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The origin of the definitive ova in the white rat (Mus norvegicus albinus).

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Laboratory of Histology and Embryology, G o r n d l University, Itltlnca, New Pork
Numerous contributions have appeared within recent years
dealing with the problems of the origin and history of the
definitive germ cells in the mammalian ovary. As a result of
these investigations, many authors maintain that those germ
cells which are found previous to the time of birth are the
progenitors of the definitive ova in the adult. Others believe
that most, if not all, of those sex cells which are present at
the time of parturition degenerate, and the definitive ova are
derived from cells of the germinal epithelium. Conflicting
views are, likewise, held by the different workers concerning
the time of the formation of the definitive ova during prepubertal and postpubertal life.
In the literature much information may be found regarding these problems. I shall, however, mention only a few
opinions concerning the formation of the definitive ova after
birth and during sexual maturity.
In a statistical study of the number of ova at various ages
in the albino rat, Arai (’20) states that the process of proliferation of new ova occurs most rapidly from fifteen to sixty
days and may continue f o r a year after birth although it
proceeds at a slower rate as puberty is attained.
Allen (’23), in his study of the white mouse, concludes
that “ a cyclical proliferation of the germinal epithelium gives
rise to a new addition of ova to the cortex of the adult ovary
at each normal oestrous period." The ova arise from dividing cells in the epithelium through mitosis. When the long
axis of the dividing cell is perpendicular to the surface, or
at an angle of more than 20" t o 30°, one of the daughter cells
is cut off under the epithelium, and then is soon surrounded
by cells completing a small follicle.
Consid6ring the occurrence of synapsis as a necessary prerequisite for the determination of a definitive ovum, Cowpertliwaite ( '25) concludes that oogenesis is not continued
during prepubertal or postpubertal life in the rat. Synapsis
has been completed at the end of the fourth day, and no transitional stages are to be found between the undifferentiated
germinal epithelial cells and the definitive germ cells.
I n the mouse, Kingery ('17) reaches the conclusion that
germ cells derived from an embryonic proliferation of the
germinal epithelium before birth and a few days afterward
degenerate, and the definitive ova arise from the germinal
epithelium during a proliferation extending from three days
after birth t o puberty. Concerning postpubertal oogenesis,
he states that the potentiality of the germinal epithelium is
lost as the ovary becomes mature and no more egg cells o r
follicle cells are produced.
Papanicolaou ( '24), studying a large number of ovaries
from guinea-pigs, including embryos and adults, states that
there is a continuous process of oogenesis from the time of
the gonadal differentiation in the embryos up to the time
of cessation of sexual activity in the older females, the process decreasing as the individual becomes older.
The source of the definitive ova still remains, then, an
unsettled question in many mammals. In the white rat, Miis
norvegicus albinus, I have made this study1 with. the aim of
determining whether ova are formed after birth and during
sexual maturity from the epithelial covering of the ovary.
I desire to express my appreciation f o r the helpful suggestions and eneouragement of Doctor Kingsbury, whose interest h a s been of great help in this work.
I wish also t o acknowledge my indebtedness to the Department of Histology and
Embryology for materials and facilities for this study.
The material for this study consisted of a series of ovaries
taken from rats ranging from newborn to old adult females.
From birth to twenty days afterward, the ovaries were
removed and fixed at intervals of one day, and less in some
instances. These were selected in order t o observe the occurrence of chromatin changes and any degeneration that might
appear in the germ cells. I f a transition of peritoneal cells
into sex cells occurred at this period, it could, likewise, be
observed. From twenty to ninety days after birth, the ovaries
were fixed at intervals of five days. Thus, this second series
included ovaries from individuals at the time of puberty and
from young adults.
A third series of ovaries was collected from adult females
(the litters having been removed in all cases) at varying
intervals after parturition, in order to detect any periodic
proliferation which might occur during the oestrous cycle.
A few ovaries were also taken from pregnant females as well
as some from very old rats.
