C63
Molecular and Cellular Endocrinology, 13 (1990) C63-C61 Elsevier Scientific Publishers Ireland. Ltd.
MOLCEL 02388
At the Cutting
Pituitary gonadotropin
Edge
gene regulation
Julie E. Mercer Division of Genetics, Department
Key worak
Follicle-stimulating
of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 021 IS, U.S.A.
hormone;
Luteinizing
hormone;
Pituitary gland; Gene expression
regulation;
Gonadotropins,
pitui-
tary Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are synthesised in the anterior pituitary gonadotropes and act on the testis and ovary to direct steroidogenesis and gametogenesis. These two gonadotropin hormones are members of the glycoprotein family of hormones which also includes the pituitary hormone thyroid-stimulating hormone (TSH) and placental chorionic gonadotropin (CG). The glycoprotein hormones each consist of two different subunits. The a-subunits are the same for all the glycoproteins within a species and are encoded by a single gene; the /3-subunits are different for each hormone, and it is these which determine the biological specificity of each hormone (Pierce and Parsons, 1981). The structure of the gonadotropin genes has recently been comprehensively reviewed by Gharib et al. (1990). The regulation of secretion of the gonadotropins from the pituitary gland has been studied extensively, but it is only relatively recently that the regulation of synthesis at the pre-translational level has been investigated. It is well established that hypothalamic gonadotropin-releasing hormone (GnRH) is secreted in a pulsatile manner to stimulate the release of LH and FSH (Clarke and Cummins, 1982). Gonadal steroid hormones have both positive and negative feedback effects on release of gonadotropins, and the gonadal peptide hormones inbibin, activin and follistatin have ef-
Address for correspondence: Dr. Julie E. Mercer, Division of Genetics, Brigham and Women’s Hospital, Thorn 917, 20 Shattuck St., Boston, MA 02115, U.S.A.
0303-7207/90/$03.50
fects largely on FSH secretion; the role of inhibin in regulating LH secretion is a matter of some dispute in the literature at present. All these regulatory factors interact to exert a complex pattern of control over gonadotropin secretion. The synthesis of the gonadotropin hormones has now been examined by molecular biological techniques to explore the differential regulation of expression of the a-subunit, LHj3 and FSHB genes. These studies have provided additional insights into the complex regulation of gonadotropins by GnRH and gonadal hormones. It is not the intention of this review to discuss all aspects of the regulation of gonadotropin hormone gene expression, which has recently been done in considerable detail by Gharib et al. (1990), but to focus on particularly interesting and perhaps even challenging aspects of the regulation of secretion of these hormones. The role of pulsatileGnRH Pulsatile delivery of GnRH to the pituitary gland is necessary to maintain LH and FSH secretion and there is a close temporal relationship between G&I-I and LH pulses (Clarke and Cummins, 1982; Levine and Rarnirez, 1982). Further, it has been demonstrated that both GnRH pulse amplitude and GnRH pulse frequency affect the secretion of gonadotropins (reviewed in Clarke,’ 1987). In addition to effects on secretion, GnRH also regulates the synthesis of pituitary gonadotropins. In ovariectomized, hypothalamo-pituitary discon-
8 1990 Elsevier Scientific Publishers Ireland, Ltd.
