The study for the mechanism of sex determination by mitochondrial and genomic DNA in amphibians

本研究成功地从皱纹蛙（Rana rugosa）中分离到了Dmrt 1、WT 1、CYP 17、CYP 19、Sox 9、AR、SF 1和Sox 3等性别相关基因。我们通过FISH技术将它们定位在玫瑰染色体上，并检测了它们在性腺性别决定过程中的表达。发现SF 1、Sox 3和AR定位于X、Y、Z和W性染色体的非重组区。雌核发育获得ZZ和WW胚胎后，我们检测了SF 1、Sox 3和AR在胚胎中的表达。结果发现，SF 1和Sox 3在ZZ和WW胚胎中均匀表达。然而，W染色体上的AR（W-AR）由于对未知转录因子无反应性而几乎不表达，而Z-AR表达正常，表明W-AR处于变性中间。接下来，我们研究了其他基因的表达R. rugosa的常染色体。在基因检测中。CYP 17和Z-AR表达在性别决定过程中的雄性中性性腺中上调，而CYP 19表达在性别决定过程中的雄性中性性腺中上调。 ...更多信息 CED.有趣的是，我们通过ChIP和报告基因检测发现，Sox 3直接结合到CYP 19启动子区，并上调其在非洲爪蟾肾上皮细胞衍生的A6细胞中的表达。众所周知，类固醇激素可以逆转许多动物的性别，但性别逆转并不需要性别决定基因，因此，我们假设类固醇激素是R. rugosa性别决定的关键因素。非常有趣的是，我们发现，类固醇基因表达在中性性腺的R.rugosa蝌蚪在性别决定过程中，类固醇合成酶的生物活性在这一时期。雄性蝌蚪的中性性腺产生更多的睾酮，而雌性蝌蚪的中性性腺产生更多的雌激素。这些结果表明，类固醇激素可能是在R. rugosa性别决定的主要因素。以前的研究表明，在生活在日本西部的皱纹蛙卵母细胞的细胞质中存在雄性化因子，而在生活在日本北方的青蛙卵母细胞的细胞质中存在雌性化因子。然后，我们确定了生活在日本四个不同地区的这个物种的线粒体DNA的所有核苷酸序列。结果表明，日本北方的皱叶杜鹃线粒体DNA长度为21 kbp，而其他三个地区的皱叶杜鹃线粒体DNA长度为17 kbp。不幸的是，我们没有找到任何编码这些因子的基因，这表明线粒体DNA可能不编码这些因子。相反，基因组DNA可能编码那些女性化和男性化的因素。最后，通过使用层粘连蛋白（基底膜中的一种成分）的抗体进行免疫组织化学染色，确定了R.rugosa的性别，远远早于先前报道的阶段。少

We isolated sex-related genes such as Dmrt1, WT1, CYP17, CYP19, Sox9, AR, SF1 and Sox3 successfully from the frog Rana rugosa. We mapped them on R.rugosa chromosomes by FISH and examined their expression in the gonad during sex determination. We found that SF1,Sox3 and AR were localized to non recombinant region of X, Y, Z, and W sex chromosomes. After producing ZZ and WW embryos gynogenetically, we examined the expression of SF1,Sox3 and AR in the embryos. As a result, we found that SF1 and Sox3 were expressed evenly between ZZ and WW embryos. However, AR on the W chromosome (W-AR) was barely expressed due to unresposiveness to unknown transcription factor(s), whereas Z-AR expression occurred normally, indicating that W-AR is in the middle of degeneration. We next examined the expression of other genes on autosomes of R.rugosa. Among genes examined. CYP17 and Z-AR expression was up-regulated in the indifferent gonad of males during sex determination, whereas CYP19 expression was enhan … More ced. Interestingly, we found by ChIP and reporter assays that Sox3 directly bound to the CYP19 promoter region and up-regulated its expression in A6 cells derived from Xenopus kidney epithelial cells. It is well-known that the sex of many animals can be reversed by steroid hormones, but a sex-determining gene is not required for the sex-reversal, Thus, we hypothesized that steroid hormones are the key factor for sex determination in R.rugosa. Of great interest, we found that steroigogenic genes were expressed in the indifferent gonad of R.rugosa tadpoles during sex determination, and that steroidogenic enzymes were biologically active in that period. In addition, testosterone was produced more in the indifferent gonad of male tadpoles, whereas estrogen was produced more in that of female tadpoles. These results suggest that steroid hormones can be the main factor for sex determination in R.rugosa. The previous study showed that there is a masculinizing factor in the cytoplasm of oocytes in R.rugosa living in western Japan, whereas a feminizing factor in that of oocytes in the frog living in northern Japan. Then, we determined all nucleotide sequences of mitochondrial DNA in this species living in four different regions of Japan. As a result, mitochondrial DNA of R.rugosa from northern Japan was 21kbp long, whereas that from ones living in other three different regions was 17kbp long. Unfortunately, we could not find any gene edcoding those factors, indicating that mitochondrial DNA may not encode the factors. Instead, genomic DNA probably encodes those feminizing and masculinizing factors. Finally, by immunohistological staining using the antibody of laminin, a component in the basement membrane, the sex of R.rugosa is determined far before the stage previously reported. Less