碩士 / 國立臺灣大學 / 動物學系 / 85 / Cystatin為cysteine proteinase之inhibitor。本實驗室先前曾發
現cystatin (12 KDa) 為鯉魚卵巢專一表現之基因，為了了解其生理功能
，本文對cystatin在鯉魚卵巢的生合成及組織分布情形加以研究。原位雜
交的結果顯示，cystatin mRNA在發育早期的卵母細胞即已出現，主要分
布於細胞質。此外，成熟卵母細胞的表層囊泡(cortical alveoli)及濾泡
細胞也有cystatin基因之表現。由免疫點墨分析發現，cystatin的含量
(包括PBS可溶與不可溶部分)隨著卵母細胞之發育而逐漸增加。而且由免
疫組織化學的觀察發現，cystatin主要分布於卵母細胞的表層囊泡與卵
殼(zona pellucida) 以及卵母細胞外圍的濾泡層中。又從西方轉印(
western blotting)的結果顯示，cystatin和卵殼蛋白之ZP2呈現相同的分
子形式 (分子量34 KDa以上)，顯示它們可能在卵發育的過程中聚合在一
起，參與卵殼之形成。此外，在成熟卵的活化(activation)過程中，表層
囊泡之cystatin會釋放到圍卵腔(perivitelline space)。同時cystatin
之化學性狀亦發生變化，即卵細胞PBS可溶部分的cystatin逐漸減少，而
PBS不可溶部分之cystatin則逐漸地增加。但若此時加入MDC
(monodansylcadaverine, transglutaminase的抑制物) 便可阻止此項化
學性狀之改變。而且將Transglutaminase(TG) 加入鯉魚卵之水洗液可使
cystatin由12KDa 聚合成不可溶。因此我們推論，當cystatin由表層囊泡
釋放時，一部分之cystatin可能經由TG的作用，而和卵殼上的蛋白聚合而
成卵殼的一部分。至於卵母細胞發育過程中，cystatin組成卵殼蛋白之機
制如何？TG是否有參？則有待日後研究。
In carp, the cystatin, a cysteine proteinase inhibitor, was
found expressed in ovary only. To better understand its
physiological role on reproduction, the biosynthesis and tissue
distribution of carp cystatin was studied. In situ hybridization
showed that the cystatin mRNAs were found both in oocytes and
follicle cells. The cystatin mRNAs of previtellogenic oocytes
were present in cytoplasm while those of vitellogenic oocytes
were in cortical alveoli. Immunodot assay showed that the
amounts of cystatins increase as vitellogenesis proceeds. By
immunohistochemical study, the ovarian cystatins were found
located on cortical alveoli and zona pellucida of oocytes, and
also the follicular wall. Western blot assay of PBS-insoluable
fraction of oocytes showed that cystatin and ZP2 have the same
pattern, suggesting that they may cross-link together during
oogenesis. By immunocytochemical assay cystatins were found
released from cortical alveoli to perivitelline space during the
activation of carp egg. As the progress of egg activation, the
PBS-soluable cystatins are gradually decreased while the PBS-
insoluable cystatins are gradually increased. This change in PBS
soluability of cystatin was inhibited by the addition of MDC
(monodansylcadaverine, a transglutaminase inhibitor) during the
egg activation. Futhermore, the cystatins released from the
activated carp egg could be conjugated by transglutaminase in
vitro. Therefore, we deduced that as cystatin is released from
cortical alveoli during egg activation, it may be cross-linked
by transglutaminase with ZP proteins to become PBS-insoluable
forms. Whether the cross-linking of cystatin and ZP proteins
such as ZP2 during oogenesis is mediated by the same mechanism
is presently unknown.
| Identifer | oai:union.ndltd.org:TW/085NTU00312007 |
| Date | January 1997 |
| Creators | Weng, Jing-Wei, 翁靖偉 |
| Contributors | Fore-Lien Huang, Yea-Sha Chang, 黃火鍊, 張月霞 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 67 |
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