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Home , Activin and inhibin, Hormone, Luteinizing hormone

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 5114-5117, July 1989 Medical Sciences Inhibin and activin modulate the release of -releasing , human chorionic gonadotropin, and from cultured human placental cells (foliculostatin/follicle-stimulating hormone-releasing /follitropin/foliicle-stimulating hormone) FELICE PETRAGLIA*, JOAN VAUGHAN, AND WYLIE VALEt Clayton Foundation Laboratories for , The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037 Communicated by C. H. Sawyer, March 20, 1989 (receivedfor review September 23, 1988)

ABSTRACT Although it is clear that human chorionic cotropin], supports the hypothesis that placental hormono- gonadotropin (hCG) and progesterone play fundamental roles genesis may be regulated in part by locally produced in , the regulation of placental production of these (13-22). remains to be defined. Recent evidence suggests that A recent report (11) showed that the addition of inhibin the human expresses related to inhibin (afi antiserum increased hCG production in primary human pla- subunits) or activin (8P subunits). Inhibin and activin (follicle- cental cultures, suggesting that endogenous inhibin might stimulating hormone-releasing protein) possess opposing ac- tonically inhibit of the placental gonadotropin. To tivities in several biological systems including pituitary follicle- evaluate the possible roles of inhibin-related proteins in the stimulating hormone (follitropin) secretion, erythroid differ- regulation of placental hormones more directly, we have entiation, and gonadal sex- production. The actions of investigated the effects-ofinhibin and activin on the secretion purified inhibin and activin on hormonogenesis by primary ofGnRH, hCG, and progesterone by cultured placental cells. cultures of human placental cells were studied. The addition of Moreover, because inhibin and activin are structurally re- activin increased gonadotropin-releasing hormone (GnRH) lated to the transforming /3 (TGF-,3) family of and progesterone production and potentiated the GnRH- growth factors (23) and because TGF-,8 and its receptors are induced release of hCG. Inhibin by itself did not modify present in human placenta (24, 25), we also evaluated the placental immunoreactive GnRH, hCG, and progesterone se- possible action of TGF-f3 on placental hormone release. cretion but reversed the activin-induced changes. Neither inhibin nor activin influenced the release of human . Furthermore, transforming growth factor (3, struc- MATERIALS AND METHODS turally related to inhibin/activin, did not signifcantly influ- Preparation of Placental Cultures. Placentae were ence hormone release from cultured placental cells. These obtained from pregnant women (n = 7) undergoing elective results support the hypothesis that inhibin and activin may play caesarean section at term. Permission to obtain the was a role in regulating the release of GnRH, hCG, and proges- granted by the Human Investigation Committee ofUniversity terone from placenta and implicate inhibin-related proteins in of California, San Diego and The Salk Institute. the endocrine of human pregnancy. Immediately after the collection, a placenta was placed on ice and chunks of tissue were minced (50-70 g). After being Inhibins are heterodimeric proteins, consisting of an a sub- rinsed three times in cold Hepes dissociation buffer (HDB; unit and one oftwo ,8 subunits (13A or PB), that were originally ref. 26), chunks were dissected free of membranes, and identified based upon their abilities to selectively suppress connective tissue and the soft tissues were minced. Cells follicle-stimulating hormone (FSH) secretion (1-6). FSH- were dissociated with 50 ml of HDB containing collagenase releasing proteins or activins were subsequently isolated and solution [0.1% bovine albumin/0.4% collagenase (type characterized as dimers comprised of inhibin (3 subunits II, Cooper Biomedical)/3000 Kunitz units of DNase II (type APA