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Preovulatory changes in cumulus-oocyte complex of the hamster.

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T H E ANATOMICAL RECORD 202:339-348 (1982)
Preovulatory Changes in Cumulus-Oocyte Complex of the
Hamster
M. LORRAINE LEIBFRIED A N D NEAL L. FIRST
Endocrinology-Reproductive Physiology Progrum, Department of Meat and
Animal Science, University of Wisconsin-Madison, Madison, Wisconsin S3706
ABSTRACT
An orderly series of associations was found between the cumulus
and the stages of nuclear development in the hamster oocyte as the oocytecumulus complex underwent preovulatory maturation. Ten hours prior to ovulation meiotic resumption was first seen in the oocyte. The onset of cumulus dissociation with corresponding morphological changes in cumulus cells was observed
eight hours prior to ovulation. Completion of the first meiotic division in the
oocyte and full cumulus dissociation occurred 2 hours prior to ovulation. Cumulus
dissociation was found to occur only in vesicular follicles destined to ovulate and
not in follicles undergoing atresia. Cells with large vacuolar inclusions with
distorted, hyperchromatic nuclei were found in the maturing cumulus.
Resumption of the first meiotic division in
the oocyte and dissociation of the cumulus
oophorus from the membrana granulosa are
two major events occurring during preovulatory maturation of the follicle in most mammals. The importance of nuclear maturation as
a prerequisite for fertilization and its causation
by the preovulatory gonadotropin surge has
been recognized (Lindner et al., 1974).Dissociation of the cumulus-oocyte complex from the
underlying granulosa, although long noted,
has been studied very little in relation to ovulation. Dissociation involves loss of cohesiveness
both between cumulus cells and between cumulus cells and oocyte, and entrapment of the
cells in the vicinity of the oocyte by a viscous,
intercellular cement. Loss of cohesiveness
between cumulus cells is due to the disappearance of intercellular junctions (Gilula et al.,
1978;Dekel et al., 1976b).The process of mucification can be due to either de novo synthesis
and accumulation of mucinous material during
preovulatory maturation (Dekel and Kraicer,
1978)or enzymatic liquification of an existing
intercellular matrix.
The result of dissociation of the cumulus is
an oocyte surrounded by single cells maintained in loose association with each other and
the egg by a viscous matrix. Dissociation has
been thought of as a process that allows release
of the oocyte from the follicle at ovulation. The
greater mass added to the oocyte by expansion
of the cumulus is thought to ensure transport
of the ovum from the point of rupture to the
0003-276X/82/2023-0339$03.00
0 1982 Alan R. Liss, Inc.
ostium abdominale. The stickiness of the mucinous material could facilitate entrapment of
the oocyte by the fimbriated infundibulum and
transport of the ovum to the site of fertilization. Cumulus cells may also aid in the capacitation of spermatozoa prior to fertilization
(Gwatkin et al., 1972; Gwatkin and Andersen,
1973);hence the need for the viscous matrix to
retain cumulus cells in the vicinity of the
oocyte after ovulation.
Changes in the cumulus related to dissociation may be the outward manifestation of a
function that is antecedent to ovulation.
Cumulus dissociation in the rat occurs after
the preovulatory gonadotropin surge (Dekelet
al, 1976b) and can be stimulated in vitro in
several mammalian species by gonadotropins
(Dekel and Kraicer, 1978; Thibault et al, 1975;
Eppig, 1979) and adenosine monophosphates
(Dekel and Kraicer, 1978; Eppig, 1979). The
disappearance of cell-to-cell junctions during
cumulus dissociation, occuring coincidentally
with preovulatory maturation of the oocyte
(Dekel et al., 1976b Gilula et al., 1978), has
been implicated as a mechanism whereby
resumption of the meiotic process prior to ovulation can be controlled.
