Effects of Unilateral Ovariectomy of the Pregnant Hamster on the Remaining Ovary

AMAR CHATTERJEE AND GILBERT S. GREENWALD Departments of Obstetrics and Gynecology and Anatomy. Universi ty of Kansas Medical Center, Kansas City , Kansas 661 03

ABSTRACT Unilateral ovariectomy of hamsters on day 1 of pregnancy re- sulted 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 as- sessed by injecting either progesterone ( 2 mg) or estradiol cyclopentylpropionate (1 rg) 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, com- pensatory ovarian hypertrophy occurs in the pseudopregnant or pregnant rat (Chat- terjee 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 estro- gen or progesterone.

The present paper deals with the effects of semispaying the pregnant hamster and the possible regulatory mechanisms in- volved. 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 sec- ond 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 go- nadotropin (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, i t was considered worth- while to determine whether the mecha- nisms 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 postovula- tory vaginal discharge occurred. The ani- mals were maintained on a 14 hour light: ten hour dark schedule (lights on: 5 AM-

Received Jan. 7, '71. Accepted Feb. 18, '71.

22 1

222 AMAR CHATTERJEE AND GILBERT S. GREENWALD

7 PM). Females were caged with males of proven fertility on the evening of day 4 (proestrus); the morning of a sperm posi- tive vaginal smear was designated as day 1 of pregnancy. Unilateral ovariectomy (or semispaying) was performed on day 1 by removing either the left or right ovary, via a Bank incision, from alternate hamsters.

Semispayed or intact pregnant hamsters were killed with an overdose of ether on day 4, 8, 12 or 16 of pregnancy (table 1). Ovaries from both groups were dissected free of adherent tissue, blotted and weighed. The five largest corpora lutea of both the intact and the semispayed animals were dissected out and weighed on a tor- sion balance. For histologic studies ovaries were fixed in Bouin’s fluid, sectioned seri- ally at 10 and stained with hematoxylin and eosin. The slides were examined at X25 magnification and the numbers of healthy antral follicles (regardless of size) and corpora lutea in each ovary were counted (results in table 3 ) .

The ability of exogenous steroids to pre- vent ovarian compensation was tested by injecting progesterone or estrogen (table 2). Progesterone and estradiol cyclopentylpro- pionate (ECP) were diluted with sesame oil so that 1.0 ml of the vehicle contained either 10 pg of ECP or 2.0 mg of proges- terone. Groups of semispayed or intact pregnant hamsters were injected daily sub- cutaneously with either steroid from days 8 through 11 of pregnancy and killed on day 12.

In other experiments ovulation induced by exogenous gonadotropin was used as an endpoint (table 4). Intact or semispayed animals were injected subcutaneously on day 12 of pregnancy between 8:30 and 9:30 AM with 20 IU of human chorionic

gonadotropin (HCG) in 0.1 ml of saline. The hamsters were killed the next morn- ing and the oviducts were flushed with physiological saline via the ostium tubae abdominale. Hyaluronidase was used to re- move the granulosa cells adhering to the ova, and the eggs were counted with the aid of a dissecting microscope at X 15 mag- nification.

The significance of the data was deter- mined by the Student’s t test.

RESULTS Unilateral ovariectomy and ovarian

weight during pregnancy. Mean ovarian weight increased progressively in the in- tact pregnant hamster; by day 4 the differ- ence from the day 1 ovary was significant ( p < 0.0005) (table 1). The most striking increase in ovarian weight occurred be- tween days 8 and 12 of pregnancy (p < O . O O l ) , representing growth of the corpora lutea and an increased number of antral follicles (table 3) . Ovarian weight stabi- lized between days 12 and 16 of gestation (table 1 ) and both luteal weight and the number of corpora lutea and of antral fol- licles were unchanged during this period (table 3 ) . Following unilateral ovariec- tomy at day 1, the remaining ovary was significantly heavier by day 4 of pregnancy than the ovary of the intact animal (p < 0.005) (table 1). The ovary of the semi- spayed hamster on days 4, 8, 12 and 16 weighed respectively 17.9, 26.4, 52.7 and 43.4 per cent more than its counterpart in the intact animal. Mean luteal weight did not differ significantly at any stage of preg- nancy between the intact and semispayed hamsters.

