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目的探讨叶酸(folic acid,FA)对非洲爪蟾卵母细胞体外成熟的影响及作用机制。方法将健康卵母细胞移入含不同浓度FA(分别为125μmol/L、250μmol/L、500μmol/L和1 000μmol/L)的ND96培养液中体外培养,同时设立阴性对照组和孕酮阳性对照组。加FA后每隔1 h观察1次细胞生发泡破裂(germinal vesicle break-down,GVBD),记录发生GVBD的卵母细胞数量,计算GVBD率,同时用10%三氯乙酸溶液固定后,切开进行确认。观察6 h后收集各组卵母细胞,采用蛋白免疫印迹实验(Western Blotting)检测CyclinB2,Mos,p-ERK1/ERK1蛋白的表达。结果除FA 125μmol/L、250μmol/L、500μmol/L、1 000μmol/L组2 h时与空白对照组GVBD率之间差异无统计学意义,其余各记录点的GVBD率与空白对照组间差异均有统计学意义(均有P<0.05);Western Blotting分析显示,FA和孕酮处理后卵母细胞的p-ERK1,CyclinB2,Mos蛋白表达水平明显升高。结论 FA可促进爪蟾卵母细胞体外成熟过程中GVBD发生,提示FA对爪蟾卵母细胞的成熟具有促进作用,其作用机制可能与干扰成熟促进因子和丝裂原活化蛋白激酶有关。
Objective To investigate the effect of folic acid (FA) on the in vitro maturation of Xenopus laevis oocytes and its mechanism. Methods Healthy oocytes were cultured in ND96 medium containing different concentrations of FA (125μmol / L, 250μmol / L, 500μmol / L and 1000μmol / L respectively), and negative control group and progesterone positive control group . After every 1 h of FA, germinal vesicle break-down (GVBD) was observed and the number of GVBD-producing oocytes was recorded to calculate the GVBD rate. The cells were fixed with 10% trichloroacetic acid and cut open Undergo verification. Oocytes were harvested after 6 hours. The expression of CyclinB2, Mos and p-ERK1 / ERK1 protein were detected by Western Blotting. Results There was no significant difference in GVBD rate between control group and control group at 125μmol / L, 250μmol / L, 500μmol / L, 1 000μmol / L FA for 2 hours. (All P <0.05). The results of Western Blotting showed that the expressions of p-ERK1, CyclinB2 and Mos protein in oocytes treated with FA and progesterone were significantly increased. Conclusions FA can promote the development of Xenopus oocytes during GVBD maturation, suggesting that FA can promote the maturation of Xenopus oocytes. The mechanism may be related to interfering with maturation promoting factor and mitogen-activated protein kinase.