- Email: [email protected]
Pregnant Mare’s Serum gonadotropin. III. Hemispaying and the Reversal of the Antifertility Faculty of Pregnant Mare’s Serum Gonadotropin*
FERTILITY AND STERILITY Copyright' 1977 The American Fertility Society
Vol. 28, No. 10, October 1977 Printed in U.S.A.
PREGNANT MARE'S SERUM GONADOTROPIN. III. HEMISPAYING AND THE REVERSAL OF THE ANTIFERTILITY FACULTY OF PREGNANT MARE'S SERUM GONADOTROPIN*
AMAR CHATIERJEE, PH.D., D.Sc.t ALOK K. PAL, PH.D. TARUN GUPTA, PH.D.
Department of Physiology, Raja Peary Mohan College, Uttarpara, Hooghly, West Bengal 712258, India
A single injection of 10 1U of pregnant mare's serum gonadotropin (PMSG) on day 5 of pregnancy caused wastage of the fetoplacental unit by day 16 of pregnancy. Semispaying, which seems to subtract approximately 50% of the ovarian steroid contribution, at 24, 48, and 72 hours following the PMSG schedule prevented the antifertility faculty of the hormone preparation. However, hemicastration at 96 hours following the gonadotropin regimen was found to be ineffective, and 100% of the test animals showed complete termination of pregnancy. Experimental data collectively tempt us to propose that an estrogen excess, particularly of follicular origin in both ovaries, is essential for more than 72 hours following gonadotropin sensitization before the antifertility faculty of PMSG can be demonstrated.
Pregnant mare's serum gonadotropin (PMSG), a long-acting follicle-stimulating hormone (FSH)like hormone, poss~sses the biologic properties of both FSH and luteinizing hormone (LH). Because of its FSH predominancy, PMSG acts primarily at the ovarian follicular level and stimulates an enhanced production of estrogen, since induced superovulation by PMSG is known to be caused by an ovulatory surge of LH through a positive feedback effect of estrogen. I The luteolytic characteristic of PMSG has recently been shown to be associated with an ovarian polyfollicular state. 2 Estrogen is evidently luteolytic in a number of mammalian species, including humans,3-6 and reduces the weight as well as the progesterone content of the corpus luteum. 7-9 In the present paper an attempt has been made to determine whether negation of approximately 50% of the ovarian steroid contribution by surgical manipu-
lation can prevent the pregnancy wastage faculty ofPMSG. MATERIALS AND METHODS
Received April 18, 1977; accepted May 6, 1977. *Supported by a research grant from the Indian Council of Medical Research, University Grants Commission, New Delhi (to T. G.); Grant M 76.18 from The Population Council, New York, N. Y.; and a grant from the World Health Organization (Human Reproduction Unit/Small Supplies Programme). tTo whom reprint requests should be addressed.
1101
Laboratory-bred albino rats weighing 180 to 200 gm were used. The animals were housed under a controlled lighting schedule of 12 hours of light/ day and had free access to food pellets and water. The constancy of the estrous cycle was confirmed before the animals were subjected to experimentation. Females were caged with males of proven fertility on the evening of vaginal proestrus. Insemination was confirmed the next morning by the presence of either spermatozoa or a vaginal plug. The day on which spermatozoa or vaginal plugs were found was designated day 1 of pregnancy. Pregnant rats were placed into groups according to the treatment schedule (Table 1). PMSG (Anteron), donated by Schering AG, Berlin, Germany, was dissolved in 0.9% NaCI solution and injected subcutaneously at a dose of 10 IV (0.2 ml) on day 5; control animals received only the saline vehicle. Surgical hemispaying was performed on left and right ovaries alternatel,,- at
1102
CHATI'ERJEE ET AL.
