雄性激素於人類卵巢顆粒細胞增殖及接合素43(Connexin 43)表現之研究

Abstract

Proliferation and differentiation of granulosa cells are involved in the folliculogenesis. Gap junctions of granulosa cells are thought to modulate numerous physiological processes, including folliculogenesis and oogenesis. Connexins (Cx) are membrane-spanning proteins that assemble to form the intercellular channels of gap junctions. At least 21 human connexins have been identified. Among them, Cx43 is identified in every stage of folliculogenesis and the expression level is decreased as the follicle goes to atresia. Although it is well established that overproduction of androgens in polycystic ovarian syndrome (PCOS) results in anovulation, the underlying mechanism is less well understood. We hypothesize that high level of androgen may influence Cx 43 expression and proliferation of human granulosa cells. Human granulosa cell line (HO-23) was treated by various dosages (0, 1, 10, or 100 ng/ml) of dihydrotestosterone (DHT), a 5αreduced metabolite of testosterone. Western blot for PCNA, p27kip1, Cx 43 protein and quantitative RT-PCR for Cx 43 mRNA were performed. Gap junctional intercellular communication (GJIC) was analyzed by using the scrape-loading dye transfer (SLDT) technique. Androgen receptor (AR) antagonist, flutamide , was used to test the specificity of the observed androgen responses. We found that human granulosa cell proliferation was not influenced by DHT. Western blot analysis of DHT-treated granulosa cell protein samples showed a decreased amount of Cx 43 protein in dose-dependent manner. Cx43 mRNA levels determined by quantitative RT-PCR analysis in HO-23 cells grown in DHT medium indicated significant downregulation compared with control group. Flutamide significantly blocked the inhibitory effects of DHT on Cx 43 protein and mRNA expression. DHT-treated cells demonstrated a decreased enhancement of GJIC as assessed by SLDT technique. In conclusion, while both androgen and gap junctions are reported to have important roles in follicular development, this is the first report describing a potential regulation of Cx 43 expression by DHT in cultured human granulosa cells. High level androgen reduces Cx 43 expression and impairs GJIC between human granulosa cells through androgen receptors .It may induce the follicles atresia with the impairment of the folliculogenesis and explain part of the underlying mechanism of anovulation in PCOS women.