Abstract：A certain concentration of lysobisphosphatidic acid (LPA) can promote oocyte maturation, while cumulus cells and oocyte-secreted factors (OSFs) can guarantee the normal development and function of oocytes. The purpose of this study was to investigate the expression of oocyte-secreted factors (OSFs) regulated by exogenous lysophosphatidic acid (LPA) in cumulus cells. The cumulus cells of Bos grunniens cultured in vitro were used as the research object, and exogenous LPA (0, 5, 15, 30 and 50 µmol/L) was supplemented into the cultured cumulus cells. PCR, qPCR, immunofluorescence tech and Western blot (WB) were used to evaluate the expression status of OSFs after exogenous LPA regulation. qPCR results showed that when the concentration of exogenous LPA was 15 µmol/L, the relative expression levels of growth differentiation factor 9 (GDF9) and fibroblast growth factors (FGF10) mRNA were the highest. And when the concentration was greater than 15 µmol/L, the expression levels of GDF9 and FGF10 mRNA decreased. The relative expression levels of bone morphogenetic proteins 1 (BMP1) and BMP15 mRNA were the highest when the concentration of LPA was 30 µmol/L. However, when the concentration was greater than 30 µmol/L, the expression of BMP15 mRNA decreased, and the relative expression level of BMP1 mRNA did not change significantly. Western blot results showed that when the concentration of LPA was 30 µmol/L, the relative expression level of GDF9, BMP1 and BMP15 protein was the highest. However, when the concentration of LPA was more than 30 µmol/L, the expression levels of BMP1 and BMP15 proteins were decreased, while the expression of GDF9 protein was inhibited. Compared with the response of other OSFs proteins to LPA, the relative expression level of FGF10 protein and the concentration of added LPA were dose-dependent within a certain range of LPA concentrations. Immunofluorescence results showed that the fluorescence labeling of OSFs in the cumulus cells of 0 µmol/L treatment group was mainly concentrated in the cytoplasm and nucleus, while the fluorescence labeling of OSFs in the cytoplasm of cumulus cells was significantly (P<0.05) enhanced after exogenous LPA treatment. This study confirmed that exogenous LPA can regulate the expression of OSFs in yak cumulus cells at the gene and protein levels during in vitro culture, and the addition of appropriate concentration of LPA can increase the secretion of related cytokines in yak cumulus cells. In turn, the development of oocytes is regulated. This study can be used as a reference for yak and other mammalian oocyte maturation in vitro, and is helpful to the development of related cell research.
刘斌, 王靖雷, 高丽青, 许瑞华, 张倩, 王萌, 王立斌, 余四九, 徐庚全, 潘阳阳. LPA 对牦牛卵丘细胞主要卵源因子表达的影响[J]. 农业生物技术学报, 2023, 31(5): 989-1000.
LIU Bin, WANG Jing-Lei, GAO Li-Qing, XU Rui-Hua, ZHANG Qian, WANG Meng, WANG Li-Bin, YU Si-Jiu, XU Geng-Quan, PAN Yang-Yang. Effect of LPA on the Expression of Main Oocytesreted Factors in Cumulus Cells of Yak (Bos grunniens). 农业生物技术学报, 2023, 31(5): 989-1000.
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