Prostaglandins and follicular functions

Transcription

1 Prostaglandins and follicular functions David T. Armstrong M.R.C. Group in Reproductive Biology, Departments of Obstetrics & Gynaecology and Physiology, University of Western Ontario, London, Canada N6A 5A5 Ovulation The involvement of prostaglandins (PGs) in the regulation of ovarian follicular function was first postulated on the basis of the demonstration that inhibitors of prostaglandin synthesis, such as aspirin and indomethacin, were capable of blocking ovulation in rats (Armstrong & Grinwich, 1972; Orczyk & Behrman, 1972). These initial findings were soon confirmed in several other species, including mice (Lau, Saksena & Chang, 1974), rabbits, (Grinwich, Kennedy & Armstrong, 1972; O'Grady, Caldwell, Auletta & Speroff, 1972), rhesus and marmoset monkeys (Wallach, de la Cruz, Hunt, Wright & Stevens, 1975; Maia, Barbosa & Coutinho, 1978), pigs (Ainsworth et al., 1979) and goldfish (Stacey & Pandey, 1975). In two of these species (rabbits and goldfish), the inhibitor was effective when applied locally to the follicle, indicating that the blockade was exerted directly upon the follicle, rather than being mediated via some indirect mechanism, such as through inhibition of gonadotrophin secretion. Further evidence of a role of prostaglandins at the follicular level was provided by the findings that intrafollicular levels of prostaglandins of both the E and F series increased markedly in several of these species shortly before ovulation (Yang, Marsh & LeMaire, 1974; Armstrong, Moon & Zamecnik, 1974; Bauminger & Lindner, 1975; Ainsworth, Baker & Armstrong, 1975; Tsang, Ainsworth, Downey & Armstrong, 1979a); indomethacin, at dosages which prevented ovulation, effectively prevented these increases. The observation that injection of antiserum against PGs blocked the LH-induced ovulation in oestrous rabbits, whether administered systemically (Lau et al., 1974) or via intrafollicular injection (Armstrong et al., 1974), added support to the concept of a role of prostaglandins in ovulation. Antiserum to PGF-2\g=a\appeared to be more effective than that to PGE in these experiments, suggesting that PGF-2\g=a\was the prostaglandin of greater importance in ovulation. Luteinization Numerous subsequent studies raised the possibility that prostaglandins of the E series may play a role in other follicular functions. In the studies of Yang et al (1974) with rabbits, PGE levels remained elevated somewhat longer than PGF levels after ovulation, leading the authors to suggest that while PGF-2a may be most important for follicular rupture, PGEs may play a role in the luteinization process which normally follows ovulation. Further support for this idea was provided by the demonstration in vivo that intrafollicular injection of PGE-2 induced luteinization of rabbit follicles (Phi, Moon & Armstrong, 1977), and by reports of the effects of PGE-2 on progesterone production by follicles or granulosa cells undergoing 'luteinization' in culture (Channing, 1972; Ellsworth & Armstrong, 1974; Neal, Baker, McNatty & Scaramuzzi, 1975). Surprisingly, blockade of ovulation by indomethacin appeared not to be accompanied by blockade of progesterone secretion and corpus luteum formation (Armstrong et al, 1974; /81 / S02.00/ Journals of Reproduction & Fertility Ltd

2 Ainsworth et al, 1979), even when the indomethacin treatment was continued well beyond the time of ovulation (Phi et al, 1977). In addition, indomethacin appeared not to interfere with the normal preovulatory LH surge. These findings that ovulation could be blocked without any apparent alteration of gonadotrophin and steroid secretion led to hopes that a new class of anti-fertility drugs could be developed that possessed the desired anti-ovulatory effect but not side-effects associated with the more general endocrine disturbances caused by steroidal contraceptives. Prostaglandin synthesis by human follicles Before embarking on a widespread search for anti-prostaglandin agents more acceptable than indomethacin for ultimate use as ovulation inhibitors in women, it seemed important first to attempt to ascertain whether the concepts developed from the above-mentioned studies in experimental animals were applicable to women. To this end, we began investigation of prostaglandin production by the human follicle, as well as of effects of prostaglandins on human follicle cells. Results of studies with cultured human follicle wall tissue (theca and granulosa cells) indicated significant production of prostaglandin F, which was stimulated by addition of gonadotrophins (human menopausal gonadotrophin and human chorionic gonadotrophin, hcg) to the culture media (Plunkett, Moon, Zamecnik & Armstrong, 1975). Investigations with isolated follicle cell types have indicated that both the theca and granulosa cells have the ability to produce substantial amounts of prostaglandins in culture (unpublished observations). Prostaglandin effects on isolated cell types from human follicles Oestrogen biosynthesis In subsequent studies, human follicles were separated into their two principal