RFRP-3对猪卵母细胞体外成熟的影响

doi:10.11843/j.issn.0366-6964.2022.04.013

摘要/Abstract

摘要： 旨在研究RFRP-3对猪卵母细胞体外成熟的影响。本研究采用从屠宰场收集健康母猪的卵巢中挑取的GV期卵母细胞，随机分为3组，在培养基中分别添加0、10^-6和10^-8mol·L^-1 RFRP-3培养猪卵母细胞44 h后统计各组卵母细胞的成熟率；在后续试验中将收集到的卵母细胞随机分为2组，在培养基中分别添加0和10^-8 mol·L^-1 RFRP-3培养猪卵母细胞44 h，观察猪卵母细胞的卵丘扩展情况并计算各组的卵丘扩展指数和各组卵母细胞的成熟率；利用qRT-PCR检测卵丘扩展因子(PTGS2、HAS2、PTX3)、卵母细胞分泌因子(GDF9、BMP15)和周期蛋白相关基因(CCNB1和CDK1)的表达变化；利用ELISA试剂盒检测MPF和cAMP的含量；采用放射免疫法检测孕酮和雌激素的浓度，每组卵母细胞量不少于100枚，每个试验重复3次。结果表明，与对照组相比，添加10^-8mol·L^-1 RFRP-3培养猪卵母细胞可极显著降低卵母细胞的成熟率(P<0.01)；通过显微镜观察并计算卵丘扩展指数发现，试验组中卵丘扩展无明显变化(P>0.05)，但卵丘扩展因子(PTGS2、HAS2、PTX3)的表达极显著下降(P<0.01)；RFRP-3可以极显著降低猪卵母细胞MPF的含量(P<0.01)，对cAMP的含量无显著影响(P>0.05)；添加RFRP-3可促进GDF9(P<0.01)和BMP15(P<0.05)的表达，抑制CCNB1(P<0.05)和CDK1(P<0.05)的表达；同时试验组培养基中孕酮、雌激素的浓度也极显著下降(P<0.01)。综上，RFRP-3通过调控猪卵母细胞成熟相关因子和卵丘扩展因子的表达以及类固醇激素的分泌，从而抑制卵母细胞的体外成熟。本研究为阐明RFRP-3对哺乳动物卵母细胞的调控作用奠定理论基础。

关键词: RFRP-3, 猪, 卵母细胞, 体外成熟

Abstract: The aim of this study was to investigate the effect of RFRP-3 on in vitro maturation of porcine oocyte. In this study, GV stage oocytes from the ovaries of healthy sows collected from slaughterhouses were randomly divided into 3 groups. The maturation rate of porcine oocytes in each group was calculated after incubating with 0, 10^-6 and 10^-8 mol·L^-1 RFRP-3 for 44 h, respectively. In the subsequent experiment, the collected oocytes were randomly divided into 2 groups. Porcine oocytes were cultured in medium with 0 and 10^-8 mol·L^-1 RFRP-3 for 44 h, respectively. The cumulus expansion of porcine oocytes was observed and the cumulus expansion index and maturation rate of oocytes in each group were calculated. The expression of cumulus expansion factors (PTGS2, HAS2, PTX3), oocyte secretory factors (GDF9, BMP15) and cyclin related genes (CCNB1 and CDK1) were detected by qRT-PCR. The contents of MPF and cAMP in oocytes were detected by ELISA kit. The concentrations of progesterone and estrogen were measured by radioimmunoassay. Oocytes in each group were no less than 100, and each test was repeated 3 times. Compared with the control group, the maturation rate of porcine oocytes cultured with 10^-8mol·L^-1 RFRP-3 was extremely significantly reduced (P<0.01). No significant change in cumulus expansion rate and cumulus expansion index were observed(P>0.05), but the expression of cumulus expansion factors (PTGS2, HAS2, PTX3) was extremely significantly decreased (P<0.01). RFRP-3 could extremely significantly reduce MPF content in porcine oocytes (P<0.01), and had no significant effect on cAMP content (P>0.05). After the addition of RFRP-3, the expression levels of GDF9 (P<0.01) and BMP15 (P<0.05) were promoted, while the expression levels of CCNB1 (P<0.05) and CDK1(P<0.05) were inhibited; meanwhile, the concentrations of progesterone and estrogen in medium of experimental groups were extremely significantly decreased (P<0.01). In conclusion, RFRP-3 inhibits in vitro maturation of porcine oocytes by regulating the expression of maturation related factors and cumulus expansion factors and the secretion of steroid hormones. This study will lay a theoretical foundation for elucidating the regulatory effect of RFRP-3 on mammalian oocytes.

Key words: RFRP-3, porcine, oocyte, IVM

中图分类号:

S828.3

贾金美, 冯建豪, 阳美霞, 张羽芳, 张虹亮, 王水莲. RFRP-3对猪卵母细胞体外成熟的影响[J]. 畜牧兽医学报, 2022, 53(4): 1133-1142.

JIA Jinmei, FENG Jianhao, YANG Meixia, ZHANG Yufang, ZHANG Hongliang, WANG Shuilian. Effect of RFRP-3 on in Vitro Maturation of Porcine Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1133-1142.

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