Acute stimulatory effects of luteinizing hormone-releasing hormone (LHRH) and LHRH analogues on the preovulatory rat follicle

0022·4731/83 S3.00+0.00 Copyright © 1983 Pergamon Press Ltd

J . ." eroiJ 8io,""rlll. Vol. 19. No. I. pp. 119 ·125. 1983 Prinled in Great Britain. All rights reserved

ACUTE STIMULATORY EFFECTS OF LUTEINIZING HORMONE-RELEASING HORMONE (LHRH) AND LHRH ANALOGUES ON THE PREOVULATORY RAT FOLLICLE KURT AHREN*, HAKAN BILlIG*, MARTIN R. CLARKt, CARL EKHOLM*, TORBJORN HILLENSJO*, WILLIAM 1. LEMAIREt, CLAES MAGNussoN* and C. S. SHEELA RA It *Department of Physiology, University of Goteborg, Sweden, and Department of Obstetrics and Gynecology and tEndocrine Laboratory, University of Miami, Medical School, Miami, U.S.A.

INTRODUCTION

It is now well established that LHRH and its potent synthetic analogues can have extra-pituitary actions, among them effects on the gonads, when administered in pharmacological doses. An evidence for a direct effect on the ovary was first given by Rippel and Johnson[ I J when they demonstrated a dose-dependent inhibition by LHRH of ovarian growth in immature rats treated with hCG. More conclusive evidence of a direct effect of LHRH and its agonistic analogues was given in 1979 when it was reported that LHRH inhibited FSH-stimulated progesterone production by cultured granulosa cells [2]. Several other inhibitory effects on ovarian function (e.g. on steroidogenesis, gonadotrophin receptors, cyclic AMP formation) have since then been reported from various laboratories [for Ref. see 3,4]. A stimulatory effect of LHRH and its agonistic analogues on the ovary was first reported by Clark et al.[5]. They demonstrated that LHRH and two analogues markedly stimulated prostaglandin synthesis by granulosa cells from preovulatory rat follicles. Studies were therefore designed to investigate whether these peptides have stimulatory effects also on other functions of the preovulatory follicle and, if so, how these effects might be related to the inhibitory effects reported by other groups. The results of some of these studies are summarized and discussed in this paper.

were cultured in Eagle's minimal essential medium with Hepes and 10% fetal calf serum. For the ill !'iva experiments the rats were hypophysectomized on the morning of day 28 and the hormones injected 1400--1500 h. When oocyte maturation or prostaglandin E levels were determined the rats were sacrificed 6-10 h after the injections, while ovulations were recorded on the morning of day 29. The completeness of hypophysectomy was determined in all animals by assaying pituitary sites and plasma for presence of gonadotrophins and growth hormone by RIA. The following hormones have been used: LH-NIH-S18, SI9 and S21, FSH-NIH-SI3, LHRH (Ferring and Sigma), [o-Ala 6 , desGly'ONH 2 J LHRH ethylamide (LHRHa, Sigma), [o-Glu', o-Phe 2, o_Trp 3.6] LHRH (antagonist, Boehringer and Mannheim). Oocyte meiosis was staged by Nomarski interference contrast microscopy. Steroids and prostaglandin E were assayed by RIA[6,5] and cAMP according to Gilman[17]. Oocyte oxygen consumption was determined with a microspectrophotometric method employing hemoglobin as indicator of oxygen tension [8, 9]. Data are presented as mean ± SE. Statistical significances were determined by ANOVA followed by Student-Newman-Keuls' multiple range test.

RESULTS

In vitro effects on whole follicles MATERIALS AND METHODS

Immature Sprague-Dawley rats, 26 days old, were treated with \0 IV PMSG. This treatment results in ovulation of 12-18 ova early on the morning of day 29. Ovarian follicles or granulosa cells were isolated on the morning of day 28. The follicles were incubated in Krebs Ringer bicarbonate buffer with glucose and I % bovine serum albumin. Granulosa cells Correspondence to: Professor Kurt Ahren, Department of Physiology, University of Goteborg, P.O. Box 33031, S-400 33 Goteborg, Sweden. t Present address: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.