October 1977
TABLE 1. Reversal of PMSG-Induced Pregnancy Wastage by Hemispaying in Rats Treatment (day of pregnancy)
Control PMSG PMSG PMSG PMSG
(day (day (day (day
5) 5) 5) 5)
+ + + +
ULO ULO ULO ULO
(day (day (day (day
6) 7) 8) 9)
Day of sacrifice ll
Fetal wt.' mg
mg
16 (6)
296.4 ± 7.8
299.8 ± 13.6
53.0 ± 4.5
4.2 ± 0.1
16 16 16 16
279.5 ± 4.2 310.8 ± 7.1 297.9 ± 6.1 Resorbed
285.3 ± 8.8 344.8 ± 23.6 318.1 ± 9.4 Resorbed
77.0 62.5 58.7 58.8
4.2 4.5 4.6 2.3
(8) (8) (10) (8)
Placental wt.'
CL wt.'
Ovarian wt.'
mg
± ± ± ±
mg
9.9" 5.6 3.7 4.9
± ± ± ±
0.2 0.2 0.3 0.6"
"ULO, Unilateral ovariectomy. "Numbers in parentheses are numbers of animals. '"Mean ± standard error of the mean. "P < 0.05. "P < 0.01.
24, 48, 72, and 96 hours after the hormone regimen with an idea that, following the distribution of gonadotropic hormone in both ovaries, surgical extirpation of one ovary could eliminate approximately 50% of the ovarian hormonal contribution-particularly of the stimulated follicular elements. Control animals were subjected to an identical operative procedure. Implanted blastocysts were counted through a midventral incision on day 8 of pregnancy with the animals under light ether anesthesia. Animals were killed on day 16 of pregnancy, and at autopsy the weights of the fetuses, placentas, ovaries, and microdissected corpora lutea (CL) were statistically analyzed with Student's t-test. RESULTS AND DISCUSSION
Preliminary data from our laboratory revealed that the administration to rats of 10 IU of PMSG on day 5 of pregnancy leads to complete resorption of the fetoplacental unit by day 16. 2 However, in the present study surgical extirpation of one ovary 24 hours after the injection of PMSG resulted in the absolute maintenance of pregnancy in all of the test animals. The fetal survival rate on the day of sacrifice (day 16) was recorded to be 91.9% as compared with the control value of 95.8% (Table 2). The gravimetric growth status of fetuses, placentas, and CL was also maintained in parallel with that of controls (Table 1). Hemispaying performed at 48 and 72 hours following the hormone regimen similarly resulted in pregnancy maintenance in aU of the animals; however, fetal survival rates were reduced to 98.8% and 68.3%, respectively (Table 2). The average weights of the surviving fetuses, placentas, andCL in both groups of animals were found to be statistically identical with those of controls (Table 1). Conversely, ablation of one ovary 96 hours after gonadotropin administration failed to continue
pregnancy in 100% of the test animals. The intrauterine embryos were completely resorbed and the degree of luteal regression was highly significant (P < 0.01). The collective data of the present experiments indicate that an altered hormonal contribution by PMSG-sensitized ovaries is essential for more than 72 hours in order to demonstrate the antifertility faculty ofPMSG. However, studies of the ovarian hormone status following PMSG sensitization have demonstrated the stimulation of an appreciable quantity of estrogen, 10 probably offollicular origin. 2 The luteolytic property of this increased level of estrogen has been established in a variety of mammalian species, including humans. 3 -6 Moreover, cyclic luteolysis has been shown to be dependent on the availability of follicular estrogen, since x-irradiation which destroys ovarian follicles retards the rate of luteal involution." The successful use of a specific antiestrogenl2 or progesterone 2 to prevent the antifertility faculty of PMSG tempts us to speculate that PMSG induces luteolysis by activating ovarian estrogen.IO This estrogen, in turn, may act either by stimulating '3 or inhibiting 14 endogenous LH. LH constitutes one of the important components of the luteotropic complex in rats l5 ; however, its excessive availability is often luteolytic '6 through the stimulation of ovarian prosTABLE 2. Effect of Unilateral Ovariectomy (ULO) on the Fetal Survival Rate in PMSG-Treated Pregnant Rats No. of implantation Treatment (day of pregnancy)
sites at laparotomy
No. of fetuses at sacrifice
(day 8)
(day 16)
48
46
95.8
74 76 82 72
68 69 56 0
91.9 90.8 68.3 0
Survival rate %
Control PMSG (day PMSG (day PMSG (day PMSG (day
5) 5) 5) 5)
+ ULO + ULO + ULO + ULO
(day (day (day (day
6) 7) 8) 9)
PREGNANT MARE'S SERUM GONADOTROPIN. III
Vol. 28, No. 10
taglandin activityl7 and thus luteal 20a-hydroxysteroid dehydrogenase activity.18 Estrogen has also been suggested to act at the luteal level, blocking the LH action 8 on steroidogenesis III animals as well as in humans. 9 Acknowledgments. The encouraging cooperation of Dr. S. Tejuja, Dr. R. T. Mahoney, Dr. S. J. Segal, and Dr. J. M. Spieler is greatly appreciated. We are grateful to Roche Products Ltd., Bombay, for providing the vitamin preparation used.
