褪黑素对水牛卵母细胞体外成熟的影响及其受体介导机制的探究

doi:10.11843/j.issn.0366-6964.2019.02.009

摘要/Abstract

摘要：

旨在研究外源性褪黑素（melatonin，MT）对水牛卵母细胞体外成熟的影响及其受体介导机制。进行以下试验：1）通过免疫荧光技术检测了水牛卵丘细胞和卵母细胞上褪黑素的两种受体MT1和MT2的表达情况。2）在体外成熟培养液中添加不同浓度褪黑素（0、10^-9、10^-8、10^-7 mol·L^-1），观察褪黑素对水牛卵母细胞体外成熟及其随后体外受精胚胎发育的影响。3）根据褪黑素最优浓度，成熟液中添加不同处理组合：未处理组、褪黑素（10^-8 mol·L^-1 MT）、褪黑素受体拮抗剂（10^-8 mol·L^-1 LZU）、褪黑素受体激动剂（10^-8 mol·L^-1 ⅡK7）、同时添加褪黑素受体拮抗剂和褪黑素（10^-8 mol·L^-1 LZU+10^-8 mol·L^-1 MT），处理卵母细胞24 h后，统计卵母细胞第一极体排出率，并对第一极体的卵母细胞进行ELISA Kit检测，同时，成熟后的卵母细胞分别进行体外受精，并统计其分裂率和囊胚率。结果显示：1）在水牛卵丘细胞和卵母细胞上均发现有褪黑素受体MT1和MT2；2）褪黑素处理的各组卵母细胞成熟率均显著高于0 mol·L^-1组（P<0.05）。随后，各组受精后的分裂率也显著高于0 mol·L^-1 MT组（P<0.05），而且10^-8和10^-7 mol·L^-1 MT组的囊胚率显著高于0 mol·L^-1 MT组（P<0.05）；3）褪黑素受体拮抗剂（10^-8 mol·L^-1 LZU）组的卵母细胞成熟率、体外受精胚胎分裂率和囊胚率与未添加组相比，差异均不显著（P>0.05）；褪黑素受体激动剂（10^-8 mol·L^-1 ⅡK7）组的水牛卵母细胞成熟率、体外受精胚胎分裂率和囊胚率均显著高于未添加组（P<0.05），但与褪黑素组（10^-8 mol·L^-1 MT）相比差异不显著（P>0.05）；同时添加褪黑素受体拮抗剂和褪黑素（10^-8 mol·L^-1 LZU+10^-8 mol·L^-1 MT）组的水牛卵母细胞成熟率、体外受精后胚胎的分裂率和囊胚率与未添加组相比，差异不显著（P>0.05）。4）褪黑素处理组的卵母细胞内cAMP的含量显著低于未处理组，而cGMP含量显著高于未处理组（P<0.05）。综上表明，褪黑素通过与细胞膜G蛋白偶联受体MT1和MT2结合，从而抑制了cAMP合成，提高cGMP含量，进而促进卵母细胞成熟及早期胚胎发育。

Abstract:

The purpose of this study was to investigate the effects of exogenous melatonin on in vitro maturation of buffalo oocytes and their receptor mediated mechanisms. The following tests:1)The expression of melatonin receptors MT1 and MT2 in cumulus cell and oocytes of buffalo were detected by immunofluorescence.2)The effects of different concentrations of melatonin (10^-9, 10^-8, 10^-7 mol·L^-1) added in the maturation medium on the efficiency of buffalo oocytes IVM and the development of the embryos after in vitro fertilization (IVF) were explored.3)According to the optimal concentration of melatonin, different treatment combinations were added into the mature liquid:untreated,melatonin (10^-8 mol·L^-1 MT),melatonin receptor antagonist (10^-8 mol·L^-1 LZU),melatonin receptor agonist (10^-8 mol·L^-1 ⅡK7),melatonin+ melatonin receptor antagonist (10^-8 mol·L^-1 MT+10^-8 mol·L^-1 LZU).The first polar body excretion rate of oocyte was calculated and the first polar body oocytes were detected by ELISA Kit after 24 h,and then mature oocytes were fertilized in vitro and their rates of division and blastocyst were calculated.The results showed that:1)The both of the MT1 and MT2 receptors were presented in the buffalo cumulus cells and oocytes; 2)The first polar body exhaust rates of oocytes cultured in the maturation medium added with different concentrations of melatonin (10^-9,10^-8,10^-7 mol·L^-1) were significantly higher than the 0 mol·L^-1MT group and the embryonic cleavage rate in each treatment group was also significantly higher than in 0 mol·L^-1 MT group (P<0.05),moreover,the blastocyst rate of 10^-8 and 10^-7 mol·L^-1 MT groups was significantly higher than that of control group (P<0.05); 3)The oocyte maturation rate, division rate and blastocyst rate of embryo in the melatonin receptor antagonist group (10^-8 mol·L^-1 LZU) were not significantly different from those in the untreated group (P>0.05).The oocyte maturation rate, embryo division rate and blastocyst rate after in vitro fertilization were significantly higher in the group of melatonin receptor agonist group(10^-8 mol·L^-1 ⅡK7) than these in the untreated group(P<0.05),however, the difference was not significant compared with the melatonin group (10^-8 mol·L^-1 MT) (P>0.05); The oocyte maturation rate,division rate and blastocyst rate of embryo in melatonin+melatonin receptor antagonist group (10^-8 mol·L^-1 MT+10^-8 mol·L^-1 LZU) were not significantly different compared with that in untreated group (P>0.05). 4) The concentration of cAMP in the oocyte of melatonin group was significantly reduced compared with that of the untreated group, but the concentration of cGMP was significantly increased than the untreated group (P<0.05). It can be seen from this study, by binding to the cell membrane G protein coupled receptor MT1 and MT2, melatonin inhibits cAMP synthesis and increases cGMP content, thereby promoting the mature and early embryonic development of oocytes.

中图分类号:

S823.8⁺3.2

郭振伟, 黄永军, 范威宏, 文冬梅, 沈朋雷, 潘尔科, 陆凤花, 石德顺. 褪黑素对水牛卵母细胞体外成熟的影响及其受体介导机制的探究[J]. 畜牧兽医学报, 2019, 50(2): 314-322.

GUO Zhenwei, HUANG Yongjun, FAN Weihong, WEN Dongmei, SHEN Penglei, PAN Erke, LU Fenghua, SHI Deshun. The Effect of Melatonin on the Maturation of Oocyte in vitro and Its Receptor-mediated Mechanism in Buffalo[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(2): 314-322.

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