Effect of in Vivo Gonadotropin Treatment on the Ability of Progesterone, Estrogen, and Cyclic Adenosine 5′-Monophosphate to Inhibit Insulin-Dependent Granulosa Cell Mitosis in Vitro1

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Abstract
The ability of progesterone (P4), estradiol-17 beta (E2), and 8-bromo (br)-cAMP to inhibit small granulosa cells (GCs) from undergoing insulin-dependent mitosis was examined. Small GCs were isolated from immature and eCG-primed rats and separated by Percoll fractionation. Small GCs were cultured for 24 h with various combinations of insulin, steroids, steroid receptor antagonists, and 8-br-cAMP. Before and after culture, the number of GCs was counted. Small GC proliferation was expressed as a percentage increase over the initial value P4 inhibited insulin-dependent mitosis of small GCs isolated from both immature and eCG-primed rats. The effects of P4 were dose-dependent, steroid-specific, and reversed by the progesterone antagonist RU486. E2 inhibited insulin-dependent mitosis of small GCs isolated from immature but not eCG-primed rats. The action of E2 was dose-dependent and inhibited by the estrogen antagonist tamoxifen. Additional studies were conducted in which small GCs from immature rats were cultured with insulin in the presence of both P4 and E2 and their respective antagonist. Both antagonists were required for insulin to induce GC mitosis in the presence of P4 and E2. Further, the ability of P4 to suppress insulin-dependent mitosis was reduced if it was not present during the first 6 h of culture. In contrast, E2 could be added up to 12 h after insulin exposure and still completely prevent GC mitosis. 8-br-cAMP also prevented insulin-dependent GC proliferation. The actions of 8-br-cAMP could not be reversed by aminoglutethimide or RU486.(ABSTRACT TRUNCATED AT 250 WORDS)

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