Reversal of the e f f ec t s of unilateral ouariectomy by steroids. If semispaying

TABLE 1 Effects of unilateral ovariectomy on ovarian and luteal weight in pregnant hamsters 1

Intact control hamsters 2 Unilaterally ovariectomized hamsters 8

Day of Mean Mean Mean Mean pregnancy ovarian wt luteal wt ovarian wt luteal wt

(mg -c SE) (mg +- SE) (mg c SE) (mg f SE) 1 12.3 k 0.25 (29) - - - 4 14.6 k 0.63 (12) 0.42 f 0.02 ( 10) 17.3f0.57 (10) 0.4320.03 (6) 8 15.9k0.41 (14) 0.79f0.02 (14) 20.121.3 (7) 0.86f0.03 (5) 12 24.1f0.82 (16) 1.1020.02 (14) 36.721.2 (9) 1.20&0.02 (5) 16 24.250.83 (12) 1.20-CO.03 (14) 34.721.4 (9) 1.30k0.05 (6)

1 All operations were done on day 1 of pregnancy (day of sperm in vaginal smear). 2 Numbers in parentheses represent number of animals on which averages are based.

OVARIAN COMPENSATION - PREGNANT HAMSTER 223

decreases peripheral levels of ovarian steroids which regulate gonadotropin se- cretion, it should be feasible to reverse the effects by giving appropriate amounts of estrogen and/or progesterone. This possi- bility 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 compensa- tion (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 follow- ing unilateral ovariectomy. The intact hamster showed a pronounced increase in the mean number of antral follicles be- tween days 8 and 12 of pregnancy ( p <

0.01) (table 3 ) and the number of follicles was maintained until day 16. Following unilateral ovariectomy at day 1 of preg- nancy, the remaining ovary contained sig- nificantly more follicles at days 4 through 16 than the ovary of the corresponding intact animal (table 3). The number of corpora lutea remained constant through- out 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 hor- mone altered the number of follicles in the intact controls (table 3) .

Unilateral ovariectomy and compensa- tory ovulation during pregnancy. Ovula- tion can be induced by HCG in the preg- nant 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 Treatment (days 8-11 of pregnancy)

Mean Mean ovarian wt luteal wt

y o n day 12 of pregnancy) (mg + SE) (mg +- SE)

A Intact control 1 24.1 2 0.82 (16) 1.1 ? 0.02 (16) B ULO day 1 1 36.72 1.20 (9) 1.2k0.02 (5) C ULO + 1 fig estradiol cyclopentyl- 27.3 2 0.64 ( 7 ) 1.220.01 ( 6 )

propionate (ECP) D UL0+2 mg progesterone E Intact + 1 pg ECP F Intact + 2 mg progesterone

25 .82 1.30 ( 8 ) 1.220.03 ( 6 ) 27.72 0.98 (12) 1.2C0.07 ( 8 ) 26.92 1.10 (10) l .lfO.04 ( 6 )

Data from table 1; intact control or unilaterally ovariectomized (ULO) hamsters killed on day 12.

TABLE 3 Number of antral follicles and corpora lutea in intact or unilaterally

ovariectomized (ULO) pregnant hamsters

Treatment Mean no. of Mean no. of

Day of antral follicles corpora lutea pregnancy t+- SE) (+ SE)

Intact control

ULO day 1

4 16.722.91 ( 3 ) 3.021.00 ( 3 ) 7.02 1.15 (3) 8 19.72 1.47 ( 3 )

12 40.723.94 ( 3 ) 7 .0f2 .00 ( 3 ) 16 39.028.00 ( 3 ) 6 . 0 2 1.73 ( 3 ) 4 27.720.72 ( 3 ) 4.7e0.89 (3) 8 34.0k0.00 ( 3 ) 5 . 0 2 1.00 (3)

