0013-7227/91/1283-1647$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 3 Printed in U.S.A.
Expression of Ovarian Inhibin during Pregnancy in the Rat* TERESA K. WOODRUFF, JACQUELINE ACKLAND, JASON 0. RAHAL, NEENA B. SCHWARTZ, AND KELLY E. MAYO Departments of Biochemistry, Molecular Biology, and Cell Biology (T.K. W., J.O.R., K.E.M.) and Neurobiology and Physiology (J.A., N.B.S.), Northwestern University, Evanston, Illinois 60208
ABSTRACT. We have examined the expression of the rat inhibin genes in the maternal ovary during pregnancy. RNA blot analysis indicates that the inhibin-a chain mRNA is expressed in the ovary throughout gestation at levels comparable to those observed in cycling rats. In situ hybridization shows that the inhibin-a and -/?A mRNAs are produced in the granulosa cells of developing antral follicles; little or no hybridization to functional corpora lutea is observed. Early in pregnancy, a large number of follicles hybridize to both a- and /3A-inhibin cDNA probes. Many of these follicles undergo atresia during the first half of pregnancy, and the number of inhibin-expressing follicles reaches a nadir on day 15. This is followed by an increase in inhibin-
producing follicles, which peaks just before parturition. The increase in inhibin-expressing follicles observed in late pregnancy corresponds to a small rise in serum inhibin levels, as measured using an a chain-specific RIA. After the first postpartum ovulation, few hybridizing follicles are observed. Ovariectomy in either early (day 6) or mid (day 15) pregnancy results in a significant fall in serum inhibin levels and a robust increase in serum FSH levels 9 h after surgery. These results suggest that inhibin is produced by the maternal ovary during pregnancy, that its synthesis is modulated during late gestation, and that inhibin may play a role in regulating FSH secretion during pregnancy. (Endocrinology 128: 1647-1654,1991)
T
HE TRANSITION from regular estrous cycles to a prolonged 22-day gestation period occurs as a consequence of fertilization and implantation in the rat. To achieve this change, the hypothalamus, pituitary, adrenals, and ovaries must make significant shifts in the release of tropic hormones, alter steroidogenesis, modify the follicular and luteal population, and regulate the availability of cellular receptors (1-5). Furthermore, the embryonic endocrine system must rapidly develop response mechanisms to incoming hormonal cues. There is evidence that this interaction may occur as early as the time of implantation (6). These complex interactions between maternal and fetal systems must be highly integrated and are not completely understood. As a result of these changes, the hormonal profiles of circulating gonadotropins and steroid hormones during pregnancy are dramatically different from those found during the estrous cycle. The pituitary gonadotropins LH and FSH remain low throughout pregnancy (2). Received August 14,1990. Address all correspondence and requests for reprints to: Dr. Kelly E. Mayo, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, Illinois 60208. * This work was supported by NSF Presidential Young Investigators Award DCB-8552977 (to K.E.M.), Chicago Community Trust Searle Scholars Program Grant 87-G-113 (to K.E.M.), NIH Grant HD-07507 (to N.B.S.), and NICHHD Program Project Grant HD-21921.
Serum steroids, however, attain levels greater than those found at any time during the normal estrous cycle. Luteal tissues play a predominant role in producing these steroids. During midpregnancy, serum estradiol and testosterone levels rise, and progesterone production is greatly elevated until the corpora lutea undergo functional luteolysis on day 18 in preparation for parturition and postpartum ovulation (7). It is clear that feedback systems must be established from the maternal ovary to maintain the reproductive status of pregnancy. One hormone that may be important in the modulation of FSH secretion during pregnancy is inhibin. Inhibin is a heterodimer composed of an a and a ]0 chain that selectively suppresses FSH secretion from the anterior pituitary (8-11). Two related forms of inhibin (A and B) (12) are generated by combination of the unique a chain with one of two highly related /? chains (j8A and j8B). Inhibin-a and -jS cDNA clones have been isolated from rat ovary (13, 14), and we and others have previously characterized the expression of the inhibin mRNAs in the ovary during the rat estrous cycle (15, 16). Inhibin mRNAs were found to be localized predominantly to the granulosa cells of maturing antral follicles in the cycling rat, and inhibin mRNA levels were found to change as antral follicles developed, matured, and ovulated. We wished to extend these studies to examine the
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INHIBIN EXPRESSION DURING PREGNANCY
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pattern of inhibin expression in the ovary during gestation for several reasons. Firstly, the patterns of follicular development and atresia differ from those during the estrous cycle; there is no clear wave-like change in the population of antral follicles, and the follicles of early pregnancy undergo atresia (4). Secondly, the corpora lutea of pregnancy are functional, producing high levels of progesterone (2). Finally, FSH is maintained at a low level throughout the 22-day gestation period (1, 4), in contrast to the marked dynamic changes in FSH levels that occur during the estrous cycle. To begin to address these issues, we have examined the localization and regulation of rat inhibin-a and -/3A mRNAs in the maternal ovary during pregnancy. Materials and Methods Animals and collection of tissue Timed pregnant rats were obtained from