Ooe vte maturation. Mammalian oocytes remain

arrest~d in the prophase stage of the first meiotic division until meiosis is resumed in the preovulatory follicle following the LH surge. Oocyte meiosis can also be initiated in vitro by the addition of gonadotrophin to isolated preovulatory rat follicles [10, 11). Surprisingly, LHRH and two potent LHRH analogues were found to induce meiosis in follicle-enclosed rat oocyte in vitro [12]. Figure I demonstrates this effect and also that the stimulatory effect of an LHRH agonist on meiosis was abolished by an antagonistic LHRH analogue. LH induction of meiosis was not affected by the LHRH antagonist. LHRH also induced expansion and mucification of the cumulus oophorus sur119

KURT AHREN et al.

120

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~ ~20

i

(,:)

0....---'---''--................---''---''--....-.----.......'-----C

LH

LHRHo

Ant

LH

A~t

LHRHo

~t

Fig. I. Effect of the LHRH antagonist (10 jlg/ml) on the meiosis-inducing effect of LHRHa (10 ng/m!) and LH (10 ng/ml) on follicle-enclosed oocytes incubated for 4 h. Number of oocytes examined given in the bars [17].

rounding the follicle-enclosed oocyte [13J, as was previously shown for gonadotrophins [14]. After induction of meiosis by LH or FSH in vivo or in in vitro the rat oocyte increases its oxygen consumption by 20-40% [9]. Oocyte respiration was stimulated also after induction of meiosis in vitro by an LHRH agonist [15], as i1lustrated in Fig. 2. Thus, LHRH can mimic LH both regarding induction of meiosis, cumulus mucification and stimulation of oocyte metabolism. Follicular lactate production. It had earlier been found that the rate 0tflcolysis, as measured by glucose uptake and lactat production, in the preovulatory follicle is marke y and dose-dependently stimulated by LH [II, 16 : Addition ofLHRH to incubated follicles also stim ated lactate production, although the maximal e ct was not as great as for LH [12]. This LHRH e ect could be blocked by the LHRH antagonist, while the antagonist did not influence the effect of LH on lactate production [17], as illustrated in Fig. 3.

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Follicular steroidogenesis. LH increases the secretion of progesterone, androgens and estrogens by preovulatory rat follicles (during 4-6 h). This general stimulation is later (after 4-6 h) fol1owed by an inhibition of androgen and estrogen synthesis while progesterone synthesis continues [6,18,19]. An agonistic LHRH analogue stimulated fol1icular progesterone production during a 4 h incubation period (Fig. 4). This stimulation was, however, quantitatively much less tha'n the effect of LH. Another difference was that the effect of LH on progesterone secretion could be observed much earlier than the effect of the LHRH analogue, 2 and 4 h respectively [20]. Also androgen production was stimulated by LHRHa during the initial incubation period (0-6 h). When a second incubation was continued (6-8 h), neither LH nor LHRHa stimulated androgen formation while progesterone secretion still was stimulated [21]. This indicates that LHRHa after a lag period inhibits the e21-side chain cleavage enzyme in the preovulatory follicle, and that LHRH, therefore, also in this respect has an "LHlike" activity. Follicular cyclic AMP and prostaglandins. Addition of LH to preovulatory rat follicles produces a rapid and pronounced increase in fol1icular cyclic AMP [22, 23]. Under the conditions when the agon5

+

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15

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LHRHa

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Fig. 2. Oxygen consumption of oocytes isolated from follicles that were incubated for 4 h with or without LHRH (1 jlg/ml) or LH (10 Jlg/ml) or not incubated at all. Number of measurements indicated within the bars [15].

c

LH

AM

LHRHa

A~t

Fig. 3. Effect of LHRHa (10 ng/ml) and LH (10 ng/ml) alone or in combination with the LHRH antagonist (10 jlg/ml) on lactate accumulation in media from a 4 h incubation of rat follicles [17].

121

LHRH stimulates rat preovulatory follicles TISSUE LEVELS

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Fig. 4. The effect of LH (I JIg/mil and LHRHa (I Jlg/ml), alone or in combination, on progesterone secretion (right panel) and tissue progesterone (left panel) in a 4 h incubation of whole preovulatory follicles [20].

