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Immunocytochemical localization of inhibin in rat and human reproductive tissues
Molecular and Cellular Endocrinology,
54 (1987) 239-243
239
Elsevier Scientific Publishers Ireland, Ltd. MCE 01790
Rapid Coruscation
Imrnunocytochemical localization of inhibin in rat and human reproductive tissues Istvan Merchenthaler
I, Michael D. Culler 2, Peter Petrusz 3 and And&
Negro-Vilar 2
’ ~eFartmenf of Anatomy, Uniuersiiy Medical School, P&s, Hungcuy, 2 Loboratory of 3ehovzorai and Neurological Toxicology, Natlonul Institute of Enuironmenta~ Health Sciences, Research Triangle Park, NC X709, U.S.A. and ’ department Universiiy of North Carohna, Chapel Hill, NC 27514, U.S.A.
of Anatomy,
(Received 13 October 1987; accepted 13 October 1987)
Key words: Inhibin; Immunocytochemistry;
Sertoli cell; Granulosa cell; Placenta
Recently, the structures of two forms of in&bin present in human follicular fluid were elucidated from the corresponding cDNA sequences. Using specific antisera generated against the a-chain common to both forms, we have examined the cellular localization of inhibin in the male and female rat gonads and in human placental tissue. Specific a-inhibin immunoreactivity was localized within the Sertoli cells of a number of tubules in each testes section. However, other adjacent tubules were unstained suggesting a stage-specific production of inhibin. Intense immunostaining was observed in the granulosa cells of ovarian follicles at various stages but not in the thecal cells. I~~ost~~ng was present in the human placenta and limited to the cytotrophoblast cells, suggesting a role of inhibin during pregnancy. The present study demonstrates the probable site of production of inhibin in the gonads and placenta and further implicates this important factor as a key regulator of reproductive functions.
Introduction The production of inhibin by the testis was first postulated by McCullagh (1932) who described a non-steroidal, water-soluble gonadal principle of testicular origin capable of preventing the appearance of castration cells in the pituitary of gonadectomized animals. Subsequent studies demonstrated that inhibin was a selective inhibitor of FSH secretion in both sexes (deJong, 1979; DePaolo et al., 1979; Franchimont et al., 1979; Grady
Address for correspondence: Andres Negro-Vilar, M.D., Ph.D., Reproductive Neuroendocrinology Section, Laboratory of Behavioral and Neurological Toxicology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC, U.S.A.
et al., 1982; Negro-Vilar and Lumpkin, 1983; Steinberger, 1983). Early pu~fication studies described inhibin activity in testicular and ovarian extracts, follicular fluid from several species, as well as in rete testis fluid and seminal plasma (deJong and Sharpe, 1976; Schwartz and Channing, 1977; Setchell et al., 1977; Chari et al., 1978; Sairam et al., 1983). More recently, inhibin was successfully isolated and partially characterized from porcine fol~cular fluid (Ling et al., 1985; Miyamoto et al., 1985; Rivier et al., 1985) and from bovine follicular fluid (Robertson et al., 1985; Fukuda et al., 1986). The complete amino acid sequences of two human ovarian inhibins have been deduced from their cDNA sequences (Mason et ai., 1985) and recently cDNA cloning and sequence analysis was
240
reported for rat ovarian inhibins (Esch et al., 1987). It is now obvious that at least two inhibin molecules are produced that consist of an a-subunit linked to either one of two similar but distinct P-subunits. These heterodimers possess potent FSH inhibiting activity while other homoor heterodimers that consist of two /3-subunits are active stimulators of FSH secretion (Ling et al., 1986; Vale et al., 1986). The latter substances have homology with transforming growth factor p, suggesting that this family of proteins, produced by Sertoli and granulosa cells, may be involved in different biological processes regulating reproductive functions. We have recently developed and characterized antisera against an N-terminal fragment of the human sequence of cy-i&bin. These antisera, which crossreact with inhibin from human, porcine and rat sources, were used in this study to provide the first immunocytochemical demonstration of the cellular localization of a-inhibin in the rat testis and ovary and in the human placenta. Materials and methods Tissue preparation. Adult male and 21-day-old and adult female Sprague-Dawley rats were perfused under deep phenobarbital anesthesia through the abdominal aorta with cold saline (50 ml) followed by ice-cold 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PBS), pH 