摘要
目的:探讨血清和糖皮质激素诱导的蛋白激酶3(SGK3)在小鼠卵母细胞第一次减数分裂恢复中的作用,初步阐明SGK3在哺乳动物卵母细胞早期发育中的调控机制。方法:利用超排卵技术获取生发泡(GV)期小鼠卵母细胞,利用显微注射技术将表达质粒体外转录获得的SGK3 mRNA注射至GV期卵母细胞,分为对照组、Tris-EDTA缓冲液(TE)组和SGK3 mRNA组,采用Western blotting法检测各组小鼠卵母细胞中SGK3蛋白表达水平,显微注射SGK3 mRNA后1、2、3和4 h观察并计算各组卵母细胞生发泡破裂(GVBD)率,采用SGK3抗体稀释抑制实验观察各组卵母细胞形态表现,采用Western blotting法检测体外培养不同时间点卵母细胞中磷酸化SGK3(pSer48)(SGK3-pSer48)和磷酸化细胞分裂周期蛋白2(CDC2)(pTyr15)(CDC2-pTyr15)蛋白表达水平。结果:与对照组和TE组比较,SGK3 mRNA组小鼠卵母细胞中SGK3蛋白表达水平升高(P<0.01),显微注射后1和2 h时GVBD率升高(P<0.01)。SGK3抗体稀释抑制实验,随着SGK3抗体浓度增加,各组小鼠卵母细胞GVBD率呈浓度依赖性降低。过表达SGK3后,与对照组比较,SGK3 mRNA组小鼠卵母细胞中检测不到CDC2-pTyr15蛋白表达的时间至少提前1 h。不同稀释浓度SGK3抗体作用后,与对照组比较,随着SGK3抗体浓度升高和时间的延长,小鼠卵母细胞中CDC2-pTyr15蛋白表达水平逐渐降低(P<0.01),SGK3-pSer486蛋白表达水平逐渐升高(P<0.01)。结论:过表达SGK3可以增加小鼠卵母细胞GVBD率,加快CDC2-pTyr15的脱磷酸化,而CDC2-pTyr15的脱磷酸化晚于SGK3-Ser486的磷酸化。SGK3可能作为CDC2上游调节因子参与调控小鼠卵母细胞第一次减数分裂的恢复。
Objective:To discuss the role of serum and glucocorticoid-induced protein kinase 3(SGK3)in the resumption of the first meiotic division in the oocytes of the mice,and to preliminarily clarify the regulatory mechanism of SGK3 in the early development of mammalian oocytes.Methods:The germinal vesicle(GV)stage mouse oocytes were obtained by superovulation techniques.The SGK3 mRNA,obtained from in vitro transcription of expression plasmids,was injected into the GV stage oocytes by microinjection techniques.The oocytes were divided into control group,Tris-EDTA buffer(TE)group,and SGK3 mRNA group.Western blotting method was used to detect the expression levels of SGK3 protein in the oocytes in various groups;the germinal vesicle breakdown(GVBD)rates of the oocytes in various groups were observed and calculated at 1,2,3,and 4 h after microinjection of SGK3 mRNA;the morphological appearance of the oocytes in various groups was observed by SGK3 antibody dilution inhibition experiment;Western blotting method was used to detect the expression levels of phosphorylated SGK3(pSer48)(SGK3-pSer48)and phosphorylated cell division cycle protein 2(CDC2)(pTyr15)(CDC2-pTyr15)proteins in the oocytes cultured in vitro at different time points.Results:Compared with control group and TE group,the expression level of SGK3 protein in the oocytes in SGK3 mRNA group was increased(P<0.01),and the GVBD rates at 1 and 2 h after microinjection were increased(P<0.01).The SGK3 antibody dilution inhibition experiment results showed that as the increasing of concentration of SGK3 antibody,the GVBD rates of the oocytes in various groups were decreased in a concentration-dependent manner.After overexpressing SGK3,compared with control group,the time when CDC2-pTyr15 protein expression could not be detected in the oocytes in SGK3 mRNA group was advanced by at least 1 h.After treated with different concentrations of SGK3 antibody,compared with control group,as the increasing of concentration of SGK3 antibody and the extending of treatment time,the expre
作者
郭文秀
庄妍
张慧灵
何文宁
孟峻
GUO Wenxiu;ZHUANG Yan;ZHANG Huiling;HE Wenning;MENG Jun(Department of Clinical Laboratory Diagnosis,Inner Mongolia Medical University,Hohhot 010059,China;Department of Clinical Laboratory,Affiliated Hospital,Inner Mongolia Medical University,Hohhot 010050,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2024年第4期891-899,共9页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金项目(81360109,81660267)
内蒙古自治区科技厅自然科学基金项目(2021MS08158)。