Charles Rivers Breeding Laboratory (Wilmington, MA). The day of insemination was designated day 1. Cycling rats were obtained from the same source and were monitored daily for vaginal cytology to determine cycle stage. Animals were used only after at least two consecutive 4 day cycles. All rats were maintained in separate cages on a 14-h light, 10-h dark cycle (lights on at 0500 h), with water and food provided. Animals were decapitated on each of the indicated days of gestation or of the estrous cycle at 1000 h, and trunk blood was collected. Ovaries, livers, and placentas were excised, frozen directly on dry ice, and stored at -70 C. Blood samples were allowed to clot overnight at 4 C, centrifuged at 800 X g for 20 min, aliquoted, and stored at -20 C until assayed for serum gonadotropins, progesterone, or inhibin. Endocrine ablation experiments Day 6 and day 15 pregnant animals were anesthetized at 0800 h using metaphane. Lateral abdominal incisions were made under sterile conditions, and ovaries were removed (or simply touched in the sham-operated animals). The abdominal wall was sutured, and the animals were allowed to recover under heated lights. Nine hours after surgery (1700 h), the animals were decapitated, and trunk blood was collected. Four animals were used per group, except for the sham-operated group on day 15 in which only three animals were used. RNA blot analysis RNA was prepared from frozen ovaries by homogenization in guanidine isothiocynate, as described by Chomczynski and Sacchi (17). Twenty micrograms of each total RNA were electrophoresed on a denaturing agarose-formaldehyde gel (18) with RNA size markers from Bethesda Research Laboratories (Besthesda, MD), and the RNA was transferred to a nylon membrane by blotting in 20 X SSC (standard saline citrate) for 16 h. The gel was stained with acrydine orange after electrophoresis and after blotting to check for equivalent loading and complete transfer of RNAs. Hybridization was to the 1500-
Endo • 1991 Voll28«No3
basepair (bp) insert of rat inhibin-a cDNA clone rINA-13 radiolabeled by nick translation (13). In situ hybridization Twenty-micron frozen sections of ovary or liver (control) were cut on a Reichart 820 cryostat (Buffalo, NY) and mounted alternately between two microscope slides for eventual hybridization to a and /3A probes. Cross-sections of the ovary were taken with respect to the attached oviduct to maintain a standard orientation. Sections were fixed in 5% paraformaldehyde, treated with acetic anhydride in 0.1 M triethanolamine for 10 min, dehydrated through an ethanol series, and stored in a vacuum desiccator until they were hybridized (350 /xm), is similar to that found during the estrous cycle (15, 16). The luteal tissue of pregnancy is particularly interesting, in that corpora lutea produce progesterone during most of gestation (2). In either functional (day 6-18) or nonfunctional (day 19-21) corpora
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INHIBIN EXPRESSION DURING PREGNANCY
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a Follicle
100X
500X
Corpus Luteum 100X
500X
FIG. 3. Expression of inhibin mRNAs in follicular and luteal tissues. Sequential sections from the ovary of an animal on day 18 of gestation were analyzed using in situ hybridization. The left panels were hybridized to an inhibin-a chain probe; the right panels to an inhibin-/3A chain probe. In the upper panel, a healthy antral follicle is shown at both XlOO and X500 magnifications, as indicated. Arrow 1 points to the granulosa cell layer; arrow 2 points to the thecal cell layer. In the lower panel, a functional corpus luteum is shown at both XlOO and X500 magnifications, as indicated. Arrow 3 points to luteal cells. The X500 magnifications are from the boxed area on the XlOO magnification photographs.
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Endo • 1991 Vol 128 • No 3
INHIBIN EXPRESSION DURING PREGNANCY
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1500
30
750 i A • Intact •IOVX
EHD # Follicles Serum Inhibin
1000 E
I
2
a
500-
I
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iibin
16E
.5 co
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o g 10
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FlG. 4. Inhibin expression during pregnancy. On each indicated day of gestation, the number of follicles hybridizing to both the a and /3A chain inhibin probes per 100 ovarian sections (which essentially represents 1 ovary) was determined using in situ hybridization. Sera from these same animals as well as several additional animals were analyzed for inhibin by RIA (filled symbols). Values are presented as the mean ± SE for 2 different animals on each day (in situ hybridizations) or 5 different animals on each day (inhibin RIA). Parturition was on day 22 or 23.
}•
400 Day 6
Day 15
FIG. 6. Effects of ovariectomy on serum FSH and inhibin levels during pregnancy. Intact, sham-operated, or bilaterally ovariectomized rats were analyzed. Sham-operated animals were not different from intact animals and are not shown. Surgery was on either day 6 or day 15 of gestation, and serum hormone values were measured 9 h later. FSH values are shown in the top panel (A), and inhibin values in the bottom panel (B). Values are the mean ± SE. By two-way analysis of variance: FSH ovariectomy, P < 0.001; and inhibin ovariectomy, P < 0.001. 5 10 Day of Gestation
20
Day of 25 Delivery
FlG. 5. Serum gonadotropin and progesterone levels during pregnancy. Sera from the same animals represented in Fig. 4 were analyzed for FSH, LH, and progesterone by RIA. The limit of detection of the LH assay was 0.3 ng/ml. Values are presented as the mean ± SE for five different animals on each day. Parturition was on day 22.