The purpose of this study is to describe the
time relationship between meiotic resumption
in the oocyte and cumulus dissociation during
the preovulatory period. The hamster was
Received M a y 7. 19x1: accepted September 15. 19x1
340
M.L. LEIBFRIED AND NEAL L. FIRST
chosen as an animal model owing to the constancy of its estrous cycle.
MATERIALS AND METHODS
Preparation o f animal materal
Female golden hamsters between 4 and 6
weeks of age and maintained on a lighting
schedule of 1 4 hours of light and 10 hours of
dark were used. Only animals exhibiting three
or more consecutive 4-day estrous cylces were
included in the study. Day 1 of the cycle was
defined as the day of the proestrous vaginal
discharge (Ward, 1946).
Starting at 15:OO on Day 4 of the cycle and
ending at Day 1,four animals were killed every
two hours. This time interval corresponds to
the period just prior to or at the beginning of
the endogenous gonadotropin surge preceding
ovulation (Sheela Rani and Moudgal, 1977)and
lasting until ovulation is completed in our colony. In addition, four animals were killed at
9:OO on Day 1to obtain the average number of
ovulations expected for hamsters killed earlier.
At autopsy the ovaries and oviducts were removed and fixed in Bouin’ssolution, embedded
in paraffin, serially sectioned at 7 pm, and
stained with Mallory’s triple stain (Humason,
1976).
Handling of histological material
Since we wished to consider oocyte-cumulus
relationships only in Graafian follicles, examination of an animal‘s follicular population was
restricted to vesicular follicles 2 400 pm in
diameter, the smallest expected diameter for
normal antral follicles in a cycling hamster
(Greenwald, 1973). Size was estimated by
counting the number of serial sections in which
the follicle appeared and multiplying by the
thickness of a section.
Using pyknosis, sloughing of cells, and karyorrexis as indicators of atresia, antral follicles
were divided on the basis of their granulosa into
two categories: normal or atretic. A follicle was
classified as atretic if the granulosa showed
one or more signs of atresia.
The categories used for evaluating stage of
meiosis in the oocytes were:
(1) Dictyate- An intact germinal vesicle
was still present.
(2) Prometaphase I-The germinal vesicle
had dissappeared and the chromatin was condensing in the cytoplasm.
(3) Metaphase I-The bivalents were in
association with the spindle apparatus.
(4) Anaphase to telophase I-The bivalents
were separating.
(5) Metaphase 11-The first polar body and
second metaphase plate were present.
(6) Other-Either no discernible chromatin
was present or the configuration did not fit the
preceding categories.
Cumulus dissociation was evaluated according to the following categories:
(1) Compact - Cumulus cells were closely
associated in layers around the oocyte. The
outline of the discus proligerus was smooth.
(2) Loose-The hillock assumed an irregular outline. Cells were losing their epitheliallike
arrangement and, beginning centripetally,
were showing intercellular gaps.
(3) Expanding - Cumulus cells were individually isolated from one another except for
those forming the corona radiata. Large
deposits of a mucopolysaccharide-staining
material were seen as a granular precipitate between the cells.
(4) Free-The description noted for the
category termed expanding applied to this
category but intercellular distances were further accentuated. The original area of the
discus proligerus was no longer discernible and
the oocyte had been pushed toward the center
of the antrum
(5) Ouiductal- Cumulus masses and associated oocytes were found in the oviducts.
(6) Degenerate - Pyknosis andior karyorrexhis was apparent in the cumulus. Cell
sloughing had caused thinning of the cumulus
layers (five to eight cellular layers normally
surround the oocyte).
(7) Nude -Cellular degeneration and
sloughing had left the oocyte free in the
antrum surrounded by the zona pellucida.