or AJB) (7, 8). Although inhibin and activin were first IV, Sigma)] in a water-jacketed Spinner suspension flask isolated from the , various tissues including the pla- (Wheaton Scientific). The dissociation mixture was main- centa contain inhibin subunit mRNAs (9-12). Whereas the tained at 370C and continuously stirred at 200-300 rpm for 1 gonads contain a large excess ofa- over (B-chain mRNAs, the hr. Trypsin (0.4%; Sigma) was then added, and the solution placenta has an excess of p-subunit mRNAs; such observa- was stirred for an additional 10 min. The tissue fragments tions raise the possibility that the placenta might have a were filtered through a 53-gum mesh filter (Spectrum Medical preponderance of,,8 dimers (12). However, there is not yet Industries). Filtered cells were transferred to a sterile plastic conclusive evidence that intact activin is synthesized by tube and centrifuged at 300 x g for 10 min. The resultant human placenta. supernatant was further centrifuged at 400 x g for 10 min. Placental inhibin a-subunit immunoreactivity is localized The pelleted cells from both centrifugations were resus- in the cytotrophoblast layer of the villi (11), a region shown pended in culture medium (J3-PJ containing 10%o fetal bovine earlier to contain other regulatory peptides including - serum; ref. 16). The remaining unfiltered cells and tissue otropin-releasing hormone (GnRH; refs. 13-20), somato- fragments were further digested with trypsin for 10 min and (21), and corticotropin-releasing factor (22). The evi- subjected to the above procedure. All cells obtained were dence that the placenta produces these hypophysiotropic peptides, which modulate the secretion of various hormones Abbreviations: hCG, human chorionic gonadotropin; GnRH, gonad- related to those in the pituitary [human chorionic gonado- otropin-releasing hormone; FSH, follicle-stimulating hormone; tropin (hCG), (hPL), and corti- TGF-p, transforming growth factor ,B; hPL, human placental lacto- gen; irGnRH, immunoreactive GnRH. *Present address: Department of and Gynecology, Uni- The publication costs ofthis article were defrayed in part by page charge versity of Modena School of Medicine, Via del Pozzo 71, 41100 payment. This article must therefore be hereby marked "advertisement" Modena, Italy. in accordance with 18 U.S.C. §1734 solely to- indicate this fact. tTo whom reprint requests should be addressed.

5114 Downloaded by guest on September 30, 2021 Medical Sciences: Petraglia et al. Proc. Natl. Acad. Sci. USA 86 (1989) 5115 resuspended in the culture medium and plated in 35-mm followed by the multiple-range test ofDuncan and by Tukey's six-well multiwell dishes (Costar) previously coated with test. poly(D-lysine) (Sigma). This procedure yielded 3 x 106 cells per g of tissue, and 5.0-6.5 X 105 cells were plated per well. RESULTS Experimental Procedures. The cultures were maintained at The addition ofpurified ovine inhibin to the cultured placen- 370C in a water-saturated atmosphere containing 5% C02; the tal cells did not alter the basal secretion rate of hCG but culture medium was changed every 2 days. The secretion reduced the GnRH-induced release of hCG (Fig. 1). Activin, experiments were conducted between 5 and 10 days after which only slightly and not significantly increased the release plating. The incubation medium consisted of fi-PJ medium of hCG when added alone, strongly potentiated the stimula- supplemented with 0.1% bovine serum albumin. Treatments tory action of GnRH on hCG secretion (Fig. 2). This effect were added in a small volume (<50 1.d) to triplicate wells was dose-related (Fig. 2) and was reversed by inhibin (Fig. 1). containing 1.0 ml ofincubation medium. The incubation time While inhibin did not modify the immunoreactive GnRH was 48 hr. (irGnRH) concentrations in placental cell cultures, the high- Peptide and Protein Hormones. Purified ovine inhibin (in- est doses of activin (105 pM and 350 pM) significantly hibin aPA heterodimer) was isolated from rete testis fluid by increased the secretion ofirGnRH (from 