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Follicular histology and physiological correlates in the preovulatory hamster.

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Follicular Histology and Physiological Correlates
in the Preovulatory Hamster
REID L. NORMAN' AND GILBERT S. GREENWALD
Departments of Obstetrics and Gynecology and Anatomy, University of
Kansas Medical Center, Kansas City, Kansas, 66103
ABSTRACT
The surge in plasma luteinizing hormone (LH) in the proestrous
hamster begins at 1430 (Turgeon and Greenwald, '72) and this is followed by
a sharp increase in follicular and interstitially derived progesterone at 1500
(Norman and Greenwald, '71). The purpose of the present study was to relate
various histologic events in the ovary of the preovulatory hamster to these physiological changes, dating from the LH increase between 1430 and 1500. The earliest
maturational changes in the oocyte occurred at 1600 as the nuclear membrane
began to disappear, correlating with an increase in the number of pycnotic nuclei
in the surrounding cumulus cells and an abrupt reduction in mitotic activity in
the membrana granulosa. It is possible that the latter event is related to increased
progesterone secretion by the follicle at 1500. The most rapid increase in follicular diameter occurred between 2000 and 2200 - five to seven hours after the
LH surge and was accompanied by a pronounced stromal edema especially of the
medullary portion of the ovary. During this same time period, meiosis proceeded
to the metaphase stage and the cumulus cells began to disperse to form the corona
radiata. Of the eight hours required for the first meiotic division, four to six hours
are spent in metaphase. The majority of animals (75% ) ovulated by 0100 and
all animals ovulated by 0200. Therefore, ovulation occurred 10 to 11 hours after
the LH surge at 1500.
Ovaries from eight groups of animals
(4 animals/group) were collected at two
hour intervals beginning at 1000 on day
4. With the animal under ether anesthesia,
one ovary from each animal was removed,
trimmed and placed in Bouins' fixative for
at least 48 hours. Hematoxylin and eosin
preparations were made from ovaries
serially sectioned at 10 p. Follicular size
was determined by measuring the two diameters at right angles to each other in the
largest section of the follicle including the
nucleus of the oocyte. The third diameter
was measured by counting the number of
MATERIALS AND METHODS
sections in which granulosa cells were
Adult female hamsters with at least present. Follicular volume was calculated
three consecutive four day estrous cycles using the formula 4 / 3 d where r is onewere maintained on a 14:lO light-dark half the average of the three diameters.
schedule with the lights on from 0500 to Only the four largest, healthy antral fol1900 (CST). Day 1 designates the morn- licles were measured in each ovary.
ing of a copious vaginal discharge, indicative of ovulation during the previous night.
Received Oct. 1, '71. Accepted Dec. 17, '71.
Present address: Department of Anatomy, School
The afternoon of day 4 corresponds to of 1Medicine.
The Center for the Health Sciences, Los
proes trus.
Angeles, California 90024.
Follicular growth has been previously
described during the estrous cycle of the
hamster (Greenwald, '60; '61). The present paper is concerned with the rapid
changes occurring in antral follicles during
proestrus and the purpose is to correlate
ovarian histology with other parameters
established in this laboratory for the
hamster, such as the duration of the critical period (Greenwald, '71 ), ovarian progesterone secretion (Norman and Greenwald, '71), and plasma levels of LH
(Turgeon and Greenwald, '72).
ANAT. REC., 173: 95-108.
95
96
REID L. NORMAN AND GILBERT S. GREENWALD
frequent in the cumulus cells than in the
membrana granulosa.
