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Expression of inhibins and activins in the equine placenta
Animal Reproduction Science 94 (2006) 413–416
Abstract
Expression of inhibins and activins in the equine placenta夽 K. Taya a,c,∗ , K.Y. Arai b , Y. Tanaka a , H. Taniyama d , N. Tsunoda e , Y. Nambo f , N. Nagamine a , G. Watanabe a,c a
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan b Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan c Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan d Department of Veterinary Pathology, Rakuno Gakuen University, Hokkaido 069-8501, Japan e Shadai Corporation, Hokkaido 059-1432, Japan f Hidaka Training Research Center, Japan Racing Association, Hokkaido 057-171, Japan Available online 25 April 2006
1. Introduction Inhibins were originally isolated as gonadal peptides that inhibit FSH secretion from the pituitary gland (Ying, 1988). Two inhibins, inhibin A and inhibin B, have been identified; each is composed of a common ␣-subunit and either a A subunit or a B-subunit (Ying, 1988). Homo- and hetero-dimers of the -subunits of inhibin are called activins (Ying, 1988), multifunctional growth factors. It has been proposed that placental function and development are regulated by local activin production, because activin has been shown to influence human placental cell function (Petraglia et al., 1989; Steele et al., 1993; Caniggia et al., 1997). The presence of Asubunit mRNA and the absence of ␣-subunit mRNA in the equine placenta have been shown by Yamanouchi et al. (1997). However, it is not known whether the equine placenta also expresses B-subunit or secretes inhibin/activin dimers. Neither is it known whether the placenta contributes to the high concentrations of inhibins in equine fetal plasma (Nambo et al., 1996; Tanaka et al., 2002, 2003). In this study, we examined expression patterns of inhibins and activins in the equine placenta. 夽 This paper is part of the special issue entitled Proceedings of the Ninth International Symposium on Equine Reproduction, Guest Edited by Margaret J. Evans. ∗ Corresponding author. Tel.: +81 42 367 5767; fax: +81 42 367 5767. E-mail address: [email protected] (K. Taya).
0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.03.036
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K. Taya et al. / Animal Reproduction Science 94 (2006) 413–416
2. Materials and methods 2.1. Tissue samples Placentae were collected from normal pregnant Thoroughbred and Anglo-Arab mares at Days 100–312 of gestation after euthanasia with an overdose of a mixture of barbiturate and suxamethonium chloride. Fetal gonads were also collected. All procedures were carried out in accordance with the guidelines established by the Rakuno Gakuen University for use of laboratory animals. Homogenates of the tissue samples were prepared for ELISAs as previously described (Tanaka et al., 2002, 2003). The homogenized samples were divided into the following gestational age groups: Days 100–140 (n = 3); Days 140–180 (n = 11); Days 180–220 (n = 8); and Days 220–260 (n = 3). For histochemical studies, six placentae were recovered at different stages of gestation (Days 130, 132, 162, 208, 227 and 312 of gestation) and fixed in freshly prepared 4% (w/v) paraformaldehyde in 0.01 M phosphate buffered saline (PBS) and embedded in paraffin. 2.2. ELISA Using two-site ELISA kits (Serotec, Oxford, UK), concentrations of inhibin pro-␣C and inhibin A were measured in tissue homogenates as previously described (Tanaka et al., 2002, 2003). Purified human inhibin pro-␣C and purified 32-kDa-bovine inhibin A were used as standards. Concentrations of activin A in tissue homogenates were measured using a two-site ELISA kit (Serotec) (Knight et al., 1996). Recombinant activin A was used as a standard. 2.3. Immunohistochemistry Immunohistochemistry was performed as previously described (Tanaka et al., 2003). Primary antibodies used in this study were a rabbit polyclonal antibody to [Tyr30]-porcine inhibin ␣ chain (1–30), a monoclonal antibody raised against the synthetic A-subunit (E4), and a monoclonal antibody raised against the synthetic human B-subunit (C5). 