cellular components, granulosa and theca cells, in order to examine their possible responsiveness to exogenous prostaglandins. For comparison, their responsiveness to the two classes of gonadotrophins, follicle stimulating hormone (FSH) and luteinizing hormone (hcg), were also determined. A comparison of the ability of granulosa and thecal preparations from a representative pool of 3-5 mm human follicles to secrete oestradiol-17ß when cultured without or with FSH and hcg, is presented in Text-fig. 1(a). In contrast to these low rates of oestrogen secretion by both cell types when cultured in the absence of an aromatizable substrate, addition of testosterone to the culture medium caused a striking 10- to 25-fold stimulation of oestradiol production by granulosa cells from the same pool of follicles (Text-fig. lb). In the presence of testosterone, an ability of purified FSH to stimulate oestradiol secretion by granulosa became evident. HCG was ineffective in stimulating oestrogen production by granulosa cells either in the absence or presence of testosterone. In the absence of testosterone, hcg appeared to stimulate oestradiol production by some thecal preparations, but this was not statistically significant, and in contrast to the results with granulosa cells, addition of testosterone to thecal preparations did not markedly alter oestradiol production, nor did it permit expression of effects of either gonadotrophin on oestradiol production (Moon, Tsang, Simpson & Armstrong, 1978). Cyclic AMP production Since there is abundant evidence that the gonadotrophic hormones exert their actions on their target cells via stimulation of specific receptor-linked adenylate cyclase, with resulting production of cyclic adenosine monophosphate (camp) acting as an intracellular 'second messenger' (Marsh, 1976), camp production was monitored in granulosa cells exposed to FSH

3 (a) (b) "ja X I I Control EU FSH (250 ng/ml) ^ hcg (1.u./ml) Granulosa rru; Theca 50 h fm- } Granulosa Theca Text-fig. 1. Oestradiol-17ß secretion by granulosa and theca preparations from human follicles during culture for 24 h (a) without steroid substrate, and (b) with an aromatizable substrate (0-5 µ -testosterone). Values are mean ± s.e.m. for (a) duplicate and (b) triplicate cultures per treatment, from granulosa and theca preparations from a representative pool of 3-5-mm human follicles. FSH: Papkofflot G4-150C, ovine; hcg: Ayerst. (Data from Moon et al, 1978.) Control FSH _ 1 10 hcg PGE-2 (ng/ml) (ng/ml) (i.u./ml) Text-fig. 2. Cyclic AMP production by granulosa cells during incubations for 2 h. Values are mean + s.e.m. for quadruplicate incubations per treatment, from granulosa cells isolated from a representative pool of 4-6-mm human follicles. and to hcg. As is evident in Text-fig. 2, FSH, but not hcg, stimulated camp production by human granulosa cells. The failure of these cells to respond to hcg with increased production of camp or oestradiol suggested that the cells were too immature to have acquired LH (hcg) receptors. Investigations with granulosa cells from numerous species have revealed that LH receptors are acquired late in follicular development, and are indicative of granulosa cell maturity (Zeleznik, Midgley & Reichert, 1974; Richards, 1979). Prostaglandin E-2 at concentrations which appeared to be within the physiological range for other species was highly effective in stimulating camp production by these granulosa cells. PGE responsiveness is therefore apparently acquired at an earlier stage of maturity than is LH responsiveness. That the PGE-stimulated camp production by granulosa cells is probably of physiological significance is indicated by its effectiveness in stimulating production of both oestradiol (in the

4 presence of testosterone only) and progesterone (both in the absence and presence of testosterone) (Table 1). Testosterone increased the production of progesterone by granulosa cells cultured with or without the stimulatory agents, FSH and PGE-2. Table 1. Effect of prostaglandin E-2 (PGE-2) on steroid production by human granulosa cells during culture for 24 h Hormone cone, (ng/mg protein) Treatment Progesterone Oestradiol-17ß Control Testosterone (0-5 um) ±8-8 FSH (0-25 µ / 1) 108 FSH + testosterone 325 PGE-2 (10 µ / 1) 718 PGE-2 + testosterone ± ± ± ± ± ± ± ±61-6 Values are means ± s.e.m. from triplicate cultures of granulosa cells isolated from a representative pool of 4-6-mm human follicles. Androgen biosynthesis Production of androgens by granulosa cells was negligible both in the absence and in the presence of FSH, hcg or PGE-2 (data not shown). This is in agreement with findings in most other species, and is consistent with the apparent lack of the 17<x-hydroxylase and C172o-lyase enzymes in granulosa cells (see Armstrong & Dorrington, 1977). As mentioned above, oestradiol production by the thecal preparations investigated was very low and of dubious significance. In contrast, the thecal preparations produced large amounts of androgens. As illustrated in Text-fig. 3, hcg stimulated androgen secretion (testosterone + DHT) in a dose-dependent fashion (Tsang, Moon, Simpson & Armstrong, 1979b). Production of androstenedione was substantially greater than that of testosterone (Tsang, Armstrong & Whitfield, 1980). FSH failed to stimulate androgen production by thecal preparations, consistent with findings in other species that theca cells lack significant levels of FSH receptors (Zeleznik et al, 1974). HCG, but not FSH, stimulated camp production by human thecal preparations, ' fe*4h FSH Cone, of FSH ^g/m Text-fig. 3. Effect of gonadotrophins on androgen (testosterone + DHT) and camp production by human theca tissues. Values are mean + s.e.m. of triplicate incubations of theca preparations isolated -from a pool of 4-6-mm follicles obtained from a representative patient. (Data from Tsang et al. 1979b.)