REFERENCES 1. Zarrow MX, Caldwell AL Jr, Hafez ESE, Pincus G: Superovulation in the immature rat as a possible assay for LH and HCG. Endocrinology 63:748,1958 2. Pal AK, Gupta T, Chatterjee A: Pregnant mare's serum gonadotrophin. I. Progesterone or prolactin and the reversal of antifertility efficacy of pregnant mare's serum gonadotrophin. Acta Endocrinol (Kbh) 83:506, 1976 3. Sen K: Studies on some aspects of estrogen-induced luteolysis in pregnant rats. Ph.D. thesis, Calcutta University, Calcutta, India, 1977 4. Stormshak F, Kelley HE, Hawk HW: Suppression of ovine luteal function by 17f3-estradiol. J Anim Sci 29:476, 1973 5. Karsch FJ, Krey LC, Weick RF, Dierschke DJ, Knobil E: Functional luteolysis in the rhesus monkey. Endocrinology 92:1148, 1973 6. Gore BZ, Caldwell BV, Speroff L: Estrogen-induced human luteolysis. J Clin Endocrinol Metab 36:615, 1973 7. Brunner MA, Donaldson ME, Hansel W: Exogenous hormone and luteal function in hysterectomized and intact heifers. J Dairy Sci 52:1849, 1969 8. Cook B, Karsch FJ, Foster DL, Nalbandov AV: Estrogen-induced luteolysis in ewe. Possible site of action. Endocrinology 94:1197, 1974
1103
9. Karsch FJ, Sutton GP: An intra-ovarian site for the luteolytic action of estrogen in the rhesus monkey. Endocrinology 98:553, 1976 10. Armstrong DT, Greep RO: Failure of deciduomal response to uterine trauma and effects ofLH (luteinizing hormone) on estrogen secretion in rats with ovaries luteinizing by exogenous gonadotropins. Endocrinology 76:246, 1965 11. Karsch FJ, Novaroske JW, Roche JF, Norton HW, Nalbandov AV: Maintenance ofovine corpora lutea in the absence of ovarian follicles. Endocrinology 87:1228, 1970 12. Chatterjee A, Pal AK, Gupta T: Pregnant mare's serum gonadotrophin. II. Reversal of the antifertility faculty of pregnant mare's serum gonadotrophin by using clomiphene citrate or reserpine in rats. Contraception 15:571, 1977 13. Everett JW: Hormonal factor responsible for deposition of cholesterol in the CL of the rat. Endocrinology 41:364, 1974 14. Mepharson JC, Eldridge JC, Costoff A, Mahesh VB: Pituitary-gonadal axis before puberty. Effects of various estrogen steroids in the ovariectomized rat. Steroids 24: 41, 1974 15. Greenwald GS, Rothchild I: Formation and maintenance of corpora lutea in laboratory animals. J Anim Sci 27:139, 1968 16. Chatterjee A: Combination of prolactin and human chorionic gonadotropin: an evidence of luteotrophic or luteolytic complex in rats (abstr FP7). Presented at the Symposium on Advances in Chemistry, Biology and Immunology of Gonadotropin, Bangalore, India, 1973 17. Bauminger S, Leiberman ME, Linder HR: Steriod independent effect of gonadotropins on prostaglandin synthesis in rat Graafian follicles in vitro. Prostaglandins 9:753, 1975 18. Kidwell WR, Balogh K Jr, Wiest WG: Effect ofluteinizing hormone on glucose-6-phosphate and 20a-hydroxysteroid dehydrogenase activity in superovulated rat ovary. Endocrinology 79:352, 1966
Vol. 28, No. 10, October 1977 Printed in U.S.A.
