- 學位論文
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
Study of androgen on the connexin 43 expression and proliferation of human granulosa cells
摘要
卵巢顆粒細胞的增殖與分化受到促性腺激素連續且合併作用的調節，並影響濾泡的形成與凋亡。在不同種類的動物中，雄性激素對於顆粒細胞的增殖有不同的影響，而對於人類卵巢顆粒細胞的作用，則較少有相關報告。另外，顆粒細胞的間隙結合(gap junction), 經由細胞與細胞之間離子與小分子的傳遞, 可以調節很多生理的過程，包括濾泡形成和卵子形成。 Connexins(Cx)是組成間隙結合的膜蛋白質, 目前在人類至少有21種Cx被發現, 其中 Cx43存在於每個階段的濾泡形成裡，當濾泡停止發育時，Cx43表現程度就會減少。在多囊性卵巢症候群(PCOS)的病人裡, 雖然已經確定生產過剩的雄性激素會導致不排卵，但是真正的機制還不是很清楚。因此，我們假設高濃度的雄性激素會改變人類卵巢顆粒細胞的增殖與Cx 43 的表現, 進而影響濾泡形成。 研究中, 人類卵巢顆粒細胞株(HO23)以不同濃度 (0，1，10，100 ng/ml) 的dihydrotestosterone (DHT) (一種5αreduced 睪丸酮的代謝物)一起培養。在顆粒細胞增殖的研究，我們使用西方點墨法定量增生細胞核抗原(PCNA）及 p27kip1(細胞週期的抑制蛋白)之蛋白質，以流式細胞儀來分析顆粒細胞的凋亡。另外，以西方點墨法測定 Cx 43的蛋白質, 以定量逆轉錄核酸聚合酵素連鎖反應定量Cx 43 mRNA，並且利用刮痕充填/染劑轉移(SLTD)技術來分析顆粒細胞間隙結合間的交流(GJIC)。 為了明瞭雄性激素影響Cx 43的表現, 是否經由雄性激素接受器, 我們加入flutamide (一種雄性激素接受器拮抗劑) 來觀察其變化。 結果發現, 人類卵巢顆粒細胞的增殖不受DHT的影響。在經過不同濃度的DHT培養後, Cx 43蛋白質表現量隨著DHT濃度增加而呈現遞減的現象, 並在培養24小時後, Cx 43蛋白質量達到有意義的減少。定量RT-PCR的分析顯示, 經由高濃度DHT培養後的顆粒細胞, Cx 43 mRNA的表現量與對照組比較會呈現有意義的減少。此外, 這種高濃度DHT抑制Cx 43 表現的現象, 會因為 flutamide 的加入而逆轉回來。至於SLDT的研究也顯示經由高濃度DHT處理過的顆粒細胞, 與對照組比較會呈現較少的GJIC。 過去的報告顯示, 雄性激素和間隙結合在濾泡形成過程中扮演著重要的角色，而本篇研究則是在人類卵巢顆粒細胞, 有關於雄性激素調控Cx 43 表現的第一篇報告。高濃度的雄性激素會透過雄性激素接受器, 來降低人類顆粒細胞間Cx 43 的表現，並減少 GJIC。而上述的結果會引起濾泡形成障礙, 導致濾泡生長停頓, 這可能可以解釋為什麼在PCOS婦女裡, 因為高濃度的雄性激素導致不排卵的部分原因。
並列摘要
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.