Both mitotic figures and formation of large
lipid-containing vacuoles were observed in the
cumulus cells. For this study two sample sections of the cumulus were rated as having
mitotic figures present or absent. The sections
chosen were those on either side of the oocyte
where both zona pellucida and ooplasm are
first encountered. A follicle was scored: 0 if
neither of the samples possessed a mitotic figure, 1 if one of the sections had one or more
mitotic figures, or 2 if both sections had one or
more mitotic figures present. This scoring system was also used to score the presence or
absence of the vacuolar inclusions in cumulus
cells.
Statistical methods
Analysis of variance (Barr et al., 1976) was
performed on both number and size of follicles.
Cramer’s v (Dixon and Brown, 1977)was used
34 1
PREOVULATORY CHANGES IN CUMULUS-OOCYTECOMPLEX
t o obtain a measure of association for cumulus
and chromatin categories within a time period.
Log linear analysis (Dixon and Brown, 1977)
was used to analyze the data concerning mitotic figures. After preliminary analysis showed
no effect of replicate for the categorical data on
formation of vacuoles in the cumulus cells,
numbers were collapsed across replicates and a
chi square contingency table analysis was performed (Snedecor and Cochran, 1967).
RESULTS
Means for number and size of preovulatory
follicles for each time period are shown in Table
1. After eliminating the values for animals
killed at 15:OO. there were no differences between time periods in total number of follicles
or the subcategories of normal and atretic follicles. Graafian follicles, therefore, were committed to ovulation or atresia at the time interval chosen for the study. Follicular diameter
was different between time periods (P <
0.001), indicating that follicles are still
increasing in size during the 12-hour period
preceding ovulation (Table 1).Normal follicles
were larger than atretic follicles during the
preovulatory period (P < 0.001). From the
results of the analyses for size and number of
follicles, we concluded that classification of
follicles based on appearance of the granulosa
allowed separation of two distinct types of
follicles. All further discussion and analysis
will treat normal and atretic follicles
separately.
The univariate frequency distributions for
chromatin and cumulus categories in normal
follicles are shown in Figure 1. The cumulus
category termed nude was deleted since follicles classified as normal never showed this
character. With the use of Cramer’s v (Table l),
a high degree of association was found between
the appearance of these two characteristics
within time. The sequence of associations
between chromatin and cumulus will be
described by individual time periods.
15:OO
The discus proligerus had the smooth,
rounded outline characteristic of the oocytic
hillock (Fig. 2). With few exceptions the cumulus category found was that termed compact.
The cells forming the cumulus were cuboidal
and tightly packed in layers around the oocyte
in a pseudoepithelial arrangement (Fig. 3). All
of the oocytes remained in the dictyate stage
(Fig. 3) with an intact nuclear membrane and
nucleoli present. The exceptions to the compact cumulus were cumuli classified as degenerate (Fig. 12). Analysis of variance on the
number of follicles present across all observation times (Table 1) indicated that a greater
number of normal, vesicular follicles (P < 0.05)
were present at 15:OO yet the total number of
follicles was not different. The presence of folli-
TABLE 1. Means and statistics for follicular data’
Time
Mean No. of follicles
2 400pm’lanimal
Total No. of normal2
follicles observed
Mean No. of normal
follicles2/animd
Mean diameter of
normal follicles (pm)
Total No. of atretic
follicles observed
Mean No. of atretic
folliclesianimal
Mean diameter of
atretic follicles (pm)
Cramer’s v3
Mean score for mitotic
index3 (x2 = 329.0)
Mean score for index of
vocuolated cells’
l:oo
3:OO
900
19.25
18.50
21.25
18.25
54
63
57
13.50
15.75
14.25
735.00
-
-
17:OO
19:OO
19.25
19.50
20.50
21.75
69
53
59
57
17.25
13.25
14.75
14.25
13.50
497.04
631.32
661.56
700.29
707.78
10
25
23
30
23
20
5.00
480.28
1.00
495.78
0.709
528.85
0.317
1.37
1.52
1.34
1.12
1.24
1.96
1.a9
1.90
6.25
414.00
490.68
1.00
475.35
0.523
0.71
0.44
o,O1
0
7.50
54
5.75
5.75
2.50
-
21:OO
23:OO
15:OO
22
16
5.50
4.00
475.36
470.31
-
-
-
-
-
-
‘Data are from four animals within a time period.