lutea we did not detect expression of inhibin-a or -j8A mRNA during gestation. During the estrous cycle we have observed transient expression of inhibin-a mRNA in the forming corpus luteum early on estrous morning (29), but have not seen any expression of either a or /?A mRNA in these structures at other times. Others have also observed inhibin-a mRNA in the corpus luteum of the cycling rat (16, 30) and in the corpus luteum of the primate ovary (31). We have shown that the number of follicles expressing inhibin-a and -/?A mRNAs changes during pregnancy. During the first half of pregnancy there is a slow decline in the number of expressing follicles, which appears to
be associated with follicular atresia. The atretic follicles of pregnancy initially show a loss of inhibin-/3A mRNA expression and later lose inhibin-o: mRNA expression. This decrease in inhibin gene expression is observed before definitive histological signs of atresia (22). These results agree with earlier findings on atretic follicles in cycling rats (16, 23). By day 15 of gestation, both the number of inhibin mRNA-expressing follicles and the amount of ovarian a chain mRNA reach their lowest levels. After day 15 of pregnancy, there is a steady increase in the number of inhibin-expressing follicles as the follicles that will be ovulated shortly after parturition mature. This increase in inhibin-expressing follicles during late pregnancy is associated with a small increase in serum inhibin levels as measured by a chain RIA; inhibin levels are significantly elevated on day 19 compared to all other times during pregnancy. The relationship between the number of inhibin-expressing follicles and serum inhibin levels during early pregnancy is less pronounced. In another study of inhibin levels during pregnancy, Taya and co-workers (32) showed that levels of
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i V-
INHIBIN EXPRESSION DURING PREGNANCY
inhibin in peripheral blood rise significantly on days 2 and 4 of pregnancy, decrease between days 6 and 14, then rise gradually from day 18 until day 20. This agreed with earlier measurements of bioactive inhibin in ovarian venous plasma by these investigators (33). From studies during both pregnancy and pseudopregnancy, Taya and co-workers concluded that serum inhibin concentrations varied with the number of healthy antral follicles, in good agreement with our measurements of inhibin mRNA-expressing follicles during gestation. Despite the changes we observed in inhibin expression during pregnancy, no striking relationship between inhibin expression and serum FSH levels was apparent. We, therefore, asked whether there was a specific role for ovarian inhibin in regulating FSH secretion during pregnancy. We found that serum FSH was significantly and specifically elevated when released from the influence of the ovary after ovariectomy either early or late in pregnancy. This rise in FSH was associated with a significant decrease in serum inhibin levels as measured by RIA. Although decreases in serum progesterone and estrogen after ovariectomy might also play a role in modulating FSH secretion, replacement of these steroids to ovariectomized animals is not sufficient to decrease FSH to basal levels (34). In addition, the changes we observed after ovariectomy during pregnancy are very similar to those seen after ovariectomy in cycling animals, where functional corpora lutea or a placenta are absent (21). We, therefore, conclude that inhibin is likely to play a role in regulating FSH secretion during pregnancy, and that sufficient inhibin is available throughout gestation to maintain FSH at near-basal levels. Folliculogenesis during pregnancy has been studied by Greenwald and co-workers (4), who concluded that there is no cyclic change in follicular development during pregnancy based on follicular size criteria. Extending these data, we find that although the overall size classes of follicles progressing through pregnancy do not dramatically change, the maturational status of these follicles, as measured by inhibin mRNA expression, is not static. The gonadotropin receptor contents of granulosa and thecal cells of antral follicles also change throughout pregnancy, increasing from days 3-6, steadily decreasing between days 8-19, then dramatically increasing until term (5). This indicates that while no cyclic changes in follicular size classification are evident, there are clear functional variations in the developmental status of the antral follicles during pregnancy. The control of FSH levels and follicular development during the second half of the fertile cycle is not well understood. Although FSH is known to be the major regulator of follicular development during the estrous cycle, Bogovich and co-workers (35) have suggested that LH, and not FSH, is responsible for recruitment of
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follicles late in gestation, and Taya and Sasamoto (36) have demonstrated that LH administration during midpregnancy can induce the development of ovulatory follicles without changing basal FSH levels. In contrast, Greenwald (4) has shown that hCG does not induce ovulation before day 21 unless the ovary is previously primed with PMSG. In our current experiments we observed a significant increase in serum FSH levels and in the number of follicles expressing the inhibin mRNAs after day 15, while serum LH levels did not rise until day 22 of gestation. It seems likely that this enhanced FSH secretion might provide the driving force for increased inhibin gene expression and inhibin secretion during late pregnancy. After the first postpartum ovulation and the concurrent decrease in inhibin negative feedback, FSH levels rise (the secondary FSH surge), and new follicles are recruited, reinitiating the estrous cycle. The general increase in inhibin-expressing follicles seen in late pregnancy temporally corresponds to the decline in serum progesterone by days 18-19, although further experiments will be necessary to determine whether serum progesterone itself has any direct role in regulating inhibin production during late pregnancy. It is unclear what function this increase in inhibin during late gestation might have, since the lower inhibin levels observed during most of gestation are effective in maintaining FSH at basal levels. Interestingly, we have recently found that the placental decidua expresses very high levels of /?A chain, but essentially no a chain, mRNA late in gestation (Rahal, J. 0., and K. E. Mayo, manuscript in preparation). Because the /3 chains of inhibin are able to dimerize to form a FSH-releasing protein called activin (37, 38), activin is likely to be produced in the placenta late in gestation. Clearly, further study of the complex interactions among inhibin, activin, and FSH will be required to fully elucidate the roles of these hormones in regulating follicular development during pregnancy. Acknowledgments We would like to thank the NIDDK for immunological reagents, our colleagues for comments on the manuscript, Brigitte Mann for help with the RIAs, and Wylie Vale and Catherine Rivier for supplying the inhibin antiserum.
