Oocyte loss, either directly through attrition (germ cell death) or indirectly through follicular atresia (somatic or granulosa cell death), is a fundamental event associated with defining the time of normal or premature reproductive senescence in females. Although apoptosis has been reported to function as the underlying mechanism responsible for death of both germ cells and somatic cells in the ovary, the final molecular steps which commit ovarian cells to death have not been fully elucidated. To examine if death repressor activity of the bcl-2 gene product is important for germ cell survival, we conducted studies using a Bcl-2 loss-of-function (bcl-2 -/-) transgenic mouse model. Histological analyses revealed that ovaries collected from bcl-2 -/- mice possessed numerous aberrantly formed primordial follicle-like structures containing a single layer of granulosa cells without an oocyte. Additionally, the total number of primordial follicles present which contained a healthy oocyte was markedly reduced in bcl-2 -/- mice as compared to heterozygote (bcl-2 -/+) or wild-type (bcl-2 +/+) mice, suggesting that expression of the bcl-2 death repressor gene is critical for endowment of a normal complement of germ cells and primordial follicles in the mammalian ovary.

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Previous studies have indicated that developing avian granulosa cells collected from follicles 2-3 wk prior to ovulation (e.g. 6-8-mm in diameter) are steroidogenically incompetent, apparently due to a lack of functional cytochrome P450 side-chain cleavage (P450scc) enzyme activity. The present studies were designed to test this hypothesis by determining the absence or presence of P450scc messenger RNA, immunoreactive protein, and enzyme activity in granulosa tissue of developing hen ovarian follicles. Additionally, the interactive roles of FSH, the adenylyl cyclase-cAMP system, and the protein kinase C pathway in granulosa cell differentiation were investigated. Granulosa cells collected from developing, 6-8-mm follicles were found to contain extremely low but detectable levels of a single, 2.0-kb P450scc mRNA transcript, as well as immunoreactive P450scc protein (53 kDa). However, this protein was apparently incapable of converting 25-hydroxycholesterol to pregnenolone in a cell-free system. Preincubation of granulosa cells with ovine FSH or forskolin for 24 h rendered the cells capable of converting cholesterol precursor to pregnenolone during a subsequent 3-h incubation. Inclusion of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), in the preincubation medium blocked the stimulatory actions of FSH and forskolin on the induction of P450scc activity; however, PMA-preincubation did not alter the ability of granulosa cells to convert exogenous pregnenolone to progesterone compared to vehicle-pretreated cells. These data suggest that steroidogenic incompetency in differentiating avian granulosa cells is primarily due to a lack of active P450scc enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies were conducted to evaluate the effects of vasoactive intestinal peptide (VIP) on steroidogenesis and plasminogen-activator (PA) activity in isolated granulosa cells of the largest preovulatory (F1) follicle of the hen. Vasoactive intestinal peptide, but not avian pancreatic polypeptide, the chicken VIP fragment (16-28) or the VIP congener, PHM-27, induced a dose-related increase in progesterone and androgen secretion, with an apparent median effective dose (ED50) of 5.9 X 10(-7) and 5.7 X 10(-7) M, respectively. The effects of VIP were, at least in part, mediated by the adenylyl cyclase system in that cotreatment of cells with VIP and the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), potentiated the steroidogenic effects. However, the time course of action for VIP on steroidogenesis was considerably slower than that for the gonadotropin, luteinizing hormone (LH), and this was attributed to a slower induction of cyclic adenosine 3',5'-monophosphate (cAMP) formation within granulosa cells. Finally, VIP was found to be a potent inhibitor of PA activity, and this inhibition was potentiated by coincubation of VIP with IBMX. We suggest that, in the hen, VIP has a direct and specific action on both steroidogenesis and PA activity, and that these actions are mediated, at least in part, by the adenylyl cyclase system. The comparatively slow induction of cAMP formation by VIP suggests that this peptide is involved in the control of cell differentiation and development rather than the ovulatory process.