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 presen t in ovarian follicles at all stages of development, and VIP, acting p rimarily via the cAMP pathway, has been reported to modulate many aspe cts of granulosa cell function. Herein we examined the effects of VIP and its potential mechanisms of action on apoptosis in antral follicle s isolated from ovaries of gonadotropin-primed immature rats and incub ated in vitro under serum-free conditions. Additionally, the effects o f VIP on apoptosis in isolated avian granulosa cells incubated in vitr o were used as a comparative model system to determine whether the abi lity of VIP to modulate apoptosis in the ovary has been conserved thro ugh evolution. Genomic DNA extracted from incubated rat antral follicl es exhibited extensive levels of internucleosomal DNA cleavage charact eristic of cell death via apoptosis. Treatment of follicles with VIP ( 1-1000 nM) caused a dose-dependent reduction in the extent of apoptoti c DNA breakdown, with a maximal effect achieved with 100 nM VIP. Provi sion of the adenylyl cyclase activator, forskolin (10 mu M), mimicked the inhibitory effect of VIP on apoptosis and concomitantly increased intrafollicular cAMP accumulation, suggesting a role for the cAMP path way in mediating the immediate actions of VIP on follicular cell survi val, Moreover, treatment of rat antral follicles with insulin-like gro wth factor-binding protein 3 (3 mu g/ml) partially antagonized the abi lity of VIP (100 nM) to suppress apoptosis, suggesting involvement of endogenous insulin-like growth factor I in mediating the downstream ac tions of VIP in incubated rat antral follicles. To further confirm tha t 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 mu M) were fixed, embedded, and section ed for morphological analysis. Follicles fixed immediately after isola tion from equine CG-primed rat ovaries were classified as morphologica lly healthy, consistent with the absence of biochemical evidence for a poptosis (e.g. oligonucleosomes) in this pool of follicles. Follicles incubated for 24 h in the absence of tropic support displayed extensiv e granulosa cell pyknosis and disorganization characteristic of follic les at a moderate stage of atresia. Inclusion of VIP or forskolin main tained the morphological health status of incubated follicles at that resembling healthy follicles fixed immediately after isolation from ov aries of equine CG-primed rats. Lastly, extensive levels of internucle osomal DNA cleavage were also detected in avian granulosa cells incuba ted for 6 h under serum-free conditions. Moreover, the extent of apopt otic DNA breakdown was significantly reduced by treatment with VIP (10 00 nM) or the membrane-permeable cAMP analog, 8-bromo-cAMP (1 mM) sugg esting that the role of VIP in promoting follicular cell survival has been conserved through evolution. From these data, we propose that int rafollicular 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 wh ich VIP suppresses apoptosis in granulosa cells, and the antiatretogen ic actions of VIP in the rat follicle may also be mediated at least in part via increased intrafollicular levels or the bioactivity of insul in-like growth factor I.