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Morphological changes in atretic graafian follicles during induced atresia in the hamster.

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THE ANATOMICAL RECORD 212:353-357 (1985)
Morphological Changes in Atretic Graafian Follicles
During Induced Atresia in the Hamster
C.J. HUBBARD AND G.S. GREENWALD
Department of Physiology, University of Kansas Medical Center, Kansas City, KS 66103
ABSTRACT
Hypophysectomized female hamsters primed with 30 i.u. pregnant
mare serum gonadotropin (PMSG) were injected with anti-PMSG serum 72 h later
to induce atresia in Graafian follicles. The earliest histological evidence of atresia
was seen in the cumulus oophorus at 4 h with a significant increase in pyknotic
nuclei. The mural granulosa cells, however, did not show pyknoses until 12 h
following anti-PMSG serum. The highest number of pyknotic nuclei in the cumulus
oophorus was seen a t 24 h, when pseudomaturation of the oocyte occurred. Breakdown of the mural granulosa cells was greatest by 48 h. Morphometric measurements of the follicles showed a significant decline in the thickness and area by the
granulosa layer a t 48 h following injection of anti-PMSG serum.
The thecal compartment showed a decline in number of mitoses at 4 h and 12 h
and in area at 48 h. However, no pyknotic thecal cells were observed during the 72 h
following injection of anti-PMSG serum. The results support previous work using
this model, which showed major biochemical changes in the granulosa cells a t 12
and 48 h. The theca, however, was comparatively unaffected during atresia. The
various studies using this model show changes in atretic follicles similar to those
seen in other models and in the intact animal. The major advantage of this model is
that it produces a large synchronous population of follicles that rapidly becomes
atretic.
mediately following surgery. Animals in the control
group (0 h) were injected i.p. with 100 pl of saline a t
0900 h on day 4 and decapitated. All other groups were
injected i.p. with 100 p1 of anti-PMSG serum at 0900 h
on day 4 and decapitated 4, 8, 12, 24, 48, or 72 h later.
Both ovaries were immediately removed from each animal and placed in Bouin’s fixative for 1 h. Following
fixation the ovaries were dehydrated in a graded series
of alcohol baths ending in xylene. The ovaries from each
animal were then embedded in paraffin, sectioned serially at 5 pm, placed on glass slides, and stained with
hematoxylin and eosin stains. A total of 10 Graafian
follicles from the ovaries of each animal was examined
with the light microscope according to the following
criteria:
1. Only mature Graafian follicles were measured. Sections were chosen that included a n oocyte containing a
nucleus.
MATERIALS AND METHODS
2. Area-The total area occupied by the thecal and
Mature female hamsters (Mesocricetus auratus) were granulosa cell compartments in addition to the antrum
maintained on a 14L:lOD cycle (lights on a t 0500 h) with of a follicle were determined by tracing the projected
food and water provided ad libitum. The day of estrus microscopic image using a Sumagraphics digitizer (Fair(day 1) was determined by the presence of the characteristic vaginal discharge. The animals were maintained
for at least three consecutive cycles prior to use. The
hamsters were hypophysectomized between 0900 and
Received October 25, 1984; accepted March 15, 1985.
1000 h (four animals/group) on day 1 using a paraphar- C.J. Hubbard’s present address is Department of Anatomy, Univeryngeal approach. Thirty international units of PMSG sity of South Dakota, School of Medicine, Vermillion, SD 57069.
(Pemix; Sankyo Co., Tokyo, Japan) was injected i.p. im- Address reprint requests to Dr. G.S. Greenwald.
Previous studies by Bill and Greenwald (1981) developed a model for follicular atresia that produced a large
synchronous population of atretic follicles. Atresia was
produced in hypophysectomized hamsters injected with
pregnant mare serum gonadotropin (PMSG) on day 1 of
the cycle followed 2 days later with a n injection of antiPMSG serum. Removal of gonadotropin support produced a rapid decline in serum and tissue estradiol and
testosterone with a subsequent decrease in CAMP. In
the final stages of atresia, production of progesterone
also declined (Hubbard and Greenwald, 1981). Total
physiological collapse of the follicles occurred between
48 and 72 h following injection of the antiserum. Based
on the rapid decline in the steroidogenic capacity of
these follicles, a morphometric study was undertaken to
document the histological changes occurring during progressive follicular atresia of the follicles.