7.4 (250 ml). Testes and ovaries were removed and immersed overnight in the same fixative. Samples of two human, term placentas were fixed by immersion in 4% paraformaldehyde. After washing in PBS, the specimens were rehydrated, embedded in paraffin, and cut into 5-8 pm serial sections. Antisera. Several antisera were raised in rabbits and sheep against the l-32 N-terminal sequence of the a-chain of human inhibin (Peninsula Laboratories, San Carlos, CA). The antigen was coupled to either human serum albumin (hSA) or bovine thyroglobulin (bTG) with 1-ethyl-3(3-dimethyl aminopropyl)carbodiimide and injected into a sheep (hSA conjugate: MC-4) or rabbits (bTG conjugate: MC-6, MC-7 and MC-8) as described previously (Culler and Negro-Vilar, 1986). Immunocytochemistry. The paraffin-embedded sections were deparaffinized and treated with
trypsin for 10 min (Towle et al., 1984). The double PAP technique, as described by Ordronneau et al. (1981), was used to localize h-a-inhibin-like immunoreactivity in the gonads and the placenta. Sheep anti-rabbit immunoglobulin (IgG), rabbit anti-sheep IgG, rabbit and sheep PAP were obtained from Amel Products (New York). After completing the diaminobenzidine (DAB) reaction, the sections were washed in Tris-PBS and exposed to 0~0, vapors for 10 min. Following a wash in PBS the sections were dehydrated and mounted. The specificity of the antisera was tested using several criteria. Absorption of the antisera with the respective carrier proteins (hSA or bTG) did not affect staining. Absorption of the antisera with as low as 1 pg of the h-a-inhibin (l-32) fragment or with porcine follicular fluid, however, completely abolished the immunostaining. The specificity of the staining reaction was further substantiated by serially diluting the primary antisera, resulting in a gradual decrease in the intensity and finally in the diappearance of the staining. The antisera were also characterized by radioimmunoassay. All antisera showed parallel inhibition of ‘251-h-a-inhibin binding when either h-ainhibin l-25, porcine or human follicular fluid, highly purified porcine follicular fluid (W.H.O. Standard WGL4-55B) or rat Sertoli cell culture media (kindly provided by Dr. A. Kierszenbaum, University of North Carolina) were used as competitors (Culler and Negro-Vilar, in preparation). Results With the combined application of trypsin pretreatment and our modified double PAP technique, we were able to detect cr-inhibin-like immunoreactivity in both the male and female rat gonad and in the human placenta. Without trypsin pretreatment and with the conventional PAP staining, a less intense staining was observed. In the testis, staining was observed in different tubules (Fig. la) and clearly localized to the cytoplasm of Sertoli cells (Fig. lb), indicating the presence of a-inhibin-like immunoreactivity in these cells. No staining was observed among germ cells in the different stages of spermiogenesis or in interstitial cells. Immunoreactive Sertoli cells were observed in numerous seminiferous tubules in each
Fig. 1. Irnmunocytcchemical staining for inhibin in different reproductive tissues. (a) Low-magnification photomicrograph of a section of adult rat testes immunostained using ovine anti-n-inhibin serum MC-4 (1 : 20000). Only Sertoli cells show immunopositive staining. (b) Higher magnification of a tubule from the same animal as Fig. la. (c) Control testes section. The preabsorption of MC-4 anti-a-inhibin serum with 10 gg h-a-inhibin 1-32/m] 1:20000 dilution completely abolished the Sertoli cell staining. (d) Photomicrograph of a section of a 21-day rat ovary showing a-inhibin immunopositive granulosa cells in a tertiary follicle as revealed by rabbit anti-a-inhibin serum MC-8 (1: 15000). (P) Secondary follicles from a section of a 21-day rat ovary with a-inhibin immunopositive granulosa cells. (f) Control ovary section. Preabsorption of the MC-8 anti-a-inhibin serum with 10 pg h-a-inhibin l-32/ml 1: 15000 dilution completely abolished the granulosa cell staining. (g) Photomicrograph of a section of human placenta. Only one villus is shown. The cytotrophoblast cells are immunopositive for a-inhibin. Some of the mesenchymal cells within the villus are also stained. Anti-a-inhibin serum MC-8 was used at a dilution of 1: 15000 to stain this tissue. Absorption with h-a-inhibin l-32 as in Fig. lc and lf legend completely abolished the immunostaining (not shown). Magnifications: 0, c-g = 170 X ; b = 400 X
242
