柠檬苦素对小鼠卵母细胞体外成熟及其体外受精胚胎发育的影响

doi:10.16431/j.cnki.1671-7236.2022.10.021

Abstract

Abstract: 【Objective】 The aim of this study was to investigate the effects of limonin (Lim) on the development potential of in vitro maturation (IVM) mouse oocytes and subsequent in vitro fertilization (IVF) embryos,in order to provide reference for the optimization of in vitro maturation culture system.【Method】 Different concentrations of Lim (0,10,20,50 μmol/L) were added into the culture medium of mouse IVM culture medium.After 12 h of maturation culture,the excretion rate of the first polar body (PBI) of mouse oocytes was calculated,and the optimal concentration of Lim was selected.The optimal concentration of Lim was added to IVM culture medium,0 μmol/L Lim was used as control group,after mature culture for 12 h,the levels of reactive oxygen species (ROS),glutathione (GSH) and mitochondrial membrane potential (MMP) were detected by immunofluorescence staining.The mRNA expression levels of antioxidant and apoptosis-related genes in oocytes were detected by Real-time quantitative PCR.The embryo cleavage rate and blastocyst rate were calculated at 24 h and 3.5 d after in vitro fertilization.The total number of blastocyst cells and the ratio of apoptotic cells in blastocysts were detected by Fluorescein-dUTP staining and Hoechst 33342 staining.【Result】 Compared with 0 μmol/L Lim group,PBI excretion rate of oocytes in 20 μmol/L Lim group was significantly increased (P＜0.05),and 20 μmol/L Lim was used in the subsequent experiments.Compared with control group,ROS levels in oocytes of mice in 20 μmol/L Lim group were significantly decreased (P＜0.05),GSH and MMP levels were significantly increased (P＜0.05).The expression of antioxidant related genes (GPx3,CAT and Prdx3) and anti-apoptotic related genes (Bcl-2 and Bcl-xl) were significantly up-regulated (P＜0.05),while the expression of pro-apoptotic related gene (Caspase-3) was significantly down-regulated (P＜0.05).After in vitro fertilization,the cleavage rate,blastocyst rate and blastocyst total cell number of embryos were significantly increased (P＜0.05),and the apoptosis rate of cells in blastocyst was significantly decreased (P＜0.05).【Conclusion】 Adding 20 μmol/L Lim to in vitro maturation medium could improve the quality of mouse oocytes by inhibiting oxidative stress and cell apoptosis and increasing MMP level,thus improving the development potential of in vitro fertilization embryos.

Key words: mouse; oocytes; limonin; in vitro maturation; oxidative stress

CLC Number:

JIAO Anhui, ZHANG Xiaomeng, ZHAO Yuhan, WANG Yu, GAO Qingshan. Effects of Limonin on in vitro Maturation of Mouse Oocytes and Embryo Development of in vitro Fertilization[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3891-3900.

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Effects of Limonin on in vitro Maturation of Mouse Oocytes and Embryo Development of in vitro Fertilization

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