12.2 ± 2.5 to 42.2 ± immunoaffinity chromatography using an antiserum to the 3.1 and 53.3 ± 4.6 pg ofirGnRH per ml, respectively). Inhibin amino-terminal portion of the porcine inhibin a subunit (27). (90 pM) completely reversed the activin-induced irGnRH On NaDodSO4/PAGE, purified nonreduced ovine inhibin increase. Moreover, activin stimulated progesterone release shows a single band of Mr 32,000; after reduction, two bands from placental cultures in a dose-related manner (Fig. 3). of Mr 21,000 and Mr 14,000 are observed. Porcine activin, a Ihhibin did not significantly change progesterone concentra- homodimer of inhibin PAPA, was purified to homogeneity tion but completely reversed (90 pM) the effect of activin on from porcine follicularfluid as described by Vale et al. (7). On progesterone release (Fig. 3). NaDodSO4/PAGE, purified porcine activin shows a single The addition of moderate doses of TGF-,j (from 4 to 400 band ofMr 28,000 nonreduced and a single band ofMr 14,000 pM) neither altered the release of hCG, irGnRH, and pro- upon reduction. GnRH was prepared by solid-phase meth- gesterone from cultured placental cells nor influenced the odology (28). hCG, a-subunit hCG, and the antiserum against action of activin and/or of inhibin on the secretion of these hCG were gifts of the National Hormone and Pituitary hormones (data not shown). Only the highest concentrations Program of the National Institute of and Digestive ofTGF-fi (4 nM) significantly increased secretion ofirGnRH and Diseases. TGF-,B was purchased from R & D (from 11.5 ± 2.6 to 24.7 ± 3.3 pg of irGnRH per ml). Systems, (Minneapolis). Neither inhibin nor activin nor TGF-f3 affected the release Placental Hormone RIAs. The GnRH RIA used a rabbit of hPL from cultured placental cells (data not shown). anti-[Lys8]GnRH as first (1:400,000 final dilution) and a sheep antiserum to rabbit gamma globulins as precip- DISCUSSION itating antibody (16). Synthetic GnRH was used as the standard and for preparation of tracer (I25I-labeled by the These results show that inhibin and activin influence hor- chloramine-T method and HPLC-purified). The assay sensi- mone secretion from cultured human placental cells. Activin tivity was 0.5 pg per tube; the ED50 was 20 ± 0.5 pg per tube. The inter- and intraassay coefficients of variation were 7.5% 100 and 3.3%, respectively. hCG was measured by double-antibody RIA (Diagnostic + ACTIVIN Products, Los Angeles). Tracer bound to antibody was precipitated with sheep anti-rabbit gamma globulins and 10%o 80 polyethylene glycol. The assay was standardized against two World Health Organization preparations: the First Interna- tional Reference Preparation for Immunoassay 75/537 and the Second International Standard for Bioassay 61/6. This EI assay did not recognize GnRH, hPL, corticotropin, or cor- 60- CONTROL ticotropin-releasing factor and showed 0.2% cross-reactivity with . The assay sensitivity was 1 inter- national milliunit/ml; the inter- and intraassay coefficients of variations were 4% and 2%, respectively. 0 + ACTIVIN U) The hPL concentrations were measured by using a Coat- 0 40. + INHIBIN A-Count solid-phase RIA kit (Diagnostic Products). This C) assay did not cross-react with hCG, GnRH, releasing factor, or growth hormone and only 0.005% with . The assay was standardized against the World + INHIBIN Health Organization First International Reference Prepara- 20 tion for Immunoassay 73/545. The assay sensitivity was 25 ng/ml. The inter- and intraassay coefficients of variations were 5% and 2.5%. Progesterone concentrations were measured by using a - *I .. ... II1 I . Coat-A-Count solid-phase RIA kit (Diagnostic Products). 