小白鼠子宮液之絲胺酸蛋白酶移除結合於精子表面P12的研究

Abstract

胰蛋白酶抑制因子P12由57個胺基酸所組成，是一個分子量6126 Da的鹼性不含醣基的蛋白，具有3對雙硫鍵，屬於Kazal-type胰蛋白酶抑制因子。在生殖系統及附屬性腺中，P12只有在儲精囊、前列腺和凝固腺有表現，並且其表現受到雄性激素的調控，成熟的雄性小白鼠才開始有mRNA和蛋白表現。在射精之後，P12會專一性結合於精子頂體而影響精子的活性。P12已被證實具有抑制精子吸收鈣離子的功能，以及精子的獲能作用、頂體反應、活動力和高度活化作用也會受到P12抑制。本論文目標研究是在精卵結合之前，結合於精子頂體的P12如何被移除的生理意義。 以酶譜法及蛋白酶活性的測定來分析子宮液中與P12有交互作用的蛋白酶，發現子宮液中有一個可以與P12結合的蛋白酶，分子量約33 kDa，不同於胰蛋白酶並且此蛋白酶的活性可以受到P12的抑制。此蛋白酶活性也會受到PMSF（phenylmethylsulphonyl fluoride）所抑制，而得知是一個絲胺酸蛋白酶。由質譜儀的定序，推測該蛋白酶可能是airway trypsin-like protease。經由間接免疫染色分析法的結果，證實子宮液中的絲胺酸蛋白酶可以移除結合於精子頂體的P12。

Trypsin inhibitor P12 consists of 57 amino acid residues in which six cysteines are crosslinked into three disulfide bonds. It is a protein of Mr 6126 Da with no glycoconjugate and belongs to Kazal-type trypsin inhibitor family. Among the reproductive tracts of male and female mice, P12 is only present in seminal vesicle, prostate and coagulating gland of adult male. P12 can specifically bind to sperm acrosome region to affect sperm activaty after ejaculation and it’s gene expression in these accessory sexual glands is androgen-dependent. It has verified that P12 can suppress a great extent of Ca2+-uptake by spermatozoa and has the ability to suppress capacitation, acrosome reaction, motility and hyperactivation of mouse spermatozoa. This work aims to study how to remove P12 on sperm acrosome before sperm-egg encounter. Results of Zymography and protease activity assay demonstrated a 33 kDa P12-inhibited protease in the mouse uterine fluid. It was a serine-type protease, because it could be inhibited by PMSF (phenylmethylsulphonyl fluoride). It was likely to be airway trypsin-like protease, as suggested by the partial peptide sequences determined by MS/MS analysis. Result of indirect immunofluorescence statining assay demonstrated the removal of P12 on sperm acrosome by the serine protease in uterine fluid.