. On the prolifera-
tive changes taking place in the epithehum of vagina and cervix of mice with
advancing age and under the influence of experimentally administered estrogenic
hormones. Am. J. Cancer 32: 256.

320 BIOLOGY OF THE LABORATORY MOUSE

83. Tanzer, R. C. 1936. Efifect of prolan on transplantable mouse sarcoma. Am.
J. Cancer 26: 102.

84. TwoMBLY, G. H. 1940. Breast cancer produced in male mice of the C57 (Black)
strain. Proc. Soc. Exp. Biol, and Med. 44: 617.

85. Wagner, A. 1932. The effects of castration on resistance to cancer. Hos-
pitalsted. 75: 817.

86. WooLLEY, G. W. 1940. Unpubhshed material.

87. WooLLEY, G. W., E. Fekete and C. C. Little. 1939. Mammary tumor devel-
opment in mice ovariectomized at birth. Proc. Nat. Acad. Sc. 25: 277.

88. Zondek, H., B. Zondek and W. Hartoch. 1932. Prolan and tumor growth.
Klin. Wchnschr. 11: 1785.

Cnapter 9

THE MILK INFLUENCE IN TUMOR FORMATION

^A* John J. Bittxer, Roscoe B. Jackson Memorial Laboratory.

New approaches to the problem of breast cancer etiology in mice devel-
oped following the advancement of the extra-chromosomal theory. Inde-
pendent work was published almost simultaneously by the Staff of the
Jackson Memorial Laboratory (28) and Korteweg (20) using strains which
had been sent from the Jackson Laboratory. This work has been confirmed
in several other experiments with different strains of mice (25-27. 29, 21-23,
19, 5. 8-9. 17-18).

In these experiments reciprocal matings were made between high and low
breast tumor strains of inbred mice. When the maternal parents were
members of the high tumor strains it was observed that the Fi and Fo
generation hybrids had a higher breast tumor ratio than if the paternal
parents were representatives of the high tumor line. This maternal
influence has three possible explanations:

A. That some influence is transmitted in the milk of the potentially
breast cancerous females to their young while nursing.

B. That some influence is transferred to the progeny of breast cancerous
mothers during uterine development.

C. Cytoplasmic inheritance.

To date all the experimental evidence emphasizes the role played by
the milk influence. In all. at least three "influences" must be taken into
consideration in the development of breast cancer, at least in mice. These
are:

1. A "breast cancerous producing influence" present in the milk of
cancer stock mothers.

2. A breast cancer susceptibility due to one or more dominant factors
transmitted by breast cancer strain mice.

3. An ovarian or hormonal influence which may or may not be asso-
ciated with breeding, depending upon the strain of mice studied.

The evidence supporting this explanation follows.
To test the eft'ects of foster nursing on the breast tumor incidence, young
born to high breast tumor females were removed soon after birth and were

321

322 BIOLOGY OF THE LABORATORY MOUSE

nursed by females of low tumor strains. The fostered females and
their progeny were later used as breeders. The breast tumor incidence
in such fostered mice was very low (4, 6-8, 12-18). Similar results
have been obtained when females of other high tumor strains were fostered
(2, 18).

In later work it was determined that the time interval between birth
and the transfer of the young to the foster mother was very important (13).
If the young are permitted to nurse their high tumor mother for twenty-
four hours or longer there is no reduction in the breast tumor incidence.
Progeny of these mice were not observed.

In inbred strains of mice showing a high breast tumor incidence the
ratio of this type of cancer is similar among the progeny of the non-cancerous
and the cancerous mothers (30, 9). If fostered high tumor females develop
breast cancer, the incidence for the first generation progeny is comparable
to the control group. With each succeeding generation of progeny there is
a decrease in the breast tumor incidence. If the progeny of tested non-
breast cancerous fostered females develop mammary cancer, the tendency
is not transmitted. An increase in the breast tumor percentage may be
obtained by giving the progeny to females of high tumor strains during
the nursing period (13).

No significant increase in the breast tumor incidence may be obtained
by fostering the young of resistant strain females to high cancer mothers
(6, 2, 17). Sub-line differences may account for the variations which
have been noticed (3, i, 17).

The breast tumor incidence in virgin females of high tumor strains
depends on the stock. Some stocks have a high virgin incidence (25) while
others are very low (11). The foster nursing of young from all types of
high tumor strains, tested thus far, resulted in a reduced tumor percentage
for females which were used as breeders (3, 18, 2).

If mice of low breast tumor strains are crossed to representatives of
high breast tumor stocks, first generation females, used as breeders, which
were nursed by females from the high breast tumor line showed a high
incidence regardless of the maternal parent. Low ratios were observed in
hybrids which had low tumor strain maternal parent and were not fostered,
or high tumor maternal parent and were nursed by low tumor strain females.
The evidence secured in the reciprocal first and second generation mice is in
accord with the theory that breast cancer susceptibility is transmitted as a
dominant (8, 17). Foster nursing has no apparent effect on lung cancer
development (16).

THE MILK INFLUENCE IN TUMOR FORMATION 323

Additional work with first generation females has demonstrated that the
influence usually obtained in the milk may be transferred to some individuals
by the inoculation of normal tissue from young potentially cancerous mice
(15). This influence may be transferred through the milk to the second
generation mice, as expressed by their increased tumor incidence. As
stated, females of a resistant strain do not show a high breast tumor ratio
if they are nursed by high tumor strain females. Such females, however,
receive the "milk influence" which they in turn may pass on by nursing
with the subsequent development of breast cancer in animals having the
breast cancer constitution.

The nature of the breast cancer producing influence has not been deter-
mined. That it occurs in many of the internal organs of high breast cancer
strain animals has been demonstrated.

BIBLIOGRAPHY

1. AxDERVONT, H. B., AND W. J. McEleney. IQ37. Incidence of spontaneous
tumors in a colony of strain C3H mice. Pub. Health Rep., U.S.P.H.S. 52 : 772-780.

2. Andervont, H. B., and W. J. McEleney. 1938. The influence of nonbreeding
and foster nursing upon the occurrence of spontaneous breast tumors in strain C3H
mice. Pub. Health Rep., U.S.P.H.S. 53: 777-783.

3. BiTTNER, J. J. 1930. The experimental determination of an invisible mutation.
Mich. Acad. Arts and Letters 11: 349-351.

4. BiTTNER, J. J. 1936. Some possible effects of nursing on the mammary gland
tumor incidence in mice — preliminary report. Science 84: 162.

5. BiTTNER, J. J. 1936. The spontaneous tumor incidence in mice. I. "Z" stock
X "I" stock. J. Hered. 27: 391-393.

6. BiTTNER, J. J. 1937. Mammary tumors in mice in relation to nursing. Am. J.
Cancer 30: 530-538.

7. BiTTNER, J. J. 1937. Some possible effects of nursing on the mammary gland
tumor incidence in mice. Am. J. Clin. Path. 7: 430-435.

8. BiTTNER, J. J. 1937. Relation of nursing to the theory of extra-chromosomal
causation of breast cancer in mice — preliminary report. J. Hered. 28: 363-365.

9. BiTTNER, J. J. 1938. The genetics of cancer in mice. Quart. Rev. Biol. 13:
51-64.

10. BiTTNER, J. J. 1939. Relation of nursing to the extra-chromosomal theory of
breast cancer in mice. Am. J. Cancer 35: 90-97.

11. BiTTNER, J. J. 1939. Breast and lung carcinoma in "A" stock mice. Pub.
Health Rep., U.S.P.H.S. 54: 380-392.

12. BiTTNER, J. J. 1939. Breast cancer in breeding and virgin "A" and "B " stock
females and their hybrids. Pub. Health Rep., U.S.P.H.S. 54: 1113-1118.

13. BiTTNER, J. J. 1939. Breast cancer and the pedigree relationship of fostered
"A" stock mice. Pub. Health Rep., U.S.P.H.S. 54: 1642-1650.

14. BiTTNER, J. J. 1939. "Influences" of breast cancer development in mice. Pub.
Health Rep. 54: 1 590-1 597.

324 BIOLOGY OF THE LABORATORY MOUSE

15. BiTTNER, J. J. 1939. The influence of transplanted normal tissue on breast
cancer ratios in mice. Pub. Health Rep. 54: 1827-1831.

16. BiTTNER, J. J. 1940. Foster nursing and lung cancer in "A" stock mice. Am.
J. Cancer 38: 95-102.

17. BiTTNER, J. J. 1940. The possible method of transmission of breast cancer
susceptibility in mice. Am. J. Cancer 39: 104-113.

18. BiTTNER, J. J., .\ND C. C. LiTTLE. 1937. The transmission of breast and lung
cancer in mice. J. Hered. 28: 117-121.

19. Cloudm.an, a. M., .4ND C. C. Little. 1936. The genetics of tumor formation in
mice in relation to the gene T for brachyury. J. Genet. ;i2: 487-504.

20. KoRTEWEG, R. 1934. Proefondervindelijke onderzoekingen aangaande erfij-
kheid van kanker. Ned. Tijdschr. Geneesk. 78: 240-245.

21. KoRTEWEG, R. 1936. On the manner in which the disposition to carcinoma of
the mammary gland is inherited in mice. Genetica 18: 350-371.

22. KoRTEWEG, R. 1936. De erfelijke factoren welke de dispositie voor kanker van
borstklier bij de muis befalen. Xed. Tijdschr. Geneesk. 80: 4008-4014.

23. KoRTEWEG, R. 1937. Les facteurs hereditaires determinant la predisposition au
cancer de la mamelle chez la souris. Acta Union Internationale contre le Cancer
2: 136-143.

24. MURR.A.Y, W. S. 1934. The breeding behavior of the dilute brown stock of mice
(Little dba). Am. J. Cancer 20: 573-593.

25. Murray, W. S., and C. C. Little. 1935. The genetics of mammary tumor
incidence in mice. Genetics 20: 466-496.

26. Murray, W. S., AND C. C. Little. 1935. Further data on the existence of extra-
chromosomal influence on the incidence of mammary tumors in mice. Science 82:
228-230.

27. Murray, W. S., and C. C. Little. 1936. Extrachromosomal influence in rela-
tion to the incidence of mammary and non-mammary tumors in mice. Am. J.
Cancer 27: 516-518.

28. Staff, Jackson Memorial Laboratory. 1933. The existence of non-chromoso-
mal influence in the incidence of mammary tumors in mice. Science 78: 465-466.

29. Staff, Jackson Memorial L.ABORATORY. 1936. The constitutional factor in the
incidence of mammary tumors. Am. J. Cancer 27: 551-555.

30. Strong, L. C. 1935. The genetic appearance of spontaneous carcinoma of the
mammary gland in the C3H mice. Am. J. Cancer 25: 599-606.

Cnapter 10

INBRED AND HYBRID ANIMALS AND THEIR
VALUE IN RESEARCH

By W. Lawson Russell, Roscoe B. Jackson Memorial Laboratory.

Introduction, 325. Genetic effects of inbreeding, 327. Random mating, 327,
Inbreeding, 327. Phenotypic effects of inbreeding, 330. General causes of pheno-
typic variation, 331. Measurement of phenotypic variation, 333. Effect of inbreed-
ing on the "average," t,s3- Effect of inbreeding on the variation, 334. Causes of
a change in variation, 334. Decreased variation following inbreeding, 335. Increased
variation following inbreeding, 336. Different effects in the two sexes, 336. The
value of inbred lines in research, 337. The value of the genetic effects of inbreeding,

337. Discovering major gene differences in cases obscured by variation in modifiers
or environment, 337. Estimating the relative importance of heredity and environ-
ment, 337. Estimating the relative importance of various environmental factors, 338.
Recognizing new mutations, 338. The value of the phenotypic effects of inbreeding,

338. Change in "average," 338. Reduced variation, 338. Increased variation, 338.
Uniformity in time, 339. Combination of effects, 339. Differences between inbred
lines, 339. Hybrids, 340. Genetic characteristics, 340. Phenotypic characteristics,
340. Average, 340. Variation, 341. Differences between reciprocal hybrids, 341.
The value of hybrids in research, 341. Compared with inbred hnes, 341. Hybrid
vigor, 342. The great variety available, 342. Special uses, 342. The building and
maintenance of inbred lines, 343, Selection, 343. Preserving vigor, 343. Tests of
genetic uniformity, 343. Sublines, 344. Risk of contamination, 344. Effect of relax-
ing inbreeding, 345. Fallacies, 345. Misinterpretation of variation within strains,
345. Misinterpretation of differences between strains, 346. Bibliography, 347.

Introduction

During the past few years there has been a tremendous increase in the
number of inbred animals, particularly mice, used in research. A large part
of this increase can be attributed to the efforts of Dr. C. C. Little, who has
not only repeatedly advocated the use of inbred material, e.g. (7), but has,
with the aid of students and colleagues, established many inbred strains
of mice and made them available in large quantity to other research workers.
Thus, the number of mice, mostly from inbred lines, supplied by the Jack-
son Laboratory to other laboratories has increased from 12,000 in 1933 to
120,000 in 1939.

325

326 BIOLOGY OF THE LABORATORY MOUSE

Nevertheless, any geneticist who samples the recent literature in such
fields as physiology, biochemistry, bacteriology, pathology, cancer research,
and experimental medicine in general, is struck by three points. First,
most of the workers who are still using animals of uncertain origin could
profit by the use of inbred stocks. Second, even when inbred animals are
used, they are frequently not utilized to their full value. Third, owing to a
lack of understanding of the consequences of inbreeding, erroneous conclu-
sions are sometimes drawn from the results obtained with inbred material.

As a geneticist, the author of this chapter may perhaps be permitted to
blame geneticists for the above failings. They have provided an excellent
theoretical analysis of the Mendelian consequences of inbreeding, and an
extensive series of critical experiments that have verified theory and brought
new facts to light ; but they have expended singularly little effort to sort out
and explain those results and conclusions which are of importance to
research workers in general. General discussions of inbreeding have been
concerned, on the one hand, with the genetic consequences and, on the other,
with the relation of these to evolutionary theory, improvement of livestock
and domesticated plants, and interpretation of such special phenotypic
effects as decline in vigor. Furthermore, of the six recent and better known
text-books of genetics only two mention the value of inbred animals in
research, and each of these devotes only one paragraph to this topic.

This chapter was planned to bring together and classify those effects
of inbreeding which are of general value to experimentalists who are using
mice or other laboratory mammals in their research. Much of what is
discussed applies, of course, to other organisms as well.

For this purpose the most serious gap in the literature is the lack of an
adequate treatment of the phenotypic effects resulting from inbreeding.
For example, general discussions of inbreeding have implied, if not definitely
stated, that the decrease in genetic variation following inbreeding neces-
sarily results in decreased phenotypic variation. Yet several cases have
been reported in which a particular character shows more variation in a
certain inbred line than it does in random bred stocks, or did in the stock
from which the inbred line was derived. It has been this author's experience
that this effect is a seemingly inexplicable paradox to many students and
research workers. It has, therefore, seemed desirable to discuss the pheno-
typic effects of inbreeding in more detail than the title of this book would,
at first sight, warrant.

The attempt has been made to present the material of this chapter in a
form that can be understood by those not specially trained in genetics.

INBRED AND HYBRID ANIMALS AND THEIR VALUE 327

The few technical terms and simple genetic concepts not explained can be
understood by reference to a text-book on the subject.

Genetic Effects of Inbreeding

Following Mendel's work, studies on the mechanism of heredity were
naturally focussed on mutations that produced easily recognizable effects.
This emphasis on major mutations invited the conclusion that nearly all
individuals in any one species have the same genotype (set of genes), the
remaining individuals exhibiting mutations. Such is not the case. Genetic
studies have shown that in, for example, any wild population of rodents, or
any laboratory population not closely selected or inbred, there is tremendous
genetic variation; although the population may show none of the major
mutations recorded by the geneticist. The changes effected by selection of
small phenotypic variations may be cited as one demonstration of this fact.

Before examining the effect of inbreeding on this genetic variation it is
necessary to consider how genetic variation is affected by the absence of
inbreeding, namely random mating.

Random Mating

Taking the extreme case of an indefinitely large random breeding popula-
tion, undisturbed by such factors as mutation, it has been shown theoret-
ically that, whatever the original proportions of any two alleles {A, a) may
be, the proportions of the heterozygous {Ad) and the two homozygous {A A
and ad) classes of zygotes reach an equilibrium in not more than two
generations. Further, the relative frequencies of all possible genotypes
{AABbcc . . . , AabbCc . . . , etc.) tend to approach an equilibrium in
which the different series of genes are combined at random. Linkage has
no effect on the ultimate equilibrium. With reversible or irreversible muta-
tions occurring at constant rates there will be an approach to a new
equilibrium.

In practice, the above conditions are not found. Such factors as selec-
tion and limited size of population will change the relative frequencies of the
various genotypes from generation to generation. Provided none of these
factors is intensive, however, considerable genetic variation will remain.
We can now consider what effect more or less intensive degrees of inbreed-
ing will have on that variation.

Inbreeding

The primary effect of all systems of inbreeding is an increase in the
proportion of homozygous gene pairs present in the population. With some

328 BIOLOGY OF THE LABORATORY MOUSE

systems, for example brother-sister mating, the population necessarily
breaks up into non-interbreeding lines in each of which there is a limited
number of parents in each generation. Under such systems an increasing
number of genes will become fixed in any one line. Thus, if genes A and a
are both present in the original population, some lines will become fixed
so that all individuals in that line are A A, other lines will become fixed
for aa, while others may, in a limited period, not yet have become fixed for
that particular gene pair.

This effect of inbreeding is easy to understand for a system as close
as brother-sister mating, where, in any one line, there are only two parents
for each generation. Merely by chance, matings will occur in which both
parents are homozygous for the same gene. Once this has happened all
their descendants will be homozygous for that gene so long as they are bred
only with each other and no mutation occurs.

The change in proportion of homozygosis with continued self-fertiliza-
tion was given by Jennings (6). The effects of continued brother-sister
mating were investigated by Pearl, Fish, Jennings, and Robbins, and are
reviewed by Wright (ii). The rate of increase in the proportion of homo-
zygosis, and the limit reached, under systems of less intense inbreeding are
by no means easy to see. A general method for determining them has been
devised by Wright (ii, 17) using his ingenious method of path coefficients.
For our purposes it will be sufficient to cite only a few of the results (Fig.

131)-

Figure 131 shows that with brother-sister mating (two parents in each
generation) the rate of loss of heterozygosis is much more rapid than with
double-first-cousin mating (four parents in each generation), although it
is considerably slower than that which can be obtained when self-fertiliza-
tion (one parent in each generation) is possible. The inbred strains of
laboratory mammals have been produced almost exclusively by brother-
sister mating. With this system, each generation theoretically loses
approximately 19% of its heterozygosis in the succeeding generation
(except that the fluctuation is wide of this mark in the first three genera-
tions). The actual proportions of heterozygosis in succeeding generations,
giving the curve in Fig. 131, are: (i, }4), %, %, ^{q, %2, etc. The propor-
tions can be written for any number of generations simply by following the
rule that each numerator is the sum of the two preceding, while the denomi-
nators double in each successive generation.

Mating offspring with younger parent, generation after generation, gives
the same result as brother-sister mating, with the exception that the average

INBRED AND HYBRID ANIMALS AND THEIR VALUE 329

rate of loss of heterozygosis in sex-linked genes is 29% (50% every two
generations) instead of 19%. This system is, then, slightly superior to
brother-sister mating. The more frequent use of the latter has probably
been dictated by its practical convenience.

It is sometimes required to estimate the percentage of homozygosis in
a stock that is inbred, but which has not consistently followed any one

100

(fi

ll.

LlI

fi.')

liJ

/^
S

\.^

Fig.

10 12 14 16 18 20 22 24 26 28 30

GENERATION

131. — The percentage of homozygosis in successive generations under three
different systems of inbreeding.

system of inbreeding. If pedigree records have been kept this can be done
by the use of coefficients of inbreeding (12, 15, 23).

With brother-sister mating, and any system that results in separate
lines of descent, a limit is reached, according to the above calculations, only
when complete homozygosis has been attained, that is when all individuals
in any one line are genetically identical.

Haldane (5) has discussed various factors that may affect the conclusions
reached above. He has shown that linkage may affect the distribution
of the heterozygosis left after inbreeding. Thus, the number of organisms
in which all the original heterozygosis has been lost may, as a result of

330 BIOLOGY OF THE LABORATORY MOUSE

linkage, be considerably higher than would be expected purel\' from the
original number of heterozygous gene pairs. On the other hand, the remain-
ing organisms will carry more heterozygosis than expected. In a similar
treatment Bartlett and Haldane (i) have discussed the effects of forced
heterozygosis. For brother-sister mating of yellow mice, for example, they
have estimated the probability of finding heterozygosis due to linkage with
the yellow locus.

A factor that may affect the rate of increase of homozygosis is described
by Haldane (5) as follows: "A breeder will probably select the most vigorous
individuals as parents. He will eliminate a number of weak or infertile
recessives, which will be homozygous for particular genes, and probably so
for genes closely linked with them. He may also select for vigor due to
heterozygosis as such. Hence at least during the first five to ten genera-
tions, when the population is still appreciably heterogeneous, progress
towards homozygosis will be slightly slower than the above calculations
would suggest."

Haldane gives formulae by which, under various systems of inbreeding,
the frequency of heterozygosis, at any locus, due to mutation after inbreed-
ing has begun can be estimated from the mutation rate. Unfortunately,
little is known about mutation rate in mammals. Haldane (4) estimates
that the gene for haemophilia arises by mutation in the population of
London about once in 50,000 life cycles. He concludes (5): "If this is
generally true for mammals, and the number of genes is not less than in
Drosophila, we may expect that as the result of mutation most members of
a mammalian pure line will be heterozygous for at least one gene as the result
of mutation. Since after 30-40 generations the majority of animals in such
a line have lost all their original heterozygosis, the line is then as pure as it
is every likely to be."

The last sentence sums up the practical conclusions to be drawn from
this section. A later section will show that the genetic uniformity of a
given strain in regard to a given character can usually be tested statistically.

Phenotypic Effects of Inbreeding

So far we have considered only the genetic effects of inbreeding. The
experimentaHst does not work on genotypes, however, he is concerned with
characters or phenotypes. It is important, therefore, to consider how the
genetic consequences of inbreeding may, in turn, influence phenotypic or
character variation. As an introduction to this, it seems desirable to
digress briefly on the general causes of phenotypic variation.

INBRED AND HYBRID ANIMALS AND THEIR VALUE 331

General Causes of Phenotypic Variation

The contribution that genetics has made to an understanding of these
causes has been brought out clearly by Wright (18). With the aid of
diagrams he has emphasized the fact that, however complex the network
of processes involved in the development of a character may be, all processes
trace back to a gene action somewhere or an external stimulus somewhere.

Wright's general treatment, particularly when applied to mammals, can
be extended by distinguishing two main paths by which genes can influence
a character. The more direct path is that tracing back from the character
to the genes in the individual bearing the character. A character, particu-
larly in mammals, may also be affected by the maternal environment, which,

CHARACTER

Fig. 132.

in turn, is determined partly by the genetic constitution of the mother.
Therefore, a second path traces back through the maternal environment to
the genes of the mother. The maternal environment may also be afi'ected
by the grand-maternal environment, which, in turn, is determined partly
by the genes of the grandmother. The final result of this analysis is an
indefinite number of paths, which presumably have less importance the
further back they go (Fig. 132).

The genes of the mother, grandmother, etc., are not the only genes
external to the individual that can influence a character. Nor is the
maternal environment the only medium through which such genes can act.
For example, the number of young raised to weaning is determined partly
by the genetic constitution of the young, and it has been known to aft'ect
not only such characters as gain in weight after birth, but also characters
influenced by the temperature of the nest. Variation in tangible environ-
mental factors of this nature can, of course, often be eliminated from an
experiment. To make our classification complete, however, the term
''biological environment" will be used to group maternal environment with
all other environmental factors affected by the genetic constitution of the

332

BIOLOGY OF THE LABORATORY MOUSE

stock. There are, then, two mam paths by which a character may be
influenced by genes: the direct path within the individual, and the path
through the ''biological environment" from the genetic constitution of the
stock (including, conceivably, the genes of the individual), (Fig. 133).

GENETIC

CONSTITUTION

OF STOCK

BIOLOGICAL
ENVIRONMENT

CHARACTER

GENETIC
CONSTITUTION
OF INDIVIDUAL

Fig. 133-

To the genetic factors we can now add the remaining cause determining
the phenotype, namely the "physical environment," using this term to
denote environmental influences which are not aft'ected by the genetic con-
stitution of the stock (Fig. 134).

PHYSICAL
ENVIRONMENT

GENETIC

CONSTITUTION

OF STOCK

BIOLOGICAL
ENVIRONMENT

GENETIC
CONSTITUTION
OF INDIVIDUAL

Fig. 134.

The distinction between two major paths of gene action is, perhaps, not
essential for an understanding of the rest of this chapter, but it is hoped
that it will be of use in emphasizing two points: first, that a character
may be influenced by genes other than those of the individual bearing the
character; and second, a corollary of the first, that some so-called "environ-
mental" factors may be under genetic control. Since the genetic effects of
inbreeding can act on the phenotype through the "biological environment,"
as wefl as through the individual, their potential results are greater than is
often supposed.

INBRED AND HYBRID ANIMALS AND THEIR VALUE 333

Measurement of Phenotypic Variation

In order to discuss the phenotypic effects of inbreeding it is necessary
to express phenotypic variation in terms that the mind can grasp. The two
most useful measures of a distribution of biological data are its location
and scatter. Thus, the two most significant questions that can be asked
about a set of measurements on, for example, tail length in a population of
mice are: (7) Where is the distribution of lengths located? or: What is the
average length? and (2) How much spread is there about this average? It
will be convenient to refer to these two measures in a general sense as
"average" and "variation," remembering that each can be expressed in
definite terms by several statistics, of which the mean and standard devia-
tion are respectively the most valuable.

We can now separate the phenotypic effects of inbreeding into effects on
the average and eft'ects on the variation. The discussion of some of these
will be simplified if we assume that the genetic effects have reached the
limiting condition in which there is no genetic variation left, all the indi-
viduals in the inbred line having one and the same genotype. Such a limit
has actually been reached, at least for all genes with measurable effects on
certain characters in certain inbred lines. If, through mutation or insufii-
cient inbreeding, a line is not genetically pure for a certain character, the
effects discussed below may still occur, although perhaps not to the full
extent possible.

Effect of Inbreeding on the "Average"

The average value of a character will be prescribed by the genotype
fixed. It seems extremely likely that this genotype will determine an
average that differs from that in the foundation stock from which the
inbred line was derived. There will be no change if: (i) the character
is not affected by the extent of genetic variation possible with the original
genes, or (2) the effect of the genotype fixed happens to correspond to the
average effect of the many genotypes present in the foundation stock.
Neither condition can be expected to occur very often. Observation agrees
with expectation: a change in average usually occurs with inbreeding and is,
as we shall see later, one of the most valuable results for the research worker.

With many characters the change in average may go in either direction.
For example, average amount of white spotting in a piebald stock may either
increase or decrease with inbreeding. However, with some characters the
change is in the same direction in all, or most, of several inbred lines studied.

334 BIOLOGY OF THE LABORATORY MOUSE

Thus, average vigor and average fertility usually decline with inbreeding
(13). The generally accepted explanation of this phenomenon is that genes
unfavorable to vigor, fertility, etc., are usually recessive; and, since inbreed-
ing fixes genes in homozygous condition, irrespective of whether they are
dominant or recessive, all the individuals in an inbred line are likely to have
a proportion of homozygous recessives higher than that in the average
individual in the foundation stock.

Effect of Inbreeding on the Variation

Causes of a change in variation. — It was stated in the introduction to
this chapter that inbreeding may lead either to a decrease or to an increase
in phenotypic variation. In order to explain both effects it is necessary to
distinguish as separate causes: (7) the reduction of genetic variation, and (2)
the characteristics of the resulting fixed genotype. Let us consider each of
these in turn.

1. It is apparent from Fig. 134 that reduction in genetic variation will, in
itself, tend to decrease variation in a character. In the limiting case, when
all genes affecting a character have become fixed, differences between
individuals will be determined solely by differences in the ''physical
environment."

2. The characteristics of the genotype fixed may, however, tend either
to decrease, or to increase, phenotypic variation, according as the develop-
mental processes determined by this genotype are less, or more, susceptible
to variation in the "physical environment" than are the developmental
processes of the bulk of the individuals in the foundation stock.

The total effect of inbreeding on phenotypic variation will be due to a
combination of (7) and (2). If the tendency of (2) is either to decrease
variation in a character, or not to increase it as much as it is decreased by
(7), then the character will be less variable in the inbred line. On the other
hand, if (2) increases the variation more than (7) tends to reduce it, then the
inbred line will show more variation than the foundation stock.

Here, then, we have an explanation of the paradox, mentioned in the
introduction to this chapter, that, although inbreeding causes a reduction in
genetic variation, it sometimes results in increased variation in a char-
acter. The result is due to the nature of the genotype fixed.*

* From a statistical point of view the paradox can be explained by the type of scale
on which the variation is measured. The increase in variation can occur only when
the scale is such that the magnitude of the environmental effects differs at different
points on the scale. An opportunity is then provided for inbreeding to shift the stock

INBRED AND HYBRID ANIMALS AND THEIR VALUE 335

We can now turn to examples.

Decreased variation following inbreeding. — ^This is the more commonly
observed result. At the moment we need mention only a few examples,
such as reduction of variation in: intensity of coat color, amount of white
spotting, tissue speciticity, and reaction to bacterial inoculation. The
degree of reduction differs widely and is dependent on the relative impor-
tance of heredity and environment in determining the variation in the
foundation stock. Thus, variation in tissue speciticity seems to be deter-
mined mainly by genetic factors, for it is greatly reduced by inbreeding.
In fact, if this character is measured by percentage of "takes" in transplants
between individuals, there is commonly no variation left at all in an inbred
line, all the transplants being successful. On the other hand, variation in a
character like white spotting may be determined largely by the environ-
ment and, therefore, not greatly reduced by inbreeding. Wright and Chase
(22) measuring white spotting in the guinea pig on an appropriate scale

to a point at which it is more sensitive to the environment than are the bulk of the
individuals in the foundation stock. On a scale on which environmental effects are
equal at all points, variation cannot increase as a result of inbreeding. A natural
scale of this type, with its simple logical relation to the causes of variation, is to be
preferred; and when a character does not fall easily into one it is sometimes possible
to devise such a scale and transform the data to it (16). If all characters could be
expressed in these terms the question of increased variation following inbreeding
would not arise. For many characters, however, no such scale has been found, and
in some of these there is reason to e.xpect that it would be too complicated for practical
purposes. In these cases we can only use the descriptive scales available. It must
be remembered that a measure of the variation on these scales, though it may be of
descriptive value, does not have the analytical value of statistics derived from data
recorded on natural scales.

One of these scales is necessarily quite common in biology because of the frequent
occurrence of physiological thresholds in development. On one side of the threshold
the character is recorded as "normal," with no variation, while on the other side the
character falls into a graded series of ''abnormalities." Most of the examples of
increased variation following inbreeding given later probably involve physiological
thresholds, the random bred stock falling mostly, or entirely, on the "normal" side
of the threshold, and the inbred strain falling largely, or completely, on the " abnormal "
side.

When a character is recorded only in two categories (e.g., 3-toed and 4-toed,
tumorous and non-tumorous, infected and not infected) an inbred strain is to be
regarded as more variable than its foundation stock if it falls closer to a 50:50 dis-
tribution in the two categories. Here, however, no particular value is obtained by
speaking of the "variation," for the distribution of data as recorded can be described
completely simply by stating the percentage in either category.

336 BIOLOGY OF THE LABORATORY MOUSE

found that the standard deviation of a random bred stock was decreased
only 23% by inbreeding.

Increased variation following inbreeding. — The most careful studies of
this, as of many other effects of inbreeding, have been made by Wright.
To give one example, Wright (19) obtained several inbred strains of guinea
pigs that showed more variation than the random bred control stock in
respect to number of digits. In one strain, 69% of the animals showed
various grades of development of an extra toe on the hind foot ; whereas the
random bred control stock showed less than 1% with any development of
an extra toe. Wright showed that, within each strain, inbreeding had
eliminated genetic variation influencing this character. The increased
phenotypic variation could be attributed only to fixation of genotypes that
resulted in strains more susceptible to environmental variation affecting
development of toes.

The Jackson Laboratory C57 black strain of mice shows more variation
in development of eyes than that recorded for random bred stocks. In
some sublines more than 20% of the females exhibit eye abnormalities rang-
ing from slight cataract to an eyeless condition, although it has been found
(unpublished data of the author) that within sublines there is no genetic
variation affecting this character.

Green (3) has shown that the Bagg albino strain of mice exhibits a
variation in number of presacral vertebrae that is probably greater than that
of the original stock prior to inbreeding.

In several strains of mice variation in development of tumors is greater
than that characteristic for random bred stocks.

Other cases can be found in the literature. Many more have
undoubtedly occurred. The fact that they have not been reported may
be due to two causes. First, workers have not been on the look-out for
this effect, because most geneticists have stressed the decrease in genetic
variation, but have not pointed out how increased phenotypic variation
might occur. Second, early work by geneticists was naturally focussed
on such characters as intensity of coat color and tissue specificity, that is
characters which are not much aft'ected by environmental variation, and
which are, therefore, likely to exhibit decreased variation in inbred strains.

Different Effects in the Two Sexes

It should be remembered that even when sex-linked genes have become
fixed in an inbred line the sexes will still differ genetically in their sex

INBRED AND HYBRID ANIMALS AND THEIR VALUE 337

chromosome balance. Therefore, the average and variation of a char-
acter may, and usually do, differ in the two sexes.

The Value or Inbred Lines in Research

The variety of ways in which inbred lines have already been used in
research is extensive enough to warrant an attempt at classification. In
the space available here the classification can be illustrated only by a few
examples. The two main headings (value of genetic effects and value of
phenotypic eft'ects) given below are based on a division of experiments into
those in which the emphasis is on the genetic constitution of the stock and
those which are concerned with the phenotypic nature of the stock irrespec-
tive of its genetic explanation. To date, most of the experiments in the
former group have been made by geneticists.

The Value of the Genetic Effects of Inbreeding

Discovering major gene differences in cases obscured by variation in
modifiers or environment. — The value of inbreeding, here, lies in the pos-
sibility it aftords of obtaining at least one of a pair of alleles in homozygous
condition along with a uniform set of modifiers. Wright's analysis, reviewed
m 1936 (22), of white spotting in the guinea pig provides an excellent
example. By crossing self animals with inbred spotted strains and repeat-
edly backcrossing to the spotted, he was able to show that this character
is determined by a major pair of alleles even though it is greatly affected
by modifiers and environment.

Estimating the relative importance of heredity and environment. — The
importance of environment can be judged by the amount of phenotypic
variation remaining in an isogenic inbred line. The importance of heredity
in the foundation stock can be estimated from the amount by which varia-
tion is reduced by inbreeding. This has already been discussed on p. 335,
where it was pointed out that, by this criterion, tissue specificity is deter-
mined largely, if not entirely, by genetic factors; while variation in amount
of white spotting in piebald guinea pigs is determined largely by environ-
mental factors.

Unless a suitable transformation of scale can be made (see footnote,
p. 334) the method is not applicable for an estimate of the importance of
heredity when inbreeding results in increased variation. Its use in any
case should be guarded by a consideration of the type of scale upon which
the character is measured. See Wright and Chase (22) for an example of
correct usage.

338 BIOLOGY OF THE LABORATORY MOUSE

Estimating the relative importance of various environmental factors. —

To gauge the importance of different environmental factors it is naturally
desirable to have genetic variation eliminated, or controlled as much as
possible. This can be achieved by using inbred strains. Some of the char-
acters studied in this way are: Polydactyly (19), and white spotting (22),
in the guinea pig; harelip (9), and skeletal variation (3), in the mouse.
Recognizing new mutations. — In mammals particularly, geneticists are
anxious to find new major mutations and observe the frequency with which
mutations occur. The appearance of a mutant animal is usually recog-
nized more easily in an inbred strain than in a stock in which there is con-
siderable genetic variation.

The Value of the Phenotypic Effects of Inbreeding

Change in average. This is one of the most used results of inbreeding.
An inbred line frequently provides in quantity a type of animal that is rare,
or perhaps never observed, in random bred stocks. Thus, inbred lines of
mice are available in which the incidence of certain types of tumors is very
high. To mention only two uses, these lines are of value both to experimen-
talists who want spontaneous tumor tissue in quantity and to those who
want animals known to be susceptible to tumor growths. Other examples
of valuable strains are: those with low resistance to carcinogens and those
with high susceptibility to bacterial infection. Thus, the susceptibility of
C57 black mice proved of use in testing the protective value of typhoid
vaccine (10).

Reduced variation. — Although the preceding effect probably has been
used as much as, if not more than, the reduced variation following inbreed-
ing, the reduced variation has been by far the most publicized effect. Its
value is obvious when, as frequently occurs, inbred lines are superior to
random bred animals in their uniformity of response to such experimental
treatments as: hormone injections, feeding deficient diets, administering
drugs, excision of organs, exposure to carcinogens, and immunity tests.
Results are more clear-cut, and a significant difference between experi-
mental and control can be demonstrated with fewer animals.

We have already mentioned the uniformity of tissue specificity found in
inbred lines. This has been of great value in work on transplanted tumors,
transplanted normal tissues, and parabionts.

Increased variation. — Increased variation in Polydactyly in the guinea
pig, and in eye defects and skeletal characters in the mouse, facilitated

INBRED AND HYBRID ANIMALS AND THEIR VALUE 339

studies of the factors affecting these characters; for the amount of variation
in random bred stocks was too small for practical analysis.

This effect of inbreeding will undoubtedly be used more frequently when
it becomes widely known. Thus, embryologists derive much of their
information about normal processes of development from a study of abnor-
mahties, both naturally occurring and experimentally induced. They are
already using mutant types and will doubtless appreciate the value of an
inbred strain that provides abnormalities covering a wide range.

Uniformity in time. — The genetic make-up of a random bred stock of
limited size will drift considerably from generation to generation. There-
fore, the characteristics of the stock may differ markedly at different times
and thereby cause trouble in a long-time investigation. The genetic con-
stitution of an isogenic inbred line can change only by mutation. The
phenotypic nature of the line is, therefore, less likely to vary with time.
This applies even when the phenotypic variation is greater in the inbred
strain.

Combination of effects. — ]\Iore than one of the above effects can, of
course, often be used in a single investigation. Thus, a strain combining
high susceptibility to a bacterial infection with low variation in response
to inoculation may be used in a successfully uniform series of tests over a
long period.

Differences between inbred lines. — It is to be expected that almost any
character studied will be shown to differ in different lines. Differences
have already been observed in a great many characters. They cover the
range from gene to behavior pattern, including countless biochemical,
cytological, histological and gross anatomical characters, and numerous
immunological, physiological and embr^'ological processes. We may
mention reported differences in calcium content of bones, chiasma frequency
in spermatocytes, histology of the adrenal, shape of the xiphoid process, sus-
ceptibility to yellow fever, ox}'gen consumption of excised tissues, develop-
ment of the mammary gland, and behavior response to a foreign male, as a
mere suggestion of the diversity.

The value of inbred lines exhibiting these differences has been shown
in many ways. Often they have been used to demonstrate the importance
of hereditary factors, or, by crossing strains, to analyse the genetic differ-
ences. ]More frequently they have been employed in studying intermediate
causes of a difference, or in searching for associated phenomena to which
the difference might be attributed. Thus, Fekete (2) has investigated the
histology of the mammary glands in "high" and ''low" tumor strains of

340 BIOLOGY OF THE LABORATORY MOUSE

mice as a possible clue to the nature of the factors causing mammary
cancer.

Hybrids

The only type of hybrid that will be discussed here is the one that is of
particular value in research, namely the first generation hybrid (Fi) obtained
by crossing two inbred strains.

Genetic Characteristics

Apart from the segregation of the X and Y chromosomes, all the
germ cells of an isogenic inbred line are genetically identical. It follows
that all the offspring obtained by crossing females of one isogenic strain
with males of another will have the same genotype. Thus, if one strain is
AABBccdd . . . and the other aabbccDD . . . , all the Fi hybrids will be
AaBhccDd . . . Offspring of the reciprocal cross will have the same
genotype again in the homogametic sex. Individuals of the heterogametic
sex (male, in mammals) will have the same genotype for their autosomes,
but a different set of sex-linked genes, their X and Y chromosomes being
derived from the opposite parents.

There is one important respect, however, in which hybrids differ from
their parental strains: they will not breed true. Though genetically uni-
form, they are heterozygous for all genes which differ in the two parents.
The F2 will have, therefore, as a result of segregation in the germ cells
of the Fi, the maximum genetic variation possible with the genes provided
by the parental strains. Backcrosses of the Fi to either parental strain,
and outcrosses of the Fi to any other strain, will also give considerable
genetic variation in the offspring.

Phenotypic Characteristics

Average. — The average of a character in the Fi may fall between the
parental averages, it may correspond to either parent, or it may lie beyond
the parental values.

Intermediate averages were obtained by Wright (20) in some of his
crosses of normal and polydactylous strains of guinea pigs. E. L. Green
and the author got similar results in two hybrids from strains of mice differ-
ing in number of presacral vertebrae.

The hybrid will, of course, correspond to one of its parents when the
character difference is determined by dominant genes all carried by one
parent. Thus, the Fi of a cross between agouti and black strains will be

INBRED AND HYBRID ANIMALS AND THEIR VALUE 341

agouti. There are, however, other cases which do not depend on this simple
cause. Wright's thorough analysis of Polydactyly in the guinea pig (20)
again provides a good example. A cross between a three-toed and a four-
toed strain gave all three-toed, yet Wright was able to show that at least
four, probably more, genes were involved and that there was no evidence
of dominance.

In almost any character connected with vigor or fertility the average in the
hybrid commonly exceeds both parents (14), hence the term "hybrid vigor."

Variation. — It is. frequently assumed that, because of their comparable
genetic uniformity, the parental strains and the Fi will have the same
degree of phenotypic variation. This is a common result, but it is not the
only one possible. We have already shown that the extent of the variation
in a character in an inbred line is determined by the nature of the genotype
fixed, as well as by the absence of genetic variation, for the effect of environ-
mental variation may differ with different genotypes. Similarly, pheno-
typic variation in a hybrid is dependent on the genotype of the hybrid
and may be less or greater than that in the parental strains.

