SIRT1对牦牛卵母细胞体外成熟与老化的影响

doi:10.11843/j.issn.0366-6964.2019.12.008

摘要/Abstract

摘要： 旨在探索SIRT1在牦牛卵母细胞体外成熟与老化过程中的作用。本研究在体外成熟液中分别添加SIRT1特异性激动剂SRT2104（SRT组）和特异性抑制剂Inauhzin（INZ组），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24与36 h后卵母细胞内的ROS水平；采用实时荧光定量PCR法检测体外培养24与36 h后卵母细胞内SIRT1、FOXO3a、SOD2以及Bax的表达水平；体外培养24与36 h后的牦牛卵母细胞进行体外受精，观察并统计其卵裂率与囊胚形成率。结果显示，体外培养24 h，SRT组的卵丘细胞扩展程度显著高于对照组（P<0.05），而INZ组的卵丘细胞扩展程度和第一极体排出率显著低于对照组（P<0.05）。随着体外培养时间的增加，卵母细胞内的ROS水平显著增加（P<0.05）；添加SRT2104能显著抑制卵母细胞中ROS水平的积累（P<0.05），而添加Inauhzin则显著上调卵母细胞内的ROS水平（P<0.05）。体外培养24 h后，SRT组SIRT1、FOXO3a与SOD2的表达水平显著高于对照组（P < 0.05），但Bax的表达水平显著降低（P<0.05）；INZ组的SIRT1、FOXO3a与SOD2表达均显著低于对照组（P<0.05），但Bax的表达水平显著上调（P<0.05）。牦牛卵母细胞体外培养24 h后，SRT组的卵裂率与囊胚形成率显著高于INZ组和对照组（P<0.05）；卵母细胞体外培养36 h后，INZ组的卵裂率和囊胚形成率显著低于其他组（P<0.05）。综上表明，SIRT1参与了牦牛卵母细胞的体外成熟，在体外培养液中适当添加SIRT1激动剂，有利于卵母细胞体外成熟及缓解老化，同时改善早期胚胎的发育能力。

关键词: SIRT1, 卵母细胞, 成熟, 老化, 牦牛

Abstract: The purpose of this study was to explore the role of SIRT1 on the in vitro maturation and aging of yak oocytes. The SIRT1 specific agonist (SRT2104, SRT group) and inhibitor (Inauhzin, INZ group) were added to the in vitro maturation medium of yak oocytes, respectively. After 24 h in vitro culture of yak cumulus-oocyte complexes (COCs), the expansion of cumulus cells and the excretion of the first polar body were observed. The ROS level in oocyte was measured by immunofluorescence after 24 and 36 h in vitro culture. The expression levels of SIRT1, FOXO3a, SOD2 and Bax in oocytes were detected by RT-qPCR method. Then, yak oocytes were fertilized after 24 and 36 h in vitro culture, and the cleavage and blastocyst formation rates were observed, respectively. The results showed that, after 24 h culture, the cumulus cells expansion degree in SRT group was significantly higher than those in the control group (P<0.05), while the cumulus cells expansion degree and the first polar body excretion rate in INZ group were significantly lower than those in the control group (P<0.05). With the increasing of culture time, the ROS level in oocyte increased significantly (P<0.05); SRT2104 addition significantly inhibited the ROS accumulation in oocytes (P<0.05), while Inauhzin addition significantly increased the ROS level in oocytes (P<0.05). After 24 h culture, the expressions of SIRT1, FOXO3a and SOD2 in SRT group were significantly higher than those in the control group (P<0.05), but the expression of Bax was significantly lower (P<0.05); the expressions of SIRT1, FOXO3a and SOD2 in INZ group were significantly lower than those in the control group (P<0.05), but the expression of Bax was significantly increased (P<0.05). After 24 h culture, the cleavage rate and blastocyst formation rate of oocytes in SRT group were significantly higher than those in INZ group and control group (P<0.05). While after 36 h culture, the cleavage rate and blastocyst formation rate of oocytes in INZ group were significantly lower than those in other groups (P<0.05). In conclusion, SIRT1 is involved in regulating in vitro maturation of yak oocytes. Adding SIRT1 agonist properly in in vitro culture medium can promote in vitro maturation and alleviate aging of yak oocytes, simultaneously improve the developmental capacity of early embryos.

Key words: SIRT1, oocyte, maturation, aging, yak

中图分类号:

S823.85

熊显荣, 王艳, 李键, 熊燕, 字向东, 邱翔. SIRT1对牦牛卵母细胞体外成熟与老化的影响[J]. 畜牧兽医学报, 2019, 50(12): 2440-2448.

XIONG Xianrong, WANG Yan, LI Jian, XIONG Yan, ZI Xiangdong, QIU Xiang. Effects of SIRT1 on the in vitro Maturation and Aging of Yak Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(12): 2440-2448.

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