5 ~ < I Q S en E PGE-2 cone, (pg/ml) Text-fig. 4. Effect of PGE-2 on androgen and camp production by human thecal tissue during incubations for 2 h. Values are mean ± s.e.m. of 5 incubations of theca tissues from 2 representative pools of follicles 4-8 mm in diameter. (Data from Tsang et al, 1980.) I O over approximately the same range of dosages as that required for stimulation of androgen production (Text-fig. 3) (Tsang et al, 1979b). The actions of hcg on isolated thecal preparations could be mimicked by PGE-2 in the ability to stimulate both camp and androgen production (Text-fig. 4) (Tsang et al, 1980). The ability of exogenous camp (in the form of its dibutyryl derivative) to stimulate androgen production by theca preparations provides additional evidence that the stimulatory action of PGE-2 and hcg may be mediated by camp (Table 2) (Tsang et al, 1979b). Table 2. Effect of hcg and dbcamp on androgen production by isolated theca tissues from human follicles during incubations for 2 h Follicle pool no. Diam. of follicles (mm) Androgen (testosterone + DHT) production (ng/mg protein) Control 81 ± ± ± ±0-13 hcg (1 i.u./ml) dbcamp (1 mm) 62 ± ± ± ± ±7-19 Values are means ± s.e.m. of triplicate incubations of theca preparations from led follicles. pooled follicles.

6 Discussion and Conclusions The results of these studies with isolated human follicle cells summarized here are in general agreement with models proposed for other animal species to explain cellular and gonadotrophic interactions in regulation of follicular steroid biosynthesis (see Leung & Armstrong, 1980). They support a primary role of granulosa cells in production of oestradiol and of theca cells in production of androgens. Further, they indicate that at the stage of development of follicles examined, FSH was the more important gonadotrophin in regulating granulosa cell functions (oestrogen and progesterone secretion), and LH the more important in regulating theca cell functions (secretion of androgens). Although no convincing evidence of oestrogen production by theca cells was obtained in the present studies, Channing, Anderson & Batta (1978) have reported significant amounts of oestrogen production by human theca tissues. It seems likely that the latter were obtained from follicles at more advanced stages of development. Evidence of increased thecal oestrogen production as follicles undergo maturation has been obtained from rhesus monkeys (Channing & Coudert, 1976), sheep (Armstrong, Weiss, Selstam & Seamark, 1981) and pigs (D. T. Armstrong & G. J. King, unpublished observations). As reviewed above, it is well established that prostaglandins are somehow involved in the process of ovulation. The facts that prostaglandin E-2 can mimic the action of FSH on human granulosa cells and of LH on theca cells at early stages of follicular development, and that follicular tissues (both granulosa and theca cells) are capable of production of substantial amounts of prostaglandin (Plunkett et al, 1975; Triebwasser, Clark, LeMaire & Marsh, 1978; D. T. Armstrong & G. J. King, unpublished) raise the possibility that prostaglandins may play a role in regulation of other follicular functions as well. For example, PGE-2 may substitute for the pituitary gonadotrophins at certain stages of follicular development, and complement their actions at other stages (Text-figs 5 and 6). The obligatory nature of such actions has been questioned on the grounds that inhibitors of prostaglandin synthesis do not interfere with the ability of LH to stimulate steroidogenesis by preovulatory follicles, luteinization, or oocyte maturation (Lindner et al, 1974; Armstrong et al, 1974; Ainsworth et al, 1979). However, it is of interest to speculate on other possible roles of follicular prostaglandins in ovarian regulation Theca cell Granulosa cell Text-fig. 5. Cellular and hormonal interactions in the regulation of steroid biosynthesis by cells of growing preantral and early antral follicles. Theca cells contain receptors for, and respond to, LH and PGE (but not FSH) with increased production of androgen but of little or no oestradiol. Granulosa cells contain receptors for, and respond to, FSH and PGE (but not LH) with increased conversion to oestradiol, from androgen derived from theca cells, and with increased conversion of endogenous sterol to progesterone.