C64
netted (OVX/HPD) sheep, where the pituitary is surgically isolated from any hypothalamic input, mRNA levels for LHj3, FSHj3 and cY-subunit fall to very low levels which are restored to OVX levels by administration of pulsatile but not constant infusion of GnRH (Hamernik et al., 1988; Mercer et al., 1988, 1989). Studies in rats and mice using GnRH agonist/ antagonist administration or GnRH antisera have further demonstrated the role of GnRH in maintaining gonadotropin subunit mRNA levels (Simard et al., 1988; Stanley et al., 1988; Saade et al., 1989; Wierman et al., 1989). Using a castrate, testosterone replaced rat model in which endogenous GnRH is suppressed, Marshall and co-workers have shown that both GnRH pulse frequency and amplitude regulate the synthesis of gonadotropin subunits (Haisenleder et al., 1988; Dalkin et al., 1989). Interestingly, the frequencies and amplitudes which define the maximal response of LH secretion also, in general, evoke the maximal stimulation of LHB mRNA levels; in contrast, cr-subunit and FSHB mRNA levels respond maximally to different GnRH regimens. This group has also demonstrated that GnRH pulses stimulate transcription of all three gonadotropin genes, whereas a constant infusion of GnRH does not (Haisenleder et al., 1990). In contrast with the rat, the adminstration of 25 ng pulses of GnRH (lo-fold smaller than those used to maintain physiological circulating levels of LH and FSH) to OVX/HPD sheep does not maintain secretion of LH, but is sufficient to maintain LH/!I mRNA levels. When these sheep are treated with constant G&I-I, LH secretion ceases (Clarke et al., 1986), with LHj? and FSH/3 mRNA at levels 50% of those in pulsed animals after 7 days of infusion (Mercer and Clarke, 1989). These data suggest that there is a dissociation between LH synthesis and secretion, in sheep at least, in that GnRH pulses of a particular amplitude (250 ng) are required to maintain secretion of LH, whereas synthesis can be normally maintained by lower amplitude pulses of GnRH, and persists to some extent in the face of constant G&I-I levels. Conversely, in several studies in vitro in which rat pituitary cells were treated with GnRH either in static culture or in perifusion systems, effects on release of LH were clearly in evidence, but no
decrease in LHj3 mRNA was demonstrated (Attardi et al., 1988; Weiss et al., 1990). In a further study, no effect was seen on LHP mRNA transcription rate after GnRH treatment, despite an observed effect on LH release (Salton et al., 1989). These data again suggest a dissociation between synthesis and secretion, although the failure of these studies to show an effect on LHP mRNA levels may also be a reflection of the pattern of GnRH administration. There has been one report to date of a stimulatory effect of GnRH on LHP mRNA levels in cultured pituitary cells, and that study also shows a stimulatory effect of protein kinase C on LH/3 mRNA levels (Andrews et al., 1988). Shupnik (1990) has recently shown in rat pituitary quarters that pulsatile GnRH will stimulate transcription of both the a-subunit and LHP genes, while a constant infusion of GnRH stimulates a-subunit gene transcription only; in this study GnRH stimulated LH secretion with both pulsatile and constant infusion. Several studies have examined transcriptional regulation of the a-subunit gene by cyclic AMP in cells derived from choriocarcinomas (Deutsch et al., 1987; Silver et al., 1987; Jameson et al., 1988; Mellon et al., 1989). A recent report describes a study showing regulation of the cw-subunit gene by GnRH in clonal cells derived from pituitary tumors in transgenic mice carrying an a-subunit promoter-SV40 T-antigen fusion gene (Windle et al., 1990). Further definition of the role of GnRH and its possible second messengers in regulating the expression of the LHj3 and FSHj? genes will be greatly facilitated by the development of cell lines which allow the expression of these genes, and the tumor-producing transgenic mouse model may offer some possibilities in this regard. The site of estrogen action The way in which sex steroids regulate synthesis of gonadotropins is clearly complex. Gonadectomy leads to increases in mRNA levels for all three gonadotropin subunits in male and female rats (Gharib et al., 1987) and in ewes (Nilson et al., 1985; Mercer et al., 1989). In addition, in situ hybridization studies have shown both that the number of LHP and FSHP mRNA-containing cells increases, and that the amount of LHP and
c65
FSHB mRNA per cell also increases following gonadectomy (Childs et al., 1987,199O). A great deal of effort has gone into defining the site of action of steroid hormones within the hypothahuno-pituitary unit. The OVX/HPD sheep model has enabled in vivo studies of the surgically isolated, vascular-intact pituitary to be undertaken. These studies have shown that estrogen in the sheep does not have a direct negative action at the pituitary, since estradiol has no effect on LH/3 mRNA levels in OVX/HPD, GnRH pulsed sheep; in intact animals, estrogen thus presumably down-regulates LH/3 mRNA levels via an effect on GnRH. In contrast, FSH/3 and a-subunit mRNA levels are decreased by estrogen treatment in this model, indicating a direct effect of estrogen on the pituitary to negatively regulate these two genes. Interestingly, when cultured rat pituitary fragments were incubated with estradiol for 2 or 6 h, no effects were seen on the transcription of a-subunit or FSH/3 genes but LH/3 mRNA synthesis was increased (Shupnik et al., 1989a). This effect on LH/3 mRNA synthesis is