PREGNANT MARE'S SERUM GONADOTROPIN. III. HEMISPAYING AND THE REVERSAL OF THE ANTIFERTILITY FACULTY OF PREGNANT MARE'S SERUM GONADOTROPIN*
AMAR CHATIERJEE, PH.D., D.Sc.t ALOK K. PAL, PH.D. TARUN GUPTA, PH.D.
Department of Physiology, Raja Peary Mohan College, Uttarpara, Hooghly, West Bengal 712258, India
A single injection of 10 1U of pregnant mare's serum gonadotropin (PMSG) on day 5 of pregnancy caused wastage of the fetoplacental unit by day 16 of pregnancy. Semispaying, which seems to subtract approximately 50% of the ovarian steroid contribution, at 24, 48, and 72 hours following the PMSG schedule prevented the antifertility faculty of the hormone preparation. However, hemicastration at 96 hours following the gonadotropin regimen was found to be ineffective, and 100% of the test animals showed complete termination of pregnancy. Experimental data collectively tempt us to propose that an estrogen excess, particularly of follicular origin in both ovaries, is essential for more than 72 hours following gonadotropin sensitization before the antifertility faculty of PMSG can be demonstrated.
Pregnant mare's serum gonadotropin (PMSG), a long-acting follicle-stimulating hormone (FSH)like hormone, poss~sses the biologic properties of both FSH and luteinizing hormone (LH). Because of its FSH predominancy, PMSG acts primarily at the ovarian follicular level and stimulates an enhanced production of estrogen, since induced superovulation by PMSG is known to be caused by an ovulatory surge of LH through a positive feedback effect of estrogen. I The luteolytic characteristic of PMSG has recently been shown to be associated with an ovarian polyfollicular state. 2 Estrogen is evidently luteolytic in a number of mammalian species, including humans,3-6 and reduces the weight as well as the progesterone content of the corpus luteum. 7-9 In the present paper an attempt has been made to determine whether negation of approximately 50% of the ovarian steroid contribution by surgical manipu-
lation can prevent the pregnancy wastage faculty ofPMSG. MATERIALS AND METHODS
Received April 18, 1977; accepted May 6, 1977. *Supported by a research grant from the Indian Council of Medical Research, University Grants Commission, New Delhi (to T. G.); Grant M 76.18 from The Population Council, New York, N. Y.; and a grant from the World Health Organization (Human Reproduction Unit/Small Supplies Programme). tTo whom reprint requests should be addressed.
1101
Laboratory-bred albino rats weighing 180 to 200 gm were used. The animals were housed under a controlled lighting schedule of 12 hours of light/ day and had free access to food pellets and water. The constancy of the estrous cycle was confirmed before the animals were subjected to experimentation. Females were caged with males of proven fertility on the evening of vaginal proestrus. Insemination was confirmed the next morning by the presence of either spermatozoa or a vaginal plug. The day on which spermatozoa or vaginal plugs were found was designated day 1 of pregnancy. Pregnant rats were placed into groups according to the treatment schedule (Table 1). PMSG (Anteron), donated by Schering AG, Berlin, Germany, was dissolved in 0.9% NaCI solution and injected subcutaneously at a dose of 10 IV (0.2 ml) on day 5; control animals received only the saline vehicle. Surgical hemispaying was performed on left and right ovaries alternatel,,- at
1102
CHATI'ERJEE ET AL.
October 1977
TABLE 1. Reversal of PMSG-Induced Pregnancy Wastage by Hemispaying in Rats Treatment (day of pregnancy)
Control PMSG PMSG PMSG PMSG
(day (day (day (day
5) 5) 5) 5)
+ + + +
ULO ULO ULO ULO
(day (day (day (day
6) 7) 8) 9)
Day of sacrifice ll
Fetal wt.' mg
mg
16 (6)
296.4 ± 7.8
299.8 ± 13.6
53.0 ± 4.5
4.2 ± 0.1
16 16 16 16
279.5 ± 4.2 310.8 ± 7.1 297.9 ± 6.1 Resorbed
285.3 ± 8.8 344.8 ± 23.6 318.1 ± 9.4 Resorbed
77.0 62.5 58.7 58.8
4.2 4.5 4.6 2.3
(8) (8) (10) (8)
Placental wt.'