Differences between reciprocal hybrids. — The following three factors
may cause a phenotypic difference between reciprocal hybrids. The dif-
ference may be one of average, variation or both.

1. The opposite origin of the X and Y chromosomes in the two hybrids
may result in a phenotypic difference in the heterogametic sex.

2. The cytoplasm contributed by the mother may differ in reciprocal
crosses as a result of gene action in the female germ cells prior to fertilization.

J. The "biological environments" in which the two hybrids develop
may differ. In mammals the most important influence of this type is
probably the maternal environment. A difference attributable to a
maternal effect may have had its origin before fertilization, between fer-
tilization and birth, or even postnatally.

Differences in reciprocal hybrids have been recorded, but the total
number is not large. The present author would predict that they will be
observed more commonly as geneticists turn their attention away from
characters that are not susceptible to environmental variation and hence
not subject to the effect of the third factor given above.

The Value of Hybrids in Research

Compared with Inbred Lines

Allowing for the distinction between reciprocal crosses, Fi hybrids and
inbred lines have the same degree of genetic uniformity and comparable

342 BIOLOGY OF THE LABORATORY MOUSE

phenotypic characteristics. Therefore, most of the uses which we have
Hsted for inbred strains apply equally well to hybrids. It must be remem-
bered, however, that hybrids will not breed true and that they can be
obtained only by maintaining two inbred stocks. In the following respects
they are sometimes of more value than inbred strains.

Hybrid Vigor

Some workers who are anxious to eliminate genetic variables from an
experiment have, nevertheless, rejected the use of inbred strains because
they commonly show decreased vigor. Since hybrids usually combine a
high degree of vigor with their genetic uniformity it is surprising that they
have not been used more. They are admirably suited for assay tests of
hormones and vitamins and for most experiments in which a healthy,
vigorous animal is required. The fact that they are commonly highly
resistant to disease should be of value to the bacteriologist. For research
which requires genetic uniformity, but not the special characteristics of a
certain inbred line, hybrids are to be preferred because their vigor makes
them more economical to raise.

The Great Variety Available

As the number of inbred strains being maintained increases, the number
of hybrids made potentially available increases much more rapidly. Thus,
25 pure strains can produce 300 hybrids (600, if reciprocals are listed
separately). There are probably many more than 25 inbred strains of mice
available. While many of these have been thoroughly investigated, only
a few of their hybrids have been produced and examined. Here, then, is a
wealth of genetically uniform material which is almost untapped.

Special Uses

We have already discussed the factors which may cause reciprocal
hybrids to differ. The obtaining of reciprocal hybrids is of value when
information on the importance of these factors is required. The staff of
the Jackson Laboratory (8) reported differences between reciprocal hybrids
in mammary tumor incidence in mice. This led Bittner to a discovery of
the important post-natal maternal influence on this character (Chap. 9).
In collaboration with E. L. Green the author is investigating skeletal dif-
ferences in reciprocal hybrids obtained in three different crosses between
inbred strains of mice. Here, again, it is hoped that the obtaining of a

INBRED AXD HYBRID ANIMALS AND THEIR VALUE 343

difference in reciprocal hybrids will lead to increased understanding of
variation in the character under investigation.

Another use of hybrids lies in their common ability to grow tumors of
both parental strains. This is of value in research and also provides an
economical method for maintaining transplantable tumors.

The Building and ^Maintenance of Inbred Lines

Some of the concepts of value to those workers who wish to start or
maintain their own inbred lines are apparent in earlier sections. Others
are presented below.

Selection

Inbreeding is often combined with selection for desired characteristics,
for example high or low tumor incidence. The eft'ectiveness of various
methods of selection on various types of characters is discussed by Wright
(21). It may be pointed out here that, when there is a lot of variation which
is not genetic, selection of individuals within a single inbred line (e.g..
among the offspring of a single brother-sister mating) is of little value.
Individuals which are good by accidents of environment, and not by
heredity, may be chosen and undesirable genes fixed. In these cases selec-
tion is most effective when applied to a number of separate inbred lines;
for only between them can real hereditary dift'erences be easily recognized.

Phenotypic variation may remain after a line has become isogenic, but
selection cannot change it. In a highly inbred strain selection is, there-
fore, of value only for its possible control in fixing desirable, or eliminating
undesirable, new mutations.

Preserving Vigor

Selection of lines, rather than individuals, applies particularh- to vigor
and fertility; for Wright (13) has shown that variation in these characters
is determined largely by environment. A satisfactory inbred strain can
usually be obtained only by starting a large number of strains. In fact,
if only one brother-sister line is started from a heterogeneous stock there
is a fair chance that it will die out in spite of selection of the two most
vigorous animals in each generation.

Tests of Genetic Uniformity

It is often desirable to know whether the variation remaining in a char-
acter after considerable inbreeding is due solely to environment or partly
to unfixed genes. This can be tested. If there are unfixed genes affecting

344 BIOLOGY OF THE LABORATORY MOUSE

the character, then offspring of individuals at one end of the variation
should differ from offspring of individuals at the other end. If there is no
significant difference in offspring from different types of matings (or if the
parent-offspring correlation is not significantly different from zero) , then the
strain may be assumed to be genetically uniform for the character in question.
Lack of variation in tissue specificity, indicated by ioo% "takes" in
transplants, is sometimes used as a rough measure of the likelihood of
uniformity in genes affecting other characters.

Sublines

When a strain is maintained with a large number of animals it should be
recognized that, unless matings are made up with reference to a pedigree
chart, the strain may break up into many separate lines. These lines may
have quite different characteristics if their last common ancestors were not
genetically uniform or if different mutations have become fixed.

Among mice supplied to research workers there is, as yet, no universally
accepted way of designating the extent of dissimilar ancestry of a given
group of animals. One strain may be kept so that all individuals at any one
time trace back to a common pair of ancestors as soon as possible. Another
strain, perhaps listed under a single name, may have lines of descent which
have been separate for many generations. These are often listed as "sub-
lines," but this term may be used by one breeder to indicate five generations
of separate descent, by another to mean twenty generations, or by yet
another only when he has actually observed phenotypic differences between
the branches.

The research worker who wants maximum genetic uniformity in his
material should, therefore, keep a check on the branching of his own strains
and should specify that animals supplied from other sources have a common
ancestry within a certain number of generations or exhibit no genetic varia-
tion affecting the character under investigation.

Risk of Contaminatiox

Since a high degree of homozygosis is obtained only after many genera-
tions of inbreeding, a single unfortunate outcross may undo years of work.
In a mouse colony in which different lines are maintained an accidental
outcross may occur as a result of faulty pens, into which stray animals can
enter, or to the returning of animals to the wrong pen after removal for any
purpose. Risk of the latter can be reduced to a minimum by handling
' different lines and sublines at different times and by keeping them in

INBRED AND HYBRID ANIMALS AND THEIR VALUE 345

separate parts of the laboratory or cage rack, certainly not in adjacent
sections of wooden boxes. When several inbred lines are to be started it is
desirable to mark them with different coat colors or other genetic char-
acteristics contamination of which will be readily recognized.

Effect of Relaxing Inbreeding

If a strain is to be maintained with maximum homozygosis, there should
be no relaxation of inbreeding. Relaxation of inbreeding in a population
containing different sublines would, of course, introduce heterozygosis
immediately. It should be avoided even in an isogenic line; for it would
tend to preserve heterozygosis introduced by mutation. Haldane (5) has
discussed this effect.

There is one case, however, in which relaxation of inbreeding might be
of advantage. If a large group of animals is to be set aside for experimental
purposes, it is preferable to set aside a single pair and breed their descendants
at random to obtain the experimental animals. The effect of this is to
distribute at random throughout the population any genes which are unfixed
in the pair set aside. In practice, however, it is usually adequate to take
all the animals from the inbred strain provided they have a recent common
ancestry.

Fallacies

Misinterpretation of Variation within Strains

Publicity on the genetic uniformity to be obtained by inbreeding has
apparently led some experimentalists to expect complete phenotypic uni-
formity; although their own observations on variation in such characters
as litter size, and weight at weaning, must belie this conclusion. Possibly
the extreme uniformity obtained in a few characters like tissue specificity,
and coat color, is responsible for this view. At any rate, surprise is some-
times ex-pressed when a character is found to show variation in an inbred
strain, and attempts have been made to explain the variation away, particu-
larly when it shows itself in an all-or-none effect, such as tumor or no tumor.
Thus, the fact that some tumors occur in a "low" tumor strain has been
attributed to residual heterozygosis, and the occurrence of non-tumorous
animals in a "high" tumor strain has been "explained" by stating that
these animals would have had tumors if they had lived long enough. These
explanations may be true in special cases, but the former cannot apply
when the homozygosis has been tested, and the latter will not account for
variation in time of appearance, rate of growth, region affected, etc.

346 BIOLOGY OF THE LABORATORY MOUSE

It is hoped that the earher part of this chapter has made it abundantly
clear that phenotypic variation is usually present in an inbred line as a
result of environmental causes and that, although it is usually less, it is
sometimes actually greater than that in random bred stocks.

It might be thought that this variation could be reduced by giving more
attention to the uniformity of the laboratory environment. This would
have little effect, however, on the many characters whose variation is due
largely to intangible factors in the maternal environment. Wright's elabo-
rate search for the environmental causes of variation in white spotting in
the guinea pig (22) ended with 89% of the variability due to causes which he
could classify only as "developmental accidents." Wright has suggested
that the degree of irregular asymmetry in the expression of a character
serves as a rough estimate of the importance of these factors and, therefore,
of the extent of variation to be expected after inbreeding.

Misinterpretation of Differences between Strains

There is a common belief that a character occurring only in alternate
categories (as opposed to the other extreme: a continuous distribution)
must be due to alternate genes in the same way that agouti and black coat
color are. This has sometimes led to a hunt for a single major gene differ-
ence as the cause of a character difference that shows no, or little, overlap
in two inbred strains. Thus, attempts have been made to find a single
pair of alleles responsible for the difference between "high" and "low"
tumor strains. Such a hunt is all right if it is critical. It can only be
critical if cognizance is paid to the fact that, owing to the common occur-
rence of biological thresholds, of all-or-none processes in development, many
characters are necessarily alternate in expression. Many genes may be
involved, the effects of some combinations falling below the threshold, while
the effects of the others fall above. If this fact is realized it will be appre-
ciated that apparent dominance in the Fi of a cross between strains, a
3: 1 ratio in the F2, and a i : i ratio in the backcross, are not critical criteria
of the presence of a single major pair of genes. Many genes may be involved
and the above generations happen to be cut by a threshold of effect into
approximately the above proportions. In one of his crosses between three-
toed and four-toed strains of guinea pigs, Wright (20) actually obtained
the above ratios, but was able to show that at least four factors were
involved. The critical experiment is to test the genetic nature of the types
apparently segregating in the backcross or F2 by breeding them with the
"recessive" stock.

INBRED AND HYBRID ANIMALS AND THEIR VALUE 347

It should also be borne in mind that some differences that have been
attributed to genetic causes may be due to parasites. Because of the limited
number of parents, there is a relatively high probability that an inbred line
will become uniformly infected, particularly with parasitic organisms
that are transmitted from mother to offspring. It is not impossible that
some mammary cancer differences between strains may be due partly to
this cause.

BIBLIOGRAPHY

1. Bartlett, M. S., and J. B. S. Haldaxe. 1935. The theory of inbreeding with
forced heterozygosis. J. Genet. 31: 327-340.

2. Fekete, E. 1938. A comparative morphological study of the mammary gland
in a high and low tumor strain of mice. Am. J. Path. 14: 557-578.

3. Green, E. L. 1940. Genetic and non-genetic factors which influence the type
of the skeleton in an inbred strain of mice. Dissertation, Brown University.

4. Haldaxe, J. B. S. 1935. The rate of spontaneous mutation of a human gene.
J. Genet. 31: 317-326.

5. Haldaxe, J. B. S. 1936. The amount of heterozygosis to be expected in an
approximately pure line. J. Genet. ^,2: 375-391.

6. Jexxixgs, H. S. 191 2. Production of pure homozygotic organisms from
heterozygotes by self-fertilization. Am. Nat. 46: 486-491.

7. Little, C. C. 1939. Some contributions of the laboratory rodents to our under-
standing of human biology. Am. Nat. 73: 127-138.

8. Little, C. C. and Staff of Roscoe B. Jacksox Memorial Laboratory. 1933.
The existence of non-chromosomal influence in the incidence of mammary tumors
in mice. Science 78: 465-466.

9. Reed, S. C. 1936. Harelip in the house mouse. I. Effects of the external and
internal environments. Genetics 21: 339-360.

10. SiLER, J. F. AX"D OTHERS. 1937- Protective antibodies in the blood serum of
individuals after immunization with typhoid vaccine. Mil. Surg. 80: 91-104.

11. Wright, S. 1921. Systems of mating. Genetics 6: 11 1-178.

12. Wright, S. 1922. Coefficients of inbreeding and relationship. Am. Nat. 56:
330-338.

13- Wright, S. 1922. The effects of inbreeding and crossbreeding on guinea pigs.

I. Decline in vigor. II. Differentiation among inbred families. U.S. Dept.

Agric. Bull. No. 1090.
14' Wright, S. 1922. The effects of inbreeding and crossbreeding on guinea pigs.

HI. Crosses between highly inbred famiUes. U.S. Dept. Agric. Bull. No. 11 21.
15- Wright. S. 1923. Mendelian analysis of the pure breeds of liv'estock. I. The

measurement of inbreeding and relationship. J. Hered. 14: 339-348.

16. Wright, S. 1926. A frequency curve adapted to variation in percentage occur-
rence. ■ J. Am. Statist. Assn. 21: 162-178.

17. Wright, S. 1933. Inbreeding and homozygosis. Proc. Nat. Acad. Sc. 19:
411-420.

348 BIOLOGY OF THE LABORATORY MOUSE

i8. Wright, S. 1934- Physiological and evolutionary theories of dominance. Am.
Nat. 68: 24-53.

19. Wright, S. 1934. An analysis of variability in number of digits in an inbred
strain of guinea pigs. Genetics 19: 506-536.

20. Wright, S. 1934. The results of crosses between inbred strains of guinea pigs,
differing in number of digits. Genetics 19: 537-551.

21. Wright, S. 1939. Genetic principles governing the rate of progress of livestock
breeding. Ann. Proc. Am. Soc. Animal Production ^2: 18-26.

22. Wright, S. and H. B. Chase. 1936. On the genetics of the spotted pattern
of the guinea pig. Genetics 21: 758-787.

23. Wright, S. and H. C. McPhee. 1925. An approximate method of calculating
coefficients of inbreeding and relationship from livestock pedigrees. J. Agric.
Res. 31: 377-383-

Cnapter 11

PARASITES

By Walter E. Heston, National Cancer Institute.*

Introduction, 349. Protozoa, 350. Amoebae, 350. Flagellates, 351. Haemo
llagellates, 353. Coccidia, 354. Sarcosporidia, 357. Prevention and control of
protozoan infections, 358. Helminths, 359. Trematoda, 359. Cestoda, 359.
Nematodes, 364. Arthropods, 370. Lice, 370. Fleas, 371. Bedbugs, 372. Mites,
374. Control measures for other insect pests of the laboratory, 376. BibUography,
377-

Introduction

The house mouse has not been shghted by the evolutionary processes
developing parasitic forms. Over a dozen protozoa have been described as
parasites or commensals living in its blood stream, digestive tract, and
various other internal organs. Of the nematodes, Hall (17) lists twelve
species for which the mouse may act as host, and describes from the rat a.
thirteenth which other workers have found in the mouse. A number of
species of tapeworm infest it, the adult forms of some living in its digestive
tract and the larvae of others in its tissues. Also, to this group of internal
forms might be added the mites, lice, bedbugs, and fleas which may occur
as external parasites.

Many of these parasites are of utmost importance to the research worker
who is employing mice in his experiments. Although probably compara-
tively few of the forms have much influence on the well-being of the mouse
in the natural state, under laboratory conditions and especially under
experimental conditions they may develop into serious factors not only
because of their deleterious effects upon the mouse, but also because they
may act as influencing factors introduced into the experiments. Animals
used in testing deficient diets may have their resistance so lowered that
external or even internal parasites may get out of control. The death of a
mouse bearing a large tumor may be affected not merely because of the large

* Formerly National Cancer Institute Research Fellow at the Roscoe B. Jackson
Memorial Laboratory.

349

350 BIOLOGY OF THE LABORATORY MOUSE

tumor but because with the tumor the animal was unable to keep his para-
sites in check. Deviations from the normal blood count may be due to
parasites rather than to the experimental factors under consideration.

There is not only the havoc caused directly by the parasites to be guarded
against, but also that which they may cause more indirectly by acting as
transmitters for pathogenic viruses, bacteria, and protozoa. Although not
so many mouse diseases have been proved to be spread in such manner, it
seems possible that especially the blood-sucking parasites — bedbugs, mites,
lice, and even fleas are of much greater importance as vectors than is
definitely known.

Sometimes the fact that the mouse may act as host for so many parasites
proves to be an advantage for the research worker. In many instances it
has afforded a convenient way to study phenomena of parasitism. Basic
principles discovered in studying mouse parasites can well be applied to
parasites of man or domestic animals which do not lend themselves so
readily to experimentation. Also, some forms pathogenic to man or domes-
tic animals may be caused to take up their abode in the mouse. Thus, a
very convenient living culture chamber is provided for the parasitologist.

In this chapter an attempt has been made to discuss briefly many of
the protozoon, helminth, and arthropod parasites which may be expected to
be found infesting laboratory mice. ■

Protozoa
Amoebae

Endamoeba muris (grassi, 1879). — This (Fig. 135) is probably the most
common amoeba found in the mouse. Of 85 house mice collected at
Durham, N.C., Harkema (18) found as many as 13.09 per cent harboring
this protozoan in the small intestine. It has also been reported as found
in the caecum and colon of mice and of rats.

Structurally E. muris is very similar to E. coll. Tryphozoites will
average 30 fx in diameter. They display protruding pseudopodia with
glassy covered ectoplasm and a fine granular zone. In the cytoplasm are
occasionally lustrous granules. The nuclear membrane is thick with
peripheral chromatin granules, and there is a large karyosome. Coccal
bacteria may be enclosed in the cytoplasm. Cysts measure 15 tx-20 n in
diameter, and when mature they contain eight nuclei although two and
four nucleated cysts may be found.

E. muris is probably also much like E. coli in that it is not pathogenic
but lives more as a commensal in the lumen of the intestine of the mouse.

PARASITES

351

Walker (42) describes three other amoebae from the intestinal tract of
the mouse, namely: Amoeba cyiterica, A. musculi, and A. Jecalis.

Flagellates

Trichomonas muris (grassi, 1879). — T. muris may well be classed as
one of the most common intestinal protozoa of the house mouse. In the
above mentioned work of Harkema it was found in the caecum of 30.95%
of the mice examined.

Wenyon (43) describes T. muris in the mouse as a pear-shaped organism
in the trophozoite stage varying from 3 /i to 20/x in length (Fig. 136). There

Fig. 135.

-Endamoeha muris from the rat (X1500).

W enrich.)

A, Trophozoite; B, cyst. {After

are three anterior flagella and an undulating membrane bordered by an
axoneme which continues as a posterior flagellum. In the anterior region
are located the oval nucleus, a slitlike cytosome, and two groups of closely
aggregated blepharoplasts. The flagella arise from the most anterior one
of the blepharoplasts, and posteriorly from them extends the axostyle which
terminally protrudes as a short point. Food vacuoles containing bacteria
are found in the cytoplasm. Reproduction is by longitudinal fission or
multiple segmentation.

Cysts of T. muris have been described by Wenyon as about 6 )U to 8 ^l
in diameter. He stated that it is difficult to judge whether or not the
organism is encysted since the flagellates may become spherical and quies-
cent in passed feces although not forming a cyst.

Although trichomonads cause disorders or have been accused of causing
disturbances in man and some lower animals, no pathological condition has
yet been attributed to T. muris.

Hexamita muris (grassi). — This (Fig. 136) is also a rather common
flagellate which occurs in the intestine of mice and rats. It possesses six

352 BIOLOGY OF THE LABORATORY MOUSE

anterior flagella and two posterior flagella which arise from axonemes.
There are two nuclei situated near the anterior end. Wenyon (43) describes
the trophozoite in the intestine as being from 4 /i to 7 )U in length, while a
larger form occurring in the caecum measures as much as 10 /z. The cysts are
oblong, measuring 6 ^t to 7 ^t in length and 3 ^ to 4 /x in breadth. In the cysts
nuclear division occurs, and multiplication is also by longitudinal fission
of the trophozoite.

Fig. 136. — Flagellates from the intestine of the rat as seen when alive, i, Giardia
niiiris; 2, Hcxamita muris; 3, Tricltomonas muris. {After Hegner.)

Giardia muris (grassi, 1879). — According to Hegner (20) G. muris
occurs in a considerable portion of laboratory rats and mice. The tropho-
zoites live in the small intestine while the cysts can be found in the caecum
and the colon or in the feces (Fig. 136).

The trophozoite is a flattened, pear-shaped, bilaterally symmetrical
organism measuring on the average 9.8 jx in length and 6.75 yu in breadth. A
large ventral anterior sucker attaches it to the intestinal epithelium. Two
nuclei and a pair of blepharoplasts are located in the anterior region. From
the blepharoplasts arises a pair of flagella which pass anteriorly and after
crossing near the extreme anterior margin pass laterally to emerge one on
either side of the organism. A second pair of flagella which also arise from
the blepharoplasts pass posteriorly to emerge laterally toward the posterior
end of the body. A pair of axostyles (single, according to Kofoid and

PARASITES 353

Christiansen (25)) connect the blepharoplasts with the posterior tip of the
organism where they give rise to the pair of caudal flagella. A fourth pair
of flagella arises ventrally from the axostyles just posterior to the nuclei.
Two deeply staining bodies lie dorsal to the axostyles. Both binary and
multiple fission take place in the nonencysted stage (19).

Cysts form and are passed out with the feces, and undoubtedly infection
occurs by the ingestion of the cysts in contaminated food and water.

Slight infections are apparently not greatly harmful to laboratory mice,
although more severe infections cause enteritis. Kofoid and Christiansen
(25) have noted that in mice the organism gives rise to a readily recognizable
enteritis which appears as a chronic condition in young mice.

Haemoflagellates

Trypanosoma duttoni thiroux. — The trypanosome described as occur-
ring naturally in the blood plasma of the mouse is T. duttoni.

As is typical of trypanosomes this form is a spindle-shaped organism.
The fiagellum arises near the posterior end of the body, passes anteriorly,
is connected to the body by an undulating membrane, and extends beyond
the anterior end. This species of trypanosome is quite slender, measuring
about 25 /x in length, and the fiagellum is long. Anatomically it cannot be
distinguished from the more familiar T. lewisi found in the rat.

Apparently the life cycles of the two forms are also much the same. It
is well known that T. lewisi employs the rat flea as its intermediate host,
the rat becoming infected by swallowing the feces of the infected flea; and
Brumpt (6) has shown that the swallow flea could be made to act as the
intermediate host for T. duttoni. He demonstrated a cycle development in
the swallow flea and was able to infect mice by feeding them feces of the
infected fleas. While this was obviously not the natural intermediate host,
it does suggest that fleas occurring on mice may well act as the vectors.

Trypanosoma duttoni like T. lewisi is generally considered to be non-
pathogenic, but obviously it can occasionally cause fatal infection. Roud-
sky (32), by rapid inoculations from rat to rat of the whole blood of an
animal when the trypanosomes were at the multiplication phase, was able
to raise the virulence of T. lewisi until it was not only transmissible to
the mouse but was definitely pathogenic for the mouse as shown by the
hepatic and splenic lesions caused, and the infection proved to be transmissi-
ble from mouse to mouse. Later (t,^) he was able by a similar procedure
to increase the virulence of T. duttoni until it was infective when inoculated
into the rat.

354

BIOLOGY OF THE LABORATORY MOUSE

COCCIDIA

Eimeria falciformis (eimer). — Mice are commonly infected with E.
falciformis (Fig. 137). Although it has been listed as a coccidium
of the rat (22), several workers (43) have shown that it cannot be trans-
mitted to the rat nor can mice be infected with the rat coccidium, E.
miyairii.

Development involves the schizogony and the sporogony cycles. Infec-
tion occurs by the ingestion of the mature oocysts, each of which gives rise
to eight sporozoites which enter the epithelial cells of the digestive tract and
there undergo schizogony. This occurs chiefly in the small intestine but
may also occur in the large intestine (43) or in the stomach (31). The
merozoites liberated from the schizonts may repeat the asexual cycle, or

they may develop into microgametes and
macrogametes. Fertilization results in the
formation of a zygote which later develops
into the oocyst. These occur in the feces and
can readily serve for diagnosis. They are
subspherical and measure i6^ito2ijubyii
/i to 17 At.

It is known that laboratory mice often
may carry a mild infection of E. falciformis.
Yet, Nieschulz and Bos (27) in studying the
course of infection found that mice free
themselves of an infection with E. falciformis within 26 days when prevented
from acquiring reinfection. They suggest that the chronic condition of
spontaneous infection is probably due to reinfection.

E. falciformis is pathogenic, although in mild infections the hosts are
not injured very severely. Of 50 mice which Nieschulz and Bos experi-
mentally infected with the coccidium, 40 per cent died between the fourth
and eighth day after the infection. They reported that the death was due
chiefly to the breaking down of the intestinal epithelium by the schiozogony
forms. Wenyon (43) states that in acute infections the organisms may cause
acute enteritis.

Cryptosporidium muris tyzzer, 1907; Cryptosporidium parvum tyzzer,
191 2. — Two other coccidia of the digestive tract of the house mouse are
Cryptosporidium muris Tyzzer (40) and a smaller species, C. parvum Tyzzer
(41). C. muris is found in the stomach while C. parvum lives in the small
intestine. They differ from Eimeria falciformis in that neither is intra-

A. B.

Fig. 137. — Stages in the
development of the oocyst of
Eimeria falciformis ( X 1 000) .
{From Wenyon.)

PARASITES 355

cellular, C muris being strictl}' extracellular, while C. parvum might be
classed as intermediate.

C. muris lives in the gastric glands. During growth the forms occur on
the surface of the glandular epithelium, and all forms except the merozoites
and sporozoites possess a limiting membrane and an organ bv which they are
attached to the surface of the epithelium. The schizonts, which reach a
maximum size of 7 )U by 6 ^t, give rise to eight merozoites. The mature
oocyst is approximately 7 /z by 5 yu, and it becomes a single spore containing
four sporozoites. ]Many of the sporozoites are set free before passing from
the stomach, and Tyzzer suggests that probably autoinfection may be
affected through sexual as well as through asexual reproduction.

C. parvum develops in the cuticula of the epithelium of the intestinal
villi. The forms at first bury themselves in this layer, becoming attached by
an attachment organ to the membrane limiting the cuticula from the cyto-
plasm. As they grow they protrude from the free surface of the cuticula,
but they never penetrate the cytoplasm. Like C. muris, eight merozoites
are produced by each schizont and four sporozoites by each oocyst. The
maximum diameter of the schizonts is 5 [x, and the mature oocvsts do not
exceed 4.5 /x.

Evidently both species are quite commonly and widely distributed
among laboratory mice. However, neither is of great importance path-
ologically, although Tyzzer reports that in extensive infections C. muris does
cause dilation of the gastric glands and some leucocytic infiltration of the
gastric mucosa. Xo inflammatory processes are reported resulting from
infection with C. parvum.

Klossiella muris smith and johnson, 1902. — This sporozoan, which
Kudo (26) considers under the order Coccidia, infects the kidneys of mice.
It was first discovered by Smith and Johnson in 1889, and later they made a
study of it from the kidneys of adult gray mice caught in the animal room of
the laborator}- at Harvard University (36). They describe the external
appearance of the kidneys as being slightly enlarged with a ver}- delicate
mottling of the whole surface by minute, barely visible, grayish specks.

European authors (22, 43) have since described it as a common parasite
of white mice. Cannarella (8) encountered it in his mice used for tumor
studies. In 7,7, mice of an experiment with artificially grafted tumors he
found the organism infecting the kidneys of 43.7% of the mice with a tumor
and j\.o^c of the mice lacking tumors.

The schizogony cycle takes place in the endothehal cells of the capillaries
of the glomeruli. Young gametocytes produced by this asexual cycle make

356

BIOLOGY OF THE LABORATORY MOUSE

their way into the urinary tubules, and the sexual cycle occurs in the
epithelial cells of the convoluted tubules. Sporogony stages are shown in
Fig. 138. The sporocysts pass down the tubules and escape with the urine.
Infection can be affected by the administration through the mouth of urine
from an infected mouse.

According to Jaffe (22) the parasite may also be found in the capillary
endothelial cells of the lungs and spleen. He points out that "infiltrates"
found in the cells of the kidney and especially of the lungs "suggest path-
ogenic qualities." Cannarella explains the interstitial infiltration as the
result of mechanical action and alteration of materials bv A', muris. He

Fig. 138. — Sporozony stages of KlossieUa muris within kidney cells. A, mother-
sporoblast (X1435); B, daughter-sporoblasts (X1590); C, spores (X870). {From
Smith and Johnson.)

finds that occasionally the interstitial infiltration leads to a sclerosis of the
organ accompanied by the reduction of the functional parenchyma. The
minute specks mentioned above which are seen externally represent necro-
biotic changes in the cortex. J. M. Twort and C. C. Twort (39) state that
undoubtedly in most cases this organism is the cause of nephritis in the
mouse, and that they expect to find KlossieUa nephritis in at least 90 per
cent of their animals after they have reached the age of 1 2 months. This
was concluded after about 12,000 post-mortem examinations. Other
organs where they have found the parasite include the brain, suprarenal,
lung, thyroid, spleen, lymph glands, and pituitarv- .

This organism represents an excellent example of a parasite which may
introduce confusing factors into an experiment. Cannarella well recognizes
this for he writes: "II est done indispensable que les rechercheurs et les

PARASITES

357

•^, lifi!

experimentateurs connaissent parfaitement les alterations de degene rescence
et d 'infiltration qu'on rencontre constamment dans les reins atteints de
coccidiose, afin de ne pas mettre en relation ces phenomenes avec d'autres
causes etrangeres au coccidium qui n'est pas toujours bien reconnaissable,
qui n'est pas toujours reconnu et qui, pour le passe, a constitue souvent une
cause d'erreur." This statement could well be broadened to include the
other organs infected by the parasite.

Sarcosporidia

Sarcocystis muris blanchard. — This was the first species of Sarcocystis
to be described, having been discovered by Meischer in 1843, infecting the
muscle of mice. Since that time it has
been found in the rat, and other species
have been found infecting various other
animals. However, the most extensive
studies have been with S. muris since its
hosts can be easily infected by feeding
them infected tissue.

These parasites (Fig. 139) can be seen
as tiny white streaks known as
iVIeischer's tubes" imbedded in striated
muscle tissue or less commonly in non-
striated muscle. The tubes may be as
much as 5 cm. long or they may be
so small as to be seen only with the
microscope. They are filled with sickle-
shaped spores called "Rainey's cor-
puscles." When the spores are ingested by the host a small amoeboid body
is liberated which penetrates the epithelial cells of the intestine. Here
schizogony occurs producing merozoites which make their way to the
muscle tissue where after about forty days multinucleated plasmodia can
be found. Cells may be liberated and reinfect other muscle fibers until an
intense infection is reached. Ultimately development progresses to form
the Meischer 's tubes containing the spores.

In some cases no serious results are apparent with the infection, although
death of mice occurs with heavy infection. It has been shown that Sarco-
sporidia produce a toxic substance, and this is probably responsible for the
death of the host. For a more detailed discussion see Wenyon (43).

■ ». B»htn "'

Fig. 139. — Sarcocystis muris em-
bedded in the striated muscle of the
mouse. Cross section of a " INIeis-
cher's tube." (X75.)

358 BIOLOGY OF THE LABORATORY MOUSE

Prevention and Control of Protozoan Infections

When dealing with laboratory mice, prevention and control measures are
of much greater importance than treatment of protozoan infections, since the
life span of the mouse is so short and new animals can be so quickly produced.
However, there is no reason that some of the treatments recommended for
the different protozoan diseases of higher animals could not be employed
with some success providing they were regulated to suit the smaller
animal.

With most of the intestinal protozoa, infection occurs by ingesting the
cyst forms which have passed out of the body in the feces. Therefore,
control measures should be directed toward preventing the contamination of
the food and water. A feeding and watering arrangement such as is
described by Bittner in the chapter on Care and Recording is excellent for
this reason. There is absolutely no way in which the water can become con-
taminated, and there is only a slight chance of the feces coming in contact
with the food. It is inadvisable to feed and water laboratory mice in open
containers placed on the floor of the cages for such practice lends itself
perfectly toward the perpetuation and spread of intestinal protozoan dis-
eases. Mouse food should be stored in mouse-tight containers and feed
rooms to prevent it from being overrun by stray mice which may be infected.
Insect pests such as silver-fish and cockroaches may be the means by which
the mouse food becomes contaminated. Thus, they should be eradicated.

General hygienic procedures in caring for the cages are essential in
preventing intestinal protozoan epidemics. The cages should be cleaned
frequently and well bedded, not only to keep the mice from trampling over
the fecal material, but also to keep the cages dry. Most protozoan cysts
require moisture to live. The use of ordinary disinfectants in cages is not
generally effective in destroying the cysts of various protozoa. The use of
live steam on the cages or the emersion of the cages in a steam bath is
recommended for the destruction of cystic forms.

The above control measures would also apply to the kidney coccidium,
Klossiella muris. In this case infection occurs when the food and water
have become contaminated with the urine of the infected mouse.

In cases in which the parasite requires an intermediate host to complete
its life cycle, control measures can be most effectively directed toward the
elimination of the intermediate host. Trypanosoma dulloni is suspected of
employing the flea as an intermediate host, and elimination of the flea would
probably be the easiest method of controfling the protozoan.

PARASITES

359

If Sarcosporidia appear in laborator>' mice, the infection can be prevented
from spreading by barring any cannibalism on the part of the mice. The
spores are imbedded in the muscle tissue, and they give rise to infection when
ingested by a susceptible host.

Helminths
Trematoda

Although there can be found in the literature descriptions of trematodes
occurring in the house mouse, Musmusculus, it would seem highly improbable
that any would be found infesting laboratory mice, in
view of the fact that they commonly employ some
species of snail in which to complete their life cycles.
However, laboratory mice can be infested with certain
species of trematodes, thus, in some instances supplying
convenient aids in life cycle studies. Such an example
is reported by Price (30).

In her life cycle studies of the blood fluke
Schistosomatium douthitti (Cort), Price found that the
adult would thrive in Mus musculus although she
considered Microtus pennsylvanicus as the natural host.
In her experiments in which M. ww5cw/m5 was employed,
the life cycle of the parasite was revealed. It was
found that the adults of the species live in the hepatic
portal veins of the mouse. The gravid females make
their way to the small veins of the intestinal wall
and there deposit their eggs. The eggs rupture
through the lining of the intestine and pass to the exterior with the feces.
After development in the snail, the cercariae reinfect mice by penetrating the
skin of the host and passing along the blood stream to the hepatic portal
veins where they reach maturity.

Cestoda

Probably the most important of the worms which may parasitize labora-
tory mice are the tapeworms. Some live in the mouse as adults infecting the
intestine or bile duct, while others employ the mouse as an intermediate host
and live in the liver or mesenteries. Stiles and Hassall (38) list for the house
mouse as many as 14 different species, obviously some of which occur so
rarely as to be of little importance. Five species including the more com-
mon and more interesting ones are discussed here.

Fig. 140. — Strob-
ilocercus of Taenia
taenia eformis . (From
A ngustine.)

36o BIOLOGY OF THE LABORATORY MOUSE

Taenia taeniaeformis (batsch, 1786). — Also known as T. crassicollis.
This is a tapeworm which commonly employs the mouse or rat as an inter-
mediate host. The larval stage, which bears the name Cysticercus fasciolaris ,
is a strobilocercus (Fig. 140). It develops within a cyst in the liver of the
mouse or rat. The adult is a very common tapeworm of the intestine of the
domestic cat having also been reported from wild cats (Fig. 141).

Infection in mice is effected by ingesting the eggs. In the intestine the
shells are digested off, liberating the onchospheres which penetrate the wall
of the intestine and make their way to the liver via the hepatic portal system.
On the second day after ingestion of the egg the onchosphere
has reached the capillaries of the liver. A cyst wall forms
around the larva which develops into the strobilocercus
lying free within the cyst bathed in a clear, yellowish fluid.
Cats become infected by ingesting infected mouse or rat
livers.
ii This is an especially interesting parasite in that from

Pj(j J.J the walls of the cysts sarcomata of the liver develop.

Scolex of Taenia Bullock and Curtis (7) in 1920 reported producing
taeniaeformis cysticercus tumors in some 201 rats by feeding them the
(X15). {From gggg Qf ^]^g parasite. Later it was used extensively in
a . a] er eu- ^^^^qj. experiments, especially by Dunning and Curtis (12).
wan.) II' 1 • T 1 •

Except when bemg used m tumor studies, this parasite

quite obviously would be very undesirable in laboratory mice. However,
control measures are not difficult, for if the primary hosts, i.e., cats, are
eliminated from the laboratory, the main source of infection is removed.
If it is desirable to have a cat around the building, as sometimes one proves
quite indispensable in keeping down wild mice, the cat should not have access
to the feed room or be permitted to climb over the feed or bedding at any
time. Periodic examination and treatment of infected cats constitutes a
control measure of value for preventing infection in mice.

Taenia pisiformis (block, 1780). — This is a common tapeworm of dogs
and is occasionally found in cats. The larva is a cysticercus (Cysticercus
pisiformis) which develops in the liver and mesenteries of the rabbit and has
been reported from the mouse. However, the paucity of the records of the
cysticercus occurring in the mouse minimizes the prospects of its becoming a
pest among laboratory mice. For a rather complete account of its develop-
ment see Hegner, Root, Augustine, and Huff, Parasitology, page 318 (20).

Hymenolepis fraterna (stiles, 1906). — Many authors apply the name
U.fraterna to the common "dwarf tapeworm" of the mouse and rat, thus

PARASITES

361

separating it from the form found in man. However, others feel that the
evidences are not sufficient for separating the rodent form and the human
form into two distinct species and apply the earlier name Hymenolepis nana
(von Siebold, 1852) to the forms found in mouse, rat, and man. Mor-
phologically the forms are identical and their life cycles
are the same. Also, the rodent form and the human
form are interchangeable, although in some experiments
they have not developed as readily in the alternate
host as they did in the host in which the parents
developed. However, Shorb (35) has shown a difference
between the rat form and that found in the mouse. He
has found that although strains from wild rats are
equally infective for rats and mice, strains from mice are
more infective for mice than for rats. Thus, it would
seem that while the parasites probably originated from
one form, which Augustine considers to have been that
of the mouse (20), there have since developed definite
differences in the three forms.

The adult worm is quite small (Fig. 142). Measure-
ments given by Augustine (20) are: length 10 mm. to
45 mm. ; breadth 0.5 mm. to 0.7 mm. ; diameter of scolex
0.25 mm.; and length of hooks 14/xto 18 ju. The scolex
is globular, and on the rostellum the hooks form a single
row. They number from twenty -four to thirty. The
strobila may contain as many as 200 proglottids. The
eggs (Fig. 143A), which usually occur in large numbers in
the feces of infected animals, measure from 40 fxtotofx
in diameter. There are two membranes, the inner of
which gives rise to filiform projections at each pole.

No intermediate host is required for development.
Grassi (1887) proved that infection occurred by the
ingestion of the eggs which are infective immediately
after they have passed out of the host. Upon entering the intestine
the eggs give rise to cysticerci which develop in the villi of the small
intestine, usually limiting themselves to the anterior one-half (Fig. 144).
Later the cysticerci produce adults which become attached to the epithelium
toward the posterior part of the small intestine.

Although probably quite unusual, an intermediate host may be employed
in the life cycle. Bacigalupo (3) has shown that when eggs are ingested

Fig. 142. — Adult
dwarf tapeworm,
Hy menolcpis nana .
(Enlarged.) {From
Stiles and Crane,
after Lcuckart.)

362

BIOLOGY OF THE LABORATORY MOUSE

by certain insects including the adult Tciicbrlo molilor and T. obscurus,
cysticerci will develop which in turn grow into adult worms when the infected
insect is eaten by the primary host.

A third possible way of infection is by the development of worms within
the intestine from eggs that have never passed out of the host, i.e., internal
autoinfection. However, Hunninen (21) has shown that this does not occur
in normal mice, for which he suggests two reasons: first, that the cysticerci
develop anteriorly to the region where the adults are found, and second, that
from s to 18 days after the first infection there is an absolute resistance to

Fig. 143. — Eggs of Cestodes found in the mouse. A, Hymenolcpis nana {front A nimus-
tine); B, H. dlminuta {from Augustine); C, H. microstoma {from Joyeux and Kobozicjf).

further infection. He does suggest, however, that autoinfection may occur
in mice whose resistance is lowered as with a bacterial infection.

Treatment of mice infected with H. fraterna is hardly feasible, which is
generally true with mice parasitized by helminths. Instead, it would seem
more advisable to make the attack with preventive and control methods.
With H. fraterna the fact that internal autoinfection does not normally occur
simplifies control measures for laboratory mice. Little difficulty should be
experienced with the parasite if water and food is kept so that it cannot be
contaminated with feces and if the cages are regularly cleaned. Keeping the
cages clean will also help to eliminate some of the insects which may act as
intermediate hosts.

That the tapeworm may be transferred from the mouse to man makes it
important for one working with mice to take precaution against becoming
infected. One should form the habit of washing his hands each time he has

PARASITES

363

llnishcd handling mice Icsl his hands may have become contaminated with
the eggs which might be transferred to the mouth.

Hymenolepis diminuta (rudolphi, 1819). — This (Figs. 143B, 145 and
146) is one of the most common tapeworms of the mouse and rat, and it
sometimes occurs in man. It is also cosmopolitan in its distribution, having
been reported from various places in the United States, Europe, and South
America. Bacigalupo (2) reports that 28 per
cent of 300 rats from Buenos Aires were infected.