consistent with the site of action for negative regulation by estrogen being the hypothalamus, but suggests that the site of action of positive estrogen regulation of LH/3 mRNA is directly at the pituitary. An estrogen regulatory element (ERE) consensus sequence has been identified in the 5’ untranslated region of the rat LHB gene, and has been shown in binding studies to be functionally active (Shupnik et al., 1988). Positive feedback of estrogen on the hypothalamo-pituitary unit leads to the LH surge. Landefeld and co-workers have shown that LH/3 mRNA levels are increased by 60% during the pre-ovulatory LH surge in sheep (Leung et al., 1988), and the proestrus LH peak in rats is also preceded both by a rise in steady state LH/3 mRNA levels (Zmeili et al., 1986) and LH/I transcription (Shupnik et al., 1989a). OVX ewes treated with estrogen to elicit an LH surge show either no increase (Landefeld et al., 1987) or a decrease (Mercer et al., unpublished) in LH/3 mRNA levels following steroid injection. These data in sheep suggest that positive regulation of the LH/3 gene is not a major mechanism for positive estrogen feedback in this species, but that estrogen effects
may be largely on G&I-I secretion and/or upregulation of GnRH receptors to increase the sensitivity of the gonadotrope to GnRH. The elucidation of the GnRH receptor sequence and the development of cell lines which express pituitary gonadotropin genes will add significantly to our understanding of the manner in which estrogen is able to give rise to the LH surge. Gonadal peptides The gonadal peptides inhibin, activin and follistatin were isolated on the basis of their effects on FSH secretion from the pituitary gland. The related peptides inhibin and activin have opposite effects on FSH, with the a/J heterodimer, inhibin, suppressing FSH secretion (Robertson et al., 1986), and the /3/3homodimer, activin, stimulating FSH release (Ling et al., 1986). The structurally unrelated follistatin also suppresses FSH secretion (Vale et al., 1986). All three peptides have been identified in the gonads, and mRNA for the a and & subunits of inhibin have been localised in the pituitary (Meunier et al., 1988). In addition the follicular-stellate cells of the pituitary have been reported to secrete follistatin (Gospodarowicz and Lau, 1989). FSH/3 mRNA levels are rapidly suppressed in OVX/HPD GnRH pulsed sheep treated with an inhibin-containing follicular fluid extract, demonstrating a direct pituitary site of action for inhibin in vivo. Levels of a-subunit and LH/3 mRNA were not changed by inhibin in this model (Mercer et al., 1987). In vitro studies using rat pituitary cell cultures have also demonstrated a specific effect of inhibin to reduce FSH/3 mRNA levels in both untreated (Attardi et al., 1989a; Carroll et al., 1989) and GnRH-stimulated cells (Attardi et al., 1989b). In pituitary cell cultures it has been shown that activin increases and follistatin reduces FSH/3 mRNA levels with no change in a-subunit or LH/3 mRNA levels (Carroll et al., 1989; Attardi et al., 1990). In a subsequent study it has also been shown that one mechanism by which activin increases steady state mRNA levels for FSH/3 is by altering the half-life of the mRNA species (Carroll et al., 1990). Though these peptide hormones may also alter the transcription rate of the FSH/I gene, such studies have yet to be reported.
In addition to the effect of inhibin on FSH secretion, inhibin has been reported to decrease the basal secretion of LH from rat pituitary cells in culture (Farnworth et al., 1988) and to reduce the LH pulse amplitude in OVX/HPD sheep (Clarke et al., 1984; Mercer et al., 1987). Inhibin has also been reported to both down-regulate GnRH receptors on gonadotropes (Wang et al., 1989) and, in contrast, to increase GnRH receptor levels on the same cells (Laws et al., 1990) studies which are yet to be reconciled. Regardless of the mechanism by which the decrease in LH secretion is achieved, the data discussed earlier have convincingly shown that inhibin has no effect on LHj3 mRNA levels, again evidence of the dissociation between effects on LH secretion and synthesis. Inhibin (and activin) subunits have been colocalised with ir-LH and ir-FSH cells in the rat pituitary (Roberts et al., 1989) and it is thus possible that inhibin and activin may have an autocrine regulatory effect on FSH synthesis in the pituitary. If follistatin is indeed produced by the follicular-stellate cells of the pituitary gland, it may play a paracrine regulatory role in the control of FSH synthesis. Further elucidation of the precise localisation of what until now have been considered gonadal peptide hormones may lead us to rethink our concept of pituitary-gonadal feedback loops. Finally, of further interest is the recent identification of an ovarian activin binding protein as follistatin (Nakamura et al., 1990), and the subsequent report that a pituitary protein which specifically binds activin shows sequence homology to follistatin (Sugino et al., 1990). Since activin is synthesised in the pituitary, it may even be possible that follistatin has its effect on FSH by binding to activin and thus preventing it from having a positive effect on FSH. An alternative, intriguing possibility is that follistatin may in some way be related to the activin receptor. The answers to these questions and the elucidation of the complex feedback loops involved in cyclical hypothalamicpituitary-gonadal activity, await further studies. Acknowledgements I wish to thank Drs. W.W. Chin and Carroll for critically reading the manuscript.