For fixation, Bouin’s picro-aceto-f ormol and Flemming ’s
fluid were used. The ovaries were sectioned at a thickness
of 6, 8, and l o p . Serial sections were mounted in all cases.
Heidenhain’s iron hematoxylin was employed for staining
the greater part of the sections. No counterstain was used.
The ovary of a rat at the time of birth is somewhat hemispherical in shape and is separated from the peritoneal cavity
by a connective-tissue capsule. Structurally, it consists, for
the most part, of irregular cell masses and strands of connective tissue which radiate outward from the hilus. I n these
cell masses which are often located immediately under the
cuboidal epithelial covering of the ovary two kinds of cells
may be distinguished. Those which are smaller and have an
oval nucleus are the indifferent or future follicle cells, while
the large cells with round nuclei are the sex cells. On further
T H E A N A T O M I C A L RECORD, VOL. 37, NO. 1
examination of the germ cells, at this time, the chromatin
in the nucleus is seen to be in well-defined, distinctly moniliform threads, characteristic of the pachytene stage in maturation. Evidently, early stages have occurred before this, and
from the literature (Pratt and Long, 9 7 ) , we learn that
synapsis in the white rat occurs from three and a half t o
two days before birth, although some conjugation stages may
be found at a much later period. An occasional synaptene
stage, which may be recognized by the loop arrangement of
the chromatin threads, is observed by the writer at the time
of parturition.
I n the ovaries of rats sixteen to twenty-four hours old an
increase in the amount of stroma is noticeable. I n its growth
it has divided many of the cell masses and has reached,
peripherally, a position immediately under the covering epithelium. The follicle cells are, likewise, more numerous than
in the previous stage and are seen t o have an epithelial
origin. This is indicated by similarity in structure of their
nuclei and by the fact that in various places peritoneal cells
are seen to migrate inward from the epithelial covering of
the ovary and separate the individual germ cells. The sex
cells remain as distinct elements with more or less clear cytoplasm and with large spherical nuclei. I n the latter the
chromatin threads are still in the pachytene stage and are
evenly distributed throughout. One has no trouble, at this
time, in distinguishing between the germ cells and follicle
cells, since the former have a different nuclear structure and
the much greater size of cell body and nucleus.
Most of the sex cells attain the diplotene stage during the
third day after birth, although a few may be found earlier.
The threads of the diplotene are more numerous than in the
preceding stage, somewhat contorted, about half as wide, and
stain less darkly. Pachytene stages may also be found occasionally at this time. One o r two nucleoli having the appearance of darkly staining masses of chromatin are, likewise,
observed. The follicle cells which originally had an oval
shape are now changing, in most instances, into flat crescent-
shaped structures around the germ cells. This is typical of
the first stage in follicle formation.
This stage of maturation, previously described, is relatively
short, and the sex cells soon merge into the resting stage.
The chromatin now loses its character of threads and becomes
arranged in irregular masses. The nucleoli, often two in
number, are peripherally situated in the nucleus. Each germ
cell, by this time, is surrounded by a definite layer of follicle
At the same time that such chromatin changes, as indicated above, are occurring in the nucleus, many of the germ
cells are overtaken by degeneration. This process is very
evident in ovaries of rats sixteen hours old and apparently
reaches its height during the second and third day postpartum. This degenerative condition first affects the cytoplasm
chiefly in which large vacuoles appear. The chromatin of
the nucleus is then seen to contract to some extent and gradually to take a lighter stain. The crowded conditions in the
ovary at this early stage and inadequate vascular supply may
be the causes of this regressive process.
Those which do not meet this fate continue to grow and
become surrounded with several layers of follicle cells.
Their number is gradually lessened, however, as various ones
degenerate and disappear. Particularly, this retrogressive
process is noticed about the seventeenth day, when large
cavities are evident in the follicles. From a careful study
of abundant material, the writer believes that those germ
cells which are present at birth disappear long before the
advent of sexual maturity, and none ever exist and become
the definitive ova. Arai, in his study on the ovary of the rat,
mentions a similar condition of degeneration about the twentieth day.