12 58.724.50 ( 3 ) 7.321.45 ( 3 ) 16 61.32 1.48 ( 3 ) 7.321.86 ( 3 )

ULO+ 1 pg ECP 1 12 35.02 1.52 ( 3 ) 5.720.89 ( 3 ) ULO+2 mg progesterone 1 12 37.0 2 3.60 (3) 9.320.89 (3) Intact+ 1 pg ECP 1 12 32.3k1.31 ( 4 ) 8.5-tO.95 (4) Intact+2 mg progesterone 1 12 32.322.17 ( 4 ) 7.0 2 0.40 (4)

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

Mean no. of ova

on dav 13: Treatment 2 ovulated per wary

Intact control 16,62 1.86 (9) ULO Day 1 2 9 . 4 f 1.45 (8) ULO + 1 pg ECP 19,52 1.30 ( 4 ) UL0+2 mg progesterone 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 ovariecto- mized (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 ovula- tion rate to 29 eggs (p < 0.001 compared to the intact animal) (table 4). Injection of semispayed hamsters with ECP or pro- gesterone reduced the number of eggs ovu- lated to that in the intact pregnant animal.

Uniluteral ovariectomy and gestation. About 40-50% of the semispayed ham- sters delivered viable young before 11 AM of day 16 of pregnancy; the remainder de- livered 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 re- sponse to HCG. The first two of these changes appear within three days of semi- spaying. 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 in- crease in the number of antral follicles (Chatterjee and Greenwald, '71).

Species differences between the preg- nant rat and hamster (see introduction) account for the temporal difference in max- imal ovarian hypertrophy. In the pregnant rat, the maximal increase in weight of the remaining ovary occurs at day 10, the ap- proximate 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 proges- terone and for the intact animals as well. Thus, the proportion of ovulable follicles remains constant and is unaffected by any of the experimental manipulations.

In previous studies we have shown that semispaying of the cyclic hamster (Green- wald, '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 Green- wald, '71) indicate that the system control- ling 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 dur- ing 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 find- ings 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 mech- anisms may be involved. In preliminary experiments involving the intact pregnant hamster, we have found that the injection of progesterone, followed by HCG, ovu- lates an average of 12 ova, whereas estro- gen treatment leads to the ovulation of 32 eggs (Greenwald and Chatterjee, unpub- lished). These results are especially inter- esting because the administration of pro- gesterone 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 225

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 folli- cles developing in intact pregnant hamsters (table 3). These results can be explained by assuming that neither hormone - in approximately physiological amounts - in- hibits the basal rates of secretion of the gonadotropins orr alternatively, that there is a different degree of ovarian responsive- ness 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 se- rum 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 (Labhset- war, ’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 se- cretion. In the unilaterally overiectomized cyclic pig there is no compensatory in- crease in progesterone in ovarian venous effluent (Brinkley and Young, ’69). This is also true in the semispayed rat, but, once compensatory ovulation occurs, the secre- tory 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 in- tact animals (Edgren et al., ’69) to ac- counts of augmented levels of gonadotro- pins (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 Founda- tion Fellow in Reproductive Biology. The

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 in serum FSH following unilateral ovariectomy in the rat. Endocrinology, 84:

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 Com- pensatory ovarian hypertrophy following uni- lateral 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 ham- ster. J. Endocrinol., 40: 85-90.

Greenwald, G. S. 1961 Quantitative study of follicular development in the ovary of the in- tact or unilaterally ovariectomized hamster. J. Reprod. Fert., 2: 351-361.

1966 Ovarian follicular development and pituitary FSH and LH content in the preg- nant 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 fol- licular maturation in the guinea pig. Anat. Rec., 148: 171-176.

Johnson, D. C. 1969 Elevation of plasma go- nadotrophins in unilaterally castrated male rats. J. Endocrinol., 43: 311-312.

Labhsetwar, A. P. 1969 Influence of proges- terone on the pituitary and plasma levels of LH and FSH in the female rat. Biol. Reprod.,

Peppler, R. D., and G. S. Greenwald 1970 Ef- fects 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.

369-374.

I : 189-196.