section, while other adjacent tubules were devoid of staining. Absorption of the antiserum with synthetic h-cr-inhibin (l-32) completely eliminated the immunostaining in the testis (Fig. lc). In the ovary, granulosa cells of follicles in various stages of development were intensely stained (Fig. Id, e) indicating the presence of a-inhibin-like immunoreactivity in these cells. All granulosa cells within a given follicle appeared stained. No staining was observed in the thecal cells. Ovaries from adult rats also showed immunopositive staining of the granulosa cells and, in addition, the luteal cells (data not shown). The immunopositive staining in the luteal cells, however, was much less intense than the staining found in the granulosa cells. Absorption of the antiserum with the h-a-inhibin (l-32) peptide completely suppressed the staining in these tissues (Fig. If). In the humam placenta, the immunostaining was localized in the cells of the cytotrophoblast layer of the placental villi (Fig. lg). No staining was observed in the peripherally-located syncytiotrophoblast cells (Fig. lg). As in the previous tissues, absorption of the antiserum with h-a-inhibin (l-32) abolished the staining (not shown). Discussion This study presents the first immunocytochemical localization of inhibin in the male and female gonad. The presence of a-inhibin chain-like immunoreactivity in the Sertoli, granulosa and luteal cells provides strong evidence that these cells are the source of gonadal inhibin. Our observation of immunostaining for inhibin in the human placenta confirms earlier reports localizing inhibin by immunofluorescence and radioimmunoassay (McLachlan et al., 1986a; Petraglia et al., 1987). The presence of cY-inhibin immunoreactivity in placental tissue supports the notion that these molecules play an important role in different reproductive situations, including pregnancy. Evidence for enhanced secretion during pregnancy, in correlation with changes in circulating HCG levels, has been presented (McLachlan et al., 1986b). In addition to being released into the general circulation from Sertoli, granulosa or cytotrophoblast cells, inhibin may also act locally to
modify certain parameters of gonadal or placental function (Bicsak et al., 1987; Petraglia et al., 1987). In our studies in the adult rat testis, inhibin was localized to Sertoli cells in certain tubules, while other adjacent tubules remained unstained. This observation suggests that the expression of inhibin in Sertoli cells may be stage-specific (Kierszenbaum et al., 1987) and therefore may be related to some dynamic parameter of gametogenic function. These findings strongly support a key role for inhibin in regulating reproductive functions. References Bicsak, T.A., Vale, W., Vaughn, J., Tucker, E.M., Cappel, S. and Hsueh, A.J.W. (1987) Mol. Cell. Endocrinol. 49, 211-217. Chari, S., Duraiswarmi, S. and Franchimont, P. (1978) Acta Endocrinol. 87,434-448. DeJong, F.H. (1979) Mol. Cell. Endocrinol. 13, l-10. DeJong, F.H. and Sharpe, R.M. (1976) Nature 263, 71-72. DePaolo, L.V., Wise, P.M. and Anderson, L.D. (1979) Endocnnology 104,402-408. Esch, F.S., Shimasaki, S., Cooksey, K., Mercado, M., Mason, A.J., Ying, S.Y., Ueno, N. and Lmg, N. (1987) Mol. Endocrinol. 1, 388-396. Franchimont, P., Vertraelen-Proyard, J., Hazee-Hagelstein, M.T., Renard, Ch., Demoulin, A., Bourguignon, J.P. and Hustin, J. (1979) Vitam. Horm. 37, 243-302. Fukuda, M., Miyamoto, K., Hasegawa, Y., Nomura, M., Igarashi, M., Kangawa, K. and Matsuo, H. (1986) Mol. Cell. Endocrinol. 44, 55-60. Grady, R.R., Charlesworth, M.C. and Schwartz, N.B. (1982) Recent Prog. Horm. Res. 38, 409-457. Kierszenbaum, A.L., Abdullah, M., Ueda, H. and Tres, L.L. (1987) In: Regulation of Ovarian and Testicular Function, Eds.: V.B. Mahesh, D.S. Dhindsa, E. Anderson and S.P. Kalra (Plenum Press, New York, NY) pp. 535-560. Ling, N., Ying, S.-Y., Ueno, N., Each, F., Denoroy, L. and Guillemin, R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7217-7221. Ling, N., Ying, S.-H., Ueno, N., Shimasaki, S., Each, F., Hotta, M. and Guillemin, R. (1986) Nature 321, 779-782. Mason, A.J., Hayflick, J.S., Ling, N., Esch, F., Ueno, N., Ying, S.-Y., Guillemin, R., Niall, H. and Seeburg, P.H. (1985) Nature 318, 659-663. McCullagh, D.R. (1932) Science 76, 12-20. McLachlan, R.I., Healy, D.L., Robertson, D.M., Burger, H.G. and de Kretser, D.M. (1986a) B&hem. Biophys. Res. Commun. 140,485-490. McLachlan, RI., Healy, D.L., Lutjen, P.J., Burger, H.G. and de Kretser, D.M. (1986b) 69th Annual Meeting of the Endocrine Society, Abstract No. 22. Miyamoto, H., Hasegawa, Y., Fukuda, M., Nomura, M., Igarashi, M., Kangawa, K. and Matsuo, H. (1985) B&hem. Biophys. Res. Commun. 129, 396-403.