Meanwhile, on the sixth and seventh days after birth, certain cells of the epithelial covering of the ovary, which vary
in shape from a flat elongated to a larger and more cuhoidal
type, begin to enlarge. The nucleus of the ordinary epithelial
cell, at this time, is quite large, almost filling the entire cell
in some instances, and contains frequently two nucleoli.
From this stage the cells begin t o grow and to show marked
changes in their structure. The cytoplasm is seen to increase
rapidly in amount and stains less intensely than previously,
making the nucleus mare outstanding (fig. 2).
I n the course of further growth of the cell, the nucleus
which had formerly been somewhat oval in shape begins to
enlarge and become spherical in form. The appearance of
the chromatin is, likewise, seen to change. It clumps, so t o
speak, and stains more intensely. The cytoplasm, somewhat
granular in nature, stains much more lightly than the nucleus
(fig. 1). Quite commonly, two cells are seen t o enlarge side
by side (fig. a), indicating that they might have come from
one cell through mitosis. Underlying this epithelium, at this
stage, is a very thin tunica albuginea which has resulted
from the peripheral growth of the connective tissue. As
the cell has grown and taken on characteristics of a germ
cell, it, likewise, has caused a flattening in the cells immediately adjacent (fig. 1). Often the pressure of the latter gives
the young oocyte an oval shape.
Following this stage, the adjacent epithelial cells begin to
grow up and over the germ cell. This process is quite similar
to that in the mouse, described by Kingery. This overgrowth
results in the beginning stage of proliferation and follicle
formation, the two occurring simultaneously (figs. 4 and 5 ) .
Various sizes are obtained by the germ cell before this process of proliferation occurs. I n some instances such a large
growth is attained that large bulgings are produced in the
surface epithelium (fig. 5).
As the germ cell is pushed down through the tunica, it
grows very little (fig. 3). The chromatin of the nucleus, in
most instances, loses some of its staining power, having taken
more the character of faintly staining granules.
I n the ovary of an eight-day rat, cells in various stages of
this process of proliferation may be found, those covered
over by the growth of adjacent cells, those just under the
epithelium, and those that are being pushed through the tunica
(fig. 3 ) . Centrally, in this same figure, some of the germ cells
which were found at birth may now be seen with several
layers of follicle cells. Arai states that the formation of
newly formed follicles appears about ten days after birth.
My material, however, shows that peritoneal cells begin to
enlarge on the sixth and seventh days and many are under
the tunica surrounded with a layer of follicle cells by the
eighth day postpartum.
From thirteen to twenty-four days, the rate of this process
of germ-cell formation decreases to some extent, although
the enlarging of epithelial cells and overgrowth of adjacent
cells may still be found in the ovary (fig. 6). The cause of
this retardation may be due to the great degenerative conditions found in the central part of the gonad. As previously
mentioned, many of the germ cells present a t birth degenerate during this period after having obtained a large size and
many layers of follicle cells.
Most of the young oocytes that are found originating from
the epithelium remain, each surrounded by a few layers of
follicle cells, until after twenty days, o r at the approximate
end of the regressive conditions mentioned in the preceding
paragraph. They are then seen to grow and acquire several
layers of follicle cells. At this time an increase in the number
of enlarging epithelial cells is also apparent. One difference,
however, is the fact that such a large size is not attained while
yet in the epithelium as in early stages (figs. 7 and 8). Bulgings are not common on the surface of the ovary, yet oocytes
are seen to have an epithelial origin.
I n the ovary of a twenty-nine-day-old rat (fig. 8) two small
germ cells may be seen that are being pushed down by the
overgrowth of adjacent cells. Other oocytes are noticed at
various depths in the process of being proliferated. I n all
cases the growth of the surface epithelium seems to be the
greatest factor in causing this proliferation.
A continuous proliferation and formation of germ cells
continues until about sixty-five days after birth, when the
process becomes slightly retarded. Ovulation evidently
occurs before seventy days, since large corpora lutea may be
found at this time.
