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Growth and atresia of follicles in the ovary of the hamster.

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GROWTH AND ATRESIA O F FOLLICLES I N THE
OVARY O F THE HAMSTER
KARL M. KNIGGE'
Department of Anatomy, T h e School of Nedioine,
University of Pittsburgh, Pittsburgh,
Pennsylvania
JAMES H. LEATHEM
Department of Zoology, RzLtgms University,
N e w Brunswiok, New Jersey
TWENTY-FOUR FIGURES
Despite the relatively recent introduction of the golden
hamster as a laboratory animal, a considerable literature concerning various phases of its reproductive physiology is
available. The gestation period is somewhat less than 16 days
(Bruce and Hindle, '34 ; Bond, '45 ; Knigge and Leathem, '50).
Pre-implantation divisions and other changes of the fertilized
ovum have been described (Graves, '45 ; Venable, '46a, '46b ;
Ward, '46, '48).
Ortiz ('45, '47) has described the embryonic and post-natal
development of the ovary, while LaVelle ('51) has studied the
effect of hormones on early sex development. Other aspects
of early embryology, placentation and changes associated with
pregnancy in the hamster have been presented by Adams and
Hilleman ('50), Boyer ( '53), Orsini ( '54) and Foote, Wesley
and Foote ( '54). There is general agreement concerning the
stages of estrous cycle, although differences of interpretation
exist regarding the correlation between vaginal smear and the
stage of estrous (Deanesly, '38; Peczenik, '42; Kent and
Smith, '45; Ward, '46). The cyclic changes in the vaginal
'Present address : Department of Anatomy, University of California, Los
Angeles.
679
680
KARL M. KNIGGE AND JAMES H. LEATHEM
and cervical epithelium have been investigated to aid in establishing the estrous smear (Klein, '37; Kent and Weathersby,
'46; Kent and Roberts, '49; Miller and Bacon, '50).
Although the growth of follicles in the hamster ovary appears to be similar to that in other mammals, a detailed account of follicular growth, the regression plot, pre-ovulatory
changes, and follicular atresia has not been presented. Rolle
and Charipper ('49)' studying the effect of age on the distribution of normal and atretic, vesicular follicles in the hamster
ovary, noted that atresia of vesicular follicles was similar to
that observed in the rat and mouse. Follicular atresia has been
studied extensively in several species (rabbit: Asami, '20;
mouse : Engle, '27 ; sow : Allen, Kountz and Francis, '25 ;
guinea pig: Harman and Kirgis, '38; cat: Kingsbury, '14,
'39 ; rat : Mandl and Zuckerman, '50 ; Rennels, '51 ; Dawson
and McCabe, '51; Deane, '52).
The present study was undertaken to describe follicular
growth and atresia in the hamster ovary. Evidence is presented also indicating that, in the ovary of adult animals, the
granulosa cells of atretic pre-antrum follicles are the source
of interstitial tissue.
MATERIAL AND METHODS
Ovaries of 34 adult female hamsters, two to four months
of age were used. A minimum of 6 glands was prepared by
each of the following methods: ( A ) glands fixed in Bouin's
fluid were sectioned serially at 7 p, and stained with hematoxylin and eosin or by the Masson ( '28) triacid technique; (B)
ovaries fixed in Regaud's fluid and post-chromated in 3%
potassium dichromate for two days were double embedded in
paraffin and celloidin, sectioned at 3 and 5 p, and stained with
iron hematoxylin; (C) alkaline phosphatase activity was demonstrated after fixation in cold 80% alcohol by the calciumcobalt method of Gomori ('39) as well as by the azo-dye
method employing sodium alpha-naphthyl phosphate (Gomori,
,52) ; other sections of ovaries fixed in 80% alcohol were used
to visualize non-specific esterases, according to the method
FOLLICULAR GROWTH AND ATRESIA
681
of Gomori ('52); (D) ovaries fixed in 10% formalin were
washed in running water overnight, cut on the freezing microtome, and stained with Sudan black B, Nile blue sulfate, the
Schiff reagent, and with 2-hydroxy-3-naphthoic acid hydrazide (Ashbel and Seligman, '49 ; Seligman and Ashbel, '52).
Cholesterol was demonstrated histochemically by the Schultz
('24) method, and phospholipids by the acid hematein technique of Baker ( '46) after fixation in calcium-formaldehyde.
For purposes of comparison, the ovaries of several rats were
fixed in 80% alcohol or 10% formalin and treated as were the
hamster ovaries to demonstrate alkaline phosphatase, esterase
activity and lipids.
One ovary from each of 21 immature female hamsters,
ranging from 14 to 46 days of age, was fixed in Bouin's fluid,
sectioned serially, and stained with hematoxylin and eosin.
