两种StAR复制基因在尼罗罗非鱼类固醇生成中的功能和作用机制研究

Steroidogenic acute regulatory protein (StAR) transports cholesterol, the substrate for steroid synthesis, to the inner membranes of mitochondria. Our previous research found that there were two different StAR isoforms, named as StAR1 and StAR2 in non-mammalian vertebrates. In tilapia, StAR1 was dominantly expressed in the testis and head kidney; while StAR2 was expressed exclusively in the gonads. We speculate that StAR1 is likely to be required for cortisol production in the head kidney, and StAR2 is probably involved in estrogen production during early sex differentiation in XX gonads. In contrast, both StARs might be required for androgen production in testes. To clarify the molecular mechanisms of StAR1 and StAR2 in fish steroidogenesis, a series of investigations will be performed. In vivo gene knockout of StAR1 and StAR2 by CRISPR/Cas9 will be carried out. Simultaneously, rescue assay in StAR1 knockout XY individuals by both StAR1 overexpression and steroids (androgen or cortisol) treatment, StAR1 knockout XX individuals by cortisol treatment and StAR2 knockout XX individuals by both StAR2 overexpression and estrogen treatment, StAR2 knockout XY individuals by androgen treatment will be carried out. At the same time, the serum hormone levels, gonad histological phenotype and the expression of genes involved in sex determination and differentiation will be checked. Our final goal is to clarify the molecular mechanisms that StAR1 and StAR2 regulates fish steroidogenesis and then gonadal sex determination and differentiation, growth and osmotic adjustment process. The implementation of this proposal will provide a theoretical basis for the endocrine studies for teleosts.

类固醇合成急性调节蛋白(StAR)介导类固醇合成底物-胆固醇由线粒体外膜转入内膜，因而对类固醇激素合成至关重要。我们前期研究发现非哺乳脊椎动物有两个StAR基因（StAR1和StAR2），在罗非鱼中StAR1在头肾和精巢表达，StAR2在性腺表达，我们推测StAR1可能参与可的松、雄激素合成，StAR2参与雌激素与雄激素合成。本项目拟通过敲除罗非鱼StAR1和StAR2，对StAR1敲除XY个体进行基因过表达和可的松、雄激素回救，XX个体施以可的松回救；对StAR2敲除XX个体进行过表达和雌激素回救、XY个体施以雄激素回救。检测上述实验过程中血清激素水平，性腺表型及性别决定与分化相关基因表达，深入研究两种StAR在罗非鱼类固醇激素合成中扮演的角色，阐明StAR通过调控不同类固醇激素合成，进而调控硬骨鱼类性别决定与分化、生长发育、渗透压调控等过程的分子机制，为硬骨鱼类内分泌研究提供重要参考。

类固醇激素在脊椎动物性别分化、性腺发育和内分泌稳态等方面有重要作用，类固醇合成急性调节蛋白(StAR)介导类固醇合成底物-胆固醇由线粒体外膜转入内膜，因而对类固醇激素合成至关重要。硬骨鱼类存在两个StAR基因（StAR1和StAR2），在罗非鱼中StAR1和StAR2具有不同的时空表达模式，暗示StAR1和StAR2在雌雄个体类固醇生成中具有不同作用。因此，本项目以罗非鱼（Oreochromis niloticus）为实验材料，展开系列工作，发现（1）敲除XY个体StAR1、StAR2导致雄激素水平降低，精巢发育异常，下游类固醇通路基因表达异常，但后期部分恢复，其中StAR1、StAR2缺失引起的表型并不相同，暗示两个StAR在雄激素合成中作用的通路及分子机制不同；（2）敲除XX个体StAR2基因出现两种表型：性逆转、未性逆转（不可产卵和可产卵）。其中性逆转XX个体产精液可育；未性逆转个体有两种类型：腹部胀满，生殖突似雄鱼，性腺血管明显，积绿色液体，含各级卵细胞，但卵无法排出，坏死于体内；可排卵XX个体，但血清雄激素水平高于对照XX个体，雌激素水平高于XY个体。综上，StAR2参与XX个体早期雌激素合成，因而在雌性性别决定与分化中有重要作用；StAR1和StAR2同时参与XY个体雄激素合成，在精巢发育中有重要作用，但两个StAR的作用通路可能不同。以上研究结果推进了对硬骨鱼类内分泌的认识。