| Budget Amount *help |
¥16,250,000 (Direct Cost: ¥12,500,000、Indirect Cost: ¥3,750,000)
Fiscal Year 2009: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000)
Fiscal Year 2008: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2007: ¥8,320,000 (Direct Cost: ¥6,400,000、Indirect Cost: ¥1,920,000)
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| Research Abstract |
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
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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
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