References 1. Linkie D, Niswender G 1972 Serum levels of prolactin, luteinizing hormone, and follicle stimulating hormone during pregnancy in the rat. Endocrinology 96:632-637 2. Taya K, Greenwald G 1981 In vivo and in vitro ovarian steroidogenesis in the pregnant rat. Biol Reprod 25:683-691 3. Gibori G, Chen K, Khan I, Azhan S, Reaven G 1984 Regulation of luteal cell lipoprotein receptors, sterol contents, and steroidogenesis by estradiol in the pregnant rat. Endocrinology 114:609-617 4. Greenwald B 1966 Ovarian follicular development and pituitary
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INHIBIN EXPRESSION DURING PREGNANCY
FSH and LH content in the pregnant rat. Endocrinology 79:572578 5. Richards J, Kersey K 1979 Changes in theca and granulosa cell function in antral follicles developing during pregnancy in the rat: gonadotropin receptors, cyclic AMP and estradiol. Biol Reprod 21:1185-1201 6. Hearn J 1986 The embryo-maternal dialogue in early pregnancy in primates. J Reprod Fertil 76:809-819 7. Gibori G, Richards JS 1978 Dissociation of two distinct luteotropic effects of prolactin: regulation of luteinizing hormone-receptor content and progesterone secretion during pregnancy. Endocrinology 102:767-774 8. Ling N, Ying S-Y, Ueno N, Esch F, Denoroy L, Guillemin R 1985 Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc Natl Acad Sci USA 82:7217-7221 9. Miyamoto K, Hasegawa Y, Fukuda M, Igarashi M, Danfuwa D, Matsuo H 1985 Isolation of porcine follicular fluid inhibin of 32 K daltons. Biochem Biophys Res Commun 129:396-403 10. Rivier J, Spiess J, McClintock R, Vaughan J, Vale W 1985 Purification and partial characterization of inhibin from porcine follicular fluid. Biochem Biophys Res Commun 133:120-127 11. Robertson D, Foulds L, Lerersha L, Moran F, Hearn M, Burger H, Wetenhall R, deKretser D 1985 Isolation of inhibin from bovine follicular fluid. Biochem Biophys Res Commun 126:220-226 12. Burger H, Igarashi M 1988 Inhibin: definition and nomenclature, including related substances. Endocrinology 122:1701-1702 13. Woodruff TK, Meunier H, Jones PBC, Hseuh AWJ, Mayo KE 1987 Rat inhibin: molecular cloning of a- and /3-subunit complimentary DNAs and expression in the ovary. Mol Endocrinol 1:561— 568 14. Esch F, Shimasaki S, Cooksey D, Mercado M, Mason A, Ying SY, Ueno N, Ling N 1987 Complimentary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Mol Endocrinol 1:388-396 15. Woodruff T, D'Agostino J, Schwartz N, Mayo K 1988 Dynamic changes in inhibin rnRNAs in rat ovarian follicles during the reproductive cycle. Science 239:1296-1299 16. Meunier H, Cajander SB, Roberts VJ, Rivier C, Waschenko P, Hseuh AJW, Vale W 1988 Rapid changes in expression of inhibin «-, j8A- and /3B-subunits in ovarian cell types during the rat estrous cycle. Mol Endocrinol 2:1352-1363 17. Chomczynski P, Sacchi N 1987 Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159 18. Meinkoth J, Wahl G 1984 Hybridization to nucleic acids immobilized on solid supports. Anal Biochem 138:267-284 19. Woodruff TK, D'Agostino JB, Schwartz NB, Mayo KE 1989 Decreased inhibin gene expression in preovulatory follicles requires primary gonadotropin surges. Endocrinology 124:2193-2199 20. Vaughan JM, Rivier J, Corrigan AZ, McClintock R, Campen CA, Jolley D, Vogelmayr JK, Bardin CW, Rivier C, Vale W 1989 Detection and purification of inhibin using antiserum generated against synthetic peptide fragments. Methods Enzymol 168:588617 21. Ackland JF, D'Agostino JB, Ringstrom SJ, Hostetler JP, Mann BG, Schwartz NB 1990 Circulating radioimmunoassayable inhibin during periods of transient follicle-stimulating hormone rise: sec-
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ondary surge and unilateral ovariectomy. Biol Reprod 43:347-352 22. Osman P 1985 Rate and course of atresia during follicular development in the adult cyclic rat. J Reprod Fertil 73:261-270 23. Woodruff TK, D'Agostino J, Schwartz N, Mayo K 1989 Modulation of rat inhibin mRNAs in pre-ovulatory and atretic follicles. In: Hirshfield AN (ed) Growth Factors and the Ovary. Plenum Press, New York, pp 291-295 24. Nequin L, Alvarez J, Schwartz N 1979 Measurement of serum steroid and gonadotropin levels and uterine and ovarian variables throughout 4-day and 5-day estrous cycles in the rat. Biol Reprod 20:659-670 25. Taya K, Greenwald GS 1981 In vivo and in vitro ovarian steroidogenesis in the pregnant rat. Biol Reprod 25:683-691 26. Butcher R 1977 Changes in gondadotropin and steroids associated with unilateral ovariectomy. Endocrinology 101:830-840 27. Welschen R, Dullaart J, deJong F 1978 Interrelationships between circulating levels of estrogen, progesterone, FSH and LH immediately after ULO in the cyclic rat. Biol Reprod 18:421-427 28. D'Agostino JB, Woodruff TK, Mayo KE, Schwartz NB 1989 Unilateral ovariectomy increases inhibin mRNA levels in newly recruited follicles. Endocrinology 124:310-317 29. Dykema JC, Rahal JO, Mayo KE 1990 Regulation of inhibin and activin genes in the rat ovary. In: Gibori G (ed) The Xlll Ovarian Workshop: Regulatory Processes and Gene Expression in the Ovary. Plenum Press, New York, in press 30. Davis SR, Dench F, Nikolaidis I, Clements JA, Forage RD 1986 Inhibin A-subunit gene expression in the ovaries of immature female rats is stimulated by pregnant mares serum gonadotropin. Biochem Biophys Res Commun 138:1191-1195 31. Schwall RH, Mason AJ, Wilcox JN, Bassett SG, Zeleznik AJ 1990 Localization of inhibin/activin subunit mRNAs within the primate ovary. Mol Endocrinol 4:75-79 32. Taya K, Komura H, Watanabe G, Sasamoto S 1989 Peripheral blood levels of immunoreactive inhibin during pseudopregnancy, pregnancy, and lactation in the rat. J Endocrinol 121:545-552 33. Taya K, Kimura J, Sasamoto S 1984 Inhibin activity in ovarian venous plasma during pregnancy, pseudopregnancy and lactation in the rat. Endocrinol Jpn 31:427-433 34. Schwartz NB, Milette JJ, Cohen IR 1987 Animal models which demonstrate divergence in secretion and storage of FSH and LH. In: Burger HG, DeKretser DM, Findlay JK, Igarashi M (eds) Inhibin-Non-Steroidal Regulation of Follicle-Stimulating Hormone Secretion. Raven Press, New York, pp 239-252 35. Bogovich K, Richards J, Reichert L 1981 Obligatory role of luteinizing hormone (LH) in the initiation of preovulatory follicular growth in the pregnant rat: specific effects of human chorionic gonadotropin and follicle-stimulating hormone on LH receptors and steroidogenesis in theca, granulosa and luteal cells. Endocrinology 109:860-867 36. Taya K, Sasamoto S 1989 Initiation of follicular maturation during mid-pregnancy by the administration of LH in the rat. J Reprod Fertil 85:51-60 37. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSHreleasing protein from porcine ovarian follicular fluid. Nature 321:776-779 38. Ling N, Ying S-Y, Ueno N, Shimasali S, Esch F, Hotta M, Guillemin R 1986 Pituitary FSH is released by a heterodimer of the 0subunits from the two forms of inhibin. Nature 321:779-782
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Vol. 128, No. 3 Printed in U.S.A.
