Cao Lihua, Yang Ting, Huang Shihai, Yun Xuedan, Hou Hanqi, Wang Ting, Shi Deshun, Li Xiangping
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, 530005, PR China.
College of Life Science and Technology, Guangxi University, Nanning, 530005, PR China.
Theriogenology. 2021 Feb;161:262-270. doi: 10.1016/j.theriogenology.2020.12.009. Epub 2020 Dec 13.
Zonula occludens (ZO)-1 and ZO-2 are involved in epithelial polarity maintenance, gene transcription, cell proliferation and tumor cell metastasis. Regulating ZO-1/2 expression influences the early embryonic development of mice, but whether they are involved in oocyte maturation is still poorly understood. In the present study, the expression patterns of ZO-1 and ZO-2 in porcine cumulus cells and oocytes matured in vitro and early embryos from parthenogenetic activation were detected by qRT-PCR or Western blot, and then their roles in porcine oocyte maturation and early embryo development were investigated by shRNA technology. ZO-1 and ZO-2 were found to be expressed in cumulus cells, oocytes and early embryos, while ZO-1α was expressed only in cumulus cells, morula and blastocysts. During in vitro maturation (IVM), the abundance of ZO-1 and ZO-2 in oocytes was significantly higher than that in cumulus cells at 0 h (P < 0.01), and their mRNA and protein levels displayed relatively higher expression at 0 and 18 h, respectively. Compared with the control groups, cumulus cell expansion, oocyte nucleus maturation, and subsequent cleavage were not influenced by treatment of the cumulus-oocyte complexes (COCs) with ZO-1-shRNA1, ZO-2-shRNA2 or combined ZO-1-shRNA1 and ZO-2-shRNA2 lentivirus (P > 0.05). However, the blastocyst rate was reduced by treatment of COCs with ZO-1-shRNA1 but not ZO-2-shRNA2. The total cell number of blastocysts was decreased by downregulation of ZO-1 and ZO-2 (P < 0.05). Downregulation of ZO-1 and ZO-2 also resulted in a significant decrease (P < 0.05) in the expression of Cx43, Cx45, PTX3 and PTGS2 in cumulus cells, Cx45, BMP15, ZP3 and C-KIT in MII oocytes, and Nanog in blastocysts, with the exception of HAS2 expression in cumulus cells and Oct4 expression in blastocysts (P > 0.05). Altogether, the above results indicate that ZO-1 and ZO-2 display similar expression patterns during porcine oocyte IVM and are critical to porcine oocyte maturation and early embryonic development.
紧密连接蛋白（ZO）-1和ZO-2参与上皮极性维持、基因转录、细胞增殖和肿瘤细胞转移。调节ZO-1/2的表达会影响小鼠的早期胚胎发育，但它们是否参与卵母细胞成熟仍知之甚少。在本研究中，通过qRT-PCR或蛋白质免疫印迹法检测了ZO-1和ZO-2在猪卵丘细胞、体外成熟卵母细胞以及孤雌激活早期胚胎中的表达模式，然后利用shRNA技术研究了它们在猪卵母细胞成熟和早期胚胎发育中的作用。研究发现，ZO-1和ZO-2在卵丘细胞、卵母细胞和早期胚胎中均有表达，而ZO-1α仅在卵丘细胞、桑葚胚和囊胚中表达。在体外成熟（IVM）过程中，卵母细胞中ZO-1和ZO-2的丰度在0小时时显著高于卵丘细胞（P < 0.01），其mRNA和蛋白质水平分别在0小时和18小时时表达相对较高。与对照组相比，用ZO-1-shRNA1、ZO-2-shRNA2或联合使用ZO-1-shRNA1和ZO-2-shRNA2慢病毒处理卵丘-卵母细胞复合体（COCs）对卵丘细胞扩展、卵母细胞核成熟及随后的卵裂没有影响（P > 0.05）。然而，用ZO-1-shRNA1处理COCs会降低囊胚率，而用ZO-2-shRNA2处理则不会。下调ZO-1和ZO-2会使囊胚的总细胞数减少（P < 0.05）。下调ZO-1和ZO-2还导致卵丘细胞中Cx43、Cx45、PTX3和PTGS2的表达显著降低（P < 0.05），MII期卵母细胞中Cx45、BMP15、ZP3和C-KIT的表达显著降低，囊胚中Nanog的表达显著降低，但卵丘细胞中HAS2的表达和囊胚中Oct4的表达除外（P > 0.05）。总之，上述结果表明，ZO-1和ZO-2在猪卵母细胞IVM过程中表现出相似的表达模式，对猪卵母细胞成熟和早期胚胎发育至关重要。
