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Observations on pre- and postpubertal oogenesis in the white rat Mus norvegicus albinus.

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OBSERVATIONS ON PRE- AND POSTPUBERTAL
OOGENESIS IN THE WILIITE RAT, MUS
NORVEGICUS ALBINUS
MARION H. COWPERTHWAITE
Washington University, St. Louis, Missouri
ONE PLATE (EIGHT FIGURES)
INTRODUCTION
This investigation was suggested by recent studies on the
origin and history of germ cells in the mammalian ovary,
particularly by the conclusion arrived at by Allen ( ’23) from
his study of the white mouse, 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. ”
A further impetus toward this investigation was received
from the study of oogenesis in the white mouse, made by
Kingery ( ’17), in which the conclusion is reached that all
the germ cells derived from an embryonic proliferation of the
germinal epithelium degenerate, and that the definitive ova
arise from the germinal epithelium during a proliferatioii
which extends over a period from three days after birth
until the time of puberty, at which it ceases.
The observations made by Allen, upon which he bases his
conclusions, do not include those striking meiotic phenomena
that in general have been observed t o occur in the nucleus
of the primary oocytes at the beginning of their growth
period, and which are considered by most cytologists to be
of fundamental significance, since by them is brought about
the pairing of the maternal and paternal chromosomes preparatory to the maturation divisions.
69
TO
MARION H. C0WPEE:THWAITE
T<iiigery, on the other hand, notes the occurrence of meiotic
phcnomena iii the germ cells of tlie first proliferation arid the
absence of such phases in the germ cells of the second or jwepubertal proliferation. Since lie is convinced that all of the
(*ells of the first proliferation degenerate, lie considers tlie
absence of aiiy phases resemhling meiosis, in the defiiiitive
ova of the second proliferation, a matter of slight importance.
If tlie pairing of tlie matt~ritalaiid paternal cIii-omosomes
is a fundamental ant1 essential process in the formatioil of
true germ cells, its presence or ahseiice should be the decisive
factor in the determination of the origin of the definitive ova.
Because if aiiy coiiclnsion seems to rest on a firm liasis, both
experimental arid morpholngical, this one-that paternal and
maternal chromosomes must pair before the maturation dirisioris-seems to be so established. It is, therefore, assumed
that if synapsis caiiiiot be demonstrated in postpubertal
oocytes, they cannot be considered as representing the definit i r e ova. T i t h such a criterion in mind, this investigation of
oogeiiesis iii the white rat, Rlus iiorvegicus alhiiius, w a s made,
comparing a series of ovaries taken from animals before the
attainment of puberty, with a series which more than covered
an oestrous cycle of sexually mature animals.
MATEHIAL A N I ) METHODS
The first series of ovaries studied was taken from a group
of rats wliieli ranged i n age from those newly horn to those
eight days old. This group mas selected because the later
phases of meiosis a r e kriow-ri to occur iii oocytes during this
period. A study of the nuclear structure of tlie germ celIs
of sncli ovaries provides a hasis for a comparative study of
iiuclear plreiiomciia, size of germ cells, ant1 details of follicle
foi-mation in any possi1)le formatioil of new germ cells in
adult ovaries. A series of ovaries was likewise taken ever;\teii dwys heginning with tlie tenth clay after birth and eridiiigoii the ninetieth clay-an
age corresponding to the arerage
time of tlie first breediiig period of alhiiio rats. This series
iiicludetl, therefore, ovaries from individuals at the time of
woaiiiiig, of piil)erty, arid a t tlir attainment of sexual maturity.
OOGENESIS I N T H E WHITE R A T
71
Still a third series was taken in the following maiiiier:
MBino r a t s were mated and the birth date of the first litter
notetl. Since i t is known that tlie r a t usually ovulates twenty
to forty-eight hours after parturitioii (Donaltlson, ’13), particnlarly if her litter be removed from her, the litters were
removed as soon a s possihle from these females. The females
were then allowed to mate again and to produce a second
litter, which again was taken away immediately at birth.
This wits done because it is known that lactation inhibits
ovarian activity, and it was desired that the females might
go through a normal oestrous cycle in the presence of the
males. It is kilowin (Long arid Evans, ’22) that the oestrons
cycle of the rat has a duration of four days, on the average,
and that ovulation occurs at the height of this cycle, or at the
turning point of ovarian metabolism from aiiabolic to cataholic activity. Therefore, by taking ovaries over a period
of time more than equal to tlie length of a normal oestrous
period, any cyclical proliferation of oocytes would surely
be detected. Ovaries from females which had produced a
second litter were taken a t these times: at tlie time of the
second parturition and on the first, second, third, fourth,
fifth, and sixth days thereafter. In this way an eiitire oestrous cycle was more than covered.
