Molecular cloning and characterization of anti-Müllerian hormone (AMH) from the Japanese wrinkled frog, Rana rugosa.

The role of anti-Müllerian hormone (AMH) during gonad development has been studied extensively in many species of mammal, bird, reptile, and fish but remains unresolved in amphibians. In male mammalian embryos, Sox9 activates AMH expression, which initiates regression of the Müllerian ducts. However, Sox9 (Sry-related HMG box 9) is unlikely to initiate AMH in chicken, because AMH precedes Sox9 expression in this species. To clarify whether AMH is involved in testicular differentiation in amphibians, we cloned the full-length AMH cDNA from the Japanese wrinkled frog, Rana rugosa. The AMH gene, which appears to be autosomal, is exclusively expressed in the testis of adult frog among 8 different tissues examined; Sertoli cells are probably responsible for its expression. AMH expression was found in the undifferentiated gonad of both male and female tadpoles, increasing in the differentiating testis. Moreover, we observed consensus binding sites for Sox9 in the 5'-flanking region of the AMH gene. Sox9 stimulated statistically significant AMH expression in luciferase reporter assays when coexpressed in Xenopus kidney-derived A6 cells. However, Sox9 expression showed no sexual dimorphism when AMH expression was up-regulated in the developing testis. These results, taken together, suggest that AMH is probably involved in testicular differentiation in R. rugosa, although an additional, perhaps tissue-specific, transcription factor may be required for the regulation of AMH transcription.

Isolation and characterization of the CYP17A1 gene and its processed pseudogene in Rana rugosa.

CYP17A1 expression is up-regulated in the gonad in Rana (Glandirana) rugosa tadpoles treated with androgens to induce female-to-male sex-reversal. In this study, we isolated the CYP17A1 gene and its processed pseudogene from R. rugosa. The former was found to consist of 8 exons, and the latter a single-exon gene, designated CYP17A1P. The sequence of the promoter region of CYP17A1 differed from that of CYP17A1P. We found several consensus binding-sites for candidate transcription factors including androgen receptor (AR), Sox and FoxL2 in the CYP17A1 promoter region, but an AR-binding site was absent from CYP17A1P. When AR was over-expressed in Xenopus A6 cells, it did not increase CYP17A1 transcription in luciferase assays. CYP17A1 was strongly expressed in indifferent male gonads during sex determination and exclusively in testis, among eight adult tissues of R. rugosa. By contrast, CYP17A1P was expressed at very low, and similar levels in the adult tissues of both sexes. Fluorescent In-Situ Hybridization (FISH) analysis showed that CYP17A1P is localized to chromosome 4, while CYP17A1 is on chromosome 9. These results collectively suggest that CYP17A1, but not CYP17A1P is involved in male sex-determination in R. rugosa, and that androgens may not have a direct effect on the CYP17A1 transcription.

Up-regulation of FSHR expression during gonadal sex determination in the frog Rana rugosa.

In vertebrates, gonadal production of steroid hormones is regulated by follicle-stimulating hormone (FSH) and luteinizing hormone (LH) via their receptors designated FSHR and LHR, respectively. We have shown recently that steroid hormones are synthesized in the differentiating gonad of tadpoles during sex determination in the frog Rana rugosa. To elucidate the role of gonadotropins (GTHs) and their receptors in the production of gonadal steroid hormones during sex determination, we isolated the full-length FSHß, LHß, FSHR and LHR cDNAs from R. rugosa and determined gonadal expression of FSHR (FSH receptor) and LHR (LH receptor) as well as brain expression of FSHß and LHß during sex determination in this species. The molecular structures of these four glycoproteins are conserved among different classes of vertebrates. FSHß expression was observed at similar levels in the whole brain (including the pituitary) of tadpoles, but it showed no sexual dimorphism during gonadal sex determination. By contrast, LHß mRNA was undetectable in the whole brain of tadpoles. FSHß-immunopositive cells were observed in the pituitary of female tadpoles with a differentiating gonad. Furthermore, FSHR expression was significantly higher in the gonad of female tadpoles during sex determination than in that of males, whereas LHR was expressed at similar levels in males and females. The results collectively suggest that FSHR, probably in conjunction with FSH, is involved in the steroid-hormone production during female-sex determination in R. rugosa.

Molecular cytogenetic characterization of telomere-specific repetitive DNA sequences in Rana rugosa.

