Laboratory of Molecular Biology, Research Centre for Nutrition and Development (CIAD), Avenida Sábalo-Cerritos s/n, Mazatlán, Sinaloa 82010, Mexico.
Institute for Biomedical Research, Universidad Nacional Autónoma de México (UNAM), Mexico City 04510, Mexico.
Genes (Basel). 2013 Jun 7;4(2):293-305. doi: 10.3390/genes4020293.
The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination.
常染色体 Sry 相关基因 Sox9 编码一种转录因子，在哺乳动物的睾丸分化中发挥重要作用。在一些爬行动物中，Sox9 在性腺中差异表达，在雄性促进温度的热敏期（TSP）表现出显著的上调，这与 SOX9 在雄性途径中发挥核心作用的观点一致。然而，尽管进行了大量研究，但 SOX9 在这一事件中的作用仍不清楚。在本工作中，我们开发了一种基于 RNAi 的方法，用于沉默海龟 Lepidochelys olivacea 体外性腺培养系统中的 Sox9。一旦胚胎进入 TSP，就将性腺分离出来，并在器官培养中维持。siRNA 的转染导致 Sox9 mRNA 和蛋白的减少。此外，我们发现 Sox9 和抗缪勒管激素基因 Amh 的表达模式协调一致，表明 SOX9 可以直接或间接调节 Amh 的表达，就像在哺乳动物中一样。这些结果证明了一种在海龟性腺中敲低内源性基因的体外方法，这为研究温度依赖性别决定物种中参与性别决定或分化途径的重要基因的作用提供了一种新方法。