Not all of the oocytes formed from this continuous proliferation, which begins about six days after birth, ever
become functional. Many of them are seen to degenerate in
various stages of development. The first indication is in the
granulosa which shows a degenerative appearance and is not
intact. Further evidence that these follicles are in a regressive state is provided by the vacuolization of the oocyte, producing a crenated cell wall. The chromatin of the nucleus
arranges itself in small knots and balls (chromatolysis), and
the oocyte continues to degenerate by unequal fragmentation,
the fragments carrying with them pieces of the nucleus.
I n these definitive oocytes no stages of synapsis and synizesis are observed. I am quite certain, however, that the
germ cells in the epithelium are not oocytes which have
already passed through the earlier stages of development,
because intermediate forms are seen between ordinary cells
of the epithelium, the germ cells, and the oocytes in mature
graafian follicles. No better evidence is required that these
are true oocytes than the fact that one is able to trace them
through the first maturation division. Maturation spindles
may be found occasionally in normal oocytes with the granulosa intact.
Furthermore, it is apparent that the oogenetic potentiality
of the epithelium is not lost at puberty, as Kingery has
described it in the mouse. I n fact, at some stages during
postpubertal life the process of the formation of germ cells
is very marked. It would seem that such a condition must
exist, since evidence, as interpreted by the writer, is steadily
increasing that all follicles are relatively short-lived, and it
is now considered by some untenable to conceive of eggs lasting the lifetime of the individual. Allen points out that large
numbers of degenerating follicles in immature ovaries and
the presence of maturation spindles in ova before puberty
probably give the best evidence that such a concept is true.
There seems to be no reason why the formation of germ cells
should stop at puberty. Neither does it seem logical that
some eggs should develop, while others tend to remain in
a retarded state throughout life.
I n the rat, formation of germ cells from the epithelial covering of the ovary seems t o continue t o take place until the
fecundity is lost in old age. The process is not markedly
different from that occurring before puberty, however, and
it is quite evident that the cells do not attain such a large
size while still in the epithelium as they do'before sexual
The presence of mitotic figures in the epithelium is
increased during oestrus. Their occurrence is nearly as common in the cells with the long axis perpendicular to the
surface as those with their long axis parallel to the surface
of the ovary. It does not seem, however, that the cells cut
off by a mitotic division occurring in those cells with their
long axis perpendicular to the surface necessarily become
egg cells, as Allen describes in the mouse. Some of those
resulting from mitosis, in this case, may become the oocytes,
but the greater portion, if not all, begin to enlarge slightly
in the epithelium before being pushed down by the overgrowth of adjacent epithelial cells.
Clearly staining cytoplasm with a large, dark-staining
nucleus is the first indication that a cell possesses germinal
potentiality during sexual maturity (fig. 10). The egg cell
is then crowded down by the overgrowth of adjacent epithelial
cells as it continues to enlarge. I n most instances, as was
previously mentioned, the germ cell does not attain a large
size while still in the epithelium. Occasionally, however, one
enlarges considerably before being proliferated. As the cell
grows, the chromatin becomes less chromophilic, and is attenuated at the intersections of the reticulum. A layer of follicle
cells is soon acquired, and more layers are added as the
oocyte is pushed down through and below the tunica albuginea.
A notable increase in the formation of germ cells is evident
during oestrus. Cells enlarging in the epithelium are usually
more common as the oestrous period is approached than
immediately following. The cells, likewise, come to lie deeper
in the cortex as time progresses after an oestrous period.
The oocytes migrate or are pushed down into the interior
and degenerate if they do not have the chance to develop.
Others which do not meet this fate continue to grow and, in
many cases, are overtaken by degeneration. Degenerative
stages are often as common as in immature ovaries.
Many problems arise concerning the formation of germ
cells after birth. The greatest one, perhaps, is the question
of what should decide the status of a germ cell. Several
workers use the ultimate fate as this criterion, while others
use maturation phenomena. Whether a stage of synapsis
represents a real condition in the development of germ cells
is considered quite fully by previous investigators and will
be discussed only briefly here. The occurrence of synapsis
in the germ cells is both affirmed and denied, yet most investigators think that a synaptic stage is present and a pseudoreduction is accomplished during the conjugation. Evidence
points to the fact that, where it has been observed and
described, parasynapsis has been adopted in most cases
rather than telosynapsis.