Expression of Ovarian Inhibin during Pregnancy in the Rat* TERESA K. WOODRUFF, JACQUELINE ACKLAND, JASON 0. RAHAL, NEENA B. SCHWARTZ, AND KELLY E. MAYO Departments of Biochemistry, Molecular Biology, and Cell Biology (T.K. W., J.O.R., K.E.M.) and Neurobiology and Physiology (J.A., N.B.S.), Northwestern University, Evanston, Illinois 60208
ABSTRACT. We have examined the expression of the rat inhibin genes in the maternal ovary during pregnancy. RNA blot analysis indicates that the inhibin-a chain mRNA is expressed in the ovary throughout gestation at levels comparable to those observed in cycling rats. In situ hybridization shows that the inhibin-a and -/?A mRNAs are produced in the granulosa cells of developing antral follicles; little or no hybridization to functional corpora lutea is observed. Early in pregnancy, a large number of follicles hybridize to both a- and /3A-inhibin cDNA probes. Many of these follicles undergo atresia during the first half of pregnancy, and the number of inhibin-expressing follicles reaches a nadir on day 15. This is followed by an increase in inhibin-
producing follicles, which peaks just before parturition. The increase in inhibin-expressing follicles observed in late pregnancy corresponds to a small rise in serum inhibin levels, as measured using an a chain-specific RIA. After the first postpartum ovulation, few hybridizing follicles are observed. Ovariectomy in either early (day 6) or mid (day 15) pregnancy results in a significant fall in serum inhibin levels and a robust increase in serum FSH levels 9 h after surgery. These results suggest that inhibin is produced by the maternal ovary during pregnancy, that its synthesis is modulated during late gestation, and that inhibin may play a role in regulating FSH secretion during pregnancy. (Endocrinology 128: 1647-1654,1991)
T
HE TRANSITION from regular estrous cycles to a prolonged 22-day gestation period occurs as a consequence of fertilization and implantation in the rat. To achieve this change, the hypothalamus, pituitary, adrenals, and ovaries must make significant shifts in the release of tropic hormones, alter steroidogenesis, modify the follicular and luteal population, and regulate the availability of cellular receptors (1-5). Furthermore, the embryonic endocrine system must rapidly develop response mechanisms to incoming hormonal cues. There is evidence that this interaction may occur as early as the time of implantation (6). These complex interactions between maternal and fetal systems must be highly integrated and are not completely understood. As a result of these changes, the hormonal profiles of circulating gonadotropins and steroid hormones during pregnancy are dramatically different from those found during the estrous cycle. The pituitary gonadotropins LH and FSH remain low throughout pregnancy (2). Received August 14,1990. Address all correspondence and requests for reprints to: Dr. Kelly E. Mayo, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, Illinois 60208. * This work was supported by NSF Presidential Young Investigators Award DCB-8552977 (to K.E.M.), Chicago Community Trust Searle Scholars Program Grant 87-G-113 (to K.E.M.), NIH Grant HD-07507 (to N.B.S.), and NICHHD Program Project Grant HD-21921.
Serum steroids, however, attain levels greater than those found at any time during the normal estrous cycle. Luteal tissues play a predominant role in producing these steroids. During midpregnancy, serum estradiol and testosterone levels rise, and progesterone production is greatly elevated until the corpora lutea undergo functional luteolysis on day 18 in preparation for parturition and postpartum ovulation (7). It is clear that feedback systems must be established from the maternal ovary to maintain the reproductive status of pregnancy. One hormone that may be important in the modulation of FSH secretion during pregnancy is inhibin. Inhibin is a heterodimer composed of an a and a ]0 chain that selectively suppresses FSH secretion from the anterior pituitary (8-11). Two related forms of inhibin (A and B) (12) are generated by combination of the unique a chain with one of two highly related /? chains (j8A and j8B). Inhibin-a and -jS cDNA clones have been isolated from rat ovary (13, 14), and we and others have previously characterized the expression of the inhibin mRNAs in the ovary during the rat estrous cycle (15, 16). Inhibin mRNAs were found to be localized predominantly to the granulosa cells of maturing antral follicles in the cycling rat, and inhibin mRNA levels were found to change as antral follicles developed, matured, and ovulated. We wished to extend these studies to examine the
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INHIBIN EXPRESSION DURING PREGNANCY
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pattern of inhibin expression in the ovary during gestation for several reasons. Firstly, the patterns of follicular development and atresia differ from those during the estrous cycle; there is no clear wave-like change in the population of antral follicles, and the follicles of early pregnancy undergo atresia (4). Secondly, the corpora lutea of pregnancy are functional, producing high levels of progesterone (2). Finally, FSH is maintained at a low level throughout the 22-day gestation period (1, 4), in contrast to the marked dynamic changes in FSH levels that occur during the estrous cycle. To begin to address these issues, we have examined the localization and regulation of rat inhibin-a and -/3A mRNAs in the maternal ovary during pregnancy. Materials and Methods Animals and collection of tissue Timed pregnant rats were obtained from Charles Rivers Breeding Laboratory (Wilmington, MA). The day of insemination was designated day 1. Cycling rats were obtained from the same source and were monitored daily for vaginal cytology to determine cycle