O-GlcNAc修饰水平变化对牛卵母细胞体外成熟的影响

doi:10.11843/j.issn.0366-6964.2022.11.012

摘要/Abstract

摘要： 旨在探究O-β-N-乙酰葡糖胺（O-linked beta-N-acetylglucosamine，O-GlcNAc）修饰水平变化对牛卵母细胞体外成熟的影响。本研究以牛卵母细胞为研究对象，检测O-GlcNAc转移酶（O-GlcNAc transferase，OGT）、O-GlcNAc糖苷酶（O-GlcNAcase，OGA）及O-GlcNAc蛋白在牛体外成熟卵母细胞中的分布；将卵丘-卵母细胞复合体分别在添加4 mmol·L^-1 OGT抑制剂BADGP和100 μmol·L^-1 OGA抑制剂PUGNAc的体外成熟液中进行体外成熟，将未添加组作为对照组，分别统计各组卵母细胞第一极体排出率和体外受精胚胎发育率，并采用荧光定量PCR和Western blot检测O-GlcNAc蛋白、OGT、OGA、GFAT和TXNIP的mRNA和蛋白表达。结果表明，OGT和O-GlcNAc蛋白共定位于牛体外成熟卵母细胞的细胞质和细胞核中，而OGA和O-GlcNAc蛋白共定位于体外成熟卵母细胞的细胞质中，且相对集中在卵母细胞的皮质区。与对照组相比，BADGP处理组和PUGNAc处理组卵母细胞第一极体排出率（（52.8±5.1）%&（60.9±1.9）%vs.（70.8±5.4）%）和体外受精囊胚发育率（（9.6±4.9）%&（10.0±5.8）%vs.（21.5±4.3）%）均显著降低（P<0.05）。BADGP处理显著降低了OGT的表达，使卵母细胞的O-GlcNAc修饰水平发生下调，OGA的表达降低；而在PUGNAc处理组中，卵母细胞OGA的表达显著下调，O-GlcNAc修饰水平升高，OGT的蛋白表达显著减少；抑制OGT显著上调己糖胺生物合成途径关键限速酶GFAT mRNA的表达，而抑制OGA则显著下调GFAT mRNA的表达；此外，O-GlcNAc修饰水平改变显著上调了葡萄糖调控关键因子TXNIP的表达。研究结果表明，O-GlcNAc修饰水平改变会降低牛卵母细胞体外成熟及发育能力，卵母细胞通过反馈调节OGT、OGA、GFAT以及TXNIP的表达，应对O-GlcNAc修饰水平的波动。

关键词: 牛, 卵母细胞, O-GlcNAc修饰, OGT, OGA

Abstract: The aim of this study was to explore the effect of altered O-GlcNAc modification on in vitro maturation (IVM) of bovine oocytes. Bovine in vitro-matured(IVM) oocytes were used to detect the distribution of OGT, OGA and O-GlcNAc proteins. Then cumulus-oocyte complexes were matured in IVM medium supplemented with 4 mmol·L^-1 OGT inhibitor BADGP or 100 μmol·L^-1 OGA inhibitor PUGNAc, respectively. The none supplementation group was used as control. The first polar body extrusion rate and subsequent embryonic development in vitro were detected, the O-GlcNAc level as well as the mRNA and protein expression levels of OGT, OGA, GFAT and TXNIP were detected by RT-qPCR and Western blot. Results showed that OGT and O-GlcNAc proteins were colocalized in the cytoplasm and nucleus of oocytes, while OGA and O-GlcNAc proteins were colocalized in the cytoplasm and primarily enriched at the cortex. Compared with control group, the first polar body extrusion rate ((52.8±5.1)% & (60.9±1.9)% vs. (70.8±5.4)%) and the blastocyst formation rate ((9.6±4.9)% & (10.0±5.8)% vs. (21.5±4.3)%) significantly reduced in BADGP and PUGNAc groups (P<0.05). BADGP treatment significantly reduced the expression of OGT, the level of O-GlcNAc modification in oocytes was down-regulated, and the expression of OGA was decreased; In the PUGNAc-treated group, the expression of OGA was significantly down-regulated, the modification level of O-GlcNAc was increased, and the protein expression of OGT was significantly decreased. Inhibition of OGT significantly up-regulated the expression of GFAT mRNA, a key rate-limiting enzyme in the hexosamine biosynthesis pathway, whereas inhibition of OGA significantly down-regulated the expression of GFAT mRNA. In addition, the altered level of O-GlcNAc modification significantly up-regulated the expression of TXNIP, a key factor in glucose regulation. The results indicated that changes in the level of O-GlcNAc modification would reduce the maturation and developmental ability of bovine oocytes in vitro, and oocytes responded to the fluctuation of O-GlcNAc modification levels by feedback-regulating the expressions of OGT, OGA, GFAT and TXNIP.

Key words: bovine, oocyte, O-GlcNAc modification, OGT, OGA

中图分类号:

S823.3

王腾飞, 冯志强, 孙雅雯, 赵善江, 郝海生, 邹惠影, 杜卫华, 赵学明, 朱化彬, 庞云渭. O-GlcNAc修饰水平变化对牛卵母细胞体外成熟的影响[J]. 畜牧兽医学报, 2022, 53(11): 3856-3865.

WANG Tengfei, FENG Zhiqiang, SUN Yawen, ZHAO Shanjiang, HAO Haisheng, ZOU Huiying, DU Weihua, ZHAO Xueming, ZHU Huabin, PANG Yunwei. Effect of Altered O-GlcNAc Modification on in vitro Maturation of Bovine Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3856-3865.

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