According to Cowperthwaite, in the rat, no synapsis occurs
after the fourth day postpartum. She concludes that the
small peripheral follicles which are found in young ovaries
after four days represent a retarded growth, since meiotic
phenomena are not found in the germinal epithelium at any
stage. The cells found enlarging in germinal valleys during
sexual maturity are, likewise, not considered to be true
oocytes, since they present no evidence of synapsis. Papanicolaou, in the guinea-pig, states that epithelial cells, which are
to be considered as resting oogonia, begin to grow and display the morphological changes that characterize the growth
of spermatogonia into primary spermatocytes. The exact
stages cannot be determined, however, since the process is
not described in detail. No indication of synapsis is found
by Duesberg ('08) in his investigation of the spermatogenesis
in the rat. Kingery, in his study of the mouse, likewise
observed no conjugation of chromatin threads occurring in
either the primitive or definitive germ cells. This last author
regards the ultimate fate as the determining factor for the
definitive ova.
In the rat I have observed a conjugation stage in some of
the germ cells at the time of birth. The threads were seen
to be composed of distinctly moniliform halves, giving evidence of a double origin. I n the formation and development
of other germ cells, from the epithelium which begins six days
after parturition and continues throughout life, no synaptene
stage was observed. Neither have I observed a stage with
characteristics suggestive of synizesis.
If a transition of peritoneal cells into germ cells can be
demonstrated during prepubertal life, and then one is able
to trace them through the first maturation division, there
should be no doubt about their true oogenetic properties.
By no means should the absence of a demonstrable synaptic
stage be correlated with a loss of germinal potentiality. Of
course, there is the possibility that chromosomes representing
both parent contributions might be sorted out at the time of
maturation, producing the same result as that attained
through conjugation. If this should occur, then it is not necessary to find a former synaptic stage. Evidence that this
possibility might exist has never been demonstrated, however.
During sexual maturity, the fact that successive stages can
be found from the ordinary germinal epithelial cell t o
graafian follicle stage provides sufficient evidence that germ
cells arise by differentiation of epithelial cells. Furthermore,
if germ cells do not arise from the epithelium at this time,
how would one account for the occurrence of young follicles
at various depths in the tunica? All conditions point to the
fact that the young follicle passes through the albuginea
rather rapidly and reaches a position under the latter as
time progresses. The only exception to this is in the case
where a small oocyte is crowded into the tunica by the large
growth of a graafian follicle. I n most cases, then, all the
germ cells of prepubertal formation would be under the albuginea only in the exceptional case previously mentioned.
The observations in the rat show, however, that young
follicles may be frequently found in various depths of the
tunica for no apparent reason other than that they have
originated from the epithelium and are gradually being proliferated inward. Furthermore, it seems to be more logical
to use the ultimate fate of a cell, which can be demonstrated,
as its criterion rather than a synaptic stage, of which the
actual existence is uncertain in some cases.
The process of germ-cell formation in the rat from the
germinal epithelium, therefore, seems t o be a continuous
process throughout life, although there are certain periods,
especially during sexual maturity, when there appears to be
more marked periodicity of proliferation than at others. This
periodicity is undoubtedly due to various nutritive and
hyperemic conditions, as Papanicolaou has suggested. The
writer, however, is willing to admit that there is a gradual
decline jn the activation of the epithelium with an increase
in age.
Davenport ('25) removed the ovaries of white mice, and
then, after a lapse of a few weeks, found a regeneration
occurring in nearly two-thirds of the cases. This demonstrates. if correct, that some of the cells of the stalk of the
ovary and peritoneum may differentiate into germ cells. This
rather startling result needs, however, confirmation. From
all observations, it is not evident just what the factors are
which determine the eventual fate of cells, that is, whether
they develop into oocytes or follicle cells. This latter point
will continue to be a question until greater insight is gained
into the problem of differentiation.