Table 1. Effect of LH and LHRH agonist on follicular cAMP Exp II: Incubation time

Exp I: Incubation time Treatment Control LH LHRHa

30 min 66 1343 66

±4 ± 152 ± 15

60 min 57 2872 68

±9 ± 396 ±5

120 min 50 1488 65

120 min

±5 ± 179 ±5

106 1351 124

±8 ± 74 ± 14

240 min 137 1194 267

± 20 ± 174 ± 23

Preovulatory follicles were incubated the time indicated in the presence of LH (I Jlg/ml) or LHRHa (I JIg/mI). Follicular levels of cAMP expressed as pmoljmg protein [20].

istic LHRH analogue induced oocyte maturation and increased follicular lactate and steroid production, no changes in cAMP were detected during incubations up to 120 min (Table 1). This is in sharp contrast to the dramatic effect of LH. After 4 h of incubation with the LHRH analogue a slight but significant increase in cyclic AMP was seen. Prostaglandin E was determined after a 4 h incubation of follicles. The LHRH analogue and LH increased the level of prostaglandin E markedly and to the same extent (Fig. 5). Indomethacin abolished the effect of LH and the LHRH analogue on prostaglandin accumulation. Indomethacin, however, did not inhibit the effects of LH or LHRH agonist on oocyte maturation or follicular progesterone and cyclic AMP accumulation [20]. In vitro effects on granulosa cells

Steroidogenesis. It is well established that both LH and FSH can stimulate steroidogenesis in isolated granulosa cells. The pattern and extent of stimulation by the two gonadotrophins are determined by the developmental stage of the granulosa cells [for Ref. see 24]. Figure 6 illustrates that addition of an LHRH agonist stimulated the formation of progesterone and 20iX-OH-progesterone by granulosa cells from preovu-

latory follicles during a 24 h culture period. This effect was blocked by the LHRH antagonist (Fig. 6). Both LH and FSH stimulated progesterone secretion more markedly than the LHRH agonist in this culture system. When the LHRH analogue was added together with FSH it did not influence the stimulatory effect of the gonadotrophin during the initial 10 h of culture. After extended periods of culture (24-48 and 48-72 h), however, the LHRH analogue reduced the stimulation by LH and FSH, and did not by itself stimulate 300 s::.

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Fig. 5. The effect of LHRHa (1 Jlg/ml) and LH (I JIg/mil alone or combined with indomethacin (3 x 10- 4 M) on prostaglandin E accumulation in the medium in a 4 h incubation of preovulatory follicles [20].

122

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Fig. 6. Progesterone and 20a-OH-progesterone secretion by granulosa cells isolated from preovulatory follicles. The cells were cultured for 24 h in the presence of LHRHa (100 ng/ml) and LHRH antagonist (10 jlg/ml) alone or in combination [25].

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Fig. 7. The effect of single LHRHa (12 jlg/rat) or LH (25 jlg/rat) injections on resumption of meiosis (germinal vesicle breakdown = GVB, Polar Body = PB) in rats, hypophysectomized on the morning of proestrus. The injections were given at 1400 h and the rats were sacrificed 6 or 10 h later and oocytes were isolated from the large follicles. Number of oocytes examined is given within the bars [27].

progestin production any longer [25], thus demonstrating a change in the effect of the LHRH analogue with culture time. A similar biphasic effect of the LHRH agonist on cultured granulosa cells has been observed on lactate production [26]. LHRH alone stimulated granulosa cell glycolysis. In combination with FSH an additive effect was obtained during the first day of culture, which gradually disappeared and was replaced by an inhibition of the FSH response during the third day of culture.

treated 'with indomethacin 30min prior to the injection of hormones (Fig. 8). Most animals that had received an injection of LHRH analogue (12 or 1.2 /lg)

60 50

40 lJJ

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In vivo effects When LHRH or LHRH agonists were injected to PMSG-treated rats, hypophysectomized on the morning of proestrus, several "LH-like" effects were observed [27, 28]. Figure 7 demonstrates that injection of an LHRH-agonist caused resumption and completion of meiosis in the oocytes of preovulatory follicles and that the timing of this effect was similar to that obtained with exogenous LH. Also plasma progesterone levels were increased by injection of the LHRH analogue and LH (Fig. 9). Increased levels of prostaglandin E in the ovaries were seen 6-8 h after the injection of either LH or the LHRH agonist, and this effect was completely blocked when the animals were

'6>I: .. 00 -> 0'0 0 ......