R.S.
References Andrews, W.V., Maurer, R.A. and COM, P.M. (1988) J. Biol. Chem. 263, 13755-13761. Attardi, B. and MikIos, J. (1990) Mol. Endocrinol. 4, 721-726. Attardi, B., Keeping, H.S., Winter, S.J., Kotsuji, F., Maurer, R.A. and Troen, P. (1989a) Mol. Endocrinol. 3, 280-287. Attardi, B., Keeping, H.S., Winter, S.J., Kotsuji, F. and Troen, P. (1989b) Mol. Endocrinol. 3, 1236-1242. Carroll, R.S., Conigan, A.Z., Gharib, S.D., Vale, W. and Chin, W.W. (1989) Mol. Endocrinol. 3, 196991976. Carroll, R.S., Corrigan, A.Z. and Chin, W.W. (1990) U.S. Endoc. Sot. Meeting, Atlanta, GA, abstract 777. Childs, G.V., Lloyd, J.M., Unabia, G., Gharib, SD., Wierman, M.E. and Chin, W.W. (1987) Mol. Endocrinol. 1, 926-932. Childs, G.V., Unabia, G., Wierman, M.E., Gharib, SD. and Chin, W.W. (1990) Endocrinology 126, 2205-2213. Clarke, I.J. (1987) in Seminars in Reproductive Endocrinology (Asch, R., ed.), pp. 345-352, Thieme Medical Publishers, New York. Clarke, I.J. and Cummin s, J.T. (1982) Endocrinology 111, 1737-1739. Clarke, I.J., Cummins, J.T., Findlay, J.K., Burman, K.J. and Doughton, B.W. (1984) Neuroendocrinology 39, 214-221. Clarke, I.J., Findlay, J.K., Cummins, J.T. and Ewens, W.J. (1986) J. Reprod. Fertil. 77, 575-585. Dalkin, A.C., Haisenleder, D.J., Ortolano, G.A., Ellis, T.R. and Marshall, J.C. (1989) Endocrinology 125, 917-924. Deutsch, P.J., Jameson, J.L. and Habener, J.F. (1987) J. Biol. Chem. 262, 12169-12174. Famworth, P.G., Robertson, D.M., de Kretser, D.M. and Burger, H.G. (1988) Endocrinology 122, 207-213. Gharib, S.D., Bowers, S.M., Need, L.R. and Chin, W.W. (1986) J. Clin. Invest. 77, 582-589. Gharib, S.D., Wierman, M.E., Badger, T.M. and Chin, W.W. (1987) J. Clin. Invest. 80, 294-299. Gharib, S.D., Wierman, M.E., Shupnik, M.A. and Chin, W.W. (1990) Endocr. Rev. 11,177-199. Gospodarowicz, D. and Lau, K. (1989) Biochem. Biophys. Res. Commun. 165, 292-298. Haisenleder, D.J., Katt, J.A., Ortolano, G.A., El-Gewely, M.R., Duncan, J.A., Dee, C. and Marshall, J.C. (1988) Mol. Endocrinol. 2, 338-343. Haisenleder, D.J., DaIkin, AC., Ortolano, G.A., Marshall, J.C. and Shupnik, M.A. (1990) U.S. Endoc. Sot. Meeting, Atlanta, GA, abstract 474. Hamernik, D.L. and Nett, T.M. (1988) Endocrinology 122, 959-966. Jameson, J.L., Jaffe, R.C., Deutsch, P.J., Albanese, C. and Habener, J.F. (1988) J. Biol. Chem. 263, 9879-9886. Lalloz, M.R.A., Detta, A. and Clayton, R.N. (1988) Endocrinology 122, 1681-1688. Landefeld, T.D., Bagnell, T. and Levitan, I. (1989) Mol. Endocrinol. 3, 10-14. Laws, S.C., Bergs, M.J., Webster, J.C. and Miller, W.L. (1990) Endocrinology 127, 373-380. Leung, K., Kim, K.E., Maurer, R.A. and Landefeld, T.D. (1988) Mol. Endocrinol. 2, 272-276.