2.4. In situ hybridization Using DIG-labeled RNA probes, in situ hybridization for inhibin/activin subunits was performed as previously described (Tanaka et al., 2003). 2.5. Statistical analysis Values are presented as means ± S.E.M. To compare the mean values, one-way analysis of variance was carried out and the significance of the difference between means was determined by a Duncan multiple range test (P < 0.05). 3. Results Inhibin pro-␣C was not detected in fetal placental tissues. Maternal endometrial homogenates contained lower concentrations of inhibin pro-␣C than homogenates of fetal gonads. Inhibin A was detected in both maternal endometrium and fetal allantochorion, but the concentrations were much lower than those in the fetal gonads. Concentrations of inhibin A in fetal gonads
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tended to decrease as pregnancy progressed. While activin concentrations in fetal gonads were very low, high concentrations of activin A were detected in the endometrium and allantochorion. Concentrations of activin A in the allantochorion decreased significantly between Days 100 and 180, and remained low thereafter. Concentrations of activin A in the endometrium gradually decreased as pregnancy progressed, but the change was not statistically significant. Inhibin ␣-subunit mRNA was not detected in either endometrial glands or microcotyledons. In contrast, A-subunit mRNA was strongly expressed in endometrial glands. Inhibin ␣- and B-subunit proteins were not detected in either endometrial glands or microcotyledons. Immunopositive staining of inhibin/activin A-subunit was observed in endometrial glands, but not in microcotyledons. 4. Discussion The results demonstrated the presence of high concentrations of activin A, but very low concentrations of inhibins, in maternal and fetal placental tissues. As inhibin/activin A-subunit and its mRNA were detected in maternal but not in fetal placental tissues, activin A in the fetal allantochorionic homogenates had probably diffused across from the endometrial glands. While concentrations of inhibins were low in placental tissues, the fetal gonads contained high concentrations of inhibins. The results thus indicate that the high concentrations of inhibins in fetal circulation (Tanaka et al., 2002, 2003) do not originate from the placenta, but from the fetal gonads. Our finding of decreasing activin A concentrations in the placental tissues as pregnancy progresses is consistent with a decrease in A-subunit mRNA in the placenta during the last third of gestation (Yamanouchi et al., 1997). Inhibin B-subunit was not detected in the equine placenta by immunohistochemistry, indicating that the equine placenta does not express significant levels of B-subunit. Activin A has been shown to stimulate the outgrowth of human cytotrophoblast cells (Caniggia et al., 1997). As the formation of the equine placenta occurs gradually between Days 40 and 150 of gestation (Samuel et al., 1975), placental activin may have a role in promoting placentation during the first half of pregnancy. The relatively high concentrations of activin A in the placental tissues between Days 100 and 140, and its subsequent decrease support this hypothesis. In summary, the present study is the first to demonstrate secretion of activin A by the equine placenta and suggests involvement of activin A in the regulation of placental development. Acknowledgements We are grateful to Dr. N. Ling, Neuroendocrine Inc., for [Tyr30]-porcine inhibin ␣ chain (130)-NH2; Dr. N.P. Groome, Oxford Brookes University, for antibodies to human inhibin A- and B-subunits; and to Dr. K. Yamanouchi, University of Tokyo, for equine inhibin cDNAs. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (The 21st Century Center of Excellence Program, E-1) and the Equine Research Institute of the Japan Racing Association. References Caniggia, I., Lye, S.J., Cross, J.C., 1997. Activin is a local regulator of human cytotrophoplast cell differentiation. Endocrinology 138, 3976–3986.
