双酚A对鱼类精子发生的危害及其分子机制研究

Bisphenol A (BPA) is an endocrine disturbing compound (EDC) ubiquitously distributed in aquatic environment. It was highly accumulated in wild fish and has detrimental effects on reproduction of male fish. However, the mechanism is not clear. In the preliminary experiment of BPA exposure on rare minnow Gibiocypris rarus, our valuable results suggest that BPA affect spermatogenisis at several levels in fish. In the present study, G. rarus and zebrafish Danio rerio will be exposed to BPA for for short and long duration. The sperm quality will be detected and ultrastructure of spermid and spermatozoa will be observed. Investigations will be performed using techniques of molecular biology, biochemistry and immunohistochemistry to explore the potentail mechanisms underlying the detrimental effects on spermatogenesis upon BPA exposure. The investigations include analysis of disturbed cytokine expression elicited by oxidative stress affecting function of blood-testis barrier upon BPA exposure, assay on apoptosis of spermatogenic cells as the permeability of blood-testis barrier is altered by BPA exposure, and study on BPA’s effects on cytoplasmic removal of spermid during spermiation. The present findings will clarify the negative effects on reproduction and fertility of male fish upon BPA in aquatic environment and will provide theoretical basis for the negative effects on wild fish population and its ecological protection under exposure of various ubiquitous phenolic endocrine disturbing compounds.

双酚A（BPA）是水环境中广泛分布的内分泌干扰物，它在鱼类体内有较高的富集，显著危害雄鱼的生殖，但其机制还不清楚；我们在稀有鮈鲫BPA暴露的预实验中获得了部分有价值的结果，提示BPA在多个方面影响鱼类精子发生。本项目拟以稀有鮈鲫和斑马鱼为实验鱼，经BPA短期和长期暴露后，观察它们的精子细胞和精子的亚显微结构变化，并检测成熟精子的质量，然后采用分子生物学、生化和免疫组化方法从多个方面探讨这些危害效应的可能发生机制，包括BPA引发氧化胁迫后干扰细胞因子的表达最终对血睾屏障通透性和功能的影响， BPA对生精细胞凋亡的影响，及BPA对排精过程中精子细胞内胞浆消除的影响。本项目的成果将有助于阐明水环境中BPA影响雄性鱼类生殖能力的分子机制，为水环境中普遍存在的各种酚类内分泌干扰物对鱼类种群的影响及其生态保护提供理论支持。

哺乳动物中的研究表明，BPA可以通过影响雄性精子发生而导致生殖毒性，而BPA对鱼类精子发生的影响还未有深入研究。本项目以稀有鮈鲫为试验动物，研究了BPA暴露对鱼类精巢组织生精细胞凋亡的影响及其机制，解析了BPA对稀有鮈鲫精子的形态及功能的影响的分子机制，分析了鱼类精巢不同细胞间连接方式对BPA暴露的响应，探究了BPA长期暴露对稀有鮈鲫雄鱼生殖影响的潜在效应，探讨了亲代BPA暴露后对子代稀有鮈鲫精巢分化及生长发育的影响。获得如下结果：BPA能够激活内源性线粒体凋亡途径和引起精巢细胞周期阻滞，导致精母细胞凋亡；BPA可以引起精子形态异常和运动能力改变而引起雄性生殖危害；BPA暴露可影响Aqps 5’侧翼ERE与ER的相互作用而干扰基因表达；BPA暴露导致Aqps蛋白表达和分布的异常，引起精子渗透压耐受性降低，从而降低其受精能力；BPA通过激活细胞因子所诱导JNK信号通路，抑制CX43和occludin 5 '侧翼对SP1富集，干扰血睾屏障组成蛋白表达和分布，进而导致精巢血睾屏障功能和完整性的破坏；长期BPA暴露通过诱导精巢氧化应激、炎症反应和DNA/组蛋白甲基化的异常，导致精子受精能力的下降。亲代BPA可以改变子代雄性个体中类固醇激素水平，干扰精巢发育调控和生殖相关基因的表达，影响子代雄性个体性腺发育和精子质量；雄性亲本BPA暴露可引起雄鱼精子miRNA表达异常，引起子代个体骨骼发育异常。