小鼠精子細胞活性調控之研究

The Role of [Ca2+]i in Capacitation and Decapacitation Processes in Mouse Sperm

魏志純(Chih-Chun Wei)

指導教授 ： 黃彥華

臺北醫學大學/醫學院/醫學科學研究所/碩士(2005年)

摘要

精子獲能作用指的是精子經過射精之後會經過一連串生化反應的調控來使得精子具有與卵結合的能力，反之，稱為去獲能作用。精子從雄性睪丸分化到進入雌性生殖道的過程中，存在著一些獲能因子以活化精子，同時也存著一些去獲能因子使精子不被提早活化，避免在與卵受精之前就已耗盡能量，使精子能在適當的時間和適當的地點和卵結合。我們實驗室對於調控精子活性之獲能作用及去獲能作用機制深感興趣。我的論文分成兩部分：在獲能作用方面，我們承接之前已發表一專一表現於睪丸之孤兒受體（orphan receptor）鳥?酸環化?G(mGC-G)的研究，繼續進行此mGC-G可能扮演的功能性分析。RT-PCR結果得知mGC-G高度表現於性成熟的小鼠睪丸中；利用免疫組織染色法、免疫細胞染色法和共軛焦顯微技術得知，此mGC-G表現於成熟精子頭部與尾部中段。利用流式細胞儀和電腦輔助精子分析系統得知，由牛血清白蛋白(bovine serum albumin)所引發的細胞內鈣離子上升和高泳動力皆可被抗mGC-G-ECD抗體所抑制，然而，對A23187所引發的頂體反應結果並不明顯，因此我們推論其功能與精子游動力相關。在去獲能作用方面，我們發現一貯精囊分泌蛋白SVA(貯精囊自體抗原)，可以和PAF(platelet-activating factor)及富含膽鹼(choline)的脂質，如神經磷脂(sphingomyelin)和磷脂膽鹼(phosphatidylchoine)結合。此外，我們發現SVA藉由降低細胞內鈣離子濃度來調控精子活性。以免疫細胞螢光染色法與流氏細胞儀偵測得知，細胞膜PMCA (Ca2+-ATPase通道)抑制劑，CE(Carboxyleiosin-AM)，可回復SVA所降低細胞內鈣離子濃度的現象，顯示SVA的降低細胞內鈣離子機制調節與PMCA相關。此外，SVA可能藉由結合於SPM和PC在精子細胞膜lipid raft區域，進行調控PMCA的活性，以傳遞精子去獲能作用之訊息傳遞。

關鍵字

精子 ； 獲能作用 ； 鳥?酸環化? ； 貯精囊自體抗原 ； 鈣離子

並列摘要

Successful fertilization is tight regulated by sperm capacitation and decapacitation processes. During the transit of sperm from male reproductive tract to female oviduct, factors promoted or suppressed sperm activity should interplay to prevent the gamete prematurization. We are interested in the regulation of sperm activation, including capacitation and decapacitation. In capacitation process, we focus on a mouse orphan receptor, guanylyl cyclase G (mGC-G). mGC-G has been demonstrated to be highly and selectively expressed in mouse testes exclusively. The unique testis-enriched expression of mGC-G suggests the existence of physiological function mediated through mGC-G/cGMP signaling in the testis. In our results, we demonstrate that this novel receptor is dominantly expressed in mature sperm and located at anterior acrosome and midpiece. In addition, by a fluo-3 flow cytometer analysis and computer-assisted sperm assay (CASA), we found that bovine serum albumin(BSA)-induced elevation of [Ca2+]i level and hypermotility in mouse sperm can be neutralized by anti-rabbit mGC-G extracellular domain specific antibody, but not by control rabbit IgG. However, in the presence of BSA, the mGC-G antibody can not block the A23187 (a calcium ionophore)-induced acrosome reaction. These results suggest the role of mGC-G is in sperm motility, but not capacitation and acrosome reaction. In decapacitation process,we found that SVA binds to PAF, sphingomyelin and phosphatidylcholin, and effectively suppresses the level of [Ca2+]i by enhancing Ca2+ efflux through plasma membrane Ca2+-ATPase (PMCA) to downregulate capacitation signaling. Our observation suggests that SVA my bind to sphingolipid/phospholipid in lipid raft on sperm membrane, by which to mediate decapacitation signal of mouse sperm.

參考文獻

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