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Puromycin has been reported to block the meiosis of cultured mouse oocytes before extrusion of the first polar body, but to allow resumption of meiosis as revealed by germinal vesicle breakdown (GVB) (Schultz and Wassarman, 1977a). The conclusion from this was that GVB occurs independently of protein synthesis.In the present study rat oocytes were treated with puromycin during two successive culture periods. During the first period resumption of meiosis was prevented either by the concomitant presence of dibutyryl-cyclic AMP (dbcAMP) or by incubation of oocytes within their intact preovulatory follicles. Isolated oocytes, with or without cumulus cells, were then transferred to plain medium or medium with puromycin alone for a second culture period.Cumulus-surrounded oocytes matured spontaneously in culture. DbcAMP prevented GVB, if added within 1 h from the start of culture, whereas puromycin did not prevent GVB but blocked polar body formation, thus confirming earlier observations. When oocytes were first cultured for 0.5–4 h in the presence of both puromycin and dbcAMP and then cultured for 4 h with puromycin alone, GVB was prevented in a “time dependent” way: the longer the first culture period the lower the rate of GVB. The inhibition of GVB was, however, fully reversed when no drugs were present during the second culture. The block of GVB produced by puromycin was sustained even when the second culture was continued for 18 h. Cycloheximide, but not puromycin aminonucleoside (PAN), produced the same effects as puromycin. Similar results were obtained when denuded oocytes were used, suggesting that the effects of puromycin were directly on the oocyte and not mediated via the cumulus cells.When follicle-enclosed oocytes were incubated for 3 h with puromycin and the oocytes then isolated and cultured with puromycin, GVB was also inhibited. The rise in oxygen consumption seen in maturing oocytes could be prevented in oocytes maturing in medium containing puromycin. Also this effect of puromycin was reversible, indicating that puromycin did not inflict permanent damage on the oocyte.The interpretation of these data is that resumption of meiosis is dependent on some proteins with high turnover rates existing in the oocyte before the start of meiosis. Polar body formation might also be explained in a similar way.

This study was undertaken in order to analyse the role of cyclic AMP (cAMP) in the stimulatory action of LH on the resumption of oocyte meiosis (oocyte maturation) and follicular glycolysis. Follicular development was induced in immature rats by a single injection of pregnant mares' serum gonadotrophin. Pr-ovulatry follices were extirpated on the day before ovulation and incubated in a chemically defined medium for 4–10 h. The follicle-enclosed oocytes remained in the dictyate stage when incubated in hormone-free medium but in the presence of LH oocyte maturation was induced. When cAMP or butyryl derivatives of cAMP were added to the incubation medium no effect on the oocyte was seen. However, these nucleotides prevented the stimulatory action of LH on the oocytes. The phosphodiesterase inhibitors theophylline and IBMX had similar effects as cAMP on oocyte maturation, causing a reversible blockage of the LH effect. When 8-Br-cAMP and the phosphodiesterase inhibitor ICI 63.197 were tested it was found that both had inherent stimulatory effects on oocyte maturation at certain concentrations. Pre-incubation of the follicles in a high concentration of dibutyryl cAMP followed by transfer to plain medium resulted in oocyte maturation.Follicular lactate production was stimulated by all cyclic nucleotide derivatives tested, except cAMP, and by IBMX and ICI 63.197. There was no reduction by these agents of the stimulatory effect of LH on lactate production.A model to explain the role of cAMP in oocyte maturation is presented.

LH exerts a biphasic effect on rat follicular steroidogenesis: an initial general stimulation of steroidogenesis later followed by an inhibition of androgen and oestrogen synthesis. It is known that the stimulation by LH of steroidogenesis is mediated by cAMP. The present paper examines the role of cAMP in the late inhibitory phase of steroidogenesis.Pre-ovulatory follicles were isolated from PMSG treated immature rats and incubated in modified bicarbonate buffer for different time periods. The accumulation of progesterone (P), androstenedione (A), testosterone (T) and oestradiol-17β (E2) in the medium was measured. Addition of dibutyryl cAMP (dbcAMP, 1 mm) to the medium caused a prolonged increase in P accumulation for 10 h while A and E2accumulation was stimulated only for 4–6 h whereafter the levels were sustained. The pattern was identical to that produced by LH (10 μg/ml) in vitro.In a second type of experiments follicles were pre-incubated for 6 h with or without LH or dbcAMP and then transferred for a second 2 h incubation, in the presence or absence of unlabelled 17-hydroxyprogesterone (17-OHP) or T (1 μg/ml). Follicles pre-incubated in plain medium increased their formation of A and T in the presence of 17-OHP, while follicles exposed to LH or dbcAMP did not. Addition of T caused a similar increase in E2formation in all groups (control, LH and dbcAMP). It is concluded that since dbcAMP mimicked the entire response of follicular steroidogenesis normally elicited by LH, cAMP seems to be the physiologic mediator of LH, both on the early stimulatory and on the late inhibitory phase of pre-ovulatory follicular steroidogenesis.