F o r clear-cut nuclear detail arid general cytoplasmic fixation, Allen’s modification of Rouin’s picro-formol-acetic-acid
fixing fluid was most extensively used. F o r young ovaries
Bouin’s unmodified fluid gave excellent results and did not
lead to the overfixation arid obsciiring of nuclear detail which
often resulted after the use of both Flemming’s strong solution and Flemming-without-acetic. The nuclear stain employed was Heidenhain’s iron hematoxylili, with aqueous acid
fuclisin, eosin, or Biebrich scarlet a s a counterstain.
The younger ox-aries were cut a t a uniform thickness of
ci p, the older ones a t 8 o r 10 p, and serial sections were
mounted in every case.
72
MABLON H. COWPERTHWAITE
LITERATURE
I n view of the thorough review of the literature relative
to contiriued formation of germ cells after birth given by
Allen ( ’ 2 3 ) , to which reference has previously been made, no
attempt will he made to summarize the conflicting views on
this subject, but quotations will be made from some of the
more recently published papers which have discussed this
much-debated question, with particular reference to observations of white mice and rats.
P r a t t and Long ( ’17), working on synapsis in the egg of
the white r a t , found this process to extend over a period of
ten days-seven before, and three after birth-during which
time practically all of the nuclei pass through leptotene,
synaptene, pachytene and diplotene phases and terminate in
early follicle formation, with the oocyte nuclei in a typical
resting condition. While in the r a t there is absence of concentric zoning of oocytes sb characteristic of the cat, nevertheless those germ cells in which meiosis has advanced most
tend to he found toward the center of the ovary, those in the
earlier meiotic phases being found near the ovarian surface,
close under the germinal epithelium. Their work admittedly
stops with the completion of synapsis in the young ovary
and does not take up the ultimate fate of these young follicles,
nor the question of the occurrence of synapsis in oocytes in
older., pre- or postpubertal ovaries.
Though, if we assume that individual leptotene threads
represent chromosomes, there seems t o be parasynapsis during meiosis, and the individuality of these chromosomes seems
to be maintained through the diplotene stage, because of the
lack of order of the achromatic network found i n the resting iiuclei of the early growth period, Pratt and Long feel
that they have found little evidence in favor of “that theory
u-liich postulates that the individuality of the chromosome is
maintained thru the constant discreteness of the linin framework.” Further, they do not feel that the evidence that the
leptotene threads actually represent chromosomes is adequate,
because it was not possible to make any accurate count of
the threads.
OOGENESIS I N THE WHITE RAT
73
Arai ('20), in a statistical study on the postnatal development of the ovary of the albino rat, investigating particularly
the number of ova contained at various ages, finds that a
rapid decrease takes place in the total number of ova found
in both ovaries from 35,100 a t birth to 11,000 a t twenty-three
days of age. This number is maintained fairly constantly
until the sixty-third day, after which a sharp decrease takes
place, reaching, at the time of ovulation, the low number of
6600 ova. From then on until thirty-one months of age, a
slow gradual decrease takes place in the number of ova to
2000-a
number which remains fairly constant throughout
the life of the individual. This degeneration, while mainly involving primitive ova, is also augmented by the degeneration
of definitive ova. To compensate f o r this degeneration and t o
provide definitive ova, Arai states that, beginning at the second week after birth, but proceeding most rapidly from the
third t o the ninth weeks, a continuous proliferation of new
ova occurs from the germinal epithelium, some of which enlarge and, as they grow, are covered by adjacent epithelial
cells and sink through the tuiiica albuginea into the underlying stroma. This formation of so-called definitive ova, while
it may continue for a year after birth, proceeds a t a progressively slower rate as puberty is attained. No reference is
made to the condition of the chromatin in these 'definitive
ova.'
In the monograph of Long and Evans ( '22), while they
call attention to the rhythmic cyclical development of graafian
follicles during each oestrous period, nothing is said as to the
formation of new germ cells a t each oestrous cycle.
Similarly, Kirkham and Burr ( '13) describe the progressive
development of graafian follicles from one oestrous cycle t o
another, but they make no mention of any proliferation of
new ova during sexual maturity.