243
Negro-Vilar, A. and Lumpkin, M.D. (1983) In: Male Reproduction and Fertility, Ed.: A. Negro-Vilar (Raven Press, New York, NY) pp. 159-169. Grdronneau, P., Lindstriim, P.B.M. and Petrusz, P. (1981) J. Histochem. Cytochem. 29, 1397-1404. Petragha, F., Sawchenko, P., Lim, A.T.W., Rivier, J. and Vale, W. (1987) Science 237, 187-189. Rivier, J., Spiess, J., McCIintock, J., Vaughan, J. and Vale, W. (1985) B&hem. Biophys. Res. Commun. 133, 120-127. Robertson, D.M., Foulds, L.M., Leversha, L., Morgan, F.J., Heam, M.T.V., Burger, H.G., Wettenhall, R.E.H. and de Kretser, D.M. (1985) B&hem. Biophys. Res. Commun. 126, 220-226. Sairam, M.R., Manjunath, P., Ramasharma, K., Kato, K. and MadwhaRaj, H.G. (1983) In: Male Reproduction and Fertility, Ed.: A. Negro-Vilar (Raven Press, New York, NY) pp. 149-158.
Schwartz, N.B. and Charming, C.P. (1977) Proc. Nat]. Acad. Sci. U.S.A. 74, 5721-5724. Setchell, B.P., Davies, R.V. and Main, S.J. (1977) In: The Testis, Vol. 4, Eds.: A.D. Johnson, W.R. Gomes and N.L. VanDemark (Academic Press, New York, NY) pp. 189-238. Steinberger, A. (1983) In: Male Reproduction and Fertility, Ed.: A. Negro-V&r (Raven Press, New York, NY), pp. 183-192. Steinberger, A. and Steinberger, E. (1976) Endocrinology 99, 918-921. Towle, A.C., Lauder, J.L. and Joh, T.H. (1984) J. Histochem. Cytochem. 32, 766-770. Vale, W., Rivier, J., Vaughn, J., McCIintock, R., Corrigan, A., Woo, W., Karr, D. and Spiess, J. (1986) Nature 321, 776-779.
54 (1987) 239-243
239
Elsevier Scientific Publishers Ireland, Ltd. MCE 01790
Rapid Coruscation
Imrnunocytochemical localization of inhibin in rat and human reproductive tissues Istvan Merchenthaler
I, Michael D. Culler 2, Peter Petrusz 3 and And&
Negro-Vilar 2
’ ~eFartmenf of Anatomy, Uniuersiiy Medical School, P&s, Hungcuy, 2 Loboratory of 3ehovzorai and Neurological Toxicology, Natlonul Institute of Enuironmenta~ Health Sciences, Research Triangle Park, NC X709, U.S.A. and ’ department Universiiy of North Carohna, Chapel Hill, NC 27514, U.S.A.