Calculation of follicular and oocyte size was performed by
measuring, with an ocular micrometer, the diameter of oocytes
and the total diameter of 400 follicles in the adult, Bouinfixed ovaries. Each recorded diameter was the average of
two maximum measurements at approximate right angles to
one another; oocytes or follicles which were markedly oval
in shape or distorted were not measured. With each oocyte and
follicle measured, the number of layers of granulosa and
theca interna cells was recorded also. The measurements of
oocytes and follicles were used to calculate regression equations, which describe mathematically the growth of the follicle
relative to that of the oocyte (Brambell, '28; Snedecor, '53).
OBSERVATIONS
The growth of fiormal foZZicZes. The general pattern of follicular growth and the oocyte-follicle regression plot (fig. 1)
are similar t9 other placental mammals studied in this way.
The only marked, but anticipated, variation is the size of the
follicle at ovulation. I n table 1 are listed the size of oocyte
and follicle in relation to the number of layers of granulosa
and theca interna cells. The growth of a follicle is divided
into two phases, with phase I representing the time during
682
KARL M . KNIGGE AND JAMES H. LEATHEM
which the oocyte increases to its maximum diameter. At the
~ diameter,
end of phase I, the hamster oocyte is 65 to 6 8 in
with a corresponding average follicle size of 165 p (fig. 1).Durring phase 11,the oocyte does not increase in size appreciably
while the follicle continues t o increase to the vesicular stage.
Antrum formation begins when the follicle is 320 to 350 p in
diameter, and the fully mature follicle, affected by the preovulatory stimulus, attains a size of 650 to 6 8 0 ~ .For phase
I, a b value for the regression equation is 0.39 (y =14.5
+
a
w-
c
L
-
&70
0
0
ILW-
0
-
30-
c
w
-
5
10-
z
0
I
I
I
I
1
I
I
I
I
I
I
I
I
I
TABLE 1
T h e relation between the size of oocyte and follicle and the number of layers
of granulosa and theca interna cells
DIAMETER
OF
OOCYTE,
RANGE I N /L
DIAMETER
OF
FOLLICLE,
RANGE I N p
LAYERS
OF
GRANULOSA
CELLS
LAYERS
OF
THECA INTERNA
OELLS
12-20
22-34
35-46
47-65
68-70
68-70
70-72
12-20
30-65
65-105
105-1 65
165-330
330-550
550-680
0
1
2
3-4
5-9
0
0
small antrum
large antrum
0-1 (incomplete)
1
1-3
2-3
3-4
683
FOLLICULAR G R O W T H A N D ATRESIA
+
0.387~).For phase 11, the regression equation is y = 69.4
0.0025~. Table 2 lists the a and b values for phase I and I1
of follicular growth obtained by other investigations in several
placental mammals, and indicates that corresponding figures
calculated for the hamster fall into the same pattern and are
in closest agreement with values observed in the rat. FolTABLE 2
+
Compilation of the a, b values for the regression equation y = a
bx, describing
the two phases of follicular growth in various placental mammals
PHASE I
P H A S E I1
SPECIES
INVEBTIGATOR
a
b
a
b
Bat (Rhinolophus)
7.4
0.414
48.7
0.056
Matthews,
'37
Shrew (Sonex areneus)
1.2
0.64
67.26
0.013
Brambell,
'35
Shrew (Sonex minutus)
4.97
0.49
50.84
0.074
Brambell and
Hall, '36
Mouse
5.79
0.50
69.54
0.0025
Brambell,
Ferret
9.22
0.58
108.37
0.0030
Parkes, '31
Rat
17.73
0.29
63.11
0.0017
Parkes, '31
Rabbit
15.13
0.47
82.38
0.0105
Parkes, '31
Pig
35.22
0.14
74.95
0.0049
Parkes, '31
6.73
0.47
81.40
0.0016
Green and
Zuckerman,
Rhesus monkey
'28
'47
Baboon
15.98
0.37
82.43
0.0024
Zuckerman and
Parkes, '32
Human
10.28
0.59
75.11
0.0010
Green and
Zuckerman, '51
licular size at the end of phase I and at antrum formation are
in close agreement also with corresponding measurements of
follicles in the rat ovary (Parkes, '31).
Mitochomdria. I n sections of ovaries fixed in Regaud's
fluid and stained with iron hematoxylin, mitochondria of the
oocyte are readily visualized and present significant differences in size and distribution depending upon the size of
684
KARL M. KNIGGE AND JAMES H. LEATHEM
the oocyte. I n small oocytes, surrounded by a single, incomplete layer of flattened granulosa cells, mitochondria are
aggregated in a crescent-shaped cluster, capping the nucleus
at one pole of the cell (fig. 21). I n larger oocytes, measuring
up to 60p in diameter, the mitochondria are conspicuously
large, round and deeply stained (figs. 22, 23). They are
distributed in the cytoplasm in varying patterns, the most
significant being either clustered about one pole of the nucleus
(fig. 23), or distributed uniformly in a narrow zone a t the
periphery of the oocyte (fig. 22). As the oocyte attains its
maximum size (65 to 6 8 ~ the
) mitochondria become smaller,
stain lighter, and are distributed uniformly throughout the
cytoplasm (fig. 24). Frequently the small mitochondria are
arranged in chains, giving them a filamentous appearance.