That in the cat ovary the surface mesothelium o r germinal
epithelium is not the sole seat of proliferation of germ cells
in the prenatal ovary is brought out by Kingsbury ( '13). He
finds, on the contrary, that it shares with the underlying egg
cords in growth activity mid has, as a matter of fact, fewer
mitoses a t birth than tlie more periplieral portions of tlie
cords coiiiiectetl witli it. v. Wiiii~varterant1 Saiiimoiit state
that the oogonial divisioiis practically cease at birth, hut arc
~*enewedfor a short period twciity-oiie days after hirth.
Kingshury finds no eritlence of this r e i i e ~ ~ofd activity, admitting, however, that his material was less a1)uiirIaiit than
theirs. His conclnniou is that “ ‘tlie ware of synezesis’ appeared to Inc R fairly steady centrifugal progression. ” By
thirty-three (lays p s tpartum, all the deeper cells of the socalled meiiullaq-, or egg, cwrtls are postmeiotic, the more
pei.iplieral ones were still in various meiotic phases. Siiice
Kingshury finds 110 basis, from tlie ntutly of his material, foithe conclusion that a third proliferation of germ cells, just
M o r e the advcbnt of sesiial maturitF, gives rise to tlie definitive ova, iior evidence for a secoiid marked periodicity of
proliferation a t twenty-one days after birth, lie believes that
the first proliferation gives rise to the defiiiitire oocytes.
Kingery ( ’17) distiiiguislies two distinct proliferations of
germ cells from the germiual epithelium of the white-mouse
ovary. The oocytes of the embryonic pi-oliferation remain f o r
some time in the germinal epithelium in N resting condition.
As these oocytes pass from the leptoteiie, synaptene, pacliytene, and diploteiie phases of meiosis into resting primary
follicle formation, they sink into the stroma of the ovary. A t
birth the 1)roliferating process lias hecome so retarded that
late meiotic phases may occasionally he foiind in the epithelium itself instead of beneath it iii the ovarian tissue.
He finds R second prolifei-ation of cells from the germinal
epitlieliium which begins three days after birth and coiitinixes
nritil thirty to forty-five days after birth. This proliferation,
hecause of tlie fate of the germ cells formed, constitutes
oogenesis proper, in coiitraclistii;cti~,.I 1o tlie first proliferatiori of germ cells, all of which, he believes, degenerate, and
are, therefore, not iiivolred i~ c?-iie oogenesis. The epithelial (sells of tlic later gencratioii which a r e now developing, aiid wliicli mav from tliv moment of increase in size be
OOGENESIS I N THE WHITE RAT
75
considered primary oocytes, because of their future development into the definitive ova, show an absolute lack of the
meiotic phases which mark the developmental history of the
embryonic germ cells. At first indistinguishable from other
epithelial cells which may become either follicular or residual
germinal epithelial cells, the cells destined to become the definitive ova, according to Kingery, grow in size. Their nuclei
pass from a stage in which the chromatin reticulum is poorly
defined and delicate to one in which, for a time, the chromatin
seems more dense and the reticulum heavier. Eventually the
nuclei reach a stage having an attenuated and broken reticulum with the'chromatin filling the nucleus with more or
less isolated strands and granules of marked chromophobic
appearance. This latter stage resembles the resting stage
of early development somewhat, and, like it, seems to be
correlated with great increase in size and with formation of
primary follicles. Kingery feels that he has traced the definitive germ cells from their inception in the germinal epithelium until they are ready for maturation by a series beginning with egg cells of this form of nucleus up to immature
and mature graafian follicles.
Kingery takes decided issue with v. Winiwarter and Sainmont and others who make the criterion of a true germ cell
its meiotic history. According to him, the ultimate fate of
a germ cell would be the only criterion employed in such a
decision and not the resemblance of the germ cell to embryonic
developmental stages. That these later egg cells in the postnatal ovaries are not to be identified with germ cells of the
embryonic proliferation, he believes is established by a comparison of size of their nuclear structure and by the presence
of transitional stages between these cells and ordinary
germinal epithelial cells, on the one hand, and these cells and
oocytes of mature graafiar follicles on the other. Contrary
t o the theory of Allen ( ,231, which will be taken up presently,
the germinal potentiality o$ S: epithelial cells is lost as the
ovary attains maturity.
T H E AMERICAN J O U E N A L O F ANA'I'OIY,
YOL.
:?6,NO. 1
76
MARION H. COWPERTHWAITE
Kingery finds no evidence of synapsis in either primitive
o r primordial germ cells, nor of synezesis except in the
primitive germ cells, for throughout the whole course of development of the definitive oocptes no well-defined chromatin
threads appear. He feels that spnezesis merely indicates
one stage in a degeneration which is caused by a disturbance
of normal nucleocytoplasmic relationships, and that, since
claim has been made that both synaptic and synezetic stages
have been observed in somatic cells, no genetic significance
can be attached to their occurrence.
