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Effects of unilateral ovariectomy of the pregnant hamster on the remaining ovary.

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Effects of Unilateral Ovariectomy of the Pregnant
Hamster on the Remaining Ovary
AMAR CHATTERJEE AND GILBERT S. GREENWALD
Departments of Obstetrics a n d Gynecology a n d A n a t o m y . University of
Kansas Medical Center, Kansas City, Kansas 661 03
ABSTRACT
Unilateral ovariectomy of hamsters on day 1 of pregnancy resulted in an increase in weight of the remaining ovary by day 4, primarily by
increasing the number of antral follicles. The ovulability of these follicles was
tested by injecting 20 IU human chorionic gonadotropin (HCG) on day 12 of
pregnancy. Following this treatment, the unilaterally ovariectomized animals
ovulated 29.4 eggs whereas intact animals ovulated 16.6 eggs per ovary. In both
instances, the number of antral follicles ovulated by HCG was between 40%
and 50% of the number present. Luteal weight was unaltered by semispaying,
indicating that the regulation of corpus luteum growth does not fit a negative
feedback system.
The ability of exogenous steroids to prevent ovarian compensation was assessed by injecting either progesterone ( 2 mg) or estradiol cyclopentylpropionate
(1 r g ) daily from days 8 to 11 of pregnancy. Either steroid injected into the
semispayed hamsters prevented the expected increase in the remaining ovary
in weight, follicular development and the ovulatory response to HCG. The same
hormonal treatment of intact, pregnant animals slightly increased mean ovarian
weight but did not affect follicular development.
These results suggest that unilateral ovariectomy of the pregnant hamster,
by decreasing peripheral level of ovarian steroids, partially reduces the inhibition
of gonadotropin secretion and therefore leads to ovarian compensation.
Following unilateral ovariectomy, compensatory ovarian hypertrophy occurs in
the pseudopregnant or pregnant rat (Chatterjee and Greenwald, '71). The increase
in weight of the remaining ovary primarily
represents an increase in the number of
antral follicles, a compensatory response
prevented by the injection of either estrogen or progesterone.
The present paper deals with the effects
of semispaying the pregnant hamster and
the possible regulatory mechanisms involved. The pregnant hamster differs in
four important respects from the pregnant
rat: (1) only one set of corpora h t e a are
present in the pregnant hamster compared
to several generations (both corpora lutea
of the estrous cycle and pregnancy) in the
rat; ( 2 ) the hamster shows a remarkable
proliferation of antral follicles in the second half of gestation (Greenwald, '67)
which is not seen in the rat (Greenwald,
'66); ( 3 ) ovulation can be induced in the
ANAT. REC., 171: 221-226.
pregnant hamster by human chorionic gonadotropin (Greenwald, '67) but not in the
pregnant rat (Greenwald, '66); ( 4 ) there
is a significant drop in ovarian weight on
day 10 of gestation in the pregnant rat
(Chatterjee and Greenwald, '71 ) that has
no counterpart in the hamster. In view of
these differences, it was considered worthwhile to determine whether the mechanisms regulating ovarian compensation in
the semispayed rat also operate in the
pregnant hamster.
MATERIAL AND METHODS
Golden hamsters (Mesocricetus auratus)
were used after at least three consecutive
four day estrous cycles were followed.
Day 1 of the cycle was designated as the
day on which the conspicuous postovulatory vaginal discharge occurred. The animals were maintained on a 14 hour light:
ten hour dark schedule (lights on: 5 AMReceived Jan. 7, '71. Accepted Feb. 18, '71.
22 1
222
AMAR CHATTERJEE AND GILBERT S. GREENWALD
gonadotropin (HCG) in 0.1 ml of saline.
The hamsters were killed the next morn(proestrus); the morning of a sperm posi- ing and the oviducts were flushed with
tive vaginal smear was designated as day 1 physiological saline via the ostium tubae
of pregnancy. Unilateral ovariectomy (or abdominale. Hyaluronidase was used to resemispaying) was performed on day 1 by move the granulosa cells adhering to the
removing either the left or right ovary, via ova, and the eggs were counted with the
a Bank incision, from alternate hamsters. aid of a dissecting microscope at X 15 magSemispayed or intact pregnant hamsters nification.
