SGK3在小鼠卵母细胞第一次减数分裂恢复中的作用及其机制

doi:10.13481/j.1671-587X.20240402

摘要/Abstract

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。

关键词: 血清和糖皮质激素诱导的蛋白激酶3, 小鼠卵母细胞, 减数分裂, 细胞分裂周期蛋白2, 生发泡, 生发泡破裂

Abstract:

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 expression level of CDC2-pTyr15 protein in the oocytes was gradually decreased （P<0.01）， and the expression level of SGK3-pSer486 protein was gradually increased （P<0.01）. Conclusion Over-expression of SGK3 can increase the GVBD rate of oocytes of the mice and accelerate the dephosphorylation of CDC2-pTyr15， while the dephosphorylation of CDC2-pTyr15 is later than the phosphorylation of SGK3-Ser486. SGK3 likely serves as an upstream regulator of CDC2 and participates in controlling the resumption of the first meiotic division in the oocytes of the mice.

Key words: Serum and glucocorticoid-induced protein kinase3, Mouse oocytes, Meiotic division, Cell division cyclin protein 2, Germ-vesicle, Germ-vesicle breakdown

中图分类号:

Q132

郭文秀,庄妍,张慧灵,何文宁,孟峻. SGK3在小鼠卵母细胞第一次减数分裂恢复中的作用及其机制[J]. 吉林大学学报(医学版), 2024, 50(4): 891-899.

Wenxiu GUO,Yan ZHUANG,Huiling ZHANG,Wenning HE,Jun MENG. Effect of SGK3 on recovery of first meiotic division of oocytes in mice and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2024, 50(4): 891-899.

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Group	GVBD rate
Group	(t/h) 1	2	3	4
Control	53	76	90	95
TE	51	73	91	96
SGK3-mRNA	78^*△	94^*△	96	100

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