Allen ('23), after a painstaking study of the oestrous cycle
of the white mouse, concludes that a t each riormal oestroiis
period, young ova are added to the cortex of the adult ovary.
They are described to arise from dividing cells in the germinal
epithelium. If the angle of the mitotic; cell to the surface of
tlie ovary is rnorc than 30°, ilie proximal of the two daughter
cells is considered t o be cut off from the germinal epithelium
and soon surrounded by a follicular ring made of adjacent
epithelial cells. When mitosis is a t its height, a statistical
study of the proportion of very youiig ova in the cortex t o
slightly older ova leads him to conclude that the mitoses of
the germinal epithelium form ova, and not somatic ovarian
tissue. The new formation of ova is a cyclical phenomenon,
f o r he finds that a very large number of young ova are present
in any adult ovary four days or less after a normal oestrous
period has elapsed, whereas very few ova are visible after a
long dioestrous interval. No comparison was made between
the nuclei of these germ cells arid the nuclei of those which
are formed during embryonic or early prepubertal life.
OBS ERT'A T IOXS
I n order that an adequate basis of comparison might be
established in the study of postpubertal oogenesis, careful
study of meiosis was made in very young postnatal ovaries.
Siiice the phases of meiosis prior t o the pachytene phase,
accordirig t o the work of P r a t t and Long ('177, occiir in
embryonic ovaries and since tlic writer's series of ovaries
OOGENESIS I N T H E WHITE RAT
77
began at the time of birth, no study was made of the earlier
phases of meiosis, which they find begin with the completion
of the last oogonial divisions seven days prior to birth.
The young germ cells prior to the appearance of the leptotene threads have undergone fragmentation of the peripheral
chromatin blocks which characterized the preleptotene period.
Simultaneously with this fragmentation, they find numerous
linin strands appearing, which gather in a confused nonhomogeneous mass at one side of the nucleus.
According to their seriation of stages, the leptotene phase
which emerges from this condition is characterized by very
numerous, very fine, quite regular, or slightly moniliform
threads showing a tendency toward definite common orientation.
The synaptene phase, following next, shows, they find, very
much thicker threads which, by their moniliform halves give
evidence of a double origin, probably from the parasynapsis
of the leptotene threads.
With the appearance of the thick pachytene threads at
the time of birth, the writer’s observations will be presented.
The marked orientation toward one side of the nuclear wall,
present in the early pachytene phases, becomes less marked
just before this phase merges into the diplotene phase. The
pachytene threads are very chromophilic, heavy, and distinctly moniliform (fig. 1). Frequently the threads terminate
in an unusually large chromatin mass, several of which in
close approximation suggest the origin of the chromatin
nucleolus. The pachytene nuclei persist as late as three days
after birth, at which time, however, their number is proportionately few in contrast to the greater numbers of diplotene
nuclei. No evidence of concentric zoning with respect to
either time of origin o r place of distribution in the ovary
is to be observed, for these two stages, plus a few young
follicles, may exist side by side for two or three days wit11
no constant regularity of depth from the ovarian surface.
Many of the pachytene cells contain within the cytoplasm one
or more deeply staining masses-the
so-called chromatoid
78
MAItION H. COWPERTHTYAITE
bodies which characterize the greater number of the germ cells
of the immature ovaries (fig. 1).
Although evidences of the occurrence of the diplotene or
twisted-thread phase were found as early as twenty-four
hours after birth, the greater number of the germ cells do
not attain this phase until three days after birth. This phase
is characterized by a marked loss in chromophility of the
chromatin threads, which also begin to look fuzzy and to
lose the definiteness of their moniliform appearance (fig. 2).
The nucleoli, usually two in number, which have now definitely
appeared, stand out sharply as highly refractive, homogeneous, darkly staining masses of chromatin. With the
appearance of the greater number of more faintly staining,
finer chromatin threads, due to a split in the former heavy
pachytene threads, comes a noticeable loss in orientation and
an even distribution of the threads throughout the more oval
nucleus.
