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Follicular oestrogen synthesis: the ‘two-cell, two-gonadotrophin’ model revisited
51
Molecular and Cellular Endocrinology, 100 (1994) 51-54 0 1994 Elsevier Science Ireland Ltd. 0303-7207/94/$07.00
MCE 3185
Follicular oestrogen synthesis: the ‘two-cell, two-gonadotrophin’ Stephen G. Hillier *, Philippa F. Whitelaw and Christopher
model revisited D. Smyth
Reproductive Endocrinology Laboratory, Department of Obstetrics and Gynaecology, University of Edinburgh Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW UK
(Received 5 November 1993)
Key words: Estrogen; Ovary; Follicle; Two-cell hypothesis; Paracrine
Summary
The original ‘two-cell mechanism’ explained the endocrine regulation of follicular oestrogen synthesis and implied paracrine signalling in the follicle wall. It is now known that the CYP17 gene encoding 17-hydroxylase/ C,,_,,-lyase activity crucial to androgen synthesis, is expressed exclusively in thecal cells. 17-Hydroxylase/ C,,_,,lyase activity is regulated by LH and subject to local modulation by a factor(s) emanating in FSH-stimulated granulosa cells. The FSH receptor gene is expressed exclusively in granulosa cells, where FSH acts directly to induce cytoproliferation and differentiation via cyclic AMP/protein kinase-A mediated post-receptor signalling. Granulosa cells also express androgen receptors, and theta-derived androgen has the potential to modulate locally differentiative responses to FSH. When follicles are recruited to preovulatory development by FSH, their granulosa cells develop LH receptors functionally coupled to aromatase activity and inhibin production. Thereby they simultaneously undertake LH-responsive aromatization and inhibin synthesis. Inhibin has the potential to potently enhance LH-stimulated thecal androgen synthesis. Granulosa-derived inhibin may therefore participate in a paracrine mechanism that locally amplifies androgen synthesis, and hence oestrogen formation, in the preovulatory follicle(s).
system that integrates gonadotrophin of the preovulatory follicle.
Introduction
The principal steroidogenic cell types in Graafian follicles possess reciprocal histogenetic properties necessary for ovarian oestrogen biosynthesis (Short, 1962; Bjersing, 1967; Ryan, 1979; Armstrong et al., 1979). Granulosa cells and thecal cells respond differentially to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) with the development of distinctive enzymic capacities to synthesize (theta) or metabolize (granulosa) androgens to oestrogens. This is the ‘twocell, two-gonadotrophin’ model proposed by Armstrong et al. (1979). The Armstrong-Dorrington model set out the hormonal and cellular requirements for oestrogen biosynthesis to occur but did not explain the unique ability of a preovulatory follicle(s) to develop and secrete oestrogen, when all follicles in the ovary are receiving the same gonadotrophic stimulus. Basic research addressing this issue is surveyed here, focusing on the paracrine
-* Corresponding
author.
SSDZ0303-7207(93)E0295-6
Endocrine
regulation
of oestrogen
action in the wall
synthesis
Essential features of the Armstrong-Dorrington ‘two-cell’ model are that (1) granulosa cells, but not thecal cells, possess FSH receptors; (2) thecal cells, but not immature granulosa cells, possess LH receptors; (3) FSH stimulates aromatase activity, but not androgen synthesis, in granulosa cells; and (4) LH stimulates androgen synthesis, but not aromatase activity in thecal cells. At the molecular level, these cell-specific properties are becoming better understood as follows. The granulosa cell FSH receptor
Early evidence that granulosa cells possess membrane associated FSH receptors came from autoradiographic studies in which 1251-labelled FSH binding was selectively localized to these cells in sections of immature rat ovary (Zeleznik et al., 1974). Quantitative assessment of the granulosa cell FSH receptor was reported by Nimrod et al. (1976) but additional infor-
52
