本研究利用掃描式電子顯微鏡觀察草蝦卵受精時精子穿透卵黃外膜的過程，並分離草蝦精子之蛋白分解酶，進行酵素特性分析。
草蝦產卵時同時將成熟的卵及儲藏於儲精囊?堛犖諵l排出於海水中受精，剛排出的草蝦卵最外層由卵黃外膜所包覆，其下是卵細胞膜。草蝦精子以前端突出的尖芒附著於卵黃外膜，在極短的時間內精子快速活化並進行頂體反應，藉著胞泌作用將頂體囊泡內容物釋出，協助精子穿透卵黃外膜並與卵細胞膜結合。
由雌性草蝦儲精囊內所分離出的精子，經超音波震擊，離心後所得的萃取液，經gelatin SDS-PAGE電泳分析，可在電泳膠片上見到亮帶；而雄性草蝦所分離得的精子萃取液則無此現象，顯示草蝦精子蛋白分解酶需在雌蝦儲精囊中儲存一段時間，經後成熟作用之後才具活性。使用對酵素具專一性的螢光受質分析草蝦精子蛋白分解酶，發現草蝦精子主要具有類胰蛋白酶及類胺月太酶兩種酵素活性。利用蛋白酶抑制劑探討精子蛋白分解酶之特性，發現草蝦精子蛋白分解酶活性可受胰蛋白酶抑制劑如aprotinin、p-aminobenzamidine (PAB)、soybean trypsin inhibitor (SBTI)及N-α-p-tosyl-L-lysinechloromethyl ketone (TLCK)之抑制；而屬於胰凝乳蛋白酶抑制劑N-tosyl-L-phenylalaninechloromethyl ketone (TPCK)與金屬蛋白酶抑制劑1,10-phenanthroline (1,10-P)，對於酵素活性的抑制效果並不佳。推論草蝦精子發生頂體反應時，用以分解卵黃外膜的酵素極可能就是類胰蛋白酶。
草蝦精子蛋白分解酶在弱鹼性的環境有較佳的活性表現，於pH 8.0時酵素活性達最高。環境中的鈣離子及鎂離子則有加強酵素活性的作用。

This study aims to elucidate the process and mechanism of sperm penetration through eggs of Penaeus monodon. Sperm penetration of the vitelline envelopes (VEs) of P. monodon eggs were observed with the scanning electron microscope. The characteristics of sperm proteases in the sperm extracts from seminal receptacles of females were analysized.
In P. monodon, mating and sperm transfer to the thelycum of female occur soon after maturity moult. Females store the sperm in the seminal receptacles. At spawning, they release stored sperm and eggs simultaneously into the water column. The outermost investment of a newly spawned egg is the VE. Sperm bind to the VE via the tip of their anterior spike. They rapidly undergo the acrosome reaction, which composes of depolymerization of the spike and exocytosis of the acrosome vesicle, pass through the VE and become bound to the egg oolemma.
The isolated sperm suspended in artificial seawater were disrupted by sonication on ice. The supernatants after microcentrifuged were collected as sperm extracts. Sperm extracts were analyzed by gelatin SDS-PAGE. Sperm extract from sperm isolated from seminal receptacles of females showed clear bands of protease activity, whereas sperm extract from vas deferens and spermatophore of males did not. This results indicated that sperm of P. monodon do proceed capacitation in the seminal receptacles, and obtain sperm protease activity after capacitation. Using fluorescent peptidyl-MCA as sperm protease substrates, high trypsin-like and aminopeptidase-like activities were observed in sperm extracts. The sperm protease activity was inhibited by trypsin inhibitors aprotinin, p-aminobenzamidine (PAB), soybean trypsin inhibitor (SBTI), N-α-p-tosyl-L-lysinechloromethyl ketone (TLCK); but was not inhibited by chymotrypsin inhibitor N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and metalloprotease inhibitor 1,10-phenanthroline (1,10-P). The results indicated that sperm undergo the acrosome reaction and release sperm proteases including trypsin-like protease, which has been implicated in facilitating sperm passage through vitelline envelope.
Sperm proteases were highly active in the weak base environment, exhibiting maximum activity at pH 8.0. The protease activities were enhanced by addition of calcium chloride and magnesium chloride in the incubation medium.

目錄
章 次 頁 數
壹、緒論…………………………………………………………….. 1
貳、材料與方法……………………………………………………… 4
A、產卵的誘發…………………………………………………. 4
B、掃描式電子顯微鏡的觀察…………………………………. 4
C、精子樣品的製備……………………………………………. 5
(1)雄性草蝦精子樣品(♂sperm extract, ♂SE )…………… 5
(2)雌性草蝦精子樣品(♀sperm extract, ♀SE)……………. 6
D、電泳分析…………………………………………….……… 6
(1)草蝦精子萃取液及儲精囊液之電泳分析……………….. 6
(2)草蝦精子蛋白分解酶於不同pH之活性分析…………… 7
(3) Ca2+ 、Mg2+ 、Na+ 對草蝦精子蛋白分解酶
活性之影響………………………………………………… 8
(4) 蛋白酶抑制劑對草蝦精子蛋白分解酶活性
之影響……………………………………………………… 8
E、螢光受質分析……………………………………………… 9
(1)雌性草蝦精子蛋白分解酶活性分析…………………….. 9
(2)草蝦精子蛋白分解酶於不同pH之活性分析…………… 9
(3) Ca2+ 、Mg2+ 、Na+ 對草蝦精子蛋白分解酶
之影響…………………….……………………………… 10
(4) 蛋白酶抑制劑對草蝦精子蛋白分解酶活性
之影響………………………….………………………… 10
參、結果………………………………………….………………… 11
A、草蝦精子穿透卵黃外膜之掃描式電顯觀察……………… 11
B、電泳分析…………………………………….……………… 11
(1) 草蝦精子萃取液及儲精囊液之電泳分析……………… 11
(2) pH對草蝦精子蛋白分解酶活性之影響.………….…… 12
(3) Ca2+、Mg2+、Na+ 對草蝦精子蛋白分解酶
活性影響………………………………………….…… 12
(4) 蛋白酶抑制劑對草蝦精子蛋白分解酶
活性之影響………………………………………….… 12
C、利用螢光受質分析草蝦精子蛋白分解酶之特性………… 13
肆、討論……………………………………………………………… 15
伍、參考文獻………………………………………………………… 20






表目錄

表 次 頁 數
表1、精子萃取液對受質的活性分析……………………….…………. 39
表2、蛋白酶抑制劑對草蝦精子蛋白分解酶活性的影響….…………. 42

圖目錄

圖 次 頁 數
圖1、草蝦卵活化時卵外基質變化之掃描式電子顯微鏡圖………….. 25
圖2、草蝦精子穿透卵黃外膜之掃描式電子顯微鏡圖……………….. 27
圖3、草蝦精子萃取液之電泳分析…………………………………….. 29
圖4、草蝦精子萃取液及儲精囊液之電泳分析……………………….. 31
圖5、pH對草蝦精子蛋白分解酶活性之影響…………………………. 33
圖6、Ca2+、Mg2+、Na+對草蝦精子蛋白分解酶活性之影響………… 35
圖7、蛋白酶抑制劑對草蝦精子蛋白分解酶活性之影響…………….. 37
圖8、精子萃取液對受質的活性分析………………………………….. 40
圖9、蛋白酶抑制劑對草蝦精子蛋白分解酶活性的影響…………….. 43
圖10、pH對草蝦精子蛋白分解酶活性的影響……………………….. 45
圖11、Ca2+、Mg2+、Na+對草蝦精子蛋白分解酶活性的影響……….. 47

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