C67 Levine, J.E. and Ramirez, V.D. (1982) Endocrinology 111, 1439-1448. Ling, N., Ying, S.Y., Ueno, N., Esch, F., Denoroy, L. and Guillemin, R. (1985) Proc. Natl Acad. Sci. U.S.A. 82, 7217-7221. Ling, N., Ymg, S.Y., Ueno, N., Shimasaki, S., I&h, F., Hotta, M. and GuiIlemin, R. (1986) B&hem. Biophys. Res. Commun. 138, 1129. Mellon, P.L., C&g, C.H., Correll, L.A. and M&night, G.S. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4887-4891. Mercer, J.E. and Clarke, LJ. (1989) J. Neuroendocrinol. 1, 327-331. Mercer, J.E., Clements, J.A., Funder, J.W. and Clarke, I.J. (1987) Mol. CeII. Endocrinol. 53, 251-254. Mercer, J.E., Clements, J.A., Funder, J.W. and Clarke, I.J. (1988) Neuroendocrinology 47, 563-566. Mercer, J.E., CIements, J.A., Funder, J.W. and Clarke, I.J. (1989) Neuroendocrinology 50,280-285. Meunier, H., Rivier, C., Evans, R.M. and Vale, W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 247-251. Miyamoto, K., Hasegawa, Y., Fukuda, M., Nomura, M., Igarashi, M., Kangawa, K. and Matsuo, H. (1985) B&hem. Biophys. Res. Commun. 129, 396-403. Nakarnma, T., Takio, K., Eto, Y., Shibai, H., Titani, K. and Sugino, H. (1990) Science 247, 836-838. Nilson, J.H., Nejedhk, M.T., Virgin, J.B., Crowder, ME. and Nett, T.M. (1983) J. Biol. Chem. 258, 12087-12090. Pierce, J.G. and Parsons, T.F. (1981) Amm. Rev. Biochem. 50, 465-495. Rivier, J., Speiss, J., McCIintock, R., Vaughan, J. and Vale, W. (1985) B&hem. Biophys. Res. Commun. 133, 120-127. Roberts, V., Meunier, H., Vaughan, J., Rivier, J., Rivier, C., Vale, W. and Sawchenko, P. (1989) Endocrinology 124, 552-554. Robertson, D.M., FouIds, L.M., Leversha, L., Morgan, F.J., Heam, M.T.W., Burger, H.G., Wettenhah, R.E.H. and de Kretser, D.M. (1985) B&hem. Biophys. Res. Commun. 126,220-226. Saade, G., London, D.R. and Clayton, R.N. (1989) Endocrinology 124,1744-1753.
Sahon, S.R.J., Blum, M., Jonassen, J.A., Clayton, R.N. and Roberts, J.L. (1988) Mol. Endocrinol. 2, 1033-1042. Shupnik, M.A. (1990) U.S. Endoc. Sot. Meeting, Atlanta, GA, abstract 450. Shupnik, M.A., Gharib, SD. and Chin, W.W. (1988) Endocrinology 122,1842-1846. Shuqnik, M.A., Gharib, S.D. and Chin, W.W. (1989a) Mol. Endocrinol. 3, 474-480. Shupnik, M.A., Weimnarm, C.M., Notides, A.C. and Chin, W.W. (1989b) J. Biol. Chem. 264, 80-86. Silver, B.J., Bokar, J.A., Virgin, J.B., VaIlen, E.A., MiIsted, A. and N&on, J.H. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2198-2202. Simard, J., Labrie, C., Hubert, J.F. and Labrie, F. (1988) Mol. Endocrinol. 2, 775-784. Sugino, H., Nakamura, T., Kogawa, K., Takio, K. and Titani, K. (1990) U.S. Endoc. Sot. Meeting, Atlanta, GA, abstract 778. Ueno, N., Ling, N., Ying, S.-Y., Esch, F., Shimasaki, S. and GuiIlemin, R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8282-8286. Vale, W., Rivier, J., Vaughan, J., McCIintock, R., Corrigan, A., Woo, W., Karr, D. and Spiess, J. (1986) Nature 321, 776-779. Wang, Q.F., Famworth, P.G., Findlay, J.K. and Burger, H.G. (1989) Endocrinology 124, 363-368. Wang, Q.F., Famworth, P.G., Burger, H.G. and Findlay, J.K. (1990) Endocrinology 126, 3210-3217. Weiss, J., Jameson, J.L., Bunin, J.M. and Crowley, W.F. (1990) Mol. Endocrinol. 4, 557-564. Wierman, M.E., Rivier, J. and Wang, C. (1989) Endocrinology 124, 272-278. Windle, J.J., Weiner, R.I. and Mellon, P.L. (1990) Mol. Endocrinol. 4, 597-603. Zmeih, S.M., Papavasihou, S.S., Thomer, M.O., Evans, W.S., Marsha& J.C. and Landefeld, T.D. (1986) Endocrinology 119, 1867-1869.