of Anatomy,
(Received 13 October 1987; accepted 13 October 1987)
Key words: Inhibin; Immunocytochemistry;
Sertoli cell; Granulosa cell; Placenta
Recently, the structures of two forms of in&bin present in human follicular fluid were elucidated from the corresponding cDNA sequences. Using specific antisera generated against the a-chain common to both forms, we have examined the cellular localization of inhibin in the male and female rat gonads and in human placental tissue. Specific a-inhibin immunoreactivity was localized within the Sertoli cells of a number of tubules in each testes section. However, other adjacent tubules were unstained suggesting a stage-specific production of inhibin. Intense immunostaining was observed in the granulosa cells of ovarian follicles at various stages but not in the thecal cells. I~~ost~~ng was present in the human placenta and limited to the cytotrophoblast cells, suggesting a role of inhibin during pregnancy. The present study demonstrates the probable site of production of inhibin in the gonads and placenta and further implicates this important factor as a key regulator of reproductive functions.
Introduction The production of inhibin by the testis was first postulated by McCullagh (1932) who described a non-steroidal, water-soluble gonadal principle of testicular origin capable of preventing the appearance of castration cells in the pituitary of gonadectomized animals. Subsequent studies demonstrated that inhibin was a selective inhibitor of FSH secretion in both sexes (deJong, 1979; DePaolo et al., 1979; Franchimont et al., 1979; Grady
Address for correspondence: Andres Negro-Vilar, M.D., Ph.D., Reproductive Neuroendocrinology Section, Laboratory of Behavioral and Neurological Toxicology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC, U.S.A.
et al., 1982; Negro-Vilar and Lumpkin, 1983; Steinberger, 1983). Early pu~fication studies described inhibin activity in testicular and ovarian extracts, follicular fluid from several species, as well as in rete testis fluid and seminal plasma (deJong and Sharpe, 1976; Schwartz and Channing, 1977; Setchell et al., 1977; Chari et al., 1978; Sairam et al., 1983). More recently, inhibin was successfully isolated and partially characterized from porcine fol~cular fluid (Ling et al., 1985; Miyamoto et al., 1985; Rivier et al., 1985) and from bovine follicular fluid (Robertson et al., 1985; Fukuda et al., 1986). The complete amino acid sequences of two human ovarian inhibins have been deduced from their cDNA sequences (Mason et ai., 1985) and recently cDNA cloning and sequence analysis was
240
reported for rat ovarian inhibins (Esch et al., 1987). It is now obvious that at least two inhibin molecules are produced that consist of an a-subunit linked to either one of two similar but distinct P-subunits. These heterodimers possess potent FSH inhibiting activity while other homoor heterodimers that consist of two /3-subunits are active stimulators of FSH secretion (Ling et al., 1986; Vale et al., 1986). The latter substances have homology with transforming growth factor p, suggesting that this family of proteins, produced by Sertoli and granulosa cells, may be involved in different biological processes regulating reproductive functions. We have recently developed and characterized antisera against an N-terminal fragment of the human sequence of cy-i&bin. These antisera, which crossreact with inhibin from human, porcine and rat sources, were used in this study to provide the first immunocytochemical demonstration of the cellular localization of a-inhibin in the rat testis and ovary and in the human placenta. Materials and methods Tissue preparation. Adult male and 21-day-old and adult female Sprague-Dawley rats were perfused under deep phenobarbital anesthesia through the abdominal aorta with cold saline (50 ml) followed by ice-cold 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PBS), pH 7.4 (250 ml). Testes and ovaries were removed and immersed overnight in the same fixative. Samples of two human, term placentas were fixed by immersion in 4% paraformaldehyde. After washing in PBS, the specimens were rehydrated, embedded in paraffin, and cut into 5-8 pm serial sections. Antisera. Several antisera were raised in rabbits and sheep against the l-32 N-terminal sequence of the a-chain of human inhibin (Peninsula Laboratories, San Carlos, CA). The antigen was coupled to either human serum albumin (hSA) or bovine thyroglobulin (bTG) with 1-ethyl-3(3-dimethyl aminopropyl)carbodiimide and injected into a sheep (hSA conjugate: MC-4) or rabbits (bTG conjugate: MC-6, MC-7 and MC-8) as described previously (Culler and Negro-Vilar, 1986). Immunocytochemistry. The paraffin-embedded sections were deparaffinized and treated with