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K. Taya et al. / Animal Reproduction Science 94 (2006) 413–416
Knight, P.G., Muttukrishna, S., Groome, N.P., 1996. Development and application of a two-site enzyme immunoassay for the determination of total activin-A concentrations in serum and follicular fluid. J. Endocrinol. 148, 267–279. Nambo, Y., Nagata, S., Oikawa, M., Yoshihara, T., Tsunoda, N., Kohsaka, T., Taniyama, H., Watanabe, G., Taya, K., 1996. High concentrations of immunoreactive inhibin in the plasma of mares and fetal gonads during the second half of pregnancy. Reprod. Fertil. Dev. 8, 1137–1145. Petraglia, F., Vaughan, J., Vale, W., 1989. Inhibin and activin modulate the release of gonadotropin-releasing hormone, human chorionic gonadotropin, and progesterone from cultured human placental cells. Proc. Natl. Acad. Sci. U.S.A. 86, 5114–5117. Samuel, C.A., Allen, W.R., Steven, D.H., 1975. Ultrastructural development of the equine placenta. J. Reprod. Fert. Suppl. 23, 575–578. Steele, G.L., Currie, W.D., Yuen, B.H., Jia, X.C., Perlas, E., Leung, P.C., 1993. Acute stimulation of human chorionic gonadotropin secretion by recombinant human activin-A in first trimester human trophoblast. Endocrinology 133, 297–303. Tanaka, Y., Taniyama, H., Tsunoda, N., Shinbo, H., Nagamine, N., Nambo, Y., Nagata, S., Watanabe, G., Herath, C.B., Groome, N.P., Taya, K., 2002. The testis as a major source of circulating inhibins in the male equine fetus during the second half of gestation. J. Androl. 23, 229–236. Tanaka, Y., Taniyama, H., Tsunoda, N., Herath, C.B., Nakai, R., Shinbo, H., Nagamine, N., Nambo, Y., Nagata, S., Watanabe, G., Groome, N.P., Taya, K., 2003. Localization and secretion of inhibins in the equine fetal ovaries. Biol. Reprod. 68, 328–335. Yamanouchi, K., Hirasawa, K., Hasegawa, T., Ikeda, A., Chang, K.T., Matsuyama, S., Nishihara, M., Miyazawa, K., Sawasaki, T., Tojo, H., Tachi, C., Takahashi, M., 1997. Equine inhibin/activin A-subunit mRNA is expressed in the endometrial gland, but not in the trophoblast, during pregnancy. Mol. Reprod. Dev. 47, 363–369. Ying, S.Y., 1988. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr. Rev. 9, 267–293.
Abstract
Expression of inhibins and activins in the equine placenta夽 K. Taya a,c,∗ , K.Y. Arai b , Y. Tanaka a , H. Taniyama d , N. Tsunoda e , Y. Nambo f , N. Nagamine a , G. Watanabe a,c a
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan b Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan c Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan d Department of Veterinary Pathology, Rakuno Gakuen University, Hokkaido 069-8501, Japan e Shadai Corporation, Hokkaido 059-1432, Japan f Hidaka Training Research Center, Japan Racing Association, Hokkaido 057-171, Japan Available online 25 April 2006
1. Introduction Inhibins were originally isolated as gonadal peptides that inhibit FSH secretion from the pituitary gland (Ying, 1988). Two inhibins, inhibin A and inhibin B, have been identified; each is composed of a common ␣-subunit and either a A subunit or a B-subunit (Ying, 1988). Homo- and hetero-dimers of the -subunits of inhibin are called activins (Ying, 1988), multifunctional growth factors. It has been proposed that placental function and development are regulated by local activin production, because activin has been shown to influence human placental cell function (Petraglia et al., 1989; Steele et al., 1993; Caniggia et al., 1997). The presence of Asubunit mRNA and the absence of ␣-subunit mRNA in the equine placenta have been shown by Yamanouchi et al. (1997). However, it is not known whether the equine placenta also expresses B-subunit or secretes inhibin/activin dimers. Neither is it known whether the placenta contributes to the high concentrations of inhibins in equine fetal plasma (Nambo et al., 1996; Tanaka et al., 2002, 2003). In this study, we examined expression patterns of inhibins and activins in the equine placenta. 夽 This paper is part of the special issue entitled Proceedings of the Ninth International Symposium on Equine Reproduction, Guest Edited by Margaret J. Evans. ∗ Corresponding author. Tel.: +81 42 367 5767; fax: +81 42 367 5767. E-mail address: [email protected] (K. Taya).
