红耳龟Amh基因在温度依赖型性别决定中的功能  

FUNCTIONAL ANALYSIS OF AMH IN TEMPERATURE DEPENDENT SEX DETERMINATION IN TRACHEMYS SCRIPTA

作　　者：陈佳文 孙伟[1,2] 金琳[1,2] 惠航博 雷建东 胡佳暄 王宗吉 葛楚天 CHEN Jia-Wen;SUN Wei;JIN Lin;HUI Hang-Bo;LEI Jian-Dong;HU Jia-Xuan;WANG Zong-Ji;GE Chu-Tian(Institute of Animal Sex and Development,Zhejiang Wanli University,Ningbo 315100,China;College of Biological and Environmental Sciences,Zhejiang Wanli University,Ningbo 315100,China)

机构地区：[1]浙江万里学院动物性别与发育重点研究所,宁波315100 [2]浙江万里学院生物与环境学院,宁波315100

出　　处：《水生生物学报》2023年第11期1858-1868,共11页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金(U22A20529、31922084和31872960);浙江省杰出青年科学基金(LR19C190001);宁波市自然科学基金(2022J193);浙江省高校基础科研经费项目;浙江省“生物工程”一流学科学生创新项目(CX2020001)资助。

摘　　要：为研究Amh基因在TSD(Temperature-dependent sex determination,温度依赖型性别决定)中的功能,文章以红耳龟Trachemys scripta为TSD动物模型,分析了Amh在胚胎性腺中的精细表达特征和细胞定位;通过基因功能缺失和获得研究手段,验证了Amh在TSD中的具体功能。表达分析结果显示,Amh基因在性腺分化启动前的第15期便已呈现产雄温度(Male-producing temperature,MPT)性腺高表达;AMH蛋白主要定位在MPT性腺sertoli前体细胞上,而在各个发育时期的FPT(Female-producing temperature,产雌温度)性腺中仅检测到极其微弱的Amh mRNA和蛋白表达信号。RNA干扰实验显示,敲低Amh后的MPT性腺出现了雄性向雌性完全性逆转,雄性分化因子Sox9明显下调,雌性分化因子Foxl2和Cyp19a1显著上调;相反地,异位表达Amh后的FPT性腺则转向睾丸方向分化,Foxl2和Cyp19a1表达被抑制,Sox9表达上升。上述研究表明,Amh是启动红耳龟早期睾丸分化必需且充分的关键因子,处于TSD雄性分化分子通路上游。The regulatory role of Amh in early sex determination and gonadal differentiation has been studied in some GSD(genetic sex determination) animals,such as fish,however,the biological function of this gene in species with temperature-dependent sex determination(TSD) has not been reported.In this study,the red-eared slider turtle Trachemys scripta was used as an animal model of TSD to analyze the expression characteristics and cellular localization of Amh in embryonic gonads.Then,the specific function of Amh in TSD was identified by means of gene loss-and gain-of-function studies(LV-Amh-shRNA and LV-Amh-OE vector systems).The expression analysis showed that Amh was highly expressed in MPT(male-producing temperature) gonads of stage 15,prior to the onset of gonadal differentiation.AMH protein was mainly localized in the precursor sertoli cells of MPT gonads,however,the FPT(female-producing temperature) gonads exhibited extremely low expression levels of Amh mRNA and protein throughout the embryogenesis.RNA interference experiments showed complete male-to-female sex reversal in MPT gonads with Amh knockdown,characterized by obvious feminization of gonads,female-styled germ cell distribution,significant down-regulation of testicular marker Sox9,and remarkable upregulation of ovarian markers Foxl2 and Cyp19a1.Conversely,ectopic expression of Amh led to largely masculinized FPT gonads,a decline in Foxl2 and Cyp19a1,and production of Sox9.The statistical results showed that the sex reversal rates induced by LV-Amh-shRNA and LV-Amh-OE were92.86%(26/28) and 66.67%(26/39),respectively.These data provided evidence that Amh is both necessary and sufficient to initiate early testicular differentiation in T.scripta,and it is located upstream of the male molecular pathway of TSD,laying the foundation for the analysis of the TSD molecular mechanism.

关 键 词：Amh基因 温度依赖型性别决定 性腺分化 性逆转 红耳龟 

分 类 号：Q344.1[生物学—遗传学]