The significance of the data was deterwere killed with an overdose of ether on
day 4, 8, 12 or 16 of pregnancy (table 1). mined by the Student’s t test.
Ovaries from both groups were dissected
RESULTS
free of adherent tissue, blotted and
Unilateral
ovariectomy
and ovarian
weighed. The five largest corpora lutea of
both the intact and the semispayed animals weight during pregnancy. Mean ovarian
were dissected out and weighed on a tor- weight increased progressively in the insion balance. For histologic studies ovaries tact pregnant hamster; by day 4 the differwere fixed in Bouin’s fluid, sectioned seri- ence from the day 1 ovary was significant
ally at 10 and stained with hematoxylin ( p < 0.0005) (table 1). The most striking
and eosin. The slides were examined at increase in ovarian weight occurred beX25 magnification and the numbers of tween days 8 and 12 of pregnancy (p <
healthy antral follicles (regardless of size) O . O O l ) , representing growth of the corpora
and corpora lutea in each ovary were lutea and an increased number of antral
follicles (table 3 ) . Ovarian weight stabicounted (results in table 3 ) .
The ability of exogenous steroids to pre- lized between days 12 and 16 of gestation
vent ovarian compensation was tested by (table 1 ) and both luteal weight and the
injecting progesterone or estrogen (table 2). number of corpora lutea and of antral folProgesterone and estradiol cyclopentylpro- licles were unchanged during this period
pionate (ECP) were diluted with sesame (table 3 ) . Following unilateral ovariecoil so that 1.0 ml of the vehicle contained tomy at day 1, the remaining ovary was
either 10 pg of ECP or 2.0 mg of proges- significantly heavier by day 4 of pregnancy
terone. Groups of semispayed or intact than the ovary of the intact animal (p <
pregnant hamsters were injected daily sub- 0.005) (table 1). The ovary of the semicutaneously with either steroid from days spayed hamster on days 4, 8, 12 and 16
8 through 11 of pregnancy and killed on weighed respectively 17.9, 26.4, 52.7 and
day 12.
43.4 per cent more than its counterpart in
In other experiments ovulation induced the intact animal. Mean luteal weight did
by exogenous gonadotropin was used as an not differ significantly at any stage of pregendpoint (table 4). Intact or semispayed nancy between the intact and semispayed
animals were injected subcutaneously on hamsters.
Reversal of the e f f e c t s of unilateral
day 12 of pregnancy between 8:30 and
9:30 AM with 20 IU of human chorionic ouariectomy by steroids. If semispaying
7 PM). Females were caged with males of
proven fertility on the evening of day 4
TABLE 1
E f f e c t s of unilateral ovariectomy o n ovarian and luteal weight in pregnant hamsters 1
Intact control hamsters 2
Unilaterally ovariectomized hamsters 8
Day of
pregnancy
Mean
ovarian wt
(mg -c SE)
Mean
luteal wt
(mg +- SE)
Mean
ovarian wt
(mg c SE)
1
4
8
12
16
12.3k 0.25 (29)
14.6k 0.63 (12)
15.9k0.41(14)
24.1f0.82(16)
24.250.83(12)
0.42f 0.02( 10)
0.79f0.02(14)
1.1020.02(14)
1.20-CO.03(14)
17.3f0.57(10)
20.121.3 (7)
36.721.2 (9)
34.721.4 (9)
1
2
-
Mean
luteal wt
(mg f SE)
-
0.4320.03(6)
0.86f0.03(5)
1.20&0.02 (5)
1.30k0.05(6)
All operations were done on day 1 of pregnancy (day of sperm in vaginal smear).
Numbers in parentheses represent number of animals on which averages are based.
OVARIAN COMPENSATION - PREGNANT HAMSTER
decreases peripheral levels of ovarian
steroids which regulate gonadotropin secretion, it should be feasible to reverse the
effects by giving appropriate amounts of
estrogen and/or progesterone. This possibility was tested by injecting 2 mg of
progesterone or 1 pg of ECP from days 8
to 11 of pregnancy. It is evident for the
semispayed animals that either hormone
successfully prevented ovarian compensation (Groups C and D versus Group B;
p < 0.001) (table 2). On the other hand,
the same hormonal regimens in intact
pregnant animals actually increased mean
ovarian weight (Groups E and F versus
Group A; p < 0.01, < 0.05, respectively).