trypsin for 10 min (Towle et al., 1984). The double PAP technique, as described by Ordronneau et al. (1981), was used to localize h-a-inhibin-like immunoreactivity in the gonads and the placenta. Sheep anti-rabbit immunoglobulin (IgG), rabbit anti-sheep IgG, rabbit and sheep PAP were obtained from Amel Products (New York). After completing the diaminobenzidine (DAB) reaction, the sections were washed in Tris-PBS and exposed to 0~0, vapors for 10 min. Following a wash in PBS the sections were dehydrated and mounted. The specificity of the antisera was tested using several criteria. Absorption of the antisera with the respective carrier proteins (hSA or bTG) did not affect staining. Absorption of the antisera with as low as 1 pg of the h-a-inhibin (l-32) fragment or with porcine follicular fluid, however, completely abolished the immunostaining. The specificity of the staining reaction was further substantiated by serially diluting the primary antisera, resulting in a gradual decrease in the intensity and finally in the diappearance of the staining. The antisera were also characterized by radioimmunoassay. All antisera showed parallel inhibition of ‘251-h-a-inhibin binding when either h-ainhibin l-25, porcine or human follicular fluid, highly purified porcine follicular fluid (W.H.O. Standard WGL4-55B) or rat Sertoli cell culture media (kindly provided by Dr. A. Kierszenbaum, University of North Carolina) were used as competitors (Culler and Negro-Vilar, in preparation). Results With the combined application of trypsin pretreatment and our modified double PAP technique, we were able to detect cr-inhibin-like immunoreactivity in both the male and female rat gonad and in the human placenta. Without trypsin pretreatment and with the conventional PAP staining, a less intense staining was observed. In the testis, staining was observed in different tubules (Fig. la) and clearly localized to the cytoplasm of Sertoli cells (Fig. lb), indicating the presence of a-inhibin-like immunoreactivity in these cells. No staining was observed among germ cells in the different stages of spermiogenesis or in interstitial cells. Immunoreactive Sertoli cells were observed in numerous seminiferous tubules in each
Fig. 1. Irnmunocytcchemical staining for inhibin in different reproductive tissues. (a) Low-magnification photomicrograph of a section of adult rat testes immunostained using ovine anti-n-inhibin serum MC-4 (1 : 20000). Only Sertoli cells show immunopositive staining. (b) Higher magnification of a tubule from the same animal as Fig. la. (c) Control testes section. The preabsorption of MC-4 anti-a-inhibin serum with 10 gg h-a-inhibin 1-32/m] 1:20000 dilution completely abolished the Sertoli cell staining. (d) Photomicrograph of a section of a 21-day rat ovary showing a-inhibin immunopositive granulosa cells in a tertiary follicle as revealed by rabbit anti-a-inhibin serum MC-8 (1: 15000). (P) Secondary follicles from a section of a 21-day rat ovary with a-inhibin immunopositive granulosa cells. (f) Control ovary section. Preabsorption of the MC-8 anti-a-inhibin serum with 10 pg h-a-inhibin l-32/ml 1: 15000 dilution completely abolished the granulosa cell staining. (g) Photomicrograph of a section of human placenta. Only one villus is shown. The cytotrophoblast cells are immunopositive for a-inhibin. Some of the mesenchymal cells within the villus are also stained. Anti-a-inhibin serum MC-8 was used at a dilution of 1: 15000 to stain this tissue. Absorption with h-a-inhibin l-32 as in Fig. lc and lf legend completely abolished the immunostaining (not shown). Magnifications: 0, c-g = 170 X ; b = 400 X
242