Stiles and Crane (37) give the following
complete description of the species: "Strobila
10 to 60 millimeters in length, 2.5 to 4
millimeters in maximum breadth; composed of
800 to 1300 segments. Head small, almost
globular; 200 to 600 /jl in width; rostellum
rudimentary, pyriform, only slightly protractile;
hooks absent; suckers globular, near the apical
portion of the head, 80 to 160 fx in diameter.
Neck usually short. Segments throughout
strobila broader than long. Genital pores on left
margin, near the junction of the anterior and
middle thirds of each segment. Three testes in
each segment; vas deferens dilates into a
prominent seminal vesicle before entering the
cirrus pouch, within which also is a vesicle.
Gravid uterus occupies most of the proglottids ;
its cavity is subdivided into a large number of
incompletely separated compartments tilled
with eggs. Eggs round or slightly oval; outer
membrane 54 to 86 jj, in diameter, yellowish in
color, may be radially striated; inner membrane
24 by 20 /i to 40 by 35 )U in diameter, with mammilate projection at each
pole often not apparent; between outer and inner membranes a prominent
third layer of albuminous substance, often appearing as two delicate smooth
membranes, with intervening space filled by a granular coagulum; embryonal
hooks II to 16 /i in length."

The completion of the life cycle requires an intermediate host. This
may be one of quite a number of insects, although probably the adult
Tenebrio niolitor and the rat fleas, Nosopsyllus fasciatus and Xenopsylla
cheopis, are the more natural intermediate hosts.

Fig. 144. — Longitudinal
section of the intestinal villus
of a rat containing cystic
stage of Hymoiolcpis nana.
(Enlarged.) {From Stiles
and Crane, after Grassi and
Rovelli.)

364

BIOLOGY OF THE LABORATORY MOUSE

Infection of the primary host occurs by ingesting the infected inter-
mediate host. The cysticercoid which has developed in the intermediate
host is Hberated in the intestine of its new host and within 18 days it has
become attached to the epitheUum of the intestine and has developed into an
adult worm.

Control measures might profitably be directed toward eliminating any
fleas which might be living as parasites on the animals and also toward keep-
ing the feed room or bins free from meal worms, the
larvae of the Tenebrio beetle.

Hymenolepis microstoma (dujardin, 1845). — This
species of mouse and rat tapeworm (Fig. 143C)
probably occurs much more rarely than the other two
species of Hymenolepis that have been described, but it
is an interesting form in that the adult is not limited to
the intestine. The adults live in the duodenum and
the common bile duct which, as described by Joyeux
and Kobozieff (24), in intense infestation may be
distended until it is as large as the duodenum. The
gall bladder also may be completely filled.

Joyeux and Kobozieff (24) have given a complete
description of the adult worm. The measurements
which they give are: length 80 mm. to 350 mm.;
breadth of scolex 200 n; breadth of rostellum 100 ju;
neck 600 IX from base of scolex to first sign of segmenta-
tion. There is a simple corona of 27 hooks.
Development occurs in several insects, notably in the
Tenebrio and in the rat flea, Nosopsyllus fasciatus.

Dobrovolskaia-Zavadskaia and Kobozieff (10)
have described lesions produced by the parasites in
the liver and bile ducts. If the ductus choledocus is heavily infected, its wall
becomes chronically inflamed and irregularly thickened, and the mucous
membrane becomes hyperplastic. However, they state that the hyperplasia
of the mucous membrane has never presented a neoplastic character. When
the parasite penetrates the liver, it causes destruction of the parenchymatous
cells and focal necrosis. In more advanced cases large abscesses develop.

Fig. 145. — Head
and anterior portion
of H. diminuta from
the rat. (Enlarged.)
{From Stiles and
Crane, after Zschokke.)

Nematodes

Many species of nematodes have been reported as parasites of the house
mouse. However, a number of these probably seldom would be of impor-

PARASITES

365

tance in laboratory mice. Only those which occur commonly or have
received special attention in the held of experimentation are described here.

Syphacia obvelata (rudolphi, 1802). — Also known as Oxyiiris obvelata.
The caecum of the laboratory mouse is commonly infected with this small
oxyurid (Fig. 147) which may also occur in the colon. Upon special
examination of the caecae of 34 experimental mice, J. M. Twort and C. C.
Twort (39) found 17 infected with this species, while
of the colons of 57 of their animals 9 were found to
be infected.

Description of the genus, for which S. obvelata is
the type species, is given by York and Maplestone
(46) as follows: "]Mouth bounded by three lips;
small cervical alae present; vestibule absent;
oesophagus club-shaped with a posterior bulb con-
taining a valvular apparatus and separated from the
rest by a constriction. Male: with 2 or 3 cuticular
''mamelons" on the ventral surface; posterior
extremity bent ventrally, body cut away ventrally
behind the cloaca and then suddenly narrows and
ends in a long pointed tail; narrow caudal alae
present limited to the first part of the tail; two pairs
of preanal papillae and one pair of postanal
pedunculated papillae supporting the alae behind;
spicule relatively long and very obvious; guber-
naculum directed transversely. Female: tail long
and pointed; vulva in the anterior region of the
body, behind the excretory pore, and communicat-
ing by a short vagina, frequently protruded, with a
cuticle-lined ovejector remarkable for the thickness
of its muscle coat; uterus single, very long; receptacula seminis parallel and
narrow; two ovaries. Oviparous."

Measurements given for S. obvelata are: male 1.3 mm., female 3.5 to 5.7
mm., eggs 10 ^t to 142 jj. in length and 30 /j. to 40 jjl in breadth.

Not much inflammatory reaction is caused by this parasite unless it
occurs in large numbers; and if general hygenic conditions are maintained for
the laboratory animals, it is doubtful if heavy infection will occur.

Aspicularis tetraptera (xitzsch, 182 i). — Also known as Oxyuris tetrap-
tera. A . tetraptera like S. obvelata is very commonly found in the intestine of
the laboratory mouse, but while 5". obvelata tends to be more limited to the

Fig. 146. — Cysticer-
coid of Hymenolepis di-
mi)uita. {From Augus-
tine.)

366

BIOLOGY OF THE LABORATORY MOUSE

caecum, ,1. Iclniplcni is more commonly found in the colon. Of the mice
examined by J. M. Twort and C. C. Twort, 43 of the colons of 57 animals
contained this parasite while only 3 of the caecae of 34 mice did. The two
parasites may be found together.

These parasites can be distinguished in that the uterus or oviduct of
A. tetraptcra extends posterior to the anus, and also the tail of A. telraptera

Fig. 147. — Syphacia obvelata. A, egg; B, female from the caecum of a mouse.
(Enlarged.) {From Augustine, after Ripley.)

is short and bluntly pointed posterior to the reproductive organs, while that
of 5. obvelata is long, extending for some distance posterior to the anus. The
male of A. tetraptera measures from 2 to 2.5 mm. in length, and the female
from 2.58 to 4 mm. The eggs range from 84 /jl to go jjl in length by 34 fx to
40 jjL in breadth.

This species is similar to S. obvelata in its pathogenesis and control.

PARASITES 367

Longistriata musculi dikivlvnts, 1935. — The trichostrongylc, L. musculi,
parasitic in the intestine of the mouse, was lirst described by Dikmans (9)
in 1935, and in the same year its life history was reported by Schwartz and
AHcata (34). It is a small worm, the adult males measuring 3.25 to 4.5 mm.
long and the adult females 4.25 to 6.75 mm. The anterior end of the body is
usually coiled in a loose spiral.

In life-history studies Schwartz and Alicata found that the eggs soon after
being eliminated from the host hatched into larvae which after but one molt
reached the infective stage. Thus, there occurred a deviation from the
usual four molts characteristic of the development of nematodes generally.
Both infection through the mouth and through the skin resulted in the
appearance of worms in the small intestine which developed with two molts
into adults. The usual migration through the lungs was not essential for
development. In some cases the skin-penetrating larvae did pass through
the lungs, but these were considered exceptional.

Nippostrongylus muris (yokogawa, 1920). — Yokogawa (44) first
described this parasite from wild Norway rats caught near Baltimore. Of
26 rats taken, 24 were infected. It has also been found in the house mouse
caught in the same locality. Although Porter (28) found in a comparative
study of N . muris in rats and mice that mice were quite susceptible to infec-
tion, he concluded that the mouse is a somewhat abnormal host, as demon-
strated by the longer prepatent period, smaller percentage development,
lower egg production, and shorter duration of infestation in mice than in
rats. The parasite has been used extensively for studies of resistance in
which the rat has been employed.

The worms appear red, filiform, and somewhat narrowed anteriorly.
The adult males are 3 to 4 mm. in length with a maximum thickness of 0.085
to 0.1 mm., and the females are 4 to 6 mm. long with a maximum thickness
of 0.09 to 0.12 mm. They are usually found in clumps or nests in the
anterior half of the intestine. These nests appear red due to the excess blood
in the villi of the region. Yokogawa (45) found that the infective larvae
could enter the host both via the mouth or through the skin, the latter being
most eft'ective. They go to the lungs where they undergo a part of their
development, and later complete their development to maturity in the intes-
tine. The eggs are ellipsoidal with very thin shells. They average
58 /x X 33 IX.

N . muris is decidedly pathogenic if present in large numbers. Africa (i)
described heavily infected animals as having been manifestly ill as shown by
their emaciated condition. Their eves were dull and their hair ruffled.

368 BIOLOGY OF THE LABORATORY MOUSE

They would shun food placed before them. The stools of the infected
animals were soft and mucoid. He found clumps of the adult worms in the
intestine usually pinned to the mucosa for considerable depths. Porter (29)
found that death of heavily infected animals was due to lobar pneumonia
resulting from the migration of the larvae through the lungs similar to that
produced by A scar is larvae. He states that in cases of mild infection macro-
scopically the lungs show small haemorrhagic areas in which the larvae can
usually be found, while in severe cases the lungs may be entirely haemor-
rhagic, congested, and edematous. Microscopically there appear areas of
marked consolidation and diffuse haemorrhage.

A compensatory emphysema may be seen in areas in proximity to the
migrating larvae. Deposits of pigment were found near the larvae, usually
free, but sometimes within the mononuclear leucocytes. He observed that
in the intestine the worms migrate extensively in and out among the
vilH, causing local destruction and shrinking of the tissue, and hinted
that the adults may feed upon the glandular secretion or tissues of the
host.

A period of at least 5 or 6 days after the egg is passed from the host is
required for the infective larva to develop. Thus, it is quite unlikely that
heavy infection with this parasite would occur among laboratory animals
if the cages are frequently cleaned.

Protospirura muris (gmelin, 1790). — This species of Spiruridae infests
the stomach of mice and rats. It apparently occurs commonly, and often in
large numbers. Hall (17) states that he has seen a case in which the empty
stomach was distended by a number of these worms which ballooned the
stomach walls as so many clock springs might have done. The parasite is
quite cosmopohtan in its distribution.

These are rather thick worms with relatively small heads. The males
are from 14 to 28 mm. long with a maximum diameter of over i mm., and the
females are 15 to 40 mm. long with a maximum diameter of 1.75 mm. As
with other Filarioidea, an intermediate host is employed in the Hfe cycle, the
eggs developing into infective larvae in the body of the meal worm, Tenehrio.
Thus, control measures should be directed toward the eradication of meal
worms.

Gongylonema neoplasticum (fibiger and ditlevsen, 1914). — Also
known as Spiroptera neoplastica. This Spiruridae, which has been reported
from Denmark, the Danish West Indies, and the United States, occurs in the
squamous-celled anterior portion of the digestive tract of the mouse and rat.
Insects including the cockroach and Tenebrio serve as intermediate hosts.

PARASITES 369

The fully developed larvae may be found coiled up in the muscles of the
prothorax and limbs. Infection occurs in the primary host by the ingestion
of the infected insects.

Fibiger (16) in Denmark has reported extensively on this parasite in
relation to its induction of neoplasms in the fundus of the stomach of the
mouse and rat and in the tongue of the rat. He describes these neoplasms
as possessing exactly the same histological structure as epitheliomata in man
and animals.

Trichinella spiralis (owen, 1835). — Also known as Trichina spiralis.
The "trichina worm," well-known as the organism which causes trichinosis,
is a parasite of hogs, rats, mice, and other mammals, including man. The
adults live in the small intestine and the larvae in the muscle tissue.

The adult is a small worm with the body somewhat tapering anteriorly.
The male is from 1.4 to 1.6 mm. long, and the female from 3 to 4 mm. The
adult male and female copulate in the intestine of the host, after which the
female burrows into the mucosa of the intestine. The female is viviparous,
the larvae being deposited in the lymph spaces. The embryos make their
way to the voluntary muscle and into the sarcolemma, developing into the
infective larvae which assume the spiral form within the lemon-shaped cyst.
Muscles with the richest blood supply are said to be most heavily para-
sitized. Infection occurs by eating muscle tissue containing these infective
larvae.

It is highly possible that trichinosis could become a serious disease among
laboratory mice if the infestation were permitted to become intense. How-
ever, under usual laboratory conditions where few mice are kept in the same
cage and where animals are seldom permitted to die in the cages and be eaten
by their mates, there is slight chance for intense infection.

Other nematodes of the mouse. — The following are species described for
the mouse but of less importance than the above forms.

Capillaria bacillata (eberth, 1863). — Reported from the oesophagus of
the mouse.

Ollulanus tricuspis leuckart, 1865. — Adults live in the gastric mucosa
of the cat. Larvae develop in the musculature and connective tissue of the
mouse.

Gongylonema musculi (rudolphi, 1819). — Reported from the liver and
on the external walls of the stomach.

Heterakis spumosa Schneider, 1866. — Usually reported from the
caecum of the rat, but Harkema (18) found it in the house mouse.

For further description of these species see Hall (17).

370

BIOLOGY OF THE LABORATORY MOUSE

Arthropods

Lice

Several different species of lice have been reported as being found on the
house mouse. However, the most common louse of the laboratory mouse is

probably Polyplax serrata (Burmeister) (Fig.
148), recently redescribed by Jancke (23).
Specimens taken from the mice at the Jackson
Memorial Laboratory have been identified as
this species by Dr. H. E. Ewing, U.S. Bureau
of Entomology and Plant Quarantine. P.
serrata is a common parasite of the house mouse
in Europe, but has been found only on laboratory
mice in this country (14). Hoplo pleura
lies per omydis (Osborn, 1891) has been reported
from the house mouse in California, and H.
acanthopus (Burmeister) occurs on the house
mouse in Europe (38, 15). The common species
of the rat is Polyplax spimdosa (Burmeister)
which is cosmopolitan in its distribution (5).

Lice are permanent ectoparasites. They
move slowly and usually pass from one animal
to another only when the animals are in contact
with each other. Those found on the mouse are
of the type with sucking mouthparts, and feed
by piercing the skin and sucking the blood of
the host. The eggs are elongate and are
fastened to the hairs of the host most commonly
on the dorsal neck region or on the belly.
They can be seen readily by parting the hair,
and they afford one of the easiest ways of discovering an infestation. The
young are similar to the adults in structure, but are paler in color. After
several molts they reach the mature state. Under laboratory conditions
with the temperature controlled there is no interruption in their activity,
and many generations can be produced each year.

Mice infested with lice usually display a general unhealthy appearance.
This is somewhat due to the loss of blood, but probably more to the irritation
which the parasites cause making the animals restless and constantly
scratching.

Fig. 148. — Polyplax ser-
rata, adult female. Dorsal
view (X75).

PARASITES 371

Aside from the above detrimental qualities, lice are undesirable because
of the danger of spreading disease among the animals. Their method of
feeding by sucking the blood of the host facilitates the spread of any organ-
ism living in the blood of the host. Eliot (13) has shown that P. serrata
transmits the blood organism Eperythrozoon coccoides. P. spinulosa of the
rat transmits Bartonella muris, and in rabbits, lice act as transmitting agents
for tularemia.

Eradicative measures against lice must be applied directly to the mice
since the parasites do not commonly leave the host. Insecticides such as
sodium fluoride and pyrethrum, both of which are included in many com-
mercial products, are efTective. These may be applied by dusting the dry
product into the coat of the mouse, or by spraying or dipping the animal into
a solution of the insecticide. Spraying is more advisable than dipping, since
mice often become chilled following dipping and pneumonia may develop.
Small atomizers (perfume atomizers) can be well adapted to spraying mice.
Different oils including kerosene are effective, although, especially in the case
of kerosene, the amount applied should be limited so as not to irritate the
skin of the mouse. Whatever treatment is used should be repeated one or
two weeks after the first treatment in order to eradicate any lice which may
have hatched after the first application.

Conditions which in general tend toward producing healthy mice are of
value in louse control. If a mouse is otherwise in good condition it usually
can free itself of any lice. Animals experimentally subjected to adverse
conditions, as being fed a deficient diet, are more commonly infested.

Fleas

Fleas do not tend to be as restricted to a particular host species as do
some of the other insect parasites. Thus, it would not be surprising to find
any of a number of different species of fleas attacking laboratory mice.
However, the species commonly known as the mouse flea is Leptopsylla
musculi Duges. This species occurs abundantly on mice and rats in Europe
and has been reported from mice and rats in the U.S. (14). The common rat
flea, Nosopsyllus fasciata Bosc, which is often concerned in plague transmis-
sion, is the flea most commonly found on rats in Europe and North America.
It also occurs on mice. The Oriental rat flea, Xenopsylla cheopis (Roths-
child), likewise well known for its role in the transmission of bubonic plague,
is cosmopolitan in its distribution, having established itself in several
localities in the Midwestern States of the United States.

372 BIOLOGY OF THE LABORATORY MOUSE

One can readily recognize fleas as such by their wingless, laterally com-
pressed bodies and their remarkable jumping ability. They feed entirely
from the blood of the host, but do not necessarily remain on the host all the
time for they are often found in the nest of the host or they may even be
found throughout the laboratory. The female lays her eggs in the nesting
material or among the hairs of the host. In the latter case the eggs usually
have dropped to the bedding of the host before they have hatched. The
worm-like larvae are not parasitic but feed on any organic material in the
debris in which they live. After remaining in the larval stage for a week or
ten days, during which time they molt three times, they pupate within silken
cocoons. Under conditions favorable for development such as would be
found in a laboratory, the adults emerge from the pupae after another period
of a week or ten days.

Fleas irritate their hosts considerably, and danger in flea infestation also
lies in the possibility of their spreading disease among laboratory animals.
It is well known that fleas transmit bubonic plague and endemic typhus and
that they serve as the vector for the rat trypanosome and probably also that
of the mouse. Just how many more pathogenic organisms they transmit is
not known, but there are probably many.

Regular weekly cleaning of the cages of laboratory mice automatically
controls flea infestations as such treatment destroys the developing larvae
and pupae. However, the source of flea infestations in buildings can some-
times be traced to a dog or a cat which frequents the building. In such
cases the infestation can be controlled by cleaning and disinfecting the bed
in which the dog or the cat sleeps.

Bedbugs

In some laboratories the bedbug, Cimex lectularius Linne, has adapted
itself to feeding on the experimental animals and has proved to be a very
disagreeable pest. With the ideal conditions presented by the laboratory
and with a constant, abundant supply of food, bedbugs grow vigorously and
breed rapidly.

They are dark, flat insects with vestigial wings (Fig. 149). They feed
entirely on the blood of the host, being active at night and retiring for the
day to cracks and crevices in the cages and racks. Each female lays from
75 to 200 eggs which she conceals in the crevices where she hides. The
young bedbugs, or nymphs, are similar to the adults but are paler yellow in
color. They too feed on the blood of the host. After molting five times the
adult with the rudimentary wings appears.

PARASITES

373

Just how important bedbugs are in transmitting disease is not known, but
it would seem that they might act as transmitters for almost any blood
infection of the host or even for those diseases spread through the waste
products of the body for they readily travel from one cage to another. In
laboratory experiments they have been shown to be capable of transmitting
relapsing fever, bubonic plague, and tularemia.

It is putting it mildly to say that the eradication of a bedbug infestation
from a laboratory is a dififiicult task. Fumigation by the use of hydrocvanic
acid gas or by burning sulfur is effective in
homes, but such treatment in the labora-
tory necessitates putting all the experi-
mental animals in sterilized cages and
removing them to another building or
room which would obviously be impracti-
cal unless the colony of animals were
small. About the best attack is to clean
each room systematically and thoroughly,
removing the cages of animals from the
racks, transferring the animals to
sterilized cages and returning them to the
racks only after the racks have been
painted with kerosene with special atten-
tion given to the crevices where the bugs
may be hiding. All the crevices in the
walls of the room and the cracks in the
floor should be thoroughly treated with
kerosene. The racks should then be
placed so as not to come in contact with the walls. It is even advisable to
stand the legs of the racks in cans of the oil. A few days following this
procedure an application of a solution of i ounce corrosive sublimate to
I pint alcohol and }'i pint turpentine can profitably be applied. The solu-
tion should be painted into the cracks of the racks or about the room and
other places where the bugs are likely to come in contact with it in their
attempt to make their way to the animals. This solution retains its eft'ec-
tiveness for some time after the application. Corrosive sublimate is
extremely poisonous, and great care should be taken in handling it. The
solution should not be permitted to come in contact with the skin. It also
has a corroding eft'ect on metals, necessitating the handling of the solution in
glass or wooden containers.

Fig.
tulariiis
(Xio.)

149. — Bedbug, Cimcx Icc-
Linne, from the mouse.

374

BIOLOGY OF THE LABORATORY MOUSE

Laboratories can well be constructed so as to inhibit the establishment of
bedbug infestations. Brick walls, concrete floors, and an absence of wooden
partitions all tend to eliminate the abundance of cracks in which the bugs
might hide. For this reason, metal racks are more desirable than wooden
ones.

Other suggestions for bedbug control are given by Back (4).

Mites

Liponyssus bacoti (hirst). — The mite found most commonly infesting
the experimental mice in the Jackson Memorial Laboratory is Liponyssus

Fig.

-Tropical rat mite, Liponyssus bacoti (X50). A, dorsal view of female; B,
ventral view of female. {From Dove and Sliiljuirc.)

bacoti* (Fig. 150). This is the tropical rat mite, which was lirst described
from Egypt, but which has since been found to occur in many widely sepa-
rated countries of the world. It has been found infesting rats quite com-
monly in some of the Southern States where it even frequently attacks man.
Dr. F. C. Bishopp of the U.S. Bureau of Entomology and Plant Quaran-
tine believes that this is the first time that this species has been reported as a
pest of laboratory animals. This is especially interesting in view of the fact
that the mice from which the present strains at the Jackson Laboratory
originated have been known to have been infested with mites since before
19 19, and although this is the first time that a specific determination has
been made, it seems quite possible that the same species has prevailed. The

* Identified by Dr. H. E. Ewing of the U.S. Department of Entomology and Plant
Quarantine.

PARASITES 375

question arises as to whether or not the species is a rather common pest of
laboratory mice throughout the country having thus far escaped the
taxonomist's attention.

The mites occur on the hosts for the most part only while they are feed-
ing, and after they have become engorged with blood they retreat to crevices
in the cages or racks where they breed. They migrate freely from cage to
cage and sometimes even from one room to another. Heavily infested
animals develop a scabby skin and rough coat accompanied by a generally
poor health condition.

Although it has not been shown that L. hacoti transmits diseases of
laboratory mice, certainly such possibility exists. Dove and Shelmire (ii)
have reported that they were able to transmit endemic typhus from guinea-
pig to guinea-pig through bites of this parasite.

In the laboratory, unless control measures are applied to these pests,
their numbers will reach epidemic proportions. They can be attacked most
effectively by thorough cleaning, and by disinfecting the cages regularly. It
is well also to spray or paint kerosene into the crevices of the racks. Dusting
the animals with pyrethrum powder or spraying them lightly with pyrethrum
extract aids in the eradication of the parasite. Using metal cages and metal
racks which do not afford good breeding places is a worth-while preventive
measure.

Echinolaelaps echidninus (berlese). — This is the common rat mite
which is found on rats in various parts of the world and especially in warmer
countries. It has been reported from the house mouse in the United States.
This species has been found to be the vector of the pathogenic haemogregar-
ine (Hepatozoon muris) of the rat.

Myobia musculi (schrank). — This mite (Fig. 151) has also been found
infesting the experimental mice at the Jackson Memorial Laboratory.
(Identification by Dr. H. E. Ewing.) These are small mites less than 3-2
mm. in length. They can be found clinging tightly to the bases of the hairs
of the host by the specially adapted front legs which are enlarged and
shortened with a terminal hook for grasping the hair. A pair of long stout
bristles extends from the posterior end of the body. They evidently cause
considerable irritation to the host especially around the face regions, for
infested mice will scratch those regions until the whole area is raw.

These parasites are so small and they cling so closely to the host that it is
difficult to discover an infestation before the mouse has mutilated itself until
it must be discarded. However, pyrethrum extract sprayed on the other
members of the same cage has beenTound to be effective.

376 BIOLOGY OF THE LABORATORY MOUSE

Myocoptes musculinus koch. — This Acarina has been reported as fre-
quently found on mice, each mite tightly clutching a single hair at its base.
In this species it is the last two pair of legs that are modified for hair clasping.

Fig. 151. — Myobia musculi, adult, dorsal view.

Ewing (14) notes that after the infested mice are dead these mites will crawl
to the tips of the hairs where they are observed as tiny white specks.

Control Measures for Other Insect Pests of the L.\boratory

Cockroaches. — One of the most effective ways of eradicating cockroaches
is by the use of sodium fluoride. The dry powder should be dusted into
cracks of the partitions, behind baseboards, around sinks, under drain-
boards, and around the pipes and other such places frequented by the
insects. The roaches get the powder on their appendages and when cleaning
them get it into their mouths, thus becoming poisoned. This treatment is

PARASITES 377

slow and should be continued for some time or until all roaches are elimi-
nated. Care should be taken not to get the poison into the animal cages.

Silverfish. — These pests can be eliminated by dusting pyrethrum
powder or sodium fluoride about the places where they occur. Fresh
pyrethrum powder is the more effective in this case.

Meal worms and other grain pests. — These can readily eradicated
by fumigating the grain bin or room with carbon bisulfide. One pound of
carbon bisulfide to each loo cu. ft. of space is recommended. It is well to
pour the liquid on a cloth and place it in a container in the bin or room. The
heavy fumes sink and penetrate the grain thoroughly. The sides and
bottoms of the rooms or bins should be as nearly air tight as possible, and it
is well to cover the bin, leaving it closed for from 36 to 60 hours. The
temperature should be between 75 and 90°F. for best results.

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3. Bacigalupo, J. 1928. Estudio sobre la evolucion biologica de algunos parasitos
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6. Brumpt, E. 1913. Evolution de Trypanosoma lewisi, duttoni, nabiasi, blan-
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9. Dikmans, G. 1935. New nematodes of the genus Longislriata in rodents. J.
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10. Dobrovolskaia-Zavadskaia, N., and N. Kobozieff. 1927. Sur les lesions des
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11. Dove, W. E., AND B. Shelmire. 1932. Some observations on tropical rat mites
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12. Dunning, W. F., AND AL R. Curtis. 1939. Malignancy induced by Cy5//terc«i
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378 BIOLOGY OF THE LABORATORY MOUSE

13. Eliot, C. P. 1936. The insect vector for the natural transmission of Epery-
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14. EwiNG, H. E. 1929. A manual of external parasites. Charles C. Thomas,
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16. FiBiGER, J. 1919. On spiroptera carcinomata and their relation to true malig-
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17. Hall, M. C. 1916. Nematode parasites of mammals of the orders Rodentia,
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18. Harkema, R. 1936. The parasites of some North Carolina rodents. Ecol.
Monogr. 6: 151-232.

19. Hegner, R. W. 1923. Giardias from wild rats and mice and Giardia caviae n.
sp. from the guinea-pig. Am. J. Hyg. 3: 345-349.

20. Hegner, R. W., F. M. Root, D. L. Augustine and C. G. Huff. 1938. Parasi-
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21. HuNNiNEN, A. V. 1936. An experimental study of internal auto-infection with
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23. Jancke, O. 1930. Polyplax serrata (Burmeister) 1839. Zool. Anz. 92: 105-108.

24. Joyeux, C, and N. I. KoBOZiEFF. 1928. Recherches sur VHymenolepis micro-
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25. KoFOiD, C. A., and E. B. Christiansen. 1915. On the life-history of Giardia.
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26. Kudo, R. R. 1931. Handbook of Protozoology. Charles C. Thomas, Spring-
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27. Neischulz, O., and a. Bos. 193 1. tjber den Infektionsverlauf der Mausekok-
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28. Porter, D. A. 1935. A comparative study of Nippostrongylus muris in rats
and mice. Am. J. Hyg. 22: 444-466.

29. Porter, D. A. 1935. Studies on the pathology of Nippostrongylus muris in
rats and mice. J. Parasitol. 21: 226-228.

30. Price, H. F. 193 i. Life history of Schistosomatium douthitli (Cort). Am. J.
Hyg. 13: 685-727.

31. Reich, F. 1913. Das Kaninchencoccid Eimeria stiedae (Lindemann, 1865) nebst
einem Bietrage zur Kenntnis von Eimeria fakiformis (Eimer, 1870). Arch.
Protistenk. 28: 1-42.

32. Roudsky, D. 191 1. Action pathogene du Trypanosoma lewisi Kent renforce,
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7,7,. Roudsky, D. 191 2. Sur la receptivite du Trypanosoma duttoni Thiroux.
Compt. rend. Soc. biol. 72: 221-223.

34. Schwartz, B., and J. E. Alicata. 1935. Life history of Longistriata muscidi,
a nematode parasitic in mice. J. Wash. Acad. Sci. 25: 128-146.

35. Shore, D. A. 1933. Host parasite relations of Hymenolepis fraterna in the rat
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PARASITES 379

36. Smith, T., and H. P. Johnson. 1902. On a coccidium {Klossiella murls, gen.
et spec, nove.) parasitic in the renal epithelium of the mouse. J. Exp. Med. 6:
303-316.

37. Stiles, C. W., and C. G., Crane. 1910. The internal parasites of rats and mice
in their relation to diseases of man. U.S. Pub. Health and Mar. Hosp. Serv. Bull.
1910: 87-110.

38. Stiles, C. W., and A. Hassall. 1910. Compendium of animal parasites
reported for rats and mice (Genus Mus). U.S. Pub. Health and Mar. Hosp. Serv.
Bull. 1910: 111-122.

39. TwoRT, J. !M., and C. C. Tvvort. 1932. Disease in relation to carcinogenic
agents among 60,000 experimental mice. J. Path, and Bact. 35: 219-242.

40. Tyzzer, E. E. 1910. An extracellular coccidium, Cryptosporidium maris (gen.
et sp. nov.), of the gastric glands of the common mouse. J. Med. Res. 23: 487-509.

41. Tyzzer, E. E. 191 2. Cryptosporidium parvum (sp. nov.) a coccidium found in
the small intestine of the common mouse. Arch. Protistenk. 26: 394-412.

42. Walker, E. L. 1908. The parasitic amebae of the intestinal tract of man and
other animals. J. Med. Res. 12: 379-459.

43. Wenyox, C. ^I. 1926. Protozoology. 2 volumes. William Wood and Co.,
New York.

44. YoKOGAWA, S. 1920. A new nematode from the rat. J. Parasitol. 7: 29-33.

45. YoKOGAWA, S. 1922. The development of Heligmosomum maris, Yokogawa, a
nematode from the intestine of the wild rat. Parasitology 14: 127-166.

46. YoRKE, W., AND P. A. Maplestone. 1926. The nematode parasites of verte-
brates. P. Blakiston's Son and Co., Philadelphia.

Cnapter 12

INFECTIOUS DISEASES OF MICE

By John H. Dingle, Harvard Medical School.

Introduction, 380. Bacterial disease, 381. Mouse typhoid, 381. Septicemic dis
eases of mice: pasteurellosis, pseudotuberculosis, mouse septicemia, 391. Diseases
due to infection with Streptohacillus and pleuropneumonia-like organisms — arthritis
of mice, 399. Epidemic pneumonia in mice, 409. Infectious catarrh of mice, 413.
Pyogenic infections, botriomycosis, 415. Infection with Bacillus piliformis, 416.
Infection due to Bartonella, Eperythrozoon and Grahamella, 419. Miscellaneous
bacterial infections, 429. Fungus diseases, 429. Spirochetosis and leptospirosis
in mice, 431. Spirillum minus, 4:3,1. Leptospira ictcrolicmorrhagiac, 4^^. Virus dis-
eases of mice, 434. Infectious ectromelia, 434. Lymphocytic choriomeningitis, 438.
Encephalomyelitis of mice (Theiler), 443. Virus pneumonia in mice, 448. Inclusion
bodies in the salivary glands and liver of the mouse, 451. Factors influencing the
production of experimental and natural disease in mice, 452. Prevention of dis-
ease and control of outbreaks, 455. Bibliography, 457.

Introduction

Mice have been employed experimentally since the early days of bacteri-
ology. Until fairly recently, however, there has been a lack of extensive
information regarding their anatomy and physiology, and considerable
confusion with respect to the nature, etiological agents, and pathology of
their infectious diseases.

It is essential for any investigator who utilizes mice to become familiar
with the well animal. Anatomical descriptions are available in this book
and elsewhere (133, no), as well as reports concerning operative techniques
(133), induction of narcosis (133), the blood picture (201, 247), temperature
(247, 130), intestinal flora (307), and death rate (299) of normal mice, and
the effect of starvation on temperature, blood, etc. (130).

Equally imperative is it that he be acquainted with the clinical signs and
pathological features of their spontaneous diseases. Such knowledge may
avoid embarrassing confusion by the recognition of a latent infection whose
manifestations might otherwise be misleading in the evaluation of experi-
mental data or in an attempt to isolate an etiological agent from other hosts.
Moreover, with early recognition of disease in valuable stock, such as genet-

380

INFECTIOUS DISEASES OF MICE 381

ically pure strains, steps may be taken to prevent spread of the infection
and destruction of the colony. The mouse must also be regarded as a
reservoir of certain animal and human diseases. These considerations have
led to the inclusion in this volume of a summary of the literature concerned
with the natural diseases to which mice are subject. Other general descrip-
tions of diseases in mice may be found in the works of Jaffe (no), Meyer
(172), and others (299, 87, 170).

Bacterial Diseases
Mouse Typhoid

In 1890, a highly fatal epidemic occurred in the laboratory mice of the
Hygienic Institute at Greifswald, 69 per cent of the animals succumbing
(154) to the infection. Loeflier noted the large, brownish-red spleens and
the small yellow lesions in the livers of dead animals. Groups of organisms
in capillaries reminded him of typhoid bacilli in human tissues. His studies
of this bacillus showed that it was closely related to the colon-typhoid group
and accordingly the name '^B. typhi murium'' was proposed. Subsequent
investigations have revealed that this strain and related organisms of the
paratyphoid {Salmonella) group produce one of the most important bacterial
diseases of mice.

Occurrence. — Mouse typhoid occurs so commonly in rodents that only
with elaborate precautions can a colony be maintained free from infection.
Animals suffering from the chronic form of disease may harbor and excrete
the organism for months, thus maintaining infection in the colony; or the
infection may be introduced from without in the food or from wild rodents
gaining access to the animal rooms. Salmonella typhimurium and S.
enteritidis are found with about equal frequency in apparently healthy wild
and laboratory mice and as the cause of epidemics (47, 235, 335, 236, 113, 10,
159, 336, 26). The incidence of carrier infection in stock mice may vary
from I to 20 per cent and rarely to 100 per cent (307, 172, 330). Other
paratyphoid strains are uncommon, although epidemics due to IMorgan's
bacillus (Proteus morgani) (337) and an unknown species of Salmonella (265)
have been reported.

The natural disease. — Salmonella infections in general are bacteremic
diseases and may run acute, subacute, or chronic courses. The various
bacilli produce essentially the same clinical picture. Infection as a rule
takes place by the oral route and the incubation period extends from 3 to
6 or more days. The first sign of illness is a loss of normal activity and

382

BIOLOGY OF THE LABORATORY MOUSE

industry — the animal sits quietly in a corner of the cage, frequently hunched
over with his head bent down. His hair becomes ruffled and loses its normal
gloss; anorexia develops to a varying degree though usually not complete,
and loss of weight occurs. Later, a conjunctivitis may develop, the eyelids
become glued together, and the respiratory rate is accelerated. The feces
are usually formed, but are softer and lighter in color. A few animals
become hyperexcitable shortly before death. The temperature usually

Table i
The Effect of Infection with 5. typhimurium on the Leukocytes

OF THE Mouse

Total
Leuko-
cytes

Differential (%)

Lympho-
cytes

Mono-
cytes

Neutro-
philes

Eosino-
philes

Imma-
ture
Granulo-

•

cytes*

Normal Mice (Average)

7 SCO

66.5

5-5

26.4

1-4

Infected Mice

Mouse No. I

I St day

7200

78.4

5-2

150

1-4

6.6

yth day

5200

17. 1

5-7

77.2

21.7

Mouse No. 2

I St day

7000

73-5

4-5

20.9

I . I

6.6

yth day

5400

24-3

5-4

70-3

18. 1

* This figure represents the percentage of granulocytes which are immature.
Taken from Seiffert, Jahncke, and Arnold (247).

remains within normal limits, although a terminal rise and fall may occur
(247). The blood picture shows a leukopenia with an increase in granulo-
cytes, many of which are young forms, and a decrease in lymphocytes (Table
i). Increased polychromatophilia and slight poikilocytosis of the erythro-
cytes are present.

In the acute form the disease progresses rapidly and death may occur
within a week, the above signs occurring almost simultaneously or in rapid
succession. In the chronic form, there may be no signs of illness, or only
those of a mild infection followed by apparent recovery, or of a slowly pro-
gressive cachexia. The presence of infection can be verified, however, by
isolation of the specific organism from feces and organs of the animals months

INFECTIOUS DISEASES OF MICE 383

later (280, 281, 283). All degrees between these extremes are met with,
depending upon such variables as the dosage, resistance of the individual
mouse or strain of mice, virulence of the organism, and external environ-
mental factors such as temperature (129), and diet (329, 172).

The experimental disease. — In part because of its similarity to human
typhoid fever, the pathogenesis of Salmonella infection in mice has been
extensively studied experimentally (247, 336, 177, 139, 300, 190, 192, 191,
255). After oral administration, a transitory excretion of the bacilli occurs
in the feces. The organisms do not multiply and many are excreted from
or destroyed in the gastro-intestinal tract, since a period follows in which no
organisms can be recovered from the stools. Invasion of the lymphatic
system then occurs, with involvement of the intestinal lymph follicles,
mesenteric lymph nodes, and less often the tracheobronchial and cervical
nodes. Here multiplication presumably occurs, bacteria are carried by
lymphatic channels such as the thoracic duct to the blood stream, and a
transitory bacteremia ensues (second to fourth day), terminated by removal
of the bacilli through action of the reticulo-endothelial cells, particularly in
the liver and spleen. Bacterial proliferation occurs in the lymph nodes,
liver, and spleen for the next 2 to 4 days, as evidenced by the increase in
numbers of bacilli found in these organs, but the blood remains sterile.
Finally, a progressive re-invasion of the blood stream occurs with generaliza-
tion of the infection throughout the body and secondary invasion of the
intestine. Bacilli are found in such tissues as muscle, gall bladder, bile, and
urine after the septicemia has become established. Direct invasion of the
blood stream occurs only when the infecting strains are of highest virulence
and toxicity or when an overwhelming dose is given. Following the second
bacteremia, the bacilli multiply rapidly in the intestine and may over-
whelm the normal flora (307). In cases of chronic infection, such as is
present in mice which have survived natural or experimental infection,
organisms persist in the spleen, liver, lymph nodes, and gall bladder for
months, and are intermittently or continuously discharged in the feces (see
work of Amoss, Neufeld, Topley, and Webster). The various strains of
Salmonella behave similarly (177, 5, 6). Fatal infections may also be pro-
duced by applying bacilli to the depilated intact or lightly scarified skin, to
the mucous membranes of the conjunctiva by dropping a liquid suspension
into the eye, or by inhalation.

The role played by ''toxins," lytic products, or specific substances derived
from the bacteria is difficult to define, but presumably such products of
the organism account for the leukopenia and focal necrosis in the liver.

384 BIOLOGY OF THE LABORATORY MOUSE

Cameron, Delafield, and Wilson (43) have recently demonstrated that a toxic
substance obtained from 6'. typhimuriiim by tryptic digestion produces
congestion, disappearance of glycogen, and focal necrosis of the liver, and
early necrosis of Malpighian bodies and of lymphoid follicles in the lymph
glands.

The mortality in spontaneous and in experimental epidemics varies from
20 to 80 per cent, and is influenced by the strain of organism, dosage or
multiple exposures, resistance of the stock, age of animals, season, and other
factors (159, 308, 316, 329, 206, 207, 211, 277, 279, 181).

Pathology. — The pathology of this infection has been studied in mice
dying of the spontaneous disease, but, more satisfactorily, in mice experi-
mentally infected by mouth under controlled conditions (154, 177, 190, no,
247, 87, 26, 196). In this way it has been possible to compare the findings
in acute and chronic infections, and to follow the course of chronic disease
by daily examinations (247).

In very acute infections such as result from massive doses, the patho-
logical findings are not characteristic but resemble those of any septicemic
disease. Gross examination reveals congestion of all blood vessels and
viscera, some enlarge nent of liver and spleen which are usually dark red in
color, occasionally serosanguineous fluid in the peritoneal cavity, slight to
moderate enlargement of the lymph nodes, and redness, injection, and
swelling of the intestinal mucous membrane. Microscopically, the findings
are those of hyperemia and congestion of all the organs, fatty degeneration
in the liver, and severe catarrhal inflammation of the intestinal mucous
membrane. Bacteria may be found in large numbers in the blood, peri-
toneal exudate, and the various tissues. Focal lesions are infrequently
found in animals dying before the fifth day.

Animals living i or 2 weeks or longer show more typical lesions. Ema-
ciation is usually pronounced and the abdomen appears enlarged due to
increase in size of the liver and spleen and to intestinal distention. On
opening the body, the vascular congestion is seen to be less pronounced
than in the acute infection. The liver is enlarged and the spleen may extend
down to the level of the pelvic bones. The peritoneal and thoracic cavities
may be free from fluid or contain smafl amounts of bacilliferous serous,
serofibrinous or sanguineous (154) exudate. The intestinal serosa is usually
reddened and injected, and the content of the bowel varies from thin, watery,
yellowish material containing mucus to soft or normal scybalae.