Molecular and Cellular Endocrinology, 13 (1990) C63-C61 Elsevier Scientific Publishers Ireland. Ltd.
MOLCEL 02388
At the Cutting
Pituitary gonadotropin
Edge
gene regulation
Julie E. Mercer Division of Genetics, Department
Key worak
Follicle-stimulating
of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 021 IS, U.S.A.
hormone;
Luteinizing
hormone;
Pituitary gland; Gene expression
regulation;
Gonadotropins,
pitui-
tary Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are synthesised in the anterior pituitary gonadotropes and act on the testis and ovary to direct steroidogenesis and gametogenesis. These two gonadotropin hormones are members of the glycoprotein family of hormones which also includes the pituitary hormone thyroid-stimulating hormone (TSH) and placental chorionic gonadotropin (CG). The glycoprotein hormones each consist of two different subunits. The a-subunits are the same for all the glycoproteins within a species and are encoded by a single gene; the /3-subunits are different for each hormone, and it is these which determine the biological specificity of each hormone (Pierce and Parsons, 1981). The structure of the gonadotropin genes has recently been comprehensively reviewed by Gharib et al. (1990). The regulation of secretion of the gonadotropins from the pituitary gland has been studied extensively, but it is only relatively recently that the regulation of synthesis at the pre-translational level has been investigated. It is well established that hypothalamic gonadotropin-releasing hormone (GnRH) is secreted in a pulsatile manner to stimulate the release of LH and FSH (Clarke and Cummins, 1982). Gonadal steroid hormones have both positive and negative feedback effects on release of gonadotropins, and the gonadal peptide hormones inbibin, activin and follistatin have ef-
Address for correspondence: Dr. Julie E. Mercer, Division of Genetics, Brigham and Women’s Hospital, Thorn 917, 20 Shattuck St., Boston, MA 02115, U.S.A.
0303-7207/90/$03.50
fects largely on FSH secretion; the role of inhibin in regulating LH secretion is a matter of some dispute in the literature at present. All these regulatory factors interact to exert a complex pattern of control over gonadotropin secretion. The synthesis of the gonadotropin hormones has now been examined by molecular biological techniques to explore the differential regulation of expression of the a-subunit, LHj3 and FSHB genes. These studies have provided additional insights into the complex regulation of gonadotropins by GnRH and gonadal hormones. It is not the intention of this review to discuss all aspects of the regulation of gonadotropin hormone gene expression, which has recently been done in considerable detail by Gharib et al. (1990), but to focus on particularly interesting and perhaps even challenging aspects of the regulation of secretion of these hormones. The role of pulsatileGnRH Pulsatile delivery of GnRH to the pituitary gland is necessary to maintain LH and FSH secretion and there is a close temporal relationship between G&I-I and LH pulses (Clarke and Cummins, 1982; Levine and Rarnirez, 1982). Further, it has been demonstrated that both GnRH pulse amplitude and GnRH pulse frequency affect the secretion of gonadotropins (reviewed in Clarke,’ 1987). In addition to effects on secretion, GnRH also regulates the synthesis of pituitary gonadotropins. In ovariectomized, hypothalamo-pituitary discon-
8 1990 Elsevier Scientific Publishers Ireland, Ltd.
C64
netted (OVX/HPD) sheep, where the pituitary is surgically isolated from any hypothalamic input, mRNA levels for LHj3, FSHj3 and cY-subunit fall to very low levels which are restored to OVX levels by administration of pulsatile but not constant infusion of GnRH (Hamernik et al., 1988; Mercer et al., 1988, 1989). Studies in rats and mice using GnRH agonist/ antagonist administration or GnRH antisera have further demonstrated the role of GnRH in maintaining gonadotropin subunit mRNA levels (Simard et al., 1988; Stanley et al., 1988; Saade et al., 1989; Wierman et al., 1989). Using a castrate, testosterone replaced rat model in which endogenous GnRH is suppressed, Marshall and co-workers have shown that both GnRH pulse frequency and amplitude regulate the synthesis of gonadotropin subunits (Haisenleder et al., 1988; Dalkin et al., 1989). Interestingly, the frequencies and amplitudes which define the maximal response of LH secretion also, in general, evoke the maximal stimulation of LHB mRNA levels; in contrast, cr-subunit and FSHB mRNA levels respond maximally to different GnRH regimens. This group has also demonstrated that GnRH pulses stimulate transcription of all three gonadotropin genes, whereas a constant infusion of GnRH does not (Haisenleder et al., 1990). In contrast with the rat, the adminstration of 25 ng pulses of GnRH (lo-fold smaller than those used to maintain physiological circulating levels of LH