Effects Of Lipopolysaccharide On Maturation Of Bovine Oocyte In Vitro And Its Possible Mechanisms

Lipopolysaccharide(LPS)disturbs the secretion of gonadotropin and steroid hormone,impairs endometrial function and decreases implantation efficiency.However,there is little information regarding the effects of LPS on cyclic ovary activity,especially oocyte maturation.Therefore,we aimed to investigate the effects of LPS on the maturation potential of bovine oocyte and the relevant mechanism involved.The results were as follows:1.Experiment I studied the immune response of bovine cumulus-oocyte complexes(COCs)exposed to LPS.The results showed that:(1)The first polar body extrusion rate was decreased to some extent in LPS treatment groups(1,10μg/m L)compared with the control group.The rate of 10μg/m L LPS-treated group was significantly lower than that of the control group(P < 0.05).Thus,10μg/m L was selected as the LPS treatment concentration for subsequent experiments.(2)LPS treatment significantly elevated the expression levels of TLR4 m RNA and TLR4 protein in COCs(P < 0.05).(3)The intracellular signaling protein p-p38 MAPK level and NF-κB activity were markedly increased in LPS-treated COCs(P < 0.05).(4)The concentrations of proinflammatory cytokines,including IL-1β,TNF-α and IL-6,were significantly increased in the maturation media-supplemented with LPS(P < 0.05).2.Experiment II investigated the effects of LPS on bovine oocyte maturation potential and parthenogenetic developmental competence.The results showed that:(1)LPS treatment did not affected the cumulus cell expansion index,but the cytoplasmic maturation,characterized by peripheral cortical granules distribution,was significantly impaired in LPS-treated oocytes(P < 0.05).(2)LPS exposure significantly increased intracellular reactive oxygen species(ROS)levels in matured oocytes,and the expression levels of antioxidant-associated genes thioredoxin(Trx),thioredoxin 2(Trx2)and peroxiredoxin 1(Prx1)were significantly decreased compared to the control group(P < 0.05).(3)The incidence of early apoptotic nuclei and the release of cytochrome c were significantly increased in LPS-treated oocytes(P < 0.05).(4)LPS treatment significantly reduced the cleavage(73.3 ± 1.76% vs.82.3 ± 0.88%,n = 230),morula(30.7 ± 0.88% vs.47.3 ± 2.40%)and blastocyst formation rates(14.8 ±1.96% vs.34.2 ± 0.8%)in parthenogenetically activated oocytes,whereas the apoptotic rate in the resultant blastocysts was markedly increased(P < 0.05).3.Experiment III explored the underlying mechanism by which LPS impairs the maturation potential of bovine oocyte.The results showed that:(1)LPS exposure significantly delayed the cell cycle progression,as the percentage of oocytes arrested at metaphase I(MI)was significantly increased compared to the control group(P < 0.05).(2)The abnormal spindle rate was significantly higher in LPS-treated oocytes(12.40 ± 0.95%,n = 138)than that in the control group(4.87 ± 0.29%,n = 129),accompanied by disrupted localization and level of phosphorylated mitogen-activated protein kinase(p-MAPK)(P < 0.05).(3)LPS treatment significantly increased intracellular dihydroethidium(DHE)and ROS levels in MI oocytes(P < 0.05).(4)LPS exposure significantly increased the early apoptotic rate in MI oocytes(P < 0.05).The pro-apoptotic caspase-3 and Bax m RNA levels and caspase-3 proteinlevel were significantly increased(P < 0.05),whereas the anti-apoptotic Bcl-2 and XIAP transcript abundance were decreased in LPS-treated oocytes(P < 0.05).(5)The global dimethyl-histone H3 lysine4(H3K4me2)level was significantly increased(P < 0.05),while the levels of global DNA methylation(5-m C)and dimethyl-histone H3 lysine 9(H3K9me2)were markedly decreased in oocytes treated with LPS(P < 0.05).