摘要
在一些爬行动物中,个体的性别完全取决于胚胎发育过程中的环境温度,称之为温度依赖型性别决定(temperaturedependent sex determination,TSD).TSD的分子机制长期是个谜,特别是调控早期性腺分化的分子基础仍不清楚.本文通过表达分析和基因敲低手段研究了Sox9基因在红耳龟雄性性腺分化中的生物学功能,为TSD动物的性别决定和性腺发育的分子机制的研究奠定了基础.qRT-PCR显示,从性腺分化前的17期起,Sox9呈现产雄温度(male-producing temperature,MPT)性腺特异性高表达,而在产雌温度(female-producing temperature,FPT)性腺中表达水平极低.免疫组化进一步证实了SOX9蛋白的MPT特异性表达趋势,其定位于Sertoli前体细胞核中.温度置换实验显示,与MPT性腺相比,MPT→FPT性腺中(16期置换)的Sox9表达量从17期起就显著降低,表明Sox9能快速响应温度变化.同时MPT性腺经过雌激素处理后,Sox9表达量亦快速下调.功能缺失研究显示,经过Sox9-RNAi处理后,90.9%(20/22)的MPT性腺结构明显雌性化,皮质区高度发育,髓质区退化,揭示Sox9的敲低能导致雄性向雌性性逆转.上述研究表明,Sox9是红耳龟早期睾丸分化的关键调控因子,参与TSD的雄性分化通路.
In reptiles with temperature-dependent sex determination（TSD）, the individual＇s gender depends completely on the incubation temperature of the eggs. The molecular mechanism underlying temperaturedependent sex determination has been a long-standing mystery, in particular, the factors responsible for triggering the differentiation of biopotential gonads either into a testis or an ovary are unknown. In this study, the regulatory role of Sox9 on male gonadal differentiation in Trachemys scripta was investigated through expression analysis and gene-modulating approach, providing important reference for the study of sexual differentiation and gonadal development mechanism of reptiles with TSD and an effective molecular techniques for controlling the sex of animals. Real-time PCR showed that the MPT-specific expression of Sox9 in embryonic gonads initiated early at stage 17, preceding the onset of gonadal differentiation, however, the FPT embryonic gonads exhibited extremely low expression level of Sox9 throughout the embryogenesis. Immunofluorescence further confirmed the SOX9 protein was abundantly expressed and mainly localized in the nuclei of precursor Sertoli cells in MPT gonads. The temperature shifts experiments showed that the expression of Sox9 in MPT→FPT（which was shifted at stage 16）gonads was significantly reduced from the stage 17, indicating that Sox9 can respond rapidly to the new temperature. Meanwhile, the expression of Sox9 was also down-regulated rapidly in the MPT gonads treated with estrogen. Loss-of-function analysis showed that 90.9% MPT embryos with Sox9 knockdown exhibited male to female sex reversal, characterized by obvious feminization gonads with well organized outer cortex and degradated medullary area. These above results indicate that Sox9 is required for testicular differentiation in a TSD system.
作者
张海艳
孙伟
周英杰
包海声
蔡晗
钱国英
葛楚天
ZHANG Hai-Yan;SUN Wei;ZHOU Ying-Jie;BAO Hai-Sheng;CAI Han;QIAN Guo-Ying;GE Chu-Tian(College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China)
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2018年第6期653-662,共10页
Progress In Biochemistry and Biophysics
基金
浙江省自然科学基金(LY14C190008)
浙江省农业(水产)新品种选育重大科技专项(2016C02055-4-1)
宁波市农业重大项目(2017C110012)
浙江省重中之重学科"生物工程"开发基金(ZS2016007
ZS20170011)资助项目~~
关键词
SOX9基因
性逆转
温度依赖型性别决定
红耳龟
RNA干扰
Sox9 gene
sex reversal
temperature-dependent sex determination（TSD）
Trachemys scripta
RNAinterference