Histologic changes were not definitely
evident in the cumulus cells until 1600
when pycnotic nuclei increased in numbers (fig. 2). Although some pycnotic
RESULTS
nuclei are present at 1400, there is an upFollicular changes. The most obvious swing in degenerating cumulus cells at
change during proestrus was the growth 1600 (fig. 8). By 2000, the cumulus cells
of the antral follicle (table 1). Between started to disperse leaving behind a com1000 and 1600, follicular diameter and pact unilaminar ring surrounding the
volume were essentially unchanged. How- oocyte (fig. 3 ) . At this time, granuever, from 1600 to 2200, follicular volume losa cells bordering on the antral cavity
increased rapidly, and was 42% larger just also began to slough off into the liquor folprior to ovulation than at 1600. There was liculi (fig. 3 ) . The follicular changes at
no significant increase in the size of the 2000 coincided with a massive stromal
follicle between 2200 and 2400. The in- edema which was most evident in the
crease in follicular size was attributable to medullary portion of the ovary.
The oocyte was essentially isolated from
the content of liquor folliculi (see figs. 1-4).
An abrupt decrease in mitotic activity the membrana granulosa by 2400 and
was found in both the cumulus oophorus there were obvious changes in the consistand membrana granulosa between 1400 ency of the liquor folliculi enmeshed in the
and 1600 (table 2). Mitoses were rarely corona radiata (fig. 4). The basement
encountered thereafter, but they were more membrane separating the membrana granulosa from the theca interna was still intact at 2200. By 2400 tufts of thecal cells
TABLE 3
began
to invade the granulosa layer folFollicular growth during proestrus
lowing the dissolution of the basement
membrane; this event preceded ovulation
Follicular
Follicular
Time
diameter
volume
by 1 to 2 hours (see below).
( 1 0 6 ~ 3 +- SE
( f i +. SE)
Oocyte changes. Up to 1400, all oocytes
1000
96.1 * 3.2
5 6 7 r 6 . 1 (20)
were nucleated and contained several nu95.6 * 2.9
1200
566k6.0 (20)
cleoli of varying sizes (figs. 5-7). At 1600,
1400
98.0-t 3.0
57126.1 (16)
101.1C2.9
1600
57725.6 (16)
the nuclear membrane and nucleoli be109.5 k 4 . 7
1800
5921-8.5 (13)
came much more indistinct and the chro122.8 2 5.1
2000
6141-8.0 (18)
matin began to condense (fig. 8 ) . The nu142.0 2 5.5
2220
645k9.0 (19)
clear membrane disappeared by 1800 and
1 4 3 . 0 t 5.0
2400
6471-7.5 ( 1 7 )
75% (15/20) of the nuclei were in pro1 Numbers in parentheses are the number of follicles
phase I (fig. 9 ) ; the remaining 25% had
measured in that group.
Mitotic activity in the membrana granulosa and cumulus oophorus was determined by counting the number of mitotic
figures in the section of the follicle containing the nucleus of the oocyte.
TABLE 2
Mitotic activity in t h e m e m b r a n a granulosa a n d c u m u l u s oophorus during proestrus 1
Granulosa cells
~i~~
No. positive 2 foll.
Total no. foll.
Cumuius oophorus
Mean no.
mitoses
S.E.)
(*
~
1000
1200
1400
1600
1800
2000
2200
2400
12/12
12/12
12/12
0/10
1/10
6/12
6/11
3/12
~~~
5.7e0.37 (12)
3.6 4 0.33 (12)
4.8* 0.36 (12)
- 0-
1.0
2.020.25
1.520.22
1.0
( 1)
( 6)
( 6)
( 3)
No. positive 2 foll.
Total no. foll.
Mean no.
mitoses ( -e S.E.)
~~
12/ 12
9/ 12
11/12
2/10
6/10
11/12
8/11
3/12
2.8 2 0.39
2.0C0.37
2.520.43
1.5
1.7e0.33
2.1 % 0.34
1.8e0.24
1.340.33
(12)
( 9)
(11)
( 2)
( 6)
(11)
( 8)
( 3)
1Mitotic figures were counted only in the section of the follicle containing the nucleus of the
oocyte.
2 Positive follicles contain at least one mitotic figure.
~
97
OVARIAN HISTOLOGY: HAMSTER
advanced to the metaphase stage. During
the next four hours (1800-2200) mitotic
activity was arrested at metaphase I (figs.