stage. Animals were used only after at least two consecutive 4 day cycles. All rats were maintained in separate cages on a 14-h light, 10-h dark cycle (lights on at 0500 h), with water and food provided. Animals were decapitated on each of the indicated days of gestation or of the estrous cycle at 1000 h, and trunk blood was collected. Ovaries, livers, and placentas were excised, frozen directly on dry ice, and stored at -70 C. Blood samples were allowed to clot overnight at 4 C, centrifuged at 800 X g for 20 min, aliquoted, and stored at -20 C until assayed for serum gonadotropins, progesterone, or inhibin. Endocrine ablation experiments Day 6 and day 15 pregnant animals were anesthetized at 0800 h using metaphane. Lateral abdominal incisions were made under sterile conditions, and ovaries were removed (or simply touched in the sham-operated animals). The abdominal wall was sutured, and the animals were allowed to recover under heated lights. Nine hours after surgery (1700 h), the animals were decapitated, and trunk blood was collected. Four animals were used per group, except for the sham-operated group on day 15 in which only three animals were used. RNA blot analysis RNA was prepared from frozen ovaries by homogenization in guanidine isothiocynate, as described by Chomczynski and Sacchi (17). Twenty micrograms of each total RNA were electrophoresed on a denaturing agarose-formaldehyde gel (18) with RNA size markers from Bethesda Research Laboratories (Besthesda, MD), and the RNA was transferred to a nylon membrane by blotting in 20 X SSC (standard saline citrate) for 16 h. The gel was stained with acrydine orange after electrophoresis and after blotting to check for equivalent loading and complete transfer of RNAs. Hybridization was to the 1500-
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basepair (bp) insert of rat inhibin-a cDNA clone rINA-13 radiolabeled by nick translation (13). In situ hybridization Twenty-micron frozen sections of ovary or liver (control) were cut on a Reichart 820 cryostat (Buffalo, NY) and mounted alternately between two microscope slides for eventual hybridization to a and /3A probes. Cross-sections of the ovary were taken with respect to the attached oviduct to maintain a standard orientation. Sections were fixed in 5% paraformaldehyde, treated with acetic anhydride in 0.1 M triethanolamine for 10 min, dehydrated through an ethanol series, and stored in a vacuum desiccator until they were hybridized (350 /xm), is similar to that found during the estrous cycle (15, 16). The luteal tissue of pregnancy is particularly interesting, in that corpora lutea produce progesterone during most of gestation (2). In either functional (day 6-18) or nonfunctional (day 19-21) corpora
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INHIBIN EXPRESSION DURING PREGNANCY
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a Follicle
100X
500X
Corpus Luteum 100X
500X
FIG. 3. Expression of inhibin mRNAs in follicular and luteal tissues. Sequential sections from the ovary of an animal on day 18 of gestation were analyzed using in situ hybridization. The left panels were hybridized to an inhibin-a chain probe; the right panels to an inhibin-/3A chain probe. In the upper panel, a healthy antral follicle is shown at both XlOO and X500 magnifications, as indicated. Arrow 1 points to the granulosa cell layer; arrow 2 points to the thecal cell layer. In the lower panel, a functional corpus luteum is shown at both XlOO and X500 magnifications, as indicated. Arrow 3 points to luteal cells. The X500 magnifications are from the boxed area on the XlOO magnification photographs.
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Endo • 1991 Vol 128 • No 3
INHIBIN EXPRESSION DURING PREGNANCY
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1500
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750 i A • Intact •IOVX
EHD # Follicles Serum Inhibin
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I
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a
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I
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iibin
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o g 10
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-
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Day 15
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FlG. 4. Inhibin expression during pregnancy. On each indicated day of gestation, the number of follicles hybridizing to both the a and /3A chain inhibin probes per 100 ovarian sections (which essentially represents 1 ovary) was determined using in situ hybridization. Sera from these same animals as well as several additional animals were analyzed for inhibin by RIA (filled symbols). Values are presented as the mean ± SE for 2 different animals on each day (in situ hybridizations) or 5 different animals on each day (inhibin RIA). Parturition was on day 22 or 23.
}•
400 Day 6
Day 15
FIG. 6. Effects of ovariectomy on serum FSH and inhibin levels during pregnancy. Intact, sham-operated, or bilaterally ovariectomized rats were analyzed. Sham-operated animals were not different from intact animals and are not shown. Surgery was on either day 6 or day 15 of gestation, and serum hormone values were measured 9 h later. FSH values are shown in the top panel (A), and inhibin values in the bottom panel (B). Values are the mean ± SE. By two-way analysis of variance: FSH ovariectomy, P < 0.001; and inhibin ovariectomy, P < 0.001. 5 10 Day of Gestation
20
Day of 25 Delivery
FlG. 5. Serum gonadotropin and progesterone levels during pregnancy. Sera from the same animals represented in Fig. 4 were analyzed for FSH, LH, and progesterone by RIA. The limit of detection of the LH assay was 0.3 ng/ml. Values are presented as the mean ± SE for five different animals on each day. Parturition was on day 22.
