KDM1A对牦牛卵母细胞减数分裂成熟及其发育潜能的影响

doi:10.11843/j.issn.0366-6964.2020.10.011

摘要/Abstract

摘要： 旨在探讨KDM1A对牦牛卵母细胞减数分裂成熟及其发育潜能的影响。本研究在体外成熟液中添加不同浓度的KDM1A特异性抑制剂GSK-KDM1A，牦牛卵丘-卵母细胞复合体（COCs）体外培养24 h后，观察卵丘细胞的扩展和第一极体的排出情况；利用免疫荧光检测体外培养过程中卵母细胞内KDM1A的表达模式；采用实时荧光定量PCR检测体外培养卵母细胞内Kdm1a、Oct-4、Sox-2以及Nanog的表达水平；体外培养成熟后的牦牛卵母细胞进行体外受精，观察其卵裂率与囊胚形成率。结果显示，体外培养24 h后，GSK-KDM1A组的卵丘细胞扩展程度显著低于对照组（P<0.05），而320 nmol·L^-1组的卵丘细胞扩展程度和第一极体排出率均显著低于160 nmol·L^-1组（P<0.05）。在卵母细胞体外成熟过程中，Kdm1a呈现动态表达模式，MⅠ期的表达水平显著低于GV和MⅡ期（P<0.05）；添加GSK-KDM1A能显著抑制卵母细胞中KDM1A蛋白的表达（P<0.05），320 nmol·L^-1组各时间点KDM1A的表达量均显著低于160 nmol·L^-1组（P<0.05）。GSK-KDM1A组卵母细胞内Oct-4与Sox-2的表达水平显著高于对照组（P<0.05），但Nanog的表达水平无显著差异（P>0.05）。牦牛卵母细胞体外成熟后，GSK-KDM1A组的卵裂率显著低于对照组（P<0.05），但囊胚形成率无显著变化（P>0.05）。综上表明，KDM1A参与调控牦牛卵母细胞减数分裂成熟过程，GSK-KDM1A能有效抑制KDM1A的表达，影响卵母细胞减数分裂成熟及其发育潜能，揭示KDM1A在此过程中扮演重要角色。

关键词: 牦牛, 卵母细胞, KDM1A, 成熟, 发育

Abstract: The purpose of this study was to explore the effects of KDM1A on the meiotic maturation and developmental potential of yak oocytes. The specific inhibitor GSK-KDM1A of KDM1A was added into in vitro maturation medium of yak oocytes. After 24 h in vitro culture of yak cumulus oocyte complexes (COCs), the expansion of cumulus cells and the extrusion of the first polar body were observed. The expression level of KDM1A in oocyte was measured by immunofluorescence during in vitro culture. The expression levels of Kdm1a, Oct-4, Sox-2 and Nanog in oocytes were detected by RT-qPCR. Then, yak oocytes were fertilized after in vitro culture, and the cleavage rate and blastocyst formation rate were observed, respectively. The results showed that the cumulus cells expansion in GSK-KDM1A groups were significant lower than those in control group (P<0.05) after 24 h culture, and the cumulus cells expansion and the first polar body extrusion rate in 320 nmol·L^-1 group were significantly lower than those in 160 nmol·L^-1 group (P<0.05). During oocyte in vitro maturation, Kdm1a showed dynamic expression profile, and the expression level of Kdm1a in MⅠ-stage was significantly lower than that in GV-stage and MⅡ-stage (P<0.05). The GSK-KDM1A could significantly inhibit the expression of KDM1A protein in oocytes (P<0.05), and the expression of KDM1A in 320 nmol·L^-1 group was significantly lower than that in 160 nmol·L^-1 group (P<0.05). The expression levels of Oct-4 and Sox-2 in GSK-KDM1A group were significantly higher than that in control group (P<0.05), but there was no significant difference in the expression of Nanog (P>0.05). After 24 h culture, the cleavage rates of oocytes in GSK-KDM1A groups were significantly lower than those in control group (P<0.05), while the blastocyst formation rate was not significantly different (P<0.05). In conclusion, KDM1A is involved in regulating the meiotic maturation process of yak oocytes. GSK-KDM1A can effectively inhibit the expression of KDM1A, affect the meiotic maturation and developmental potential of oocytes, which reveal that KDM1A plays an important role in this process.

Key words: yak, oocyte, KDM1A, maturation, development

中图分类号:

S823.85

韩杰, 熊显荣, 熊燕, 吴锦波, 李键. KDM1A对牦牛卵母细胞减数分裂成熟及其发育潜能的影响[J]. 畜牧兽医学报, 2020, 51(10): 2433-2442.

HAN Jie, XIONG Xianrong, XIONG Yan, WU Jinbo, LI Jian. Effects of KDM1A on the Meiotic Maturation and Developmental Potential of Yak Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10): 2433-2442.

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