TET去甲基化酶对猪卵母细胞发育的影响

doi:10.16431/j.cnki.1671-7236.2023.05.018

Abstract

Abstract: 【Objective】 The purpose of this study was to investigate the effects of ten-eleven translocation (TET) demethylase on the development of porcine oocytes.【Method】 The collected porcine cumulus-oocyte complexes (COCs) were cultured in vitro maturation medium with different concentrations (0 (control group),100,200 and 400 μmol/L) Bobcat339 inhibitors for 42 h.Cumulus expansion diameter was measured,maturity rate was calculated,and the minimum effective concentration was determined.After 27 h culture in in vitro mature medium,the signal strength of 5mC and 5hmC and the spindle assembly were detected by immunofluorescence (IF).Reactive oxygen species (ROS) were detected by DCFH-DA dye.Mitochondrial membrane potential (MMP) was detected by JC-1 staining.ATP level of oocyte was detected by ATP detection reagent.The expression levels of cumulus expansion related genes at 42 h and antioxidation-related genes in oocytes at 27 h were detected by Real-time quantitative PCR.【Result】 Compared with control group,the cumulus expansion level of 100,200 and 400 μmol/L groups were extremely significantly decreased (P＜0.01),the maturity rate of 100,200 and 400 μmol/L groups were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The minimum effective concentration was determined to be 100 μmol/L,which was used in subsequent tests.IF test results showed that compared with the control group,5mC signal intensity in 100 μmol/L group was significantly increased at 27 h (P＜0.05),while 5hmC signal intensity was not significantly decreased (P＞0.05).Compared with control group,ROS levels were extremely significantly increased (P＜0.01),while MMP and ATP levels were extremely significantly decreased (P＜0.01).Compared with control group,the expression level of mound expansion related gene hyaluronic acid synthetase 2 (Has2) in the COCs of 100 μmol/L group was significantly decreased at 42 h (P＜0.05),and the expression level of penetrin 3 (Ptx3) gene was extremely significantly decreased (P＜0.01).The expression of antioxidation-related genes superoxide dismutase 1 (Sod1) and Sod2 in 100 μmol/L group were significantly decreased at 27 h (P＜0.05).【Conclusion】 TET demethylase could guide the normal assembly and arrangement of spindles,maintain the normal expansion of COCs and ensure the normal maturation of porcine oocytes by clearing 5mC in porcine oocytes,reducing ROS level and increasing MMP level.

Key words: porcine oocyte; TET demethylase; methylation; spindle assembly; reactive oxygen; mitochondrial membrane potential

CLC Number:

S814.6

LI Mingguo, LI Qingchun, CHEN Xin, LU Shihao, HE Fan, QI Mengfan, ZHANG Huapeng, REN Yujun, ZHANG Qingze, FU Binbin, XU Mengsi, AI Zikai, YAN Kun, FENG Yun, HUA Zaidong, HUANG Tao, BI Yanzhen. Effects of TET Demethylase on Porcine Oocyte Development[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1907-1917.

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Effects of TET Demethylase on Porcine Oocyte Development

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