Contribution of progesterone, follicle stimulating hormone and glucocorticoids in survival of serum-free cultured granulosa cell explants

Abstract

To investigate the role of progesterone (P4) as a survival factor in quail granulosa cell explants, P4 content was determined under various conditions and correlated with apoptotic indexes (AIs) evaluated by 2',6'-diamidino-2-phenylindole (DAPI)-staining. Analysis of serum-free cultures from 24 to 96 h shows decreased P4 levels in the medium paralleled by increasing AI. Inhibiting apoptosis by gonadotropic support (FSH, 100 ng/ml) stimulates a 3-fold increase of the P4 level in the medium (83.49+/-8.69 vs 26.31+/-1.61 ng/ml in serum-free controls) together with a significant decrease in AI from 8.81+/-1.06% in serum-free controls to 3.50+/-0.72%. Substantial evidence for P4 as an autocrine/paracrine survival factor can be inferred from experiments with aminoglutethimide (AG, 1 mM) and RU486 (20 microM). Blocking P4 synthesis by AG causes a 2-fold increase in apoptosis from 6.08+/-0.67% in serum-free controls to 12.53+/-1.60%. Blocking P4 receptors by RU486 causes a similar increase in AI (3.02+/-0.98% in serum-free controls to 17.07+/-3.20%) and about a 50% decrease in P4. The effect of RU486 could be attenuated by exogenous P4 but not by dexamethasone indicating selective binding of P4 to the progesterone receptor. Dexamethasone treatment promotes survival without affecting P4 levels. In further support of an autocrine/paracrine action for P4 in the granulosa cells, both the A and B form of the avian P4 receptor (PR) are identified in vivo and in vitro by Western blotting. Exogenous administration of P4 only affects survival when endogenous P4 synthesis is blocked or after 48 h of serum-free culture when endogenous P4 production is very low. Because FSH also affects survival when its stimulatory effect on P4 synthesis is blocked by AG (AI decrease from 6.08+/-0.67% in serum-free controls to 1.64+/-0.71% in FSH+AG treated) it is proposed that (1) P4 is an autocrine/paracrine survival factor in the preovulatory granulosa and (2) FSH mediates both P4-dependent and P4-independent survival pathways.