mation was not forthcoming until cDNA clones encoding the rat FSH receptor were isolated (Sprengel et al., 1991) and used to examine the tissue distribution of FSH receptor mRNA expression (Camp et al., 1991). That work confirmed ovary-specific expression of an abundant N 2.6 kb-sized FSH receptor mRNA and a less abundant - 5.0 kb transcript. In situ hybridization showed FSH receptor mRNA to be localized exclusively in the granulosa cells of healthy follicles, persisting throughout preovulatory follicular development. The thecal cell LH receptor
The presence of specific hCG/LH binding sites on thecal/interstital cells was also revealed by autoradiography, using i2’I-hCG as a surrogate LH to label specifically hCG/LH binding sites in rat ovarian sections (Zeleznik et al., 1974). Importantly, it was also shown that treatment of animals with FSH in vivo led to increased binding of lz51-hCG to granulosa cells, providing the first direct evidence that FSH induces the granulosa cell hCG/LH receptor. Quantitative assessment of the hCG/LH receptor on thecal-interstitial cells (Magoffin and Erickson, 1982) and induction of the hCG/LH receptor on granulosa cells by FSH (Erickson et al., 1979) were subsequently confirmed in vitro. Following the cloning of the rat LH receptor (McFarland et al., 1989), multiple ovarian LH receptor transcripts were identified with two major transcripts of - 6.5 kb and - 2.6 kb (Segaloff et al., 1990). Application of in situ hybridization showed LH receptor mRNAs to be located exclusively in the thecal cells of immature follicles but present in both thecal and granulosa cells of mature, antral follicles (Camp et al., 1991; Peng et al., 1992). Direct FSH induction of granulosa cell LH receptor mRNA was confirmed in vitro (Piquette et al., 1991) and in vivo (Segaloff et al., 1990). FSH and granulosa cell aromatase activity
FSH-induction of rat granulosa cell aromatase activity was demonstrated by Dorrington et al. (1975) and Erickson and Hsueh (1978). The importance of intracellular cyclic AMP in mediating this and all other development-related granulosa cell responses to FSH, including the induction of LH receptors, readily became apparent (reviewed by Hsueh et al., 1984). Early evidence that FSH action was subject to paracrine modulation came from the finding that FSH-induction of aromatase activity in vitro was augmented by androgen via an androgen-receptor regulated mechanism (Hillier and De Zwart, 1981). Once the human cDNA encoding aromatase cytochrome P450 (P450arom) had been cloned (Corbin et al., 19881, rat P450arom cDNA was obtained (Hickey et al., 1990) and used to reveal the expression of a major (- 3.3 kb) mRNA transcript in rat granulosa cells. This proved to be inducible by
FSH and up-regulated by androgen in vitro (Hickey et al., 1988; Fitzpatrick and Richards, 1991). The question remained if granulosa cells are unique sites of aromatase expression in the follicle, in view of sporadic accounts that aromatase activity could be detected in thecal cells (El-Maasarany, 1991). This was answered by in situ hybridization, revealing granulosa-specific expression of the P450arom gene in the wall of the Graafian follicle (Whitelaw et al., 1992). Induction of granulosa cell P450arom mRNA in the ovaries of hypophysectomized rats treated with recombinant human (rh) FSH effectively ruled out an obligatory contribution of LH (and of oestrogen itself) to this process (Whitelaw et al., 1992). LH and thecal androgen formation
Direct evidence that thecal/ interstitial cell androgen synthesis is selectively regulated by LH came from studies in which LH, but not FSH, enhanced androgen production by isolated thecal fragments in vitro (Fortune and Armstrong, 1977). The regulatory action of LH was shown to be mediated by cyclic AMP at the level of increased 17-hydroxylase/ C,,_,,-lyase activity (Bogovich and Richards, 1982; Erickson et al., 1985). Both rate-limiting enzymic steps in androgen synthesis are present in a single steroidogenic cytochrome P45Oc17 (P45Oc17) encoded by the CYP17 gene (Fevold et al., 1989). With the isolation and cloning of rat P45Oc17 DNA (Fevold et al., 1989), the encoded mRNA proved to be an - 2.0-kb transcript (Richards et al., 1987) expressed exclusively in thecal/ interstitial cells of the ovarian follicle (Smyth et al., 1993b). Paracrine
regulation
of oestrogen synthesis