0 1985 ALAN R. LISS, INC
C.J. HUBBARD AND G.S.GREENWALD
354
MURAL GRANULOSA
granulosal layers. Two measurements for the follicle
were taken perpendicular to each other with the first a t
the widest region of the follicle. Four different regions
were chosen at random (excluding the cumulus oophorus) for measurements of thecal and granulosal thicknesses. Each group of measurements was averaged to
yield a diameter and granulosal and thecal thickness for
each follicle.
4.Pyknosis and mitosis-The total number of pyknotic
nuclei and dividing cells (mitoses) was determined separately for the cumulus oophorus, mural granulosa, and
thecal compartments of each follicle. The area of the
cumulus oophorus was divided into two separate regions
for analysis. Region I consisted of cumulus cells surrounding the oocyte (Fig. 1.). Cells in region I1 comprised
the base of the cumulus oophorus. The presence of germinal vesicle breakdown and pseudomaturation (as indicated by the presence of a metaphase plate) of the
oocyte was also noted.
Statistical procedures consisted of analysis of variance
and least-square means test. The data were processed
using the SAS statistical analysis program (Cary, NC).
Experimental values were considered to be significantly
different from each other at a level of P < 0.05. Means
were expressed as f S.E.M.
RESULTS
The area of the granulosa cells did not decline significantly until 48 h following the injection of anti-PMSG
serum (Table 1).This was paralleled by decreased area
of the theca interna; however, the decline in the theca
was not as marked or consistent. The thickness of the
granulosa and overall follicle diameter also declined
significantly at 48 h. Thecal thickness, however, failed
field, CT) calibrated to a microscopic calibration slide. to show any change prior to the 72 h period.
In the early stages of atresia, the greatest number of
Combined areas were determined for the antrum, granulosa, and theca. The area of granulosa cells was then pyknotic cells was found surrounding the oocyte in recalculated by subtracting the areas of the antrum and gion I followed by region 11. This is demonstrated in the
theca; similarly, theca area was determined by subtract- representative section seen in Figures 2 (0 h) and 3 (12
ing the granulosal and antral areas from the whole. h). By 12 h some degenerate and pyknotic nuclei could
be seen floating freely within the antrum. Atresia assoAreas were expressed as square millimeters.
3. Linear measurements-Using the same digitizer ciated with the cumulus region was most advanced a t
system, linear dimensions were determined for the di- 24 h (Table 2, Fig. 4).In the 48-h and 72-h groups the
ameter of the follicle and thickness of the thecal and cumulus cells were entirely absent, leaving the oocyte
Fig. 1. Regional division of follicles analyzed for mitotic and pyknotic
cell nuclei during atresia. Regions I and I1 are divisions of the cumulus
oophorus which were analyzed separately. The mural granulosa includes all other cells within the granulosa compartment.
TABLE 1. Morphological changes in Graafian follicles during 72 h following induction of atresia by injection of anti-PMSG serum'
0
Area of
granulosa(mm2)
Area of
theca (mm2)
Diameter
offollicle(mm)
Thickness of
granulosa (mm)
Thickness
of theca (mm)
0.100 t .006
8
4
0.115
,009
0.105
.006
Time (h)
12
24
48
72
0.104 t .006
0.106 & .005
0.030 t .003*
0.016 k .001*
+ .010
0.068 & .006
0.058 rf: .009
0.067 f ,004
0.082 -I .007
0.042 f .005*
0.024 + .001*
0.610 k ,030
0.670 + .025
0.580 k .023
0.611 k ,032
0.607
.030
0.450+ .023*
0.373 f .008*
0.037 k .004b 0.042 ir .002'
0.048& .002d
0.043
* .003e
.OOlf
0.019 f .001g
0.033 t .003
0.053 k .003
0.036 5 .002
0.070
0.050
+ .002a
0.038 k ,002
0.029 k ,002
0.02
0.030 k .003
0.023 & .002*
'Values marked with an asterisk (*) are significantly different from all others within the same line but not from each other. The following values are
significantly different from each other to at least P < 0.05: a vs. all others except d; f and g vs. all others. The values listed above are the means of ten
Follicles taken from at least four animals each.
MORPHOLOGICAL CHANGES IN ATRETIC FOLLICLES
Fig. 2. A follicle from a PMSG-treated and hypophysectomized anima1 was injected with saline and killed at 0900 h on day 4 (controls).
Cells appear in good condition without pyknotic nuclei (see inset).
x 194.