The pathology of the gastro-intestinal tract can be correlated quite well
with the stages in the pathogenesis of the infection given above. During

INFECTIOUS DISEASES OF MICE 385

the first 2 to 4 days, slow enlargement of the solitary lymphoid follicles,
Peyer's patches, and mesenteric lymph nodes occurs, with catarrhal inflam-
mation of the mucosa appearing during the latter part of this period.
After blood stream invasion and the appearance of symptoms, the mucosa
becomes progressively red and swollen, mucus appears in increasing amounts
and hemorrhages into the mucosa and lumen are found. Ulceration of
lymph follicles appears. Similar but less marked changes are found in the
stomach, especially in the pyloric portion. Microscopically, the picture is
that of a severe .enteritis — capillary injection, denuding of epithelium,
ulceration of lymphoid follicles, and infiltration with polymorphonuclear
leukocytes and histiocytes. The mesenteric lymph nodes are swollen, con-
gested, hemorrhagic, and often show focal necrosis. Cellular infiltration
and bacteria may be found from the first few days of the infection.

The spleen is regularly enlarged to three or four times its normal size.
In color, it is dark red or reddish-purple; its capsule is tight and its consist-
ency firm. Rarely, yellowish-white nodules may be seen under the capsule.
On sectioning, the pulp protrudes and the cut surface is mottled with
irregular hemorrhagic and gray areas. Histologically, congestive hypere-
mia, increase in pulp cells, degeneration of lymphoid cells in Malpighian
corpuscles, infiltration with inflammatory cells, and occasionally focal
areas of necrosis are the chief findings. Intra- and extracellular bacteria
are present after the fourth day.

The liver enlarges progressively up to twice its normal size during the
course of the disease. It varies from a deep red to a brownish-yellow color
and is friable in consistency. The capsule is usually smooth but, when peri-
tonitis is present, it may be covered with a fibrinous exudate containing
bacteria. From the fifth or sixth day on, small, yellow, pin-head sized
lesions appear and increase in size and number. Microscopically these
consist of foci of lymphoid-like cells which begin to form about the second
day. The foci increase in size and then become necrotic. Liver cells sur-
rounding them also become necrotic, but retain their normal structural
alignment. The areas remain fairly well circumscribed, but are infiltrated
at the periphery with granulocytes and histiocytes. Occasionally, fibrinous
thrombi are found in the hepatic capillaries, and capillary hemorrhage into
the parenchyma is irregularly present. Bacteria are found extracellularly
in the tissues, intracellular}^ in neutrophiles and histiocytes, and frequently
in clumps in the capillaries.

The lungs may be entirely free from involvement, may show punctate
hemorrhages, or may be congested and hyperemic. Histologically, the

386 BIOLOGY OF THE LABORATORY MOUSE

capillaries are not infrequently distended with mononuclear cells. Pneu-
monia has been produced experimentally by aspiration of 5. enteritidis (335).

The bone marrow shows considerable damage, apparently due to the
depressant action of a toxic substance liberated from the bacilli. Maturation
of the granulocytes is disturbed, and in severe cases a practically com-
plete granulocytic aplasia may be present. It is possible that these organ-
isms elaborate a leukopenia-producing substance such as has been isolated
from the typhoid bacillus by Morgan (174). Complex polysaccharide-
phospholipids which are highly toxic and produce hyperglycemia have
been isolated from Salmonella strains, but their effect on bone marrow and
leukocytes has not been reported. (See Topley and Wilson (287), pp.
566-568 for discussion.)

Pathological changes in the remaining organs are inconstant and are
mainly the result of congestion or hyperemia. Occasionally, bacillary
emboli can be found in the glomerular tufts of the kidney and elsewhere in
capillaries. Focal inflammatory areas may be present in the myocardium.
Bacilli are almost always seen in considerable numbers in smears of the
blood, liver, spleen, and lymph nodes.

Etiology. — :As already pointed out, the organisms most commonly found
in cases of mouse typhoid are Salmonella typhimurium and Salmonella
enteritidis. Other strains of the paratyphoid group occasionally cause
sporadic deaths in laboratory or wild mice, but epidemics due to them are
rare.

In general, the organisms of this group are similar in morphology and
biochemical reactions which accounts for the several names given to various
strains and much of the confusion concerning them in the older literature.
The early work of Smith (254), Bainbridge (16), TenBroeck (266), and
others (134, 243, 282, 284) did much to clarify the situation. More recent
studies of antigenic structure have provided a logical basis for the classifica-
tion which has been accepted by the Salmonella Subcommittee of the
Nomenclature Committee of the International Society for Microbiology
(232).

The Salmonella* organisms are gram-negative rods, usually motile,
which grow aerobically on ordinary media, and form acid and gas from the
carbohydrates which they ferment. They do not attack lactose, sucrose,

* The classification and nomenclature here used are taken from Bergey's "Manual
of Determinative Bacteriology" (22), and are based on those of the Salmonella
Subcommittee (232).

INFECTIOUS DISEASES OF MICE

387

or salicin, nor do they ordinarily form indol or liquefy gelatin. 5. typhi-
murium is considered to be identical with B. typhi murium, B. aerlrycke,
B. pestis-caviae, B. paratyphosus B, Mutton type, and B. enteritidis Breslau
of the German literature. Of the several varieties of S. enteritidis, only
two are important as mouse pathogens: S. enteritidis Gaertner {B. enteritidis

Table 2

Comparison or the Chief Differential Biochemical Reactions and

Antigenic Components of Organisms Causing Mouse Typhoid or

Enteritis*

Bit

chemical Reactions

Antigenic Components

H Antigen

Organism

•2

•d
0

Antigen

Specific

Nonspecific

"(fl

u
0

0
•a

Phase

0
+

Cfi

5. Ivphirnurium

Alk.

IV, V,

I. 2, 3

XII

S. enteritidis

Var. Gaertner

Alk.

®|
ffi/

IX XII

gom

Var. Danysz

Alk.

Salmonella sp.

Acid

ffi

Syverton and

Olitsky (265)

Proteus morgani

0**

N or

(Morgan's Ba-

Alk.

cillus No. i)

Esch. colt.

Acid

■?

® = Acid and gas or positive reaction.

+ = Variable or delayed reaction.

o = Negative reaction.

? = Not recorded.

D = Doubtful, probably negative.

+ = Acid, no gas.

** = Occasional strains show delayed fermentation.

Alk. = Alkaline.

N = Neutral.
* For more detailed information and literature, see Bergey (22) and Topley and Wilson (287).

Gaertner) and S. enteritidis var. Danysz (47). The two types are identical
serologically and differ only in that the Gaertner variety ferments glycerol
in Stern's medium. Although frequently confused in the literature, both
types are pathogenic for rodents. In Table 2, the chief differential bio-
chemical reactions of the organisms discussed in this section are tabulated,
together with the antigenic structure of the Salmonella types.

388 BIOLOGY OF THE LABORATORY MOUSE

The simplest method for tentative diagnosis is to culture the heart's
blood, spleen, liver, or intestinal contents on one of the selective media
which differentiate the non-lactose-fermenting from the lactose-fermenting
colonies (Endo or eosin-methylene blue agar plates) or which inhibit the
coliform organisms (McConkey, bismuth sulphite, or desoxycholate agar
plates). Single colonies of gram-negative bacilli may then be hshed to
Russell's double sugar slants and subsequently to lead acetate medium.
The production of acid and gas in the stab or butt portion of the former
and of black lead sulphide in the latter tentatively identifies the organism
as one of the Salmonella group. Further identification may be accomplished
by the biochemical reactions (as outlined above) and the determination of
the antigenic structure either by cross-absorption tests with known aggluti-
nating antisera or by agglutinations with antisera previously absorbed to
remove all but specific antibodies.

Infections with related organisms. — Four unusual epidemics in mice due
to Salmonella or related organisms have been reported. Sangiorgi (233)
noted a spontaneous disease which involved approximately 20 per cent of
his white mice and was apparently due to a coliform bacillus. The affected
animals showed ruftled hair, shallow respirations, subnormal temperature,
weakness or paralysis of the hind extremities, marked emaciation, and
profuse diarrhea with staining of the perianal region. The pathological
findings were not unlike those described above: grayish-white necrotic
lesions in the liver, hypertrophied and congested spleen, pale kidneys,
intestines filled with yellowish, foamy, liquid contents, and serous exudate
in the abdominal cavity. The organisms cultured from the heart's blood
and spleen were short, gram-negative rods which produced acid and gas in
glucose, maltose, lactose, and saccharose and formed indol. Inulin and
starch were not fermented. Colonies on Drigalski medium showed the
typical red color of colon bacilli. These characteristics placed the organism
in the colon group. White mice fed cultures of the bacillus died in 5 to 6
days with a profuse diarrhea. The pathological findings were identical
with those of the spontaneous disease.

Spontaneous infections with Morgan's bacillus have occurred in labora-
tory mice fed on a diet of oats and raw beef (337). The mice displayed an
appearance of " unthriftiness and lethargy" which was unlike that of mice
in other epidemics. The chief signs were a rough and shaggy coat, hunched
up posture, retracted abdomen, anorexia, and occasionally antemortem
convulsions. At autopsy, there was a general pallor and dryness of the
tissues, the heart was dilated and flabby, the liver nutmeg, and the kidneys

INFECTIOUS DISEASES OF MICE 389

pale and swollen. The disease resembled a chronic intestinal infection in
which the etiological agent was unable to penetrate the intestinal mucosa
and invade the body tissues. The mortality among infected animals was
100 per cent. A motile, gram-negative bacillus isolated at autopsy was
identified as Morgan's bacillus. This organism closely resembles those of
the Salmonella group in many of its properties, although it is now classified
in the proteus group {Proteus morgani) (287, 22). Attempts to feed mice
and reproduce the infection experimentally almost completely failed, so
that a part, at least, of the etiological factors remains unknown.

Syverton and Olitsky (265) have carefully studied an unusual epidemic
of acute intestinal infection sharply limited to suckling and newly weaned
Swiss mice. The clinical signs were those of profuse diarrhea with apparent
tenesmus and marked inanition, rapid loss of weight and tone, complete
prostration in 24 to 72 hours, and death in about one-half of the affected
animals. Obstipation and cessation of suckling sometimes supervened to
produce a state of dehydration that was almost uniformly fatal. If recovery
occurred, necrosis and sloughing of the perianal tissues frequently followed.
In the absence of obstipation, recovery was usually complete. The age
incidence was from 7 to 24 days.

The gross pathological changes varied from slight h5^eremia to necrosis
of the lower ileum and colon. The spleen was dark red and contracted;
in recovered cases it frequently showed gross cicatrization. Microscopically,
characteristic changes were present in the lower ileum and colon. The
involvement of the intestines was extensive and consisted chiefly of leuko-
cytic and erythrocytic infiltration and generalized ulceration of the mucosa
to such an extent that the latter was often found as a slough in the lumen of
the bowel. Hypertrophy of Peyer's patches was particularly marked.
The spleen was hemorrhagic with varying degrees of necrosis of the pulp
cells. DifTuse parenchymatous and fatty degeneration and localized
hemorrhages were noted in the liver. Renal congestion, localized hemor-
rhages, and degeneration of tubular epithelium were present. In the brain,
minute focal hemorrhages were frequently found.

From the intestine, and less frequently from the heart's blood, liver, and
spleen, organisms of the genus Salmonella were cultured. Of 20 strains,
13 were serologically identical; the other 7 behaved serologically as a single
variant. All were culturally identical. Significant cross-agglutinations
were not obtained with antisera against known Salmonella strains.
Specific agglutinins for both types were present in the sera of one-third
of the recovered mice, but not in those of normal animals. The disease

390 BIOLOGY OF THE LABORATORY MOUSE

could be reproduced experimentally in young mice, but not in adult animals
unless massive doses were employed. Infection by contact was possible.
Virus or parasitic agents were not demonstrable. Fecal carriers appeared
probable, since the organism was found in the intestinal contents of the
recovered mice.

The organism differed from known Salmonella species in the formation
of indol, the fermentation of carbohydrates, and its specific serological
reactions, and was tentatively classified in the Asiaticus group of the genus

(44).

Antoine and Regnier (lo) have reported an epidemic of a septicemic

Salmonella infection which was unusual because of the presence of ocular
lesions. Following a conjunctivitis, the ocular and periocular tissues
became involved, producing a characteristic facies (ram's face — "Museau
de belier")- Hemorrhagic visceral lesions were present and both types of
lesions were reproduced experimentally. The organism was not further
identified.

Prevention. — Attempts to evaluate immunization against this disease
have chiefly been carried out in conjunction with studies of experimental
epidemics in mice (Neufeld, Topley, Webster, and their coworkers). In
general, it may be said that the use of killed vaccines (286, 315, 289) or
bacteriophage (290, 288, 186) is not effective in preventing fatal infection
or spread of the disease when vaccinated and normal mice are in close
contact. In many cases the chief evidence of increased resistance is merely
a prolongation of life (182, 140, 107). The vaccination itself may be fatal
(336). Furthermore, the presence of agglutinating antibodies in sera of
recovered or vaccinated mice is not necessarily correlated with resistance
(308, 289). Surviving animals may, however, be more resistant to a
subsequent inoculation by virtue of specific (289) or nonspecific (308)
protective factors.

Vaccination may be of some value in preventing epidemic spread in a
valuable animal stock which is already infected (159, 205, 105). In the
Rockefeller Institute's cancer stock, the survivors of two epidemics of
mouse typhoid due to 5. enteritidis and S. typhimurium {B. aertrycke) were
vaccinated with a killed suspension of both organisms. No further out-
breaks occurred, although a high fecal carrier rate persisted for 5. enteritidis
and a low rate for S. typhimurium. It is possible that vaccination con-
tributed, in part at least, to the disappearance of epidemic outbreaks. A
similar contaminated stock would be wholly unsatisfactory, however, for
such experimental studies as protection tests and virulence determinations.

INFECTIOUS DISEASES OF MICE 391

since the introduction of test material might be sufficient to light up the
latent Salmonella infection, with erroneous and misleading results.

There are no satisfactory methods at the present time for the eradica-
tion of the disease from an infected colony. In some cases the entire herd
must be destroyed and a new stock obtained; in others, a Salmonella-iree
stock may be obtained by quarantining the entire colony, dividing it into
small units of 5 or 6 mice, destroying all the mice in any unit in which a
death occurs from mouse typhoid, and finally destroying units in which
fecal carriers are present as determined by repeated cultures. The second
procedure is expensive and in many cases impracticable because of the labor
involved. Once a stock is obtained free from infection, it can be maintained
by adherence to general preventive measures which will be outlined later
in the chapter.

Septicemic Diseases of Mice: Pasteurellosis, Pseudotuberculosis,

Mouse Septicemia

Mice are highly susceptible to at least three types of septicemic dis-
eases, namely, pasteurellosis. pseudotuberculosis iCorynehacteriiim) and
"mouse septicemia" (Erysipelolhri.x). Spontaneous epidemics, however,
are uncommon, although sporadic deaths are not infrequently encountered.
In general, the diseases run an acute course and thus may not produce
characteristic morbid changes, so that the diagnosis must usually be made
by isolation and identification of the etiological agent. The three t>3)es will
be discussed separately, in conjunction with diseases related by virtue of
their character or the nature of the infecting agent.

Pasteurellosis. — Diseases due to Pasteiirella organisms are primarily
endemic in wild animals and include the so-called "hemorrhagic septicemia"
group, pseudotuberculosis of rodents, and plague. All three types may
occur in mice, but only the first is of much importance.

Hemorrhagic septicemia in mice. — Of all the septicemic diseases of
animals, one tj-pe may be differentiated, since it is characterized by septi-
cemia, capillary hemorrhage, serous, fibrinous, or sanguineous exudation,
and the presence of short bipolar-staining organisms. The disease is found
in a wide variety of animal species and occurs in spontaneous epidemics in
mice (285, 278, 94, 93, 19).

Detailed clinical and pathological descriptions of the spontaneous dis-
ease in mice are lacking. The illness usually is acute, death occurring a few
hours after the onset of signs which are nonspecific — apathy, ruffled coat,
anorexia, conjunctivitis, rapid respiration, etc. The disease is contagious,

392 BIOLOGY OF THE LABORATORY MOUSE

spreads readily to normal animals by contact, presumably by means of
respiratory and conjunctival secretions, and is dependent on carriers for its
continuation. The mortality varies between 75 and 100 per cent. Post-
mortem findings consist of subserosal and submucous hemorrhages, fibrino-
purulent exudations in the pleural, pericardial, and peritoneal cavities, and
hemorrhagic consolidation of variable extent in the lungs. The spleen is
seldom enlarged and other visceral organs show httle or no gross change
beyond the exudate over their surfaces. In the more chronic disease,
enlargement of the lymph nodes and small necrotic foci in the liver may be
present. Pasteurella organisms are found in large numbers in the blood,
spleen, and inflammatory exudates in acute cases, but may be obtained only
with difficulty from animals surviving for several days or longer.

Experimentally, mice are readily infected by Pasteurella of this group
regardless of the animal source of the organism. Numerous routes of
administration are effective — conjunctival, dermal, subcutaneous, intra-
peritoneal, intravenous, oral, and respiratory (139, 300, 173). Parenteral
administration produces an acute, fulminating septicemia terminating by
death in i or 2 days. Postmortem findings consist of local edema and con-
gestion, fibrinous exudate over the serous surfaces, enlargement of the spleen,
and pulmonary edema and congestion. Administration by other routes
results in a more chronic infection, lasting up to a week or longer and char-
acterized by more pronounced local reactions depending somewhat on the
route of administration. Pathological changes are similar to those found
in the spontaneous disease.

The causative agent is Pasteurella muricida (B. fnurisepticus) (22, 173).
Morphologically, the organism is a short, oval, bipolar gram-negative rod,
which is non-motile and measures about 0.3 ix in width and 1.25 /i in length.
Growth occurs aerobically on ordinary media at a wide range of tempera-
tures (20° to 37°C.). No growth occurs on bile media. Dextrose, levulose,
galactose, sucrose, and mannose are fermented with the production of acid;
indol is formed and nitrates reduced. Serologically, this organism cannot
be distinguished satisfactorily from other members of the hemorrhagic
septicemia group isolated from different animal species and named accord-
ingly. Topley and Wilson (287), however, have found two distinct types
of P. muricida, distinguishable by agglutination and maltose fermentation.

Diagnosis of the disease in the acute form can usually be made only by
identifying the organism, since the clinical and pathological findings are not
specific. In chronic cases, recovery of the organism by culture often fails
and inoculation of a normal mouse or guinea pig with tissue emulsions

INFECTIOUS DISEASES OF MICE

393

(spleen, lung, blood, mediastinal lymph nodes, etc.) may be necessary.
Differentiation of the organism from P. pseudotuberculosis and P. pestis is
based on the characteristics given in Table 3, although considerable individ-
ual variation in reactions occurs.

A similar disease occurrmg as a spontaneous epidemic among mice in
the outskirts of Astracan has been reported (67). The causative organism
resembled the Pastcurella morphologically, but produced acid and gas in
glucose, acid in lactose, mannitol, and dextrin, and failed to form indol.
It was highly pathogenic only for mice.

Table 3
DiFFERENTi.\L Ch.\racteristics OF Pasteurella muricida, P. pseudotuberculosis,

AXD P. pestis

Production of Acid from

Growth

Patho-

Litmus

Indol

in Bile

Motility

genicity

Organism

4-»

Milk

Forma-

Salt

at 20-C.

for

u
0

'.3

c
c

0
"3

tion

Medium

White
Rat

P. muricida

±*

+ **

Neutral

P. pseudotuberculosis

+ **

Alkaline

P. peslis

±*

Neutral

+ = Positive.

o = Negative.
± = Variable.

* Usually positive.
** Usually negative.

Control of the disease is accomplished chiefly by general preventive
methods. Animals vary in their individual resistance to the disease and
survivors of epidemics are relatively immune (93). Although some
immunity can be produced by vaccination with heat- or chemically-killed
organisms, it is doubtful whether such a measure would be effective in
eliminating the disease from a stock.

Pseudotuberculosis of rodents. — Spontaneous infection due to Pasteur-
ella pseudotuberculosis (B. pseudotuberculosis rodentium) occurs but rarely
and sporadically in the mouse (202, 195), although it is common in other
animals (203, 244, 176). This disease is not to be confused with pseudo-
tuberculosis of mice due to Corynebacterium pseudotuberculosis (see following
section). Mice are susceptible to experimental infection, death occurring
within I to 3 weeks after inoculation. The course may be rapidly fatal with
septicemia, or chronic with signs of enteritis. Natural infection occurs

394 BIOLOGY OF THE LABORATORY MOUSE

by the enteral route and the pathological lesions consist of whitish-grey
nodules in the intestinal lymph follicles, swelling and caseation of mesenteric
nodes, and enlargement of the liver and spleen which contain numerous
nodules varying in size. After subcutaneous inoculation, caseation develops
locally and in the regional glands. Grossly the lesions may resemble those
of tuberculosis or Salmonella infections. Microscopically, however, the
lesions are exudative in character and consist of central necrotic material
and bacilli surrounded by a zone of leukocytes and histiocytes. In the
liver foci of degenerated hepatic cells may be found. The organism is dis-
tinguished from P. pestis with difficulty, both culturally and serologically.

Plague in mice. — Infection with Pasteur ella pestis is very rare in mice.
Sporadic cases and epidemics, however, have been reported among field
mice in Mongolia and in the Kirghiz Steppes (339), where the disease is
maintained by rodent host-reservoirs. Typical hemorrhages and buboes
were found at autopsy, and the pest bacillus was isolated from nodules in
the viscera.

Pseudotuberculosis of mice. — Pseudotuberculosis is a term applied to
a number of diseases in which the gross lesions resemble those produced by
the tubercle bacillus. Its etiology is varied and includes such agents as
Salmonella organisms, P. pseudotuberculosis (see preceding section), para-
sitic infections, and others. The form described here is limited to mice and
is produced by an organism of the genus Corynehacterium. It was first
reported in 1894 by Kutscher (135), who isolated the bacillus from the lung
of a mouse dying spontaneously.

The natural disease. — Sporadic infection is usual in this disease, but
mild epidemic spread may take place in laboratory stocks of mice (25, 8,
216, 286, 94). Its occurrence is relatively infrequent; its course chronic in
character. Existence of the infection is frequently suspected by the dis-
covery of a caseous lesion of the lung or a lymph node of an otherwise
apparently normal animal. Infection presumably occurs by the respiratory
or enteric route, carriers and the rodent habit of cannibalism serving to
maintain the disease. Distribution of the organism in the animal's body is
by way of the blood stream.

Kutscher's original description gives an excellent picture of the usual
postmortem findings. The upper lobe of the right lung was transformed
into a greyish- white, friable, caseous mass, with marked inflammatory
change in the remainder of the lung. Multiple small nodules, resembling
tubercles in appearance and consisting of inflammatory foci, were present
in the left lung. The only other significant findings were a massive right

INFECTIOUS DISEASES OF MICE

395

pleural effusion and a slightly enlarged spleen. Organisms were abundant
in the caseous mass in the right lung and in the nodules of the left lung.

Pulmonary lesions are almost always found in the severe disease, and,
in fact, may frequently be the only signs of the disease (287, 25). Involve-
ment of the lung varies from pin-head sized lesions to caseation of an entire
lobe associated with pleural effusion. Recent mihary lesions have a trans-
parent greyish-blue center surrounded by a dark red inflammatory zone
which is sharply outlined. Microscopically, the normal pulmonic architec-

FlG.

-Pseudotuberculosis of mice. Spontaneous infection. Viscera of mouse
{in situ) showing lesions. {From Bongert.)

ture is not visible in the nodules. At the periphery of the lesions numerous
bacilli are seen, both intra- and extracellularly. The liver infrequently
contains yellowish-white caseous nodules which are raised when located in
the subcapsular region, thus diiTering from the necrotic foci characteristic
of Salmonella infection. Glandular enlargement and caseation, either focal
or generalized, may be found particularly in the mediastinal, mesenteric,
and cervical nodes. Isolated nodules may occur in the spleen and kidneys.
Occasionally, only the abdominal viscera are involved (Fig. 152).

The experimental disease. — Experimentally, the disease is specific for
mice, and can be produced by subcutaneous, intraperitoneal, intrathoracic,
and oral routes, and by inhalation. Death usually occurs in 3 to 5 days
and rarely later than 14 days, even following infection per os. The patho-
logical findings vary somewhat with the route of infection. Generalized
infection follows parenteral inoculation, a local abscess occurring after

396 BIOLOGY OF THE LABORATORY MOUSE

subcutaneous injection. The lesions are similar to those in the spontaneous
disease, except that the lungs are seldom infected. Inoculation into a serous
cavity results in a rather characteristic granular exudate over the serosal
surfaces, which may take the form of tiny, discrete nodules or coalesce to
form a membrane. The liver is infrequently affected, but lesions occur in
the spleen and kidneys. Perinephric abscesses and pyonephritis may
rarely be present. Diaphragm, heart, voluntary muscles, and subcutaneous
tissues may all show nodules. It is interesting, in view of the polymorphism

of this organism and its similarity to

the streptothrix, that involvement of

,^ »- , -« f. ^- ^ the joints may occur (216). Infec-

^ «,, %• "» . , , '• C " „ . tion by mouth produces lesions

i^J"Z ^ i. '•*.'J •'. :^ .7' ^ chiefly in the mesenteric glands and

y J. Lin , -sC^-*- occasionally in the abdominal viscera.

"* '/ *f .'-*"' ,^- * Microscopic examination reveals

4. ^fV "t" "rf ■ ** ^* that the nodules or "tubercles" are

4^ , ***-«' *if'^. .. composed of bacteria with varying

%"* * "*•** '•'•*' ''' - degrees of cellular infiltration. The

"' ^'' "% f^ '"*>.. ,'^t j*^" picture is not one of cellular prolifera-

^ Tt **^^J ' *]?' ' tion. The serosal nodules and mem-

"^ ^^^ branes consist of bacteria and a small

Fig. 153.— Pseudotuberculosis of mice, number of cells, but no fibrin. In

Morphology of Corynehacleriitm kutscheri lesions of longer duration, the appear-

grown on agar. Loeffler's stain. Photo- ^j-,^,^ ^g ^^at of a pyogenic abscess with

micrograph (X1500). {From Andrcwcs, suppuration and necrosis. Bacteria,

which are abundant, group them-
selves into definite colonies in the tissues and particularly on serous surfaces,
appearing as masses of interlacing filaments. Thrombi and organisms
are found within blood vessels.

Occasionally at autopsy the only lesion is a small abscess at the site
of inoculation. Bongert (25) postulated the production of a toxin to explain
these instances, and demonstrated that filtrates of broth cultures or heat-
killed organisms could cause death in 10 to 14 days without obvious lesions.
Topley and Wilson (287) have confirmed this finding and consider that the
organism produces an exotoxin which is lethal for mice.

Etiology. — The etiological agent is Corynehacterium kutscheri Bergey (22)
{B. pseudotuberculosis murium Kutscher, Corynethrix pseudotuberculosis
murium Bongert) (Fig. 153). It is a true diphtheroid, occurring as slender
granular rods with some club forms in young cultures, but showing a great

INFECTIOUS DISEASES OF MICE 397

deal of polymorphism in older cultures. It stains irregularly with the
aniline dyes, and is gram-positive and non-motile. Growth occurs aerobi-
cally on ordinary media and on Loeffler's serum medium at 37°C. Acid is
produced from dextrose, sucrose, and maltose but not from galactose,
lactose, mannite, and dextrin. Litmus milk is not changed; nitrates are
reduced; no indol is formed, and gelatin is not liquefied. Final differentia-
tion of this organism from other members of the group may be made on
the basis of its specific pathogenicity for mice. True diphtheria bacilli
(C. diphtheriae) have not been found in mice (95).

Injections due to related organisms. — Condrea (46) has described an
extremely contagious but benign disease which spread through his mouse
colony, attacking 200 mice. Only a few deaths occurred from secondary
infection. The disease was characterized by the appearance of small,
movable nodules in the subcutaneous tissue of the back or thighs. These
foci increased in size, became adherent to the skin, and then ulcerated.
Yellowish, serous fluid and necrotic caseous material could be expressed
from the nodule after removal of the crust. Microscopically, the exudate
showed leukocytes and many gram-positive bacilli. The organisms were
easily cultured aerobically on media enriched with ascitic fluid or serum,
and resembled the diphtheria bacillus in morphology. The organism dif-
fered markedly from C. kutscheri in its fermentation reactions — acid but no
gas was formed in dextrose, levulose, sucrose, maltose, mannose, arabinose,
sorbite, dextrin, inulin, and salicin. Experimentally, the disease could be
reproduced by subcutaneous or intramuscular injection without generalized
infection. Intravenous administration produced a fatal septicemia with
localized abscesses in the lungs and kidneys. Rabbits were not susceptible.
Condrea classified the organism in the genus Corynebacterium and proposed
the name "Corynebacterium miiris.^'

A somewhat similar organism was isolated by Holzhausen (loi) from
white mice injected with the brain emulsion of a dog suspected of being
rabid. Paralysis occurred on the second day, followed by death during
the course of the next day. The organism apparently produced a septicemia
without gross lesions and was readily cultured from the blood and organs.
Morphologically, it had the appearance of a diphtheroid which differed
from Condrea's organism in fermenting galactose and lactose but not
attacking arabinose. It produced hydrogen sulphide and, in litmus milk,
acid without coagulation. No exotoxin was detected. The organism has
been classified by Bergey (22) as Corynebacterium murisepticum. The
experimental disease was specific for white and grey mice.

398 BIOLOGY OF THE LABORATORY MOUSE

Mouse septicemia (Erysipelothrix). "Mouse septicemia" is the name
given to an infection tirst reported in 1880 by Koch (132) in mice which
had been injected subcutaneously with putrefying blood. Although infre-
quent, the disease has been encountered both sporadically and epidemically
(94, 173, 153, 305, 17, 204, 61) and has been the subject of experimental
investigation (82, 139, 300). The etiological agent is Erysipelothrix
muriseptica.

The most complete description of the natural disease is given by Way-
son (305), who studied an epidemic in migrating California meadow mice
(Microtus Calif ornicus estuarensis) and house mice {Mus musculiis). The
infected animals "... sat about with roached backs, roughened pelage,
labored breathing, and with eyelids glued together with purulent exudate,
and were easily caught by hand."

The gross pathology was that of a septicemia with purulent conjunc-
tivitis and congestion of the subcutaneous vessels producing a deep reddish-
pink color in the subcutaneous tissues, particularly about the lymph nodes.
Dark red patches of pneumonic infiltration were present in the lungs, with a
small amount of effusion in the pleural cavity. The spleen was enlarged
and, together with the lymph nodes and liver, was congested and showed
occasional tiny white areas of necrosis. Scattered subserous petechiae
were noted in the intestinal walls. Organisms were present in large numbers
in the blood and viscera. Wayson considered that the infection was spread
by cannibalism and by excreta.

The disease may also occur in stock laboratory mice. In performing
routine examinations on dead mice from a normal stock, Balfour- Jones (17)
noted purulent conjunctivitis, a peculiar gelatinous appearance of the
abdominal organs, enlargement of the spleen, and small discrete greyish-
white areas about i to 2 mm. in diameter in the liver. The lesions appeared
as pits on the surface of the liver, and microscopically consisted of round
areas of necrosis surrounded by an outer zone of leukocytes. During a
4-month period, 59 of 393 mice showed the above picture — chiefly mice
weighing between 12 and 15 grams. The organism isolated reproduced
the disease and was identified as an Erysipelothrix strain.

Experimentally, the disease may be reproduced by parenteral injection,
by oral, dermal, and conjunctival routes, and by inhalation. A septicemia
results from parenteral injection, fatal in 2 to 5 days. By other routes the
infection progresses more slowly. The first sign of illness is conjunctivitis,
at first serous, then purulent, gluing the eyelids together. Lassitude
follows; the animal sits with arched back and becomes anorexic and con-

INFECTIOUS DISEASES OF MICE 399

.stij)atcd. Ivcspiralion decreases in rate, and deatli occurs almost im-
l)erceptibly. The pathological lindings are essentially those described
above. Mice and rats are susceptible to infection, guinea pigs and rabbits
resistant .

Erysipclolhrix muriscptica {B. murisepticus) is one of three organisms
{E. rliusiopat/iiae, E. erysi peloides) which are indistinguishable morphologi-
cally, culturally, and serologically. Occasional differences in pathogenicity
occur — the murine organism, for example, usually does not infect hogs as
does the swine erysipelas strain {E. rhusiopatJiiae) — but are not constant
enough to permit classification on that basis. The organisms appear as
slender, gram-positive, non-motile rods and as long filaments of threads
with irregular thickenings and branching. They are facultative aerobes
and grow readily in dew-drop colonies on ordinary agar. In gelatin stab
cultures a characteristic "test tube brush" appearance is seen after 3 to
5 days at room temperature- fine threads radiate horizontally into the
medium from a central mass of growth along the needle track. No liquefac-
tion is produced. In broth, a slimy viscous growth occurs which settles to
the bottom of the tube. The fermentative reactions vary widely, but in
general acid is produced in dextrose, lactose, sucrose, maltose, galactose,
and rafiinose after 48 hours' incubation. Hydrogen sulphide is formed;
nitrate reduction is variable. Indol is not produced. Serologically, the
organism agglutinates with commercial swine erysipelas serum or antiserum
produced with any strain of the group.

The infrequent occurrence of the disease in mice renders its control of
little practical importance, but active and passive immunization should be
feasible.

Diseases Due to Infection with the Streptobacillus and Pleuro-

PNEUMONIA-LIKE ORGANISMS ArTHRITIS OF MiCE

In 1929 Levaditi and Selbie (151) isolated a strain of Streptobacillus
moniliformis from two mice which had been injected with an emulsion of
the brain and spinal cord from an apparently normal mouse. Similar
organisms had been isolated previously from human patients in France
(148) and in America {Haverhillia multiformis) (198). Subsequent work
has shown that these organisms are identical with the older Streptothrix
muris ratti, and the name of Actinomyces muris has been proposed by Topley
and Wilson (287). In view of common usage, however, the name of
Streptobacillus moniliformis will be retained here. The organism has been
found not only as an inhabitant of the nasopharynx and tissues of apparently

400 BIOLOGY OF THE LABORATORY MOUSE

normal rodents, but has also been identified as the etiological agent of
sporadic and epidemic illnesses.

The etiological situation in respect to these diseases, however, is not
an uncomplicated one. From cultures of Streptohacilliis moniliformis and
from mouse and rat tissues, Klieneberger and her co-workers (125, 126,
127, 72) have isolated a pleuropneumonia-like organism, termed Li, which
alone is relatively avirulent, but in combination with the Streptobacillus
is markedly pathogenic for mice. The relationship between the two organ-
isms is not clearly established; symbiosis (125, 126) and bacterial variation
(48) have both been advanced as explanations. Other pleuro-pneumonia-
like organisms, distinct from Li, have been isolated from diseased mice,
and apparently play an etiological role. It therefore seems desirable to
discuss together the diseases produced by these two groups of organisms.

Infection with Streptobacillus moniliformis. — This disease, which is
primarily an arthritis in the subacute and chronic cases, occurs both
sporadically and epidemically (152, 261, 161, 301). The origin of the
infection and the factors responsible for epidemic spread remain unknown.
Presumably carriers may exist within a stock or may gain access to it from
without (wild rodents) and thus serve as the source. The mortality is
usually high, but varies in different genetic strains of mice. In one epidemic
(161, 301) lasting 4 months, 414 of 650 Simpson-Marsh albino mice suc-
cumbed, whereas only 61 of about 300 Little dilute brown (dba) mice died.
Death may occur in a few days or at any time within 6 months or longer
after infection.

The natural disease. — In the acute form the disease is septicemic in
character. Signs of the infection are nonspecific— the animal appears ill,
its coat is dull, a semipurulent conjunctival discharge is present, and occlu-
sion of the palpebral fissures may occur. No characteristic changes are
found post mortem either grossly or microscopically, but the organism may
be cultured from the blood and organs. In blood smears the organisms
appear as quite regular bacilli.

A more characteristic clinical picture is presented in the subacute and
chronic cases. Polyarthritis, edema and cyanosis of the extremities and tail
suggestive of cardiac failure, conjunctivitis, and emaciation are outstanding
signs. Occasionally there occurs involvement of the vertebral column
resulting in paralysis of the hind legs, ulceration of the feet with serous
exudation and crusting but rarely gangrene, enlargement of the axillary and
inguinal lymph nodes, keratitis progressing to destruction of the eye,
arrested gestation, subcutaneous nodules, submaxillary abscesses, and

INFECTIOUS DISEASES OF MICE

401

enteritis. Deformity and ankylosis of affected joints occur and are visible
by roentgenological examination (301) (Figs. 154, 155, 156, 157).

Pathological examination of the viscera reveals marked enlargement of
the spleen with numerous, often confluent, areas of necrosis throughout the
pulp. Similar lesions are found in the liver, though to a less extent. Con-

FiG. 154. — .\rthritis and enlarge-
ment of right ankle joint in a mouse
naturally infected with Streptobacillus
moniliformis. {From van Rooyen. J.
Path, and Bad.)

Fig. 155. — Infection with Strep-
tobacillus moniliformis. Mouse
showing edema of legs and tail.
{From van Rooyen.)

gestion is present in the intestines with enlargement of the lymphatic struc-
tures. The kidneys and lungs are not affected. Involvement of the heart
is frequent and may vary from a serofibrinous pericarditis to a pancarditis.
Microscopically, endocarditis may be evidenced by small vegetations on the
valves and endocardium. The myocardium may show numerous foci of
inflammation, either acute or undergoing repair. Thrombosis of arterioles
and emboli of organisms in the capillaries occur. The articular and osseous
lesions vary from acute inflammatory to necrotic or proliferative processes.

402

BIOLOGY OF THE LABORATORY MOUSE

In other subacute cases, the picture is that of a hbrinopurulent synovitis
with extension of the inflammation into surrounding structures. Organ-
isms may be visuahzed in thrombi and inflammatory foci as pleomorphic
bacilli, filaments, etc. In chronic cases, walled-off necrosis, proliferation of
cartilage, or granulations and adhesions may replace the destroyed tissues.
Organisms are found extracellularly and within the cytoplasm of large

Fig. 156. — Infection with Strep-
tobacillus moniliformis. Three
bulbous swellings of tail shown in
mouse on left; normal mouse on
right. {From van Rooyen.)

Fig. 157. — Infection
with Streptohacillus monil-
iformis. Mouse showing
moist coat, occlusion of
palpebral fissure, and par-
alysis of hind legs.
{From van Rooyen.)

mononuclear cells. Cultures of blood, organs, and articular fluid yield the
organism even in the most chronic cases.

The mode of transmission of the spontaneous disease is not clear.
Spread by contact and cohabitation presumably occurs, and bites of infected
animals probably transfer the infection. Although organisms may be
present in the urine, infection does not appear to be transmitted by con-
tamination of food or water and all attempts to infect animals experi-
mentally by the enteral route have failed (198, 152). Similar but non-
pathogenic organisms may be found in the lungs of normal animals and in
the dust of hay and straw (112).

INFECTIOUS DISEASES OF MICE 403

The experimental disease. — Types of infection entirely similar to the
spontaneous disease may be produced by inoculation of pure cultures.
Intravenous or intraperitoneal injection produces the acute septicemic form
of the disease; subcutaneous or intra-articular administration, or instilla-
tion into the conjunctival sac results in the chronic disease described above.
Mice show considerable variation in their susceptibility, not only individ-
ually but genetically. Albino strains are more susceptible than the wild
brown mouse or hybrid black-coated stock. Other animals in general are
relatively resistant to infection.

Etiology. — The organism is a facultative aerobe which grows on liquid
or solid media containing 40 per cent or more of serum. Loeffler's medium

Fig. 158. — Film from a culture of StrzptobacUhis moniliformis immediately after
isolation. Basic fuchsin. (X900.) {From van Rooyen.)

or scrum agar is very satisfactory for its isolation. Morphologically, marked
pleomorphism occurs in recently isolated cultures; slender gram-negative
bacilli, coccoid bodies, and interlacing filaments are present. Large fusi-
form, oval, or club-shaped swellings may occur at any point in the filaments.
After repeated transplants, the morphology becomes more regular and
bacillary in form (Fig. 158).

On the surface of solid media, the colonies are of fair size (i to 2 mm.),
greyish, translucent, but not particularly characteristic. Around the colony,
often imbedded in the agar, tiny microscopic colonies with dark centers may
be found (125, 126, 48). These are the Li colonies (referred to above),
morphologically characterized by large bodies, granules, and filaments.

404 BIOLOGY OF THE LABORATORY MOUSE

Their presence in cultures of the streptobacillus was not confirmed by van
Rooyen (301).

In ascitic fluid or serum broth the organism typically produces separate
cotton-ball or flake-like colonies which are frequently attached to the sides
of the tube but settle down to the bottom if disturbed. A clear supernatant
fluid is left. Surface growth does not occur. Filtrates of cultures are not
infective (301).

Identification of the organism is made chiefly by the type of growth in
liquid media and the morphology. Biochemical reactions are of little
help — acid is produced from glucose, salicin, and sometimes lactose and
maltose. Serologically, the strains thus far studied are antigenically the
same (161, 301).

Differential diagnosis. — Diagnosis of this disease clinically involves
differentiation from three diseases, pasteurellosis, mouse pseudotuberculosis,
and ectromelia. Animals infected with Pasteurella may show a rapidly
fatal systemic infection, conjunctivitis, paralysis of the hind limbs, and
rarely arthritis, but no edema or cyanosis occurs. Mouse pseudotuber-
culosis may be differentiated by the absence of conjunctivitis and edema
and by the pathological findings. In ectromelia a dry gangrene of the feet
and legs is characteristic, paralysis and conjunctivitis are absent, and
recovery usually occurs. Final differentiation, however, is made by cul-
ture, except in the case of ectromelia where inclusion bodies and the demon-
stration of a filterable virus establish the diagnosis.

Immunity. — Immunity to the disease occurs naturally, as already pointed
out. Animals recovered from the spontaneous disease or injected with
heat-killed organisms are resistant to further infection. It is interesting to
note that neither infection nor the development of immunity has followed
the feeding of cultures (198).

Infection due to pleuropneumonia-like organisms. — Minute pleuro-
pneumonia-like organisms have recently been reported as the etiological
agents of experimental disease in mice by investigators both in England
(70, 72) and in this country (224, 225, 226, 227, 49, 264, 230). The organ-
isms are of particular importance to those studying viruses because the
agents are filterable and do not grow in ordinary culture media. Thus far,
no cases of spontaneous illness in mice have been reported, although the
micro-organisms have been found in instances of pulmonary disease in
rats (128). The appearance of pleuropneumonia-like organisms has
followed inoculation of mice for other purposes with such agents as yellow
fever virus, lymphocytic choriomeningitis virus, and the toxoplasma, or by

INFECTIOUS DISEASES OF MICE 405

serial intranasal passage of suspensions of mouse lung. Seven distinct
strains, termed Li to L7 in accordance with Kliencberger's nomenclature,
have been isolated in England, four of which were found in mice. Similarly,
five separate types, A, B, C, D, and E of Sabin (227, 230), have been dis-
covered in laboratory mice in America. Illness produced by these agents
is important in that it may appear in animals inoculated with other materials
and must therefore be recognized. Since the organisms are natural
inhabitants of mice, it is probable that under certain conditions they may
incite spontaneous disease.