The granulosa cells in preovulatory follicles aromatize androgen - hence produce oestrogen - at rates that are orders of magnitude higher than in non-ovulatory follicles. It follows that the thecal cells in preovulatory follicles produce commensurate amounts of precursor androgen. Paracrine (granulosa on theca) signalling has been invoked to explain this phenomenon (Hillier, 1991). Since oestrogen synthesis is absolutely dependent on the expression of P450arom, it follows that the granulosa-derived paracrine factor(s) that upregulates precursor androgen synthesis is likely to be encoded by an FSH-regulated gene. LH receptor and inhibin-c-ware two FSH-regulated genes that may have particular relevance to the paracrine regulation of follicular oestrogen. The importance of the LH receptor gene is that, once LH receptors are expressed, binding of LH can activate cyclic AMP mediated post-receptor signalling in granulosa cells. The importance of the inhibin-a gene is that it encodes the a-subunit of dimeric ((-up) inhibin (Ying, 1988). Inhibin is a potent up-regulator of thecal andro-
53
gen synthesis in vitro, and hence a candidate for this action in vivo (see below). Similar to P450arom (Hickey et al., 1990), expression of the inhibin-a gene is regulated via intracellular cyclic AMP (Pei et al., 1991). Thus FSH coordinately upregulates P450 arom (Fitzpatrick and Richards, 1991) and inhibin-cw (Turner et al., 1989) expression, and both gene activities are functionally coupled to the LH receptor via cyclic AMPmediated intracellular signalling (Wang et al., 1981; Bicsak et al., 1986). Inhibin potently augments LH-stimulated thecal androgen synthesis in vitro (Hsueh et al., 1987; Hillier, 1991; Smyth et al., 1993b). Other granulosa-derived factors, notably insulin-like growth factor-I, are also potential paracrine modulators of thecal androgen synthesis, based on stimulatory effects observed in vitro (Adashi et al. 1992). However, both inhibin synthesis and aromatase activity in mature granulosa cells are regulated by LH, and increased inhibin production parallels oestradiol by the preovulatory follicle. All of which suggests a physiologically significant paracrine function for inhibin in the regulation of oestrogen synthesis. A local amplification of thecal androgen synthesis by a paracrine factor(s) produced in relatively large amounts by the preovulatory follicle suggests a mechanism through which follicular dominance is sustained in the reproductive cycles of monovulatory species such as man (Hillier, 1991). Direct experimental evidence that granulosa-derived paracrine signalling influences thecal androgen synthesis in vivo has come from experiments in which thecal/interstitial cell function was assessed in immature rats treated with rh-FSH, completely devoid of LH bioactivity (Smyth et al., 1993b). Injection of rh-FSH in vivo dose-dependently increased the expression of P45Oc17 mRNA in thecal/interstitial cells and enhanced the ability of these cells to undertake LH-responsive androgen production in vitro. Since thecal/interstitial cells do not possess FSH receptors, this result was interpreted as evidence for paracrine signalling emanating in FSH-stimulated granulosa cells. This experimental approach does not reveal the identity of the signalling factor(s) involved. More recently it was shown that oestrogen synthesis induced in individually cultured rat follicles by treatment with an LHcontaining FSH preparation in vitro is blocked by the presence of a neutralizing antibody to the inhibin-cu subunit. Blockade of oestrogen synthesis by anti-inhibin serum was overcome by adding exogenous inhibin or an aromatase substrate (androstenedione) to the cultures (Smyth et al., 1993a). This is the most convincing evidence to date of a paracrine role for inhibin in regulating follicular oestrogen synthesis. A paracrine role for inhibin in modulating LH action on thecal cells would reciprocate the previously suggested role of theta-derived androgen in modulat-
ing FSH action on granulosa cells (Hillier and Turner, 1991). Inhibin-a mRNA expression (Turner et al., 1989) and inhibin protein secretion (Bicsak et al., 1976) are among many FSH-stimulated responses of immature granulosa cells known to be up-regulated by androgen in vitro (Hillier, 1987).