and FSH) to OVX/HPD sheep does not maintain secretion of LH, but is sufficient to maintain LH/!I mRNA levels. When these sheep are treated with constant G&I-I, LH secretion ceases (Clarke et al., 1986), with LHj? and FSH/3 mRNA at levels 50% of those in pulsed animals after 7 days of infusion (Mercer and Clarke, 1989). These data suggest that there is a dissociation between LH synthesis and secretion, in sheep at least, in that GnRH pulses of a particular amplitude (250 ng) are required to maintain secretion of LH, whereas synthesis can be normally maintained by lower amplitude pulses of GnRH, and persists to some extent in the face of constant G&I-I levels. Conversely, in several studies in vitro in which rat pituitary cells were treated with GnRH either in static culture or in perifusion systems, effects on release of LH were clearly in evidence, but no
decrease in LHj3 mRNA was demonstrated (Attardi et al., 1988; Weiss et al., 1990). In a further study, no effect was seen on LHP mRNA transcription rate after GnRH treatment, despite an observed effect on LH release (Salton et al., 1989). These data again suggest a dissociation between synthesis and secretion, although the failure of these studies to show an effect on LHP mRNA levels may also be a reflection of the pattern of GnRH administration. There has been one report to date of a stimulatory effect of GnRH on LHP mRNA levels in cultured pituitary cells, and that study also shows a stimulatory effect of protein kinase C on LH/3 mRNA levels (Andrews et al., 1988). Shupnik (1990) has recently shown in rat pituitary quarters that pulsatile GnRH will stimulate transcription of both the a-subunit and LHP genes, while a constant infusion of GnRH stimulates a-subunit gene transcription only; in this study GnRH stimulated LH secretion with both pulsatile and constant infusion. Several studies have examined transcriptional regulation of the a-subunit gene by cyclic AMP in cells derived from choriocarcinomas (Deutsch et al., 1987; Silver et al., 1987; Jameson et al., 1988; Mellon et al., 1989). A recent report describes a study showing regulation of the cw-subunit gene by GnRH in clonal cells derived from pituitary tumors in transgenic mice carrying an a-subunit promoter-SV40 T-antigen fusion gene (Windle et al., 1990). Further definition of the role of GnRH and its possible second messengers in regulating the expression of the LHj3 and FSHj? genes will be greatly facilitated by the development of cell lines which allow the expression of these genes, and the tumor-producing transgenic mouse model may offer some possibilities in this regard. The site of estrogen action The way in which sex steroids regulate synthesis of gonadotropins is clearly complex. Gonadectomy leads to increases in mRNA levels for all three gonadotropin subunits in male and female rats (Gharib et al., 1987) and in ewes (Nilson et al., 1985; Mercer et al., 1989). In addition, in situ hybridization studies have shown both that the number of LHP and FSHP mRNA-containing cells increases, and that the amount of LHP and
c65
FSHB mRNA per cell also increases following gonadectomy (Childs et al., 1987,199O). A great deal of effort has gone into defining the site of action of steroid hormones within the hypothahuno-pituitary unit. The OVX/HPD sheep model has enabled in vivo studies of the surgically isolated, vascular-intact pituitary to be undertaken. These studies have shown that estrogen in the sheep does not have a direct negative action at the pituitary, since estradiol has no effect on LH/3 mRNA levels in OVX/HPD, GnRH pulsed sheep; in intact animals, estrogen thus presumably down-regulates LH/3 mRNA levels via an effect on GnRH. In contrast, FSH/3 and a-subunit mRNA levels are decreased by estrogen treatment in this model, indicating a direct effect of estrogen on the pituitary to negatively regulate these two genes. Interestingly, when cultured rat pituitary fragments were incubated with estradiol for 2 or 6 h, no effects were seen on the transcription of a-subunit or FSH/3 genes but LH/3 mRNA synthesis was increased (Shupnik et al., 1989a). This effect on LH/3 mRNA synthesis is consistent with the site of action for negative regulation by estrogen being the hypothalamus, but suggests that the site of action of positive estrogen regulation of LH/3 mRNA is directly at the pituitary. An estrogen regulatory element (ERE) consensus sequence has been identified in the 5’ untranslated region of the rat LHB gene, and has been shown in binding studies to be functionally active (Shupnik et al., 1988). Positive feedback of estrogen on the hypothalamo-pituitary unit leads to the LH surge. Landefeld and co-workers have shown that LH/3 mRNA levels are increased by 60% during the pre-ovulatory LH surge in sheep (Leung et al., 1988), and the proestrus LH peak in rats is also preceded both by a rise in steady state LH/3 mRNA levels (Zmeili et al., 1986) and LH/I transcription (Shupnik et al., 1989a). OVX ewes treated with estrogen to elicit an LH surge show either no increase (Landefeld et al., 1987) or a decrease (Mercer et al., unpublished) in LH/3 mRNA levels following steroid injection. These data in sheep suggest that positive regulation of the LH/3 gene is not a major mechanism for positive estrogen feedback in this species, but that estrogen effects