’Ovulated follicles were included in the number of folliclescategorized as normal and total number of follicles. The diameter of ruptured follicles
was not measured.
’Statistics derived from data on normal. unruptured follicles to obtain a measure of association hetween stages of nuclear development of the
m y t e and cumulus dissociation for an individual time period.
342
M.L. LEIBFRIED AND NEAL L. FIRST
=
CHROMATIN
ESSI CUMULUS
50
CHROMATIN
CUMULUS
OTHER
DEGENERATE
0
100
50
MET
P
OVIDUCT
0
100
50
WA-TELO I
FREE
MET I
EXPANDING
PROMET I
LOOSE
DICTYATE
COMPACT
0
100
50
0
15:oo
1700
19:00
23:OO
300
21:OO
1:00
TIME
Fig. 1. Univariate distribution of the chromatin and cumulus oophorus categories of normal follicles.
cles classified as normal but containing degenerating cumuli account for this difference at
1500 compared to the other time intervals.
These may represent the last follicles to begin
atresia in the wave of follicles stimulated for
this ovulation. The most accurate identification of a follicle as atretic should combine
appearance of both the granulosa and cumulus
oophorus.
17:m
Again the majority ( > 90%)of cumuli in nor
ma1 follicles were compact (Fig. 4). Although
about 60% of the oocytes were still in the dicty-
PREOVULATORY CHANGES IN CUMULUS-OOCYTE COMPLEX
ate stage, the germinal vesicle was characterized by chromatin condensation and decreasing clarity of the nucleoli and nuclear membranes
(Fig. 5). The remainder of the oocytes had
undergone germinal vesicle breakdown with
chromatin condensation continuing in the
cytoplasm. The relatively low measure of association obtained at 17:OO hours (Table 1)indicated that resumption of meiosis began before
discernible changes in the cumulus occurred.
Such has been reported previously for the rat
(Hillensjo et al., 1976).
343
3:OO
Animals in our colony have completed ovulation by this time. The follicles remaining on the
ovary were all atretic. The number of ruptured
follicles counted on the pairs of ovaries corresponded to the number of oocytes found in the
oviducts.
Atretic follicles
Cumulus stage and chromatin stage of the
oocyte were not associated consistently nor
sequentially in atretic follicles. Cumulus dissociation like that in normal follicles was never
19:oo
seen in atretic follicles. The oocyte investCumulus dissociation was first observed by ments in atretic follicles were classified exclulight microscopy at this time period. The sively as degenerate (Fig. 12) and nude (Fig.
majority of the cumuli were divided between 13). At all experimental time periods, oocytes
the categories termed loose (Fig. 6) and ex- could be found in any stage of the first meiotic
panding (Fig. 8).In loose cumuli the discus pro- division and also in the chromatin category
ligerus had a rough outline and the cellular termed other. This category included oocytes
layers had lost their epitheloid arrangement with no stainable chromatin or scattered
(Fig. 7). Expanding cumuli were characterized clumps of chromatin and those possessing one
by increased intercellular distances and or two female pronuclei. At no time period were
increased density of the intercellular material oocytes in atretic follicles found consistently in
demonstrated by the staining procedure (Figs. the stage of nuclear development considered
8,9).The majority of the oocytes were found in predominant for oocytes in normal follicles. Of
the stage of chromatin development classified 169 oocytes observed in atretic follicles, only
as prometaphase I. One-fourth of the oocytes ten remained in the dictyate stage of nuclear
had reached metaphase I with a distinct development.
spindle structure present.