10-11). At 2000, 23% (5/22) of the ova
had progressed to anaphase I and at 2400
all 21 ova observed were in telophase I or
had already extruded the first polar body
(fig. 12).
The lights are turned off in our hamster
colony at 1900. The oocyte events can
therefore be expressed in terms of hours
relative to the onset of darkness. The first
nuclear changes were observed three hours
before; metaphase I lasted from one hour
before to three hours after; telophase was
completed five hours after the onset of
darkness.
By 1800, 87% of the hamsters in our
colony show behavioral estrus (Norman
and Greenwald, '71). Using the onset of
heat as an endpoint, the first nuclear
changes occurred four hours before; prophase, two hours before; metaphase, from
the onset to four hours after; telophase,
six hours after the onset of behavioral
estrus.
Time of ovulation (table 3). The earliest
ovulation, determined by flushing the oviducts, was at 2400 when one hamster ovulated one egg. By 0100 of day 1, 75% of
the animals ovulated; and all animals examined at 0200 had a full complement of
tuba1 ova. Ovulation therefore occurred
nine to ten hours following the first histologic signs of maturation in the oocyte.
DISCUSSION
Figure 13 correlates the present histologic findings with various important
physiological events in the proestrous
hamster. The critical period - when hypothalamic activation affects the pituitary
- extends from 1300 to 1415 (fig. 13; 1 ) .
This is followed by an ovulatory surge of
LH beginning at 1430 with high peripheral
levels apparent by 1500 (fig. 13;2). In the
following discussion various events will be
dated arbitrarily from the increased LH
present at 1500. Hamsters hypophysectomized after 1530 ovulate in appreciable
numbers (75% ) and 100% will ovulate
following pituitary extirpation at 1630
(fig. 13;3) despite the fact that high peripheral levels of LH are maintained until
1730 (Turgeon and Greenwald, '72). The
half-life of LH in hamster plasma is approximately 33 minutes (Turgeon and
Greenwald, '72). Collectively, this suggests that once the ovary is exposed to critical levels of LH for one-half to one and
one-half hours, the processes culminating
in ovulation are initiated and the continued
presence of the hormone is no longer required. The alternative possibility is that
sufficient LH to exert physiological effects
is tightly bound to the follicles and therefore persists even after hypophysectomy.
As shown in the present study, rapid
growth of the preovulatory follicle of the
hamster occurs between 1800 and 2200
with an initial increase beginning at 1600.
The rapid growth is initiated five hours
after the start of the LH surge (fig. 13;4).
The diameter of the follicle just prior to
ovulation is 647 which is similar to the
650-680 sL value reported by Knigge and
Leathem ('56). Greenwald ('60, '61)
measured follicular growth throughout the
hamster estrous cycle. On the morning of
day 4 the volume of the six largest follicles was 77 X 106p3.This is somewhat
less than the 96 X 106p3 reported in the
present study possibly because we measured only the four largest follicles. The
preovulatory follicle in the hamster at
2400 (143 X lOapS)is considerably smaller
TABLE 3
Spontaneous ovulation in the cyclic hamster
Time
Number of animals
ovulating
Average number
of ova
found per animal
1
Number of ova found in
individual animals 2
1
2
3
4
5
6
1
0
10
11
0
1
11
9
0
0
11
11
~~~
2400
t
0200 d a y 1
0300
0100
1/6
4/6
6/6
6/6
1
8
10.7
11.0
1 Lighting schedule was 14: 10, light-dark.
2 Ova were obtained by flushing the oviducts.
0
0
0
8 1 3 1 1
10
10
12
11
13
11
98
REID L. NORMAN AND GILBERT S. GREENWALD
than the size reported for the rat: 267 X
lo6$ (Boling, Blandau, Soderwall and
Young, '41) and the guinea pig: 900 X
lOeP3 (Myers, Young and Dempsey, '36).