0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.03.036
414
K. Taya et al. / Animal Reproduction Science 94 (2006) 413–416
2. Materials and methods 2.1. Tissue samples Placentae were collected from normal pregnant Thoroughbred and Anglo-Arab mares at Days 100–312 of gestation after euthanasia with an overdose of a mixture of barbiturate and suxamethonium chloride. Fetal gonads were also collected. All procedures were carried out in accordance with the guidelines established by the Rakuno Gakuen University for use of laboratory animals. Homogenates of the tissue samples were prepared for ELISAs as previously described (Tanaka et al., 2002, 2003). The homogenized samples were divided into the following gestational age groups: Days 100–140 (n = 3); Days 140–180 (n = 11); Days 180–220 (n = 8); and Days 220–260 (n = 3). For histochemical studies, six placentae were recovered at different stages of gestation (Days 130, 132, 162, 208, 227 and 312 of gestation) and fixed in freshly prepared 4% (w/v) paraformaldehyde in 0.01 M phosphate buffered saline (PBS) and embedded in paraffin. 2.2. ELISA Using two-site ELISA kits (Serotec, Oxford, UK), concentrations of inhibin pro-␣C and inhibin A were measured in tissue homogenates as previously described (Tanaka et al., 2002, 2003). Purified human inhibin pro-␣C and purified 32-kDa-bovine inhibin A were used as standards. Concentrations of activin A in tissue homogenates were measured using a two-site ELISA kit (Serotec) (Knight et al., 1996). Recombinant activin A was used as a standard. 2.3. Immunohistochemistry Immunohistochemistry was performed as previously described (Tanaka et al., 2003). Primary antibodies used in this study were a rabbit polyclonal antibody to [Tyr30]-porcine inhibin ␣ chain (1–30), a monoclonal antibody raised against the synthetic A-subunit (E4), and a monoclonal antibody raised against the synthetic human B-subunit (C5). 2.4. In situ hybridization Using DIG-labeled RNA probes, in situ hybridization for inhibin/activin subunits was performed as previously described (Tanaka et al., 2003). 2.5. Statistical analysis Values are presented as means ± S.E.M. To compare the mean values, one-way analysis of variance was carried out and the significance of the difference between means was determined by a Duncan multiple range test (P < 0.05). 3. Results Inhibin pro-␣C was not detected in fetal placental tissues. Maternal endometrial homogenates contained lower concentrations of inhibin pro-␣C than homogenates of fetal gonads. Inhibin A was detected in both maternal endometrium and fetal allantochorion, but the concentrations were much lower than those in the fetal gonads. Concentrations of inhibin A in fetal gonads
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tended to decrease as pregnancy progressed. While activin concentrations in fetal gonads were very low, high concentrations of activin A were detected in the endometrium and allantochorion. Concentrations of activin A in the allantochorion decreased significantly between Days 100 and 180, and remained low thereafter. Concentrations of activin A in the endometrium gradually decreased as pregnancy progressed, but the change was not statistically significant. Inhibin ␣-subunit mRNA was not detected in either endometrial glands or microcotyledons. In contrast, A-subunit mRNA was strongly expressed in endometrial glands. Inhibin ␣- and B-subunit proteins were not detected in either endometrial glands or microcotyledons. Immunopositive staining of inhibin/activin A-subunit was observed in endometrial glands, but not in microcotyledons. 