绵羊磷脂酶C-γ1对绵羊卵母细胞体外成熟的影响

doi:10.11843/j.issn.0366-6964.2022.10.018

摘要/Abstract

摘要： 卵母细胞成熟率可作为衡量体外培养卵母细胞质量和发育能力的指标。本研究旨在探讨PLC-γ1是否参与了绵羊卵母细胞的体外成熟及其影响。将绵羊卵母细胞在含有不同浓度的U73122（PLC抑制剂））及m-3M3FBS （PLC激活剂）的成熟培养液中进行培养，每个浓度150个细胞，重复试验3次，统计细胞成熟率、卵裂率及桑葚胚率以供筛选出PLC-γ1的最佳抑制及促进浓度。qPCR检测0.5 μmol·L^-1 U73122及0.5 μmol·L^-1 m-3M3FBS处理后48 h的卵母细胞中PLC-γ1、BAK、BAX、CASP3、CASP8、P53、BCL6基因mRNA水平表达情况，Western blot检测0.5 μmol·L^-1 U73122及0.5 μmol·L^-1 m-3M3FBS处理后48 h的卵母细胞中PLC-γ1、BAK、BAX、CASP3、CASP8、P53、BCL6蛋白的表达情况。每个浓度150个细胞，重复试验3次。在绵羊卵母细胞中，0.5 μmol·L^-1 U73122是促进成熟的最佳浓度，0.5 μmol·L^-1 m-3M3FBS是抑制成熟的最佳浓度。0.5 μmol·L^-1 U73122处理组卵裂率和囊胚率均显著低于对照组。0.5 μmol·L^-1 m-3M3FBS处理组卵裂率和囊胚率均显著高于对照组。通过qPCR检测结果显示，与对照组相比，U73122组中BAK、BAX、CASP3、CASP8、P53基因mRNA表达量显著上升，PLC-γ1、BCL6基因mRNA表达量显著下降；m-3M3FBS组则与之相反。Western blot结果显示，与对照组相比，U73122组中BAK、BAX、CASP8蛋白表达量显著增多，PLC-γ1、BCL6蛋白表达量显著减少，P53和CASP3蛋白表达量无明显变化。m-3M3FBS组中PLC-γ1、BCL6蛋白表达量显著增多，BAX、CASP3、P53蛋白表达量显著减少，BAK、CASP8蛋白表达量无明显变化。综上所述，本研究表明PLC-γ1在绵羊卵母细胞的体外成熟培养中发挥重要作用，其可调节卵母细胞的成熟以及早期胚胎的发育能力。

关键词: PLC-γ1, 绵羊卵母细胞, U73122, m-3M3FBS

Abstract: The maturation rate of oocyte can be used as the index to measure the quality and development ability of oocytes cultured in vitro. The purpose of this study was to investigate whether PLC-γ1 was involved in in vitro maturation of sheep oocytes and its effects. Sheep oocytes were cultured in mature medium containing different concentrations of U73122 (PLC inhibitor) and m-3M3FBS (PLC activator), with 150 cells per concentration. The experiment was repeated 3 times. The cell maturation rate, cleavage rate and morula rate were calculated to screen out the optimal inhibitory and promoting concentration of PLC-γ1. The mRNA levels of PLC-γ1, BAK, BAX, CASP3, CASP8, P53 and BCL6 in oocytes in treatment of 0.5 μmol·L^-1 U73122 and 0.5 μmol·L^-1 m-3M3FBS were detected by qPCR for 48 h. Western blot was used to detect the expression of PLC-γ1, BAK, BAX, CASP3, CASP8, P53 and BCL6 proteins in oocytes in treatment of 0.5 μmol·L^-1 U73122 and 0.5 μmol·L^-1 m-3M3FBS for 48 h. The experiment was repeated 3 times with 150 cells per concentration. In sheep oocytes, 0.5 μmol·L^-1 U73122 was the optimal concentration to promote maturation, and 0.5 μmol·L^-1 m-3M3FBS was the optimal concentration to inhibit maturation. The cleavage rate and blastocyst rate in 0.5 μmol·L^-1 U73122 treatment group were lower than those in control group. Cleavage rate and blastocyst rate in 0.5 μmol·L^-1 m-3M3FBS treatment group were higher than those in control group. The qPCR results showed that, compared with the control group, the mRNA expression levels of BAK, BAX, CASP3, CASP8 and P53 genes in U73122 group significantly increased, while the mRNA expression levels of PLC-γ1 and BCL6 genes significantly decreased, these were contrast in m-3M3FBS group. Western blot results showed that, compared with the control group, the protein expression levels of BAK, BAX and CASP8 significantly increased in U73122 group, while the protein expression levels of PLC-γ1 and BCL6 significantly decreased, while the protein expression levels of P53 and CASP3 did not change significantly. In the m-3M3FBS group, the protein expression levels of PLC-γ1 and BCL6 significantly increased, the protein expression levels of BAX, P53 and CASP3 significantly decreased, while the protein expression levels of BAK and CASP8 did not change significantly. In conclusion, this study suggests that PLC-γ1 plays an important role in in vitro maturation culture of sheep oocytes and regulates oocyte maturation and early embryo development.

Key words: PLC-γ1, sheep oocyte, U73122, m-3M3FBS

中图分类号:

S826.3

刘欣杰, 吴晓雪, 刘素平, 袁利明, 陈宁, 赛务加甫. 绵羊磷脂酶C-γ1对绵羊卵母细胞体外成熟的影响[J]. 畜牧兽医学报, 2022, 53(10): 3459-3469.

LIU Xinjie,WU Xiaoxue,LIU Suping,YUAN Liming,CHEN Ning,SAIWU Jiafu. Effects of Sheep Phospholipase C-γ1 on in vitro Maturation of Sheep Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3459-3469.

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