The rapid preovulatory growth of the follicle is probably due to the increase in
liquor folliculi (Blandau, '66). In the rabbit, enzymatic depolymerization of mucopolysaccharides in the follicular fluid
occurs just prior to ovulation (Zachariae,
'59). A similar change occurs in the liquor
folliculi of mouse follicles in which ovulation is impending (Byskov, '69). In the
rabbit, the same enzyme is apparently responsible for an increase in permeability
of the blood-follicular barrier. The hypertonicity of the follicular fluid due to the
dispersion of large mucopolysaccharide
polymers would tend to draw fluid across
the weakened blood-follicular barrier.
In the hamster, mitotic figures in the
granulosa cells decrease sharply at 1600
which is one hour after the LH surge (fig.
13;6). Similarly, when 3H thymidine is
perfused directly into the ovary of estrous
rabbits mitotic activity is apparent in maturing follicles but not in large vesicular
follicles. One hour after mating or the
administration of human chorionic gonadotropin, there is decreased labelling of
granulosa cells even in the maturing follicles (Boucek, Telegdy and Savard, '67).
Using the colchicine technique, Bullough
('42-'44) found in the mouse ovary that
vesicular follicles on days 2 and 3 of the
cycle show most of the labelled cells situated around the antral cavity and in the
cumulus but by proestrus mitotic figures
have almost vanished from the membrana
granulosa. This finding has been confirmed in the cycling mouse by the use of
autoradiography (Pedersen, '70) ; the most
peripherally located cells of the membrana granulosa cease to proliferate and
there is a progressive decline in the labelling index from estrus through proestrus.
The sharp drop in mitotic figures at
1600, encountered in the present study, is
related temporally to the onset of the LH
surge (fig. 13;2); and it appears likely
that increased steroidogenesis by the follicle may be responsible for the cessation
of mitosis. There is a significant increase
in follicular progesterone between 1400
and 160.0 (fig. 13;5) paralleling an even
more dramatic increase in interstitially
derived progesterone (Norman and Greenwald, '71). Several lines of evidence show
that the increase in ovarian progesterone
is a response to LH and not to FSH (Norman and Greenwald, '71 ). Histochemical
studies of the hamster ovary indicate that
3p-hydroxysteroid dehydrogenass increases
in the granulosa cells on the afternoon of
proestrus (Wingate, '70; Blaha and Leavitt,
'71); this is also consistent with a changing role for the membrana granulosa in
the preovulatory follicle.
In the present study, maturation of the
nucleus of the oocyte begins about one
hour after the increase in peripheral levels
of LH (fig. 13;7). Edwards and Gates
('59) found that nuclear maturation in the
mouse oocyte commences about two hours
following gonadotropin (HCG) stimulation. The timing and progression of nuclear maturation of the hamster oocyte is
similar to that observed in the mouse
(Edwards and Gates, '59). Within two
hours following the disappearance of the
nuclear membrane, all hamster oocytes had
progressed to metaphase I and remained
in this phase for four to six hours. Only
a few nuclei were observed in anaphase,
presumably because it is a very rapid
stage. Telophase figures or extruded polar
bodies were seen in all follicles at 2400.
Ward ('48) reported that hamster ova are
discharged at any stage from anaphase I
to metaphase I1 contrary to other species in
which maturation proceeds to the second
metaphase where it is then arrested (rat:
Odor, '53; Mandl, '63; rabbit: Chang, '55;
mouse : Donahue, '58). Since ova were not
examined after 2400 in our study, we do
not know how far the oocytes progressed
past the first maturation division.
The first histologic signs of oocyte maturation were evident at 1600 coinciding
with increased numbers of pycnotic nuclei
in the cumulus cells (fig. 13;s). It appears likely that these two events are both
linked to the release of LH occurring at
1500. By 1800 the cumulus cells began to
disperse and the rapid growth phase of the
follicle commenced at 2000. This coincides
with a very conspicuous interstitial edema
in which the tissue has a waterlogged appearance. Thus, five hours after the onset
of the LH surge there is histologic evidence
OVARIAN HISTOLOGY: HAMSTER
of maximal ovarian blood flow. Foote and
Thibault ('69) have shown that nuclear
maturation of the oocyte depends upon
physiological or mechanical isolation of the
oocyte from the granulosa cells. In the
hamster, a physiological mechanism is implicated since physical isolation of the
oocyte does not occur until 2400.
