Cao Jinling, Cao Zhemin, Wu Tingting
Fishery College, Nanjing Agricultural University, Wuxi 214081, China.
J Genet Genomics. 2007 Jun;34(6):497-509. doi: 10.1016/S1673-8527(07)60055-1.
Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing (Doublesex/Mab-3 DNA-binding motif) gene, is highly conserved across species. Vertebrate DMRT1 (DM-related transcription factor 1) expression occurs predominantly in the testis. Here, however, isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4 (DM-related transcription factor 4) and DMRT1 by BLAST, respectively. Despite high homology in the DM-domain, there is little similarity outside the DM-domain. To better understand the structure, function, and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination, the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression. After purification and cleavage, DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols. Consequently, it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity. The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with beta-actin as the internal standard. DMRT1 was expressed only in testis, whereas DMRT4 was over expressed in the ovary, but in both female and male, a slight expression in the brain was also detected. Statistical analysis showed that in the brain, mean DMRT4 mRNA levels in female were significantly higher than in male. Meanwhile, the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis. Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.
性别决定由体细胞和生殖系性别分化层级组成，它们之间的相互作用尚不清楚。已知一个控制体细胞性别决定的单一基因，即含有DM结构域（双性/ Mab - 3 DNA结合基序）的基因，在物种间高度保守。脊椎动物DMRT1（DM相关转录因子1）主要在睾丸中表达。然而，在此通过BLAST从奥利亚罗非鱼的卵巢和睾丸中分离出两个不同的DM结构域cDNA，分别命名为DMRT4（DM相关转录因子4）和DMRT1。尽管在DM结构域中具有高度同源性，但在DM结构域之外几乎没有相似性。为了更好地理解DMRT4和DMRT1作为性别分化和性别决定潜在候选者的结构、功能及可能作用，通过PCR获得的编码DMRT4和DMRT1的完整区域被亚克隆到载体pMAL - c2x中，并导入大肠杆菌TB1细胞以进行高效融合表达。纯化和切割后，按照标准方案用DMRT4和DMRT1蛋白免疫成年兔。因此，通过蛋白质印迹分析发现，针对DMRT4和DMRT1的多克隆抗体具有高特异性。以β - 肌动蛋白为内参，通过荧光实时RT - PCR测定雌性和雄性奥利亚罗非鱼中DMRT4和DMRT1 mRNA的相对表达水平。DMRT1仅在睾丸中表达，而DMRT4在卵巢中过量表达，但在雌性和雄性中，在脑中也检测到轻微表达。统计分析表明，在脑中，雌性的平均DMRT4 mRNA水平显著高于雄性。同时，还使用纯化的抗体通过蛋白质印迹和免疫组织化学分析了DMRT4和DMRT1蛋白的表达。发现DMRT4仅在卵巢中表达，而DMRT1在睾丸中表达。对DMRT4和DMRT1表达的研究有助于阐明它们的作用以及对鱼类性别分化的理解。