7 Theca cell Granulosa cell Text-fig. 6. Cellular and hormonal interactions in the regulation of steroid biosynthesis by cells of more mature follicles, approaching the preovulatory state. Theca cells increase their rate of production of androgens and attain the ability to produce significant amounts of oestradiol. Androgen, acting synergistically with gonadotrophins, results in an increased rate of progesterone production by the granulosa cells. Granulosa cells acquire receptors for, and responsiveness to, LH. Non-proliferating follicle Growing or mature follicle theca cell Theca / cem camp jranulosa I / cell / camp -PGE-2 PGF-2a- Atresia Text-fig. 7. Some hypothetical roles of prostaglandins in ovarian follicular regulation. Theca cells of growing follicles produce PGE-2 which can stimulate camp production both in theca and granulosa cells. This may provide the stimulus, of ovarian origin, responsible for initiation of growth of non-proliferating follicles by exerting gonadotrophin-like actions on granulosa and theca cells which do not yet possess receptors for the pituitary gonadotrophins. Prostaglandin F-2a, of follicular origin, may initiate, or otherwise participate in, the processes of luteolysis and atresia, through interaction with specific receptors on luteal and granulosa cells, respectively. (Text-fig. 7). The possibility that they may be of importance at very early stages of follicular differentiation, before the appearance of receptors for the pituitary hormones, cannot be ignored. For example, PGE produced by theca cells may, upon diffusion through the basement membrane, assist in the induction of FSH receptors and thus of FSH responsiveness of granulosa cells in immature follicles. Prostaglandins may also diffuse short distances through the ovarian stroma to adjacent follicles, to act in an LH-like manner on theca cells, possibly assisting in the initiation of growth of follicles in the non-proliferating pool. Prostaglandins may be worthy of consideration as intra-ovarian factors of follicular origin, suggested by Peters (1979) to be involved in control of growth of non-proliferating follicles, with the pituitary gonadotrophins becoming the major regulatory agents after growth has begun and they have acquired FSH and LH receptors. The observations of Lamprecht, Zor, Tsafriri & Lindner (1973) that PGE-2 will

8 stimulate adenylate cyclase activity in fetal and neonatal rat ovaries whereas LH is not effective until the second week of life are consistent with this hypothesis. Prostaglandin F-2a of uterine origin has been widely accepted as a luteolytic agent in many animal species (reviewed by Horton & Poyser, 1976). Although this appears not to be so for women, PGF-2a receptors have, nevertheless, been demonstrated in human corpus luteum tissues (Powell, Hammerstrom, Samuelsson & Sjoberg, 1974; Rao, Griffin & Carman, 1977). Prostaglandin F-2a of follicular origin may also be involved in the luteolytic process, perhaps of secondary importance in those species possessing a uterine component to this luteolytic mechanism, but of primary importance in women, who lack such a component. The ability of PGF-2ct to inhibit progesterone production by cultured human granulosa cells (McNatty, Henderson & Sawers, 1975) offers support for such an hypothesis; alternatively, this inhibitory effect may suggest a role for PGF-2a in follicular atresia. The importance of the above, or other, roles of prostaglandins in ovarian follicular regulation must await further research, particularly with tissues obtained from follicles over a wider range and at more precisely timed stages of differentiation. The development of suitable animal models