30

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Fig. 8. Ovarian prostaglandin E levels 6-8 h after injection of LHRHa (12 jlg/rat) or LH (25 jlg/rat) with or without previous injection of indomethacin (l mg/rat) in hypophysectomized rats [28].

LHRH stimulates rat preovulatory follicles 20

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Fig. 9. Plasma progesterone levels in hypophysectomized proestrous rats 6 h after injection of LHRHa (12 ,Ug/rat) or LH (25 ltg/rat) [27].

ovulated as was seen by the presence of ovulated eggs in the oviducts 16-18 h after the injections (Fig. 10). Also this effect could be abolished by indomethacin. DISCUSSION

The results presented in this review demonstrates that LHRH and its agonistic analogues can produce several stimulatory effects on the preovulatory rat follicle, both when added in dIm to whole follicles or isolated granulosa cells and when injected into rats hypophysectomized on the proestrus day. The effects of LHRH on meiosis, ovulation, and prostaglandin synthesis closely mimic the effect of LH on these parameters. On other parameters such as steroidogenesis and glycolysis the maximal effects of LHRH are considerably smaller than the effects of LH. The acute stimulatory effects of LHRH and a number of LHRH analogues on progesterone synthesis in granulosa cells from preovulatory follicles have been characterized [29]. Furthermore Jones and Hsueh[30] reported stimulatory effects of LHRH on basal progestin synthesis in granulosa cells from pre-antral follicles. In both these studies as well as in the experiments described in this paper the stimulation obtained by LHRH was small compared to the stimulation produced by gonadotrophins. LHRH has recently been reported to stimulate three key steroidogenic enzymes (cholesterol side-chain cleavage enzyme, 3fJ-hydroxysteroid dehydrogenase and aromatase) during a 24-h culture period of granulosa cells from diethylstilbestrol-treated immature rats

123

LHRH for period of 2 or more days. An exception to this is the rat luteal cell in which steroidogenesis was acutely inhibited [32]. In our experimental system with granulosa cells from preovulatory follicles LHRH also inhibited gonadotrophin stimulated steroidogenesis and glycolysis when the culture period was extended to 2-3 days. Initially (for 10--24 h), however, the LHRH analogue stimulated the cells to increase the production of steroids and lactate. These results demonstrate a biphasic effect of LHRH with initial stimulatory effects which after some time change into inhibition of gonadotrophin-stimulated events. A comparison with the effects of LHRH on the testis is adequate since many regulatory systems are, in principle, the same in the ovary and testis. Treatment of hypophysectomized rats with LHRH or LHRH agonists results in inhibitory effects on Leydig cell function. e.g. androgen production [33]. At least 2 days of treatment were, however. necessary for the development of this inhibitory effect. Androgen biosynthesis could also be inhibited i/1 uitro in Leydig cells when these cells were cultured with LHRH for at least 2-3 days [34]. Also, other laboratories have failed to show inhibitory effects in short-term incubations of Leydig cells [e.g. 35]. It is, however, of considerable interest in relation to our findings on the ovary that Sharpe and Cooper[36] reported that an LHRH agonist stimulated testosterone production 2 to 3-fold in short-term (4 h) incubation of collagenase-dispersed Leydig cells. It seems therefore likely that LHRH and its agonistic analogues have biphasic effects not only on the ovary but also on the testis. The cellular mechanisms behind the acute stimulatory "LH-like" effect of LHRH remain to be elucidated. High affinity binding sites (receptors) have been demonstrated both in the ovary and the testis, and the characteristics of these receptors are very 100

-

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[31].