References Adashi, E.Y., Resnick, C.E., Hurwitz, A., Ricciarelli, E., Hernandez, E.R., Roberts, C.T., Leroith, D. and Rosenfeld, R. (1991) Hum. Reprod. 6, 1213-1219. Armstrong, D.T., Goff, A.K. and Dorrington, J.H. (1979) in Ovarian Follicular Development and Function (Midgley, A.R. and Sadler, W.A. eds.), pp. 169-182, Raven Press, New York. Bicsak, T.A., Tucker, E.M., Cappel, S., Vaughan, J., Rivier, J., Vale, W. and Hsueh, A.J.W. (1986) Endocrinology, 119, 2711-2719. Bjersing, L. (1967) Acta Endocrinol. (Copenhagen), Suppl. 125. Bogovich, K. and Richards, J.S. (1982) Endocrinology 111,1201-1208. Camp, T.A., Rahal, J.O. and Mayo, K. (1991) Mol. Endocrinol. 5, 1405-1417. Corbin, C.J., Graham-Lorence, S., McPhaul, M., Mason, J.E., Mendelson, C.R. and Simpson, E.R. (1988) Proc. Natl. Acad. Sci. USA 85, 8948-8952. Dorrington, J.H., Moon, Y.S. and Armstrong, D.T. (1975) Endocrinology 97, 1328-1331. El-Maasarany, S., Brandt, M.E., Majercik, M.H., Zimniski, S.J. and Puett, D. (1991) Biol. Reprod. 44, 550-560. Erickson, G.F., Magoffin, D.A., Dyer, CA. and Hofeditz, C. (1985) Endocr. Rev. 6, 371-399. Erickson, G.F., Wang, C., and Hsueh, A.J.W. (1979) Nature (London) 279, 336-338. Esch, F.S., Shimasaki, S., Cooksey, K., Mercado, M., Mason, A.J., Ying, S.-Y., Ueno, N. and Ling, N. (1987) Mol. Endocrinol. 1, 388-396. Fevold, H.R., Lorence, M.C., McCarthy, J.L., Trant, J.M., Kagimoto, M., Waterman, M.R. and Mason, J.I. (1989) Mol. Endocrinol. 3, 968-975. Fitzpatrick, S.L. and Richards, J.S. (1991) Endocrinology 129, 14521462. Fortune, J.E. and Armstrong, D.T. (1977) Endocrinology 100, 13411347. Hickey, G.J., Chen, S., Besman, M.J., Shively, J.E., Hall, P.F., Gaddy-Kurten, D. and Richards, J.S. (1988) Endocrinology 122, 1426-1436. Hickey, G.J., Krasnow, J.S., Beattie, W.G. and Richards, J.S. (1990) Mol. Endocrinol. 4, 3-12. Hillier, S.G. (1987) J. Steroid Biochem. 27, 351-357. Hillier, S.G. (1991) J. Endocrinol. 131, 171-175. Hillier, S.G. and Turner, I.M. (1991) in Signaling Mechanisms and Gene Expression in the Ovary, Proceedings of the VIIIth Ovary Workshop (Gibori, G. ed.), pp. 84-96, Springer-Verlag, New York. Hillier, S.G. and De Zwart, F.A. (1981) Endocrinology, 109, 13031305. Hsueh, A.J.W., Adashi, E.Y., Jones, P.B.C. and Welsh, T.J. Jr. (1984) Endocr. Rev. 5, 76 126. Hsueh, A.J.W., Dahl, K.D., Vaughan, J., Tucker, E., Rivier, J., Bardin, C.W. and Vale, W. (1987) Proc. Natl. Acad. Sci. USA 84, 5082-5086. Magoffin, D.A. and Erickson, G.F. (1982) J. Biol. Chem. 257, 45074513 McFarland, K.C., Sprengl, R., Phillips, H.S., Kohler, M., Rosemblit,
54 N., Nikolics, K., Segaloff, D.L. and Seeburg, P.H. (1989) Science 245, 495-499. Nimrod, A. Erickson, G.F. and Ryan, K.D. (1976) Endocrinology 98, 56-64. Pei, L., Dodson, R, Schoderbek, W., Maurer, R. and Mayo, K. (1991) Mol. Endocrinol. 5, 521-534. Piquette, G.N., LaPolt, P.S., Oikawa, M. and Hsueh, A.J.W. (19911 Endocrinology 128,2449-2456. Richards, J.S., Jahnsen, T., Hedin, L., Lifka, J., Ratoosh, S.L., Durica, J.M. and Goldring, N.B. (1987) Recent Prog. Horm. Res. 43,231-270. Ryan, K.J. (1979) J. Steroid B&hem. 11, 799-800. Segaloff, D.L., Wang, H. and Richards, J.S. (1990) Mol. Endocrinol. 4, 1856-1865. Short, R.V. (1962) J. Endocrinol. 24, 59-63.