We performed a molecular cloning of the glutamic oxaloacetic transaminase (GOT1) gene from R. rugosa, and determined its chromosomal location. This gene was reportedly localized near the sex-determining region of the ZW sex chromosomes in the frog Buergeria buergeri; however, the GOT1 gene was mapped to the distal end of chromosome 9 in R. rugosa using a GOT1 cDNA FISH probe. This was also the case when a 46.3 kb genomic clone containing exon 8 and 9 and the 3'-neighboring region of the GOT1 gene, designated clone B, was used as probe. However, weak signals were also detected at the telomeric ends of other autosomes and the Z sex chromosome, and near the centromeric region of the W sex chromosome. To intensify the signals, we used eight internal fragments in clone B and applied them to chromosome mapping. Consequently, only two fragments containing repeated sequence blocks produced hybridization signals; those signals were observed on autosomes and ZW sex chromosomes. The 3'-neighboring region contained two types of repeated sequence elements: a 41 bp element, designated 41-REL, localized to telomeric ends of autosomes and a 31 bp element, designated 31-REL, localized to telomeric ends of all autosomes and the ZW sex chromosomes, and also near the centromere on the W long arm. The results collectively suggest that the two repeated sequence elements were independently amplified around the chromosomal telomeres in R. rugosa, indicating that they will be useful cytogenetic markers for studying karyotypic evolution-especially the W chromosome differentiation-in this species.

Androgen receptor of the frog Rana rugosa: molecular cloning and its characterization.

The androgen receptor(AR) gene is located on the Z and W sex chromosomes in the frog Rana rugosa, designated Z- and W-AR, respectively. Among various tissues of an adult frog, AR expression levels were highest in the testis and brain. In the testis, AR was expressed in germ cells. AR expression occured in developing embryos from stage 21 and was very high in the gonad of a male tadpole before the onset of sex determination. When Z- and W-AR were expressed in Xenopus A6 cells, they activated androgen-dependent transcription of a luciferase reporter gene. By contrast, estrogen receptor (ER) alpha and beta showed no sexually dimorphic expression during sex determination, but their expressions became much higher in the gonad of a female tadpole after sex determination. In addition, AR transcripts in the ZZ-tadpoles were twice as abundant as in the ZW genotype. In contrast, W-AR expression was extremely low although when W-AR was expressed in A6 cells, it activated transcription in the luciferase assay. In this regard it is worth noting that the promoter regions of Z- and W-AR are not identical. The results suggest that Z-AR plays an important role in the testis formation in a R. rugosa tadpole, whereas ERbeta is involved in ovary differentiation. Very low expression of W-AR may be due to its promoter region having mutations in key transcription factor binding sites, although these remain to be identified. Thus, it is proposed that AR could be a candidate for a male-determining gene in R. rugosa.

Steroidogenic gene expression during sex determination in the frog Rana rugosa.

Rana rugosa is unique among frog species in that it has two distinct types of sex chromosomes in two separate forms (XX/XY and ZZ/ZW). Treatment with sex steroids can reverse its gender from female to male or male to female. This phenomenon makes it a novel model for studying gonadal differentiation. The physiological role of sex steroids in sex differentiation in amphibians is yet unclear, however. To address this issue, we cloned the cDNAs of 17betaHSD types 8 (17betaHSD8) and 12 (17betaHSD12), 5alpha-reductase type 1 (5alphaRed1), and the steroidogenic acute regulatory protein known as StAR in the steroidogenic pathway. Then, we measured the mRNA levels of these genes during sex differentiation by real-time RT-PCR. The levels of CYP11A1, 3betaHSD, CYP17 and CYP19 mRNA were also measured by real-time RT-PCR. As a result, we detected transcripts of all such genes except for that of 17betaHSD8 in the indifferent gonad before the onset of sex determination. The expression of CYP17 occurred in indifferent gonads in both sexes; and its transcript levels were much higher in the male gonads. By contrast, the levels for CYP19 were much higher in the female gonads. CYP11A1, 3betaHSD, 17betaHSD12, 5alphaRed1 and StAR showed no sexually dimorphic expression during gonadal sex differentiation. Taken together, the results suggest that CYP17 has a major influence on testis development and that CYP19 plays a similar role in ovary development. However, the factors that up-regulate their expression remain to be identified.