Abstract. The effects of GnRH and a potent GnRH analogue (D-Ala6-des-gly-NH2-GnRH-ethylamide) on steroidogenesis in isolated preovulatory follicles of PMSG-treated immature rats were examined in short-term incubations and compared to the effect of LH. Steroids were analyzed by RIA. GnRH stimulated the accumulation of pregnenolone (2-fold), progesterone (4-fold), 20α-OH-progesterone (38-fold), androstenedione (4-fold), testosterone (3-fold), and oestradiol-17β (2-fold) during a 6 h incubation. The time-course of stimulation was the same for each of the steroids analyzed, with a significant effect at 4 and 6 h, but not at 2 h of incubation. Dose-response curves were similar for each steroid, and the GnRH analogue and GnRH gave parallel curves with minimal effective concentrations being 1 and 10 ng/ml, respectively. The stimulatory effect of LH was more pronounced and rapid (2 h) than that of GnRH. During prolonged incubation (6–8 h) with GnRH or LH there was evidence for an inhibition of testosterone production, probably due to suppression of the C21-side chain cleavage enzyme. Thus, the qualitative response to GnRH on follicular steroidogenesis resembled that of LH in some but not all respects. The differences in time-course and maximal steroid secretion between GnRH and LH are compatible with different mechanisms of action in the follicle.

Abstract. The effect of a GnRH analogue ((D-Ala6, des-Gly10-NH2)-GnRH-ethylamide,GnRHa) on granulosa and cumulus cell glycolysis in presence or absence of FSH was studied. Cumulus complexes and granulosa cells from PMSG-treated rats were cultured in Eagle's minimal essential medium (MEM) for a period of 72 h. Media were changed at 24 and 48 h and lactate content was assayed by fluorimetry.GnRHa alone stimulated lactate production in granulosa cells. GnRH combined with FSH increased lactate production in granulosa cells during the 0–24 h period and decreased it during the 48–72 h period as compared to FSH alone. GnRHa did not stimulate lactate production in cumulus complexes during 72 h culture in MEM, while FSH did. In a less complex culture medium, BMOC, GnRHa caused a small increase in lactate production and slightly enhanced the FSH effect.In conclusion, GnRHa has a direct stimulatory effect on granulosa cell glycolysis. GnRHa also modulates the FSH stimulation of granulosa cells biphasically, i.e. early enhancement (0–24 h) and late inhibition (48–72 h). GnRHa has no consistent direct effects on cumulus cell glycolysis.

The adenylate cyclase activator forskolin was used to study the role of cAMP for oocyte meiosis and follicular steroid secretion. Follicular and cumulus cAMP production was stimulated dose-dependently by forskolin, as was the follicular secretion of progesterone, testosterone and estradiol. Forskolin induced meiosis in follicle-enclosed oocytes with a maximal effect at 1 μM, with lower and higher concentrations being less effective. The spontaneous resumption of meiosis in isolated cumulus-enclosed oocytes was dose-dependently retarded by forskolin. Meiosis of cumulus-free oocytes was also retarded but only slightly. These data support the earlier hypothesis that a limited increase in follicular cAMP levels triggers meiosis, whereas sustained levels of cAMP in the oocyte itself prevent meiosis.

The secretion of steroids and the release of cAMP in response to repeated luteinizing hormone (LH) stimulation were examined during superfusion of isolated preovulatory rat follicles. A high dose of ovine LH (1 µg/ml for 20 min) caused a prolonged increase in the secretion of progesterone (P) and 20α-dihydroprogesterone (20α-OHP) and a transient increase in the secretion of testosterone (T) and estradiol-17β(E2), and was accompanied by a peak of cAMP release. A single pulse of LH at a low dose level (10 mg/ml for 20 min) gave a limited increase in T secretion, but no clear change in P, 20α-OHP and E2secretion or cAMP release. When the follicles were challenged with a second pulse of LH (at 1 µg/ml), the response varied according to the dose of LH delivered in the preceding pulse. Following exposure to the high dose of LH, the follicles were partially refractory to the second LH challenge in terms of cAMP and P and the secretion of T and E2remained low. The low dose of LH, however, had a conditioning effect on the follicles since the response to the second LH challenge was amplified in terms of P, 20α-OHP and cAMP. In this case a secondary increase in T and E2secretion was found.The differential response to varying doses of LH are likely to reflect the physiological control of steroidogenesis during final follicular maturation.