Smyth, C.D., Mir6, F., Whitelaw, P.F., Howles, C.M. and Hillier, S.G. (1993) Endocrinology 133, 1532-1538. Smyth, C.D., Gosden, R.G., McNeiIly, A.S. and Hillier, S.G. (1994) J. Endocrinol., in press. Sprengl, R., Braun, T., Niiolics, K., Segaloff, D.L. and Seeburg, P.H. (1990) Mol. Endocrinol. 4, 525-530. Turner, I.M., Saunders, P.T.K., Shimasaki, S. and Hillier, S.G. (1989) Endocrinology 125, 2790-2792. Wang, C., Hsueh, A.J.W. and Erickson, G.F. (19811 Mol. Cell. Endocrinol. 20, 136-144. Whitelaw, P.F., Smyth, C.D., Howles, CM. and Hillier, S.G. (1992) J. Mol. End~rinoi. 9, 309-312. Ying, S.Y. (1988) Endocr. Rev. 9, 267-293. Zeleznik, A.J., Midgley, A.R., Jr. and Reichert, L.E., Jr. (1974) Endocrinology 95, 818-825.
Molecular and Cellular Endocrinology, 100 (1994) 51-54 0 1994 Elsevier Science Ireland Ltd. 0303-7207/94/$07.00
MCE 3185
Follicular oestrogen synthesis: the ‘two-cell, two-gonadotrophin’ Stephen G. Hillier *, Philippa F. Whitelaw and Christopher
model revisited D. Smyth
Reproductive Endocrinology Laboratory, Department of Obstetrics and Gynaecology, University of Edinburgh Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW UK
(Received 5 November 1993)
Key words: Estrogen; Ovary; Follicle; Two-cell hypothesis; Paracrine
Summary
The original ‘two-cell mechanism’ explained the endocrine regulation of follicular oestrogen synthesis and implied paracrine signalling in the follicle wall. It is now known that the CYP17 gene encoding 17-hydroxylase/ C,,_,,-lyase activity crucial to androgen synthesis, is expressed exclusively in thecal cells. 17-Hydroxylase/ C,,_,,lyase activity is regulated by LH and subject to local modulation by a factor(s) emanating in FSH-stimulated granulosa cells. The FSH receptor gene is expressed exclusively in granulosa cells, where FSH acts directly to induce cytoproliferation and differentiation via cyclic AMP/protein kinase-A mediated post-receptor signalling. Granulosa cells also express androgen receptors, and theta-derived androgen has the potential to modulate locally differentiative responses to FSH. When follicles are recruited to preovulatory development by FSH, their granulosa cells develop LH receptors functionally coupled to aromatase activity and inhibin production. Thereby they simultaneously undertake LH-responsive aromatization and inhibin synthesis. Inhibin has the potential to potently enhance LH-stimulated thecal androgen synthesis. Granulosa-derived inhibin may therefore participate in a paracrine mechanism that locally amplifies androgen synthesis, and hence oestrogen formation, in the preovulatory follicle(s).
system that integrates gonadotrophin of the preovulatory follicle.
Introduction
The principal steroidogenic cell types in Graafian follicles possess reciprocal histogenetic properties necessary for ovarian oestrogen biosynthesis (Short, 1962; Bjersing, 1967; Ryan, 1979; Armstrong et al., 1979). Granulosa cells and thecal cells respond differentially to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) with the development of distinctive enzymic capacities to synthesize (theta) or metabolize (granulosa) androgens to oestrogens. This is the ‘twocell, two-gonadotrophin’ model proposed by Armstrong et al. (1979). The Armstrong-Dorrington model set out the hormonal and cellular requirements for oestrogen biosynthesis to occur but did not explain the unique ability of a preovulatory follicle(s) to develop and secrete oestrogen, when all follicles in the ovary are receiving the same gonadotrophic stimulus. Basic research addressing this issue is surveyed here, focusing on the paracrine
-* Corresponding
author.