These characteristics of the mitochondria are present in all
normal oocytes of larger pre-antrum and vesicular follicles.
The acquisition and progressive increase in thickness of the
zona pellucida is demonstrated clearly in iron hematoxylinstained sections (figs. 21 to 24), this membrane being seen
first when the oocyte is approximately 30 p diameter.
I n granulosa cells of normal follicles, mitochondria are
markedly smaller, fewer in number, and less intensely stained
than are those in the oocyte. No significant difference is apparent between the mitochondria in granulosa cells of preantrum and vesicular follicles.
The theca interna is organized from the continuous ovarian
stroma prior to the beginning of antrum formation, but the
complete differentiation of the constituent cells of this layer
proceeds slowly as judged by study of mitochondria1 preparations. Follicles as large as 165 p may have only a single
layer of cells (table l), their mitochondria being small and
relatively few in number. Follicles of 165 to 330 p in diameter
show the greatest range in the amount of theca present (table
l), with the mitochondria of these cells varying widely in
size, number, and staining intensity. I n some cells, spherical
mitochondria are numerous and deeply stained, while in many
cells the mitochondria appear lightly stained and are few
FOLLl’CULAR GROWTH A N D ATRESIA
685
in number. Only in follicles measuring 600 to 700 p in diameter
are the theca cells uniformly large, polyhedral in shape, and
crowded with densely staining mitochondria. Vacuolation of
thecal cells is observed rarely, being seen clearly only in the
largest, pre-ovulatory vesicular follicles. The slow differentiation of the theca interna of follicles in the hamster ovary is
in contrast to the condition observed in the rat, where the
theca interna begins to exhibit many of the histochemical reactions characteristic of steroid-producing tissue as early as the
stage of antrum formation (Dempsey and Bassett, ’43).
The stroma of the hamster’s ovary is composed of characteristic whorls of spindle-shaped cells as well as numerous
clusters of fairly large rounded cells. I n the latter cells
particularly, mitocondria are surprisingly numerous and intensely stained.
Alkalime phosphatuse. I n the hamster ovary, periods of incubation up to 24 hours with sodium glycerophosphate fail
to reveal significant alkaline phosphatase activity in cytoplasm
or nuclei of the germinal epithelium, stroma, the walls of
blood vessles o r lymphatic sinusoids. The theca interna, the
walls of blood vessels in it, and the granulosa cells of normal
follicles exhibit intense enzyme activity after one to three
hours of incubation (figs. 2, 17). Recently formed corpora
lutea present a moderate activity. The marked enzyme activity of granulosa cells of normal follicles is in contrast to
the granulosa ceils of follicles in the rats’ ovary, which are
devoid of enzyme activity (fig. 3). Using the calcium-alphanaphthyl phosphate method, staining of nuclei is reduced
markedly but the localization of enzyme activity is otherwise
similar to that revealed by cobalt sulfide.
Esterase. The ovarian stroma and the germinal epithelium
of the hamster’s ovary exhibit marked non-specific esterase
activity (fig. 5). A zone of the ovarian stroma immediately
beneath the germinal epithelium is conspicuously free of
enzyme activity (fig. 5). The granulosa cells of normal follicles, the theca interna, the oocyte, corpora lutea, interstitial
686
KARL M. KNIGGE AND JAMES H. LEATHEM
tissue and the endothelium of blood vessels present faint or
negative reactions for this enzyme.
Lipids. I n formalin-fixed ovaries, the theca interna of large
vesicular follicles, corpora lutea, and clusters of interstitial
cells present positive lipid reactions when stained with Nile
blue sulfate or Sudan black B (fig. 7 ) . The same structures
are reactive with the Schiff reagent or 2-hydroxy-3-naphthoic
acid hydrazide. Because of the prominent mitochondria in
many cells of the ovarian stroma, this tissue is stained
moderately also with aldehyde reagents (fig. 4 ) . Cholesterol,
demonstrated histochemically by the Schultz method, is present in notable amounts only in the granulosa cells of preantrum atretic follicles. Newly formed corpora lutea, clusters
of interstitial cells, and the theca interna of large vesicular
follicles exhibit faint t o moderate reactions f o r this sterol.
Phospholipids, as revealed by the Baker ('46) acid hematein
method, are present as blackened granules in the cytoplasma
of the oocyte, granulosa and theca cells, cells of the ovarian
stroma, corpora lutea, and interstitial tissue. Sections of
ovaries stained with acid hematein (fig. 6) are similar in
appearance to those stained with iron hematoxylin for mitochondria, and it appears that, as in the case of staining of
the ovarian stroma with aldehyde reagents, these bodies are
the major sites of positive reactions for phospholipids in the
hamster ovary. I n the theca interna cells of large vesicular
follicles, particularly, acid hematein reveals the mitochondria
in a manner identical to that observed in Regaud-fixed sections
stained with iron hematoxylin.