CL wt.'
Ovarian wt.'
mg
± ± ± ±
mg
9.9" 5.6 3.7 4.9
± ± ± ±
0.2 0.2 0.3 0.6"
"ULO, Unilateral ovariectomy. "Numbers in parentheses are numbers of animals. '"Mean ± standard error of the mean. "P < 0.05. "P < 0.01.
24, 48, 72, and 96 hours after the hormone regimen with an idea that, following the distribution of gonadotropic hormone in both ovaries, surgical extirpation of one ovary could eliminate approximately 50% of the ovarian hormonal contribution-particularly of the stimulated follicular elements. Control animals were subjected to an identical operative procedure. Implanted blastocysts were counted through a midventral incision on day 8 of pregnancy with the animals under light ether anesthesia. Animals were killed on day 16 of pregnancy, and at autopsy the weights of the fetuses, placentas, ovaries, and microdissected corpora lutea (CL) were statistically analyzed with Student's t-test. RESULTS AND DISCUSSION
Preliminary data from our laboratory revealed that the administration to rats of 10 IU of PMSG on day 5 of pregnancy leads to complete resorption of the fetoplacental unit by day 16. 2 However, in the present study surgical extirpation of one ovary 24 hours after the injection of PMSG resulted in the absolute maintenance of pregnancy in all of the test animals. The fetal survival rate on the day of sacrifice (day 16) was recorded to be 91.9% as compared with the control value of 95.8% (Table 2). The gravimetric growth status of fetuses, placentas, and CL was also maintained in parallel with that of controls (Table 1). Hemispaying performed at 48 and 72 hours following the hormone regimen similarly resulted in pregnancy maintenance in aU of the animals; however, fetal survival rates were reduced to 98.8% and 68.3%, respectively (Table 2). The average weights of the surviving fetuses, placentas, andCL in both groups of animals were found to be statistically identical with those of controls (Table 1). Conversely, ablation of one ovary 96 hours after gonadotropin administration failed to continue
pregnancy in 100% of the test animals. The intrauterine embryos were completely resorbed and the degree of luteal regression was highly significant (P < 0.01). The collective data of the present experiments indicate that an altered hormonal contribution by PMSG-sensitized ovaries is essential for more than 72 hours in order to demonstrate the antifertility faculty ofPMSG. However, studies of the ovarian hormone status following PMSG sensitization have demonstrated the stimulation of an appreciable quantity of estrogen, 10 probably offollicular origin. 2 The luteolytic property of this increased level of estrogen has been established in a variety of mammalian species, including humans. 3 -6 Moreover, cyclic luteolysis has been shown to be dependent on the availability of follicular estrogen, since x-irradiation which destroys ovarian follicles retards the rate of luteal involution." The successful use of a specific antiestrogenl2 or progesterone 2 to prevent the antifertility faculty of PMSG tempts us to speculate that PMSG induces luteolysis by activating ovarian estrogen.IO This estrogen, in turn, may act either by stimulating '3 or inhibiting 14 endogenous LH. LH constitutes one of the important components of the luteotropic complex in rats l5 ; however, its excessive availability is often luteolytic '6 through the stimulation of ovarian prosTABLE 2. Effect of Unilateral Ovariectomy (ULO) on the Fetal Survival Rate in PMSG-Treated Pregnant Rats No. of implantation Treatment (day of pregnancy)
sites at laparotomy
No. of fetuses at sacrifice
(day 8)
(day 16)
48
46
95.8
74 76 82 72
68 69 56 0
91.9 90.8 68.3 0
Survival rate %
Control PMSG (day PMSG (day PMSG (day PMSG (day
5) 5) 5) 5)
+ ULO + ULO + ULO + ULO
(day (day (day (day
6) 7) 8) 9)
PREGNANT MARE'S SERUM GONADOTROPIN. III
Vol. 28, No. 10
taglandin activityl7 and thus luteal 20a-hydroxysteroid dehydrogenase activity.18 Estrogen has also been suggested to act at the luteal level, blocking the LH action 8 on steroidogenesis III animals as well as in humans. 9 Acknowledgments. The encouraging cooperation of Dr. S. Tejuja, Dr. R. T. Mahoney, Dr. S. J. Segal, and Dr. J. M. Spieler is greatly appreciated. We are grateful to Roche Products Ltd., Bombay, for providing the vitamin preparation used.