SSDZ0303-7207(93)E0295-6
Endocrine
regulation
of oestrogen
action in the wall
synthesis
Essential features of the Armstrong-Dorrington ‘two-cell’ model are that (1) granulosa cells, but not thecal cells, possess FSH receptors; (2) thecal cells, but not immature granulosa cells, possess LH receptors; (3) FSH stimulates aromatase activity, but not androgen synthesis, in granulosa cells; and (4) LH stimulates androgen synthesis, but not aromatase activity in thecal cells. At the molecular level, these cell-specific properties are becoming better understood as follows. The granulosa cell FSH receptor
Early evidence that granulosa cells possess membrane associated FSH receptors came from autoradiographic studies in which 1251-labelled FSH binding was selectively localized to these cells in sections of immature rat ovary (Zeleznik et al., 1974). Quantitative assessment of the granulosa cell FSH receptor was reported by Nimrod et al. (1976) but additional infor-
52
mation was not forthcoming until cDNA clones encoding the rat FSH receptor were isolated (Sprengel et al., 1991) and used to examine the tissue distribution of FSH receptor mRNA expression (Camp et al., 1991). That work confirmed ovary-specific expression of an abundant N 2.6 kb-sized FSH receptor mRNA and a less abundant - 5.0 kb transcript. In situ hybridization showed FSH receptor mRNA to be localized exclusively in the granulosa cells of healthy follicles, persisting throughout preovulatory follicular development. The thecal cell LH receptor
The presence of specific hCG/LH binding sites on thecal/interstital cells was also revealed by autoradiography, using i2’I-hCG as a surrogate LH to label specifically hCG/LH binding sites in rat ovarian sections (Zeleznik et al., 1974). Importantly, it was also shown that treatment of animals with FSH in vivo led to increased binding of lz51-hCG to granulosa cells, providing the first direct evidence that FSH induces the granulosa cell hCG/LH receptor. Quantitative assessment of the hCG/LH receptor on thecal-interstitial cells (Magoffin and Erickson, 1982) and induction of the hCG/LH receptor on granulosa cells by FSH (Erickson et al., 1979) were subsequently confirmed in vitro. Following the cloning of the rat LH receptor (McFarland et al., 1989), multiple ovarian LH receptor transcripts were identified with two major transcripts of - 6.5 kb and - 2.6 kb (Segaloff et al., 1990). Application of in situ hybridization showed LH receptor mRNAs to be located exclusively in the thecal cells of immature follicles but present in both thecal and granulosa cells of mature, antral follicles (Camp et al., 1991; Peng et al., 1992). Direct FSH induction of granulosa cell LH receptor mRNA was confirmed in vitro (Piquette et al., 1991) and in vivo (Segaloff et al., 1990). FSH and granulosa cell aromatase activity
FSH-induction of rat granulosa cell aromatase activity was demonstrated by Dorrington et al. (1975) and Erickson and Hsueh (1978). The importance of intracellular cyclic AMP in mediating this and all other development-related granulosa cell responses to FSH, including the induction of LH receptors, readily became apparent (reviewed by Hsueh et al., 1984). Early evidence that FSH action was subject to paracrine modulation came from the finding that FSH-induction of aromatase activity in vitro was augmented by androgen via an androgen-receptor regulated mechanism (Hillier and De Zwart, 1981). Once the human cDNA encoding aromatase cytochrome P450 (P450arom) had been cloned (Corbin et al., 19881, rat P450arom cDNA was obtained (Hickey et al., 1990) and used to reveal the expression of a major (- 3.3 kb) mRNA transcript in rat granulosa cells. This proved to be inducible by
FSH and up-regulated by androgen in vitro (Hickey et al., 1988; Fitzpatrick and Richards, 1991). The question remained if granulosa cells are unique sites of aromatase expression in the follicle, in view of sporadic accounts that aromatase activity could be detected in thecal cells (El-Maasarany, 1991). This was answered by in situ hybridization, revealing granulosa-specific expression of the P450arom gene in the wall of the Graafian follicle (Whitelaw et al., 1992). Induction of granulosa cell P450arom mRNA in the ovaries of hypophysectomized rats treated with recombinant human (rh) FSH effectively ruled out an obligatory contribution of LH (and of oestrogen itself) to this process (Whitelaw et al., 1992). LH and thecal androgen formation
