O-GlcNAc修饰水平变化对牛卵母细胞体外成熟的影响

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100μmol·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...

Effect of Altered O-GlcNAc Modification on in vitro Maturation of Bovine Oocytes

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.