0 0.001 0.01 0.1 1 10 100 This assay did not recognize , , , or . The assay sensitivity GnRH, pM was 50 pg/ml. The inter- and intraassay coefficients of FIG. 1. The effect of GnRH (o) on hCG release from cultured variations were 4% and 2%, respectively. human placental cells is increased by 105 pM activin (,) and inhibited Statistical Analysis. Each data point represents the mean + by 90 pM inhibin (a). The activin-induced potentiation of GnRH SEM of three wells assayed in duplicate. Statistical analysis effects on hCG is also reversed by the addition of inhibin (c). Each of the results was performed by using analysis of variance, point represents the mean ± SEM ofthree wells assayed in duplicate. Downloaded by guest on September 30, 2021 5116 Medical Sciences: Petraglia et al. Proc. Natl. Acad Sci. USA 86 (1989) 80 tion of f3f rather than a8 dimers (31). The presence of + GnRH mRNAs for inhibin subunits in human placenta (9, 12) indicates that inhibin and activin could be locally produced. The effects of inhibin and activin on placental hormone 60x - . release from cultured cells may reflect autocrine or paracrine effects of the locally produced peptides. Although an endo- ._3 crine effect of circulating inhibin or activin cannot be ex- .6 40- cluded, the placenta itself has been shown to be the major oo source of circulating inhibin during pregnancy (32). 0 a) Even though the actions of these dimers on placental 20o - + ffiCONTROL functions remain to be explored in vivo, the present studies suggest that , by modulating the secretion ofplacental GnRH, hCG, and progesterone, could play a role 0 in the regulation of pregnancy. I fal 4 I w 0 10 1 00 1 000 We gratefully acknowledge the nursing staff of the Surgery Room and the Labor and Delivery Room at Scripps Memorial Hospital, La Activin, pM Jolla, CA, for their kind assistance. We thank Dr. Jean Rivier for FIG. 2. Activin increases 0.1 jLM GnRH-induced hCG secretion providing GnRH. We acknowledge the expert technical assistance of from cultured human placental cells in a dose-related manner (*) but Anne Corrigan, Gayle Yamamoto, David Hutchinson, Steve Sutton, does not change the basal release of hCG (a). Each point represents Ron Kaiser, and Richard McClintock; and we thank Bethany Coyne the mean ± SEM of three wells assayed in duplicate. for manuscript preparation. This research was supported by the National Institute of Health and Human Development increases the release of GnRH and progesterone and aug- (HD13527). Ovine inhibin and porcine activin were purified with ments the release of hCG induced by GnRH. Although support from the Population Council/AID and the National Institute inhibin alone does not directly affect the spontaneous secre- of Child Health and Human Development Contraceptive Develop- the ment Branch (HD-3-2826). This research was conducted in part by tion of the various placental hormones measured, effects the Clayton Foundation for Research, California Division. W.V. is a ofactivin on GnRH, hCG, and progesterone production were senior Clayton Investigator. F.P. was partially supported by the reversed by inhibin. These results agree with previous ob- Metropolitan Life Foundation and by the Consiglio Nazionale delle servations showing functional antagonism between inhibin Ricerce (position 350.542). and activin in various tissues. Indeed, the two dimers exert opposite effects on FSH release from pituitary (7), on 1. Mason, A. J., Hayflick, J. S., Ling, N., Esch, F., Ueno, N., production by the gonads (29), on the induction of Ying, S.-Y., Guillemin, R., Niall, H. & Seeburg, P. H. (1985) in a human cell Nature (London) 318, 659-661. hemoglobin synthesis erythroleukemic line, 2. Robertson, D. M., Foulds, L. M., Leversha, L., Morgan, and on cell proliferation in human marrow (30). The F. J., Hearn, M. T. W., Burger, H. G., Wettenhall, R. E. H. evidence that neither dimer influenced the secretion of hPL & deKretser, D. M. (1985) Biochem. Biophys. Res. Commun. and that moderate doses of the related protein, TGF-p, did 126, 220-222. not influence the release of the hormones from cultured 3. Miyamoto, K., Hasegawa, Y., Fukuda, M., Nomura, M., placental cells supports the specificity of the actions of Iganashi, M., Kangaw, V. & Matsuo, H. (1985) Biochem. inhibin and activin in this system. Biophys. Res. Commun. 129, 396-399. There is immunologic and chromatographic evidence for 4. Rivier, J., Spiess, J., McClintock, R., Vaughan, J. & Vale, W. the presence of inhibin in placental extracts and cultures (11). (1985) Biochem. Biophys. Res. Commun. 