lutea we did not detect expression of inhibin-a or -j8A mRNA during gestation. During the estrous cycle we have observed transient expression of inhibin-a mRNA in the forming corpus luteum early on estrous morning (29), but have not seen any expression of either a or /?A mRNA in these structures at other times. Others have also observed inhibin-a mRNA in the corpus luteum of the cycling rat (16, 30) and in the corpus luteum of the primate ovary (31). We have shown that the number of follicles expressing inhibin-a and -/?A mRNAs changes during pregnancy. During the first half of pregnancy there is a slow decline in the number of expressing follicles, which appears to
be associated with follicular atresia. The atretic follicles of pregnancy initially show a loss of inhibin-/3A mRNA expression and later lose inhibin-o: mRNA expression. This decrease in inhibin gene expression is observed before definitive histological signs of atresia (22). These results agree with earlier findings on atretic follicles in cycling rats (16, 23). By day 15 of gestation, both the number of inhibin mRNA-expressing follicles and the amount of ovarian a chain mRNA reach their lowest levels. After day 15 of pregnancy, there is a steady increase in the number of inhibin-expressing follicles as the follicles that will be ovulated shortly after parturition mature. This increase in inhibin-expressing follicles during late pregnancy is associated with a small increase in serum inhibin levels as measured by a chain RIA; inhibin levels are significantly elevated on day 19 compared to all other times during pregnancy. The relationship between the number of inhibin-expressing follicles and serum inhibin levels during early pregnancy is less pronounced. In another study of inhibin levels during pregnancy, Taya and co-workers (32) showed that levels of
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i V-
INHIBIN EXPRESSION DURING PREGNANCY
inhibin in peripheral blood rise significantly on days 2 and 4 of pregnancy, decrease between days 6 and 14, then rise gradually from day 18 until day 20. This agreed with earlier measurements of bioactive inhibin in ovarian venous plasma by these investigators (33). From studies during both pregnancy and pseudopregnancy, Taya and co-workers concluded that serum inhibin concentrations varied with the number of healthy antral follicles, in good agreement with our measurements of inhibin mRNA-expressing follicles during gestation. Despite the changes we observed in inhibin expression during pregnancy, no striking relationship between inhibin expression and serum FSH levels was apparent. We, therefore, asked whether there was a specific role for ovarian inhibin in regulating FSH secretion during pregnancy. We found that serum FSH was significantly and specifically elevated when released from the influence of the ovary after ovariectomy either early or late in pregnancy. This rise in FSH was associated with a significant decrease in serum inhibin levels as measured by RIA. Although decreases in serum progesterone and estrogen after ovariectomy might also play a role in modulating FSH secretion, replacement of these steroids to ovariectomized animals is not sufficient to decrease FSH to basal levels (34). In addition, the changes we observed after ovariectomy during pregnancy are very similar to those seen after ovariectomy in cycling animals, where functional corpora lutea or a placenta are absent (21). We, therefore, conclude that inhibin is likely to play a role in regulating FSH secretion during pregnancy, and that sufficient inhibin is available throughout gestation to maintain FSH at near-basal levels. Folliculogenesis during pregnancy has been studied by Greenwald and co-workers (4), who concluded that there is no cyclic change in follicular development during pregnancy based on follicular size criteria. Extending these data, we find that although the overall size classes of follicles progressing through pregnancy do not dramatically change, the maturational status of these follicles, as measured by inhibin mRNA expression, is not static. The gonadotropin receptor contents of granulosa and thecal cells of antral follicles also change throughout pregnancy, increasing from days 3-6, steadily decreasing between days 8-19, then dramatically increasing until term (5). This indicates that while no cyclic changes in follicular size classification are evident, there are clear functional variations in the developmental status of the antral follicles during pregnancy. The control of FSH levels and follicular development during the second half of the fertile cycle is not well understood. Although FSH is known to be the major regulator of follicular development during the estrous cycle, Bogovich and co-workers (35) have suggested that LH, and not FSH, is responsible for recruitment of
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follicles late in gestation, and Taya and Sasamoto (36) have demonstrated that LH administration during midpregnancy can induce the development of ovulatory follicles without changing basal FSH levels. In contrast, Greenwald (4) has shown that hCG does not induce ovulation before day 21 unless the ovary is previously primed with PMSG. In our current experiments we observed a significant increase in serum FSH levels and in the number of follicles expressing the inhibin mRNAs after day 15, while serum LH levels did not rise until day 22 of gestation. It seems likely that this enhanced FSH secretion might provide the driving force for increased inhibin gene expression and inhibin secretion during late pregnancy. After the first postpartum ovulation and the concurrent decrease in inhibin negative feedback, FSH levels rise (the secondary FSH surge), and new follicles are recruited, reinitiating the estrous cycle. The general increase in inhibin-expressing follicles seen in late pregnancy temporally corresponds to the decline in serum progesterone by days 18-19, although further experiments will be necessary to determine whether serum progesterone itself has any direct role in regulating inhibin production during late pregnancy. It is unclear what function this increase in inhibin during late gestation might have, since the lower inhibin levels observed during most of gestation are effective in maintaining FSH at basal levels. Interestingly, we have recently found that the placental decidua expresses very high levels of /?A chain, but essentially no a chain, mRNA late in gestation (Rahal, J. 0., and K. E. Mayo, manuscript in preparation). Because the /3 chains of inhibin are able to dimerize to form a FSH-releasing protein called activin (37, 38), activin is likely to be produced in the placenta late in gestation. Clearly, further study of the complex interactions among inhibin, activin, and FSH will be required to fully elucidate the roles of these hormones in regulating follicular development during pregnancy. Acknowledgments We would like to thank the NIDDK for immunological reagents, our colleagues for comments on the manuscript, Brigitte Mann for help with the RIAs, and Wylie Vale and Catherine Rivier for supplying the inhibin antiserum.