The experimental disease. — The type of experimental disease produced
in mice varies with the strain employed and with the route of inoculation.
The most striking illness results from intracerebral injection of the L5 or
Type A strain, isolated originally from mice which developed "rolling dis-
ease" following inoculation with the viruses of yellow fever or lymphocytic
choriomeningitis (70) or with the toxoplasma (224). After an incubation
period of 2 or 3 days — occasionally as long as 10 days — signs of illness
appear. Some of the animals show little beyond roughening of the fur and
irritability; others show a variety of nervous signs and symptoms, often
choreiform in type. Characteristically, many afflicted animals turn in
circles with their tails as a fixed axis.

According to Findlay et al. (70), approximately 10 per cent of the animals
showed the "rolling" phenomenon; the head was rotated slowly, the foreleg
raised from the ground, and with a jerk the animal rolled over in one direc-
tion for fifty or more revolutions. Death usually followed within 24 hours
after rolling began. Three-fourths of the animals died in 2 to 7 days, and
of the one-fourth surviving, about half developed acute hydrocephalus i to
2 weeks later. No attempt was made to separate the virus of lymphocytic
choriomeningitis from the L5 organisms, but it was found that other strains
of pleuropneumonia-like organisms mixed with the virus did not produce
the disease. Interestingly enough, animals which survived did not show
symptoms of choriomeningitis and were no longer susceptible to that virus —
possibly another instance of the "interference phenomenon" (218). Patho-
logically, an intense inflammatory reaction with polymorphonuclear infil-
tration was found in the substance of the cerebral cortex, the floor of the
lateral ventricles, the choroid plexus, and the meninges, frequently resem-
bling acute abscesses. In cases of hydrocephalus, the ventricles were
markedly dilated with a corresponding decrease in thickness of the cortex.
Smears stained with Giemsa's stain revealed many extracellular and intra-
cellular granules approximately twice the size of the elementary bodies of

4o6 BIOLOGY OF THE LABORATORY MOUSE

the virus of vaccinia. The L5 organism isolated from these animals was
culturally and immunologically identical with the type A of Sabin, although
the production of an exotoxin by the L5 strain has not yet been established.

Sabin's findings differ from those described above in that the majority
of the animals recovered in a few days; some, however, showed a relapse or
continued to exhibit choreiform movements for months. Variations in the
clinical picture were noted with the age of the mice — no signs occurred in
the majority of animals younger than 15 days or older than 2 months,
although infection occurred; and arthritis developed in about 30 per cent
of mice more than 2 months of age. The characteristic lesion was acute
necrosis of the caudal pole of the cerebellum and of the tissues around the
lateral ventricles. Cerebellar involvement was absent in animals showing
no signs of infection, but periventricular involvement was regularly present.

Intraperitoneal or intrathoracic injection of the L5 or A strain produces
convulsions or other signs of involvement of the central nervous system
in 20 to 40 per cent of mice (225). Death usually follows in 17 to 48 hours.
At autopsy, lesions are found only in the brain, while organisms are demon-
strable in the viscera but not in the blood or brain. The explanation of
this finding was revealed by the demonstration of a toxin, which passes
through a Seitz filter and, injected intravenously, produces nervous signs
within I or 2 hours. Most of the animals die in a few hours, but those
surviving for longer periods exhibit the same acute degeneration of the
cerebellum described above. No organisms can be demonstrated in such
animals (225).

Serial intranasal inoculations of an emulsion of lung from a "normal"
mouse by the method of bhnd passage, that is, employing the pulmonic
tissue of one animal as the inoculum for the next, may result in a pneu-
monia apparently due to pleuropneumonia-like organisms after a variable
number of passages (264). The infection progresses rapidly with ruffling of
the fur, anorexia, weight loss, and dyspnea. Death occurs after 4 or 5 days
in about one-third of the mice; if the animals survive for 7 days recovery
usually takes place. At autopsy, purple areas of pneumonic consolidation
are found involving one or more lobes or an entire lung. Pleuritis may
occur. Microscopically, the picture is one of an interstitial pneumonia,
congestion, and infiltration chiefly with mononuclear phagocytes. In
recovered mice, pneumonic areas may persist for as long as 3 weeks, or a
cystic degeneration, similar to that occurring in rats naturally infected with
pleuropneumonia-like organisms, may take place. Organisms can be
isolated by culture of the emulsified lesions. In addition to the Type A

INFECTIOUS DISEASES OF MICE 407

organisms which produced the infection just described, both Types B and C
have been found in pneumonic lungs (227, 103, 104). A conclusive etiologi-
cal relationship has not yet been established for the pleuropneumonia-like
organisms found in the lungs of these animals. Sullivan (263) has found
that after six consecutive passages of the organism on killed egg membranes,
pneumonic lesions were produced by a single inoculation, which makes
probable but does not prove a causal relationship. Further work is neces-
sary to differentiate this type of pneumonia from that caused by viruses
(54, 89, 103, 104).

The other pleuropneumonia-like organisms isolated from mice (Li, L3,
Le and Types B, C, D, and E) produced arthritis in a variable number of
inoculated animals. The B strains (226) caused a migratory polvarthritis
in almost 100 per cent of mice injected intravenously or intraperitoneally.
The disease thus produced is chronic, non-fatal, and often results in ankylosis
with a pathological picture of proliferation of joint structures. The Le
strain causes arthritis in about 30 per cent of mice, whereas the Li strain
only occasionally affects the joint (72). Dienes and Sullivan (50), however,
have not succeeded in producing infection in mice with the Li organism.
Production of toxin by these strains has not been demonstrated.

The experimental disease (72) produced in mice by inoculation of the L7
strain, obtained from rats showing polyarthritis, is of considerable interest
because of its similarity to that described above as caused by Streptohacillus
moniliformis. In some animals swelling of the tibiotarsal joint, edema of the
subcutaneous tissues, and death followed inoculation of the foot pad with
cultures mixed with agar. With intracerebral inoculation, weakness of
the hind legs, hunched back, tremors, turning in circles, and occasionally
conjunctivitis developed. Intravenous and intraperitoneal administration
resulted in pleural or peritoneal exudation and arthritis in animals surviving
48 hours or longer. Intranasal instillation produced pneumonia in 5 to 8
days. In smears of animal tissues rings, granules, and comma-shaped
structures were seen. In spite of the fact that the Li strain, apparently
associated with StreptobaciUus moniliformis, and the L7 strain are distinct
and separate organisms, the points of similarity between the two diseases
provide an adequate basis for etiological confusion.

In general, animals other than the mouse are resistant to infection with
mouse strains, and even among mice considerable variation in susceptibility
is found between individuals and breeds. Mice surviving experimental
inoculation are resistant to reinjection with the same strain although no
humoral antibodies are demonstrable.

4o8 BIOLOGY OF THE LABORATORY MOUSE

The carrier incidence in stock mice has not yet been adequately deter-
mined, but may be as high as 40 to 80 per cent. This state probably
develops after birth from contact with the mother and persists throughout
life. Organisms have been cultured from the conjunctiva, nasopharynx,
lungs, and brain, but not from the blood, liver, spleen, kidneys, or intestinal
contents (72, 230). Natural antibodies have not been demonstrated in
such animals.

Etiology. — Pleuropneumonia-like organisms may be cultivated on special
agar media* directly from the lesions in mice, or from the conjunctiva and
nasopharynx, although they are more difficult to obtain from tissues of
normal animals. After 24 to 48 hours' incubation tiny colonies, 20 to
100 ju in diameter, may be seen under the microscope. Frequently they
have clear margins and dark centers and consist of granules, globules, and
fine filaments. Examination may most simply be made by staining the
colonies directly in the agar (48) with methylene blue or azure II, although
other special techniques have been employed (125, 126).

Growth of the organisms ordinarily occurs in liquid media as a faint
opalescence appearing after 36 to 48 hours of incubation at 37°C. Meat
infusion or nutrient broth containing 30 per cent ascitic fluid or sterile
serum is satisfactory. Addition of 0.5 per cent glucose is apparently
advantageous with some strains. Dark field examination reveals tiny
granules and occasionally globules and small filaments. On subculture to
solid media, the characteristic microscopic colonies appear. The various
strains may be differentiated to some extent by culture, but more satisfac-
torily by immunological methods. Sabin (229) has stated that the members
of the pleuropneumonia group of organisms found in mice are immunologi-
cally and pathogenetically different from those found in the rat.

The organisms are approximately 250 to 300 m/x in size, as determined
by filtration through gradocol membranes (225), and pass a Berkefeld V
filter. They are inactivated at 45°C. for 15 minutes but remain infective
for more than 30 days in 50 per cent buffered glycerin and for months if
frozen and dried by the Flosdorf-Mudd lyophile method. Toxin production
is thus far demonstrable with only one strain (Type A). The toxin appears
early during growth, lasts only about 2 days after its appearance, is inacti-

* A satisfactory medium may be prepared as follows: 5 per cent defibrinated blood
is added to 2 per cent meat infusion agar (pH 7.6 to 8.0), the mixture brought to the
boiling point, immediately cooled to about 5o°C., and the clear supernatant removed
after the coagulated blood has settled out. To this is added about 30 to 40 per cent
ascitic fluid before pouring into Petri plates.

INFECTIOUS DISEASES OF MICE 409

vated at 50°C. for 30 minutes, and is antigenic, producing an antitoxin
which neutralizes its action (225, 227). Antisera specific for the various
strains may be produced in rabbits. Organic gold preparations are bac-
tericidal for these organisms in vitro and are highly active in preventing
experimental infection (71).

Epidemic Pneumonia in Mice

Pneumonic lesions in mice may be found in several of the bacterial dis-
eases already described and in certain of the virus diseases. In addition,
apparently distinct epidemic respiratory infections associated with at
least three other bacteria have been reported. In each instance pneumonic
involvement has been a prominent part of the clinical and pathological
picture. A brief description of these epidemics follows.

Infection associated with Brucella bronchiseptica. — In 1920, Keegan
(118) reported an epidemic which occurred in an animal room containing
150 mice and 86 guinea pigs. The disease appeared first in the mice as a
prolonged illness causing death in a few of the animals. It was char-
acterized by a profuse purulent conjunctivitis with swelling of the eyelids
and desquamation and depilation of the surrounding skin, roughening of the
fur, nasal discharge, and occasionally death. A few weeks later the condi-
tion increased in severity, with the additional signs of rapid, labored breath-
ing and weight loss. Mice killed at this time showed a firm, greyish-white,
lobular consolidation of one or more lobes without pleuritis. The bronchi
were dilated and filled with thick purulent exudate. Microscopic sections
revealed purulent bronchitis and bronchopneumonia. The bronchi were
filled with polymorphonuclear exudate; the mucosa was thickened or
desquamated in some areas and mononuclear infiltration was present in the
walls and about the bronchi and blood vessels. Alveolar lesions consisted
of areas of polymorphonuclear exudate and partial or complete atelectasis.
During the latter part of the epidemic some of the animals succumbed
rapidly instead of after a prolonged course. At autopsy, hemorrhagic
lesions were present in the lungs, which presented a microscopic picture vary-
ing from marked capillary engorgement and serous alveolar exudation to
frank hemorrhage. Fifty of the 150 mice developed signs of illness. The
mortality was low but the incidence high since many mice, apparentlv
normal, showed pulmonary lesions when killed. Infection in the guinea
pigs developed late in the course of the epidemic, appearing first in those
cages closest to the mice.

4IO BIOLOGY OF THE LABORATORY MOUSE

Cultures from 6 of 25 mice autopsiecl and from 12 of 15 guinea pigs
showed Brucella hronchiseptica {B. hronchise pticus) . This organism is a gram-
negative, motile, cocco-bacillus which grows on ordinary media but does not
produce acid or gas from carbohydrates. An alkaline reaction is produced in
litmus milk, and ammonia is formed from urea and asparagin. Neither
hydrogen sulphide nor indol is formed.

As Keegan points out, the low incidence of positive cultures in mice
may have been due to the fact that tracheal cultures were not made. On
the other hand, B. hronchiseptica is not a highly pathogenic organism and is
frequently associated with other agents such as viruses — in canine distemper,
for example.

A pathologically similar condition which occurred spontaneously in
chronic form in approximately 3 per cent of stock mice has been reported b}'
Branch and Stillman (28, 29). No attempt was made to isolate the etio-
logical agent. The chief lesion is one of pulmonic consolidation occurring
irregularly in the various lobes but most often involving the right medial
lobe. Lesions may be multiple or may involve the whole of a single lobe.
Early in the disease the affected areas appear red, firm, dry, and hepatized;
later they become greyish, gelatinous, and translucent in appearance, and
the surface is irregularly granular and puckered. Pleurisy is rare. The
peribronchial lymph nodes are enlarged and the lesions tend to follow and
persist in the peribronchial and perivascular tissue. Microscopically, the
bronchial exudate contains many polymorphonuclear leukocytes, whereas
the areas of alveolar consolidation consist largely of mononuclear cells.
Fibrin is rarely found. Focal areas of necrosis are occasionally found.

The same disease occurs sporadically in the stock animals at the Jackson
Memorial Laboratory. It progresses slowly and is recognized in the late
stages by failure of the animals to thrive and breed, roughening of the fur,
weight loss, rapid labored breathing, and finally death. Investigations are
being carried out to determine the etiological agent and the possible rela-
tionship of B. hronchiseptica.

Infection due to a Friedlander-like bacillus. — During the course of his
investigations on experimental epidemics of mouse typhoid, Webster (318,
319) encountered two outbreaks of respiratory infection due to a Friedlander-
like bacillus. The disease first appeared in the summer (August) , recurred
in successive waves of decreasing severity, and disappeared in the spring.
The morbidity and mortality were high.

The clinical manifestations varied; some animals developed pulmonary
involvement, others septicemia, nasal infection, or the carrier state. The

INFECTIOUS DISEASES OF MICE 411

incubation period was about 48 hours. Transmission occurred via the
nasal passages by contact. When carriers were added to a group of 100 or
more mice, some died within 5 days, 50 to 70 per cent succumbed within
2 weeks, some contracted the disease and recovered, and others were refrac-
tory. Organisms were not found in the bedding, food, or feces. Intra-
nasal inoculation reproduced the pulmonic lesions with as few as 200 to
600 organisms.

At autopsy, subserous petechial hemorrhages characteristic of septicemia
and bilateral pneumonia were noted. The lungs were red and moist, on
section contained little air, and the fluid expressed from the cut surface
was viscid and stringy. Pleurisy was frequently present. The micro-
scopic picture varied with the duration of the lesion from interstitial conges-
tion, hemorrhage, edema, and serous alveolar exudate in the early lesions
to a cellular exudate of polymorphonuclear cells filling the alveoli. A
fibrinous and cellular exudate covered the pleural surfaces. In general, the
pathology was similar to that following experimental infection of mice with
Friedlander's bacillus (29).

Cultures of the nasal passages, lungs, and blood yielded a large, gram-
negative, capsulated bacillus which w^as morphologically and culturally
indistinguishable from Friedlander's bacillus {Klebsiella pneumoniae).
Better growth occurred at 23°C. than at 37°C. The organism, however, was
entirely distinct antigenically from five known types of Friedlander's bacillus.

Infection due to an organism resembling the influenza bacillus. — Kairies
and Schwartzer (116) have described a sporadic and epidemic disease due to
an organism resembling the influenza bacillus. Sporadically ill animals
showed weakness, shaggy coats, adherent eyelids, frequent abscesses on the
head, rapid respirations, and bronchopneumonic lesions in the lungs. Dur-
ing the epidemic in which 2 to 4 mice of a stock of 500 died daily, diarrhea
was an additional feature. Leukopenia resulted from infection with the
organism, the leukocyte counts varying from 1200 to 4000 with a decrease
in lymphocytes and an increase in segmented and stab forms. Cultures of
the pharynx, nose, eyes, abscesses, heart's blood, and lungs of diseased mice
yielded a tiny gram-negative cocco-bacillus showing bipolar staining in
young cultures but involution and thread forms in old cultures. Approxi-
mately 35 to 40 per cent of healthy animals were found by culture to harbor
the bacillus. Occasionally a hemolytic streptococcus was recovered with
the cocco-bacillary organisms from all parts of the body; rarely the strep-
tococcus alone was found in the respiratory tract. Although not strongly
hemoglobinophilic, the bacillus morphologically and culturally was almost

412 BIOLOGY OF THE LABORATORY MOUSE

impossible to distinguish from Pfeiffer's bacillus and the name ''Bacterium
influenzae murium'''' was accordingly proposed. Certain aspects of the
spontaneous illness, as well as the hemorrhagic exudation produced by
experimental inoculation, resemble mouse pasteurellosis (hemorrhagic
septicemia), from which it should be differentiated. In view of the fact
that some cultures in liquid media contained tiny bodies passing a membrane
with a pore diameter of 400 to 600 m^l, a relationship may exist between
these forms and the pleuropneumonia-like organisms or the cocco-bacilli-
form bodies of infectious catarrh of mice (178).

Spontaneous pneumonia due to the pneumococcus has not been reported,
although the isolation of this organism from mice has been claimed (194).
A partial explanation may be found in experimental attempts to produce
pneumonic lesions. Following inhalation of virulent Type I pneumococci,
which killed rapidly by other routes of inoculation, the organisms were
found in the lungs only up to 3 hours after administration (256). Pneu-
monia did not result unless the animals were partially immune (258, 259)
nor did systemic infection follow (139, 256) unless the animals were intoxi-
cated with alcohol (257). Experimental pneumonia readily occurred
following inhalation of virulent hemolytic streptococci and Friedlander's
bacillus (256, 29). Later work, however, has demonstrated that certain
strains of pneumococci, inoculated intranasally, produced in some mice a
fatal respiratory and general infection with septicemia, pneumonia, pleurisy,
empyema, pericarditis, and cervical lymphadenitis (325). By balancing the
virulence of the organism and the resistance of the mice, it was possible to
produce pneumonia with a consistency which permitted study of the pathol-
ogy and pathogenesis of the infection (213, 214).

Cultures of the lungs of normal mice (112) have yielded organisms of the
streptothrix type {Streptobacillus), B. subtilis, and various kinds of cocci.
Similar organisms were found in the dust from hay and straw, and by with-
holding such substances the number of contaminating organisms was
reduced. These bacteria were non-pathogenic when injected subcuta-
neously, however, and probably are of little importance in the production
of disease.

Control measures to be undertaken in the event of an outbreak of
respiratory infection are simply those of isolation and the usual precau-
tionary procedures to prevent spread. In an unusually valuable stock,
chemotherapy with one of the sulfonamide drugs (sulfanilamide, sulfa-
pyridine, or sulfathiazole) might be attempted, although no information is
available regarding the efhcacy of any of them in these infections.

INFECTIOUS DISEASES OF MICE 413

Infectious Catarrh of Mice

This disease has been described by Nelson (178, 179, 180) as an epidemic
occurring in an isolated group of Swiss mice and their offspring, together
totaling approximately 800 mice. The condition was chronic in nature,
but spread so widely through the colony that after 10 months all but 75 of
the animals were killed. During the next 11 months 72 of the 75 mice
died — a mortality of 96 per cent. An endemic type of the disease has been
noted in one colony in which only sporadic cases were observed.

Signs of illness appeared after an incubation period of 10 or more days.
Intermittent "chattering" was commonly the first evidence of infection

Fig. 159. — Infectious catarrli of mice; characteristic posture, ruffing of hair, and
abrasions about the ear. {From Nelson, J . Exp. Med.)

and became more constant as the disease progressed. This sound is dis-
tinctive, apparently is produced in the lower part of the respiratory tract,
and resembles that made by rapid, gentle clicking of the teeth. Rhinitis,
found at autopsy but not associated with visible nasal discharge, appeared
early, as did snuffling. Some of the animals showed ruffled fur, rapid, shal-
low respiration, weight loss, and death 3 to 5 weeks after the appearance of
chattering. Others appeared to be normal, except for chattering, for many
weeks but eventually developed considerable loss of hair and scabby skin,
occasionally marginal necrosis of the ear, and terminal respiratory diffi-
culty (Fig. 159). True conjunctivitis infrequently occurred. The disease
was invariably transmissible to normal animals by direct contact and by
intranasal instillation of exudate from the respiratory tract and middle ears
of naturally infected mice.

Postmorten examination of 45 animals, either naturally or experi-
mentally infected, revealed rhinitis in 43, otitis media in 43, and pneumonia
in 35. A thick, semi-fluid, mucopurulent exudate was present in the nasal
passages. Stained films of the discharge showed the " coccobacilliform "
bodies to be described later, many leukocytes, and mucus strands. The

414 BIOLOGY OF THE LABORATORY MOUSE

tympanic cavity was tilled with a copious, purulent exudate containing
many white blood cells. The pneumonia, which was progressive and finally
resulted in death of the animal, was usually lobar in distribution. The
involved area was consolidated, contracted, and red, grey, or mottled in
color. In advanced cases, all lobes were sometimes involved. Bronchial
exudate of polymorphonuclear cells, secondary alveolar extension as indi-
cated by leukocytes, erythrocytes, large mononuclear cells, and fluid in the

alveoli, and hyperplasia of the
peribronchial lymphoid tissue were
the chief features microscopically.

In stained films of exudate from
the nares, middle ears, and lungs of
diseased animals were found small
gram-negative cells, termed "cocco-
bacilliform bodies" because of their
* similarity to those of fowl coryza
{ (Fig. 160). The organisms were

generally spherical, but rod-shaped
cells and ring forms were seen.
-^k rhey occurred singly, in pairs, and

1 • in loose clumps. Although pre-
dominantly extracellular, they were
Fig. 160. -Infectious catarrh o! mice; ^j^^ f^^j^^ -^^ polymorphonuclear
scattered extracellular coccobacilhform j^^j^^^^^ ^^^ epithelial cells,
bodies in nasal exudate. Gram stain. -^ 1 • 1

(X920.) {From Nelson.) Attempts to cultivate the organism

on "ordinary nutrient media
enriched with blood" were unsuccessful, although growth occurred in tissue
culture and in the supernatant fluid of tissue culture media. In several
instances, pure cultures of the coccobacilliform bodies were obtained,
especially from the middle ear; in others, the organism was found in associa-
tion with staphylococci, streptococci, or an unidentified short, non-motile
gram-negative bacillus, termed the "X" bacillus.

The average diameter of the coccobacilliform bodies by direct micro-
scopy was between 0.3 and 0.4 ^t. The bodies passed through a collodion
membrane with an average pore size of 640 m^t, which indicates an average
particle diameter of 480 m^. Such filtration, however, did not separate the
organism from the X bacillus.

The etiological relationship of the coccobacilliform bodies to mouse
catarrh seems to be established since the disease was reproduced by pure

INFECTIOUS DISEASES OF MICE 415

cultures after as many as twelve subcultures in tissue medium. Filtration
through Berkefeld V candles apparently removed the organism and excludes
the possibility of a filterable virus, since filtrates were not infective. The X
bacillus was not pathogenic.

The possibility has not been excluded that this disease is related to that
described by Kairies and Schwartzer (116) as being due to an influenza-like
bacillus. It seems probable, however, that the mouse catarrh is a distinct
entity. The similarity of the coccobacilliform bodies to the pleuropneu-
monia-like organisms is striking and warrants further investigation.

Pyogenic Infections, Botriomycosis

Pyemic and suppurative lesions are frequent in mice as well as in other
laboratory animals. From subcutaneous abscesses may be cultured such
organisms as Staphylococcus aureus or albus, Gafkya tetragena {Micrococcus
tclragenus), hemolytic or non-hemolytic streptococci, rarely Bacillus pyo-
cyancus, and others of less importance. The lesions may arise at the site
of incarcerated worm rests and show a variable bacterial flora (299).
Abscesses in the heart and lungs, from which pure cultures of both white and
yellow micrococci were usually obtained, have been found in 3 per cent of
12,000 autopsies on mice (299). Certain of the cocci — Gajfkya tetragena,
for example — are very pathogenic for white mice, septicemia and death
occurring within 2 or 3 days after experimental inoculation by almost any
route (73).

Abscesses about the head and neck have been observed not infre-
quently in the mice at the Jackson Memorial Laboratory and have been
found in one instance by Tyzzer (297). Pathologically, the condition
resembles that termed ''botriomycosis." The lesions are walled oft" and
composed of areas of granulation tissue enmeshed in fibrous strands with
numerous areas of dense polymorphonuclear exudation. Scattered through
the lesions are granules, irregular in outline, and surrounded usually by
polymorphonuclear exudate. Many show a rather homogenous outer rim,
staining pale blue with hemotoxylin and eosin, while the remainder of the
granule varies from deep blue to pink. Occasional club-like excrescences
may be seen. The structure of the granules may be granular and amor-
phous, or may suggest a central cellular appearance, likened by some inves-
tigators to cocci embedded in zooglial substance (287, p. 1181). Although
the disease in mice has not been adequately investigated, considerable
evidence is accumulating which indicates that in other animals the lesions
are due to staphylococci (287, pp. 1180--81; 124, 123).

4i6 BIOLOGY OF THE LABORATORY MOUSE

Kutschera (136) observed a spontaneous epidemic in white mice due to
a streptococcus, although a staphylococcus was present as well in many
animals. The affected animals appeared ill, their eyelids were adherent,
hair roughened, and breathing rapid. At autopsy the spleen was enlarged
to three or four times normal size and studded with yellowish, pin-head
sized abscesses. Of 30 mice studied bacteriologically, most showed a
double infection with streptococci and staphylococci. Organisms were
seen in smears of the liver, kidney, spleen, heart, and bone marrow. Experi-
mental inoculations of normal mice with organ suspensions of infected
animals produced death in i to 2 days, the findings resembling those of the
spontaneous disease without abscesses. Subcutaneous inoculation with a
pure culture of the streptococcus resulted in death in 3 days. A local
abscess was formed at the injection site, about which the tissues were
hemorrhagic; punctiform hemorrhages were present in the peritoneum,
intestines, and testicles, and the spleen was swollen. Similar experimental
infections with streptococci have been obtained by other workers (139, 300).

Although such epidemics are rare, apparently normal mice may harbor
streptococci (341). Cultures of the blood of 35 white mice were positive
for streptococci in two instances. After injection with sterile milk, adren-
alin, or plague vaccine, 6 of 35 mice showed streptococci by blood culture.
The strains obtained were not identical in their cultural reactions and only
one produced hemolysis, although all were gram-positive cocci, growing in
long chains.

Infection with Bacillus Piliformis

A highly fatal bacterial disease in Japanese waltzing mice has been
reported by Tyzzer (295). The disease spread among this inbred stock in
epidemic fashion, affected a few hybrids of the first and second filial genera-
tions (Fi and Fo), but did not involve the common laboratory mice. It
presumably originated from the common mouse during cross-breeding
experiments, although the organism was not found in stained sections of the
intestines of many laboratory and wild mice.

Signs of infection appeared 24 to 48 hours before death and consisted
of roughened fur, ataxia, and watery or slimy diarrhea. In young animals
the disease was more acute, with diarrhea the prominent feature. Diagnosis
could be made in some animals by removing the fur from the abdomen and
viewing the lesions on the ventral surface of the liver through the transparent
abdominal wall. The time of death varied from 6 to 44 days after exposure,
with an average of 10 to 20 days.

INFECTIOUS DISEASES OF MICE 417

At autopsy the only gross lesions were found in the liver, which was
enlarged and contained a varying number of opalescent, grey or yellowish
nodules. These nodules were usually discrete, varying from less than 0.5
mm. to more than 2.0 mm. in diameter (Fig. 161). Microscopically, the
lesions were situated in close proximity to the portal vein, suggesting an
embolic distribution. They consisted of necrotic tissue with an extensive
peripheral polymorphonuclear infiltration. Hepatic cells about the lesions
contained many long, slender bacilli, lying roughly parallel to one another,
each organism separated from the adjoining one (Fig. 162). Numerous
spores were present as well as vegetative forms apparently undergoing
sporulation. Organisms were frequently
present in the gall bladder and bile.
Although no visible lesions were present
in the alimentary tract, microscopic sec-
tions of the cecum and first portion of
the large intestine revealed many bacilli
and spores within epithelial cells,
phagocytes, lymphatics, and in the
depths of the glands (Fig. 163). Almost
no host reaction was present.

The organism was a slender, non- Fig. 161.— 5. pUijormis infec-

motile, non-acid fast, gram-negative tion in mice; gross appearance of
bacillus, showing considerable pleomor- lesions in liver. {From Tyzzer, J.
phism. Some organisms presented a ^ ^ '

granular, band-like appearance. Spores were situated sub terminally.
One attempt to demonstrate the heat resistance of the spores was incon-
clusive, but a contaminated cage remained infective after one year at room
temperature. All attempts to grow the organism in pure culture on enriched
culture media failed, except for one occasion when it grew briefly in symbiosis
with a streptococcus.

Infection could be best produced experimentally by contact of susceptible
mice with diseased ones or with a contaminated cage, and by ingestion of
infected tissue or intestinal contents. Common laboratory mice, rabbits,
and guinea pigs were resistant. Intravenous injection of waltzing mice
with large doses produced the t>'pical liver lesions and death, but minimal
lesions and immunity followed small doses. Intraperitoneal and subcuta-
neous administration did not result in systemic disease. On the basis
of these findings, together with the pathological picture, Tyzzer
postulated that the fatal disease was produced by a secondary embolic

4i8

BIOLOGY OF THE LABORATORY MOUSE

invasion of the liver following primary infection of the gastro-intestinal
tract.

This disease is particularly interesting because of its limited host sus-
ceptibility. Further study from the point of view of heredity (go) has

^^.ri

Fig. 162. — Banded bacilli at the pcrii)hL'ry of a liver lesion in a mouse <lying of B.
piliformis inlection. Stained section (X1400). {From Tyzzer.)

Fig. 163. — Smear of intestinal epithelium from a mouse dying of B. pUiformis
infection. Note intracellular bacilli having the appearance of spore-formation.
Spore stain. (X1400.) {From Tyzzer.)

indicated that predisposition to infection is independent of the waltzing
factor, the dominant white factor, and sex. The condition, moreover,
may become of greater practical importance, since Tyzzer (297) has recently
found it in highly inbred stocks used for cancer studies.

Van Rooyen (301) has inferred that this disease may be the same as
that produced by Streptobacilliis moniliformis. Apart from clinical difTer-

INFECTIOUS DISEASES OF MICE 419

ences, it is difficult to reconcile the identity of the two organisms because
of the morphological appearance of the spores of B. piliformis, the failure of
this organism to grow in serum-enriched media on numerous attempts, and
the persistence of infective material in a contaminated cage for a year.

Infection Due to Bartonella, Eperythrozoon and Grahamella

Three types of organisms which parasitize red blood cells have been
found in mice, as well as in other rodents. None produces obvious clinical
disease under natural conditions. Infection with two of them, Bartonella
and Eperythrozoon, remains latent until manifested by splenectomy, expo-
sure to x-ray, or infection with an unrelated agent. The grahamellae, on
the other hand, are less notably affected by such events. In Table 4 are
summarized the chief characteristics of these three organisms.

The exact classification of these organisms has been subject to con-
siderable doubt and disagreement. Bartonellae and eperythrozoa have
been thought by some to be protozoa, by others to be bacteria or Rickettsia-
like organisms. Certain authors, in addition, have considered the graha-
mellae to be basophilic granulations in the erythrocytes. Although final
classification cannot yet be made, it seems probable that the organisms are
closely related to bacteria in view of their morphology and behavior. An
excellent review of bartonella and eperythrozoon infections up to 1935 may
be found in the monograph by Weinman (331).

Infection with Bartonella. — Bartonellosis as a disease is perhaps best
known in man (Oroya fever) and in the rat (infectious or "pernicious"
anemia of rats), but it occurs in many other species of animals, especially
in small rodents (164, 331, 333). Bartonellae or bartonella-like organisms
have been reported in splenectomized white mice (185, 238, 171, 239, 63,
121, 167, 60), white-footed deer mice, Peromyscus leucopus (296), and wood
mice, Mus sylvaticus (34) . Infection has also occurred following the inocula-
tion of mice with trypanosomes (164, 156), although it is not clear whether
the strains so obtained had their origin in the mouse or were introduced
with the trypanosomes. In spite of the fact that some investigators (303,
157, 158, 155) have apparently found spontaneous infection in mice with
considerable consistency, the condition must be rare, if not absent, in many
mouse stocks. In a combined series of over 100 splenectomized mice (165.
2, 302, 298), no bartonellae were found.

The natural disease. — No instances of spontaneous clinical disease have
been reported in mice, although on rare occasions bartonellae may be seen
in the blood of naturally infected animals (171). Following splenectomy,

420

BIOLOGY OF THE LABORATORY MOUSE

however, latent infections become apparent, and organisms may be seen
in great numbers in the blood after an incubation period varying from i to
4 days. The organisms increase in number for 5 or 6 days, then gradually

Table 4
Comparison of Bartonella, Eperythrozoon, and Grahamella*

Bartonella

Eperythrozoon

Grahamella

Morphology

Bacillary and coccoid;
occasionally ring-like

Delicate rings or disks;
fine short rods

Coarser, bacillary; no
coccoid forms

Staining:
Giemsa

Other aniline dyes

Red to reddish-blue;
less intense

Poorly, if at all

Bluish-red or violet;
faintly except for or-
ganisms applied to
margins of erythro-
cytes

Poorly, if at all

Blue; more intense
More easily stained

Location

Often epiglobular or
free

Epiglobular or free;
usually a preference
for polychromato-
philic cells

Endoglobular; uni-
formly spaced in
cells

Number of organ-
isms on or in affec-
ted cells

Few

Numerous

Few or numerous

Effect of splenec-
tomy

Latent infections be-
come apparent

Without marked effect

Chemotherapy with
arsenical c 0 m -
pounds

Tends to sterilize

No effect

Natural transmission

Arthropod vector

Unknown

Pathogenicity

Pathogenic

Usually non-patho-
genic

Non-pathogenic

Cultivation on arti-
ficial media

Sometimes successful

Not proved

Not yet determined

* Modified from Bruynoghe and Vassiliadis (34) and Tyzzer and Weinman (298).

disappear until at the end of 3 weeks they may be found only with diffi-
culty (238, 171, 158). Smears of the blood at the height of the infection
reveal organisms in connection with a relatively high proportion of the

INFECTIOUS DISEASES OF MICE 421

erythrocytes. The organisms are few in number per red blood cell and
appear to be on or possibly in some instances in the cell itself. A few may
be found at times free in the plasma, possibly released by destruction of
infected erythrocytes. Ordinarily, splenectomized mice show no signs of
infection (238, 171), although some animals develop anemia (303, 155, 167).
Pathological examination of splenectomized mice reveals little beyond
possible hyperplasia of endothelial cells and foci of lymphocytes in the liver
(55). Carrier mice may show some degree of splenomegaly which, how-
ever, may occur in the absence of infection of this t>pe. The manner of
spread in mice has not yet been established. Arthropods may be the
vectors since in rats the flea {Hacmatopinus spinulosis) can transmit the
infection (64, 331).

iMixed infections with bartonella and Eperythrozoon coccoides may follow
splenectomy (238, 157, 158. 121, 60). Lwoff and Vaucel (157, 158) believe
that under these conditions the virulence of the Bartonella may be increased.

The administration of arsenicals to both rats and mice is effective in
eradicating latent bartonellosis or in treating the more acute infection which
follows splenectomy. Dosages recommended (34, 122) are approximately
12.5 mg. of neosalvarsan or 100 mg. of tryparsamide per 100 grams of body
weight. A combination of arsenic and antimony (Bayer's "sdt 386") is
stated to be particularly efficacious (122). Sulfanilamide has been found
to be unsatisfactory in the rat disease (65). Domagk and Kikuth (55) have
emphasized that the effective dose of the chemotherapeutic agent must not
injure the reticulo-endothelial system if the results are to be satisfactory.

The experimental disease. — Numerous attempts have been made to
transmit the mouse strain of bartonella to both normal and splenectomized
mice free from the disease, with varying degrees of success. In some
instances, transmission has failed (185, 121, 60); in others, massive and at
times fatal infection with anemia has resulted even in normal mice (156,
60). The work of Lw^off, Provost, and Vaucel (156, 303, 158, 155) is of
some interest in this regard. A non-splenectomized mouse, inoculated
with Trypanosoma cruzi. developed a bartonella infection, which was then
transferred successively in normal mice by inoculation of blood, and was
ultimately separated from the trypanosome. The trypanosome had been
passed through two rats and one dog before inoculation into the mouse.
None of the mice was resistant to the bartonella infection. The organisms
appeared usually on the second day, increased in number until the fifth or
sixth day, and disappeared about the ninth day. Anemia and splenomegaly
were the only significant pathological changes reported. No recurrences

422 BIOLOGY OF THE LABORATORY MOUSE

were noted even in those animals in which a fatal infection with the trypan-
osomes subsequently developed. Inoculation of rats produced an infec-
tion of varying severity, but not all rats were susceptible. The organism
was cultivated in Noguchi's medium (184), and was termed "virus
spontane." A second strain of bartonella, termed "virus provoque," was
obtained following splenectomy of normal mice. The clinical course of the
two infections was similar, and no morphological differences were noted in
the two strains. Other dift'erences were described, however. The "virus
provoque " was not cultivated. It did not infect normal mice (non-splenec-
tomized) or splenectomized mice which had recovered from the homologous
infection, although the latter animals were susceptible to the "virus spon-
tane." Mice, recovered from the "virus spontane," acquired the "virus
provoque" after spelenectomy without alteration in the incubation period
or duration of the infection. On the basis of these data, the two strains
were considered to be different immunologically.

Evaluation of this work is difficult. The possibility exists that the
"virus spontane" may originally have been derived from the rat. Normal
mice are susceptible to Bartonella muris of the rat (238, i), and although
many authors (331) consider the rat and mouse strains to be identical,
definite differences have been reported (238, 167). In favor of the mouse
origin of the "virus spontane" is the fact that a similar strain of high
virulence was obtained from a splenectomized mouse having a mixed infec-
tion with bartonella and Eperythrozoon coccoides (157, 158). Kikuth (121),
however, encountered a similar mixed infection but was able to transmit
only the eperythrozoon to other splenectomized mice.

As already mentioned, Bartonella muris of the rat may be transmitted
to mice by inoculation of infected rats' blood. Although many animals
show no signs of infection (122), a fatal anemia may develop both in normal
and in splenectomized animals (238, i, 2, 239, 167, 3). Repeated passage of
the organism in young normal mice may apparently increase its virulence
until most of the animals succumb from the infection (i). In such animals
all the erythrocytes may be infected by the fourth or fifth day and the num-
ber of red blood cells may fall from 9,000,000 per cu. mm. at the time of
infection to 1,000,000 per cu. mm. at the time of death on the fifth or
seventh day. Hemoglobinuria, common in rats, rarely occurs in mice.
In animals which recover, the destruction of the organisms is sudden, con-
stituting a crisis in which the number of infected cells falls from 100 per
cent to less than i per cent within 24 hours. The spleen is uniformly
enlarged (three to four times normal size) , and in recovered animals there is

INFECTIOUS DISEASES OF MICE 423

extensive phagocytosis of red blood cells by the pulp cells. In splenec-
tomized mice (2) the course is generally more acute than in normal mice, but
varies with the age of the animal. In those younger than 3 months, infec-
tion is chronic for a period as long as 4 months, with intermissions and
relapses. The red cell count may fall to approximately 2,000,000 per
cu. mm., and mononucleosis up to 18 per cent may occur. Most of the
animals recover. On the other hand, infection in splenectomized mice
6 months or more of age is usually acute and terminates fatally from 3 to
12 days after B. miiris appears in the blood.

The function of the spleen in relation to immunity in bartonellosis is of
considerable interest and has been the subject of much investigation in rats
^75- 76. 77. 64, 166, 4, 332), but a satisfactory explanation has not yet
been obtained. Weinman (332) has shown that the anemia has no apparent
relationship to immune bodies in the serum, but is hemolytic in type and
due to direct action of the organism on the erythrocytes. In mice, as in
rats, the spleen seems to offer some barrier to infection, but here also its
role in immunity is not clear. By partial ablation, Adler (2) has demon-
strated that about 30 per cent of the spleen is sufficient to control infection.
Repeated injections of B. muris cause splenomegaly, but an immunity per-
sisting indefinitely after splenectomy does not result. It seems likely that
the immunological aspects of this disease will not be clarified until cultural
methods are employed and larger numbers of virulent organisms are used for
immunization.

Tyzzer (296) has recently demonstrated that the natural bartonella
infection in white-footed deer mice {Peromyscus leucopus) can be trans-
mitted to splenectomized normal white mice. A severe and occasionally
fatal anemia results. If the splenectomized mice suffer from infection with
Eperythrozoon coccoides, however, the bartonella infection is prevented from
developing or suppressed if already established. On the other hand, the
mouse eperythrozoon may be transmitted to the splenectomized vole (Micro-
ius pennsylvanicus) , but does not interfere with natural bartonellosis in this
animal. Such phenomena of interference, first noted with viruses, have as
yet been observed in relatively few instances and the mechanism is not
known. The phenomenon must be kept in mind, however, since, as Tyzzer
points out, experimental results may be altered by the presence of unrec-
ognized infection.

Etiology. — The bartonellae of rodent origin are small, pleomorphic,
gram-negative bacteria which vary in width from o.i to 0.5 ^i and in length
from 0.5 to 2.0 ^l. Variations from coccoid to bacillary form mav be seen

424 BIOLOGY OF THE LABORATORY MOUSE

(185, 34, 122). Ring forms occur in certain species, as well as long rods
segmented along their axes in a manner suggestive of division. Cultivation
has been successful on Noguchi's leptospira medium and on other media
(184, 158, 155, 142, 143, 304, 331, 333) after a period of i to 2 weeks of
incubation. The optimum temperature is between 25° and 28°C. Cul-
tivated strains may be motile. Growth has also been obtained in egg
embryos (304).