Antral follicles and corpora lutea following unilateral ovariectomy. The intact
hamster showed a pronounced increase in
the mean number of antral follicles between days 8 and 12 of pregnancy ( p <
223
0.01) (table 3 ) and the number of follicles
was maintained until day 16. Following
unilateral ovariectomy at day 1 of pregnancy, the remaining ovary contained significantly more follicles at days 4 through
16 than the ovary of the corresponding
intact animal (table 3). The number of
corpora lutea remained constant throughout pregnancy in both the semispayed and
intact hamster. Both progesterone and ECP
prevented the increase in number of antral
follicles that normally developed following
unilateral ovariectomy, but neither hormone altered the number of follicles in the
intact controls (table 3) .
Unilateral ovariectomy and compensatory ovulation during pregnancy. Ovulation can be induced by HCG in the pregnant hamster (Greenwald, '67) but not in
the pregnant rat (Greenwald, '66). The
former observation was also made in this
TABLE 2
Effect of exogenous steroids on ovarian weight in intact and unilaterally
ovariectomized pregnant hamsters
Group
A
B
C
D
E
F
Treatment
(days 8-11 of pregnancy)
Intact control 1
ULO day 1 1
ULO 1 fig estradiol cyclopentylpropionate (ECP)
UL0+2 mg progesterone
Intact 1 pg ECP
Intact 2 mg progesterone
+
+
+
Mean
Mean
luteal wt
ovarian wt
(mg +- SE)
(mg + SE)
y o n day 12 of pregnancy)
24.1 2 0.82 (16)
3 6 . 7 2 1.20 (9)
27.3 2 0.64 ( 7 )
1.1 ? 0.02 (16)
1.2k0.02 ( 5 )
1.220.01 ( 6 )
2 5 . 8 2 1.30 ( 8 )
2 7 . 7 2 0.98 (12)
2 6 . 9 2 1.10 (10)
1.220.03 ( 6 )
1.2C0.07 ( 8 )
l.lfO.04 ( 6 )
Data from table 1; intact control or unilaterally ovariectomized (ULO) hamsters killed on day 12.
TABLE 3
Number o f antral follicles and corpora lutea i n intact or unilaterally
ovariectomized (ULO)pregnant hamsters
Treatment
Intact control
ULO day 1
ULO+ 1 pg ECP 1
ULO+2 mg progesterone 1
Intact+ 1 pg ECP 1
Intact+2 mg progesterone 1
Day of
pregnancy
4
8
12
16
4
8
12
16
12
12
12
12
Mean no.of
antral follicles
Mean no. of
corpora lutea
16.722.91 ( 3 )
19.72 1.47 ( 3 )
40.723.94 ( 3 )
39.028.00 ( 3 )
27.720.72 ( 3 )
34.0k0.00 ( 3 )
58.724.50 ( 3 )
6 1 . 3 2 1.48 ( 3 )
35.02 1.52 ( 3 )
37.0 2 3.60 ( 3 )
32.3k1.31 ( 4 )
32.322.17 ( 4 )
3.021.00 ( 3 )
7.02 1.15 ( 3 )
7.0f2.00(3)
6 . 0 2 1.73 ( 3 )
4.7e0.89 ( 3 )
5 . 0 2 1.00 ( 3 )
7.321.45 ( 3 )
7.321.86 ( 3 )
5.720.89 ( 3 )
9.320.89 ( 3 )
8.5-tO.95 ( 4 )
7.0 2 0.40 (4)
t+- SE)
(+ SE)
1 Estradiol cyclopentylpropionate (ECP) or progesterone injected subcutaneously on days 8 to 11
of pregnancy.
