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

我们成功地从蛙蛙中分离到了Dmrt1、WT1、CYP17、CYP19、Sox9、AR、SF1和Sox3等性别相关基因。我们利用FISH技术将它们定位到rugosa染色体上，并在性别决定过程中检测了它们在性腺中的表达。我们发现SF1、Sox3和AR定位于X、Y、Z和W性染色体的非重组区。在产生ZZ和WW胚后，我们检测了胚胎中SF1、Sox3和AR的表达。结果表明，SF1和Sox3在ZZ胚和WW胚中表达均匀。然而，W染色体上的AR （W-AR）由于对未知转录因子的不响应而几乎不表达，而Z-AR则正常表达，表明W-AR处于变性中期。接下来，我们检测了其他基因在rugosa常染色体上的表达。在被检测的基因中。在雄性性腺中，CYP17和Z-AR表达上调，而CYP19表达增强。有趣的是，我们通过ChIP和报告基因检测发现，Sox3直接结合到CYP19启动子区域，并在爪蟾肾上皮细胞衍生的A6细胞中上调其表达。众所周知，许多动物的性别可以通过类固醇激素逆转，但性别逆转不需要性别决定基因，因此，我们假设类固醇激素是rugosa性别决定的关键因素。令人感兴趣的是，我们发现在性别决定过程中，甾体生成基因在rugosa蝌蚪的不同性腺中表达，并且甾体生成酶在这一时期具有生物活性。此外，雄性蝌蚪的冷漠性腺分泌睾酮更多，而雌性蝌蚪的冷漠性腺分泌雌激素更多。这些结果表明，类固醇激素可能是红毛鼠性别决定的主要因素。先前的研究表明，生活在日本西部的rugosa蛙的卵母细胞细胞质中存在雄性化因子，而生活在日本北部的蛙的卵母细胞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，来自日本北部的rugosa的线粒体DNA长度为21kbp，而生活在其他三个不同地区的rugosa的线粒体DNA长度为17kbp。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可能没有编码这些因子。相反，基因组DNA可能编码了那些女性化和男性化的因素。最后，通过使用层粘连蛋白（一种基底膜成分）抗体的免疫组织学染色，rugosa的性别在先前报道的阶段之前就被确定了。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