REFERENCES 1. Zarrow MX, Caldwell AL Jr, Hafez ESE, Pincus G: Superovulation in the immature rat as a possible assay for LH and HCG. Endocrinology 63:748,1958 2. Pal AK, Gupta T, Chatterjee A: Pregnant mare's serum gonadotrophin. I. Progesterone or prolactin and the reversal of antifertility efficacy of pregnant mare's serum gonadotrophin. Acta Endocrinol (Kbh) 83:506, 1976 3. Sen K: Studies on some aspects of estrogen-induced luteolysis in pregnant rats. Ph.D. thesis, Calcutta University, Calcutta, India, 1977 4. Stormshak F, Kelley HE, Hawk HW: Suppression of ovine luteal function by 17f3-estradiol. J Anim Sci 29:476, 1973 5. Karsch FJ, Krey LC, Weick RF, Dierschke DJ, Knobil E: Functional luteolysis in the rhesus monkey. Endocrinology 92:1148, 1973 6. Gore BZ, Caldwell BV, Speroff L: Estrogen-induced human luteolysis. J Clin Endocrinol Metab 36:615, 1973 7. Brunner MA, Donaldson ME, Hansel W: Exogenous hormone and luteal function in hysterectomized and intact heifers. J Dairy Sci 52:1849, 1969 8. Cook B, Karsch FJ, Foster DL, Nalbandov AV: Estrogen-induced luteolysis in ewe. Possible site of action. Endocrinology 94:1197, 1974
1103
9. Karsch FJ, Sutton GP: An intra-ovarian site for the luteolytic action of estrogen in the rhesus monkey. Endocrinology 98:553, 1976 10. Armstrong DT, Greep RO: Failure of deciduomal response to uterine trauma and effects ofLH (luteinizing hormone) on estrogen secretion in rats with ovaries luteinizing by exogenous gonadotropins. Endocrinology 76:246, 1965 11. Karsch FJ, Novaroske JW, Roche JF, Norton HW, Nalbandov AV: Maintenance ofovine corpora lutea in the absence of ovarian follicles. Endocrinology 87:1228, 1970 12. Chatterjee A, Pal AK, Gupta T: Pregnant mare's serum gonadotrophin. II. Reversal of the antifertility faculty of pregnant mare's serum gonadotrophin by using clomiphene citrate or reserpine in rats. Contraception 15:571, 1977 13. Everett JW: Hormonal factor responsible for deposition of cholesterol in the CL of the rat. Endocrinology 41:364, 1974 14. Mepharson JC, Eldridge JC, Costoff A, Mahesh VB: Pituitary-gonadal axis before puberty. Effects of various estrogen steroids in the ovariectomized rat. Steroids 24: 41, 1974 15. Greenwald GS, Rothchild I: Formation and maintenance of corpora lutea in laboratory animals. J Anim Sci 27:139, 1968 16. Chatterjee A: Combination of prolactin and human chorionic gonadotropin: an evidence of luteotrophic or luteolytic complex in rats (abstr FP7). Presented at the Symposium on Advances in Chemistry, Biology and Immunology of Gonadotropin, Bangalore, India, 1973 17. Bauminger S, Leiberman ME, Linder HR: Steriod independent effect of gonadotropins on prostaglandin synthesis in rat Graafian follicles in vitro. Prostaglandins 9:753, 1975 18. Kidwell WR, Balogh K Jr, Wiest WG: Effect ofluteinizing hormone on glucose-6-phosphate and 20a-hydroxysteroid dehydrogenase activity in superovulated rat ovary. Endocrinology 79:352, 1966