The clear stimulatory effects of LHRH and LHRH analogues on a number of various functions of the ovarian follicle may seem difficult to reconcile with the many reports from other laboratories of direct inhibitory effects of LHRH on ovarian function [for Ref. see 3,4]. Inhibitory LHRH effects have mostly been seen on gonadotrophin stimulation of granulosa cells from hypophysectomized diethylstilbestroltreated rats and cells have been cultured together with 5.8. 19-I(A)-J

«3) (3)

«0 (4)

021 CII

0-'-----'-----'.........- Indomethacin. - + - + - + Hormone:

LH

LHRHa

Fig. 10. Ovulation in PMSG-treated rats hypophysectomized in the morning of proestrus and injected with LHRHa (12 j.tg/rat) or LH (25,Ug/rat) at 1430 h with or without previous injection of indomethacin (1 mg/rat). Number of rats indicated within parentheses. Ovulation was recorded 16--18 h after the hormone injections [28].

124

KURT AHREN

similar to those of the LHRH receptors in the anterior pituitary [for Ref. see 3]. Direct functional coupling between the LHRH receptor and the cyclic AMP system seems unlikely, at least in the ovarian follicular cells where cyclic AMP was not increased during the first 2 h of exposure to LHRH while LH increased follicular cyclic AMP levels within few minutes. An attractive hypothesis is that Ca 2 + is involved as a second messenger in the same way as it seems to be involved in LHRH stimulation of gonadotrophin secretion from the anterior pituitary [for Ref. see 37]. Other interesting facts related to the mechanism of action of LHRH are the recent observations that both LHRH and LH stimulate phosphatidylinositol metabolism in isolated rat granulosa cells [38] and that the ovary contains a cyclic AMP-independent, calcium-sensitive, phospholipid-dependent protein kinase [39]. The fact that there are specific high affinity binding sites for LHRH in both ovary and testis and that exogenous addition of these peptides exerts several effects on both gonads open the possibility, but does not prove, that LHRH or LHRH-like peptides constitute a local regulatory system for the gonadal function. Reports of LHRH-like biological activity in extracts of testicular tissue [40,41] and ovaries [42] are of great interest in this connection. It has, however, been difficult to reproduce the experiments where LHRH-like activity was found in ovarian extracts [43]. Further work is undoubtedly needed to elucidate the possible physiological significance both of the stimulatory and inhibitory effects of LHRH on gonadal function.

Acknowledgements-These studies were supported by grants from the Swedish MRC (27, 5650. 6154, 6350), Magnus Bergvall's and Hjalmar Svensson's Foundations, The Swedish Society of the Medical Sciences, the Medical Faculty, University of Goteborg and by the NIH (HD08747 to W. J. LeMaire). Parts of the studies were conducted while W. 1. LeMaire was in receipt of a Fogarty Senior International Fellowship (TW 003118) and a Visiting Scientist research grant from the Swedish MRC (5732) and C S. Sheela Rani was in receipt of a Research Training Grant from the WHO. LH and FSH were kindly supplied by the NIAMMD and the antisera for steroid RIA by Drs Hans Lindner and Fortuna Kohen, The Weizmann Institute of Science, Israel. We wish to thank Mrs Harriet Thelander for technical assistance and Mrs Gun Derevall and Mrs Britt-Marie Helder for preparing the manuscript. REFERENCES

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et al.