In blood smears colored by Giemsa's method the organisms stain a light
reddish-blue color. They appear to be on or within the red blood cells
(Fig. 164). At the height of the infection, the majority of the cells contain

Fig. 164. — Bartonella in the peripheral blood of a spontaneously infected, splen-
ectomized white mouse. (From Schilling.)

organisms, although the number of organisms on a given cell is usually
small — from i to 10. Placement on the margin of the erythrocytes is com-
mon. Organisms have not been demonstrated in endothelial cells apart
from those present in phagocytized erythrocytes. The strains found in mice
are reported (238, 167) to be smaller and finer than the rat strains, with a
greater tendency to the formation of long, thin, bacillary forms.

Schilling (238) has proposed the name Bartonella muris musculi for the
mouse organism to differentiate it from B. muris {B. muris ratti) of the rat.
Since such nomenclature becomes cumbersome and because of definite
differences in the human and animal bartonelloses, Tyzzer and Weinman
(298) have proposed two genera for bartonella organisms: Bartonella, type
species B. bacilliformis, to include bartonella which multiply within cells
(vascular endothelium) other than erythrocytes and which produce wart-
like or nodular cutaneous eruptions, and Haemobartonella, type species
H. muris, to include bartonellae in which there is no demonstrable mul-
tiplication outside the blood and which do not produce cutaneous eruptions.
The known animal strains would thus be classified in the genus Haemo-
bartonella.

Eperythrozoon infection in mice. — In 1928, Schilling (237) and Dinger
(51) almost simultaneously discovered a new ring-like organism in the
blood of splenectomized mice and concluded that it differed from the

INFECTIOUS DISEASES OF MICE 425

bartonellae. These observations have been amply confirmed, and Schil-
ling's name of Epcrythrozoon coccoides has been adopted for the mouse
strain on the basis of priority.

Occurrence. — The infection is apparently widely distributed geo-
graphically, and has been described in various strains and stocks of mice in
Europe (52, 238, 32. 120, 167), in Africa (302), and in America (63, 77, 163).
Both laboratory and wild mice may harbor the organisms. Not all stocks of
mice carry the infection, however, since Marmorston (163) found that 5 of 8
inbred strains were, free from the disease. The incidence of infection in
carrier stocks is usually high, varying from 50 to 100 per cent.

The natural disease. — As in bartonellosis, severe spontaneous infection
with E. coccoides does not occur. Splenectomy is usually required to permit
detection of the organisms in the blood, although rare organisms may be
seen in animals having latent infections (237, 32, 33, 167). Other insults to
the host, such as x-ray irradiation or experimental lymphatic leukemia (163),
may be followed by the appearance of eperythrozoa in the blood stream.
The only significant pathological change in carrier mice is an increase in the
weight of the spleen to approximately twice normal (163). Histological
examination may reveal phagocytosis of infected erythrocytes, but does not
show a concentration of organisms in the spleen (52, 63).

Following splenectomy, the organisms appear in the blood after an
interval of from i to 19 days, but the usual period is 2 to 4 days (237, 51, 52,
32, 63, 120, 122, 167, 163, 331). During the next 5 days they increase in
numbers rapidly, showing a definite preference for polychromatophilic
erythrocytes. At the height of the infection, almost all the red cells may be
involved, but certain cells show an extreme degree of parasitism, containing
20 to 40 or more organisms which entirely cover the surface or form cap-like
colonies. Free organisms are present in the plasma. The organisms then
rapidly diminish in number in the course of 2 or 3 days, but may persist in
small although variable numbers up to 6 months. The great majority of
mice have no clinical signs of disease. A few may show milled fur and slight
weight loss at the height of the infection. The blood changes are slight —
mild anemia, increase in reticulocytes and polychromatophilic cells, and
inconstant leukocytosis. Xo significant pathological changes are found.
One exception to the usual clinical course has been reported by Galli-\'alerio
(83). Nine months after splenectomy and the initial infection the mouse
sickened, lost much of the hair about the head, and died. Autopsy revealed
emaciation, viscid conjunctival secretion, pale and edematous muscles, large
soft liver, and dull red kidneys. Large numbers of Eperythrozoon coccoides

426 BIOLOGY OF THE LABORATORY MOUSE

were found in the red cells and free in the plasma. No other cause of death
was determined, although it is doubtful if an adequate search for other
infectious agents was made.

A strain of Eperythrozoon, termed E. dispar and differing from E.
coccoides in morphology and pathogenicity, has been found in field mice
(Arvicola arvalis) and dwarf mice {Mus minutus) by Bruynoghe and Vas-
siliadis (32, 33, 37), and in the vole by Tyzzer and Weinman (298). No
clinical signs or special alterations of the blood were noted.

Several instances of mixed infection with bartonellae and eperythrozoa
have been described (157, 158, 121, 163, 60). In view of the interference
which Tyzzer (296) found in white mice between E. coccoides and the deer
mouse strain of bartonella, it is interesting to note that Marmorston (163)
encountered the same phenomenon in splenectomized mice spontaneously
developing infection with natural strains of both organisms. In each of
4 animals showing mixed infections, the bartonellae became evident only
after the eperythrozoa had disappeared. Moreover, when blood of these
animals was injected into young splenectomized mice, only the E. coccoides
developed.

Transmission of E. coccoides from infected to uninfected splenectomized
mice by the mouse louse, Polypax serrata, appears to be a natural method
of spread (62). Attempts to induce infection by other vectors, by contact,
by a deficient diet, and by hereditary transmission have all been unsuccessful
(52,63, 167,331).

Chemotherapy with arsenical compounds is effective in preventing or
eliminating infection with E. coccoides (32, 33, 120, 122).

The experimental disease. — Infections with E. coccoides may be readily
transmitted to splenectomized white mice free from the disease by sub-
cutaneous or intraperitoneal inoculation of blood from an actively or
latently infected mouse (237, 52, 32, 33, 331). The resulting disease is
entirely similar to the natural infection. Injection of normal, disease-free
mice produces a latent infection which may at any time be activated by
splenectomy (331, 163). Reinoculation of chronically infected splenec-
tomized mice is without effect (52, 331), but if the organisms are eradicated
by arsenical therapy a second inoculation will reproduce the original
infection with little or no evidence of immunity (32, 33). Attempts to
infect by the oral route have been unsuccessful (331). Citrated blood
retains its infectivity for 5 days but not for 10 days at 5°C. (331).

Inoculation of other species of animals with E. coccoides has given con-
flicting results. In normal rats a latent infection, becoming evident after

INFECTIOUS DISEASES OF MICE 427

splenectomy, has been produced (63, 77), whereas hi splenectomized rats
immediate infection appeared (52, 32, ^^, 63, 122). McCluskie and Niven
(167), however, failed to confirm these results. Infection of splenectomized
rabbits has been successful in some hands (35, 37), but not in others (120).
Certain individual splenectomized voles {Microtus pennsylvanicus) have
been found to be susceptible (296).

•J ^**m.V<

►•% • ® **

• • ' A

Fig. 165. — Epcrytlirozooii coccoidcs. Spontaneous infection in splenectomized
laboratory mouse. Note organisms on the margins of the erythrocytes and free in
the plasma. Giemsa. (X900.) {Courtesy of Dr. D. Weinman.)

The role of the spleen in this infection appears to be entirely similar to
that in bartonellosis. No satisfactory attempts to demonstrate immunity
have as yet been made.

Etiology. — Eperythrozoon coccoides characteristically appears in the
blood of infected animals as a small gram-negative ring- or disk-shaped
organism (Fig. 165), occurring either attached to the surface of erythrocytes
or free in the plasma (237, 51, 52, 167, 63, 163, 331). It may be visualized
by dark field technique or in dry blood smears stained with Wright's or
Giemsa's stains. The rings are fairly uniform, approximately i ju in diam-
eter, and have an unstained central zone with a bluish-red or violet periphery.
Masses of cytoplasm at one or two points in the circumference may stain
more intensely, giving the appearance of polar bodies. Some variation in
shape occurs and racquet- or club-like forms may be seen. In addition, fine
bacillary and tiny coccoid forms occur, quite distinct from the rod-like
appearance of the rings which are viewed on edge. Organisms attache? to

428 BIOLOGY OF THE LABORATORY MOUSE

erythrocytes typically stain more intensely than those free in the plasma.
Other stains, such as hemotoxyhn, cresyl-blue, and azure II, stain them
poorly if at all. No motility has been noted by darkfield examination.

E. dispar (32, 33, 37, 298), found in Mus minutus, Arvicola arvalis, and
Microtus pennsylvanicus, is predominantly discoid in its morphology but is
differentiated from E. coccoides chiefly by its animal pathogenicity. E.
coccoides does not infect Mus minutus and Arvicola arvalis, whereas E. dispar
fails to infect rats and white mice.

Attempts to cultivate the organism have in most instances been unsuc-
cessful. Dinger (52) has reported cultivation on a coagulated egg medium in
Locke's solution. Although no ring forms were seen in the culture, inoculation
of a splenectomized mouse after three transfers produced the typical disease.

Grahamella infection in mice. — In 1905, Graham-Smith (91) described a
new type of organism situated within the red blood cells of the mole. The
organisms stained blue with Giemsa's dye, and appeared as long or short,
curved, irregular rods, occasionally wedge- or club-like in shape. Dark
chromatin masses sometimes occurred at one or both ends of the rods. The
only pathological changes found were areas of focal necrosis in the livers of
the most severely infected animals. This observation was soon confirmed
by Thomson (272), who further noted that the bodies were non-acid-fast and
gram-negative. He could not transmit them to the rabbit, guinea pig, rat,
or mouse. Brumpt (31) in 191 1 proposed the generic name of Grahamella for
these organisms. Similar structures have subsequently been found in the
erythrocytes of the common laboratory and wild mice and related species
(212, 45, 21, 34, 36, 234). Classification of these organisms has not been
settled (31, 96, 141, 36), and probably will not be established until cultiva-
tion has been achieved.

The incidence of the condition in mice may be high (34) , but no adequate
statistics are available. Infection does not result in clinically apparent
disease or in recognizable pathological changes. Splenectomy is without
pronounced effect (34). Examination of the blood of infected animals
reveals that the organisms are generally restricted to the erythrocytes
(45, 298) and may appear or disappear irregularly in a given animal (36).
The percentage of affected cells is low, even in heavy infections (34, 2, 122),
but the number of organisms in such cells is usually high, varying from about
5 to 20 or 30. The grahamellae differ from the bartonellae and eperythrozoa
in being more or less uniformly spaced within the erythrocyte. They are
considered to be non-pathogenic (2, 122), and attempts to transmit them to
other animals have failed (141, 36), although final evaluation must await

INFECTIOUS DISEASES OF MICE 429

further work. Chemotherapy with arsenical compounds has no apparent
effect (34).

The mouse grahamellae, termed CrahameUa musculi by Benoit-Bazille
(21), somewhat resemble bacillary bodies in their morphology (45, 21, 34,
120, 122). They appear as rather coarse, irregular rods which may be club-
shaped or beaded (Fig. 166). Variation in length (0.5 to 2 /x) is greater than
that in width (0.2 to 0.3 fx). They are stained a more intense blue by
Giemsa's method and are more azurophilic than the bartonellae and epery-
throzoa. Other aniline dyes also stain
them well. No adequate attempts to
culture the organisms have as yet been
reported.

Miscellaneous Bacterial Infections

Mice are highly susceptible to a
considerable number of pathogenic
bacteria which but rarely, if at all,
cause spontaneous illness in these
animals. Thus the anaerobic spore-
forming bacilli {Clostridia) produce
rapidly fatal diseases following adminis- Fig. 166. — GrahamcUa musculi.

tration of toxin or cultures. The Spontaneous infection in a laboratory
organisms are commonly found in the "^°^^^*^- Giemsa. (Xqoo.) {Cour-
r r ■ , , , 1 • r i- tcsv of Dr. D. Weinman.)

feces of animals, yet natural infection ' -

has not been reported. Spontaneous tuberculosis caused by the avian
tubercle bacillus has been found in mice (no, p. 633), infection apparently
being acquired through the ingestion of avian feces. The enterococci and
proteus organisms other than Proteus morgani (see p. 388) may infrequently
produce disease in mice (94). Of more interest and importance, however,
are two epidemics of tularemia in mice (200, 117). One occurred in Contra
Costa County, California, among meadow mice (Microtus calijornicus
aestuarinus) ; the other in the Kotelnikovo region of the Stalingrad district,
Russia, among common mice (Mus musculus). In both instances the death
rate was high and Pasteurella tularemia was isolated from afflicted animals.
The disease was readily reproduced in mice experimentally (see also 78).

Fungus Diseases

Infection of the skin with fungi, commonly called "favus" or "ring-
worm," is not uncommon in mice (no, p. 627; 299, 170, 249), and may even

430 BIOLOGY OF THE LABORATORY MOUSE

spread epidemically (58, 197). Sporadic cases may be recognized by Uic
denuded plaques usually present on the head or trunk. The skin in these
areas is thickened; and disk-like, whitish-yellow crusts or scales cover the
lesions. At the margins, the hair is of poor texture and easily pulled out.
Diagnosis may be made by culture of the infected tissue on Sabauraud's
agar and by microscopic examination of hairs or scales mounted on slides in
10 to 20 per cent sodium or potassium hydroxide solution. After 20 or 30
minutes mycelia and spores of the fungus may be seen both inside and out-
side the hairs. Various types of fungi have been reported : Achorion quincke-
anum, A . Schoenleinii, Trichophyton ectothrix megalosporium, and T. gypseum.
Achorion quinckeanum {Sabouraudites quinckeanus, Micros porum quincke-
anum) is generally considered to be the common cause of favus of mice.
There is considerable disagreement concerning classification of the fungi and
identification of the various species is difficult. Details may be found else-
where (215, 66, 340, 85). In general, spontaneous recovery occurs but treat-
ment can be effected by the application of tincture of iodine, Whitfield's
ointment, or a mercurial ointment. Care must be taken in handling
infected animals, since the fungi readily infect man.

DuBois (58) has described an infection with T. gypseum granulatum
involving 5 to 9 mice in a single cage. The first 3 animals affected showed
an inflammatory type of cutaneous reaction with follicular suppuration.
The lesions progressed slowly over the skin, producing complete loss of hair
and toxic cachexia resulting in death. No visceral involvement was found
at autopsy. The 2 other mice developed only focal areas of involvement
without suppuration and recovery took place in 4 weeks. Microscopic
examination and cultures of both types of lesions revealed the same organ-
isms. Experimental inoculations produced only the attenuated type of
infection.

Parish and Craddock (197) encountered an extensive epidemic of
trichophytosis due to T. gypseum aster oides among a breeding stock of 2500
mice. The onset was sudden, 400 mice developing signs of the disease
within a few days. Although the spread of the disease during the next
6 weeks was slow, recrudescence occurred and over 1000 mice of all ages were
affected, necessitating destruction of the colony.

The lesions most frequently were situated on the neck, but were common
on the back and rump. They consisted of bald patches with inflammatory
thickening and scaliness of the skin. At the margins the hair had lost its
luster, was brittle, easily detached, and in the worst cases the entire coat had

INFECTIOUS DISEASES OF MICE 431

an "unhealthy, bristhng" appearance. The tendency of the infection
seemed to be towards recovery. Spread occurred by contact.

Examination of hairs from the periphery of the lesions after treatment
with potassium hydroxide revealed chains of spores in the medulla of the
hairs. Individual spores were oblong or square with rounded corners, and
measured 3 to 6 ^t in diameter. After about 3 days of cultivation on
Sabauraud's maltose agar, tufts of growth appeared, enlarging to form white
disks with a chalky, central opaque mass and a large powdery areola. After
subculture, the margins presented uneven, ray-like prolongations and the
reverse side of the colonies was brownish-red in color. The disease was
reproduced experimentally by rubbing cultures into depilated and scarified
skin. Further studies on transmission and immunity were terminated
because of infection in the laboratory attendants. Ringworm of a different
type was discovered in 4 of several hundred mice from a different stock,
housed in another department of the laboratory.

Spirochetosis and Leptospirosis in Mice

Spiral organisms are not of significance as the etiological agents of fatal
disease in mice. They are of importance, however, because mice may be
carriers of the organisms which, in turn, may manifest themselves during the
course of experimental or diagnostic procedures. Spirochetes have been
found in spontaneous and transplanted tumors of mice (27, 294, 86, 41), and
in the blood of animals inoculated with trypanosomes (334, 30, 99). The
identity of these strains has not been estabUshed satisfactorily and various
names have been applied to them: Spirochaeta miiris or BorreUa muris (22),
S. mlcrogyrata var. Gaylordi, 5. laverani, S. naganophilia, and others. Some
of the organisms encountered probably were identical with Spirillum minus;
others may have been saprophytic intestinal forms, or strains introduced by
contamination of blood or tissue used for inoculation. Two forms, namely
Spirillum minus and Leptospira icier ohemorr ha giae, are worthy of further
discussion because of their relationship to human disease.

Spirillum minus. — This organism, which was first found in a rat by
Carter in 1887, has subsequently been shown to cause one type of rat-bite
fever. It is identical with Spirochaeta morsus muris, and probably with
5. laverani and S. muris. Varying figures have been given for the occurrence
of the organism in laboratory (221) and wild mice. It has been found in the
blood of one of two field mice (38), in the mammary glands of 31 of ^^

432 BIOLOGY OF THE LABORATORY MOUSE

lactating albino mice (149, 150), in the seminal vesicles* of t,^ of 34 male
mice, and in Bartholin (clitoral) glands* of 6 female mice (260), in the blood
of 15 of 31 apparently normal white mice (131), in 6 of 8 white mice of one
stock and 65 of 150 mice (two examinations) of a different stock (79).
Other investigations give the incidence as i to 4 per cent (338, 241).

Mice infected spontaneously or experimentally usually remain healthy,
showing no signs of illness and only slight splenic enlargement if killed.
With repeated passage, however, the spirillum may become more virulent
and produce death in approximately 14 days (221). Organisms appear in
the blood 9 to 15 days after inoculation, gradually increase in number
for I or 2 weeks, then slowly decline (241, 267, 222). At the height of
the infection one or more spirilla may be found per microscopic field by
dark field examination. Rarely, they may be demonstrable as long as
II months after injection. The susceptibility of the mouse and the ease of
demonstrating the organisms would make this animal an ideal one for
diagnosis of the disease in other animals were it not for the above incidence
of natural infection.

Transmission of infection in wild or stock animals presumably occurs by
biting and contamination of food and water with urine (241). Hereditary
transmission is both affirmed (11, 150) and denied (267). Infection of
suckling mice by ingestion of organisms in the milk (149, 150) and from
mouse to mouse by sexual contact (260) has been proposed.

Spirillum minus is a rapidly motile, rigid organism, having from two to
six regular spirals. The ends taper and are provided with one or more
flagella. It is perhaps best observed by dark field examination, but it can
be stained by aniline dyes or by silver impregnation methods. The
organism probably should be classified as a bacterium in the same family
as the vibrio group, although common usage includes it with the
Spirachaeta.

Various strains isolated from mice, rats, and cases of human infection
have been studied rather extensively in an attempt to differentiate them
(338, 223, 240, 241). Some differences in virulence and serological reac-

* The identification of the spiral organisms found by Stroesco (260) in the seminal
vesicles and Bartholin's glands as Spirillum minus was based on morphological appear-
ance in stained sections of tissue. Dubois (59) has shown that this organism is more
like a spirochete in morphology and motility and further differs from S. minus in
pathogenicity and resistance to chemotherapeutic agents. Both Dubois and ISIack-
enzie (160) consider it to be a new, non-pathogenic species which must be differentiated
from 5. minus.

INFECTIOUS DISEASES OF MICE 433

tions have been found by various investigators, but the detailed work of
Schockaert (240, 241) indicates that virulence varies regardless of source and
that the human, rat and mouse strains constitute a single species. Human
cases resulting from the bite of a mouse (in, 115, 217), or from inoculation
of a mouse strain of the spirillum (131, 240), are indistinguishable clinically
from those due to human or rat strains.

Recovery from infection and disappearance of the organisms in mice
probably is due to an immunological mechanism. Although lytic antibodies
have never been satisfactorily demonstrated in mice, active immunization
to homologous and heterologous strains does occur. Arsenic preparations,
such as arsphenamine or neoarsphenamine, are effective in treating human
infection and might be tried if it were desirable to rid infected mice of the
organisms.

Leptospira icterohemorrhagiae.* — Following the identification of Lepto-
spira ictcrohcmorrhagiae as the causative agent of infectious jaundice (Weil's
disease) and its demonstration in rats, Miyajima [quoted by Ido, et al. (108)]
found the organism on several occasions in the kidneys of the field mouse,
Microtus montehelloi. The leptospira was subsequently found in i of 6 field
mice (108), and in i of 2 field mice but not in 2 house mice from the Edin-
burgh area of Scotland (38). Packchanian (193) has recently reported the
occurrence of Leptospira icterohemorrhagiae in laboratory white mice {Mus
musculus) and the susceptibility of certain species of American deer mice
{Peromysciis) to experimental infection. White mice ordinarily show no
signs of infection. Inoculation of infected mouse blood or tissues into
guinea pigs or American deer mice, however, results in fever, jaundice,
hemorrhages, and death. The organisms are demonstrable in the blood and
urine.

Morphologically, Leptospira icterohemorrhagiae is a delicate organism
having closely wound, rigid spirals and secondary wavy curves. It varies
from 0.1 to 0.2 ^^ in width and from 6 to 12 ^t in length, occasional specimens
being as long as 25 /x. The organism is flexible and one or both ends may be
curved or hooked, giving an S or C shape. During movement, the hooked
ends whirl around rapidly. Darkfield examination and silver impregnation
are best for demonstration of the organism. Bile salts (10 per cent) but not
saponin dissolve it. Cultivation may be effected in dilute serum media at
25°C.

* Sellards (248) has recently proposed Leptospira interrogans as the correct name
for this organism.

434 BIOLOGY OF THE LABORATORY MOUSE

A somewhat similar organism, termed Leptospira aquicole pseudoictero-
genica, has been reported in the kidneys and urine of stock mice (23). The
strain apparently is identical with one found in water.

Virus Diseases op Mice

Filterable viruses have been known to produce a variety of diseases in
plants and animals since 1892. Only in the past 10 years, however, have
spontaneous diseases due to the viruses been recognized in mice. Some of
these diseases may spread within a mouse colony, producing highly fatal
results. Others may produce no visible signs of infection but are equally
important, since they may evidence themselves in the course of experimental
procedures.

The virus diseases to be discussed are infectious ectromelia, lymphocytic
choriomeningitis, encephalomyelitis of mice (Theiler), virus pneumonia, and
salivary gland disease. The etiological agents are acceptable as viruses
since they fulfill one or more of the following criteria: transmissibility,
filterability, failure to grow on non-viable culture media, absence of culti-
vable bacteria, presence of inclusion bodies in the cells of the host, and
production of immunity.

Infectious ectromelia. — In 1930 Marchal (162) described a new virus
disease of mice. It occurred principally in young mice and was noted most
frequently when the animals were separated from their mothers and placed
together in groups of 50. Subsequently, the spontaneous disease has been
found in England (169) and on the Continent (24, 242, 102), but has not yet
been reported from this country. It occurs in laboratory mice of differ-
ent stocks and has been found in wild mice caught in the laboratory.
McGaughey and Whitehead (169) found the disease to be so widespread in
England that difficulty was experienced in obtaining healthy mice. Some
animals apparently harbor the virus and develop the disease only when
subject to experimental inoculation (169, 102).

The natural disease. — Qinically, the disease occurs in two forms. The
acute or abdominal type usually appears first in a stock of infected mice and
is evidenced by loss of normal activity and ruffled, lustreless coats without
other significant signs. No skin lesions are present. Death may occur
after an illness as short as 4 hours, and the fatality rate may reach 80 to 90
per cent in certain lots of mice. Recognition of this form may be difficult
unless careful autopsies are performed.

The chronic or cutaneous form of the disease appears later in animals
surviving the acute type or in those previously uninfected. Here skin

INFECTIOUS DISEASES OF MICE 435

manifestations form a prominent part of the clinical picture and are almost
pathognomonic. Enlargement is noted in one foot — usually a hind foot —
which presents a swollen translucent appearance due to edema of the sub-
cutaneous tissues. As the edema increases, exudation of serous fluid occurs
through the skin and crusting takes place over superticial ulcers. \'esicles
may also form. The diseased skin is usually sharply demarcated from
healthy tissue by a line of constriction, and gangrene of a toe or the foot may
follow, with ultimate separation of the foot at this line. Recovery ordinarily
occurs in these cases, and the animals are then immune to subsequent expo-

FiG. 167. — Infectious ectromelia: left, advanced lesions of foot and leg; right, normal
mouse. {From McGaughey and Whitehead, J . Path, and Bacf.)

sure or inoculation. Should the disease spread to involve any or all of the
other feet, the tail, or the skin around the mouth or over the body, death
invariably results (Fig. 167).

Pathology of the natural disease. — Mice dying acutely show an increase
in peritoneal, pleural, and pericardial fluid which at times may be abundant.
Serous membranes, especially those of the intestines, are markedly con-
gested. The liver is usually pale and anemic or greyish-brown in appearance
and soft and necrotic in consistency. The spleen is ordinarily normal in size
or but slightly enlarged, and may be either studded with yellowish-grey areas
of necrosis or present an appearance of massive necrosis. Congestion is
present in the lymphatic glands, lungs, and sometimes the adrenal cortex.
Xo organisms can be found by direct smear or by culture of the exudate or
organs. Aside from generalized congestion and occasional small hemor-

436 BIOLOGY OF THE LABORATORY MOUSE

rhages, the microscopic picture is one of diffuse necrosis in the liver and
spleen. The characteristic feature, however, is the presence of intracyto-
plasmic eosinophilic inclusion bodies in the epithelial cells of the intestine
and the acinar cells of the pancreas (Fig. i68). These inclusions occur
singly or in small groups, and vary in size up to 7 /jl. Only one observer (242)
has noted them in the liver.

The lesions in the chronic or cutaneous type of infection are more exten-
sive. Those in the skin consist of crusted, superficial ulcers surrounded by
indurated, edematous tissue. Ascites, hydrothorax, and congestion are

^ present. The liver is a mottled red
MP '-'■:4% color and contains numerous greyish-

white areas of necrosis. The spleen is
enlarged and red with similar necrotic
areas. Over the peritoneum and
serosal surface of the viscera may be
found whitish areas suggestive of fat
necrosis. The kidneys are usually
normal but may be enlarged and pale,
resembling those of the second stage of

igF

*'«»!.

^ ^ *^ •^ nephritis. Microscopic examination

s^^^ confirms the gross evidence of an exten-

^^^^ sive necrotizing process which involves

Fig. 168. — Infectious ectromelia: the skin, liver, spleen, peritoneum, and

inclusion bodies (black) in cells of the other tissues. Perivascular cufhng and

pancreas. Mann's stain. (Xiooo.) r ,. j .• j •,

^^ ,, ^ , JTT-,- , JN fatty degeneration are pronounced m

(From McGaug/iev and ]] Inte/icad.) , ,. , „ 1 i-i 1

the liver. Affected kidneys show

groups of endothehal-like cells in the cortex, small hemorrhages, and fatty
degeneration of the convoluted tubules. Intracytoplasmic inclusion bodies
are most numerous in the epithelial cells of the skin, where they may be
as large as 13 /jl in diameter (Fig. 169). They are also found in connective
tissue cells and endothelial cells of vessels in the subcutaneous tissue,
epithelial cells of the intestine, acinar cells of the pancreas, secretory
cells of the salivary glands, and epitheHal cells of the tongue and
lips. The larger inclusions are round or oval in shape, stain evenly
with acid dyes, and as they enlarge cause degeneration and finally dis-
appearance of the nucleus. For demonstration of the bodies, the tissue
may be fixed in a saturated solution of bichloride of mercury containing
5 per cent glacial acetic acid, and stained with the ordinary hematoxylin-
eosin stain. Mann's methyl blue eosin or acid fuchsin and Weigert hema-

INFECTIOUS DISEASES OF MICE

437

toxylin may be used. After chromic acid fixation, methylene blue gives a
characteristic picture.

The experimental disease. — The disease can be transmitted to normal
mice bv inoculation of edema fluid, blood, or various tissue emulsions from
infected animals. Blood plasma is infective from i to 5 days after the
appearance of the lesions, the liver and spleen after 3 days. Intradermal
inoculation in the foot pad usually reproduces the cutaneous type of disease.
Intraperitoneal or intravenous injection produces the acute form, with

Fig. 169. — Infectious ectromelia: section of skin showing edema, necrosis, and
inclusion bodies in the epithelial cells. Hematoxylin-eosin. (X360.) {From
Birsch-Hirschfeld.)

death in 4 to 6 days. A disseminated pneumonia follows inhalation of the
virus. Transmission of infection also occurs by permitting contact of
normal and infected mice under ordinary conditions, which has permitted
study of experimental epidemics of the disease (92, 119).

Properties of the virus. — The virus responsible for this disease may be
isolated from the liver, spleen, central ner\-ous system, lymph nodes, lungs,
peritoneal and edema fluids, and blood. It is filterable through diatoma-
ceous or porcelain filters (Chamberland L2, Mandler, Berkefeld X)and has an
estimated size of 100 to 150 m/i ( i8). It is resistant to dr}'ing in a desiccator,
may be preser\-ed for months in 50 per cent glycerin at o°C., but is inacti-

438 BIOLOGY OF THE LABORATORY MOUSE

vated by o.oi per cent formalin in 48 hours and by a temperature of S5°C. in
30 minutes. Studies of the inclusions (18, 98, 144) have revealed the presence
of elementary bodies entirely similar to the Paschen bodies of vaccinia and
the Borrel bodies of fowlpox. The inclusions are resistant to digestion with
trypsin (24). Propagation of the virus has been successful only in tissue
culture and on the chorio-allantoic membrane of the egg (56, 57, 199, 40).

The virus is strikingly species-specific. Only mice show signs of the
disease, although inapparent infection has been demonstrated in the rat (39).
One attack of the disease confers a solid immunity upon the survivors, in
whose serum neutralizing antibodies are demonstrable. Attempts to pro-
duce immunity by the use of inactivated virus have thus far been unsuccess-
ful (24), and prevention of the disease can be accomplished only by general
measures.

An epidemic disease, somewhat similar to ectromelia, has been reported
by Thompson (273, 275). Intracytoplasmic and intranuclear inclusion
bodies were found in the parenchymatous cells of the liver, but were absent
from epithelial tissues. Further comparison of the two diseases was not
reported.

Lymphocytic choriomeningitis. — The virus of lymphocytic choriomenin-
gitis was first obtained from mice by Traub (291, 292) in 1935. It was
encountered during his work with the viruses of equine encephalomyelitis
and hog cholera and was found to be distinct immunologically and path-
ologically from both of them. In spite of the fact that he at first had
recognized no disease in his mouse colony, it seemed likely that the mouse
was the natural host of the virus. Accordingly, he inoculated a group of 60
5-week-old mice with sterile bouillon by the intracerebral route. Nine of
these animals developed symptoms in from 3 to 13 days, and 4 died. No
bacteria were obtained by culture. Inoculation of suspensions of brain into
guinea pigs reproduced the disease. From later studies, he estimated that
about 50 per cent of the mice were infected with this agent, which was
identified as the virus of lymphocytic choriomeningitis (292, 219, 220).

The original isolation of the virus was reported in 1934 by Armstrong and
Lillie (14), who accidentally encountered it in monkeys during the course of
their studies on the epidemic of encephalitis in St. Louis. The origin of this
strain was not definitely determined. Subsequently, the virus has been
isolated from cases of meningitis in man (219, 246. 69, 138, 15), and from
laboratory and house mice (69, 146, 138, 15). The latter observation is of
particular interest since the virus was found in grey mice (Mus musculus)
trapped in two houses in which human cases occurred (15). Accidental

IXFECTIOUS DISEASES OF MICE 439

infection of a laboratory worker (147) and experimental reproduction of the
disease in man by inoculation with the murine virus (145), together with the
above reports, suggest that mice constitute a natural reservoir of the disease.

The natural disease. — In most instances in which the virus of lympho-
cytic choriomeningitis has been found in mice the infected animals have
appeared to be entirely normal and healthy and the disease has existed as a
latent infection. In the colony observed by Traub ( 292), hovsever, signs of
infection were noted. He describes the animals as follows: " . . . a num-
ber of 2 to 6 week. old mice were emaciated and drowsy. Their fur was
ruffled and they were often seen sitting in corners of the cage by themselves.
Their movements were slow and stiff, and their legs appeared long in propor-
tion to their thin bodies." Other signs of infection were conjunctivitis,
photophobia, and a slow rate of growth. No signs of involvement of the
central nervous system were noted. Approximately one-half of the mice in
the colony were infected, although the morbidity was less than 20 per cent,
and the mortality less than 2 per cent of the number of infected animals.
The majority recovered completely in 3 weeks. The active agent was
isolated from the blood and brain of apparently normal mice, of those show-
ing only conjunctivitis and photophobia, and of those obviously ill or found
dead.

Xo gross lesions were found at autopsy of these animals. IMicroscopic
examination of the liver revealed slight perivascular round cell infiltration,
scattered hinphocytic infiltration in the interstitial tissue, and patchy
reticulo-endothelial hj'perplasia. Slight peribronchial and perivascular
infiltration with round cells and slight thickening of the alveolar walls were
found in the lungs of 2 of 12 mice. Only i mouse showed a slight meningeal
reaction consisting of lymphocytic cells.

Transmission of the natural disease presumably occurs by contact, since
normal mice may be infected by placing them in a cage with diseased ani-
mals. Although the route of infection is not definitely known, it has been
demonstrated that the virus is often present in the urine and nasal secretions
of diseased mice (292) . The agent has also been found in embryo, new born,
and suckling mice (^138).

The experimental disease. ^Laboratory and wild mice are susceptible
to virus introduced by almost any route of inoculation, but only by intra-
cerebral injection is a definite clinical picture produced. Following such
administration, an incubation period of 5 to 7 days elapses before the mice
appear ill. They then show signs of malaise- lassitude, ruftled fur, hunched
back, and partially closed eyes. Death may occur suddenly without other

440 BIOLOGY OF THE LABORATORY MOUSE

signs of infection, but more commonly the animals become hyperactive so
that even a slight stimulus will cause them to leap into the air or will induce
a convulsion. If the mouse is lifted by the tail, a convulsion frequently
follows, characterized by rapid clonic movements of the fore legs, terminat-
ing in a sustained tonic extensor spasm of the hind limbs, and lasting from
one to several minutes. Convulsive attacks also occur spontaneously.
Death may result in the first or subsequent attacks. If the animals survive
for 3 or 4 days after the onset of signs of the disease, complete recovery with-
out residual paralysis usually occurs. Blood counts are within normal
limits (292). This same clinical course may be seen in naturally infected
mice injected intracerebrally with sterile starch emulsion or bouillon.

Intranasal and subcutaneous inoculations produce no signs of the disease,
but the virus may be demonstrated in the blood and the animals acquire an
immunity to subsequent intracerebral inoculation. Mice inoculated intra-
peritoneally or intravenously may show labored respiration 5 to 10 days
later for a period of a week or more. Convulsions do not occur. A few of
the mice die, but the majority recover and are resistant to a second inocula-
tion. The virus may persist for weeks or months in mice recovering from
experimental infection and has been demonstrated in the brain, blood, liver,
spleen, kidneys, lungs, adrenal, nasal passages, and urine. No neutralizing
antibodies, however, have been observed in the blood of recovered mice

(293)-

The pathological picture varies with the route of inoculation. Meso-
dermal tissues are primarily involved with the production of a hyperplastic
reaction. Following intracerebral inoculation, congestion is apparent
grossly in the surface vessels of the brain, in the liver, and in the spleen,
which may be slightly enlarged. Microscopically, there is infiltration of the
meninges of the brain and spinal cord, the cellular exudate being composed
chiefly of lymphocytes, and to a less extent, of mononuclear and polymorpho-
nuclear cells. Infiltration is most marked at the base of the brain (Fig. 170),
but the choroid plexuses and the ependyma are quite constantly involved.
Perivascular round cell infiltration is present if the animals survive for 2 or
3 weeks. Involvement of the nervous tissue proper is minimal, and no
inclusion bodies are found. Changes in the other organs are minor ; irregular
hyperplasia of reticulo-endothelial (Kupffer) cells and slight lymphocytic
infiltration in the liver, and small areas of interstitial bronchopneumonia in
the lungs are the chief findings.

Mice developing signs of infection after intraperitoneal or intravenous
inoculation show visceral lesions but infrequently there is evidence of even

INFECTIOUS DISEASES OF MICE

441

slight meningitis. The significant findings are enlarged spleen, a pale or
nutmeg liver, serous pleuritis and peritonitis, lungs which may appear
normal or contain small areas of consolidation, and occasionally pale and
slightly swollen kidneys. Microscopically, there is generalized proliferation
of the reticulo-endothelial cells, interstitial and perivascular round cell
infiltration, and interstitial bronchopneumonia. Rarely is there necrosis of
i:)arenchymal cells. Blood counts in mice injected intravenously may show

Fig. 170. — Lymphocylic choriomeningitis. Marked meningitis at the base of
the brain of a mouse inoculated intracerebrally with the virus. Eosin and methylene
blue. (X130.) {From Traub.)

a leukocytosis up to 55,000 per c. mm. with a relative and absolute lympho-
cytosis and monocytosis.

Cultures of various tissues from infected animals reveal no bacteria of
possible etiological significance, and the disease may be reproduced by the
inoculation of filtrates of tissue emulsions after passage through Berkefeld
or Chamberland candles. Guinea pigs are particularly suitable for inocula-
tion since they are highly susceptible, do not themselves carry the virus, and
react with a characteristic clinico-pathological picture due to a slowly
progressing pneumonia (292). Intracerebral, subcutaneous, and intranasal
routes of inoculation may be employed.

Properties of the virus. — As already pointed out, the virus of lympho-
cytic choriomeningitis is mesodermatropic in nature and is widely distrib-

442 BIOLOGY OF THE LABORATORY MOUSE

uted in the tissues of infected animals. Different strains vary in the degree
of their virulence, but in general the virus is pathogenic for mice, rats, guinea
pigs, monkeys, and man. The serum of certain convalescent or recovered
animals contains complement-fixing and protective antibodies, as does that
of rabbits inoculated with virus suspensions (io6, 147, 251, 252, 253).

The virus withstands freezing and drying (252), and 50 per cent glycerin
for at least i month (291), but rapidly decreases in infectivity when sus-
pended in physiological saline solutions at room temperature unless protected
by the addition of 2 per cent normal inactivated serum (252). In size the
virus particles are not more than 100 to 150 m/i in diameter, as determined
by filtration through graded collodion membranes (220). From suspensions
of infected tissue, a soluble antigen — apparently protein in nature — has been
obtained (252, 253). It is capable of fixing complement and of precipitating
when mixed with immune serum. The antibodies which react with this
antigen are apparently distinct from those responsible for neutralization of
the virus in protection tests. Immunological reactions with both tissue
emulsions and soluble antigen are entirely specific, and no qualitative differ-
ences have been found between various strains of the virus.

Diagnosis of the disease. — Since the disease may be present in a mouse
colony as a subclinical, latent infection, its existence may not be suspected.
Recognition of the infection may be accomplished by several methods, (a)
The virus may be isolated by intracerebral inoculation of an "indicator
host " (7) — that is, a guinea pig or mouse known to be free from the infection
— with blood or emulsion of brain, spleen, or kidney, {b) Demonstration of
immunity in a certain number of stock mice is an indication of previous
infection. Traub, for example, found that the morbidity rate following
intracerebral inoculation of the virus into mice from the infected stock was
about 60 per cent and the mortality rate about 40 per cent, (c) Intra-
cerebral injection of a sterile, non-infectious agent, such as starch emulsion
or bouillon, may be a sufficient stimulus in some of the animals to cause a
flare-up of the inactive infection, resulting in a clinical picture similar to that
seen in normal mice inoculated intracerebrally with the virus (292, 69, 146).

Final diagnosis of the disease is made on the basis of the clinical course in
mice or guinea pigs, the pathological findings, and immunological identifica-
tion of the virus by complement fixation or protection tests, or by inoculation
of recovered animals with a known strain of the virus. Immunological
methods, for example, afford a clear distinction between the viruses of lym-
phocytic choriomeningitis and acute meningo-pneumonitis (81), although
the clinico-pathological features following intracerebral injection are similar.

INFECTIOUS DISEASES OF MICE 443

No specific measures are as yet available for effective immunization of
mice or for prevention of the disease. General preventive measures
should be taken to protect a disease-free colony or to stop spread of the
infection.

Encephalomyelitis of mice (Theiler). — Spontaneous encephalomyelitis
of mice is a virus disease which rarely produces clinical signs under natural
conditions. The active agent, however, is widespread in distribution. It
may be obtained with great regularity from normal mice of certain age
groups, or may be. encountered in animals inoculated with other agents.
Since Theiler (268) first described the disease and demonstrated its etiology
the virus has been found in several strains of mice in the United States (269,
231, 270), as well as in Germany (88), Japan, (109), and Palestine (189).

Occurrence.— Tht incidence of the natural disease is difficult to deter-
mine, but is probably very low. Various figures have been given : i in about
2000 Swiss mice purchased from various dealers (269); i or 2 per 1000 mice
of the Rockefeller strain (231) although no cases were found among a series
of 5000 animals observed later (189). The low incidence does not indicate
lack of contact with the infective agent, however, since the virus has been
demonstrated in the intestinal contents of almost all (66 to 100 per cent)
mice between the ages of i and 2 months (187, 271, 188, 189).

The natural disease. — Spontaneous illness in mice (268, 269) may
be recognized by the development of flaccid paralysis of the hind legs without
other apparent signs. No reports of the course and mortality are available.
Pathological examination of the central nervous system reveals scattered
necrosis of ganglion cells and perivascular infiltration, most marked in the
spinal cord but also present in the brain. The disease seems to become
evident chiefly in young mice — approximately 6 to 7 weeks of age — some of
which are apparently highly susceptible to invasion of the central nervous
system. There is no evident reason why certain animals should be afflicted
while the great majority escape, yet practically all at this age are carriers of
the virus. In paralyzed animals, the virus is present in the spinal cord in
highest concentration and in the brain. It has not been demonstrated in the
blood.

The virus is regularly found in the contents and walls of the gastro-
intestinal tract, in the mesenteric glands, and in the feces, but not in the
central nervous system, salivary glands, or other organs of normal mice
between the ages of 4 and 8 weeks. It is absent or irregularly present in
animals younger than 20 days or older than 6 months. Excretion of the
virus may persist up to 53 days after the first isolation (271).