References 1. Linkie D, Niswender G 1972 Serum levels of prolactin, luteinizing hormone, and follicle stimulating hormone during pregnancy in the rat. Endocrinology 96:632-637 2. Taya K, Greenwald G 1981 In vivo and in vitro ovarian steroidogenesis in the pregnant rat. Biol Reprod 25:683-691 3. Gibori G, Chen K, Khan I, Azhan S, Reaven G 1984 Regulation of luteal cell lipoprotein receptors, sterol contents, and steroidogenesis by estradiol in the pregnant rat. Endocrinology 114:609-617 4. Greenwald B 1966 Ovarian follicular development and pituitary
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FSH and LH content in the pregnant rat. Endocrinology 79:572578 5. Richards J, Kersey K 1979 Changes in theca and granulosa cell function in antral follicles developing during pregnancy in the rat: gonadotropin receptors, cyclic AMP and estradiol. Biol Reprod 21:1185-1201 6. Hearn J 1986 The embryo-maternal dialogue in early pregnancy in primates. J Reprod Fertil 76:809-819 7. Gibori G, Richards JS 1978 Dissociation of two distinct luteotropic effects of prolactin: regulation of luteinizing hormone-receptor content and progesterone secretion during pregnancy. Endocrinology 102:767-774 8. Ling N, Ying S-Y, Ueno N, Esch F, Denoroy L, Guillemin R 1985 Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc Natl Acad Sci USA 82:7217-7221 9. Miyamoto K, Hasegawa Y, Fukuda M, Igarashi M, Danfuwa D, Matsuo H 1985 Isolation of porcine follicular fluid inhibin of 32 K daltons. Biochem Biophys Res Commun 129:396-403 10. Rivier J, Spiess J, McClintock R, Vaughan J, Vale W 1985 Purification and partial characterization of inhibin from porcine follicular fluid. Biochem Biophys Res Commun 133:120-127 11. Robertson D, Foulds L, Lerersha L, Moran F, Hearn M, Burger H, Wetenhall R, deKretser D 1985 Isolation of inhibin from bovine follicular fluid. Biochem Biophys Res Commun 126:220-226 12. Burger H, Igarashi M 1988 Inhibin: definition and nomenclature, including related substances. Endocrinology 122:1701-1702 13. Woodruff TK, Meunier H, Jones PBC, Hseuh AWJ, Mayo KE 1987 Rat inhibin: molecular cloning of a- and /3-subunit complimentary DNAs and expression in the ovary. Mol Endocrinol 1:561— 568 14. Esch F, Shimasaki S, Cooksey D, Mercado M, Mason A, Ying SY, Ueno N, Ling N 1987 Complimentary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Mol Endocrinol 1:388-396 15. Woodruff T, D'Agostino J, Schwartz N, Mayo K 1988 Dynamic changes in inhibin rnRNAs in rat ovarian follicles during the reproductive cycle. Science 239:1296-1299 16. Meunier H, Cajander SB, Roberts VJ, Rivier C, Waschenko P, Hseuh AJW, Vale W 1988 Rapid changes in expression of inhibin «-, j8A- and /3B-subunits in ovarian cell types during the rat estrous cycle. Mol Endocrinol 2:1352-1363 17. Chomczynski P, Sacchi N 1987 Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159 18. Meinkoth J, Wahl G 1984 Hybridization to nucleic acids immobilized on solid supports. Anal Biochem 138:267-284 19. Woodruff TK, D'Agostino JB, Schwartz NB, Mayo KE 1989 Decreased inhibin gene expression in preovulatory follicles requires primary gonadotropin surges. Endocrinology 124:2193-2199 20. Vaughan JM, Rivier J, Corrigan AZ, McClintock R, Campen CA, Jolley D, Vogelmayr JK, Bardin CW, Rivier C, Vale W 1989 Detection and purification of inhibin using antiserum generated against synthetic peptide fragments. Methods Enzymol 168:588617 21. Ackland JF, D'Agostino JB, Ringstrom SJ, Hostetler JP, Mann BG, Schwartz NB 1990 Circulating radioimmunoassayable inhibin during periods of transient follicle-stimulating hormone rise: sec-
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ondary surge and unilateral ovariectomy. Biol Reprod 43:347-352 22. Osman P 1985 Rate and course of atresia during follicular development in the adult cyclic rat. J Reprod Fertil 73:261-270 23. Woodruff TK, D'Agostino J, Schwartz N, Mayo K 1989 Modulation of rat inhibin mRNAs in pre-ovulatory and atretic follicles. In: Hirshfield AN (ed) Growth Factors and the Ovary. Plenum Press, New York, pp 291-295 24. Nequin L, Alvarez J, Schwartz N 1979 Measurement of serum steroid and gonadotropin levels and uterine and ovarian variables throughout 4-day and 5-day estrous cycles in the rat. Biol Reprod 20:659-670 25. Taya K, Greenwald GS 1981 In vivo and in vitro ovarian steroidogenesis in the pregnant rat. Biol Reprod 25:683-691 26. Butcher R 1977 Changes in gondadotropin and steroids associated with unilateral ovariectomy. Endocrinology 101:830-840 27. Welschen R, Dullaart J, deJong F 1978 Interrelationships between circulating levels of estrogen, progesterone, FSH and LH immediately after ULO in the cyclic rat. Biol Reprod 18:421-427 28. D'Agostino JB, Woodruff TK, Mayo KE, Schwartz NB 1989 Unilateral ovariectomy increases inhibin mRNA levels in newly recruited follicles. Endocrinology 124:310-317 29. Dykema JC, Rahal JO, Mayo KE 1990 Regulation of inhibin and activin genes in the rat ovary. In: Gibori G (ed) The Xlll Ovarian Workshop: Regulatory Processes and Gene Expression in the Ovary. Plenum Press, New York, in press 30. Davis SR, Dench F, Nikolaidis I, Clements JA, Forage RD 1986 Inhibin A-subunit gene expression in the ovaries of immature female rats is stimulated by pregnant mares serum gonadotropin. Biochem Biophys Res Commun 138:1191-1195 31. Schwall RH, Mason AJ, Wilcox JN, Bassett SG, Zeleznik AJ 1990 Localization of inhibin/activin subunit mRNAs within the primate ovary. Mol Endocrinol 4:75-79 32. Taya K, Komura H, Watanabe G, Sasamoto S 1989 Peripheral blood levels of immunoreactive inhibin during pseudopregnancy, pregnancy, and lactation in the rat. J Endocrinol 121:545-552 33. Taya K, Kimura J, Sasamoto S 1984 Inhibin activity in ovarian venous plasma during pregnancy, pseudopregnancy and lactation in the rat. Endocrinol Jpn 31:427-433 34. Schwartz NB, Milette JJ, Cohen IR 1987 Animal models which demonstrate divergence in secretion and storage of FSH and LH. In: Burger HG, DeKretser DM, Findlay JK, Igarashi M (eds) Inhibin-Non-Steroidal Regulation of Follicle-Stimulating Hormone Secretion. Raven Press, New York, pp 239-252 35. Bogovich K, Richards J, Reichert L 1981 Obligatory role of luteinizing hormone (LH) in the initiation of preovulatory follicular growth in the pregnant rat: specific effects of human chorionic gonadotropin and follicle-stimulating hormone on LH receptors and steroidogenesis in theca, granulosa and luteal cells. Endocrinology 109:860-867 36. Taya K, Sasamoto S 1989 Initiation of follicular maturation during mid-pregnancy by the administration of LH in the rat. J Reprod Fertil 85:51-60 37. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSHreleasing protein from porcine ovarian follicular fluid. Nature 321:776-779 38. Ling N, Ying S-Y, Ueno N, Shimasali S, Esch F, Hotta M, Guillemin R 1986 Pituitary FSH is released by a heterodimer of the 0subunits from the two forms of inhibin. Nature 321:779-782
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