will be essential to our achievement of more complete understanding of the role and importance of prostaglandins in follicular regulation. It remains to be determined whether inhibitors of prostaglandin synthesis or actions will be found which are effective in blocking ovulation or other processes essential to reproduction in women, and if so, whether such compounds will prove of value as anti-fertility agents at acceptable dosages and modes of administration. The collaboration of Dr Y. S. Moon,. K. Tsang, C. W. Simpson and Ms M. Dobias in various aspects of the research reviewed here is gratefully acknowledged. The research was supported by grants from the Medical Research Council and the World Health Organization. D.T.A. is an M.R.C. Career Investigator. References Ainsworth, L., Baker, R.D. & Armstrong, D.T. (1975) Pre-ovulatory changes in follicular fluid prostaglan din F levels in swine. Prostaglandins 9, Ainsworth, L., Tsang, B.K., Downey, B.R., Baker, R.D., Marcus, GJ. & Armstrong, D.T. (1979) Effects of indomethacin on ovulation and luteal function in gilts. Biol. Reprod. 21, Armstrong, D.T. & Dorrington, J.H. (1977) Estrogen biosynthesis in the ovaries and testes. In Adv. Sex Hormone Res., Vol. 3, pp Eds J. A. Thomas & R. L. Singhal. University Park Press, Baltimore. Armstrong, D.T. & Grinwich, D.L. (1972) Blockade of spontaneous and LH-induced ovulation in rats by indomethacin, an inhibitor of prostaglandin biosyn thesis. Prostaglandins 1, Armstrong, D.T., Moon, Y.S. & Zamecnlk, J. (1974) Evidence for a role of prostaglandins in ovulation. In Gonadotropins and Gonadal Function, pp Ed. N. R. Moudgal. Academic Press, New York. Armstrong, D.T., Weiss, T.J., Selstam, G. & Seamark, R.F. (1981) Follicular steroid biosynthesis: hormonal and cellular interactions. J. Reprod. Fert., Suppl. 30 (in press). Bauminger, S. & Lindner, H.R. (1975) Periovulatory changes in ovarian prostaglandin formation and their hormonal control in the rat. Prostaglandins 9, Channing, C.P. (1972) Stimulatory effects of prostaglan dins upon luteinization of rhesus monkey granulosa cell cultures. Prostaglandins 2, Channing, C.P. & Coudert, S.P. (1976) Contribution of granulosa cells and follicular fluid to ovarian estrogen secretion in the rhesus monkey in vivo. Endocrinology 98, Channing, C.P., Anderson, L.D. & Batta, S.K. (1978) Follicular growth and development. Clin. Obstet. Gynaecol. S, Ellsworth, L.R. & Armstrong, D.T. (1974) Effect of indomethacin and 7-oxa-13-prostynoic acid on luteinization of transplanted rat ovarian follicles induced by luteinizing hormone and prostaglandin ^.Prostaglandins 7, Grinwich, D.L., Kennedy, T.G. & Armstrong, D.T. (1972) Dissociation of ovulatory and steroidogenic actions of luteinizing hormone in rabbits with indomethacin, an inhibitor of prostaglandin syn thesis. Prostaglandins 1, Horton, E.W. & Poyser, N.L. (1976) Uterine luteolytic hormone: a physiological role for prostaglandin F2. Physiol.Rev. 56, Lamprecht, S.A., Zor, U., Tsafriri, A. & Lindner, H.R. (1973) Action of prostaglandins E2 and of luteinizing hormone on ovarian adenylate cyclase, protein kinase, and ornithine decarboxylene activity during postnatal development and maturity in the rat. J. Endocr. 57,