444 BIOLOGY OF THE LABORATORY MOUSE

Intracerebral injection of mice with other agents (109, 270) has resulted
in the isolation of the most virulent strains of the virus yet obtained. No
relationship could be established between the strain of murine encephalo-
myelitis isolated and the agents injected (the viruses of yellow fever and
human encephalitis) , so that exacerbation of a latent infection seems to be
the most likely explanation. The signs of infection were entirely similar to
those resulting from experimental intracerebral inoculation of known strains
of the virus.

The experimental disease. — The production of clinical disease by
experimental inoculation of the virus depends on the virulence of the strain
of virus, the route of inoculation, and the age of the mice. With strains of
relatively low virulence — those obtained from intestinal contents of normal
mice or the central nervous system of naturally infected mice — intracerebral
injection of young animals gives a high morbidity and mortality, whereas by
intranasal inoculation only a low incidence of paralysis occurs. Other routes
are ineffective (268, 269, 270, 88, 189). With strains of higher virulence
(109, 270), however, signs of involvement of the central nervous system
occur following intracerebral, intranasal, and intraperitoneal injection with
greater regularity, and occasionally following subcutaneous inoculation.
The influence of age is shown by the fact that the morbidity and mortality
rates are lower and the incubation time longer in mice over 12 weeks of
age (270, 84, 109).

After intracerebral injection (268, 269, 88, 109, 270), a period of 5 to
30 days may elapse before the appearance of signs of the disease, but the
average time is 10 to 14 days. The first sign is a weakness of one limb,
rapidly followed by flaccid paralysis of that member. The paralysis may
spread to involve all four legs, but usually the hind limbs are more markedly
affected so that locomotion is possible only by use of the fore legs. Atrophy,
emaciation, and contractures of the involved members occur. Incontinence
of urine may be observed in severely aftlicted animals. In spite of the above
evidence of damage to the nervous system, the mice do not appear acutely
ill during the first stages of the disease. Finally, however, the fur becomes
ruffled, respiration labored, and the animal succumbs. Mice 4 weeks of age
or younger, however, may die without showing signs of infection. If
recovery occurs, the extent of the involvement diminishes, but residual
paralysis of the hind limbs is almost constantly present. Such recovered
animals may harbor the virus in the spinal cord for more than a year (269).
The duration of the disease from the first appearance of clinical signs to death
or recovery varies between 2 and 10 days. Following other routes of

INFECTIOUS DISEASES OF MICE 445

inoculation the clinical picture, if apparent at all, is essentially that described
above.

No significant gross changes are found at autopsy (268, 269, 88, 109).
Microscopic lesions occur primarily in the spinal cord and are characterized
by perivascular round cell infiltration, acute neuronal necrosis particularly of
the anterior horn cells, neuronophagia, and gliosis. Ganglion cells of the
posterior root are not involved. The brain shows perivascular cullting
to a lesser extent and degeneration of occasional isolated neurons. A
decrease in the number of anterior horn cells is found in the cords of recovered
mice with residual paralysis. No inclusion bodies have been demon-
strated. The virus may most easily be demonstrated in the spinal cord and
brain.

The clinical picture produced by the two more virulent strains of virus
isolated by Theiler and Gard (270) differs considerably from that described
above. The incubation period following intracerebral inoculation is much
shorter (2 to 6 days), the course more rapid (24 to 48 hours), the mortality
greater, and the titer of virus in the infected brains higher. With one strain
(FA) the signs of infection following intranasal or intracerebral injection
resemble those of an encephalitis more than a myelitis, e.g., an appearance of
being sick, hyperexcitabihty, ruflied fur, twitching, and tonic convulsions
sometimes terminating in death. Weakness of one of the legs may occur but
paralysis is rare. The histopathological appearance is that of a marked
encephalitis with a minimal meningeal reaction. Following intraperitoneal
inoculation, however, flaccid paralysis is usually the predominant sign.
With the other strain (GD VII), hyperirritability may be the first sign of
infection, but the mice appear well and the signs are referable to lesions of the
cord, i.e., flaccid paralyses. The same picture results from intracerebral,
intranasal, or intraperitoneal injection.

Properties of the virus. — Although the several strains of this virus vary
in virulence or invasiveness, they are much alike, if not identical, in their
other properties (268, 269, 270, 88, 109). The average particle diameter as
determined by filtration through graded collodion membranes is 9 to 13 m^^,
closely approximating that of the viruses of human poliomyelitis and of
foot-and-mouth disease of cattle. Filtration through all grades of Berkefeld
filters is accomplished with ease. The virus may be preserved in 50 per cent
glycerin at 2° to 4°C. for more than 150 days and is most stable at pH 8.0 or
pH 3.3. It withstands the action of ether and precipitation by ammonium
sulphate, but is destroyed or inactivated by a temperature of 50°C. with
rapidity, by 20 per cent ethyl alcohol in 45 minutes in the icebox, by i per

446 BIOLOGY OF THE LABORATORY MOUSE

cent hydrogen peroxide in 2 hours at 37°C., and by desiccation in the frozen
state at — i6°C.

The virus is Umited in its host pathogenicity. Mice are susceptible, but
guinea pigs, rabbits, and rhesus monkeys are resistant. Theiler and Gard
(270) have recently reported that one of their strains of encephalomyelitis
virus is pathogenic for the cotton rat. This work is of considerable interest,
since the cotton rat has been reported by Armstrong (12, 13) to be suscepti-
ble to the Lansing strain of human poliomyelitis virus, and the virus recov-
ered from the inoculated animals was then found to be pathogenic for mice
by the intracerebral route. An immunological relationship apparently
exists between these two viruses, since Theiler and Gard (270) found "...
that mice which had been infected with the virus of mouse encephalomyelitis
were resistant to a subsequent intracerebral inoculation of Armstrong's
Lansing strain of human poliomyelitis virus ..." Jungeblut and Sanders
(114) have also isolated a virus from a cotton rat injected with the SK
(New Haven) strain of human poliomyelitis virus. The animal died a week
after inoculation without presenting signs of disease, but subsequent passage
of the agent to cotton rats resulted in flaccid paralysis of the hind legs and
death. Inoculation of mice produced an illness clinically like mouse
encephalomyelitis. Mice from a colony immune to the spontaneous murine
encephalomyelitic agent, however, were susceptible to infection with their
virus. All later attempts to produce infection in rats or mice with the
original material were unsuccessful. Further investigation is necessary to
clarify the relationship between the above strains of virus.

Immunologically, the murine strains of this virus thus far isolated are
antigenically related (270). Recovered but paralyzed animals are resistant
to a second inoculation regardless of the route used to infect them. Mice
infected intracerebrally with a relatively avirulent strain of virus are immune
to subsequent inoculation with a highly virulent strain (GD VII) . Although
the interference phenomenon might be responsible for erroneous conclusions
in experiments of this type, the high degree of resistance to a second inocula-
tion is more suggestive of true active immunity. The two more virulent
strains (FA and GD VII) are not immunologically identical, however, since a
greater resistance is produced by immunization with the homologous than
with the heterologous strain. The greater resistance of older mice is prob-
ably due to previous contact with the virus (271, 189), but the same phe-
nomenon is seen with other infectious agents and may be the resultant of
anatomical and physiological (228), as well as immunological factors.

INFECTIOUS DISEASES OF MICE

447

Neutralization of the virus by the serum of convalescent mice has not
been satisfactorily demonstrated by the methods so far employed. The
results suggest that relative protection can be conferred, but the degree of
protection is insufhcient to permit an immunological comparison of the
various strains of virus by this method. The murine virus is not neutralized
by antiserum for the virus of human poliomyelitis.

Differential diagnosis. — The clinical course of this disease following
intracerebral inoculation is sufficiently distinctive to difTerentiate it from
other encephalitis -agents such as lymphocytic choriomeningitis, equine
encephalomyelitis, etc., whereas the diameter of the virus, pathological find-
ings, and host specificity distinguish it from acute meningo-encephalitis (8i).

Final diagnosis is made on the basis of size, host range, pathology, and
cross-protection. Parasitic meningo-encephalitis may be distinguished
pathologically, and bacterial infections by cultural methods.

Epidemiology. — ^Considerable interest is centered in epidemiological
studies (271, 189) of this disease because of its similarity to human polio-
myelitis. The significant features thus far demonstrated are the widespread
distribution of the virus as evidenced by its almost constant presence in the
intestines of young mice, the low incidence of spontaneous disease, the
prolonged period of excretion in the feces, and the gradual development of
resistance with increasing age. The exact route of natural infection is not
known, but in all probability is either nasal or oral since fecal excretion must
keep the environment almost constantly infected. That such excretion by
an infected mouse is not dependent on continuous infection, however, is
shown by isolation experiments in which the opportunity for self-infection
was minimal. Under such conditions, the intestinal wall is apparently the
site of elaboration of the virus, and invasion of the mesenteric glands may
occur secondarily. Whether the intestinal tract is the focus for distribution
of the virus when first introduced, or is but secondarily infected, has not been
determined. Theiler and Gard (271) have suggested that the development
of antibodies due to infection of the intestinal tract may be responsible for
the increasing resistance with age. The failure of an individual animal to
form antibodies might then allow invasion of the central nervous system and
the production of clinical disease. Experiments with a disease-free stock of
mice would be of value in elucidating further the epidemiology and nature
of this disease, which, though unimportant as regards mortality, is of con-
siderable importance to an investigator employing mice in the study of
viruses.

448 BIOLOGY OF THE LABORATORY MOUSE

Virus pneumonia in mice. — Mice are being widely used for the investiga-
tion of certain human respiratory infections, such as influenza, because these
animals respond to intranasal administration of the causative viruses with
the production of pneumonic consolidation. In the isolation of the virus
from nasopharyngeal washings from the patient, however, it is often neces-
sary to make several "blind" passages of lung tissue from the first animal
before the mice develop extensive lesions or die from the infection. An
infectious agent, latent in the experimental animal, could thus be carried
along during the successive passages, and increasing in virulence, could
finally produce obvious disease. In this manner three different groups of
investigators (54, 89, 103, 104) have encountered respiratory disease which
differed from that seen with known viruses. They have further shown that
the disease may be produced by repeated serial passage of lung tissue from
apparently normal healthy mice, which indicates that a certain percentage
of mice harbor the responsible agent. Two types of disease have been
found, differing somewhat in course, host susceptibility, and production of
immunity, although the possibility of immunological relationship between
the respective viruses has not yet been determined. For the sake of
simplicity, therefore, the two types will be described separately.

No instances of spontaneous illness due to either type of infection have
been reported, although the viruses have been found in albino Swiss mice as
young as 3 weeks of age, and in other albino strains obtained from a number
of different sources. Small areas of spontaneous pulmonary consolidation
occur in such animals with varying frequency: i to 2 per cent (104) and 35
per cent (54). The viruses must accordingly have a fairly wide distribution
and a low virulence under natural conditions. Increase in virulence of the
agents with successive intranasal passage would then account for the produc-
tion of extensive and often fatal pneumonic lesions.

Pneumonia described by Dochez, Mills, and Mulliken (54), and by
Gordon, Freeman, and Clampit (89). — This form of experimental pneu-
monia, first described by Dochez, Mills, and Mulliken (54), appeared after
I to 9 intranasal passages. Clinically, the signs of infection were loss of
activity, refusal of food, ruffled coat, and hunched posture, with the develop-
ment of rapid, labored respirations as the disease progressed. Deaths began
to occur after 4 to 7 passages, the mice succumbing 2 to 4 days after inocula-
tion. The mortality rate was high; in fact, all mice (5 to 10 grams in weight)
developing signs of infection died (89).

At autopsy the only significant lesions were found in the lungs. Early in
the course of the disease, sharply demarcated, greyish-pink areas of con-

INFECTIOUS DISEASES OF MICE 449

solidation were present in the apices or dorsal portions of the lung. The
consolidation spread as the disease progressed and at death the entire lung
might be involved, often having a uniform dark red or plum-colored appear-
ance resembling the lesions due to the influenza virus (262). The micro-
scopic picture was one of a patchy interstitial pneumonia, with mononuclear
inhltration and varying degrees of hemorrhage and edema. Cellular
exudate in the bronchial lumina consisted of mononuclear and polymorpho-
nuclear leukocytes. The epithelium of the bronchi was well preserved in
contradistinction to the necrosis and desquamation produced by the
influenza virus. Non-inflammatory focal necrosis was present in the liver.
A variety of organisms were cultured from the lungs in some instances, but
none reproduced the disease.

The virus was present in the lung and in the liver (89) of infected mice,
and passed Berkefeld N and V as well as Seitz filters. Mice were susceptible
only to intranasal inoculation. No spread occurred by contact. In ferrets,
administration of virus by the nasal route produced an elevation of tempera-
ture to about io5°F., occasionally associated with respiratory difficulty.
Intratracheal inoculation of rabbits resulted in pneumonia, mediastinitis,
and pericarditis, complicated, however, by the presence of secondary
bacteria (54). The guinea pig was resistant. Protective serum was not
produced in rabbits by administration of lung emulsions containing the
virus, nor was active immunization of mice successful. The agent is
apparently distinct from human influenzal virus, since mice convalescent
from infection with the latter were fully susceptible to the murine virus.
Further immunological studies are necessary, however, to achieve certain
dift"erentiation and identification of this agent.

Pneumonia described by Horsfall and Hahn (103, 104). — As already
pointed out, this t>^e of experimental pneumonia differs in certain impor-
tant respects from that described in the preceding paragraphs. The disease
was found to be latent in 3 of 8 different colonies of albino Swiss mice.
Using 3 to 4-week-old animals, infection became apparent after 2 to 7 intra-
nasal passages of the supernatant fluid from lung emulsion at an interval of
7 to 9 days, but not by rapid serial passage at 4 to 5 day intervals. The
mice appeared well for 5 to 7 days, but then showed a decrease in activity
and food consumption, loss of weight, ruiitled fur. slow, deep, and sometimes
labored respirations, and often cyanosis of the ears and tail. Death occurred
from 8 to 14 days after inoculation. The morbidity and mortality rates
varied with the amount of virus inoculated and the particular stock of mice
employed.

450 BIOLOGY OF THE LABORATORY MOUSE

The pulmonary lesions did not differ significantly from those described
above. Consolidated areas varied in extent and were hilar in distribution
with radiations outward along the bronchi. The histopathological appear-
ance was essentially the same. In the great majority of instances (85 per
cent) the lungs were sterile and such bacteria as were found had no etiological
significance. It is of interest to note that pleuropneumonia-like organisms
were isolated with ease and in approximately the same numbers from the
lungs of normal mice as from those infected with the murine and influenzal
viruses. These organisms did not reproduce the disease, nor did rabbit
antiserum containing agglutinins neutralize the murine virus.

The virus was found to be strictly pneumotropic for mice and to increase
in virulence with the first few serial passages. Routes other than intra-
nasal failed to produce infection, and the virus could not be obtained from
the brain after intracerebral inoculation, nor from the liver following intra-
peritoneal injection. Attempts to transmit the infection by contact were
unsuccessful. Ten other species of animals, including rabbits, ferrets,
guinea pigs, and rhesus monkeys, were resistant to infection. The virus
has been cultivated in tissue culture with considerable loss in virulence.

Active immunity in mice was readily obtained by two intraperitoneal
injections of living virus or by intranasal inoculation of amounts insuflicient
to produce death. All strains of this murine virus were identical immu-
nologically and were easily distinguished from the human and swine strains
of influenza by cross immunity and neutralization experiments. The virus
was neutralized, however, by approximately one-third of 67 human sera,
although in later experiments no association could be made with any of the
respiratory diseases common in humans.

In suspensions of infected mouse lung the virus was inactivated at
56°C. in 30 minutes, and decreased in titer rapidly at room temperature
unless protected by the addition of 10 per cent normal horse serum. No
decrease in activity occurred when frozen and stored at — 76°C. It was
readily filterable through Berkefeld V and N filters but not through the
Seitz filter. By the use of graded collodion membranes, its diameter was
found to be approximately 100 to 150 m/x.

It thus seems probable that experimental pneumonias in mice, though
similar pathologically, may be due to different viruses. The agents do not
cause spontaneous illness and not all mouse stocks are infected with them.
The original source is not known, but may be human (104), if neutralization
of this virus is as specific as it is with other viruses. The primary impor-
tance of this disease to investigators, however, is its similarity to that pro-

INFECTIOUS DISEASES OF MICE 451

duced in mice by the viruses of influenza (9, 80, 250, 262) and the possibiHty
of mistaking its identity.

Inclusion bodies in the salivary glands and liver of the mouse. — Cellular
inclusions have presented something of a problem to investigators for a
number of years. Certain of them occur frequently in abnormal or malig-
nant cells, but are artefacts due to intracellular necrosis and the action of
ingredients in the fixatives (42). In the past two decades inclusion bodies
of a different type have been found in cells of the salivary glands and liver of
a number of animal species, including mice (68, 137, 273, 274, 168). They
occur quite constantly in some stocks or breeds of mice. Transmission to
normal young or adult mice is readily accomplished, is species-specific, and
no bacteria or parasites are found in association with the bodies. Foci of
chronic inflammatory cells are present in the affected organs. With the
possible exception of an epidemic mentioned by Thompson (273, 275; see
section on ectromelia), animals harboring them appear to be perfectly
healthy. These characteristics suggest that an infectious agent of low
pathogenicity is responsible for their production. The nature of the
inclusion bodies — whether degenerative, metabolic, mutative, or infectious —
is not known. Since the appearance of inclusions is concomitant with
infection by many of the known viruses, however, it is logical by analogy to
consider a virus as the causal agent here. Filterability, moreover, is
reported in one instance fi68).

Inclusion bodies in the salivary glands. — The incidence of salivary
gland disease varies between 20 and 60 per cent in adult albino mice of
certain stocks. Other colonies may be entirely free from the disease regard-
less of the age of the animals, but in general mice less than i month of age
do not show the lesions. Spontaneous illness has not been described. The
natural method of transmission has not been determined, but once a colony
has been infected the disease continues for generations.

Histopathologically, lesions are found only in the salivary glands.
Acidophilic intranuclear inclusions, usually large but of varying size, occur
in acinar cells of the serous and mucous portions of the glands, occasionally
in duct cells, and rarely in alveolar cells of the parotid. Such cells arc
hypertrophied and irregular in shape with granular, basophilic cytoplasm.
The nuclear contents may be completely replaced or distorted by inclusions
which are composed of minute spherules and are often surrounded by a
halo. Scattered foci of mononuclear cells are present throughout the tissue,
often without any apparent relationship to the aft'ected acinar cells.

452 BIOLOGY OF THE LABORATORY MOUSE

The disease may be transmitted to normal adult or young mice by
inoculation of emulsions of the infected salivary glands or by filtrates of
such emulsions. Negative results are obtained with other tissues. No
clinical manifestations occur in adult animals and the virus localizes in the
salivary glands regardless of the route of inoculation. Following intra-
cerebral injection a mild meningeal reaction may result, with exudation of
mononuclear cells and occasional inclusion bodies within cells of the cerebral
tissue, endothelial cells of the choroid plexus, and mononuclear cells. In
young animals (3 weeks of age) a fatal infection may be produced by intra-
peritoneal and occasionally by intracerebral inoculation (168). Death
usually occurs in 3 to 7 days. Necrotic lesions are found most extensively
in the liver, spleen, adrenals, lymph nodes, and subperitoneal tissue. Intra-
nuclear inclusions are frequent in these tissues, but are not found in the
salivary glands unless the animal survives for 8 days or longer. Experi-
mentally, strains of mice vary in their susceptibility to the virus. Other
species of animals are resistant.

The properties of the transmissible agent have not been fully inves-
tigated. It is destroyed by a temperature of 6o°C. for 30 minutes. Filtra-
tion through a Berkefeld V filter has been accomplished.

Inclusion bodies in the liver. — Inclusions in hepatic cells are probably
very uncommon since Twort and Twort (299) did not notice them in the
course of some 12,000 postmortem examinations. Findlay (68), however,
observed acidophilic intranuclear inclusions in the livers of all the mice of
one strain (Clacton) obtained from a London dealer. They were not found
in the livers of newly born mice. Transmission to a disease-free strain of
mice was accomplished by inoculation of an emulsion of infected liver. This
observation has been confirmed by Thompson (273, 275), who noted hepatic
inclusions in 5 of 25 apparently healthy mice as well as during an epidemic
which somewhat resembled ectromelia.

Factors Influencing the Production of Experimental
AND Natural Disease in Mice

The study of any infectious disease is best carried out in its natural
host. For obvious reasons, however, an experimental study of certain dis-
eases on such a basis may be impracticable if not impossible, and it is neces-
sary to resort to a different species of animal. The disease thus obtained
may or may not be similar to the original one, but it will be dependent, as is
the natural disease, on at least three important variables: the microbe, the

INFECTIOUS DISEASES OF MICE 453

environment, and the host. The extensive work in experimental epidem-
iology (276, 74, 5, 6, 181, 313, 314, 320, 94, 279, 92) well demonstrates the
significance of these factors in natural diseases of the mouse, and numerous
other observations attest their importance in artificial infections.

Microbic factors. — The type of disease resulting from the introduction
of an infective agent into the body of the host is dependent on dosage, route
of inoculation, and virulence of the agent. Thus, increase in dosage may
alter the course from a benign subclinical infection to a rapidly fatal, over-
whelming infection; or the subcutaneous route of inoculation may be
entirely ineffective, whereas intracerebral injection produces a striking
encephalitis. The role of virulence or pathogenicity is somewhat more
difficult to assess. Strains of an organism obtained from different sources
or in various stages of dissociation undoubtedly vary in their capacity to
produce disease. Whether or not it is possible to alter the inherent virulence
of a given strain by repeated animal passage is open to question, at least
in the case of certain organisms, when all other factors are kept as nearly
constant as possible (308. 192, 309, 310, 311, 312).

Environmental factors. —Various features of the environment — tempera-
ture, diet, season, number of animals per cage, and cleaning routine — alter
the type of disease chiefly by their eft'cct on host factors, and, to a less
extent, on microbic factors. Thus, mice of the same stock reared on a
bread and milk diet without obvious dietary deficiency were found to be
more susceptible to mouse typhoid (329, 210) than those fed the more com-
plete McCollum diet. Crowding of animals in a cage may aft"ect the
microbic factors by increasing the dosage or altering the route of infection
if the organism is excreted by the inoculated animals.

Host factors. — When a group of mice, maintained under controlled
environmental conditions, is given a standard dose of an infective agent, a
certain number of them become ill and die, others recover, and still others
may show no signs of infection. The relative proportion in each group will
depend on the specific and nonspecific resistance of the host — a complex
mechanism, the individual factors of which are not easily segregated and
subjected to quantitative analysis. Considerable progress has been made in
this direction, however, chiefly as a result of the stimulating investigations in
the field of experimental epidemiology.

Specific resistance is considered to be an immunity acquired through
previous contact with the infectious agent. That such specific immunity
as a factor in resistance is operative in certain natural and experimental
infections is generally accepted, but in others its relative importance in

454 BIOLOGY OF THE LABORATORY MOUSE

comparison with nonspecific factors is questioned. Webster and Hodes
(328) have recently demonstrated that highly susceptible mice are not
immunized to a subsequent test dose by repeated, sublethal doses of mouse
typhoid bacilli or St. Louis encephalitis virus given by a natural route.
They further emphasize that reinoculation of survivors is not an adequate
test of active immunity unless the animals employed are known to be
'' ... at least 90 per cent susceptible to the test agent given by a normal
portal of entry." An animal surviving the first dose by virtue of nonspecific
resistance may withstand a second dose in the same manner without neces-
sarily having an active immunity. Moreover, in such diseases as mouse
typhoid and ectromelia, vaccination by the methods thus far employed may
give some protection but does not confer a solid immunity (306, 205, 289, 92).
Nonspecific resistance appears to be a characteristic of the individual,
dependent on heredity and probably on other factors as yet unknown, as
modified by age and environmental influences. The degree of resistance
varies among individuals in a single breed of mice as well as among different
breeds (295, 277, 312, 315, 206, 208, 209, 90, 245). Because of this fact it is
possible by selective inbreeding to develop stocks with a relatively high
resistance or susceptibility to one infectious agent but not necessarily to
another (316, 317, 289, 321, 322, 97, 326, 327, 323, 324). A study of hybrid
and backcross generations (295, 90, 321, 324) indicates that resistance is
dominant, but segregates independently of the sex and color factors. The
responsible genetic factors are considered by Hill (97) to be multiple, since
litters of long inbred lines may show more variation in reaction than can be
ascribed to chance, whereas Webster (323) supports the theory of a single
factor type of inheritance with possibly a number of small modifiers, since
mortahties in succeeding generations showed no definite progress with selec-
tion. Both Hill and Webster have emphasized the need for extreme precau-
tions in work of this kind in order to exclude the specific resistance of
acquired immunity, either active or passive.

There are numerous observations, some of which are mentioned in the
preceding pages (see also 183), to indicate that resistance varies with age,
older animals in general becoming more resistant. Nonspecific anatomical
and physiological factors (228) undoubtedly play a role, but there is also
evidence that specific factors may be involved, since immature animals are
less able than mature ones to respond to an antigenic stimulus by the forma-
tion of antibodies (20, 53, 175).

The above work is important not only because it aids in the selection and
use of animals experimentally, but also because it points out many of the

INFECTIOUS DISEASES OF MICE 455

important features to be considered in the prevention and control of natural
disease in animal colonies.

Prevention of Disease and Control of Outbreaks

The application of general preventive measures is the only satisfactory-
way, in the absence of specific prophylaxis or therapeutics, to prevent the
introduction and spread of natural disease in a mouse colony. Success will
depend to a large extent upon the strictness with which the control measures
are maintained. "Some idea of the frequency with which spontaneous
disease is encountered may be gained from the figures on occurrence given in
the preceding sections. In addition. Greenwood and Topley (94) report
that during 7 years the most important spreading diseases were due to
Sahnonella typJiimurium. S. enteritidis, Pasteurella muricida, and Erysipelo-
thrix muriseptica. whereas infection due to Proteus morgani, other tj'pes
of Proteus, enterococci, and Corynehacterium kutscheri spread to a less
extent.

It is apparent from the discussion in the last section that those procedures
which increase the environmental and host resistance factors and decrease
the microbic factor of dosage will thereby lessen the opportunity for the
spread of infection. Of these, the environmental factors are probably
most important, since they are most readily subjected to control and in
themselves modify host resistance and dosage. An attempt will be made
to present a description of the ideal physical equipment for the animal rooms,
although experience has shown that departures from this in many respects
can be made satisfactorily. The cleaning technique is modeled on that now
in use in the Jackson Memorial Laboratory.

The animal rooms should be rodent proof, light, well ventilated with
regulation of temperature and humidity, and so constructed as to permit
washing of the walls and floor. This process is facilitated by a central
drain and a rounded baseboard which obliterates the angle between the
walls and the floor. The floor should be considered to be contaminated at
all times and nothing placed thereon should be permitted to come in con-
tact with cages, racks, tables, or other equipment of the room without
sterilization. Low tables can be used to support clean cages during actual
replacement of cages. Racks should be of simple metal construction set
out from the walls to eliminate breeding places for vermin, and if possible
suspended from the ceiling. They should be sufficiently spacious to permit
arrangement of individual cages without contact between them. The cages

456 BIOLOGY OF THE LABORATORY MOUSE

themselves may be of simple metal box- or pan-like construction with a
detachable screen lid permitting replacement of water bottles and food with-
out removal of the lid. Food and bedding are best stored in metal con-
tainers, bins, or special rooms protected from vermin and stray rodents.
Each cage as a unit should house the smallest number of mice consistent
with the total number and available space. Breeders are best kept in
separate cages in a separate room.

Washing of the rooms, racks, and transfer of cages should be carried out
at least once a week. The attendant is best garbed in a coverall or gown
which can be laundered, and should scrub and dip his hands in disinfectant
between sections of racks while transferring animals from dirty to clean
cages. In a separate room the cages and water bottles are cleaned, washed,
and sterilized, preferably by steam. If chemical sterilization is employed,
a sufficiently long period of contact must be allowed to insure effective action
of the germicide. Sterilized wood shavings are most satisfactory for bed-
ding, and may be placed in the clean, dry cages before they are removed to
the animal room for the next cleaning. By following this cleaning technique,
dust in the animal room itself is reduced and disposal of waste becomes a
simple matter.

A nutritionally complete diet may be prepared on the basis of the
McCollum or Steenbock formulas or their modifications (329, 321). Ade-
quate diets are also available commercially. Under ordinary circumstances,
sterilization of the food is not necessary. Except in special instances, little
can be accomplished by attempting to alter the specific host factors.

Before being added to the general stock, new mice should be kept in
quarantine for at least 3 weeks, distributed in separate cages containing 4 to
6 mice each. Postmortem examinations, with cultures, should be made on
all dead animals. If infection is recognized, the cage-mates must be killed.
Should no cause for the death be found, the other animals in that cage are
watched for an additional 2 or 3 weeks. A second death is an indication for
destruction of the remaining animals in the unit; otherwise they may be
considered to be normal.

Measures similar to quarantine should be taken in the event of an out-
break of disease in the general stock. At the first appearance of the disease
the room should be rigidly isolated and the diseased mice cared for only by
attendants who have no contact with normal animals. Depending on
previous conditions, the animals should be redistributed into the smallest
possible number per cage unit. A specific death is then an indication for
the destruction of all the mice in that unit. If the disease is very extensive,

IXFECTIOUS DISEASES OF MICE

457

it may be necessary to kill all the animals, but in all probability the above
procedure will prevent an extensive epidemic, or at least permit a number of
survivors from which the stock can be rebuilt. Since in a number of diseases
the carrier state may be persistent, care must be taken in adding new sus-
ceptible mice or in augmenting the number of animals per unit, until
examination of a sufficient sample of apparently normal mice and of those
dying sporadically reveals no evidence of the disease.

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INFECTIOUS DISEASES OF MICE 469

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226. Sabix, a. B. 1939. Experimental proliferative arthritis in mice produced by
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470 BIOLOGY OF THE LABORATORY MOUSE

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INFECTIOUS DISEASES OF MICE 471

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472 BIOLOGY OF THE LABORATORY MOUSE

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INFECTIOUS DISEASES OF MICE 473

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474 BIOLOGY OF THE LABORATORY MOUSE

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Cnapter 13

CARE AND RECORDING

i^v John J. Bittner, Roscoc B. Jackson Memorial Laboratory.

The salient features in the care of mice are probably similar in most
laboratories. The majority of investigators naturally consider that the
methods they use are superior to those employed by others. In some
respects they may be correct as the system one must apply is often deter-
mined by unusual circumstances which only experience may correct. A
stock of mice maintained in a satisfactory condition by one worker may die
out under the care of a second with conditions practically identical. There
is also in the management of mice some indescribable "knack" which some
have and others apparently lack. There is no indication that this is due to a
lack of interest on the part of the latter individual; it is more likely to result
from too much attention and handling of the animals.

The method to be described for the care of mice is that followed at the
Jackson Laboratory with satisfactory results.

The mouse boxes (Fig. 171) are made of wood and measure 12" X 12" X
6". The bottom is covered by I4" plywood. The front, back and sides of
the box are made from ^ 2" stock and the center partition is -^4" thick. The
thick center partition gives more room for the covers to overlap and the life
of the box is lengthened as it is through this board which the mice are most
likely to gnaw. The boxes may be painted or may be dipped in a solution
of equal parts of turpentine and linseed oil containing dryer. Lionoil may
be substituted for the linseed oil.

Wooden boxes have some advantages over metal or wire cages, the most
important being that the mice are warmer than they would be in metal boxes
and are not subjected to drafts. Also, less light penetrates the boxes thus
giving a more natural habitat for the rearing of young. The initial cost is
considerably lower than for the other types, but replacement must be made
more often.

The stock for the covers measures 1I4" X f^" with the pieces cut so that
part fits down into the box and the flange overlaps the side of the box to
support the weight of the cover, the food hopper and the water bottle. The

475

476

BIOLOGY OF THE LABORATORY MOUSE
BOX TOP

A. WIRE
4 strands
to the inch

2 m m. opening

gloss size 9mm. diam

^ BASE

5:" ply board

MOUSE BOX

with one side
remove d

Fig. 171. — Design and measurements for mouse boxes, covers, hoppers, etc.

CARE AND RECORDING 477

wire is nailed on the under surface of the frame to prevent the mice from
chewing the cover and the edges of the box.

A cross-bar near the front on the covers supports the water bottle (16 oz.)
in a slanting position. A bent glass tube passing through a rubber stopper
extends from the water bottle down into the pen. The end of the gooseneck
is partially closed to form a nipple from which a drop of water hangs. There
is no leakage from the water bottle unless the opening in the tube is too large.
Ordinarily the bottles need to be filled no oftener than once a week.

A hole is cut in the wire of the cover to receive the wire food hopper which
measures, at the top, 3X2 inches. At the bottom of the hoppers the
measurements are 3 X 1^4 inches. The wire from which the hoppers are
made has four wires to the inch and the parts are sewed together with fine
copper wire. The upper parts of the hoppers are bent so that they interlace
with the wire of the cover to hold the hoppers in place and prevent openings
through which small mice might escape. The hoppers extend to within an
inch from the floor of the cage and hold suf]ficient food to last six mice one
week.

Several commercial foods in pellet form are available for use in hoppers
of this type.

The type of rack which one uses is important from the standpoint of
cleanliness and the control of vermin. Metal racks are more satisfactory
from this standpoint than are wooden shelves. The clearance between
shelves should be at least 11 inches. The shelves may be 12 inches or 24
inches wide to provide space for one or two rows of boxes. In small quarters
the shelf space may be increased considerably by careful planning and the
use of certain types of racks.

All boxes containing mice should be changed at least once a week. The
clean boxes should contain shavings or sawdust and a small amount of
cotton for bedding. Some stocks require shredded paper in place of cotton.
The soiled bedding in the used boxes should be entirely removed after
which the boxes should be thoroughly sterilized and dryed before they
are used again. The water bottles and covers should be washed at
intervals.

The mice are marked by a series of holes and notches on the ears (Fig.
172). The units are recorded on the right ear, the tens on the left ear.
Number one, two and three are represented by holes at the front, top and
back respectively; four, five and six each by a single notch starting from the
front of the ear; seven is represented by two notches close together at the
front; eight by two notches at the top and nine by two notches at the back

478 BIOLOGY OF THE LABORATORY MOUSE

of the ear. By this system it is possible to number, on the ears, from i to 99
for individual identification. The complete serial number of each animal is
kept on the individual card.

The animals are marked when they are weaned and breeding pens are

usually made up at this time. Each side of the boxes has sufhcient space for

six adult animals and thus five females may be mated to each male in strains

where the animals show high fertility. Matings in

LEFT EAR RIGHT EAR • , , , • > • , u 4^u \/ • <.

mbred strams of mice are always brother X sister

r o\ '^ ) unless a son is mated to its mother or a daughter

"^' — '^— ^ '■ to its father. Cousin matings should never be made

e/^ o) if one wishes to maintain a homozygous stock. A
— '-■ practice which will save considerable time eventu-
CN /'j ally is to keep a small pedigree chart for every strain

^ -Jiy ^- on which every individual is recorded which has

e/'~^ been mated. The development in any animal of
~._y 4. any desired character or condition should be noted
C/^^ on this chart. After every few generations the best
,^_^ 5. line may be selected from which to continue the
C/^~\. stock. Litters or sub-lines which are not wanted
.,^^^ 6. should be discontinued. Unless some method for
CjS~^ selecting the matings is used, the strain may
^^y J eventually consist of sub-lines which differ markedly
C>— V from each other. If the development of different

y g sub-lines is desired the chart will aid in assuring the
C^-. worker that all lines are being continued.

J On the individual female card are recorded the

^-^^ number, color and sex of each mouse. Space is also

V ) provided for the strain or line, the inbred generation,

"*~^' date of birth, death, development of spontaneous

Fig. 172.— -Earmark- tumor, age at death or development of tumor. The
ines for individual iden- ,. .- ^, . , , •, , ■ ■ u u ^^

.,? . . ., pedigree ot the animal may be written at the bottom

tincation; units on right ^ <=' 1 i- 1

ear tens on left ear ^^ ^^^ ^^^'^ ^^^ ^^^ mating and breeding box number

inserted. Space has been provided to enter data for
twelve litters. In successive columns, from left to right, may be given for each
htter: the generation, ledger number, date born, number born, born dead,
weaned, females, males, age of mother at the birth of each litter and the num-
ber of days between litters. These cards may be altered to catalogue any
information that a worker may want in his investigations. (Sample card will
be supplied on request.) The male card may be a ruled 4X5 library card

CARE AND RECORDING 479

on which may be Hsted the data regarding the male and the numbers of the
females to which it has been mated.

In addition to the individual cards a serial ledger should be maintained
in which all the litters are entered. A separate ledger may serve for each
stock or a continuous one for all stocks.

Pregnant females should be given an individual pen in which to have and
rear their young. Experience will show that more and better young will be
raised to weaning age if this system is followed. The female's individual
card should be changed from the breeding pen file to that corresponding to
the new box when she is separated from the male. The new box number
may be noted on the male card to assist in locating the female at any time
before she is returned to the home pen.

On the date of birth of a litter all the desired data should be recorded on
the individual card. The recording in the ledger may, if desired, be delayed
until the young are weaned.

Consideration should be given to the following details for the satisfactory
care of mice.

A well heated and ventilated room should be available. The temperature
should be approximately 72° at all times. If an automatic ventilating sys-
tem is not used, care should be used in ventilating the animal room by means
of windows. Avoid sudden changes in temperature and direct drafts on the
mice as they easily contract pneumonia.

Take rigid sanitary precautions in the care of the boxes, bottles, goose-
necks and racks. If the water bottles are all filled at one time the sterilizing
of the goosenecks is advisable to prevent the spread of disease from one box
to another. Covers and hoppers should be cleaned at regular intervals.

Food and water should be before the mice at all times. The selection of
the food may only be determined by experience as some strains will do better
on one diet than another. Soiled food remaining in the hoppers when the
boxes are changed should be discarded.

Do not place too many mice in a single pen. Overcrowded mice often
become infested with mites or lice. These may be controlled by periodic
dusting with powdered tobacco or a mixture of i part of derris root powder
and 3 parts of talcum powder (it may be necessary to sterilize the ingredients
before they are used).'

To obtain and maintain inbred strains, mate only brothers and sisters.
Mark all animals used for experimental or breeding purposes and make use of
individual cards and a ledger for complete records.