Mitotic figures and vacuole formation
21:OO
The formation of vacuolar inclusions in cells
Cramer’s v for this time period was unity, in- of the cumulus was not independent of time (P
dicating a high degree of association for the < 0.001). Vacuoles were not observed until
chromatin and cumulus categories typical for 1900, the time when the onset of cumulus disthis time. Over 85% of the follicles observed sociation is first observed (Table 1). These
had oocytes with the chromatin configuration vacuoles (Fig. 14),probably having a high lipid
at metaphase I . The predominant cumulus content, fill the cytoplasm in subsequent
type was that termed expanding (Fig. 9).
stages and seem to distort and displace the
nucleus to an eccentric location in the cell.
23:OO
Analysis of the categorical data for mitotic
Although the majority of cumulus-oocyte figures (Table 1)gave a log linear model of best
complexes remained in the same stage as the fit when rating of mitosis is considered as time
preceding time period, i.e., expanding and dependent. A quadratic expression best
metaphase I, about a third had proceeded to described the linear component of mitotic f r e
free and anaphase-telophase I categories.
quency with time. As seen in Table 1, frequency of mitotic figures increased until 21:00,
1:OO
then declined.
Hamsters in our colony begin ovulating at
this time. Two out of four animals killed at this
DISCUSSION
period had one or two ovulations. Twclthirds of
An
ordered,
sequential
relationship between
the oocytes had completed the first meiotic
division with the expulsion of the first polar cumulus dissociation and oocyte maturation
body and formation of the second metaphase has been demonstrated for the hamster. A simplate. The other oocytes were still in amphase- ilar relationship has been shown for the rat
(Dekel et al., 1979). In both our study with
telophase I (Fig. 11). The predominant
hamsters and others using rats (Hillensjoet al,
cumulus class was free (Fig. 10).
344
M.L. LEIBFRIED AND NEAL L. FIRST
Fig. 2. Normal follicle observed a t 15:OO. The cumulus is
compact and oocyte contains an intact germinal vesicle.
x 63.
Fig. 3 . Normal follicle observed at 15:OO. The nucleus of
the oocyte and epithelial arrangement of the cumulus cells
are apparent. x 160.
Fig. 4 . Normal follicle observed a t 17:OO. ' f i e cumulus
remains in the immature or compact category. x 63.
Fig. 5. Normal follicle observed a t 17:OO. Mitotic figures
are seen in the cumulus. Chromatin condensation is occurring in the germinal vesicle. x 160.
PREOVULATORY CHANGES IN CUMULUS-OOCYTE COMPLEX
Fig. 6. Normal follicle observed a t 19:OO. The cumulus is
classified as loose with the uocyte at prometaphase I. X 63.
Fig. 7. Normal follicle observed a t 19:OO. The loosening
cumulus shows the loss of epithelial arrangement and development of intercellular spaces. X 160.
345
Fig. 8. Normal follicleobserved a t 2190. The cumulus is
expanding. Increased intercellular distance and density of
staining of the intercellular matrix characterize this
cumulus category. x 63.
Fig. 9. Normal follicle observed a t 21:OO. The expanding
cumulus and metaphase Z are predominant at this time
period. x 160.
346
M.L. LEIBFRIED AND NEAL L. FIRST
Fig. 10. Normal follicle observed a t 23:OO. The ooeyte
shows expulsion of the first polar body. The cumulus is
classified as free. x 63.
Fig. 11. Normal follicle observed a t 23:OO. The oocyte is
in late telophase I. The vacuolated cells of the free cumulus
are prominent during this time period. x 160.
Fig. 12. Atretic follicleobserved a t 15:OO. Thecumulus is
degenerate. x 63.
Fig. 13. Atretic follicle observed a t 21:OO. One polar
body and a pronucleus are seen in the oocyte. The cumulus
category is that termed nude. x 63.
PREOVULATORY CHANGES IN CUMULUS-OOCYTECOMPLEX
Fig. 14. Normal follicle observed a t 23:OO. Mitotic f i g
ures and vacuolated cells are seen in the cumulus. x 160.