224
AMAR CHATTERJEE AND GILBERT S. GREENWALD
TABLE 4
Induction of ovulation by human chorionic
gonadotropin 1 following various
steroid treatments
Treatment 2
Intact control
ULO Day 1
ULO 1 pg ECP
UL0+2 mg progesterone
+
Mean no. of ova
ovulated per wary
on dav 13:
1 6 , 6 2 1.86 (9)
2 9 . 4 f 1.45 (8)
1 9 , 5 2 1.30 ( 4 )
14,322.25 ( 4 )
1 20 IU HCG on day 12 of pregnancy (SC).
2 Hormones injected subcutaneously from days 8 to
11 of pregnancy of intact or unilaterally ovariectomized (ULO) hamsters.
study; injection of 20 IU of HCG induced
the ovulation of 16.6 ova per ovary in the
intact animals; following semispaying, the
remaining ovary almost doubled its ovulation rate to 29 eggs (p < 0.001 compared
to the intact animal) (table 4). Injection
of semispayed hamsters with ECP or progesterone reduced the number of eggs ovulated to that in the intact pregnant animal.
Uniluteral ovariectomy and gestation.
About 40-50% of the semispayed hamsters delivered viable young before 11 AM
of day 16 of pregnancy; the remainder delivered by 9 AM of day 17. Intact hamsters
delivered over the same time span.
DISCUSSION
Following unilateral ovariectomy of the
pregnant hamster, compensatory changes
in the remaining ovary include increases
in: ( 1 ) ovarian weight; (2) number of
antral follicles; and ( 3 ) ovulation in response to HCG. The first two of these
changes appear within three days of semispaying. Luted weight was unaffected by
semispaying, suggesting that regulation of
the growth of the corpus luteum does not
fit a negative feedback system. Similarly,
luteal growth is unaltered in the pregnant
rat following unilateral ovariectomy, and
ovarian compensation represents an increase in the number of antral follicles
(Chatterjee and Greenwald, '71).
Species differences between the pregnant rat and hamster (see introduction)
account for the temporal difference in maximal ovarian hypertrophy. In the pregnant
rat, the maximal increase in weight of the
remaining ovary occurs at day 10, the approximate midpoint of pregnancy, whereas
in the semispayed hamster a comparable
increase is attained on day 12 of the 16
day gestation period.
The increase in ovarian weight in the
hamster represented mainly a response of
the follicular population; the effects on
interstitial development were difficult to
evaluate. Following unilateral ovariectomy,
there was an approximate doubling in the
number of antral follicles (table 3) and
between 40% and 50% of the follicles
were ovulated by HCG (table 4). The same
relationship holds true for the semispayed
animals treated with estrogen or progesterone and for the intact animals as well.
Thus, the proportion of ovulable follicles
remains constant and is unaffected by a n y
of the experimental manipulations.
In previous studies we have shown that
semispaying of the cyclic hamster (Greenwald, '61), guinea pig (Hermreck and
Greenwald, '64) and rat (Peppler and
Greenwald, '70) results in compensatory
ovulation at the end of the cycle in which
one ovary is removed. The present results
with the pregnant hamster and OUT findings
in the pregnant rat (Chatterjee and Greenwald, '71) indicate that the system controlling the secretion of FSH and LH operates
in pregnancy to the same extent that it
does in the non-pregnant state. That is, the
regulatory mechanism is as sensitive during pregnancy as it is in the estrous cycle
despite the elevation in progesterone levels.
This indicates that this aspect of follicular
development is not affected by endogenous
progesterone even though these levels are
sufficiently high to prevent ovulation.
The question raised by the above findings is: why is progesterone treatment as
effective as estrogen in preventing ovarian
hypertrophy? It is possible that although
the end result is the same, different mechanisms may be involved. In preliminary
experiments involving the intact pregnant
hamster, we have found that the injection
of progesterone, followed by HCG, ovulates an average of 12 ova, whereas estrogen treatment leads to the ovulation of 32
eggs (Greenwald and Chatterjee, unpublished). These results are especially interesting because the administration of progesterone or ECP to intact hamsters
develops the same mean number of antral
follicles (table 3). This suggests that high
levels of progesterone may somehow pre-
OVARIAN COMPENSATION - PREGNANT HAMSTER
vent the physiologic “maturation” of the
follicle to the point where i t is ovulable.
