Vasoactive intestinal peptide-mediated suppression of apoptosis in the ovary: Potential mechanisms of action and evidence of a conserved antiatretogenic role through evolution

Vasoactive intestinal peptide (VIP)-containing nerve fibers are present in ovarian follicles at all stages of development, and VIP, acting primarily via the cAMP pathway, has been reported to modulate many aspects of granulosa cell function. Herein we examined the effects of VIP and its potential mechanisms of action on apoptosis in antral follicles isolated from ovaries of gonadotropin-primed immature rats and incubated in vitro under serum-free conditions. Additionally, the effects of VIP on apoptosis in isolated arian granulosa cells incubated in vitro were used as a comparative model system to determined whether the ability of VIP to modulate apoptosis in the ovary has been conserved through evolution. Genomic DNA extracted from incubated rat antral follicles exhibited extensive levels of internucleosomal DNA cleavage characteristic of cell death via apoptosis. Treatment of follicles with VIP (1-1000 nM) caused a dose-dependent reduction in the extent of apoptotic DNA breakdown, with a maximal effect achieved with 100 nM VIP. Provision of the adenylyl cyclase activator, forskolin (10 μM), mimicked the inhibitory effect of VIP on apoptosis and concomitantly increased intrafollicular cAMP accumulation, suggesting a role for the cAMP pathway in mediating the immediate actions of VIP on follicular cell survival. Moreover, treatment of rat antral follicles with insulin-like growth factor-binding protein 3 (3 μg/ml) partially antagonized the ability of VIP (100 nM) to suppress apoptosis, suggesting involvement of endogenous insulin-like growth factor I in mediating the downstream actions of VIP in incubated rat antral follicles. To further confirm that VIP and activation of the cAMP pathway prevented atresia, individual rat antral follicles incubated for 24 h in the absence or presence of VIP (100 nM) or forskolin (10 μM) were fixed, embedded, and sectioned for morphological analysis. Follicles fixed immediately after isolation from equine CG-primed rat ovaries were classified as morphologically healthy, consistent with the absence of biochemical evidence for apoptosis (e.g. oligonucleosomes) in this pool of follicles. Follicles incubated for 24 h in the absence of tropic support displayed extensive granulosa cell pyknosis and disorganization characteristic of follicles at a moderate stage of atresia. Inclusion of VIP or forskolin maintained the morphological health status of incubated follicles at that resembling healthy follicles fixed immediately after isolation from ovaries of equine CG-primed rats. Lastly, extensive levels of internucleosomal DNA cleavage were also detected in arian granulosa cells incubated for 6 h under serum-free conditions. Moreover, the extent of apoptotic DNA breakdown was significantly reduced by treatment with VIP (1000 nM) or the membrane-permeable cAMp analog, 8-bromo-cAMP (1 mM), suggesting that the role of VIP in promoting follicular cell survival has been conserved through evolution. From these data, we propose that intrafollicular neuropeptides such as VIP can act to prevent the atresia of developing follicles in the absence of gonadotropins. Activation of the cAMP-protein kinase A pathway may function as one mechanism by which VIP suppresses apoptosis in granulosa cells, and the antiatretogenic actions of VIP in the rat follicle may also be mediated at least in part via increased intrafollicular levels or the bioactivity of insulin-like growth factor I.