Newly formed corpora lutea in the
hamster on the morning of day 1 are 546
in diameter and are highly compact with
very little trace of an antral cavity (Greenwald, '68). Their diameter is therefore
slightly less than the preovulatory follicle
(647 p ) and they are distinctly different
in histologic appearance from the fresh
corpora lutea of the rat. This suggests that
the morphological changes unfolding during ovulation may be quite different between the two species.
Ovulation occurred as early as 2400 and
was completed in all animals by 0200 in
agreement with previous reports on the
hamster (Ward, '46; Austin, '56; Strauss,
'56; Goldman and Porter, '70). Dating
from the LH surge, most animals ovulated
10 to 11 hours later (fig. 13;9) which in
most species is the time interval elapsing
between the administration of HCG and
ovulation (for references see Edwards and
Gates, '59).
ACKNOWLEDGMENTS
RLN was supported by a training grant
in reproduction from NIH (TL HD25-09).
The research was supported by grants from
NIH (HD 00596) and the Ford Foundation.
LITERATURE CITED
Austin, C. R. 1956 Ovulation, fertilization and
early cleavage in the hamster (Mesocricetus
auratus). J. Roy. Micros. SOC.,75: 141-154.
Blaha, G. C., and W. W. Leavitt 1970 The distribution of As-3P-hydroxysteroid dehydrogenase
activity in the golden hamster during the
estrous cycle, pregnancy and lactation. Biol.
Reprod., 3: 362-368.
Blandau, R. J. 1966 The mechanism of ovulation. In: Ovulation. R. B. Greenblatt, ed. Lippincott Co., Philadelphia, pp. 1-15.
Boling, J. L., R. J. Blandau, A. L. Soderwall and
W. C. Young 1941 Growth of the Graffian
follicle and the time of ovulation in the albino
rat. Anat. Rec., 79: 313-331.
Boucek, R. J., G. Telegdy and K. Savard 1967
Influence of gonadotrophin on histochemical
99
properties of the rabbit ovary. Acta Endocrinol.,
54: 295310,
Bullough, W. S. 1942-1944 The method of
growth of the follicle and corpus luteum in
the mouse ovary. J. Endocr., 3: 150-155.
Byskov, A. G. S. 1969 Ultrastructural studies
on the preovulatory follicle in the mouse ovary.
Z. Zellforsch., 100: 285-299.
Chang, M. C. 1955 The maturation of rabbit
oocytes in culture and their maturation, activation, fertilization and subsequent development
in the fallopian tubes. J. Exp. Zool., 128:
379405.
Donahue, R. P. 1968 Maturation of the mouse
oocyte in vitro: I. Sequence and timing of nuclear progression. J. Exp. Zool., 169: 237-250.
Edwards, R. G., and A. H. Gates 1959 Timing
of the stages of the maturation divisions, ovulation, fertilization and the first cleavage of eggs
of adult mice treated with gonadotropins.
J. Endocr., 18: 292-304.
Foote, W. D., and C. Thibault 1969 Recherches
experimentales sur la maturation in vitro des
ovocytes de truie et de veau. Ann. Biol. Anim.
Bioch. Biophys., 9: 329-349.
Goldman, B., and J. C. Porter 1970 Serum LH
levels i n intact and castrated golden hamster.
Endocrinology, 87: 676-679.
Greenwald, G. S. 1960 The effects of unilateral
ovariectomy on follicular maturation in the
hamster. Endocrinology, 66: 89-95.
1961 Quantitative study of follicular
development in the ovary of intact or unilaterally ovariectomized hamster. J. Reprod. Fert., 2:
351-361.
1968 Failure of hypophysectomy to
affect regression of cyclic hamster corpus
luteum. J. Reprod. Fert., 16: 495-497.
