双酚A通过Boule干扰精母细胞减数分裂的分子机制研究

Bisphenol A (BPA), an endocrine disrupting chemical, has adverse effects on male spermatogenesis. Our previous studies have found that BPA induced meiotic arrest in neonatal mice, at the same time the expression of the testis specific gene Boule and its downstream genes (Cdc25A and Cdk1) decreased significantly. Mouse testicular seminiferous tubules culture in vitro also confirmed that BPA could decrease the expression of Boule. As a key gene involving in the process of meiosis, whether Boule is an essential factor in meiotic arrest of spermatocyte induced by BPA and the mechanism by which BPA inhibits the expression of Boule are not yet known. This study aims to explore the role of Boule in BPA-induced meiotic arrest of spermatocytes. Moreover, analysis of Boule gene expression, including its transcription, mRNA localization and mRNA stability, will be carried out to explore the way of BPA inhibiting Boule expression. Time-course experiment will also be performed on neonatal mice to analyze the effect of BPA on meiosis of spermatocytes. This study will not only clarify the function and mechanism of Boule in BPA-induced meiotic arrest of spermatocyte through molecular biology, cell biology and animal work, but also have the potential to provide a new target and theoretical basis for the treatment on male infertility caused by environmental factors.

双酚A（BPA）作为环境内分泌干扰物，严重影响雄（男）性精子发生。课题组前期研究发现新生雄鼠接触BPA导致精母细胞减数分裂阻滞，降低睾丸特异基因Boule及其下游基因Cdc25A、Cdk1的表达，体外小鼠睾丸曲细精管培养模型也证实BPA能抑制Boule表达。作为生精细胞减数分裂调控基因，Boule是否是BPA诱导减数分裂阻滞的关键因子及其表达降低的调控机制尚未完全阐明。本课题拟通过睾丸曲细精管体外培养模型，明确BPA是否通过调控Boule表达导致减数分裂阻滞；再分析BPA对Boule基因的转录及其mRNA的核质转移和稳定性的影响，探索BPA调控Boule表达的机制；联合体内实验多个时间点分析BPA对新生小鼠精母细胞减数分裂的影响。该研究将从分子、细胞和动物整体水平系统研究Boule在BPA干扰生精细胞减数分裂中的作用及机制，为环境因素所致男性不育的干预提供新的靶点和理论依据。

本课题主要研究新生期暴露于双酚A（Bisphenol A，BPA）对小鼠睾丸第一个生精波生精细胞增殖分化的影响，并通过体外小鼠睾丸曲细精管培养模型，探讨BPA干扰精母细胞减数分裂的分子机制。体内实验：仔鼠出生后1-35天（PND 1-35），每天皮下注射溶于玉米油的BPA（0, 0.1, 5mg/kg.bw），分别于PND 9、18、22、35、70分批取材，称体重、睾丸重，HE染色结果显示BPA能干扰睾丸生精细胞正常的增殖分化。DNA分析结果显示BPA处理组PND 22小鼠睾丸中四倍体数目高于对照组（P＜0.05），PND 35小鼠睾丸中单倍体数目均高于对照组（P＜0.05），PND 70小鼠睾丸中单倍体细胞数目显著减少（P＜0.05）。磷酸化TMT相对定量蛋白质组学GO功能注释和KEGG通路分析发现BPA 0.1 mg/kg组Cdk1蛋白磷酸化水平明显下调，Cyclin B1-Cdk1、组蛋白激酶活性以及细胞周期的调控过程都受到显著的影响。Western Blot结果显示BPA组小鼠Boule、Cdc25、p-cdc2 P34（Tyr15）、Tnp2、Prm2蛋白表达量均显著减少（P＜0.05, P＜0.01）。体外实验：培养18日龄ICR小鼠睾丸生精细胞，加入BPA（0，1.0 µM）处理48 h，双酚A处理睾丸细胞导致圆形精子数量减少，BPA（0，1.0 µM，10 µM）处理4 h，生精细胞Boule、Wee1、Cdc25和p-cdc2 P34（Tyr15）蛋白表达均下降。荧光定量PCR法检测BPA处理组和Boule敲降组睾丸细胞中P19 mRNA水平显著升高（P<0.05），TP1 mRNA水平显著降低（P<0.05）。Boule敲降挽救组P19 mRNA和TP1 mRNA恢复到正常水平。Boule敲降组生精细胞Boule、Cdc25和p-cdc2 P34（Tyr15）蛋白表达显著下降，Boule敲降并挽救组Boule、Cdc25和p-cdc2 P34（Tyr15）蛋白表达得以恢复。以上研究表明双酚A可能通过Boule-Cdc25-cdc2通路干扰睾丸细胞的减数分裂。本研究的开展阐明了双酚A影响睾丸细胞的增殖分化的分子机制，并为环境内分泌干扰物雄（男）性生殖毒性的评价及其机制的研究提供了适宜的体内外模型。