Follicular atresia a>ndt h e origin of interstitial tissue
A t r e s i a ( t y p e I ) of p r e - a n t r i m follicles. I n the ovary of
the adult hamster, as in other species, pre-antrum (primary)
and vesicular (Graafian) follicles are continually undergoing
atresia, but the course of degeneration of pre-antrum follicles
is different from that occurring in vesicular follicles. Atresia
of pre-antrum follicles is designated as type I, with representa-
FOLLICULAR GROWTH AND ATRESIA
687
tive stages shown in figures 8 to 11. Although small follicles of
30 to 7 0 p in diameter degenerate in this way, the majority
begin atresia in the range of 95 to 160 p. The first indication
of atresia is seen in the granulosa cells and is evidenced by a
loss of chromophilia and a decrease in cellular density (fig.
8). Initially, the theca interna, composed of fibroblast-like
cells characteristic of follicles of the size, appears unmodified.
Following the initial response of the granulosa cells, degenerative changes appear in the oocyte as it becomes distorted from
its normal spherical shape, with the zona pellucida thickening
markedly and becoming deeply eosinophilic (fig. 9). Two nucleoli are frequently present, but pseudomaturation spindles,
division of the oocyte, or stainable crystalline structures in the
cytoplasm are rare. Division of the oocyte, when it occurs, is
generally associated with larger follicles (150 to 300 p).
Loss of chromophilia of the granulosa cells proceeds until
they are faintly stained, with the cell boundaries of many cells
appearing indistinct and their nuclei small and pycnotic (fig.
9). Other granulosa cells, however, containing clear vesicular
nuclei, enlarge to as much as three times their original size
(figs. 9, 10). Their cytoplasm contains vacuoles which vary
considerably in number and size. Small, yellowish, cytoplasmic granules, which increase in size and number as atresia
progresses, are present also. Following the early changes in
the oocyte and granulosa cells, a compact, theca-like layer of
considerable size appears around the degenerating follicle
(figs. 10, 11, 14, 19). This layer of cells is clearly derived
from the ovarian stroma. The constituent cells are arranged
in somewhat lamellar fashion and are more compact than are
those in the general interfollicular stroma, the individual cells,
however, appearing identical to stromal cells (figs. 9, 10, 11).
There is no evidence, from routine or histochemical preparations, that these cells undergo any kind of transformation.
As atresia progresses and the oocyte degenerates, the enlarged theca regresses until it is indistinguishable from the
surrounding stroma (figs. 11, 18). I n view of the transient
nature of this theca-like layer, because its constituent cells
688
KARL M. KNIGGE AND JAMES H. LEATHEM
do not differentiate into thecal cells, and because of its
eventual regression again to ovarian stroma, it is referred
to as a pseudotheca. The transformed, lutein-like granulosa
cells may remain clustered about the crumpled zona pellucida
or, as is common in smaller follicles, they become dispersed
among the cells of the surrounding stroma. I n the final
stages of atresia, a crumpled zona pellucida, some remnants
of the oocyte, and a cluster of interstitial cells, originating
from granulosa cells, are the only evidence of a former follicle
(figs. 12, 13).
The transformation of granulosa cells into interstitial cells
is apparent also in mitochondria1 and several histochemical
preparations. Concurrent with vacuoles which appear in the
cytoplasm of granulosa cells, mitochondria enlarge and become
prominent, with the cells clearly demarcated from the pseudotheca (fig. 19). As atresia progresses, the mitochondria exhibit increased afiinity for iron hematoxylin, staining intensely
in completely formed clusters of interstitial cells (fig. 20).
Alkaline phosphatase activity, intense in the granulosa cells
of normal follicles, declines somewhat in these cells as their
cytoplasm becomes vacuolated (fig. 14). I n the latter stages
of atresia and in older clusters of interstitial cells, enzyme
activity is quite intense (fig. 16). Phosphatase activity is
present in the pseudotheca of pre-antrum atretic follicles and
frequently also in the surrounding stroma, thus making it
difficult to demarcate clearly the boundaries of these zones
(fig. 14). Enzyme activity, however, disappears completely
from the pseudotheca as the follicle regresses in the latter
stages of atresia. Esterase activity, which is minimal or
absent in the granulosa cells of normal follicles, may be
present in moderate amounts in some of the granulosa cells
of pre-antrum atretic follicles. The intense esterase activity
of the ovarian stroma makes it difficult to follow clearly
enzyme. I n older clusters of interstitial cells, a very coarse,
changes occurring in the granulosa cells with respect to this
red granular precipitate, evidence of esterase activity, is present in many cells.
