Flaws J A, DeSanti A, Tilly K I, Javid R O, Kugu K, Johnson A L, Hirshfield A N, Tilly J L
Department of Population Dynamics, Johns Hopkins University, Baltimore, Maryland 21205-2179, USA.
Endocrinology. 1995 Oct;136(10):4351-9. doi: 10.1210/endo.136.10.7664654.
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 avian granulosa cells incubated in vitro were used as a comparative model system to determine 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 microM), 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 micrograms/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 microM) 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 avian granulosa cells incubated for 6 h under serum-free conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
含血管活性肠肽（VIP）的神经纤维存在于卵巢卵泡发育的各个阶段，据报道，主要通过cAMP途径发挥作用的VIP可调节颗粒细胞功能的多个方面。在此，我们研究了VIP对从经促性腺激素预处理的未成熟大鼠卵巢分离并在无血清条件下体外培养的窦状卵泡凋亡的影响及其潜在作用机制。此外，将VIP对体外培养的分离禽颗粒细胞凋亡的影响用作比较模型系统，以确定VIP调节卵巢细胞凋亡的能力在进化过程中是否得以保留。从培养的大鼠窦状卵泡中提取的基因组DNA表现出广泛的核小体间DNA切割水平，这是细胞凋亡导致细胞死亡的特征。用VIP（1 - 1000 nM）处理卵泡导致凋亡DNA降解程度呈剂量依赖性降低，100 nM VIP时达到最大效应。提供腺苷酸环化酶激活剂福斯可林（10 microM）模拟了VIP对凋亡的抑制作用，并同时增加了卵泡内cAMP积累，表明cAMP途径在介导VIP对卵泡细胞存活的即时作用中发挥作用。此外，用胰岛素样生长因子结合蛋白3（3微克/毫升）处理大鼠窦状卵泡部分拮抗了VIP（100 nM）抑制凋亡的能力，提示内源性胰岛素样生长因子I参与介导VIP在培养的大鼠窦状卵泡中的下游作用。为进一步证实VIP和cAMP途径的激活可防止闭锁，将在无或有VIP（100 nM）或福斯可林（10 microM）存在的情况下培养24小时的单个大鼠窦状卵泡固定、包埋并切片进行形态学分析。从经马绒毛膜促性腺激素预处理的大鼠卵巢分离后立即固定的卵泡被分类为形态学上健康，这与该组卵泡中不存在凋亡的生化证据（如寡核小体）一致。在没有促性腺激素支持的情况下培养24小时的卵泡显示出广泛的颗粒细胞固缩和紊乱，这是闭锁中期卵泡的特征。加入VIP或福斯可林可维持培养卵泡的形态学健康状态，使其类似于从经马绒毛膜促性腺激素预处理的大鼠卵巢分离后立即固定的健康卵泡。最后，在无血清条件下培养6小时的禽颗粒细胞中也检测到广泛的核小体间DNA切割水平。（摘要截断于400字）
