Jiang Dong-Neng, Mustapha Umar Farouk, Shi Hong-Juan, Huang Yuan-Qing, Si-Tu Jia-Xin, Wang Mei, Deng Si-Ping, Chen Hua-Pu, Tian Chang-Xu, Zhu Chun-Hua, Li Ming-Hui, Li Guang-Li
Guangdong Research Center on Reproductive Control and Breeding Technology of Indigenous Valuable Fish Species, Key Laboratory of Marine Ecology and Aquaculture Environment of Zhanjiang, Key Laboratory of Aquaculture in South China Sea for Aquatic Economic Animal of Guangdong Higher Education Institutes, Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China.
Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, China.
Comp Biochem Physiol B Biochem Mol Biol. 2019 Jul;233:35-45. doi: 10.1016/j.cbpb.2019.04.002. Epub 2019 Apr 10.
Gonadal soma-derived factor (Gsdf) is critical for testicular differentiation and early germ cell development in teleosts. The spotted scat (Scatophagus argus), with a stable XX-XY sex-determination system and the candidate sex determination gene dmrt1, provides a good model for understanding the mechanism of sex determination and differentiation in teleosts. In this study, we analyzed spotted scat gsdf tissue distribution and gene expression patterns in gonads, as well as further analysis of transcriptional regulation. Tissue distribution analysis showed that gsdf was only expressed in testis and ovary. Real-time PCR showed that both gsdf and dmrt1 were expressed significantly higher in testes at different phases (phase III, IV and V) compared to ovaries at phase II, III and IV, while gsdf was expressed significantly higher in phase II ovaries than those of phase III and IV. Western blot analysis also showed that Gsdf was more highly expressed in the testis than ovary. Immunohistochemistry analysis showed that Gsdf was expressed in Sertoli cells surrounding spermatogonia in the testis, while it was expressed in the somatic cells surrounding the oogonia of the ovary. Approximately 2.7 kb of the 5' upstream region of gsdf was cloned from the spotted scat genomic DNA and in silico promoter analysis revealed the putative transcription factor binding sites of Dmrt1 and Sf1. The luciferase reporter assay, using the human embryonic kidney cells, demonstrated that Dmrt1 activated gsdf expression in a dose-dependent manner in the presence of Sf1 in spotted scat. These results suggest that Gsdf could play a role in regulating the development of spermatogonia and oogonia, and also participate in male sex differentiation by acting as a downstream gene of Dmrt1 in spotted scat.
性腺体细胞衍生因子（Gsdf）对于硬骨鱼类的睾丸分化和早期生殖细胞发育至关重要。点带棘鲷（Scatophagus argus）具有稳定的XX-XY性别决定系统和候选性别决定基因dmrt1，为理解硬骨鱼类性别决定和分化机制提供了良好模型。在本研究中，我们分析了点带棘鲷Gsdf的组织分布和性腺中的基因表达模式，并进一步进行转录调控分析。组织分布分析表明，Gsdf仅在睾丸和卵巢中表达。实时PCR显示，与II、III和IV期卵巢相比，不同阶段（III、IV和V期）睾丸中的gsdf和dmrt1表达均显著更高，而II期卵巢中的gsdf表达显著高于III和IV期卵巢。蛋白质免疫印迹分析也表明，Gsdf在睾丸中的表达高于卵巢。免疫组织化学分析表明，Gsdf在睾丸中精原细胞周围的支持细胞中表达，而在卵巢中卵原细胞周围的体细胞中表达。从点带棘鲷基因组DNA中克隆了约2.7 kb的gsdf 5'上游区域，通过在线启动子分析揭示了Dmrt1和Sf1的推定转录因子结合位点。使用人胚肾细胞进行的荧光素酶报告基因检测表明，在点带棘鲷中，Dmrt1在存在Sf1的情况下以剂量依赖性方式激活gsdf表达。这些结果表明，Gsdf可能在调节精原细胞和卵原细胞发育中发挥作用，并且还通过作为点带棘鲷中Dmrt1的下游基因参与雄性性别分化。
