骨形态发生蛋白-6对小鼠腔前卵泡体外成熟的影响

Effect of bone morphogenetic protein-6 on maturity of preantral follicles of mouse in vitro

目的：观察骨形态发生蛋白（BMP-6）在小鼠体内和体外卯泡的定量表达，探讨外源性BMP-6对小鼠腔前卵泡体外成熟的影响及其可能的作用机制。方法：采用免疫印迹观察BMP-6蛋白在不同年龄小鼠体内，以及体外培养不同时间的卵泡中定量表达情况；采用腔前卵泡体外培养方法，将不同浓度的rhBMP-6添加至培养液中持续到培养8d或11d，观察腔前卵泡的生长、成熟情况；应用ELISA法测定和比较添加BMP-6不同剂量组在培养6、10、11d的培养液中雌二醇和孕酮的含量。结果：免疫印迹结果显示，在体内和体外培养的卵泡中均检测到BMP-6蛋白的表达，体外表达量明显低于体内。体外培养中，相对于对照组，50ng／mlBMP-6添加至培养8d显著增加腔前卵泡的成熟率，而添加至培养11d显著降低腔前卯泡的成熟率；100ng／mlBMP-6添加至培养8d和11d对腔前卵泡的成熟率无影响；50和100ng／mlBMP-6对于腔前卵泡的生长，形态上并无明显变化。ELISA结果显示，50ng／mlBMP-6添加至培养11d组中，6、10、11d培养液中雌二醇和孕酮含量低于对照组，而添加至培养8d组中，10、11d培养液中雌二醇和孕酮显著升高；100ng／mlBMP-6添加至培养8d和11d，培养液中孕酮含量明显低于其他组，但雌二醇却明显高于其他组。结论_夕h源性BMP-6能够促进腔前卵泡的体外成熟，这种作用可能是通过影响类固醇激素的合成和（或）分泌而产生的。

Objective： To observe the quantitative expression of BMP-6 protein in the follicles in vivo and vitro, and investigate the effect of exogenous BMP-6 on the maturity of preantral follicles in vitro and the potential mechanism of the influence. Methods： The expression of BMP-6 protein in the follicles from the ovaries of mice of different ages and from different incubation time in vitro is examined by Western blotthing technique; Different concentrations of rhBMP 6 were added to culture solution for 8 d or 11 d of culture, and to observe development and maturity of preantral follicles of mouse in vitro ; The content of estradiol and progesterone in culture solution of the 6, 10, 11 d was detected by ELISA technique. Results： BMP-6 protein was expressed in the follicles in vivo and vitro, and BMP-6 protein expression index in vivo was more than in vitro. In the culture system in vitro, contrast to control, 50 ng/ml BMP-6 was added for 8 d of culture, increasing the maturing rate of preantal follicles significantly, and augment estradiol and progesterone content of the 10, 11 d culture solution notly, but 50 ng/ml BMP-6 was added for 11 d of culture, decreasing the maturing rate of preantal follicles markedly, reducing estradiol and progesterone content in the culture solution of the 6,10,11 d~ !00 ng/ml BMP-6 added for 8 d or 11 d of culture did not affect the maturing rate of preantal follicles notly, but augment estradiol content, at the same time, decreasing progesterone content markedly. Conclusion： BMP-6 protein is expressed in follicles of mouse, and the expression is in dynamic change with the development and maturity of preantral follicles; Exogenous BMP-6 can promote maturity of preantral follicles in vitro, and this impact is connected with the concentration and time of addition. Moreover, the role of BMP-6 is produced through its influence to the synthesis and/or secretion of steroid hormone.