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LHRH stimulates rat preovulatory follicles hormone on ovarian adenylate cyclase, protein kinase and ornithine decarboxylase activity during postnatal development and maturity in the rat. J. Endocr. 57 (1973) 217-233. 23. Nilsson L., Rosberg S. and Ahren K.: Characteristics of the cyclic 3',5'-AMP formation in isolated ovarian follicles from PMSG-treated immature rats after stimulation in vitro with gonadotrophins and prostaglandins. Acta Endocr., Copenh. 77 (1974) 559-574. 24. Channing C P. and Tsafriri A.: Mechanism of action of luteinizing hormone and follicle-stimulating hormone on the ovary in vitro. Metabolism 26 (1977) 413-468. 25. Sheela Rani C S., Ekholm C, Billig H., Magnusson C and Hillensjo 1. : Biphasic effect of GnRH on progestin secretion by rat granulosa cells. Bioi. Reprod. (1983). 26. Billig H., Magnusson C, Ekholm C and Hillensjo T.: Biphasic effect of a GnRH agonist on glycolysis in cultured rat granulosa cells. BioI. Reprod. 2, Suppl. 1 (1982) 152A. 27. Ekholm C, Hillensjo 1. and Isaksson 0.: Gonadotropin releasing hormone agonists stimulate meiosis and ovulation in hypophysectomized rats. Endocrinology 108 (1981) 2022-2024. 28. Ekholm C, Clark M., Magnusson Isaksson O. and LeMaire W. J.: Ovulation induced by a gonadotropin releasing analogue in hypophysectomized rats involves prostaglandins. EndocrillOlogy 110 (1982) 288-290. 29. Clark M. R.: Stimulation of progesterone and prostaglandin E accumulation by luteinizing hormone-releasing hormone (LHRH) and LHRH analogs in rat granulosa cells. Endocrinology 110 (1982) 146-152. 30. Jones P. B. C. and Hsueh A. 1. W.: Regulation of ovarian 3p-hydroxysteroid dehydrogenase activity by gonadotropin-releasing hormone and follicle-stimulating hormone in cultured rat granulosa cells. Endocrinology 110 (1982) 1663-1671. 31. Dorrington J., McKeracher H., Munshi S. and GoreLangton R.: LHRH independently stimulates steroidogenic enzymes in granulosa cell cultures. Abstr 396 at the 64th Annual Meeting, The Endocrine Society (1982). 32. Behrman H. R., Preston S. L. and Hall A. K.: Cellular mechanism of the antigonadotropic action of luteinizing hormone-releasing hormone in the corpus luteum. Endocrinology 107 (1980) 656-664.

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33. Bambino 1. H., Schreiber J. R. and Hsueh A. 1. W.: Gonadotropin-releasing hormone and its agonist inhibit testicular luteinizing hormone receptor and steroidogenesis in immature and adult hypophysectomized rats. Endocrinology 107 (1980) 908-917. 34. Hsueh A. 1. W., Schreiber 1. R. and Erickson G. F.: Inhibitory effect of gonadotropin releasing hormone upon cultured testicular cells. Mol. Cell. Endocr. 21 (1981) 43-49. 35. Badger 1. M., Beitins I. Z., Ostrea 1., Crisafulli J. M., Little R. and Saidel M. E.: Luteinizing hormonereleasing hormone does not inhibit testosterone production in rat interstitial cells in vitro. EndocrillOlog.v 106 (1980) 1149-1153. 36. Sharpe R. M. and Cooper I.: Stimulatory effect of LHRH and its agonists on Leydig cell steroidogenesis in vitro. Mol. Cell. Endocrinol. 26 (1982) 141-150. 37. Conn P. M., Marian 1., McMillian M., Stern J., Rogers D., Hamby M., Penna A. and Grant E.: Gonadotro· pin-releasing hormone action in the pituitary: A three step mechanism. E/ldocr. Rev. 2 (1981) 174-185. 38. Davis 1. S., Farese R. V. and Clark M, R.: LH and GnRH acutely increase phosphatidylinositol levels in rat granulosa cells. Bioi. Reprod. Suppl 1 26 (\982) 41. 39. Davis 1. S. and Clark M. R.: Calcium-activated, phospholipid-dependent protein kinase activity in ovarian cytosol from PMSG-treated rats. Abstr 399 at the 64th Annual Meeting, The Endocrine Sociecy (1982). 40. Sharpe R. M., Fraser H. M., Cooper I. and Rommerts F. F. G.: Sertoli-Leydig cell communication via an LHRH-like factor. Nature 290 (1981) 785-787. 41. Sharpe R. M., Fraser H. M., Cooper I. and Rommerts F.F.G.: The secretion, measurement and function of a testicular LHRH-like factor. AIm. N. Y. Acad. Sci. (1982). 42. Ying SoY, Ling N., Bohlen P. and Guillemin R.: Gonadocrinins: Peptides in ovarian follicular fluid stimulating the secretion of pituitary gonadotropins. Endocrin· ology 108 (1981) 1206-1214. 43, Esch F., Ling N., Ying Soy and Guillemin R.: Peptides of gonadal origin involved in reproductive biology. In

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