355
antiserum. Therefore, the legends in these figures will indicate only
the time elapsed following PMSG-antiserum plus additional comments. ~ 1 9 4 .
Fig. 3.Following hypophysectomy on day 1, PMSG-antiserum was
Fig. 4. 24 h following PMSG-antiserum. The cumulus oophorus has
undergone extensive degeneration. x 194.
injected at 0900 h on day 4, and the animal was killed 12 h later. Some
granulosa cells can be seen suspended in the antrum (arrowheads, see
inset). The experimental protocol for Figures 4-6 was the same as
described above with exception of the time period following PMSG-
Fig. 5.48 h following PMSG-antiserum. The oocyte has undergone
pseudomaturation. x194.
356
C.J. HUBBARD AND G.S. GREENWALD
TABLE 2. Pyknotic and mitotic cells per follicle in the cumulus oophorus during atresia
Region of cumulus oophorus
Mitoses per follicle
Region I
Region I1
Pyknoses per follicle
Region I
Region II
0
*
Hours following injection of anti-PMSG serum
4
8
12
2.1 0.4l
1.8 f 0.3l
0.8 f 0.2'
1.4 f 0.3l
1.2 f 0.43
2.4 k 0.3'
0.3 f 0.1"
0.05 0.05"
23.4 & 1.5b
12.6 O.gb
23.8 i 3.1b
11.3 l.gb
*
*
0.1 f 0.084
0.4 f 0.1'
20.9 + l.gb
13.7 _+ 1.3b
24
0.004
0.002
*
94.9 13.0'
33.4 f 3.9"
'The following values are significantly different within the same line to at least P < 0.05: 1 vs. 2 , l and 3 vs. 4, a vs. b,
b vs. c
floating freely within the antrum Pigs. 5,6). The mural
granulosa showed a large decline in the numbers of
pyknoses at 72 h. This was due to a massive reduction
in the total number of granulosa cells present in the
follicles at this time period (Fig. 6). Germinal vesicle
breakdown and pseudomaturation of the oocyte was observed at and following the 24-h time period. The number of mitotic cells in regions I and I1 of the cumulus as
well as the mural granulosa declined significantly between 8 and 12 h (Table 2, Fig. 7). In region I, a significant drop in mitoses also occurred at 4 h when compared
with 0 h but not 8 h. The cause for this decline is
unknown. No pyknotic thecal cells were seen during the
entire 72 h period of observation. However, the numbers
of thecal mitoses declined significantly at 12 h (from
2.18 f 0.3 per follicle at 8 h to 0.85 k 0.4 at 12 h) and
reached 0 by 48 h. Thecal thickness was also significantly reduced by 72 h (Table 1).
DISCUSSION
These results support our previous work showing
changes in levels of cyclic nucleotides and steroids during
induced follicular atresia. The cumulus oophorus showed
a significant increase in pyknotic nuclei at 4 h (Table 2)
when CAMP,estradiol, and testosterone synthesis showed
a decline (Hubbard and Greenwald, 1981).By 12 h, when
the number of pyknoses had increased significantly in the
mural granulosa (Fig. 2), luteinizing hormone (LH)-stimdated CAMP, estradiol, and testosterone were at their
nadir (Hubbard and Greenwald, 1983).The number of mitoses in both the cumulus and granulosa also showed a
significant decline at 12 h. The number of pyknotic mural
granulosa cells was highest at 48 h (Fig. 71, which, based
on our previous studies, is the time when LH was unable
to stimulate progesterone in whole follicles (Hubbard and
Greenwald, 1983).It is of interest to note in our previous
studies (Hubbard and Greenwald, 1983) and the present
one that a reduction in biochemical activity and morphological breakdown of the granulosa appears to have occurred almost simultaneously. One might anticipate that
the decline in steroid-synthesizing capacity of the granulosa would precede actual morphological breakdown of
the cells. This may still be the case, but it may occur
within a shorter period than the times observed in this
study. By 48 h mural granulosal degeneration was advanced, and the highest number of pyknotic nuclei were
present (Fig. 7). A significant decrease in both area and
thickness of the granulosa also occurred at this time (Ta-
Fig. 6.72 h following PMSG-antiserum.The granulosa cell region is
only 2-3 layers thick. x 194.
ble 1). Indeed, at 48 h the diameter of the follicle was significantly reduced, and the oocyte was floating free within
the antrum (Fig. 5).