FOLLICULAR GROWTH AND ATRESIA
689
I n formalin-fixed ovaries, granulosa cells of pre-antrum
atretic follicles and interstitial cells are stained with Sudan
black B (fig. 7), Nile blue sulfate, and present positive reactions for cholesterol. These cells are stained weakly with the
Schiff reagent and with 2-hydroxy-3-naphthoic acid hydrazide.
The appearance of granulosa cells of atretic follicles and interstitial cells, when stained with the Baker method for phospholipids, suggests that, as in the case of ovarian stroma, mitochondria are responsible for the staining with acid hematein.
Atresia ( t y p e II) o f vesicular follicles. Histological and
histochemical changes which occur during atresia of vesicular
follicles are different from those observed in atresia of preantrum follicles. Type I1 atresia represents a true degeneration, with the granulosa cells becoming intensely hyperchromatic and the nuclei and elements of the cytoplasm
eventually coalescing to form blackened globules (fig. 18).
Alkaline phosphatase activity of the granulosa diminishes in
the early stages of atresia (fig. 17) and disappears completely
as the cells degenerate. As with many degenerating cells, the
granulosa cells exhibit much sudanophilic lipid (fig.7), but
cholesterol is absent and the cells stain only faintly with aldehyde reagents. The oocyte becomes deeply stained, but
remains intact for a considerable period of time, while the
stroma surrounding the degenerating follicle is not organized
into a pseudotheca as is the case with pre-antrum atretic
follicles (fig. 18). Cells of the theca interna present no
evidence that they give rise to interstitial tissue (fig. 18).
Alkaline phosphatase activity (fig. 17) and lipids (fig. 7)
disappear from the theca and its constituent cells cannot be
distinguished from stromal cells as infiltration of the follicular
cavity occurs.
An apparently atypical type of atresia is observed in
certain pre-antrum and vesicular follicles. These follicles
contain features characteristic of both types I and I1 atresia,
the theca being moderately enlarged as in type I atresia and
the granulosa cells hyperchromatic and degenerating as in
type I1 (fig. 15). Follicles of this nature are relatively in-
690
KARL 1M. KNIGGE AND JAMES H. LEATHEM
frequent and can be related t o a definite range of follicle sizes.
The smallest are 280 v in diameter and the largest 400 1-1.
Relation of type I atresia t o iHitial ovulation. I n ovaries
of animals 28 days of age o r younger, atresia of pre-antrum
follicles (type I) is not observed. Interstitial tissue is likewise
absent, the hamster ovary thus differing in this respect from
the rat, where primary interstitial tissue (Dawson and
McCabe, '51 ; Rennels, '51) forms a conspicuous component of
TABLE 3
T h e relation between atresia ( t y p e I ) of pre-antrum follicles and initial
ovulation in immature hamsters
AGE O F
ANIMALS,
DAYS
CORPORA
LUTEA
PRESENT
NUMBER O F ATRETIC
FOLLICLES PER OVARY
CONDITION O F
VESICULAR
POLLICLES
PROBABLE
NUMBER
OF QYCLES
Early
Advanced
0
0
none
0
28,28
-
0
0
all atretic
( t m e 11)
0
31, 33, 34
-
112,107,87
10,21,7
all atretic
(type 11)
0
33, 35, 35
-
107,115,110
102,81,88
normal
Initial
ovulation
35, 37, 38
+
90,120,108
193,174,203
normal
131,102,124
200,282,241
normal
102, 94, 89
416,370,420
normal
14,18,20,25
38,38,41
45,46,46
+
+
The order i n which the numbers of atretic follicles is presented corresponds
t o the order of the ages of animals (column 1) in whose ovaries the follicles
were counted.
the ovary as early as 14 days of age. Type I atresia of preantrum follicles, as well as scattered clusters of interstitial
cells, are first observed in the hamster ovary at 31 to 34 days of
age, with initial ovulation occurring at 33 to 35 days of age
(table 3 ) . I n order to determine whether a relationship exists
between type I atresia and initial ovulation, the total number
of atretic pre-antrum follicles in the serially sectioned ovaries
of animals 14 to 46 days of age were counted, the stage of
FOLLICULAR GROWTH AND ATRESIA
691
atresia of each follicle being recorded as either early or
advanced. Atretic follicles in the advanced category are
considered as beginning with those in which the oocyte is
completely degenerated and the pseudotheca is regressing
and difficult to demarcate clearly from the adjacent stroma
(figs. 11, 13). The histology of corpora lutea serves as a
guide in dating initial ovulation, for newly formed corpora
lutea are highly vascular, possess prominent thecal cones of
connective tissue and blood vessels, with the lutein cells being
loosely packed and of variable size. The cavity of a new
corpus luteum persists for about 24 hours. Older corpora lutea
are generally smaller in size and their position is not as
prominent on the surface of the ovary. The lutein cells are
densely packed and of uniform size.