1. The germ cells which are found in the ovary of a rat at
birth are thought to degenerate before sexual maturity and
not be the progenitors of the definitive ova.
2. The definitive ova are formed from cells of the germinal
epithelium covering the ovary, extending from six o r seven
days after birth until fecundity is lost in old age.
3. Observations indicate that the process is retarded after
4. During sexual maturity, an increase in the activation of
the epithelium is found during the oestrous period.
5. No synapsis was observed during the process of transition of germinal epithelial cells into germ cells. The writer
considers that the presence of successive stages from the
ordinary epithelial cell to the graafian follicle provides sufficient evidence that they are true oocytes.
ALLEN, EDGAR1923 Ovogenesis during sexual maturity. Am. Jour. Anat.,
vol. 31, no. 5.
ARAI, HAYAW 1920 Postnatal development of the ovary, with especial reference t o number of ova (albino r a t ) . Am. Jonr. Anat., vol. 27.
MARION H. 1925 Observations on pre- and postpubertal
oogenesis in the white rat, Mus norvegicus albinus. Am. Jour. Anat.,
vol. 36.
DAVENPORT,C. B. 1925 Regeneration of ovaries in mice. Jonr. Exp. Zo61.,
vol. 42.
DUESBERG,J. 1908 Les divisions des spermatoeytes chez l e rat. Arch. f .
Zellforsch., Bd. I, S. 399-449.
KINGERY,H. M. 1917 Oogenasis in the white mouse. Jour. Morph., vol. 30.
GEORGEN. 1925 Oogenesis during sexual maturity as elucidated
by experimental methods. Soc. Exp. Biol. and Med., vol. 21, p. 393.
J. A. 1917 The period of synapsis in the egg of the
white rat, Mus norvegicus albinus. Jonr. Morph., vol. 29, no. 21.
1 Ovary from rat, seven days after birth, to show the flattening of adjacent
epithelial cells by the growth of an oocyte. Picro-aceto-formol fixation ; stained
with iron hematoxylin. X 192.
2 Ovary from a rat, eight days after birth, to show the enlarging adjacent
cells in the epithelium. Picro-aceto-formol fixation; stained with iron hematoxylin. X 380.
3 Ovary from a rat, eight days after birth, to show the common occurrence
of two young adjacent oocytes, indicating that they might have originated from
the same mitosis in the epithelium. Picro-aceto-formol fixation ; stained with
iron hematoxylin. X 192.
4 Formation o f ' a small follicle while the definitive germ cell is still in the
epithelium. Ovary from a rat, seven days a f t e r birth. Picro-aceto-formol fixation; stained with iron hematoxylin. X 380.
5 Ovary from a rat, eight days after birth, to show the large bulging from
the surface which is caused by the growth of the definitive oocyte. Picro-acetoformol fixation; stained with iron hematoxylin. x 380.
G, germ cells enlarging ; D, definitive oocytes.
6 Enlargement and follicle formation of a definitive oocyte in germinal
epithelium. Ovary from a rat, seventeen days a f t e r birth. Picro-aceto-formol
fixation; stained with iron hematoxylin. X 380.
7 Oocytes being pushed down by the overgrowth of the epithelium in the
ovary of a rat, twenty-nine days old. Picro-aceto-formol fixation; stained with
iron hematoxylin. X 380.
8 Ovary of a rat, twenty-nine days a f t e r birth. Such a large increase in size
does not occur in epithelium before being proliferated as in younger ovaries.
Picro-aceto-formol fixation ; stained with iron hematoxylin. X 192.
9 Oocytes in and j u s t under epithelium. Ovary from a rat, sixty days old.
Picro-aceto-formal fixation ; stained with iron hematoxylin. X 380.
10 Ovary from a r a t during oestrus to illustrate the enlargement of a cell in
the epithelium and the overgrowth of adjacent epithelial cells in the process of
proliferation. Picro-aceto-formol fixation ; stained with iron hematoxylin. x 380.
G, germ cells enlarging; Pr, proliferation of a germ cell
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