HSPA9B在牦牛体外受精胚胎中的表达

The expression of HSPA9B in yak IVF embryos

本试验旨在研究热休克蛋白70-9B（heat shock 70 000protein-9B,HSPA9B）在牦牛早期体外受精胚胎中的表达差异及其与体外受精胚胎发育的关系。在正常生理条件下,采集青海高原牦牛卵巢,捡取质量良好的卵丘-卵母细胞复合体（cumulus-oocyte complexes,COCs）,进行体外受精和体外培养,采取实时荧光定量（RT-qPCR）和间接免疫荧光技术,检测HSPA9B在不同时期体外受精胚胎中的表达差异及其蛋白分布。实时荧光定量结果表明,HSPA9B在2~4细胞期胚胎中的相对表达量,分别是在桑椹胚和囊胚中的6.693 4、5.227 4倍,HSPA9B在4~8细胞期胚胎中的相对表达量分别是在2~4细胞期胚胎、桑椹胚和囊胚中的1.136 8、9.089 7和5.937 2倍。间接免疫荧光染色结果表明,在2~4细胞期胚胎、4~8细胞期胚胎和桑椹胚中,胞核HSPA9B的相对表达量较胞质弱,在囊胚中胞核HSPA9B的相对表达量较胞质强。由此得出结论：通过基因水平研究,发现HSPA9B在牦牛早期各时期的体外受精胚胎中存在表达差异,HSPA9B在2~4、4~8细胞期胚胎中的相对表达量与其在桑椹胚和囊胚中的相对表达量差异性极显著（P〈0.01）,提示：可能是HSPA9B在胚胎早期发育中发挥重要作用;通过蛋白水平研究,发现HSPA9B在2~4细胞期胚胎、4~8细胞期胚胎和桑椹胚中,胞核HSPA9B的表达量较胞质的表达弱,在囊胚中胞核HSPA9B的表达量较胞质的表达强,结果表明,在体外培养的环境应激条件下,牦牛体外受精胚胎发育至囊胚时,HSPA9B蛋白的分布可能发生了移位。

In order to verify the expression of heat shock 70 000 protein-9B（HSPA9B）in yak early IVF embryos. In this study,we collected yak ovaries in Qinghai Plateau, the COCs with multilay- ered compact cumulus,IVF and cultured in vitro. The expression of HSPA9B in early IVF embry- os were detected by the methods of real-time PCR and immunofluorescence at mRNA and protein levels. Real-time PCR results showed that the relative expression of HSPA9B in 2-4 cell stage em- bryos is 6. 693 4,and 5. 227 4 times as much as the HSPA9B in morulae and blastocysts respective- ly,the relative expression of HSPAgB in 2-4 cell stage embryos is 1. 136 8,9. 089 7 and 5. 937 2 times as much as in 2-4 cell embryos morulae and blastocysts,respectively; immunofluorescence staining showed that yak early IVF embryos were marked with HSPA9B fluorescence, HSPA9B protein was expressed in both nucleus and cytoplasm of embryonic cells, but the expression of nu- cleus was lower than the expression of cytoplasm in 2-4 cell stage embryos,4-8 cell stage embryos and morula,and the result is opposite in blastocystso In the gene level, the study showed the differences in expression of HSPA9B in yak early embryos fertilized in vitro in each period,the difference of the relative expression of HSPA9B between 2-4 cell stage embryos,4-8 cell stage em- bryos and morula and blastocysts is very significant（P〈0.01）,which can effectively egulate embryonicdevelopment. In the protein level, the expression of nucleus was lower than that of cytoplasm in 2- 4 cell stage embryos,4-8 cell stage embryos and morula,and the result is opposite in hlastocysts, which suggests that faced in the environmental stress conditions in vitro, distribution of HSPA9B protein is shifted between the nucleus and the cytoplasm.