The diplotene phase merges gradually, during the third
and fourth days after birth, into the succeeding one-the resting phase of the oocyte i n a primary follicle (fig. 3). The
nuclear structure of the oocytes of the early growth period
is characterized by a marked chromophobia of the granulesthe more evident again because of the prominent, eccentric
chromatin nucleoli. Although the greater number of the
young germ cells a r e in early follicle formation, a few cliplotene phases may still be found close under the germinal
epithelium in addition to a few obviously young germ cells
which have not yet hecome siirroiinded by a primary follicular
sheath.
A t the end of the fourth day postpartum, meiosis has
been completed and 110 transitional stages a r e to be found between the untliff erentiated germinal epithelial cells and the
postmeiotic germ cells. These latter cells are of two kinds--some which lie in the deeper portions of the ovarian cortex,
having a single complete follicular ring; others which lie
close to the germinal epithelium, having one or two follicle
cells only. At first the arrangement of the granules is in-
OOGENESIS I N THE WHITE RAT
79
dicative of tlie course of the former diplotene threads. After
the apparent dissolution of the linin framework, however,
the granules tend to clump together somewhat (fig. 3 ) . The
investing follicular cells form a syncytium with each other,
but not with the cytoplasm of the germ cell itself.
From this time on, a gradation of follicular size is apparent, ranging from those small follicles which lie under the
germinal epithelium, to those follicles, composed of two or
three rows of cells, which lie embedded more deeply in the
stroma of the ovary. The nuclear appearance of the smaller
and more peripheral follicles is similar to that found in
follicles of similar size in the younger ovaries-evenness of
granule distribution and lack of apparent linin (fig. 3). Comparing these follicles as to size and nuclear condition of the
oocyte with tlie larger follicles, one is led to the conclusion
that the smaller follicles represent a retarded growth condition.and that they are not derived from a new germ-cell
formation. This conclusion is reached because of a failure
t o find fresh meiotic phenomena in the germinal epithelium
either at this age o r at any succeeding period in the series
of ovaries studied. Cells in mitosis are rare in the germinal
epithelium and, when they are present, are of the same size
as adjacent epithelial cells. These dividing cells obviously
do not take part in the proliferation of new germ cells.
After the rat is two weeks old, the nuclei of some of the
oocytes of the young follicles in the ovarian cortex exhibit
a picture similar to the description previously given. Others
show by their chromatin distribution a similarity to the
central cells found in young follicles of like size and distribution in mature ovaries. The difference in appearance
is that the chromatin granules have a definite tendency to
clump in peripheral masses which are connected with each
other and with the eccentric nucleolus by faint linin strands
(fig. 4).
An interesting phenomenon was observed several times in
the ovaries of sexually mature animals, and infrequently in
the prepubertal ovaries, namely, an apparent young germ
cell in a prefollicular stage (figs. 6 and 7).
80
MARION H. COWPERTHWAITE
Such cells occur at no constant depth below the ovarian
surface, nor at any definite angle to it. A t first they a r e
marked off from adjacent cells chiefly by a n increased nuclear
aiid cytoplasmic volume rather than by a marked difference
in nuclear structure. It would appear that the change which
does occiir i n the appearance of the nucleus and which sets
i t a p a r t from the adjacent cells might be iiiterpreted to
occ~irin some such manlier a s follows: If the distinct linin
framework were to break down when the cell is but slightly
enlarged, it wonld account for the uneven distribution of
granules throughout the nucleus. As both nucleus and cytoplasm increase in size, however, remiiarits of this framework
a r e seeii radiating toward masses or clumps of discrete
chromophilic granules (fig. 7 ) . Although many of the masses
adhere closely to the nuclear membrane, the distribution of
the granules is not so markedly peripheral a s it becomes
when the cell begins to assume follicle formation. A t the
timc of follicle formation, the distribution of the granular
masses of chromatin is distinctly peripheral for the most
part, with very faint linin strands passing from one clump to
another. No iiucleolus a s such is visible at this stage in
any of the young follicles, no matter how much they may
vary in follicular development. Correlated with an increase
in the size of the follicle, there is an apparent loss in the discreteness of the granules making up the peripheral clumps,
so that more or less homogeneous masses result, connected
by extremely faint liiiiri strands (fig. 8).
As the entire follicle enlarges, the nuclear picture of the
contained cell again changes and comes to resemble exactly
the appearance of the postmeiotic nuclei of the medullary
oocytes of younger ovaries (fig. 5). Only a faint suggestion
of the peripheral blocks and clumps remains indicated by
a slight tendency toward a heavier and more irregular granule
distribution at the periphery than throughout the rest of
the nucleus. A faint liriiri network, 011 which granules a r e
ranged, is present throughout the nucleus. Some of its fibers
may be observed to be attached directly to the nucleolus.
