2013-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645377摘要：睪固酮維繫男性生殖功能，睪固酮是由細精管外間質細胞之Leydig cell製造，但是很特別的是睪丸內之睪固酮濃度高於血液濃度25至125倍，為何需要這麼高的睪丸內睪固酮濃度才能維持精子製造呢？原因還不清楚。由已知的研究，知道精子的製造需要睪固酮，雄性素受體及Sertoli cell三者存在。啟動雄性素受體所需要的睪固酮遠低於睪丸內之濃度。因此相對高濃度的睪固酮不是只經由與雄性素受體結合啟動下游DNA進行傳統轉錄及轉譯，應該還存在，需要相對較高濃度睪固酮的其他機轉以參與及啟動精子成熟。若能知道這些與精子製造有關之睪固酮訊息途徑，將可能是未來治療精子品質不良的有效方法。現在已發現睪固酮可以經由迅速活化kinase signaling pathway 改變基因表現稱為非典型途徑。現在已知的兩種Sertoli cell的睪固酮非典型途徑，一是與鈣離子有關，一是與磷酸化酶活化MAP kinase cascade 及CREB轉錄因子磷酸化有關。但以上的機轉，並不需要高濃度的睪固酮，還是不能解釋相對高濃度的睪丸內睪固酮之作用目標。過去的研究模式是經由剔除雄性素受體的模式或經由抑制腦下垂體的激素，減低睪固酮的分泌。前者只能證實雄性素受體參與控制精子生成，後者會影響FSH 及LH之回饋抑制軸，不能建立單一控制因素之低睪固酮模式。我們將建立一個可以降低睪丸內睪固酮濃度的動物模式，以EDS（ethane dimethanesulphonate）選擇性破壞Leydig cell，使睪丸內睪固酮降低。並植入睪固酮膠囊以消除LH負回饋機制及維持血清內正常睪固酮濃度。測試高低差異的睪固酮濃度，將引發何種睪丸基因表現的差異。由這些基因差異，以探討哪些訊息傳遞途徑是與高濃度睪固酮有關。此為二年研究計畫，本研究的目標分別為第一年目標1：建立低ITT濃度之動物模式目標2：建立調整ITT濃度及測量ITT之方法：經由調整EDS與睪固酮膠囊劑量，建立與睪丸內睪固酮濃度之關係。測試何種睪丸內睪固酮之濃度，可以啟動已知之睪固酮非典型訊息途徑。第二年目標3 ：微基因晶片分析不同之ITT濃度，其睪固酮相關基因表現之差異，尋找可能之訊息途徑。目標4：確認由目標3所獲得的非典型睪固酮訊息途徑。預期本研究將可尋找到控制精子製造的獨特訊息傳遞途徑，可以進一步研究該途徑作為治療不孕或避孕的標的基因，深具臨床實用價值。<br> Abstract: Testosterone is essential for spermatogenesis and male fertility and acts in the testis through the androgen receptor (AR) in the Sertoli cell (SC) to support male germ cell development and survival as well as the release of mature spermatozoa.Testosterone levels in the adult testis remain at a relatively stable and high level. The levels of intratesticular testosterone (ITT) are about 10-fold higher than in the serum of rats and 25- to 125-fold higher than in serum of men. AR is saturated and biological responses are maximal at intracellular free steroid concentrations of 1 nM or lower; however, Sertoli cells require much higher local levels of androgen (>70 nM) to fully support spermatogenesis.Why local testosterone is high for spermatogenesis? The physiological necessity for higher levels of testosterone in the testis is not yet known. Two observations suggest that testosterone acts in SC through pathways in addition to classical mechanism of androgen to regulate spermatogenesis. First, few genes have been characterized that are known to be induced with testosterone through the classical mechanism of AR binding to specific promoter elements. Second, [Ca2+]i levels are elevated in SC within seconds of androgen stimulation and thus cannot be dependent on AR-DNA interactions and initiation of gene expression.The classical mechanism of testosterone action in which testosterone activates gene transcription through AR and AREs does not appear to fully explain testosterone regulation of spermatogenesis. Two non-classical testosterone signalling pathways in Sertoli cells had been postulated. First, testosterone-mediated activation of phospholipase C and calcium influx into SC is described. Second is kinases signaling pathway that testosterone activate of Src, EGFR and ERK kinases as well as the CREB transcription factor with CREB-mediated transcription.In this 2-years study, an animal model will be setup by using a cytotoxic agent-EDS to selective destruct the LC and implants of testosterone will be administrated to alleviate the negative feedback of gonadotropic hormone. Our Aims are:1st yearAim 1：Setup an animal model of low ITT levelAim 2：Measurement and modulate the ITT level for classical and non-classical androgen signaling pathways2nd yearAim 3：Microarray analysis of androgen-related gene expression by different ITT level.Aim 4：Confirmation of the associated novel non-classical androgen signaling pathways fromAim 3.After complete the study, we are confident that we will obtain one or two novel androgen signaling pathway associated with spermatogenesis, and these will be used as the targets for improving male spermatogenesis or as a block target for male contraceptive.訊息途徑男性不孕睪固酮睪丸signaling pathwaymale infertilitytestosteronetestis