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The effect of an agonistic gonadotropin releasing hormone (GnRH) -analog (D -Ala’ ,desGly’#{176} -NH3 -GnRH -ethylamide, GnRHa) on granulosa cell steroidogenesis in the presence or absence of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) was studied. Granulosa cells, isolated from preovulatory follicles of pregnant mare’s serum gonadotropin (PMSG) -treated immature rats or from the less mature follicles of untreated immature rats, were cultured for a period of 72 h with daily changes of medium, and progesterone and its metabolite, 20a -dihydro progesterone (20o-OHP), were assayed in the medium. In granulosa cells from preovulatory follicles, LH and FSH caused a much greater stimulation of steroidogenesis than did GnRHa. There appeared to be no interaction between GnRHa and FSH during the first 10 h, but at 24 h and later the presence of GnRHa clearly inhibited the steroidogenic response to LH and FSH. Steroidogenesis in granulosa cells from immature rats was considerably lower and the effects of GnRHa and FSH alone less pronounced. In these cells, FSH #{149}stimulated progesterone secretion was inhibited by GnRHa only at 72 h. In contrast, 20o-OHP secretion in the same cultures was potentiated by the combined presence of FSH and GnRHa. In conclusion, it seems as though the effects of GnRHa on granulosa cell steroidogenesis varies with exposure time, the initial response being stimulatory and the later inhibitory. Further more, the response is also to some extent determined by the maturational stage of the granulosa cells.

Resumption of meiosis (oocyte maturation) is normally triggered by the preovulatory LH surge, and the first meiotic division is completed at the time of follicle rupture. The mature follicle has receptors for LH both in the theca and granulosa cells but no specific LH receptors have been demonstrated on the oocyte itself. Therefore, the triggering effect of LH on meiosis is likely to be mediated indirectly by an effect on the somatic cell types, probably the granulosa cells. We have studied the mechanism of LH action on meiosis with particular emphasis on the relation to the cumulus cells. In this review we will summarize some of our previous findings as well as report on some new results. We focus on studies where oocyte and cumulus maturation have been studied in relation to metabolic changes (1) of the whole isolated follicle and (2) of the isolated oocyte-cumulus complex.

Rabbit antisera against native human insulin-like growth factor I (IGF-I; somatomedin C) or a synthetic tetradeca peptide, representing the carboxyterminal amino acids 57–70 of human IGF-I, were used to map immunohistochemically the distribution of IGF-I immunoreactive material in adult rats. Both antisera were specific for IGF-I, as characterized by immunoabsorption, immunoblotting and radioimmunoassay. There was no cross-reactivity to IGF-II, relaxin or pro-insulin; substances having a high degree of structural homology with IGF-I. High IGF-I immunoreactivity was observed in spermatocytes of the testis; in oocytes, granulosa and theca interna cells of the ovary during early stages of follicle development; in some lymphocytes and in reticular cells of lymphoid and hematopoetic organs; in salivary gland duct cells; in the adrenal medulla, the parathyroid gland and the Langerhans' islets. Chondrocytes in the epiphyseal and rib growth plates and at articular surfaces showed strong IGF-I immunoreactivity. Brown but not white fat cells were stained. Nerve cells in the peripheral and autonomic nervous system showed faint to intense IGF-I immunoreactivity. In contrast, neurons and neuroglial cells in the central nervous system were generally negative; motor neurons being an exception. Erythropoeitic, trombocytopoeitic and myeloic cells in the bone marrow showed IGF-I immunoreactivity, but only at defined developmental stages. Hepatocytes showed faint IGF-I immunoreactivity, but became more intensely stained after pretreatment with colchicine. The present results suggest that IGF-I is synthetized by cells in several tissues and organs in the adult rat. There was an apparent association between the localization of IGF-I and cell differentiation. Certain cells involved in secretory processes also displayed high IGF-I immunoreactivity. The wide distribution of IGF-I indicates that the circulating pool of IGF-I has multiple origins.

Effects of adenosine analogues on adenylate cyclase activity in preovulatory rat ovarian membranes were studied. Adenosine analogues stimulated adenylate cyclase activity in the following rank order of potency: NECA (5'-(N-ethyl)carboxamidoadenosine) greater than 2-chloroadenosine greater than N6-(R-phenylisopropyl)-adenosine greater than N6-(S-phenylisopropyl)adenosine. The apparent EC50 for NECA was 0.28 microM. The adenosine receptor antagonist 8-phenyltheophylline (10 microM) displaced the dose-response curve for NECA to the right, increasing the EC50 for NECA about one order of magnitude. NECA also additively increased maximally FSH-stimulated adenylate cyclase activity. These results suggest that adenosine stimulates adenylate cyclase in rat ovarian membranes via adenosine receptors of the A2 type.

Several studies have shown differences in gonadotrophin receptor content between different granulosa cell (gc) populations within the ovarian follicle, but little is known about the gonadotrophin sensitivity in gc from different parts of the follicle. This study is an investigation of progestin synthesis and responsiveness to highly purified human and partly purified rat gonadotrophins. It involves short-term culture of rat cumulus and mural gc obtained from preovulatory follicles of PMSG-treated immature rats. The responsiveness to FSH in terms of progestin accumulation was similar in the two gc populations, whereas the responsiveness to LH was greater in the mural gc than in the cumulus. The morphological response in the cumulus cells (mucification) correlated with the steroidogenic response. The pattern of progestin synthesis differed between the two gc populations. In the cumulus gc progesterone was dominant, whereas 20α-dihydroprogesterone was the main progestin secreted in the mural gc. The difference in responsiveness to gonadotrophins correlates well with the reported differences in receptor content in the two gc populations.

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.