INDEX

A strain, breast tumoj incidence in, 259
carcinoma of skin appendage, 198
litter size in, 57
lung tumor incidence in, 2O3
tumors in hybrids of, 264
transplantable tumors of, 296
tumor transplants in, 2S4
Abscesses (see pyogenic infections)
Accessory glands, 137
A chorion qninckeanum, 430

Schoenleinii, 430
Actinomyces muris, 399
Adeno-acanthoma, 190
Adenocarcinoma, hemorrhagic cyst, 189

intracanalicular, 187

macroglandular, 188

of hypophysis, 229

of lung, 209 ^

of mammary gland, 182

of pancreas, 221

of renal pelvis, 222

papillary cyst, 185

simple, 182

variable type, 183
Adeno fibroma, 181
Adenoma, fibro, 181

of liver, 220

of lung, 209

of mammary gland, 176

of renal tubules, 221

papillary cyst, 180

polylocular cyst, 179

simple, 177
Adrenal cortex, nodular hyperplasia, 311
Adrenal glands, loi

brown degeneration of, 312
protozoan, infection in, 356
Age, role in tumor transplantation, 299
Aleukemia, 215
Allantois, 25
Alveoli, of lungs, 129

Amnion, 20

composed of somatopleure, 32
later development of, 36
relation to tail fold, 44
Amniotic cavity, 20

folds, 18
Amoeba enteric a, 351
fecalis, 351
DiHsciiIi, 351
Amoebae, 350
Ampulla, of ductus deferens, 137

of oviduct, 155
Ampullary glands, 139
Anemia, due to bartonellosis, 421

to Eperythrozoon infection, 425
Angio-endothelioma, 208
Angioma, 194, 200
Antrum, 149

first appearance of, 60
Anus, 124

development of, 28
Aorta, dorsal, 50
Archenteron, 25
Arsenicals, bartonellosis treated with, 421

Eperythrozoon infection treated with, 426
spirochete infection treated with, 433
Arteries, 90

omphalomesenteric, 50
Arthritis of mice, 399

differential diagnosis, 404
etiology, 403
immunity, 404
mode of transmission, 402
the experimental disease, 403
the natural disease, 400
Ascites, in infectious ectroraelia, 436
Aspicularis tetraptera, 365
Ataxia, due to Bacillus piliformis, 416
Atresia of follicles, 154

cyclic in young mice, 61
in newborn rats, 60
relation to estrous cycle, 74
Atria, of the heart, 90

481

482

INDEX

Bacillus piliformis, 416

pscudotubercHlosis murium, 396
pyocyaneus, 415
subtilis, 412

typhi murium, 381, 387
Bacteremia, in mouse typhoid, 383
Bacteria (see also infectious diseases)
anaerobic, 429
avian tubercle bacillus, 429
Bacillus piliformis, 416

pyocyaneus, 415

subtilis, 412
Bacterium influenzae murium, 412
Bartonella {Haenwbartonella) muris, 419
Brucella bronchiseptica, 409
coccobacilliform bodies, 414
Corynebacterium kutscheri, 396, 455

muris, 397

murisepticum, 397
enterococci, 455

Eperythrozoon coccoides, 420, 424
Erysipelothrix ■muriseptica , 399, 455
Friedlander-like bacillus, 410
Grajfkya tetragena, 415
Grahamclla musculi, 420, 428
Micrococcus cyaneus, 415
Pasteur ella muricida, 392, 455

^e5//j, 393

pseudotuberculosis, 393

tularemia, 429
pleuropneumonia-like organisms, 404,

408
Proteus morgani, 389, 455
Salmonella enteritidis, 381, 386, 455

species unknown, 389

typhimurium, 381, 386, 455
Staphylococcus albus, 415

aureus, 415
Streptobacillus moniliformis, 400, 403,

418
Streptococci, 416
Bacterium influenzae murium, 412
Bartonella muris, 419

transmitted by lice, 371
Bartonellosis, 419
etiology, 423
mixed infection with bartonellae and

eperythrozoa, 426
the experimental disease, 421

Bartonellosis, the natural disease, 419

treatment, 421
Basophils, 92
Bedbugs, 372

as transmitters of disease, 373
eradication of, 373
Bile ducts, 126

tapeworms in, 364
Birth (see parturition)
Bladder (see urinary bladder)
Blastocoele, 5
Blastula, 5
Blood, 92

embryo bathed in maternal, 39
in vagina as sign of pregnancy, 40
islands, 37
platelets, 92

protozoan infections in, 353
Blood counts, 92

deviations from normal, 350
in infected mice, 382
in lymphocytic choriomeningitis, 440,
441
Blood forming organs, 94

tumors of, 212
Blood vessels, 90

development of, 50
Body cavity (see coelom)
Body wall, tumors of, 195, 199
Bone, chondroma, 199

osteogenic sarcoma, 207
osteoma, 200
Bone marrow, 94

appearance in mouse typhoid, 386
cell count, 95
Botrio mycosis, 415
Bowman's capsule, 130
Boxes, mouse, 455, 475
Brain, protozoan infection in, 356
Breast (see mammary)
Bronchi, 128
Bronchioles, 129
Brother-sister mating, 328, 329

of yellow mice, 330
Brucella bronchiseptica, 409
Bulbo-urethral glands (of Cowper), 144

C3H strain, blood count in, 93

breast tumor incidence in, 259

INDEX

483

('sH strain, transplantable tumors of, 205
C57 black strain, angioma in, 194
blood count in, 93
eye abnormalities in, 336
gestation period in, 56
liver tumors in, 220
lung tumor incidence in, 263
tumors in hybrids of, 264
mean tumor age, 268
osteogenic sarcoma in, 207
pneumonia in, ,410
prenatal mortality in, 57
skin tumors in, 195
susceptibility to typhoid, 338
transplantable tumors of, 296
tumor incidence in, 266

incidence in hybrids of, 252
types of tumors in, 267
C57 brown strain, blood count in, 93

transplantable tumors of, 296
C57 leaden strain, blood count in, 93

transplantable tumors of, 296
C58 strain, leukemia in, 271
CBA strain, transplantable tumors of, 295
Caecum, 122
Cages, 455, 475

cleaning of, 456
Canal, pericardial-peritoneal, n
C a pillar ia bacillata, 369
Capillaries, 90

lymphatic, 91
Capsule of Bowman, 130
Carcinogens, in conjunction with estrogens,
314
sex difference in effect, 312
Carcinoma, 171

of Langerhan's islands, 221
of liver, 220

of skin appendages, 194, 197
of urinary bladder, 222
Carcinoma simplex, mammary, gland 190
of lung, 210
of uterus, 225
Carcinosarcoma, mammary gland, 192

of lung, 212
Care of mice, 358, 360, 371, 373, 376, 455, 475
Catarrh of mice, 413
Cavity, amniotic, 20
ectoplacental, 20
proamniotic, 10

Cavity, segmentation, 5

yolk, 7
Cell mass, inner, 5
Cells, chief (of gastric glands), 119

chromophil, 99

chromophobe, 98

decidual, 156

epithelial, in vaginal smear, 68

follicular, 149

giant, of embryo, 40

goblet (of intestine), 120

granulosa, 149

interstitial, of Leydig, 135

lutein, 153

macrophages, 96

mast, 96

of Kupffer, 125

of Paneth, 121

parietal, 119

plasma, 96

septal of pulmonary alveoli, 130

Sertoli, 133

spermatogenic, 133

theca lutein, 154

thymocytes, 103
ce strain, ovarian tumors in, 223

prolonged estrous smear in, 76
skin tumors in, 195
Central nervous system, tumors of, 228
Cervical tumors, induced by estrogens, 314
Cervix, of uterus, 157
Cestoda, 359

Chief cells, of stomach, 119
Chondroma, 193, 199
Chondro-osteosarcoma, 208
Chondrosarcoma, 194, 208
Chorion, 20, ;^;^
Chromophil cells, 99
Chromophobe cells, 98
Chromosomes of mouse, 243
Chronic inflammation, 171
Cimcx Icctiilariiis, ^■j2
Classification of tumors, 208
Cleavage, 4
Clitoral glands, 157

carcinoma of, 198
Clitoris, 157

Coagulating glands, 55, 141
Coccidia, 354

Coccobacilliform bodies, 414
Cockroaches, 358, 376

484

INDEX

Coefficients of inbreeding, 329
Coelom, development of, 32

extra-embryonic (see exocoelom)
pericardial, 33, 45
CoUiculus seminalis, 139

turbarius, 155
Colloid, 100
Colon, 122

nematodes in, 365
Columnar entoderm, 21

Conjunctivitis, due to mouse septicemia, 398
in Brucella infection, 409
in lymphocytic choriomeningitis, 439
in mouse arthritis, 400
typhoid, 382, 390
Convulsions, due to encephalomyehtis, 445
in lymphocytic choriomeningitis, 440
Copulatory response, 65
Corium, 105
Corona radiata, 149
Corpora lutea, 80, 151

factor in mammary cancer, 313
number of and egg number, 57
Corpus cavernosum penis, 146
urethrae, 145
uteri, 157
Corynebacteriiim kutschcri, 396, 455
muris, 397
muriscpticiim, 397
Coryncthrix pseudotuberculosis murium, 396
Cryptosporidium muris, 354

parvum, 354
Cumulus cells, 78 (see also follicular cells)

oophorus, 149
Cysticercus fasciolaris, 360

dba strain, transplantable adenocarcinoma of,
290
tumors of, 296
tumor incidence in hybrids of, 252
transplants in, 284
Decidua, 7

basalis, 39
capsularis, 38

intermediate or vascular zone, 39
Decidual cells, 156
Deciduomata, 79

induced during lactation, 81
Dental formula, 1 10
Dermis, 105

tumors of, 199
Development, postnatal, 58
Diarrhea, due to Bacillus piliformis, 416

Bacterium- influenzae murium, 41 1
coliform bacillus, 388
Salmonella infection, 389
Diestrus (see estrous cycle)
Diet, 456

efTect on disease, 453
role in tumor transplantation, 297
Digestive system, tumors of, 219
Digestive tube, layers, 116
Diseases (see infectious diseases)
Distal entoderm, 8

Reichert's membrane and, 33
Dorsal aorta, 50
Ductus deferens, 137

epididymidis, 137
Duodenal glands, 121
Duodenum, 120

tapeworms in, 364

dba strain, adrenal cortex of castrates, 311
arthritis in, 400
blood count in, 93
breast tumor incidence in, 259
gestation period in, 56
liver tumors in, 220
mean tumor age, 268
melanoma in, 194, 204
osteogenic sarcoma in, 207
prenatal mortality in, 57
rhabdomyosarcoma in, 194
skin tumors in, 195

Ear, melanoma of, 204
papilloma of, 195
Echinolaelaps ccliidniiius, 375
Ectoderm, embryonic, 8
extra-embryonic, 8
Ectoplacental cavity, 20
Ectoplacental cone, 10

as origin of giant cells, 41
lumen in, 13
maternal blood in, 10
Ectromelia, 404, 434

pathology of the natural disease, 435
properties of the virus, 437

INDEX

48:

Ectromelia, the experimental disease, 437

the natural disease, 434
Efferent ducts, of testis, 136
Egg, 2 (see also ova)

fertilization of, 2, 77

maturation of, 77

passage into uterus, 4

size of, 2

transport of, 78
Egg cylinder, 7
Eimcria fakiformis, 354

miyairii. 354
Embryonal adenoma, of ovary, 22^
Embryonal cell carcinoma, of ovary, 22^

of testes, 227
Embryonic ectoderm, 8
Embryos, bathed in maternal blood, 39

early growth of, 5

hybrid vigor in, i

implantation of, 5

later position of, 51

nourishment of, 39

orientation of, 15

seven somite, 41

turning of, 44

variation in, 2
Encephalomyelitis (of mice), 443

differential diagnosis, 447

epidemiology, 447

properties of the virus, 445

relation to poliomyelitis, 446

the experimental disease, 444

the natural disease, 443
Rndamoeha coli, 350

maris, 350
Endocardium, 45, 90
Endocrine glands, histology, 98
Endocrine secretions, lung tumors and, 264
mammary tumors and, 259
produced by tumors, 315
tumor formation and, 310
Endometrium, 156
Endothelioma, of brain, 229
Enteritis, due to E. falcifonnis, 354
Enterococci, 455
Entoderm, columnar, 21

distal, 8

formation of, 7

gut, 24

notochord classed as, 24

proximal, 8

Entoderm, Reichert's membrane and, 3;^

squamous, 21
Environment, 331, 338

relative importance of heredity and, 337
role in infectious disease, 453
Eosinophils, 92
Eperythrozoon coccoidcs, 420, 424

transmitted by lice, 371
Eperythrozoon infection, 424

etiology, 427

occurrence, 425

the experimental disease, 426

the natural disease, 425

treatment, 426
F^pidermis, 105

tumors of, 195
Epidermoid carcinoma, 196

epithelial pearls in, 196

lung metastases of, 196

lymph node metastases of, 196
Epididymis, 137
f]pi-myocardium, 45
Epithelial horn, 195
pearls, 189, 196
Epithelium, germinal, production of ova by,

59
vaginal, 69
Erysipelothrix, 398
Erysipelothrix muriseptica, 399, 455
Erythroblasts, 94
Erythrocytes, 92

Bartonella infection in, 424
Eperythrozoon infection in, 425, 427
Grahamella infection in, 428
Esophagus, 116

papilloma of, 219
Estrogens, tumor rate affected by, 313, 314
Estrous cycle, 65

bodily activity during, 75
divisions of, 65

electrical potential, changes during, 75
in x-rayed mice, 65
low temperature lengthens, 76
mammary changes during, 75
ovary, changes during, 73
oviduct, changes during, 72
time relations of, 75
tumor influence on, 315
uterus, changes during, 72
vaginal changes during, 69
smears as guide to, 67

486

INDEX

Estrous cycle, weight changes during, 75
Estrus, age at first, 58

delayed by pseudopregnancy, 78
in lactating mice, 81

external signs of, 65

onset of, 75

ovulation during, 76

postpartum, 56, 75
Exocoelom, 20

relation to coelom, 7,^
Expansion, tumor growth by, 169
External genetalia, epidermoid carcinoma of,
196
papilloma of, 195
Extra-chromosomal influence, 251, 262, 266,

273
Extra-embryonic coelom (see exocoelom)

ectoderm, 8
Extramedullary myelopoiesis, non-malignant,

215, 216
Eyelid, papilloma of, 195
Eyes, melanoma of, 204
opening of, 58

Fat, lipoma, 200

Favus, 429

Fertility, in inbred strains, 334, 343

Fertilization, 2, 77

Fibro-adenoma, 181

Fibroma, 193, 199

Fibrosarcoma, 194, 203

neurogenic, 204

of kidney, 222

of liver, 221

of mammary gland stroma, 192

of pancreas, 221

of uterus, 226
Filterable organisms, 408 (see also virus
diseases)
pleuropneumonia-like, 408
Flagellates, 351
Fleas, 371

as intermediate hosts, 363, 364

as vectors, 353, 372, 421

eradication of, 372
Follicles (see ovarian follicles)
Follicular cells, 2, 149 (see also cumulus cells)
first appearance of, 59

Fore-gut, 26

posterior motion of portal, 31
Foster nursing, tumor incidence and, 257, 321
Friedlander-like bacillus, 410
Fungus diseases, 429

Gaffkya tetragena, 415
Gall bladder, 126

tapeworms in, 364
tumors of, 219
Gastric glands, 119

protozoan infection in, 355
Gene mutations, 234 (see also mutations)
Genetic factors, in infectious disease, 400,

403, 418, 454
Genetics of leukemia, 270
of lung tumors, 262
of mammary tumors, 259
of non-epithelial tumors, 264
of tumor transplantation, 279
Genital system, female, 146

male, 133
Germ cells, primordial, 59
Germ layers (see inversion of germ layers)
Germinal epithelium, production of ova by,

59
Gestation, 55

lengthened in nursing females, 56
Giant cells, 40
Giardia inuris, 352
Glands, adrenal, loi

ampullary, 139

anal, 124

bulbo-urethral (of Cowper), 144

clitoral, 157

coagulating, 55, 141

dorsal prostates, 142

duodenal (Brunner's), 121

endocrine, 98

exorbital lacrimal, 108

gastric, 118

Harderian, 109

hibernating, 108

intestinal (Lieberkiihn's), 120

intra-orbital lacrimal, 109

lingual, 112

male accessory, 137

mammary (see mammary glands)

of female urethra, 132

INDEX

487

Glands, of the larynx, 128

palatine, no

pancreas, 1 26

parathyroid, 100

parotid, 115

pineal, 104

pituitary, 98

preputial, 146

pyloric, 120

sublingual, 115

submaxillary, 112

thymus, 103

thyroid, 100

urethral (Littre), 143

uterine, 6, 155

ventral prostates, 143

vesicular, 55, 140
Glans penis, 146
Glioma, of brain, 228
Goblet cells, of intestine, 1 20
Golgi material in sperm, 3
Gongylonema muscitli, 369

neoplasticum, 368
Graafian follicles (see ovarian follicles)
Grahamella musculi, 420, 428
Granulosa cells, 149
Granulosa-cell tumors, 224
Growth, head fold as center of, 28

primitive streak as center of, 31
Gut (see fore-gut, hind-gut and mid-gut)

entoderm, 24

IlacmobartoneUa maris, 424

Haemoflagellates, 353

Hair, 106

Harderian glands, 109

Head fold, 28

Head process, 20

gut entoderm derived from, 24
notochord derived from, 24
Heart, 90

development of, 45

lesions in mouse arthritis, 401
Heat (see estrus)
Helminths, 359
Hemangio-endothelioma, 208

of liver, 220
Hemangioma, 194, 200

of liver, 220

Hemangioma, of urinary bladder, 222
Hemoglobin, 92
Hemorrhagic septicemia, 391
mortality in, 392

similar to Bacterium influenzae infec-
tion, 412
Hepatic lesions, due to Bacillus piliformis,
417
to Trypanosoma lewisi, 353
in ectromelia, 435
in mouse septicemia, 398

typhoid, 385
in Pasteurella infection, 392
in pseudotuberculosis, 394, 395
Hepatoma, 219

Heredity, relative importance of environment
and, 337
role in infectious diseases, 400, 403, 418,
454
Hcterakis-spumosa, 369
Heterosis (see hybrid vigor)
Heterozygosis (see also homozygosis)

due to mutation in inbred strains, 330
effect of inbreeding on, 328, 329
forced, 330
Hexamita muris, 351
Hibernating glands, 108
Hind-gut, 26, 42
Histiocytoma, 214, 217
Hodgkin's disease, 218
Homozygosis (see also heterozygosis)
effect of inbreeding on, 328, 329
of relaxing inbreeding, 345
of selection on, 330
Hoplo pleura acanthropus, 370

hesperomydis, 370
Hormones (see endocrine secretions)
Hybrid vigor, 341, 342

in embryos, i
Hybrids, 340

compared with inbred lines, 341

genetic characteristics of, 340

great variety available, 342

litter size in, 57

lung tumor incidence in, 264

non-epithelial tumors in, 269

phenotypic characteristics of, 340

Polydactyly in, 340

reciprocal differences between, 341

special uses, 342

transplantation of tumors from, 286

488

INDEX

Hybrids, tumor transplants in, 285

tumors in reciprocal, 322

use in tumor transplantation, 297

useful breeding period of, 59

value in research, 341

variation in, 341
Hydrothorax, in infectious ectromelia, 436
Hymcnolepis dim'uutta, 363

fraterna, 360

transferred from mouse to man, 362

microstoma, 364

liana, 361
Hypophysis, 98 (see also pituitary gland)

adenocarcinoma of, 229

tumor rate af^ectedby, 313

Ileum, 121

Immunization, to transplanted leukemia, 291

Implantation, changes in uterus, 37

delayed in nursing females, 56

of embryo, 5

pseudopregnancy and, 79
Inbred mice, useful breeding period of, 59
Inbred strains, building and maintenance of,

343

change of "average" in, 333, 338

decreased variation in, 335, 338

differences between, 339

fallacies, 345

heterozygosis due to mutation, 330

increased variation in, 336, 338

leukemia transplantation in, 290

parasites in, 347

risk of contamination in, 344

selection in, 330, 343

sublines, 344

tests of genetic uniformity, 343

uniformity in, 339

value in research, 337

vigor and fertility in, 343
Inbreeding, 325

coefficients of, 329

different effects in two sexes, 336

effect of linkage in, 329

of relaxing, 345

on the "average," 333

on tissue specificity, 335

on variation, 334

on vigor and fertility, 334

Inbreeding, effect on white spotting, 333, 335
genetic effects of, 327
need for in tumor studies, 249
phenotypic effects of, 326, 330
value of genetic effects of, 337

of phenotypic effects of, 338
Inclusion bodies, 451

in infectious ectromelia, 436
Infectious catarrh, 413

similar to Bacterium influenzae infec-
tion, 412
Infectious diseases, 349, 380 (see also bac-
teria, fungus diseases, protozoa)

arthritis of mice, 399

bartonellosis, 419

botriomycosis, 415

due to Baccillus pilifonnis, 416

to Bacterium influenzae murium, 41 1
to Epcrythrozoon coccoides, 424
to Grahamella musculi, 428
to pleuropneumonia-like organisms,

404
to Streptohacillus moniliformis, 400

ectomelia, 404, 434

encephalomyelitis of mice, 443

environmental factors in, 453

factors influencing, 452

favus, 429

hemorrhagic septicemia, 391

host factors in, 453

inclusion bodies in livej, 452

infectious catarrh, 413

leptospirosis, 433

lymphocytic choriomeningitis, 438

microbic factors in, 453

miscellaneous, 429

mouse septicemia, 398
typhoid, 381

pasteurellosis, 391, 404

plague, 394

pneumonia (three t)T)es), 409

prevention of, 358, 390, 455, 479

pseudotuberculosis of mice, 394, 404
of rodents, 393

pyogenic infections, 415

ringworm, 429

salivary gland inclusion bodies, 451

spirochetosis 431

transmission by bedbugs, 373
by fleas, 372
by lice, 371

IXDEX

489

Infectious diseases, transmission by mites,

375
virus pneumonia, 448
Infiltration, tumor growth by, 169
Inheritance (see genetics)
Inner cell mass, 5

Interlocking, tumor growth by, 169
Interstitial cells of Leydig, 135
Intestinal portal, anterior, 27
Intestine, carcinoma of, 219

fibrosarcoma of, 219

hemangioma of, 219

large, 122

nematodes in, 365, 369

polyp of, 219

protozoan infections in, 350, 352, 354

small, 120

tapeworms in, 360
Inversion of germ layers, 10
reversed, 28

Japanese waltzing mice. Bacillus piriformis
infection in, 416
spleen transplants in, 286
tumor transplants in, 280

Jejunum, 121

Kidneys, 130

lesions in pseudotuberculosis, 395
protozoan infection in, 355
tumors of, 221

Klebsiella pneumoniae, 411

Klossiella muris, 355

control measures, 358

Kupffer, cells of, 125

Lacrimal glands, exorbital, 108

intraorbital, 109
Lactation, 81

corpora lutea of, 81, 154
mammary glands of, 160
Lactation interval, 81
Langerhan's islands, 126
carcinoma of, 221
Larynx, 127

Lateral mesoderm, 29

Leaden strain (see C57 leaden)

Leg lesions, in infectious ectromclia, 435

in mouse arthritis, 400
Legs, paralysis of, 443
Leiomyosarcoma, of uterus, 227
Leptopsylla musculi, 371
Leptospira icterohemorrhagiae , 433
Leptospirosis, 431
Leukemia, 214

genetics of, 270

immunization to transplanted, 291

in reciprocal crosses, 2 7 1

transplantation of, 290
Leukocytes, 92

in vaginal smears, 68
Leukocytosis in lymphocytic choriomenin-
gitis, 441
Leukopenia, due to Bacterium influenzae
murium, 411

in mouse typhoid, 382
Lice, 370

as transmitting agents, 371

eradication of, 371
Life, length of, 59
Lingual glands, 112
Linkage, 234

data, negative, 244

effect in inbreeding, 329

groups, 243
Lipoid tumors, in yellow mice, 269
Lipoma, 194, 200
Liponyssus bacoti, 374
Liposarcoma, 204
Lips, no

epidermoid carcinoma of, 196

papilloma of, 195
Litter size, 56
Liver, 124

inclusion bodies in, 452

lesions of (see hepatic lesions)

nematodes in, 369

tapeworms in, 360

tumors caused by tapeworms, 360

tumors of, 219
Longistriata musculi, 367
Lung, 128

adenocarcinoma of, 209

adenoma of, 209

carcinoma simplex, 210

490

INDEX

Lung, lesions due to infections (see pneumonic
lesions)
metastasis of tumors to, 212
nematodes in, 367
papillary adenocarcinoma, 210
protozoan infection in, 356
tumors of, 208
Lung tumors, dominant nature, 262
genetics of, 263

hormonal influence absent, 264
incidence in reciprocal cross, 264
metastasis of, 211
Lutein cells, 153
Lymph nodes, 95

enlarged in mouse arthritis, 401
lesions in pseudotuberculosis, 394
protozoan infection in, 356
Lymph nodules, aggregate, 122

solitary, 122
Lymph organs, tumors of, 212
Lymphangio-endothelioma, 208

of liver, 220
Lymphangioma, 194, 202
Lymphatic vessels, 91
Lymphoblastoma, 215
Lymphocytes, 92

tumors of, 215
Lymphocytic choriomeningitis, 438
diagnosis, 442
in laboratory mice, 438
in wild house mice, 438
properties of the virus, 441
the experimental disease, 439
the natural disease, 439
Lymphocytoma, 215
Lymphosarcoma, 216

more frequent in females, 312

MacDowell-Bagg albino strain, blood count

in, 93

ovulation in, 76

vaginal smear in, 69

vertebral variation in, 336
Macrophages, 96

Malignancy of tumors in mice, 168
Malpighian bodies, 97
Mammary glands, 158

changes during estrous cycle, 75

in pseudopregnancy, 79

Mammary glands, development induced by
nursing, 81

extent of, 175

grovk'th following ovariectomy, 311

histology, 158

involution, 162

retarded by irritation, 81

of lactation, 160

of pregnancy, 159

of puberty, 159

regressing, 161

resting, 162

spirochetes in, 431
Mammary region, epithelial tumors of, 176

non-epithelial tumors of, 193

tumors of, 174
Mammary tumors, 174

adeno-acanthoma, 190

adenocarcinoma, 182
variable, 183

adenofibroma, 182

adenoma, 176

carcinoma simplex, 190

carcinosarcoma, 192

dominant inheritance, 251

endocrine influence on, 259, 311

epithelial pearls in, i8g

fibro-adenoma, 181

genetics of, 259

hemorrhagic cyst adenocarcinoma, 189

in feminized males, 312

in male mice, 311

in ovariectomized females, 311

influence of milk on, 257, 321

intracanalicular adenocarcinoma, 187

macroglandular adenocarcinoma, 188

papillary cyst adenocarcinoma, 185
adenoma, 180

polylocular cyst adenoma, 179

simple adenocarcinoma, 182
adenoma, 177

transplantation of, 280
Marrow (see bone marrow)
Marsh strain, arthritis in, 400
Mast cells, 96
Maternal environment, 331
Maturation of egg, 77
Maturity, age at, 58
Meal worms, eradication, 377
Medulloblastoma, of brain, 228
Megakaryocyte, 95

INDEX

491

Meiosis in ova, 62
Meischer's tubes, 357
Melanoma, 1Q4, 204
malignant, 204
metastasis of, 204
Membrana granulosa, 149
Membrane, Reichert's, ^t,
Meningitis, in lymphocytic choriomeningitis,

441
Mesenteries, 124
Mesoderm, distribution of, 28
formation of, 15
lateral, 29

wings of, 15
paraxial, 29

relation to head process, 21
somatic, t,2
splanchnic, 32
Mesometrium, orientation of embryo in rela-
tion to, 15
Metastasis, 170
Metestrus (see estrous cycle)
Mice, care of, 358, 360, 371, 373, 376, 455, 475
Micrococcus tctragenits, 415
Mid-gut, 44

Milk, transmission of Spirillum in, 432
Milk influence, 321

transferred by tissue graft, 323
Mites, 374

control measures, 375
]\litochondria in sperm, 2
Monocyte sarcoma, 219
Monocytes, 92
Monocytoma, 214, 217
Morgan's bacillus, 388
Mortality, in mouse typhoid, 384
Morula, 4

Mouse septicemia, 398
Mouse typhoid, 381

bacteriological diagnosis of, 388
blood picture, 382
etiology, 386

infections with related organisms, 388
occurrence, 381
organisms related to, 388
pathology, 384
prevention of, 390
signs of illness, 381
the experimental disease, ^^^
the natural disease, 381
Mouth, development of, 28

Mits hactriamis, carcinoma of pancreas in, 221

tumors in, 270
Muscle, nematodes in, 369

protozoan infection in, 357

tumors of, 194, 205, 227
Mutations, causing heterozygosis in inbred
strains, 330

chromosome, 242

detection of, 338

gene, 234

in transplanted tumors, 288, 303

rules for assigning symbols to, 242

sex-linked, 244, 286

with irregular inheritance, 240
IMyeloblasts, 95
ISIyelocytes, 95
Myelocytoma, 217
Myeloid cell tumors, 216
Myelosarcoma, 217
Myobia musculi, 375
Myocardium, 45, 91
Myocoptes musculinus, 376
Myometrium, 156
Myosarcoma, 194, 205, 227

Nematodes, 349, 364

in intestine, 365, 369

in liver, 369

in lungs, 367

in muscle, 369

in stomach, 368
Nephritis, due to A", inuris, 356
Neural groove, 23
Neutrophils, 92
Nipples, 158

Nippostrongylus muris, 367
Non-epithelial tumors, 268, 269
Normoblasts, 94
Nosopsyllus fascialiis, 363, 371
Notochord, 24

classed as entoderm, 24
Nourishment of embryo, 39

Ocular lesions, in mouse typhoid, 390
Ollulanus tricuspis, 369
Omphalomesenteric artery, 50

492

INDEX

Oocyte, 149 (see also ova)

growth of, 64
Oral cavity, no

plate, 28
Orientation of embryo, 15
Os penis, 146

Osteogenic sarcoma, 194, 207
Osteoma, 193, 200

Otitis media, due to infectious catarrh, 413
Ova, 150 (see also egg, oocyte)
degeneration of (see atresia)
experimental transfer of, 258
high mortaUty among, 64
length of life of, 62
meiosis in, 62
number of, 61

parthenogenetic development of, 154
polynuclear, 61
proliferation of, 59
Ovarian follicles, atresia of (see atresia)
growth of in adult, 62, 64
in young mice, 60
prior to ovulation, 73
length of life of, 63
mature, 149, 150
polyovular, 61
primary, 149
Ovarian tumors, 222
cystic, 22^
embryonal, 225
granulosa-cell, 224
solid, 224
Ovary, 148

changes during estrous cycle, 73
in vitro growth of, 63
ligation of, 63
mitotic activity in, 74
regeneration of, 64
transplantation of, 65
x-rayed, 63
Oviduct, 155

changes during estrous cycle, 72
distension following ovulation, 77
Ovogenesis, 59

cyclic in young mice, 61
Ovulation, 76, 149

corpora lutea of, 80, 153
follicle growth prior to, 73
Oxyuris obvelata, 365
tetraptera, 365

Palate, hard, no

soft, no
Palatine glands, no
Pancreas, 126

inclusion bodies in, 436

tumors of, 221
Paneth cells, 121
Papilla, renal, 130
Papillae, of corium, 106

of the tongue, 1 1 1
Papillary adenocarcinoma, of lung, 210
Papillary adenoma, of ependymal cells, 229
Papillary cyst adenocarcinoma, of kidney,

221
Papilloma, 195

of renal pelvis, 221

of urinary bladder, 222
Paralysis, due to coliform bacillus, 388

to Coryncbactcrium murisepticum, 397
to encephalomyelitis of mice, 443

in mouse arthritis, 400
Parasites, 349 (see also helminths, nematodes,
protozoa, tapeworms)

as vectors, 350

Aspicularis tetraptera, 365

Capillaria bacillata, 369

Cimex ledularius, 372

Cysticercus fasciolaris, 360

Echinolaelaps echidnhiiis, 375

eradication of, 371, 373

Gongyloncma miisculi, 369
neoplasticum, 368

Hcterakis spumosa, 369

Hoplo pleura acanthopus, 370
hesperomydis, 370

Hymenolepis dim inula, 363
fraterna, 360
microstoma, 364

importance of, 349

Lcptopsylla musculi, 371

Liponyssus bacoti, 374

Longistriata musculi, 367

Myobia musculi, 375

Myocoptes musculinus, 376

Nippostrongylus muris, 367

Nosopsyllus fasciata ,371

Ollulanus tricuspis, 369

Polyplax serrata, 370
spinulosa, 370

INDEX

493

l'ar;isilcs, Protuspinira iniiris, jOtS

Schislosomulium doHlhitti, j5(;

Syphacia obvclata, 365, 366

Taenia pisiformis, 360
tacniaeformis, 360

transferred to man, 362

Trichindla spiralis, 36g

Xcnopsylla cheopis, 371
Parathyroid glands, 100
Paraxial mesoderm, 29
Parent-offspring mating^ 328
Parietal cells, of stomach, 119
Parotid glands, 115
Pars intermedia of hypophysis, 100

nervosa of hypophysis, 100
Parturition, time of, 56
Paste iirella muricida, 392, 455

pestis, 393, 394

pseudotuberculosis , 393

tularemia, 429
Pasteurellosis, 391, 404

control, 393

diagnosis, 392

similar to Bacterium influenzae infection,
412
Path coefHcients, 328
Penis, 145

Peri-anal papilloma, 195
Pericardial coelom, 33, 45
Pericardial-peritoneal canal, 2>i
Pericardium, 91
Perivitelline space, 2
Peyer's patches, 122
Pharynx, 112

Photophobia, in lymphocytic choriomening-
itis, 439
Pigment of skin, 106
Pineal body, 104
Pituitary gland, 98 (see also hypophysis)

adenomas, induced by estrogens, 314
protozoan infection in, 356
Placenta, 40
Plague (in mice), 394

transmission of, 371
Plasma cells, 96
Platelets, blood, 92
Pleura, 128

Pleuropneumonia-like organisms, 404
carrier incidence, 408
etiology, 408
experimental disease due to, 405

Pleuropneumonia like organisms, toxin pro

duction, 408
Pneumonia in mice, 409

Pneumonic lesions, due to Bacterium influ-
enzae murium, 411
to Friedl :nder-hke bacillus, 411
to infectious catarrh, 413
to virus pneumonia, 448
in Brucella infection, 409
in mouse septicemia, 398
in pseudotuberculosis, 394
Polar bodies, 2, 78, 150
Poliomyelitis, relation to encephalomyelitis,

446
Polydactyly in hybrids, 340

variation increased in inbred strains,
336
Polyplax serrata, 370

spinulosa, 370
Postnatal development, 58
Postpartum estrus, 75

Pregnancy, blood in vagina as sign of, 40
corpora lutea of, 80, 153
mammary glands of, 159
mucus in vagina during, 68
Prepuce, 146
Preputial glands, 146

carcinoma of, 198
Primitive streak, 15

as growth center, 31
head process derived from, 20
homology of, 32
somite formation and, 31
Primordial germ cells, 59
Proamnion, 29
Proamniotic cavity, 10
Proestrus (see estrous cycle)
Pronuclei, male and female, 3
Prostates, dorsal, 142

ventral, 143
Proteus morgani, 381, 387, 389, 429, 455
Protospirura muris, 368
Protozoa, 350

Amoeba enter ica, 351
fecalis, 351
musculi, 351
Cryptosporidium muris, 354

parvum, 354
Eimeria falciformis, 354

miyairii, 354
Endamoeba muris, 350

494

INDEX

Protozoa, Giardia muris, 352
Hexamila muris, 351
in blood, 353

infecting gastric glands, 355
kidney, 355
muscle, 357
intestinal, 350, 351, :3,S2, 354, 358
Klossiella muris, 355
Sarcocystis muris, 357
Trichomonas muris, 351
Trypanosoma didloni, 353
lewisi, 353
Protozoan infections, prevention and control,

358
Proximal entoderm, 8
Pseudo-leukemia, 215
Pseudopregnancy, 78

corpora lutea of, 80, 153
Pseudotuberculosis of mice, 394, 404

differentiation from pseudotubercu-
losis of rodents, 394
etiology, 396
infections due to related organisms,

397

the experimental disease, 395

the natural disease, 394

toxin in, 396
Pseudotuberculosis of rodents, 393
Puberty, mammary glands of, 159
Pyloric glands, 1 20
Pylorus, carcinoma of, 219
Pyogenic infections, 397, 415 (see also ulcers)

Quarantine, in control of disease, 456

Rainey's corpuscles, 357
Random mating, 327
Reciprocal hybrids (see hybrids)
Recording, method of, 475
Rectum, 122
Red pulp, 97
Reichert's membrane, t,t,
Renal papilla, 130

pelvis, 131
Respiratory system, 127
Rete testis, 135
Reticulum cell tumors, 218

Rhabdomyosarcoma, 194, 205

Rhinitis, due to infectious catarrh, 413

Rib, chondrosarcoma of, 194

Ringworm, 429

Rockefeller strain, encephalomyelitis in, 443

Rolling disease, 405

Round cell sarcomas, 212

Salivary glands, inclusion bodies in, 451
Salmonella enkritidis, 381, 386, 455

typhimuriiim, 381, 386, 455

unknown species, 381, 389
Sarcocystis muris, 357
Sarcoma, 171

induced by estrogens, 314

of liver, caused by tapeworm, 360
Sarcosporidia, 357

control measures, 359

toxic substance produced by, 357
Schistosomatium doulhitii, 359
Segmentation cavity, 5
Selection, in inbred strains, 330, 343
Seminal vesicle (see vesicular gland)
Seminiferous tubules, 133
Septal cells, of pulmonary alveoli, 130
Septicemia, due to Coryncbacterium muriscp-
ticiim', 397

mouse, 398
Septicemic diseases, 391
Sertoli cells, 133
Sex, determination at birth, 58

ratio, 57

role in tumor transplantation, 299
Sex-linked genes, 244, 286

effect of inbreeding on, 329
Silverfish, 358, 377
Simple cysts, of ovary, 223
Sinusoids, 90
Skin, 105

carcinoma of, 194, 197

epidermoid carcinoma of, 196

lesions of in ectromelia, 436

papilloma of, 195

tumors of, 195
Snuffling, due to infectious catarrh, 413
Somatic mesoderm, 32
Somatopleure, 32
Somites, 28
Spermatogenesis, 133

INDEX

495

Spermatogenic cells, 133
Spermatozoa, Golgi material in, 3

mitochondria in, 2

size of, 135

survival in oviduct, 78

transport of, 78
Spirillinm minus, 431
Spirochetosis, 431
Spiroptcra neoplastica, 368
Splanchnic mesoderm, 32
Splanchnopleure, 32

relation to yolk-sac, 36
Spleen, 96

enlarged due to streptococcus, 416
in bartonellosis, 421
in mouse septicemia, 398

protozoan infection in, 356

transplantation of, 286
Splenectomy, efifect on latent infections, 419,

425
Splenic cords, 98
Splenic lesions, due to Trypanosoma lewisi,

353
in ectromelia, 435
in mouse arthritis, 401

typhoid, 385
in pseudotuberculosis, 394, 395
Squamous entoderm, 21
Squirrel, thirteen-striped ground, 13
Staphylococci, 415
Stocks (see strains)
Stomach, 117

carcinoma of, 219
glands of, 118
nematodes in, 368
Stomodaeum, 28

Strains (see under the following: A, C3H,
C57 black, C57 brown, C57 leaden,
C58, CBA, ce, dba, Japanese
waltzing, MacDowell-Bagg albino,
Marsh, Mits bactrianus, Rockefeller,
Swiss, W, X, Y)
Strcplobacillus monilijormis, 400, 403, 407,

418
Streptococci, 416
Streptothrix, 396
Striated muscle, tumor of, 205
Subcutaneous tissues, tumors of, 195, 199
Sublingual glands, 115
Submaxillary glands, 112
female, 114

Submaxillary glands, male, 1 14

tumors of, 219
Sulfonamide drugs, 412
Susceptibility, number of genes involved, 283

to tumor transplants, 281
Swiss mice, diarrhea in, 389

encephalomyelitis in, 443

infectious catarrh in, 413

virus pneumonia in, 449
Symplasia, 41
Syphacia obvclata, 365, 366

Tarnia crassicollis, 360

pisiformis, 360

taeniaeformis, 360
Tail, lesions in mouse arthritis, 402

melanoma of, 204
Tail fold, 42
Tapeworms, 359

control measures, 360, 362, 364

dwarf, 360
Taste buds, 112
Technique, embryological, i

histological, 89

of tumor transplantation, 292
Teeth, no

Temperature, in mouse typhoid, 382
Tenebrio, as intermediate host, 362, 363, 364
Teratoma, of ovary, 225
Testis, 133

excretory ducts of, 135

tumors of, 227
Testosterone, tumor rate affected by, 313
Theca externa, 149

interna, 149

lutein cells, 154
Thirteen-striped gound squirrel, 13
Thymus, 103
Thyroid gland, 100

protozoan infection in, 356
Tissues, transplantation of, 304
Tongue, 1 10

Torsion (see turning of embryo)
Toxin, produced by C. kutschcri, 396
Trachea, 128
Transition line, 21
Transplantation of tissues, 304

of tumors, 279

age, role in, 299

496

INDEX

Transplantation of tumors, arising in hybrids,
286
biological factors, role in, 299
chemical basis of, 288
diet, role in, 297
endocrine influence on, 314
factors influencing, 297
genetics of, 297
irritating agents, role in, 298
leukemia, 290
methods, 292

number of genes involved, 283, 287
relation to individuality, 301
sex, role in, 299
useful sites, 293
value in growth studies, 297
Transplanted tumors, immunization against,
291
list of available, 294
mutations in, 288
physiological individuality of, 302
relation of to spontaneous, 301
storage in dry ice, 297
Trematoda, 359
Trichina spiralis, 369
Tricliinella spiralis, 369
Trichinosis, 369
Trichomonas miiris, 351
Trichophyton ectothrix »ic gal os pari u>n, 430
Trichophyton gypseum, 430
Trichophytosis, 430
Trophectoderm, 7

as origin of giant cells, 40
relation to decidua, ^^
Trypanosoma duttoni, 353

control measures, 358
Icu'isi, 353
Tumor incidence, affected by .1" gene, 260
by ova transfer, 258
difference in reciprocal hybrids, 251
endocrine influence on, 259, 310
estrous cycle and, 312
extra-chromosomal influence in, 251,

262, 266, 273
frequency of breeding and, 312
in backcross generations, 256
in castrate mice, 311
influence of milk on, 257, 321
lower in virgin females, 259, 311
nematode infection and, 369
tapeworm infection and, 360

Tumor incidence, tissue transplants increase,

Tumors, benign, 171

characteristics of, 169

classification of, 171, 175, 214, 223

definition of, 169

etiology, 171

formation (see tumor incidence)

hormones produced by, 315

immunization to transplanted, 291

in A strain (see A strain)

in C57 black (see C57 black)

in dba strain (see dba strain)

in Y strain, 269

inheritance of mammary, 259

linkage not found, 264

lymphatics in, 170

malignancy in mice, 168

malignant, 171

mammary (see mammary tumors)

metastasis of, 170

of primary lung, 211
morphological symptoms, 170
multiple, 168
nerves in, 170
non-epithelial, 268, 269
of blood forming organs, 212
of body wall, 199
of central nervous system, 228
of dermis, 199
of digestive system, 219
of epidermis, 195
of gall bladder, 219
of histiocytes, 217
of kidney, 221
of liver, 219

of lung, 208, 212 (see also lung)
of lymphocytes, 2 1 5
of mammary region, 174
of monocytes, 217
of myeloid cells, 216
of ovary (see ovarian tumors)
of pancreas, 221
of skin, 195

of subcutaneous tissues, 195, 199
of submaxillary gland, 219
of testis, 227
of urinary bladder, 221
of uro-genital system, 221
of uterus, 225
physiological individuality of, 302

INDEX

497

Tumors, rare sites of, 229

recurrence of, 170

relation transplantable to spontaneous,
301

somatic mutation as cause of, 303

spirochetes in, 431

stroma of, 1 70

transplantation (see transplantation of
tumors)
Turning of embryo, 44
Typhoid (see mouse typhoid)

Ulcers, 397, (see also pyogenic infections)
due to Corynchacteriiim miiris, 397
in infectious ectromelia, 436

Ureter, 131

Urethra, female, 132
male, 143

Urinary bladder, 132
tumors of, 221

Urinary system, 130

Uro-genital system, tumors of, 221

Uterine epithelium, implantation changes in,

7
Uterus, 155

changes during estrous cycle, 72
pregnancy, 37
in pseudopregnancy, 79

tumors of, 225

Vagina, 157

age at opening, 58

changes during estrous cycle, 69

epithelium of, 71

mucification of in pregnancy, 68
Vaginal plug, 55

smears, 67
mucus in, 68
Valves, of the heart, 91

of veins, 90
\'ariation, between strains, misinterpretation
of, 346

causes of, 331

effect of inbreeding on, 334

in embryos, 2

in hybrids, 341

measurement of, ^^t,

under random mating, 327

within strains, misinterpretation of, 345
Vas deferens, 137

Vasa efferentia, 136
vasorum, 90

\'eins, 90

\'entricles, of the heart, 90

\'esicular glands, 55, 140

Vibrissae, 107

Vigor (see also hybrid vigor)
in inbred strains, 334, 343

Mlli, intestinal, 120

X'irus diseases, 408, 434 (see also lilterable
organisms)
encephalomyelitis of mice, 443
inclusion bodies of liver, 452
infectious ectromelia, 434
lymphocytic choriomeningitis, 438
salivary gland inclusion bodies, 451
virus pneumonia, 448

Virus pneumonia in mice, 448

Vitellus, 2

W strain, skin tumors in, 195
White pulp, 97

X strain, osteogenic sarcoma in, 207
skin tumors in, 195

X zone, of adrenal, 103

Xcnopsylla cheopis, 371

X-rays, effect on latent infections, 425
irradiation of ovaries, 63
mice sterilized with, 65
translocations induced by, 242

Y (yellow) strain, lipoid tumors in, 269
lipoma in, 200
liposarcoma in, 204
liver tumors in, 220
osteogenic sarcoma in, 207
Yolk cavity, 7

relation to yolk-sac, 36
Yolk-sac, 36

as organ of exchange, 40
change in shape of, 51
circulation in, 51

Zcnopsyllci cheopis, 363
Zona pellucida, 2, 149

disappearance of, 7

'HMm

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