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（ o rphan 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 al bumin) 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 (Ca 2+-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。
小鼠精子細胞活性調控之研究
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 i nterested in the regulation of sperm activation, including capacitation and decapacitation. In c apacitation process, we focus on a mouse orphan receptor, guanylyl cyclase G (mGC-G). mG C-G has been demonstrated to be highly and selectively expressed in mouse testes exclusivel y. The unique testis-enriched expression of mGC-G suggests the existence of physiological f unction mediated through mGC-G/cGMP signaling in the testis. In our results, we demonstrat e that this novel receptor is dominantly expressed in mature sperm and located at anterior acr osome 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 extracel lular domain specific antibody, but not by control rabbit IgG. However, in the presence of BS A, the mGC-G antibody can not block the A23187 (a calcium ionophore)-induced acrosome r eaction. These results suggest the role of mGC-G is in sperm motility, but not capacitation an d acrosome reaction. In decapacitation process,we found that SVA binds to PAF, sphingomy elin and phosphatidylcholin, and effectively suppresses the level of [Ca2+]i by enhancing Ca 2+ efflux through plasma membrane Ca2+-ATPase (PMCA) to downregulate capacitation sig naling. 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.
The Role of [Ca2+]i in Capacitation and Decapacit ation Processes in Mouse Sperm