1971 Preovulatory changes in ovulating hormone in the cyclic hamster. Endocrinology, 88: 671-677.
Knigge, K. M., and J. L. Leathem 1956 Growth
and atresia of follicles in the ovary of the
hamster. Anat. Rec., 124: 679-707.
Mandl, A. 1963 Pre-ovulatory changes in the
oocyte of the adult rat. Proc. Roy. SOC.B, 158:
105-11s.
Myers, H. I., W. C. Young and E. W. Dempsey
1936 Graffian follicle development throughout the reproductive cycle in the guinea pig,
with special reference to changes during estrous
(sexual receptivity). Anat. Rec., 65: 381-401.
Norman, R. L., and G. S. Greenwald 1971
Effect of phenobarbital, hypophysectomy and
x-irradiation on preovulatory progesterone levels
in the cyclic hamster. Endocrinology, 89:
598-605.
Odor, D. L. 1955
The temporal relationship
of the first maturation division of rat ova to the
onset of heat. Am. J. Anat., 97: 461-491.
Pedersen, T. 1970 Follicle kinetics in the ovary
of the cyclic mouse. Acta Endocrinol., 64:
304-323.
-
100
REID L. NORMAN AND GILBERT S. GREENWALD
Straws, F. 1956 The time and place of fertili- Wingate, A. L. 1970 A histochemical studv of
zation of the golden hamster egg. J. Embryol.
the hamster ovary. Anat. Rec., 166: -399
Exptl. Morph., 4: 42-56.
(Abstract).
Turgeon, J., and G. S. Greenwald 1972 Preovulatory levels of plasma LH in the cyclic
Zachariae, F. 1959 Acid mucopolysaccharides
in the female genital system and their role in
hamster. Endocrinology, in press.
Ward, M. c. 1946 A studv of the
cvcle
the mechanism of ovulation. Periodica, Denmark: 1-64.
and breeding of the golden hamster Cricetus
auratus. Anat. Rec., 94: 139-161.
PLATES
PLATE 1
Follicle at 2000 ( X 93).
Follicle at 2400 ( x 93).
3
4
( x 93).
Follicle at 1600 ( X 93).
2
1 Follicle a t 1000
Follicles at different times on proestrus
EXPLANATION O F FIGURES
0
CL
W
OVARIAN HISTOLOGY:HAMSTER
Reid L. Norman and Gilbert S. Greenwald
PLATE 1
PLATE 2
EXPLANATION OF FIGURES
Nuclear maturation of the oocyte during proestrus
104
5
Oocyte at 1000 ( x 233).
6
Oocyte at 1200 ( X 233).
7
Oocyte at 1400 ( X 233).
8
Oocyte at 1600
( x 233).
OVARIAN HISTOLOGY: HAMSTER
Reid L. Norman and Gilbert S. Greenwald
PLATE 2
105
PLATE 3
EXPLANATION O F FIGURES
Nuclear maturation of the oocyte during proestrus
( x 233).
( x 233).
11 Oocyte at 2200 ( x 233).
12 Oocyte at 2400 ( x 233).
Oocyte at 1800
Oocyte at 2000
I
I CRITICAL PERIOD
(Grrrnrold '71 I
AFTER HYWX
( Q r m w a l d '71 I
9
10
x
25
0
,
650
4 DIAMETER OF p
FOLLICLE
I
/,
I
.-
I
I
1
B CUMULUS
,
I
CHANGES
I
I
I
I
'"
1100
9. OVULATION
'
I
. ANIMALS
!
---
i"
I -7.1200
1300 WM)
is00
16w
,700
1800 1900zdoo 2ioo 2100 2300
- -- -
2400 oiw oiZG&o
DAY I
-
Fig. 13 Correlation of histological and physiological events i n the proestrous hamster.
106
OVARIAN HISTOLOGY: HAMSTER
Reid L. Norman and Gilbert S. Greenwald
PLATE 3
107
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