Effects of unilateral ovariectomy of the pregnant hamster on the remaining ovary.код для вставкиСкачать
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. 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