Prostaglandins and Follicular Functions David T
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; 0022-4251/81 /030283-09S02.00/0 © 1981 Journals of Reproduction & Fertility Ltd
Downloaded from Bioscientifica.com at 10/01/2021 11:25:14PM via free access 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
Downloaded from Bioscientifica.com at 10/01/2021 11:25:14PM via free access (a) (b)
250 X I I Control EU FSH (250 ng/ml) 200 ^ hCG (1 ¡.u./ml) ja" 150
100
50 h fm- } rrU; 2 1 0 Granulosa Theca 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 2 50 500 1 10 10 100 FSH hCG PGE-2 (ng/ml) (i.u./ml) (ng/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
Downloaded from Bioscientifica.com at 10/01/2021 11:25:14PM via free access 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 61 0 + 7-6 1-3 ± 0-6 Testosterone (0-5 um) 155 9 ±8-8 393-6 ±49-4 FSH (0-25 µ / 1) 108 9 + 20-6 1-3 ± 0.4 FSH + testosterone 325 1 ±34-8 576-1 ±21-5 PGE-2 (10 µ / 1) 718 6 + 89-9 2-3 ± 0-5 PGE-2 + testosterone 1041 0± 171-6 569-3 ±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
20
16
12
' fe*4h FSH
0 01 01 1 10 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.)
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150
I < Q
~
en S E
0 O I 0 08 0 4 2 0 10 0 50 0 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.) 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
Androgen (testosterone + DHT) production (ng/mg protein) Follicle pool Diam. hCG dbcAMP no. of follicles (mm) Control (1 i.u./ml) (1 mM) 3-6 •81 ±0-41 •62 ±0-90 3-6 86 ±0-18 •32 ±0-26 3-6 •43 ± 016 44 ± 0-26 7-10 •06 + 0-17 •44 ±3-14 7-10 •79 ±0-13 •64 ±7-19
Values are means ± s.e.m. of triplicate incubations of theca preparations from pooledled follicles.
Downloaded from Bioscientifica.com at 10/01/2021 11:25:14PM via free access 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.
Downloaded from Bioscientifica.com at 10/01/2021 11:25:14PM via free access 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 Growing or mature follicle follicle theca cell
Theca / ceM cAMP
jranulosa I / -PGE-2 PGF-2a- cell / cAMP 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
Downloaded from Bioscientifica.com at 10/01/2021 11:25:14PM via free access 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
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