绵羊卵母细胞体外成熟过程中VEGF对DNMT3a表达的影响

doi:10.11843/j.issn.0366-6964.2016.03.009

摘要/Abstract

摘要：

旨在探究血管内皮生长因子（Vascular endothelial growth factor，VEGF）在绵羊卵母细胞体外成熟过程中对DNMT3a的影响。本研究采用RNA干扰和显微注射技术将针对VEGF两个受体FLT1和KDR/Flk-1的有效干扰片段导入到绵羊卵丘-卵母细胞复合体（Cumulus-oocytes complexes，COCs）中，检测体外成熟培养各时间卵母细胞和颗粒细胞中DNMT3a mRNA和蛋白表达水平的变化。结果表明，COCs体外成熟培养过程中，无论是否添加VEGF，卵母细胞内DNMT3a mRNA表达量都随着培养时间延续而降低，培养至8 h时DNMT3a mRNA表达量急速下降，之后下降速度放缓，至24 h后各组趋于一致，且未注射干扰片段对照组的下降速度快于其他干扰组(P＜0.01)。添加VEGF组DNMT3a mRNA表达量的下降速度快于未添加组。干扰片段导入后发现，FLT1-siRNA和KDR-siRNA复合干扰组干扰效果优于KDR-siRNA干扰组，FLT1-siRNA干扰组干扰效果最弱(P＜0.01)。无论是否添加VEGF，在COCs体外成熟培养过程中，颗粒细胞DNMT3a mRNA表达量都随着培养时间延续而降低，且各组表达量差别不大。其中未注射干扰片段对照组的下降速度在COCs成熟培养中期快于其他干扰组(P＜0.05或P＜0.01)，FLT1-siRNA干扰效果较好。无论是否添加VEGF卵母细胞内DNMT3a蛋白表达量从0~4 h上升，之后匀速下降，至16 h急速下降，24 h为零。未导入干扰片段对照组下降速度最快。其中16 h对照组、FLT1-siRNA干扰组、KDR-siRNA干扰组和FLT1-siRNA & KDR-siRNA复合干扰组DNMT3a蛋白表达量，添加VEGF后，分别下降到培养初期的23.12%、31.07%、41.47%和37.90%，未添加VEGF的组则分别下降到培养初期的37.38%、54.60%、57.58%和82.75%。颗粒细胞中，添加VEGF组DNMT3a蛋白表达量呈现逐渐下降趋势，至20 h时，已无DNMT3a蛋白表达；未添加VEGF组从0~4 h有略微上升，之后呈现逐渐均速下降趋势，20 h时，急速下降，至24 h时，无DNMT3a蛋白表达，且干扰片段对DNMT3a蛋白表达影响不大，而本身颗粒细胞中DNMT3a蛋白表达量也不高。结果提示，绵羊COCs体外成熟培养过程中，VEGF加快了卵母细胞中DNMT3a mRNA和蛋白表达量的下降速度。而干扰片段的导入则使DNMT3a mRNA和蛋白表达量下降速度放缓；颗粒细胞内VEGF的添加和受体干扰片段的导入，对DNMT3a mRNA和蛋白表达影响效果不明显。

关键词: 血管内皮生长因子, DNMT3a, 卵母细胞, 绵羊, 体外培养

Abstract:

To study the effect of vascular endothelial growth factor (VEGF) on DNMT3a expression during ovine oocytes maturation in vitro. In this research, in order to test DNMT3a mRNA and protein expression of oocytes and granule cells (GCs) at different time points during cumulus-oocytes complexes (COCs) cultured in vitro, RNAi and microinjection were used to import 2 pairs of effective double chain small interfering RNA (dsRNA) fragments which were designed for VEGF receptors FLT1 and KDR/Flk-1 into COCs. The results indicated that, during the culturing process of COCs maturation in vitro, the expression level of DNMT3a mRNA of oocytes would be decreased regardless of the presence of VEGF. DNMT3a mRNA expression quantity fell sharply at the 8 h, then, it decreased slowly and tended to be similar to each other groups at the 24 h. DNMT3a mRNA without dsRNA of control group significantly declined faster than that of the other treatment groups (P＜0.01). The expression level of DNMT3amRNA of VEGF groups in oocytes showed a faster decrease than the groups without VEGF. After dsRNA fragments microinjection, interfering effect of FLT1-siRNA & KDR-siRNA group was better than that of the KDR-siRNA group and that of FLT1-siRNA group was the lowest (P＜0.01). During the maturation process, DNMT3a mRNA of GCs would be decreased regardless of the presence of VEGF, and the difference between each group were tiny. At the mid maturity of COCs, DNMT3a mRNA without dsRNA of control group decreased faster than that of the other treatment groups (P＜0.05 or P＜0.01), and the effect of RNAi on FLT1-siRNA group was the best. Whether adding VEGF or not, the protein expression level of DNMT3a in oocytes had been increasing from initial time until the 4 h, then it started to decrease at a constant speed. A noteworthy decrease of DNMT3a protein expression was then observed at the 16 h and was completely unexpressed at the 24 h. The protein expression level of DNMT3a in control group without dsRNA had the fastest decreasing speed. At the 16 h, DNMT3a protein levels of the control group, FLT1-siRNA group, KDR-siRNA group and FLT1-siRNA & KDR-siRNA group were decreased to 23.12%, 31.07%, 41.47% and 37.90% respectively with VEGF comparing to that of the 0 h. At the same time, that of the non-VEGF groups had decreased to 37.38%, 54.60%, 57.58% and 82.75%. In GCs, DNMT3a protein expression levels of groups with VEGF decreased gradually since 0 h and could not be tested at the 20 h, and that of groups without VEGF increased slightly from 0 to 4 h, then presented the average decline gradually until the 20 h where a rapid drop appeared and went completely undetected at the 24 h. In addition, dsRNA had negligible effect on DNMT3a protein expression levels, while the original DNMT3a protein expression level in the GCs were low. In conclusion, during the process of COCs maturation in vitro, VEGF could accelerate the decreasing speed of both mRNA and protein expression levels of DNMT3a. dsRNA fragments microinjection could slow down the decreasing speed of both mRNA and protein levels of DNMT3a. VEGF and dsRNA had no obvious influences on both DNMT3a mRNA and protein expression of GCs.

Key words: vascular endothelial growth factor(VEGF), DNMT3a, oocytes, ovine, in vitro

中图分类号:

S826.2

曹忻，邹云龙，蔡勇，周平，李明，石国庆，康相涛. 绵羊卵母细胞体外成熟过程中VEGF对DNMT3a表达的影响[J]. 畜牧兽医学报, 2016, 47(3): 484-492.

CAO Xin，ZOU Yun-long，CAI Yong，ZHOU Ping，LI Ming，SHI Guo-qing，KANG Xiang-tao. The Effects of VEGF on DNMT3a Expression in Oocytes Maturation of Ovine in vitro[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(3): 484-492.

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