OOGENESIS I N THE WHITE RAT
81
Before discussing the theoretical significance of this apparent follicle formation and the question whether the cells
contained in the follicles a r e true germ cells, the series of
ovaries taken from sexually mature females at definite intervals after the second parturition may be considered.
If Allen's theory, that the definitive germ cells are those
added to the cortex of the adult ovary from the germinal
epithelium a t each normal oestrous period, is true, then we
should expect to find cyclical proliferation in other mammals,
such as he describes f o r the white mouse.
The first evidence of a proliferation of new ova he finds
in the enlargement of certain of the germinal epithelial cells,
preparatory to their mitotic division. I f the angle of the
longitudinal axis of the mitotic cells is more than 20" or 30"
to the ovarian surface, one daughter cell is contributed, he
believes, to the cortex of the ovary. This cell becomes siirrounded by follicle cells and develops into a definitive oocyte.
The frequency of the mitoses varies with the specific period of
the oestrous cycle, being greatest during the hyperemia of
oestrus proper, and falling with the anemia of the metoestrum. Consequently, the number of young ova also varies
with the periodicity of the cycle, and a correlative proportion
was observed between the number of mitoses arid the number
of young follicles close under the germinal epithelium.
No comparison is made between the appearance of the
chromatin in these young ova and that in the ova found before
puberty. But from the figures that illustrate his paper,
which show all stages of ordinary mitoses in the germinal
epithelhm, we a r e led to the inference that no meiotic phenomena were observed and that their absence was not deemed
a matter of significance.
I n the r a t a new oestrous cycle normally occurs directly
after a parturitioii. Ovulation, which takes place at the
height of the cycle, may be expected to occur from twenty
t o forty-eight hours after the birth of a litter. A series of
ovaries more than covering an entire oestrous cycle would,
therefore, furnish evidence of any periodicity in the forma-
82
MARION H. COWPERTHWAITE
tion of new germ cells. Further, if ovulation marks the end
of the anabolic period of the ovary and the inauguration of
the catabolic period, this should be reflected in the relative
freqnency of new germ cells at these periods of oestrus.
Also such new germ cells should bear a coilstaricy of relation
to depth from the ovarian surface, if they he proliferations
from the germinal epithelium.
Throughout the series of ovaries employed, no evidence
of such a proliferation of new germ cells has been found. I n
rare instances, spireme threads were found in the squamous
cells overlying a large corpus luteum or a maturing graafian
follicle. Always, however, the angle of the cell was snch
that both of the daughter cells would remain in the germinal
epithelium.
Such mitoses, theref ore, find a n adequate explanation in
the fact that the growth of the germinal epithelium is required to keep pace with the growth of the ovary in volume.
In any given germinal valley small follicles of one or two
follicular cells may be found a hundred or more micra from
the surface, whereas at a depth of two or three cells only helow the surface, other follicles a r e present composed of a
complete follicular ring. The reverso condition is as frequently true. It is difficult to correlate these facts with a
migratioii from the germinal epithelium, even allowing that
the? originated during more than one oestrous cycle.
The number of young follicles of a given size had no ohserved correlation with the stage of the oestrous cycle. Had
a cyclical proliferation occurred and had the new germ cells
heeii growing normally, the number of follicles of any average
size should change with the lciigtli of the time that had elapsed
siiicc the onset of the oestrous period in w7liicli their formation began. Follicles in corresponding stages of development
were to be found in the ovaries of each day of the period
studied.
The same traiisitional stages of follicle formation were
observed in sexually mature ovaries as in the prepubertal
ovaries and tliosc of early postpuhertal life. From a cell ell-
OOGENESIS I N T H E WHITE RAT
83
larging in situ exhibiting a picture of the nucleus comparable
to the adjacent cells (fig. 6), through a stage exhibiting a
fairly even granule distribution, development proceeds to a
phase showing peripheral masses of chromatin connected by
faint linin strands (fig. 8). This stage in which there is a
small, more or less complete single follicular sheath, is succeeded by a larger follicle. I n the nucleus of the central cell
of these larger follicles the chromatin blocks have become fuzzy-edged and indistinct, and scattered granules
appear through the nucleus on a very irregular and faint
linin network. A nucleolus for the first time has now appeared. Also, as in the younger ovaries, a follicular syncytium is present which does not extend to the cytoplasm
of the central cell except in those cases showing ingestion by
the oocyte of a follicle cell. Again, as in the larger follicles,
the nucleolus is not homogeneous as it apparently is in the
smaller follicles.