The results (table 3) indicate that type I atresia of preantrum follicles, normally not seen in the ovary of immature
animals, begins several days prior to initial ovulation and then
proceeds regularly thereafter with a relatively constant number of pre-antrum follicles undergoing atresia during each
cycle.
DISCUSSION
The regression equation of normal follicular growth in the
hamster ovary reemphasizes the fact that, as in all mammals
studied, the oocyte attains its maximum diameter long before
the follicle reaches full size (Brambell, '28; Parkes, '31; Green
and Zuckerman, '47). There is considerable evidence that
this biphasic pattern of follicular growth is an expression of
underlying differences in the physiology of young, pre-antrum
and vesicular follicles. I n many animals, including the hamster
(Knigge, '54), f ollicular development can proceed up to
antrum formation in the absence of the hypophysis (Smith,
'30 ; Swezy, '33), although gonadotropins may stimulate it
to some extent (Williams, '44;Paesi, '49). Hisaw ('47) has
summarized the evidence suggesting that a system of selfcontained organizers is responsible f o r events occurring in
early follicular growth. Thus, the oocyte is considered to play
an active role in organizing the granulosa, which in turn is
692
KARL M. KNIGGE AND JAMES H. LEATHEM
responsible f o r differentiation of a theca from surrounding
ovarian stroma. I n the atretic pre-antrum follicle of the
hamster ovary, differentiation of the large pseudotheca from
the surrounding stroma may be an expression of an abnormal
organizing effect of the oocyte.
During phase I1 of follicular growth, the oocyte is considered to be passive (Smith and Engle, '27; Brambell, '28),
further growth being stimulated by hypophyseal gonadotropins. A marked increase in mitotic activity of the granulosa
cells (Schmidt, '42; Lane and Davis, '39) and the beginning
of antrum formation (Lane and Greep, '35) are evidence of
a turning point in the physiology of the follicle. The differentiation of a theca interna and secretion of estrogen by it
are believed to play a major role in the ability of the follicle
to respond in a normal fashion to gonadotropins (Hisaw, '47).
Certain features of the mitochondria in the hamster oocyte
indicate also the biphasic nature of follicular growth. During
phase I, the oocyte is growing rapidly and its mitochondria
are large, deeply staining and distributed in the cytoplasm in
varying patterns. It is probable that the size of the mitochondria and their diverse patterns of distribution at this
time are related to the processes of growth of the oocyte,
but the possible relationship to secretory activity should not
be overlooked, since these organelles are known to be concerned with this function. At the end of phase I of follicular
growth, the mitochondria become smaller, lightly stained, and
distributed uniformly in the cytoplasm, thus reflecting a decline in metabolic activity when the oocyte has nearly attained
its maximum size. These observations on the mitochondria of
the hamster oocyte are at variance somewhat with those of
Levi ('51) and Van der Stricht ('23), whose work forms
the basis of most of our current notions on this subject.
During the period of growth of the oocyte (phase I), these
authors described a characteristic distribution of the mitochondria, being in a dense mass concentrated about one pole
of the nucleus. I n contrast, the distribution of mitochondria
in a narrow zone at the periphery of the oocyte was considered
FOLLICULAR GROWTH AND ATRESIA
693
characteristic of the final stages of growth, with this condition
persisting throughout the period of maturation and up to
to the time of fertilization. Also, the difference in size and
intensity of staining between mitochondria of pre-antrum and
vesicular follicles was not described.
Another indication of the differences in physiology of
normal pre-antrum and vesicular follicles is demonstrated in
the hamster ovary by the events occurring when these follicles
become atretic. During atresia of pre-antrum follicles, many
granulosa cells are transformed into interstitial tissue,
whereas in atresia of vesicular follicles the entire granulosa
degenerates. I n most species of animals the ovarian interstitial tissue can be traced without difficulty to the theca
interna of atretic vesicular follicles (Kingsbury, '39 ; Dawson
and McCabe, '51). The hamster, however, is not nnique in
deriving interstitial tissue from granulosa cells, for this has
been described as occurring in the dormouse (Velloso de
Pinho, '25), mouse (Brambell and Parkes, '27), some catarrihine monkeys (Corner, '28) and the guinea pig (Hartman
and Kirgis, '38). Dawson and McCabe ( '51) have described
the origin of primary interstitial tissue from granulosa cells
in the immature rat ovary.
The conditions which lead to follicular atresia are largely
unknown. Hisaw ('47) suggests that atresia results because
of a failure of proper differentiation of the theca interna,
due to inadequate stimulation by LH. On the other hand,
Burrows ('49) feels that a high level of LH at the time of
ovulation is the cause of atresia of small follicles. That atresia
of pre-antrum follicles may be related to the levels of gonadotropins is shown in the present study by the observation that
it commences in immature animals when hypophyseal gonadotropins reach sufficiently high titers to cause initial ovulation.