9 Lau, I.F., Saksena, S.K. & Chang, M.C. (1974) Prostaglandins F and ovulation in mice. J. Reprod. Fert. 40, Leung, P.C.K. & Armstrong, D.T. (1980) Interactions of steroids and gonadotropins in the control of steroidogenesis in the ovarian follicle. Ann. Rev. Physiol. 42, Lindner, H.R., Tsafriri,., Lieberman, M.E., Zor, U.. Koch, Y., Bauminger. S. & Barnea, A. (1974) Gonadotropin action on cultured Graafian follicles: Induction of maturation division of the mammalian oocyte and differentiation of the luteal cell. Recent Prog. Horm. Res. 30, Maia,., Jr, Barbosa, I. & Coutinho, E.M. (1978) Inhibition of ovulation in marmoset monkeys by indomethacin. Fert. Steril. 29, Marsh, J.M. (1976) The role of cyclic AMP in gonadal steroidogenesis. Biol. Reprod. 14, McNatty, K.P., Henderson, K.M. & Sawers, R.S. (1975) Effects of prostaglandin F2 and E2 on the production of progesterone by human granulosa cells in tissue culture. J. Endocr. 67, Moon, Y.S., Tsang, B.K., Simpson, C. & Armstrong, D.T. (1978) ß-Estradiol biosynthesis in cultured granulosa and theca cells of human ovarian follicles: stimulation by follicle stimulating hormone. J. clin. Endocr. Metab. 47, Neal, P., Baker, T.G., McNatty, K.P. & Scaramuzzi, RJ. (1975) Influence of prostaglandins and human chorionic gonadotropin on progesterone con centration and oocyte maturation in mouse ovarian follicles maintained in organ culture. J. Endocr. 65, O'Grady, J.P., Caldwell, B.V., Auletta, F.J. & Speroff, L. (1972) The effects of an inhibitor of prostaglandin synthesis (indomethacin) on ovulation, pregnancy, and pseudopregnancy in the rabbit. Prostaglandins 1, Orczyk, G.P. & Behrman, H.R. (1972) Ovulation blockade by aspirin or indomethacin: in vivo evidence for a role of prostaglandin in gonadotropin secretion. Prostaglandins 1, Peters, H. (1979) Some aspects of early follicular development. In Ovarian Follicular Development and Function, pp Eds A. R. Midgley & W. A. Sadler. Raven Press, New York. Phi, L.T, Moon, Y.S. & Armstrong, D.T. (1977) Effects of systemic and intrafollicular injections of LH, prostaglandins and indomethacin on the luteinization of rabbit graafian follicles. Prostaglandins 13, Plunkett, E.R., Moon, Y.S., Zamecnik, J. & Armstrong, D.T. (1975) Preliminary evidence of a role for prostaglandin F in human follicular function. Am. J. Obstet. Gynec. 123, Powell, W.S., Hammerstrom, S., Samuelsson,. & Sjoberg,. (1974) Prostaglandin F2 receptor in human corpora lutea. Lancet i, Rao, C.V., Griffin, L.P. & Carman, F.R., Jr (1977) Prostaglandin F2 binding sites in human corpora lutea. /. clin. Endocr. Metab. 44, Richards, J.S. (1979) Hormonal control of ovarian follicular development. A 1978 perspective. Recent Prog. Horm. Res. 35, Stacey, N.E. & Pandey, S. (1975) Effects of indometha cin and prostaglandins on ovulation of goldfish. Prostaglandins 9, Triebwasser, W.F., Clark, M.R., LeMaire, WJ. & Marsh, J.M. (1978) Localization and in vitro synthesis of prostaglandins in components of rabbit preovulatory Graafian follicles. Prostaglandins 16, Tsang, B.K., Ainsworth, L., Downey, B.R. & Armstrong, D.T. (1979a) Preovulatory changes in cyclic AMP and prostaglandin concentrations in follicular fluid of gilts. Prostaglandins 17, Tsang, B.K., Moon, Y.S., Simpson, C.W. & Armstrong, D.T. (1979b) Androgen biosynthesis in human ovarian follicles: cellular source, gonadotropic con trol, and adenosine 3',5'-monophosphate mediation. /. clin. Endocr. Metab. 48, Tsang, B.K., Armstrong, D.T. & Whitfield, J.F. (1980) Steroid synthesis by human ovarian follicular cells in vitro. Endocrinology 106A, Abstr Wallach, E.E., de la Cruz,., Hunt, J., Wright, K.H. & Stevens, V.C. (1975) The effect of indomethacin on HMG-HCG induced ovulation in the rhesus monkey. Prostaglandins 9, Yang, N.S.T., Marsh, J.M. & LeMaire, WJ. (1974) Post-ovulatory changes in the concentrations of prostaglandins in rabbit graafian follicles. Prostaglandins 6, Zeleznik, A.M., Midgley, A.R., Jr & Reichert, L.E., Jr (1974) Granulosa cell maturation in the rat. Increased binding of human chorionic gonadotropin following treatment with follicle stimulating hormone in vivo. Endocrinology 95,