The only possible evidence of periodicity in germ-cell formation in the series of ovaries studied may be found in the observation that more cells were found enlarging in situ in the
ovaries taken during the time of parturition and on the fifth
day thereafter than in those taken at other times. Each of
these periods corresponds to the beginning of a new oestrous
cycle; but with this one observation any evidence f o r cyclical
appearance of new germ cells ends.
One other point of difference between the young follicles
of the adult white mouse and the albino rat is that, in the
former, the follicles are described as rarely occurring above
a large corpus luteum o r a maturing graafian follicle. There
seems to be no such restriction of a possible oogenetic site,
in the albino rat, for medium-sized graafian follicles have
been observed in the tunica albuginea above corpora hxtea,
and younger follicles are very frequently observed in the
same region.
The contrast in the nuclear picture exhibited by the central
cells of follicles of immature and mature ovaries should not
be made the sole crilerion for the genuineness of oocyte
84
M A R I O N H. COWPERTHWAITE
formation in mature ovaries, for there a r e many factors
which may operate to produce differences in appearance of
the chromatin of a resting nucleus. Among such factors
the presence of hormones which produce secondary sexual
characters may be mentioned, as well as alterations of
vascularity and therefore of available nutrition.
However, the fact that is insurmountably difficult of explanation is the absence of any phase of meiosis, especially
the absence of evidence of the pairing of threads representing maternal and paternal chromosomes. Synapsis has been
both affirmed and denied with reference to its occurrence or
necessity in germ cells. Its genetic significance has also been
questioned on the ground of its apparent occurrence in somatic
cells. I t s genetic significance has been still further questioned
because of the doubt of the identity of the threads ~vhieli
apparently do pair in meiosis in tlie germ cells of the young
albino rat, with individual chromosomes, and because of the
doubt of the persistence of the discreteness of the linin
framework.
If the occurrence of meiosis, or synapsis, is a necessary
prerequisite for tlie determination of a definitive ovum, the
young follicles of the mature ovary cannot be considered as
containing true oocytes. Tlie possibility suggests itself that
some of the germ cells that had previously undergone meiosis
may have become dedifferentiated. The loss of a typical
postmeiotic appearance need not necessarily be correlated
with a loss of germinal potentiality. Such cells might be the
ones which apparently enlarge, become differentiated from
adjacent cells of the germinal valleys, and form the central
cells of the young follicles of mature ovaries. Nothing was
observed giving any evidence that such was the case. If, or1
the other hand, cells riot obviously derived from the germinal
epithelium nor of known meiotic history have germinal
potentiality ; if, from their chromatin content, chromosomes
representing maternal arid paternal contributions can be
sorted out at the time of the maturation divisions so that
past synaptic conditions a r e not essential, then the young
I,
OOGENESIS I N THE WHITE BAT
85
follicles which apparently start growth after the oocytes of
the prenatal proliferation of germ cells have gone into
follicle formation may be capable of developing into definitive
ova. I n view of the significance of synapsis in the history
of germ cells, such a conclusion derived from such premises
is considered to be untenable, and the conclusion derived from
this investigation is that oogenesis is not continued during
pre- or postpubertal life.
I wish to express my appreciation of the many helpful suggestions and criticisms received from Prof. Caswell Grave,
under the direction of whom this investigation was conducted.
SUMMARY
1. Synapsis has been completed by the fourth day postpartum.
2. The cortical follicles remain in a retarded state of development long after the medullary follicles have matured
or undergone atresia.
3. Before o r after puberty, no germinal epithelial cells
have been observed t o undergo meiosis or t o enlarge ill situ.
4. After the attainment of sexual maturity, no evidence
of a cyclical proliferation of new germ cells from the germinal
epithelium was found.
5 . Consequently, the relation of young follicles of mature
ovaries to the surface u7as not a constant one, nor did they
evidence in any other way a periodicity in origin and development.
6. The growth in situ of certain cells of the germinal valleys
may account for the production of young follicles in mature
ovaries. The central cells of such follicles have a nuclear
picture resembling that of those in which meiosis is known
t o have occurred.
7. It is improbable that the celk which enlarge in situ have
become dediff erentiated from postmeiotic germ cells.
8. If synapsis be the criterion of the definitive oocyte, the
central cells of the young follicles of mature ovaries cannot
be eonsidered true oocytes and oogenesis is not continued
throughout pre- and postpubertal life.
86
MARION H. CO\VPERTHWAITE
BIBLIOGRAPHY
ALLEN,EDGAR1922 Oestrous cycle in the mouse. Am. Jour. Anat., vol. 30, no. 3 .