The overall sequence of changes taking place in the
granulosal compartment is similar to that observed by
Uilenbroek et al. (1980) in the rat in which ovulation
was blocked by two injections of nembutal at proestrus
and estrus. In their studies, an increase in the number
of pyknotic cells was observed 24 h after the last injection. This was followed at 48 h by a reduction in the
total number of granulosa cells and hence reduced thickness of the mural granulosa. Pseudomaturation of the
oocyte also occcurred about midway through the process.
In the present study, we observed a much more rapid
progression through the stages of atresia than has been
observed previously (Uilenbroek et al., 1980). This is
probably because atresia is induced much more quickly
357
MORPHOLOGICAL CHANGES IN ATRETIC FOLLICLES
200
1
T
w
1501
@' PYKNOT~CNUCLEI/FOLLICLE
1004
HMITOSES/FOLLICLE
d
10
9
T
I
P
0
I-
::[
4
40
3 L
2
a
10
1
0
4
Ij
12
24
48
72
HOURS FOLLOWING I N J E C T I O N OF P M S G ANTI-SERUM
Fig. 7. Average number of pyknotic nuclei and mitoses (per follicle, + S.E.M.) observed in the
mural granulosal compartment of Graafian follicles during atresia. The following values are
significantly different from each other to at least P < 0.05: 1vs. 2; a vs. b, c, and d; d vs. e.
through the injection of anti-PMSG serum, causing a has been seen in other models using intact animals
rapid decline in gonadotropin support (PMSG). Previous (Ryan, 1981).
studies in this laboratory have shown that when aniACKNOWLEDGMENTS
mals are injected with normal rabbit serum instead of
This research was supported by a grant from the NIH
anti-PMSG, atresia proceeds at a much slower rate, (HD00596).
demonstrating the efficacv of the PMSG-antiserum
(Hubbard anZGreenwald, f98l).
LITERATURE CITED
The thecal cells remained intact with no pyknotic nu,lei apparent throughout the 72-h period. The numbers Bill, C.H., and Greenwald, G.S. (1981)Acute gonadotropin deprivation.
I. A model for the study of follicular atresia. Biol. Reprod., 24:913of mitoses declined. however. at 12 h. and thecal thick921
ness declined at 7 2 h. Unlike atresia' in the intact ani- Hubbard, C.J., and Greenwald, G.S. (1981)Changes in DNA, cyclic
nucleotides and steroids during induced follicular atresia in the
mal, no thecal h y p e ~ r o p h ywas seen. This is probably
hamster. J . Reprod. Fertil. 63:455461.
due to the
Of
gonadotrophin Hubbard,
C.J., and Greenwald, G.S. (1983)In vitro effects of luteinizing
(PMSG)from the system. Binding of 1251-labeledhuman
hormone on induced atretic graafian follicles in the hamster. Biol.
Reprod., 28:849-859.
chorionic gonadotropin to the theca has been shown not
to diminish during initial atresia (&aha and Green- Shaha, C., and Greenwald, G.S. (1982) Autoradiographic analysis of
changes in binding of FSH and hCG during induced follicular
wald, 1982). Though the thecal cells remainviable, they
atresia in the hamster ovary. J. Reprod. Fertil. 66:197-201.
lose the Overall
to produce
Silavin, S.L., and Greenwald, G.S. (1984)Steroid production by isolated
tenedione (12 h), testosterone, and 17a-hydroxyprogestheca and granulosa cells after initiation of atresia in the hamster.
J. Reprod. Fertil., 71:387-392.
terone (12-24 h) but not txogesterone when stimulated
Uilenbroek, J.Th.J., Woutersen, P.J.A., and van der Schoot, P. (1980)
by LH in vitro (Silavin a i d Greenwald, 1984).
Atresia of preovulatory follicles: Gonadotropin binding and steroisummary, the
of this study have shown a
dogenic activity. Biol. Reprod., 23:219-229.
progressive decline in flanulosal integrity accompanied Ryan, R.J. (1981)Follicular atresia: Some speculations on biochemical
bv Dseudomaturation of the oocvte midwav through the
markers and mechanisms. In: Dynamics of Ovarian Function. N.B.
Schwartz and M. Hunzicker-Dunn, eds. Raven Press, New York,
a&&c process. The theta, how&er, showed no de&nerPP. '-ll.
ative changes, but neither did it exhibit hypertrophy as
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