133, 120-122. Although activin has not yet been demonstrated to be pro- 5. Ling, N., Ying, S.-Y., Ueno, N., Esch, F., Denoroy, L. & Guillemin, R. (1985) Proc. Natl. Acad. Sci. USA 82,7217-7219. duced in the placenta, the excess of /-subunit relative to 6. Forage, G. G., Ring, J. M., Brown, R. W., McInerney, B. V., a-subunit mRNAs in that tissue (12) would favor the forma- Cobon, G. S., Gregson, R. P., Robertson, D. M., Morgan, F. J., Hearn, M. T. W., Findlay, J. K., Wettenhall, R. E. H., 40 - Burger, H. G. & deKretser, D. M. (1986) Proc. Nati. Acad. Sci. USA 83, 3091-3097. 7. Vale, W., Rivier, J., Vaughan, J., McClintock, R., Corrigan, A., Woo, W., Karr, D. & Spiess, J. (1986) Nature (London) 321, CONTROL c 30- 776-779. c 8. Ling, N., Ying, S.-Y., Ueno, N., Shimasaki, S., Esch, F., Hotta, v M. & Guillemin, R. (1986) Nature (London) 321, 779-781. 9. Mayo, K. E., Cerelli, G. M., Spiess, J., Rivier, J., Rosenfeld, M. G., Evans, R. M. & Vale, W. (1986) Proc. Nati. Acad. Sci. 0" 20 - a, USA 83, 5849-5852. c 10. McLachlan, R. I., Healy, D. L., Robertson, D. M., Burger, H. G. & DeKretser, D. M. (1986) Biochem. Biophys. Res. 0)a, Commun. 140, 485-490. 2m 10 - + INHIBIN 11. Petraglia, F., Sawchenko, P., Lim, A. T. W., Rivier, J. & Vale, W. (1987) Science 237, 187-189. 12. Meunier, H., Rivier, C., Evans, R. & Vale, W. (1988) Proc. Nati. Acad. Sci. USA 85, 247-251. 13. Khodr, G. S. & Siler-Khodr, T. M. (1978) Fertil. Steril. 30, 0- 301-304. 01 1 0 1 00 1000 14. Haning, R. V., Choi, L., Kiggens, A. J., Kuzma, D. L. & Sum- Activin, pM merville, J. W. (1982) J. Clin. Endocrinol. Metab. 55, 213-218. 15. Belisle, S., Guevin, J.-F., Bellabarba, D. & Lehoux, J.-G. FIG. 3. Activin stimulates the release of progesterone from (1984) J. Clin. Endocrinol. Metab. 59, 119-126. cultured human placental cells in a dose-related manner (r). This 16. Petraglia, F., Lim, A. T. W. & Vale, W. (1987) J. Clin. En- effect is completely reversed by the addition of 90 pM inhibin (*). docrinol. Metab. 65, 1020-1025. Each point represents the mean ± SEM of three wells assayed in 17. Khodr, G. S. & Siler-Khodr, T. M. (1980) Science 207, 315-317. duplicate. 18. Seppala, M., Wahlstrom, T., Lehtovirta, P., Lee, J. N. & Downloaded by guest on September 30, 2021 Medical Sciences: Petraglia et al. Proc. Nati. Acad. Sci. USA 86 (1989) 5117

Leppaluoto, J. (1980) Clin. Endocrinol. (Oxford) 12, 441-451. 27. Vaughan, J. M., Rivier, J., Corrigan, A. Z., McClintock, R., 19. Tan, L. & Rousseau, P. (1982) Biochem. Biophys. Res. Com- Campen, C. A., Jolley, D., Voglmayr, J. K., Bardin, C. W., mun. 109, 1061-1071. Rivier, C. & Vale, W. (1989) Methods Enzymol. 168, 588-617. 20. Seeburg, P. H. & Adelman, J. P. (1984) Nature (London) 311, 28. Rivier, C., Vale, W. & Rivier, J. (1983) J. Med. Chem. 26, 666-668. 1545-1550. 21. Watkins, W. B. & Yen, S. S. C. (1980) J. Clin. Endocrinol. 29. Hsueh, A. J. W., Dahl, K. D., Vaughan, J., Tucker, E., Rivier, Metab. 5, 969-970. J., Bardin, C. W. & Vale, W. (1987) Proc. Natl. Acad. Sci. 22. Petraglia, F., Sawchenko, P., Rivier, J. & Vale, W. (1987) USA 84, 5082-5086. Nature (London) 328, 717-719. 30. Yu, J., Shao, L.-E., Lemas, V., Yu, A. L., Vaughan, J., Rivier, 23. Massague, J. (1987) Cell 49, 437-438. J. & Vale, W. (1987) Nature (London) 330, 765-767. 24. Frolik, C. A., Dart, L. L., Meyers, C. A., Smith, D. M. & 31. Mason, A. J. (1988) in Nonsteroidal Gonadal Factors, eds. Sporn, M. B. (1983) Proc. Nati. Acad. Sci. USA 80, 3676-3680. Hodgen, G. D., Rosenwaks, Z. & Spieler, J. M. (Jones Insti- 25. Massague, J. (1983) J. Biol. Chem. 258, 13614-13620. tute Press, Norfolk, VA), pp. 19-29. 26. Vale, W., Vaughan, J., Yamamoto, G., Bruhn, T., Douglas, C., 32. McLachlan, R. I., Healy, D. L., Lutjen, P. J., Findlay, J. K., Dalton, D., Rivier, C. & Rivier, J. (1983) Methods Enzyymol. deKretser, D. M. & Burger, H. G. (1987) Clin. Endocrinol. 103, 565-577. (Oxford) 27, 663-668. Downloaded by guest on September 30, 2021