1923 Ovogenesis during sexual maturity. Am. Jour. Anat., vol. 31,
no. 5.
ARAI, HAYAW 1920 On the cause of the liypertropliy of the surviving ovary
a f t e r somispaying and the number of ova in it. Am. Jour. Anat.,
vol. 2q, no. 1.
1920 On postnatal development of the ovary (albino r a t ) , with
special reference to tlie number of ova. Am. Jour. Anat., vol. 27, no. 4.
CUNNINGHAM,
R. 8. 1922 The reaction of the cells lining tlie peritoneal cavity,
including the germinal epithelium of the ovary, to vital dyes. Am.
Jour. Anat., vol. 30, no. 4.
DONALDSON,
HENRY13. 1915 The rat. Meinoirs of Tlie Wistar Institute of
Anatomy and Biology, no. 6.
FIKKET,
J E A N 1920 On tlie origin of tlie germ cells in the higher vertebrates.
Anat. Rec., vol. 18.
HUBER.G. C . 191.5 Development of tlie albino rat, Mus norregicus nlbinus.
Jour. Morph., vol. 26.
ISHII,
0. 1922 Observations on tlie sexual cycle of tlie white rat. Anat. Rev.,
rol. 23, no. 5.
KINGERY,
H. M. 1917 Oogenesis in tlie white mouse. Jour. hforph., vol. 30,
110. 1.
KINGSBURY,
B. F. 1913 Tlie morphogenesis of tlir mamrna1i;~n ovary : Felis
tlomestica. Am. Jour. Anat., vol. 13, no. 3.
1914 Interstitial cells of mammalian ovary. Am. Jour. Anat.,
vol. 16.
KIRKHAM,
W. H. 1910 Ovulation in mammals with special reference to the
mouse and rat. Biol. Bull., vol. 18.
KIRKHAM,
W. B., A N D BURR,H. 8. 1913 T!ie breeding llnl)its, maturntion of
eggs and ovulation of tlie nlbino rat. Am. Jour. Anat., vol. 15.
1922 Tlie oestrous cycle of the rat :inti
LONG,JOSEPH
A., A N D EVANS,H. 1,.
its associated plienomena. Memoirs of the Univ. Calif., xol. 6.
LONGLEY,
W. H. 1911 Tlie maturation of the egg and ovulation in the domestic
cat. Am. Jour. Anat., rol. 1 2 , no. 2.
METCALF, MAYNARD M. 1900 Notes on niorpliology of tlie l’unic;rta.
Zoologisclie Jalirbucher (Abteilung f u r Anatoniie uiid Ontogenie der
Thiere), 73. Ed., 4. Heft.
PRATT,B. H., AND LONG,J. A. 1917 Synapsis in the egg of tlie white rat,
Mus norvegicus albinus. Jour. Morpli., vol. 29, no. 2.
WAJ,SH, 1,. 6. M. 1917 Growth of the ovarian follicle of the guinea-pig under
norni:il a n d p:itliologic:rl conditions. Jour. Exper. Medicine, vol. 26.
PLATE
87
PLATE: 1
EXPLANATION OF FIGUKEX
All the figures were drawn with the aid of a camera lucida, using a 1.8-mm.
fluorite objective and a 10..5 compensating ocular. The magnification of all the
figures is 1149 diameters. IVith the exception of figure 1, in which aqueous ;icld
fuclisin was employed as :i counterstain, the figures were all drawn froin
inaterial fixed in moiiifird Bouin’s fluid arid stained with Heidenhain’s iron
Iiematoxylin and alcoliolic eosin.
7 Pncliytene phase of primary oocyte. Twelve hours after birth.
2 Lhplotene phase of prim:u y oocyte. Forty-eight hours a f t e r birth.
3 Rrsting phase of primary oorytc in follicle formation. Eight days after
birth.
4 Prini:Lry oocyte in ret;irded state of developmciit, close under the germiiial
zpitlieliuui. Twenty-two days after birth.
.T Typic:il primary oocgtr in medium sized follicle. Twenty-two days after
hirtli.
6 Cell of gc,rniinal v:tlley en1:irging. F i v e days after second ~iarturition.
i Cell of germin:rl v : ~ I l ~ yenlarging. Six days after second parturition.
8 Young follicle of adult ovary. TIirrr days after second parturition.
88
PLATE 1
OOGENESIS I N THE WHITE RAT
MARION H . OOWPERTHWAITE
89
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