section, while other adjacent tubules were devoid of staining. Absorption of the antiserum with synthetic h-cr-inhibin (l-32) completely eliminated the immunostaining in the testis (Fig. lc). In the ovary, granulosa cells of follicles in various stages of development were intensely stained (Fig. Id, e) indicating the presence of a-inhibin-like immunoreactivity in these cells. All granulosa cells within a given follicle appeared stained. No staining was observed in the thecal cells. Ovaries from adult rats also showed immunopositive staining of the granulosa cells and, in addition, the luteal cells (data not shown). The immunopositive staining in the luteal cells, however, was much less intense than the staining found in the granulosa cells. Absorption of the antiserum with the h-a-inhibin (l-32) peptide completely suppressed the staining in these tissues (Fig. If). In the humam placenta, the immunostaining was localized in the cells of the cytotrophoblast layer of the placental villi (Fig. lg). No staining was observed in the peripherally-located syncytiotrophoblast cells (Fig. lg). As in the previous tissues, absorption of the antiserum with h-a-inhibin (l-32) abolished the staining (not shown). Discussion This study presents the first immunocytochemical localization of inhibin in the male and female gonad. The presence of a-inhibin chain-like immunoreactivity in the Sertoli, granulosa and luteal cells provides strong evidence that these cells are the source of gonadal inhibin. Our observation of immunostaining for inhibin in the human placenta confirms earlier reports localizing inhibin by immunofluorescence and radioimmunoassay (McLachlan et al., 1986a; Petraglia et al., 1987). The presence of cY-inhibin immunoreactivity in placental tissue supports the notion that these molecules play an important role in different reproductive situations, including pregnancy. Evidence for enhanced secretion during pregnancy, in correlation with changes in circulating HCG levels, has been presented (McLachlan et al., 1986b). In addition to being released into the general circulation from Sertoli, granulosa or cytotrophoblast cells, inhibin may also act locally to
modify certain parameters of gonadal or placental function (Bicsak et al., 1987; Petraglia et al., 1987). In our studies in the adult rat testis, inhibin was localized to Sertoli cells in certain tubules, while other adjacent tubules remained unstained. This observation suggests that the expression of inhibin in Sertoli cells may be stage-specific (Kierszenbaum et al., 1987) and therefore may be related to some dynamic parameter of gametogenic function. These findings strongly support a key role for inhibin in regulating reproductive functions. References Bicsak, T.A., Vale, W., Vaughn, J., Tucker, E.M., Cappel, S. and Hsueh, A.J.W. (1987) Mol. Cell. Endocrinol. 49, 211-217. Chari, S., Duraiswarmi, S. and Franchimont, P. (1978) Acta Endocrinol. 87,434-448. DeJong, F.H. (1979) Mol. Cell. Endocrinol. 13, l-10. DeJong, F.H. and Sharpe, R.M. (1976) Nature 263, 71-72. DePaolo, L.V., Wise, P.M. and Anderson, L.D. (1979) Endocnnology 104,402-408. Esch, F.S., Shimasaki, S., Cooksey, K., Mercado, M., Mason, A.J., Ying, S.Y., Ueno, N. and Lmg, N. (1987) Mol. Endocrinol. 1, 388-396. Franchimont, P., Vertraelen-Proyard, J., Hazee-Hagelstein, M.T., Renard, Ch., Demoulin, A., Bourguignon, J.P. and Hustin, J. (1979) Vitam. Horm. 37, 243-302. Fukuda, M., Miyamoto, K., Hasegawa, Y., Nomura, M., Igarashi, M., Kangawa, K. and Matsuo, H. (1986) Mol. Cell. Endocrinol. 44, 55-60. Grady, R.R., Charlesworth, M.C. and Schwartz, N.B. (1982) Recent Prog. Horm. Res. 38, 409-457. Kierszenbaum, A.L., Abdullah, M., Ueda, H. and Tres, L.L. (1987) In: Regulation of Ovarian and Testicular Function, Eds.: V.B. Mahesh, D.S. Dhindsa, E. Anderson and S.P. Kalra (Plenum Press, New York, NY) pp. 535-560. Ling, N., Ying, S.-Y., Ueno, N., Each, F., Denoroy, L. and Guillemin, R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7217-7221. Ling, N., Ying, S.-H., Ueno, N., Shimasaki, S., Each, F., Hotta, M. and Guillemin, R. (1986) Nature 321, 779-782. Mason, A.J., Hayflick, J.S., Ling, N., Esch, F., Ueno, N., Ying, S.-Y., Guillemin, R., Niall, H. and Seeburg, P.H. (1985) Nature 318, 659-663. McCullagh, D.R. (1932) Science 76, 12-20. McLachlan, R.I., Healy, D.L., Robertson, D.M., Burger, H.G. and de Kretser, D.M. (1986a) B&hem. Biophys. Res. Commun. 140,485-490. McLachlan, RI., Healy, D.L., Lutjen, P.J., Burger, H.G. and de Kretser, D.M. (1986b) 69th Annual Meeting of the Endocrine Society, Abstract No. 22. Miyamoto, H., Hasegawa, Y., Fukuda, M., Nomura, M., Igarashi, M., Kangawa, K. and Matsuo, H. (1985) B&hem. Biophys. Res. Commun. 129, 396-403.
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