Several lines of evidence suggest that LH is the gonadotropin
which plays a role in atresia of pre-antrum follicles. Greep,
van Dyke and Chow ('42)' in studying the effects of pure
L H on the rat ovary, noted that small follicles may be converted to lutein bodies, although the origin of the individual
694
KARL M. KNIGGE AND JAMES ET. LEATHEM
lutein-like cells was not considered. The probable influence of
LH in causing the conversion of granulosa cells to lutein-like
cells during atresia is supported also by the fact that this
conversion is a normal response of granulosa cells after
ovulation. The implication of LH as a factor in atresia of
pre-antrum follicles, however, does not explain why some
follicles degenerate and others remain normal.
SUMMARY
Quantitative aspects of normal follicular growth, ovarian
cytology and histochemistry, and atresia of pre-antrum and
vesicular follicles were studied in immature and adult hamsters.
The regression equations, relating growth of the oocyte to
growth of the follicle, are similar to those observed in other
mammalian forms. I n the hamster, the equations for phases
I and I1 are y = 14.5 0.387~and y =69.4 0 . 0 0 2 5 ~respec~
tively. The biphasic nature of follicular growth is reflected
also in the mitochondria of the oocyte, these organelles differing markedly in phases I and I1 with respect to size, staining intensity and distribution.
Normal granulosa cells exhibit intense alkaline phosphatase
activity, faint nonspecific esterase activity, and negative
reactions for sudanophilic lipid, aldehydes, o r cholesterol.
Mitochondria in these cells are small, being demonstrated by
Regaud’s iron hematoxylin and possibly by the Baker acid
hematein method for phospholipids.
The granulosa cells of atretic vesicular follicles undergo
a degeneration (type 11),leading to loss of alkaline phosphatase activity, positive reactions for sudanophilic lipid, and
eventual fragmentation and dissolution of the cells. During
atresia of pre-antrum follicles, the granulosa cells hypertrophy, become vacuolated, and exhibit positive reactions for alkaline phosphatase, lipids, and cholesterol. Their mitochondria enlarge and stain intensely. These transformed
granulosa cells are the source of interstitial tissue in the
hamster ovary. Atresia of pre-antrum follicles and interstitial
+
+
’
FOLLICULAR GROWTH AND ATRESIA
695
tissue are both observed first at the time of initial ovulation.
The possible role of LH in causing atresia is discussed.
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FOLLICULAR G R O W T H A N D ATRESIA
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Proc. Zool. SOC.
PLATES
PLATE 1
EXPLANATION OF FIGURES
Ovaries for all figures (2 to 24) are from adult hamsters, except figure 3 which
is from a rat.
Fixed in cold 80% alcohol and stained for alkaline phosphatase. Moderate
enzyme activity is present in the theca interna, while the stroma is negative.
Intense enzyme activity in the granulosa cells is in contrast to that seen in
the rat (fig. 3). X 50.
I n the rat, the ovarian stroma, endothelium of blood vessels, and thecae
internae exhibit alkaline phosphatase activity while granulosa cells are negative. Fixed and prepared as in figure 2. X 35.
Fixed in 10% formalin and stained with 2-hydroxy-3-naphthoic acid hydrazide. The ovarian stroma, as well as thecae internae and interstitial tissue
are reactive. X 35.
Fixed in cold 80% alcohol and stained for nonspecific esterase. The stroma
exhibits intense enzyme activity, except i n an area immediately beneath the
germinal epithelium, which is positive also. Some esterase activity is present
in the granulosa. X 90.
Fixed in calcium-formaldehyde and stained by the Baker method for phospholipids, which appear uniformly distributed throughout the stroma. The
theca interna of pre-antrum follicles is stained similarly, while the granulosa
is lighter. X 180.
Fixed in 10% formalin and stained with Sudan black B. I n the lower right,
sudanophilic lipid is present in the theca interna but absent from the granulosa of a normal, large vesicle follicle. Above, the granulosa cells of an atretic
vesicular follicle contain large quantities of lipid, the theca interna being
inconspicuous or absent. This follicle is surrounded by clusters of interstitial
tissue. At the pointer is a crumpled zona pellucida, surrounded by lipidcontaining granulosa cells of a pre-antrum atretic follicle. The pseudotheca
around this atretic follicle can still be distinguished. X 250.
700
PLATE 1
FOLLICULAIt GROWTH AND ATRESIA
K . hl. KNICIGE AND J. H. LEATHEM
701
PLATE: 2
EXPLANATION OF FIGURES
Figures 8 to 13, showing progressive stage in atresis of pre-antrum follicles;
ovaries fixed i n Bouin’s fluid, sectioned at 7 p nnd stained with hematoxylin and
eosin. X 235.
