体内因子与牦牛卵泡细胞及黄体细胞凋亡的研究

The vast majority of follicles undergo a degenerative process in reproductive life. Apoptotic cell death is the fundamental mechanism in response to follicular atresia. Moreover, the apoptosis of granulose cells (GCs) is implicated in directly mediating atretic process. Multiple studies have confirmed that the apoptotic process of GCs is not only related to apoptotic genes, but triggered by cytokines related to reproduction and Caspases family. Cell adhesion and extra cellular matrix in follicles also play an important intraovarian role. Importantly, the reproductive hormones have strongly related to control ovarian GCs apoptosis. Immense evidence implied that hormones as modulators may integrate into a network modulating apoptosis of granulose cells. However, the frame of the network has not been well outlined yet. Therefore, the present study is aimed at acquiring new evidence for the network by defining the effects of melatonin（Mel），gonadotropin-releasing hormone（GnRH），follicular-stimulating hormone（FSH），leiteininzing hormone（LH），estrodiol-17β（E2） and progesterone（P4）on apoptosis，proliferation and differentiation in vitro in GCs obtained from large preovulatory follicles of yaks in nature estrus, using flow cytometry and HE histochemical assay to investigate apoptotic incidence and proliferation index of GCs, and radioimmunoassay to evaluate the concentrations of progesterone and estrogen in culture media, and electron and light microscopes methods to observe and confirm cellar structure. The results show that the granulose cells can produce progesterone and estrogen in vitro. Trends in proliferation of GCs treated by FSH, Mel, P4 and LH are evident. FSH, Mel, P4 and E2 can lead to the suppression of an intrinsic apoptosis of GCs; FSH has stronger role in the suppression than other three hormones, and the effects can not be affected by GnRH and Mel. The effect of Mel on apoptosis of GCs could be easily affected by the co-treatments with GnRH, FSH, LH, P4 and E2, but no addition effects is found with each other. GnRH can significantly induce the apoptosis of cultured GCs in vitro, while the apoptosis in response to GnRH can be completely inverted by FSH co-treatment and partially decreased by Mel, E2 and P4 co-treatments. FSH, Mel, LH and E2 can increase the production of progesterone of cultured GCs in vitro respectively, and there is an addition effect of Mel and E2 on progesterone production. GnRH can markedly suppress progesterone secretion of cultured GCs with or without FSH and LH co-treatments. E2 can invert the suppression of GnRH completely, also has the additive action on secreting progesterone with GnRH co-treatment. Mel partially releases the suppression action of GnRH. FSH, LH, Mel, GnRH and P4 can significantly promote GCs secreting estrogen in vitro. Mel can inhibit the action of P4 on GCs secreting estrogen. Co-treatments of GnRH and FSH or LH can decrease the activity of estrogen secretion.

本项目拟采用先进的手段和方法，在研究牦牛卵泡细胞和黄体细胞凋亡的形态学变化和生化特征的基础上，探讨主要体内因子对牦牛卵泡细胞和黄体细胞凋亡的作用和可能的机制，以揭示牦牛所特有的某些生殖生理特点。其结果不仅对牦牛，而且对其它动物和人类助孕技术水平的提高以及防止流产和研制抗早早孕药物都具有重要意义。