may be largely on G&I-I secretion and/or upregulation of GnRH receptors to increase the sensitivity of the gonadotrope to GnRH. The elucidation of the GnRH receptor sequence and the development of cell lines which express pituitary gonadotropin genes will add significantly to our understanding of the manner in which estrogen is able to give rise to the LH surge. Gonadal peptides The gonadal peptides inhibin, activin and follistatin were isolated on the basis of their effects on FSH secretion from the pituitary gland. The related peptides inhibin and activin have opposite effects on FSH, with the a/J heterodimer, inhibin, suppressing FSH secretion (Robertson et al., 1986), and the /3/3homodimer, activin, stimulating FSH release (Ling et al., 1986). The structurally unrelated follistatin also suppresses FSH secretion (Vale et al., 1986). All three peptides have been identified in the gonads, and mRNA for the a and & subunits of inhibin have been localised in the pituitary (Meunier et al., 1988). In addition the follicular-stellate cells of the pituitary have been reported to secrete follistatin (Gospodarowicz and Lau, 1989). FSH/3 mRNA levels are rapidly suppressed in OVX/HPD GnRH pulsed sheep treated with an inhibin-containing follicular fluid extract, demonstrating a direct pituitary site of action for inhibin in vivo. Levels of a-subunit and LH/3 mRNA were not changed by inhibin in this model (Mercer et al., 1987). In vitro studies using rat pituitary cell cultures have also demonstrated a specific effect of inhibin to reduce FSH/3 mRNA levels in both untreated (Attardi et al., 1989a; Carroll et al., 1989) and GnRH-stimulated cells (Attardi et al., 1989b). In pituitary cell cultures it has been shown that activin increases and follistatin reduces FSH/3 mRNA levels with no change in a-subunit or LH/3 mRNA levels (Carroll et al., 1989; Attardi et al., 1990). In a subsequent study it has also been shown that one mechanism by which activin increases steady state mRNA levels for FSH/3 is by altering the half-life of the mRNA species (Carroll et al., 1990). Though these peptide hormones may also alter the transcription rate of the FSH/I gene, such studies have yet to be reported.
In addition to the effect of inhibin on FSH secretion, inhibin has been reported to decrease the basal secretion of LH from rat pituitary cells in culture (Farnworth et al., 1988) and to reduce the LH pulse amplitude in OVX/HPD sheep (Clarke et al., 1984; Mercer et al., 1987). Inhibin has also been reported to both down-regulate GnRH receptors on gonadotropes (Wang et al., 1989) and, in contrast, to increase GnRH receptor levels on the same cells (Laws et al., 1990) studies which are yet to be reconciled. Regardless of the mechanism by which the decrease in LH secretion is achieved, the data discussed earlier have convincingly shown that inhibin has no effect on LHj3 mRNA levels, again evidence of the dissociation between effects on LH secretion and synthesis. Inhibin (and activin) subunits have been colocalised with ir-LH and ir-FSH cells in the rat pituitary (Roberts et al., 1989) and it is thus possible that inhibin and activin may have an autocrine regulatory effect on FSH synthesis in the pituitary. If follistatin is indeed produced by the follicular-stellate cells of the pituitary gland, it may play a paracrine regulatory role in the control of FSH synthesis. Further elucidation of the precise localisation of what until now have been considered gonadal peptide hormones may lead us to rethink our concept of pituitary-gonadal feedback loops. Finally, of further interest is the recent identification of an ovarian activin binding protein as follistatin (Nakamura et al., 1990), and the subsequent report that a pituitary protein which specifically binds activin shows sequence homology to follistatin (Sugino et al., 1990). Since activin is synthesised in the pituitary, it may even be possible that follistatin has its effect on FSH by binding to activin and thus preventing it from having a positive effect on FSH. An alternative, intriguing possibility is that follistatin may in some way be related to the activin receptor. The answers to these questions and the elucidation of the complex feedback loops involved in cyclical hypothalamicpituitary-gonadal activity, await further studies. Acknowledgements I wish to thank Drs. W.W. Chin and Carroll for critically reading the manuscript.
R.S.
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