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原癌基因{sl c--erbB_(2)}和{sl c--myb}经丝裂原活化蛋白激酶和成熟促进因子途径调节卵母细胞的成熟

郑月慧, 郑莉萍, 李芳, 吴磊, 戴育成

南昌大学医学院生理学教研室.江西,南昌 330006；南昌大学医学院第二附属医院.江西,南昌 330006

摘要

该研究探讨了原癌基因{sl c--erbB_(2)}和{sl c--myb}对小鼠卵母细胞成熟的影响及其在调控卵母细胞成熟中与丝裂原活化蛋白激酶（mitogen--activated protein kinase，MAPK）和成熟促进因子（maturation promoting factor，MPF）的上下游关系。{sl c--erbB_(2)}反义寡脱氧核苷酸（anfisense oligodeoxynucleotide，ASODN）和{sl c--myb} ASODN均呈剂量依赖方式抑制卵母细胞的生发泡破裂（germinalvesicle breakdown，GVBD）率和第一极体（first polar body，PB1）排放率，并显著延迟其成熟时间。小鼠卵母细胞显微注射重组人c--erbB_(2)蛋白和c--myb蛋白后，培养6h其GVBD率分别比对照组上升了23.1％（{sl P}<0.05）和32.2％（{sl P}<0.05），培养12h其PB1排放率分别比对照组上升了17.3％（{sl P}<0.05）和23.5％（{sl P}<0.05）。RT--PCR结果显示，小鼠卵母细胞中存在{sl c--erbB_(2)} mRNA和{sl c--myb} mRNA表达;{sl c--erbB_(2)} ASODN能明显抑制卵母细胞中{sl c--erbB_(2)} mRNA和{sl c--myb} mRNA的表达，{sl c--myb} ASODN能明显抑制卵母细胞中{sl c--myb} mRNA的表达，对{sl c--erbB_(2)} mRNA无明显影响；MAPK抑制剂PD98059以及MPF抑制剂roscovitine在抑制卵母细胞成熟的同时，均能阻断显微注射重组人c--erbB_(2)蛋白和重组人c--myb蛋白对卵母细胞成熟的促进作用，但对卵母细胞中{sl c--erbB_(2)} mRNA和{sl c--myb} mRNA表达无明显影响。Western blot结果显示，{sl c--erbB_(2)} ASODN、{sl c--myb} ASODN、PD98059、roscovitine均使卵母细胞中MAPK磷酸化水平和cyclinB1含量下降。结果提示，原癌基因{sl c--erbB_(2)}、{sl c--myb}在卵母细胞成熟中起重要作用，可能是调控卵母细胞成熟中关键蛋白激酶如MAPK、MPF的上游激活物。

关键词： 卵母细胞成熟; 基因; c-erbB_(2); c-myb; 丝裂原活化蛋白激酶; 成熟促进因子

{sl c--erbB_(2)} and {sl c--myb} induce oocyte maturation via activation of mitogen--activated protein kinase and maturation promoting factor

Zheng Yuehui, Zheng Liping, Li Fang, Wu Lei, Dai Yucheng

Department of Physiology,School of Medicine, Nanchang University.Nanchang 330006,Jiangxi；China

Abstract

It is important to study the mechanism of oocyte maturation because oocyte maturation is essential for the female procreation. The present study was designed to observe the effects of protooncogenes c-erbB_(2) and c-myb on oocyte maturation and the upstream and downstream relationship with mitogen-activated protein kinase （MAPK） and maturation promoting factor （MPF）. The investigation was designed as follows： （1） In order to explore the effects of protooncogenes on oocyte maturation, the dose- and time-depondent effects of c-erbB_(2) antisense oligodeoxynucleotide （ASODN） and c-myb ASODN on oocyte maturation were examined, and the effects of oocyte microinjection with recombinant c-erbB_(2) and c-myb proteins on oocyte maturation were investigated; （2） In order to study the upstream and downstream relationship among protooncogenes of c-erbB_(2), c-myb and protein kinases of MAPK and MPF in regulating oocyte maturation, mouse oocytes were cultured in the medium treated with c-erbB_(2) ASODN, c-myb ASODN, PD98059 （the MAPK inhibitor） or roscovitine （the MPF inhibitor） for 8 h, respectively, and the expressions of c-erbB_(2) mRNA, c-myb mRNA, MAPK and MPF were examined. The results showed that both c-erbB_(2) ASODN and c-myb ASODN inhibited the rate of germinal vesicle breakdown （GVBD） and the first polar （PB1） extrusion of denuded oocytes （DOs） in a dose- and time-dependent way, and delayed their maturation time significantly. When recombinant c-erbB_(2) and c-myb proteins were microinjected into cytoplasm of germinal vesicle stage oocyte, we found that the GVBD rate increased by 23.1% （P〈0.05） and 32.2% （P〈0.05）, respectively, for 6-hour culture, and the PB1 extrusion rate increased by 17.3% （P〈0.05） and 23.5% （P〈0.05）, respectively, for 12-hour culture. RT-PCR showed that the mRNA expressions of c-erbB_(2) and c-myb were detected in oocytes; c-erbB_(2) ASODN inhibited c-erbB_(2) mRNA and c-myb mRNA expressions; c-myb ASODN inhibited c-myb mRNA expression but had no effect on c-erbB_(2) mRNA expression. Nonsense tat ODN had no effects on the expressions of c-erbB_(2) mRNA and c-myb mRNA. Neither PD98059 nor roscovitine changed the expressions of c-erbB_(2) mRNA and c-myb mRNA though both of them inhibited recombinant c-erbB_(2) and c-myb proteins-induced oocyte maturation. Furthermore, MAPK phosphorylation and cyclinB1 synthesis in oocytes were inhibited remarkably when oocytes were treated with c-erbB_(2) ASODN, c-myb ASODN, PD98059 and roscovitine. Nonsense tat ODN had no effects on MAPK phosphorylation and cyclinB1 content. The results suggest that protooncogenes c-erbB_(2) and c-myb play an important role in oocyte maturation; the effects of c-erbB_(2) and c-myb depend upon the action of MAPK and MPF, and their activation is the event that occurs downstream of c-erbB_(2) and c-myb in the maturation signal pathway.

Key words: oocyte maturation;Gene;c-erbB_(2);c-myb;mitogen-activated protein kinase;maturation promoting factor

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引用本文：

郑月慧, 郑莉萍, 李芳, 吴磊, 戴育成. 原癌基因{sl c--erbB_(2)}和{sl c--myb}经丝裂原活化蛋白激酶和成熟促进因子途径调节卵母细胞的成熟[J]. 生理学报 2008; 60 (1): 97-104.

Zheng Yuehui, Zheng Liping, Li Fang, Wu Lei, Dai Yucheng. {sl c--erbB_(2)} and {sl c--myb} induce oocyte maturation via activation of mitogen--activated protein kinase and maturation promoting factor. Acta Physiol Sin 2008; 60 (1): 97-104 (in Chinese with English abstract).