孕酮激素诱发斑马鱼精原细胞增殖分化的机理研究

Recently, research reports showed that 17α,20β-dihydroxy-4-pregnen-3-one (DHP), the biologically active progesterone molecule in many teleost fishes, has multiple regulatory functions during the proliferation and differentiation of spermatogonia. Our previous studies showed that, in zebrafish, exposure to DHP in vivo induced the proliferation of type A spermatogonia and their proliferation and differentiation into type B spermatogonia and also spermatocytes. Besides, after characterization of the zebrafish nuclear progestin receptor (nPR), further analyses indicated that the nPR was expressed in spermatogonia and Sertoli cells and Leydig cells in the testis. Moreover, another study indicates nPR was expressed robustly in the preoptic region of the hypothalamus in the brain. The localization of Pgr suggests that it mediates DHP regulation of spermatogonial proliferation and differentiation. The present study aims at elucidating the molecular mechanism(s) of these DHP effects. Using the zebrafish as model animal, we propose to investigate the (1) potential effect of DHP on the regulation of reproductive neuroendocrine hormones in the hypothalamus-pituitary; (2) effects of DHP directly on testis enriched in type A spermatogonia, by using a recent developmental primary testis tissue culture system; (3) the target genes of DHP in spermatogonia and Sertoli cells (to be separated from transgenic zebrafish using fluorescence-activated cell sorting), by applying suppression subtractive hybridization and proteomic approaches. The results from this study can further our understanding in reproductive endocrinology in teleost, and can also provide useful scientific knowledge for improving the biotechnology of fish propagation in China.

近年来，已报道的和我们前期开展的研究结果初步表明，鱼类特有的孕酮激素- - 17α,20β-双羟孕酮（DHP）参与了精原细胞增殖分化过程的调控。同时，应用斑马鱼开展的研究已经明确，孕酮激素核受体（nPR）在下丘脑视前区以及精巢内多种细胞上均有表达。为了进一步阐明孕酮激素诱发斑马鱼精原细胞增殖分化的机理，本项目拟在前期研究工作的基础上，开展以下研究内容：（1）应用活体暴露的方法，研究DHP是否参与下丘脑-垂体系统中生殖神经内分泌激素的调控；（2）应用以A型精原细胞为主的精巢进行体外培养实验，研究DHP对精原细胞增殖分化的作用；（3）应用荧光激活细胞分选法从转基因斑马鱼精巢中分离出精原细胞和支持细胞,再结合抑制性消减杂交技术和蛋白质组学技术，研究DHP对精原细胞和支持细胞基因表达的调控。研究结果不仅可以丰富鱼类生殖内分泌调控研究的基础理论，也可为今后我国鱼类人工繁殖技术的完善提供科学依据。

近年来，应用斑马鱼开展的研究已经明确，孕酮激素核受体（Pgr）在下丘脑视前区以及精巢内多种细胞上均有表达。为了进一步阐明孕酮激素在雄性斑马鱼生殖过程中的作用，本项目拟在前期研究工作的基础上，开展以下研究内容：（1）研究DHP是否参与下丘脑-垂体系统中生殖神经内分泌激素的调控；（2）应用精巢进行体外培养实验，研究DHP对支持细胞基因表达的调控.主要研究结果如下：.1. 本文采用Golden TALEN技术，成功构建了孕酮核受体基因敲除家系（Pgr-/-）。Pgr-/-的孕酮核受体（Pgr）DNA序列缺少7个碱基，氨基酸编码出现移码突变，蛋白质电泳技术未检测到蛋白表达。组织学显示，Pgr-/-雄鱼精巢组织中成熟精子量少于野生型雄鱼。.2. 野生型雄鱼垂体中促性腺激素β亚基基因（fshb和lhb）表达呈现周日变化趋势，高峰表为06:00。该时间点的Pgr-/-雄鱼垂体中fshb和lhb表达量则显著低于同时间点的野生型雄鱼。.3. 野生型雄鱼活体暴露于DHP水溶液后，垂体中fshb和lhb 表达量随DHP暴露浓度的增加和暴露时间的延长均出现上升趋势；而暴露于DHP+ RU486（DHP核受体拮抗剂）的水溶液后，垂体中fshb和lhb 表达量出现随RU486暴露浓度的增加而降低的趋势。.4. 野生型雄鱼和Pgr-/-雄鱼活体分别暴露于DHP水溶液中24h后，Pgr-/-雄鱼垂体中fshb和lhb表达量显著低于野生型。垂体组织离体暴露于DHP后，Pgr-/-雄鱼垂体中的lhb表达量与野生型雄鱼比较无显著性差异，但fshb表达量显著低于野生雄鱼。荧光原位共杂交技术显示，Pgr与fshb共同表达在相同的细胞中，而Pgr与lhb表达在不同的细胞中。.5.野生型雄鱼脑中th2和垂体中drd2a和drd2b 表达量出现周日变化。野生型雄鱼和Pgr-/-雄鱼活体暴露于DHP水溶液后，脑中th2表达量和垂体中drd2a和drd2b表达量均显著低于Pgr-/-雄鱼。垂体组织离体暴露于DHP后，Pgr-/-雄鱼垂体中drd2a和drd2b表达量显著低于敲除型雄鱼。.6. 野生型雄鱼精巢组织离体暴露于DHP后，RNA-seq测序结果显示，DHP处理组精巢组织中ifi44表达量极显著升高。原位杂交技术显示，ifi44在精巢组织的间质细胞和支持细胞中均有表达，且主要在间质细胞中表达。