Abstract：
Fertilization proteins JUNO and CD9 play vital roles in sperm-egg fusion, but little is known about their expression patterns during in vitro maturation (IVM) and their function during in vitro fertilization (IVF) of bovine oocytes. In this study, qRT-PCR and immunofluorescence staining were used to detect the mRNA and protein expression levels of JUNO and CD9 genes in bovine oocytes and cumulus cells. Then, fertilization rate of MII oocytes treated with (i) JUNO antibody (1, 5 and 25 μg/ml) or (ii) CD9 antibody (1, 5 and 25 μg/ml) or (iii) CD9 antibody (5 μg/ml) + JUNO antibody (5 μg/ml) were recorded. Our results showed that the mRNA and protein expression levels of JUNO and CD9 genes significantly increased from bovine GV oocytes to MII oocytes, and similar mRNA expression patterns of JUNO and CD9 were also detected in cumulus cells. All groups of oocytes treated with CD9 antibody or JUNO antibody showed significantly decreased fertilization rates (p < .05). Particularly, the fertilization ability of oocytes treated with CD9 antibody (5 μg/ml) + JUNO antibody (5 μg/ml) sharply decreased to 3.48 ± 0.11%. In conclusion, our study revealed the expression levels of JUNO and CD9 genes in oocytes and cumulus cells increased during IVM of bovine oocytes, with JUNO protein playing a major role in the fertilization of bovine oocytes.
摘要：
受精蛋白JUNO和CD9在精卵融合中起重要作用，但是对它们在体外成熟（IVM）过程中的表达模式以及在牛卵母细胞的体外受精（IVF）过程中的功能知之甚少。在这项研究中,采用qRT-PCR和免疫荧光染色检测牛卵母细胞和卵丘细胞中JUNO和CD9基因的mRNA和蛋白表达水平。然后，记录用(i)JUNO抗体(1、5和25μg/ml)或(ii)CD9抗体(1、5和25μg/ml)或(iii)CD9抗体(5μg/ml)+JUNO抗体(5μg/ml)处理的MII卵母细胞的受精率。我们的结果表明，从牛GV卵母细胞到MII卵母细胞，JUNO和CD9基因的mRNA和蛋白表达水平显着增加，在卵丘细胞中也检测到相似的JUNO和CD9的mRNA表达模式。用CD9抗体或JUNO抗体处理的所有卵母细胞组显示显著降低的受精率(p<0.05)。特别是,用CD9抗体（5μg/ml）+JUNO抗体（5μg/ml）处理的卵母细胞的受精能力急剧下降至3.48±0.11％。总之,我们的研究表明，在牛卵母细胞的IVM过程中，卵母细胞和卵丘细胞中JUNO和CD9基因的表达水平增加，JUNO蛋白在牛卵母细胞的受精中起主要作用。
