绵羊卵母细胞3种体外培养方式下外源添加血管内皮生长因子对FLT-1表达的影响

doi:10.11843/j.issn.0366-6964.2018.09.010

摘要/Abstract

摘要：

本研究旨在探究外源添加血管内皮生长因子（VEGF）对绵羊卵母细胞体外成熟过程中FLT-1表达的影响。采用3种不同的体外成熟培养方式：裸卵单独培养、颗粒细胞与裸卵共培养以及卵丘-卵母细胞复合体（cumulus-oocytes complexes，COCs）培养，外源添加5 ng·mL^-1血管内皮生长因子（vascular endothelial growth factor，VEGF）为试验组，不添加为对照组，采用qPCR、Western blot分别检测绵羊卵母细胞及颗粒细胞中FLT-1 mRNA和蛋白表达水平。结果表明：1）3种不同培养方式下，无论是否添加VEGF，颗粒细胞与卵母细胞中均能检测到FLT-1 mRNA和蛋白表达。添加VEGF能极显著降低共培养试验组12、16和20 h以及COCs试验组4、8、12、16和24 h颗粒细胞FLT-1 mRNA表达（P<0.01），能极显著降低共培养试验组12、20和24 h以及COCs试验组4、8和12 h卵母细胞FLT-1 mRNA表达（P<0.01），但会极显著增加裸卵试验组4～24 h卵母细胞FLT-1 mRNA表达（P<0.01）。2）添加VEGF能够极显著降低共培养试验组和COCs试验组中颗粒细胞FLT-1蛋白4～12 h的表达量（P<0.01），共培养试验组和COCs试验组中颗粒细胞FLT-1蛋白表达呈波动性下降趋势。COCs对照组和试验组颗粒细胞中FLT-1蛋白表达量在各时间点均高于同期共培养试验组。3）3种不同培养方式中，除裸卵试验组在20～24 h有一个急剧升高外，其余组中卵母细胞FLT-1蛋白表达量总体均呈波动性下降趋势。综上表明，卵母细胞和颗粒细胞中存在FLT-1的自分泌和旁分泌，卵母细胞中FLT-1 mRNA的表达与外围颗粒细胞的存在与否、数量多少以及包裹方式息息相关；在体外培养环境下，添加VEGF有效地激活了FLT-1 mRNA表达，但同时结合FLT-1蛋白以促进卵母细胞成熟的生物学效应，从而减少了FLT-1蛋白的表达。

Abstract:

The purpose of this research was to investigate the effect of exogenously added VEGF on the expression of the FLT-1 during the process of ovine oocytes maturation in vitro. Three different in vitro maturation culture methods were adopted:naked oocytes separate cultivation, granule cells with naked oocytes co-cultivation and cumulus-oocytes complexes (COCs) cultivation. Treatment groups were adding exogenous 5 ng·mL^-1 vascular endothelial growth factor (VEGF) while control groups were not. The FLT-1 mRNA and protein expression levels in both ovine oocytes and granule cells were detected by qPCR and Western blot, respectively. The results indicated that:1)Adding VEGF could extremely significantly reduce the FLT-1 mRNA expression in granule cells in the co-cultivation treatment group at 12, 16 and 20 h, and the FLT-1 mRNA expression in COCs treatment group at 4, 8, 12, 16 and 24 h (P<0.01), and extremely significantly reduce the FLT-1 mRNA expression in oocytes in the co-cultivation treatment group at 12, 20 and 24 h, and the FLT-1 mRNA expression in COCs treatment group at 4, 8 and 12 h (P<0.01), but extremely significantly increase the FLT-1 mRNA expression in oocytes in the naked oocytes separate cultivation treatment group at 4-24 h (P<0.01). 2)Adding VEGF could extremely significantly reduce the expression of the FLT-1 protein in granule cells in both co-cultivation treatment group and COCs cultivation treatment group at 4-12 h (P<0.01). The expression of the granule cells FLT-1 protein in both naked oocytes co-cultivation treatment group and COCs cultivation treatment group showed a fluctuating downward trend. The granule cells FLT-1 protein expression in both COCs cultivation control group and COCs cultivation treatment group were higher than that of granule cells with naked oocytes co-cultivation treatment group at all time points.3)Among the 3 in vitro maturation culture methods, except for a sharp increase in the naked oocytes separate cultivation control group at 20-24 h, the oocytes FLT-1 protein expression in the other groups showed a downward trend of fluctuation. In conclusion, the FLT-1 has autocrine and paracrine effects in oocytes and granule cells. The expression of the FLT-1 mRNA and protein in oocyte are closely related to the presence or absence of peripheral granule cells, the number and the way of encapsulation. During in vitro culture environment, the addition of VEGF can effectively activate the FLT-1 mRNA expression, but at the same time combine the FLT-1 protein in order to play a biological effect promoting oocytes maturation, thus reducing the FLT-1 protein expression.

中图分类号:

S826.2

曹忻, 邹云龙, 陆会宁, 高丹丹, 周平, 石国庆, 杨具田. 绵羊卵母细胞3种体外培养方式下外源添加血管内皮生长因子对FLT-1表达的影响[J]. 畜牧兽医学报, 2018, 49(9): 1889-1898.

CAO Xin, ZOU Yun-long, LU Hui-ning, GAO Dan-dan, ZHOU Ping, SHI Guo-qing, YANG Ju-tian. The Effects of Adding Exogenous VEGF on FLT-1 Expression during the Process of Ovine Oocytes Maturation in vitro in Three Different Ways[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(9): 1889-1898.

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