HSP27基因在牦牛卵母细胞和体外早期胚胎中的表达

为了探讨牦牛HSP27(heat shock proteins27, HSP27)基因在牦牛卵母细胞和早期孤雌激活胚胎中的表达情况及定位,本试验通过实时荧光定量PCR检测牦牛HSP27基因在未成熟卵母细胞和成熟卵母细胞以及孤雌激活不同时期胚胎中的相对表达量,用免疫荧光染色法检测HSP27蛋白在未成熟卵母细胞和成熟卵母细胞以及孤雌激活不同时期胚胎中的表达与定位情况。结果显示,HSP27基因在牦牛未成熟卵母细胞和成熟卵母细胞以及体外培养孤雌激活不同时期胚胎中均可表达,其中在未成熟卵母细胞中的表达量显著高于成熟卵母细胞;在孤雌激活胚胎中,2~4细胞期表达量最高,囊胚期胚胎表达量最低,随着胚胎的发育,HSP27的表达量呈现逐渐降低的趋势;在2~4细胞期和囊胚期胚胎中的表达与其他组表达存在显著差异性,在5~8细胞期和桑椹胚中的表达无显著差异。免疫荧光染色结果表明,HSP27蛋白在牦牛卵丘细胞和卵母细胞中均有表达,在牦牛孤雌激活2~4细胞期、5~8细胞期和桑椹期胚胎中主要表达于细胞核内和细胞质中,在囊胚期主要表达于细胞核中;而HSP27蛋白的表达水平与基因表达水平基本一致。本研究为后续研究HSP27基因在牦牛生殖生理过程中的作用机制提供了参考。

Expression of HSP27 Gene in Yak Oocytes and Early Embryos in Vitro

The aim of this study was to investigate the expression and localization of heat shock protein27 (HSP27) gene in yak oocytes and early parthenogenetic activated embryos. In this experiment, real-time fluorescence quantitative PCR (RT-qPCR) and Immunofluorescence staining techniques were used to detect the relative mRNA expression and differential distribution of yak HSP27 gene in immature oocytes, mature oocytes, and parthenogenetic activated early embryos at different stages. The qRT-PCR results indicated that yak HSP27 gene expressed in immature oocytes, mature oocytes, in vitro cultured parthenogenetic activated 2~4 cell stage embryos, 5~8 cell stage embryos, mulberry embryos, and blastocyst stage embryos. The expression of yak HSP27 at the oocyte stage in immature oocytes and mature oocytes was significantly different. The highest expression level was found in the 24 cell stage embryos in vitro, whereas the lowest expression level was detected in blastocyst stage embryos in early parthenogenetic activation stage. The expression of HSP27 decreased gradually with embryo development. In vitro cultured parthenogenetic activated 24 cell stage and blastocyst embryos, expression in parthenogenetic activated blastocyst embryos was significantly different from other experimental groups (P<0.05), but the differences in expression between 5~8 stage embryos and morula stage embryos were not significant (P>0.05). Immunofluorescence staining showed that yak HSP27 protein expressed in cumulus cells and oocytes, and it was mainly found in the nucleus and cytoplasm in parthenogenetic activated 2~4 cell stage, 5~8 cell stage, and mulberry embryos in vitro. During the blastocyst stage, HSP27 protein was expressed was mainly in the cytoplasm and was consistent with the expression of gene. These results show that the expression of HSP27 in yak immature oocytes and mature oocytes was significantly different, with the development of parthenogenetic activated embryos, the expression gradually decreased. It was speculated that HSP27 plays an important role in yak oocyte maturation, embryogenesis, and development. Our study provides theoretical reference for further exploring the role of HSP27 in yak reproductive physiology.