1976; Dekel et al., 1979),meiotic resumption in
the oocyte preceded observable cumulus disso
ciation. Electron microscopic evaluation is
needed to confirm this point since early ultrastructural changes would have been missed by
the methods used to assess the onset of cumulus dissociation in these studies.
When the onset of cumulus expansion was
first observed, morphological changes in cumulus cells were also seen. A morphological
change in cumulus cells corresponding to the
onset of cumulus dissociation was also
reported for the rat (Hillensjo et al., 1976;
Dekel et al., 1979). The change observed was
described as blebbing, or formation of pseudo
podia. The major changes found in the cells of
the hamster cumulus were the formation of
large vacuoles and the distortion and hyperchromicity of the nucleus. Such changes could
be signs of cellular degeneration. LH in the rat
decreases cumulus cell respiration and
destroys their ability to form monolayers in
culture (Dekel et al., 1976a).These also could
be taken as signs of impending cellular degen-
347
eration. Cumulus cells of oviductal eggs
recovered after ovulation from mice show
signs of cellular degeneration (Zamboni, 1970).
It is possible that the gonadotropin surge prior
to ovulation may be a terminal signal for the
cumulus cells.
Cumulus expression and associated morphological changes in cumulus cells were not
observed in follicles undergoing atresia. Cumulus dissociation must be controlled by hormonal events of the preovulatory phase of the
estrous cycle and it occurs in follicles “selected
to undergo ovulation. In atretic follicles with
advanced degeneration of the cumulus, oocytes
were commonly found to have resumed meiosis. The presumed loss of intercellular association in cumulus cells may have allowed release
of the oocyte from meiotic arrest. Hay and
Cran (1978)have already suggested this after
studying atretic, vesicular follicles of sheep.
Loss of intercellular association or communication as part of cumulus dissociation has been
shown to occur in the rat oocyte cumulus complex prior to ovulation (Gilula et al., 1978).This
termination of cell-tocell communication is
hypothesized to be the mechanism allowing
meiosis to resume in the oocyte. It is striking
that under two such disparate conditions as
follicular atresia and preovulatory follicular
development, loss of integrity of the cumulus
is thought to allow for release of the oocyte
from meiotic arrest. If meiotic arrest during
folliculogenesisis originally dependent on formation of intercellular communication with
follicle cells, as has been suggested (Ohno and
Smith, 1964; Anderson and Albertini, 1976),
then meiotic resumption during follicular
atresia or development is the reverse process
or loss of communication. Control of meiosis
would then be analogous to contact inhibition.
In summary, we have shown a sequential
series of associations between the cumulus as
dissociation occurs and the stages of nuclear
development in the hamster oocyte prior to
ovulation. Meiotic resumption was observable
prior to the onset of cumulus dissociation, as
shown by light microscopy. Corresponding to
the onset of cumulus dissociation, large vacuolar inclusions are found in the cells of the cumulus. Loss of integrity of the cumulus under the
conditions of atresia or development takes separate pathways. Cumulus dissociation was
found to occur only in preovulatory follicles
and not in follicles undergoing atresia. Cumulus dissociation, therefore, must be initiated by
hormonal events of the preovulatory phase of
the estrous cycle.
348
M.L. LEIBFRIED AND NEAL L. FIRST
ACKNOWLEDGMENTS
The authors are grateful to Dr. Fields C.
Gunsett for statistical consulting and to Julie
Busker for assistance in histologic preparation
of the tissue.
This research was supported by the College
of Agricultural and Life Sciences, University
of Wisconsin-Madison; U.S.D.A S.E.A. grant
No. 12-14-5001-313 Mod. 1; Public Health
Service training grant No. 5-T01-00104-10
from the National Institute of Child Health
and Human Development; and grant No.
630-0505B from the Ford Foundation.
This article is Department of Meat and Animal Science Paper No. 788.
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