Direct evidence that thecal/ interstitial cell androgen synthesis is selectively regulated by LH came from studies in which LH, but not FSH, enhanced androgen production by isolated thecal fragments in vitro (Fortune and Armstrong, 1977). The regulatory action of LH was shown to be mediated by cyclic AMP at the level of increased 17-hydroxylase/ C,,_,,-lyase activity (Bogovich and Richards, 1982; Erickson et al., 1985). Both rate-limiting enzymic steps in androgen synthesis are present in a single steroidogenic cytochrome P45Oc17 (P45Oc17) encoded by the CYP17 gene (Fevold et al., 1989). With the isolation and cloning of rat P45Oc17 DNA (Fevold et al., 1989), the encoded mRNA proved to be an - 2.0-kb transcript (Richards et al., 1987) expressed exclusively in thecal/ interstitial cells of the ovarian follicle (Smyth et al., 1993b). Paracrine
regulation
of oestrogen synthesis
The granulosa cells in preovulatory follicles aromatize androgen - hence produce oestrogen - at rates that are orders of magnitude higher than in non-ovulatory follicles. It follows that the thecal cells in preovulatory follicles produce commensurate amounts of precursor androgen. Paracrine (granulosa on theca) signalling has been invoked to explain this phenomenon (Hillier, 1991). Since oestrogen synthesis is absolutely dependent on the expression of P450arom, it follows that the granulosa-derived paracrine factor(s) that upregulates precursor androgen synthesis is likely to be encoded by an FSH-regulated gene. LH receptor and inhibin-c-ware two FSH-regulated genes that may have particular relevance to the paracrine regulation of follicular oestrogen. The importance of the LH receptor gene is that, once LH receptors are expressed, binding of LH can activate cyclic AMP mediated post-receptor signalling in granulosa cells. The importance of the inhibin-a gene is that it encodes the a-subunit of dimeric ((-up) inhibin (Ying, 1988). Inhibin is a potent up-regulator of thecal andro-
53
gen synthesis in vitro, and hence a candidate for this action in vivo (see below). Similar to P450arom (Hickey et al., 1990), expression of the inhibin-a gene is regulated via intracellular cyclic AMP (Pei et al., 1991). Thus FSH coordinately upregulates P450 arom (Fitzpatrick and Richards, 1991) and inhibin-cw (Turner et al., 1989) expression, and both gene activities are functionally coupled to the LH receptor via cyclic AMPmediated intracellular signalling (Wang et al., 1981; Bicsak et al., 1986). Inhibin potently augments LH-stimulated thecal androgen synthesis in vitro (Hsueh et al., 1987; Hillier, 1991; Smyth et al., 1993b). Other granulosa-derived factors, notably insulin-like growth factor-I, are also potential paracrine modulators of thecal androgen synthesis, based on stimulatory effects observed in vitro (Adashi et al. 1992). However, both inhibin synthesis and aromatase activity in mature granulosa cells are regulated by LH, and increased inhibin production parallels oestradiol by the preovulatory follicle. All of which suggests a physiologically significant paracrine function for inhibin in the regulation of oestrogen synthesis. A local amplification of thecal androgen synthesis by a paracrine factor(s) produced in relatively large amounts by the preovulatory follicle suggests a mechanism through which follicular dominance is sustained in the reproductive cycles of monovulatory species such as man (Hillier, 1991). Direct experimental evidence that granulosa-derived paracrine signalling influences thecal androgen synthesis in vivo has come from experiments in which thecal/interstitial cell function was assessed in immature rats treated with rh-FSH, completely devoid of LH bioactivity (Smyth et al., 1993b). Injection of rh-FSH in vivo dose-dependently increased the expression of P45Oc17 mRNA in thecal/interstitial cells and enhanced the ability of these cells to undertake LH-responsive androgen production in vitro. Since thecal/interstitial cells do not possess FSH receptors, this result was interpreted as evidence for paracrine signalling emanating in FSH-stimulated granulosa cells. This experimental approach does not reveal the identity of the signalling factor(s) involved. More recently it was shown that oestrogen synthesis induced in individually cultured rat follicles by treatment with an LHcontaining FSH preparation in vitro is blocked by the presence of a neutralizing antibody to the inhibin-cu subunit. Blockade of oestrogen synthesis by anti-inhibin serum was overcome by adding exogenous inhibin or an aromatase substrate (androstenedione) to the cultures (Smyth et al., 1993a). This is the most convincing evidence to date of a paracrine role for inhibin in regulating follicular oestrogen synthesis. A paracrine role for inhibin in modulating LH action on thecal cells would reciprocate the previously suggested role of theta-derived androgen in modulat-
ing FSH action on granulosa cells (Hillier and Turner, 1991). Inhibin-a mRNA expression (Turner et al., 1989) and inhibin protein secretion (Bicsak et al., 1976) are among many FSH-stimulated responses of immature granulosa cells known to be up-regulated by androgen in vitro (Hillier, 1987).
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