4. Discussion The results demonstrated the presence of high concentrations of activin A, but very low concentrations of inhibins, in maternal and fetal placental tissues. As inhibin/activin A-subunit and its mRNA were detected in maternal but not in fetal placental tissues, activin A in the fetal allantochorionic homogenates had probably diffused across from the endometrial glands. While concentrations of inhibins were low in placental tissues, the fetal gonads contained high concentrations of inhibins. The results thus indicate that the high concentrations of inhibins in fetal circulation (Tanaka et al., 2002, 2003) do not originate from the placenta, but from the fetal gonads. Our finding of decreasing activin A concentrations in the placental tissues as pregnancy progresses is consistent with a decrease in A-subunit mRNA in the placenta during the last third of gestation (Yamanouchi et al., 1997). Inhibin B-subunit was not detected in the equine placenta by immunohistochemistry, indicating that the equine placenta does not express significant levels of B-subunit. Activin A has been shown to stimulate the outgrowth of human cytotrophoblast cells (Caniggia et al., 1997). As the formation of the equine placenta occurs gradually between Days 40 and 150 of gestation (Samuel et al., 1975), placental activin may have a role in promoting placentation during the first half of pregnancy. The relatively high concentrations of activin A in the placental tissues between Days 100 and 140, and its subsequent decrease support this hypothesis. In summary, the present study is the first to demonstrate secretion of activin A by the equine placenta and suggests involvement of activin A in the regulation of placental development. Acknowledgements We are grateful to Dr. N. Ling, Neuroendocrine Inc., for [Tyr30]-porcine inhibin ␣ chain (130)-NH2; Dr. N.P. Groome, Oxford Brookes University, for antibodies to human inhibin A- and B-subunits; and to Dr. K. Yamanouchi, University of Tokyo, for equine inhibin cDNAs. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (The 21st Century Center of Excellence Program, E-1) and the Equine Research Institute of the Japan Racing Association. References Caniggia, I., Lye, S.J., Cross, J.C., 1997. Activin is a local regulator of human cytotrophoplast cell differentiation. Endocrinology 138, 3976–3986.
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K. Taya et al. / Animal Reproduction Science 94 (2006) 413–416
Knight, P.G., Muttukrishna, S., Groome, N.P., 1996. Development and application of a two-site enzyme immunoassay for the determination of total activin-A concentrations in serum and follicular fluid. J. Endocrinol. 148, 267–279. Nambo, Y., Nagata, S., Oikawa, M., Yoshihara, T., Tsunoda, N., Kohsaka, T., Taniyama, H., Watanabe, G., Taya, K., 1996. High concentrations of immunoreactive inhibin in the plasma of mares and fetal gonads during the second half of pregnancy. Reprod. Fertil. Dev. 8, 1137–1145. Petraglia, F., Vaughan, J., Vale, W., 1989. Inhibin and activin modulate the release of gonadotropin-releasing hormone, human chorionic gonadotropin, and progesterone from cultured human placental cells. Proc. Natl. Acad. Sci. U.S.A. 86, 5114–5117. Samuel, C.A., Allen, W.R., Steven, D.H., 1975. Ultrastructural development of the equine placenta. J. Reprod. Fert. Suppl. 23, 575–578. Steele, G.L., Currie, W.D., Yuen, B.H., Jia, X.C., Perlas, E., Leung, P.C., 1993. Acute stimulation of human chorionic gonadotropin secretion by recombinant human activin-A in first trimester human trophoblast. Endocrinology 133, 297–303. Tanaka, Y., Taniyama, H., Tsunoda, N., Shinbo, H., Nagamine, N., Nambo, Y., Nagata, S., Watanabe, G., Herath, C.B., Groome, N.P., Taya, K., 2002. The testis as a major source of circulating inhibins in the male equine fetus during the second half of gestation. J. Androl. 23, 229–236. Tanaka, Y., Taniyama, H., Tsunoda, N., Herath, C.B., Nakai, R., Shinbo, H., Nagamine, N., Nambo, Y., Nagata, S., Watanabe, G., Groome, N.P., Taya, K., 2003. Localization and secretion of inhibins in the equine fetal ovaries. Biol. Reprod. 68, 328–335. Yamanouchi, K., Hirasawa, K., Hasegawa, T., Ikeda, A., Chang, K.T., Matsuyama, S., Nishihara, M., Miyazawa, K., Sawasaki, T., Tojo, H., Tachi, C., Takahashi, M., 1997. Equine inhibin/activin A-subunit mRNA is expressed in the endometrial gland, but not in the trophoblast, during pregnancy. Mol. Reprod. Dev. 47, 363–369. Ying, S.Y., 1988. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr. Rev. 9, 267–293.