The injection of neither estrogen nor
progesterone affected the number of follicles developing in intact pregnant hamsters
(table 3). These results can be explained
by assuming that neither hormone - in
approximately physiological amounts - inhibits the basal rates of secretion of the
gonadotropins orr alternatively, that there
is a different degree of ovarian responsiveness in the intact hamster.
Another explanation for the ability of
progesterone and estrogen to block ovarian
compensation is that both hormones act on
the same system. This is consistent with
their efficacy in preventing increased serum FSH in the unilaterally ovariectomized
rat (Benson et al., ’69) and in preventing
the increase of plasma FSH and LH in the
bilaterally cvariectomized rat (Labhsetwar, ’69).
Theoretically, the loss of one ovary
might reduce the levels of ovarian steroids
by 50% and consequently partially reduce
their inhibitory effect on gonadotropin secretion. In the unilaterally overiectomized
cyclic pig there is no compensatory increase in progesterone in ovarian venous
effluent (Brinkley and Young, ’69). This is
also true in the semispayed rat, but, once
compensatory ovulation occurs, the secretory rate of progesterone abruptly increases
(Telegdy and Rubin, ’66).
Contradictory findings exist on the levels
of pituitary and plasma gonadotropins in
semispayed rats, varying from reports that
there are no differences from values in intact animals (Edgren et al., ’69) to accounts of augmented levels of gonadotropins (Benson, Sorrentino and Evans, ’69;
Johnson, ’69). Similar experiments in the
cyclic hamster suggest that compensatory
ovulation results from increased secretion
of FSH (Grady and Greenwald, ’68).
ACKNOWLEDGMENTS
A.C. was supported as a Ford Foundation Fellow in Reproductive Biology. The
225
research was supported by grants from the
Ford Foundation and NIH (HD-00596).
The human chorionic gonadotropin was
kindly provided by Ayerst Laboratories.
LITERATURE CITED
Benson, B., S. Sorrentino and J. S. Evans 1969
Increase i n serum FSH following unilateral
ovariectomy i n the rat. Endocrinology, 84:
369-374.
Brinkley, H. J., and E. P. Young 1969 Effects
of unilateral ovariectomy or the unilateral de.
struction of ovarian components on the follicles
and corpora lutea of the nonpregnant pig.
Endocrinology, 84: 1250-1256.
Chatterjee, A., and G. S. Greenwald 1971 Compensatory ovarian hypertrophy following unilateral ovariectomy of the pseudopregnant or
pregnant rat. Endocrinology, 88: 491-496.
Edgren, R. A,, A. F. Parlow, D. L. Peterson and
R. D. Jones 1965 On the mechanism of
ovarian hypertrophy following hemicastration
in rats. Endocrinology, 76: 97-102.
Grady, K. L., and G. S. Greenwald 1968 Studies
on interactions between the ovary and pituitary
follicle stimulating hormone in the golden hamster. J. Endocrinol., 40: 85-90.
Greenwald, G. S. 1961 Quantitative study of
follicular development in the ovary of the intact or unilaterally ovariectomized hamster. J.
Reprod. Fert., 2: 351-361.
1966 Ovarian follicular development
and pituitary FSH and LH content in the pregnant rat. Endocrinology, 79: 572-578.
1967 Induction of ovulation in the
pregnant hamster. Am. J. Anat., 121: 249-258.
Hermreck, A. S., and G. S. Greenwald 1964
The effects of unilateral ovariectomy on follicular maturation i n the guinea pig. Anat.
Rec., 148: 171-176.
Johnson, D. C. 1969 Elevation of plasma gonadotrophins in unilaterally castrated male
rats. J. Endocrinol., 43: 311-312.
Labhsetwar, A. P. 1969 Influence of progesterone on the pituitary and plasma levels of
LH and FSH in the female rat. Biol. Reprod.,
I : 189-196.
Peppler, R. D., and G. S. Greenwald 1970 Effects of unilateral ovariectomy on ovulation
and cycle length in 4- and 5-day cycling rats.
Am. J. Anat., 127: 1-8.
Telegdy, G., and B. L. Rubin 1966 Studies of
ovarian progestin in intact and hemicastrated
young rats. Steroids, 8: 441450.
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