8
Small type I atretic follicle, showing the earliest changes in the granulosa
cells, due t o a loss of eosinophilia, greater variation in nuclear size, and
slight hypertrophy.
9
Type I atretic follicle. The zona pellucida becomes prominent and degcricrativc
changes appear in the oocyte. Granulosa cells, in the process of transformation, are larger than normal and lightly stained because of increased vacuolation.
10
Advanced type I atretic follicle, several sections away from the oocyte, showing
the transformed granulosa cells in a compact cluster surrounded by a prominent pseudotheca.
11 Advanced type I atretic follicle. Transformed granulosa cells (interstitial
cells) a r e becoming dispersed. The pseudotheca, composed of stromal cells
more densely packed than normal, blends imperceptibly with the stroms
adjacent to it.
12
Border of ovary, showing a characteristic peripheral distribution of large
clusters of interstitial cells. Between the interstitial cells and the germinal
epithelium is the dense area of the stroma which is free of esterase activity
(fig. 5).
13
Enlarged, coiled, and deeply eosinophilic zonae pellucidae persist in thc stroma
f o r a considerable period of time as evidence of type I atresia.
702
FOLLICULAR QROWTH .4ND ATRESIA
PLATE 2
K . M. KNIGGE AND J. H. LEATHEM
703
PLATE 3
EXPLANATION OF FIGURES
Ovaries f o r figures 14, 36 and 1 7 are fixed in cold SO% alcohol and stained for
alkaline phosphatase. Those f o r figures 35 and 18 a r e fixed in Bonin’s fluid and
stained with hematoxylin and eosiii (fig. 15) and Masson (fig. 18).
14 Pre-antrum atretic follicle. The granulosa cells show a decline in alkaline
phosphatase activity because of marked cytoplasmic vacuolation. The pseudothcca exhibits some enzyme activity, and in some places is seen t o be con.
tinuous with the general stroma. X 200.
15
Atretic follicle showing pycnosis and dissolution of the granulosa cells, c h a r
acteristic of type I1 atresia, and pseudotheca formation which is seen in
type I atresia. Divided oocytes are frequently associated with this type of
atresia. X 235.
16 Clusters of interstitial tissue, with uniformly small cytoplasmic vacuoles and
marked alkaline phosphatase activity. X 200.
17
Large vesicular atretic follicle. Compared with the granulosa cells of normal
follicles below, those of the atretic vesicular follicle are almost devoid of
enzyme activity. The theca interna is likewise losing its enzyme activity and
i n several places cannot be demarcated from the stroma. x 200.
18
Above, a n atretic vesicular follicle showing the granulosa cells undergoing
fragmentation and dissolution. Below a pre-antrum atretic follicle showing
the granulosa cells with vesicular nuclei and lightly staining, vacuolated
cytoplasm. X 350.
704
FOLLICULAR GROWTH AND ATRESIA
PLATE 3
I<. M. KNIGGE AND J. H . LEATHEM
705
PTJATE 4
EXPLANATION O F FIGURES
Ovaries f o r figures 19 to 24 werr fixed in Regaud’s fluid, sectioned at 3 or 5 p ,
and stained with iron hematoxylin.
19
Type I atretic follicle. Granulosa cells are clearly delineated from the large
pseudotheca and contain numerous mitochondria which arc barely visible.
The oocyte is distorted from i t s normal spherical shape and its mitochondria
are aggregated in several dense clusters. X 180.
20
Cluster of newly-formed interstitial tissue. Mitochondria in these cells exhibit
an exceeding strong affinity f o r iron hematoxylin, staining even after differentiation of the iron hcmatoxylin has been carried out to decolorizc the strorna
completely. A portion of the zona pellucida, also decolorized, is present along
the upper margin of the cluster. X 350.
21
Two primary follicles, with single layers of flattened granulosa cells. I n the
follicle at the left, the oocyte measures 1 8 in~ diameter and contains mitochondria grouped i n a crescentic cluster capping the nucleus. X 1300.
22
Oocyte, measuring 45 p in diameter, of a pre-antrum follicle. The mitochondria
are large, intensely staining, and located in a zone at the periphery of the
oocyte. The zona pellucida is stained also. x 700.
23
Oocyte, measuring approximately 50 p in diameter, of a pre-antrum follicle.
The majority of the mitochondria a r e aggregated about one pole of the
nucleus, while some a r e peripherally located. X 700.
24
Oocyte, 6 5 p in diameter, from a follicle measuring 2 7 0 ~ . During phase I1
of follicular growth, mitochondria of the oocyte arc small, lightly stained,
and distributed uniformly in the cytoplasm. The zona pcllucida is thicker
than in pre-antrum follicles. The nucleus, whose membrane is barely visible,
is approximately the same size in all of the oocytes. x 700.
706
PLATE 4
FOLLICULAR GROWTH AND ATRESIA
X. M . KNIGGE AND J. H . LEATIIIM
707
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