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Abstract: The heterogametic sex is male in all mammals, whereas it is female in almost all birds. By contrast, there are two heterogametic types (XX/XY and ZZ/ZW) for genetic sex determination in amphibians. Though the original heterogametic sex was female in amphibians, the two heterogametic types were probably interchangeable, suggesting that sex chromosomes evolved several times in this lineage. Indeed, the frog Rana rugosa has the XX/XY and ZZ/ZW sex-determining systems within a single species, depending on the local population in Japan. The XY and ZW geographic forms with differentiated sex chromosomes probably have a common origin as undifferentiated sex chromosomes resulted from the hybridization between the primary populations of West Japan and Kanto forms. It is clear that the sex chromosomes are still undergoing evolution in this species group. Regardless of the presence of a sex-determining gene in amphibians, the gonadal sex of some species can be changed by sex steroids. Namely, sex steroids can induce the sex reversal, with estrogens inducing the male-to-female sex reversal, whereas androgens have the opposite effect. In R. rugosa, gonadal activity of CYP19 (P450 aromatase) is correlated with the feminization of gonads. Of particular interest is that high levels of CYP19 expression are observed in indifferent gonads at time before sex determination. Increases in the expression of CYP19 in female gonads and CYP17 (P450 17α-hydroxylase/C17-20 lyase) in male gonads suggest that the former plays an important role in phenotypic female determination, whereas the latter is needed for male determination. Thus, steroids could be the key factor for sex determination in R. rugosa. In addition to the role of sex steroids in gonadal sex determination in this species, Foxl2 and Sox3 are capable of promoting CYP19 expression. Since both the genes are autosomal, another factor up-regulating CYP19 expression must be recruited. The factor, which may be located on the X or W chromosome, intervenes directly or indirectly, in the transcriptional regulation of the CYP19 gene for feminization in amphibians. A factor up-regulating CYP17 expression remains to be identified. [Copyright &y& Elsevier]

Abstract: Complementary DNAs encoding two major osteoclastic markers, tartrate-resistant acid phosphatase (TRAP) and cathepsin K (Cath K) were cloned from the scales of a teleost, the goldfish. This is the first report of the full coding sequence of TRAP and Cath K molecules in fish. In the goldfish scale both TRAP and Cath K mRNAs were expressed in the multinucleate osteoclasts, which showed large numbers of mitochondria and lysosomes, and a well developed ruffled border. These characteristic features of osteoclasts in the scales are similar to those in mammals. Most teleosts use the scale as an internal calcium reservoir during the reproductive season. The expression of TRAP and Cath K mRNAs in the scale significantly increased in April, which is a reproductive season, compared with that in October, a non-reproductive season. Thus, both of these molecular markers should be useful for the study of osteoclasts in the teleost scale. [Copyright &y& Elsevier]

Gonadal differentiation in amphibians is sensitive to steroids. The phenotypic sex can be changed by hormonal treatments, but the molecular mechanism for gonadal differentiation is not known. Up to date, many genes involved in gonadal differentiation have been isolated in vertebrates. Dmrt1, a gene that contains the DM-domain (Doublesex/Mab-3 DNA-binding motif), is considered to be one of the essential genes involved in the testicular differentiation cascade in mammals, birds, reptiles, and fish. However, this gene has not been isolated in amphibians yet. To elucidate its role(s) for gonadal differentiation in vertebrates, a molecular cloning of Dmrt1 in amphibians is urgent. In this study, we have successfully isolated a Dmrt1 homolog from the frog Rana rugosa testes cDNA library and examined its expression during gonadal differentiation and in sex-reversed gonads. The Dmrt1 mRNA was exclusively detected in testis among adult tissues by the RT-PCR analysis. The Dmrt1 was first expressed in the differentiating testis at stage XXV in which spermatogonia are only germ cells, and became stronger at later stages. Moreover, the Dmrt1 transcript was not detected during ovarian differentiation. However, this gene was clearly expressed in XX sex-reversed gonads caused by injection of testosterone into all-female tadpoles that have well-differentiated ovaries. Taken together, the results suggest that Dmrt1 is closely implicated in testicular, but not ovarian differentiation in amphibians. [Copyright &y& Elsevier]

Abstract: 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 FSHβ, LHβ, 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 FSHβ and LHβ during sex determination in this species. The molecular structures of these four glycoproteins are conserved among different classes of vertebrates. FSHβ 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, LHβ mRNA was undetectable in the whole brain of tadpoles. FSHβ-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. [Copyright &y& Elsevier]

Abstract: In certain species of amphibians gonadal differentiation is influenced by steroid hormones. In the case of the frog Rana rugosa testosterone given to tadpoles reverses sex from female to male, while the opposite reversal – male to female – can be achieved using estradiol-17β. In this study, we investigated whether CYP19 (P450 aromatase), the enzyme responsible for a production of estradiol-17β, was present in the differentiating gonad of R. rugosa. Initially, we immunized rabbits against frog CYP19 peptides and performed immunostaining using specific antibodies purified from that serum. CYP19-reactive signals were observed in gonadal somatic cells of the female, but not male tadpoles at stage (St.) I (the stage prior to phenotypic sex determination in tadpoles of R. rugosa). Immunopositive signals were also produced in ovarian somatic cells froglets at St. XXV (just after the completion of metamorphosis). We also examined the enzymatic activity of CYP19 in the differentiating gonad of R. rugosa. Reverse-phase HPLC (high performance liquid chromatography) analysis revealed that [3H]testosterone was converted to [3H]estradiol-17β in the gonad of tadpoles at St. I. Interestingly, the rate of conversion was much higher in females than in males. To the best of our knowledge, this is the first report on the biosynthesis of estradiol-17β in the gonad of amphibians, and the co-incident identification of active CYP19 enzyme in the differentiating gonad of R. rugosa. Based on our results, we conclude that estradiol-17β may be involved in ovarian differentiation in this species. [ABSTRACT FROM AUTHOR]

Abstract: Foxl2 is a transcription factor that plays a crucial role in the regulation of the early development of the female gonad in mammals and fish. However, little is known regarding its role in ovarian differentiation in amphibians. In this study, we isolated a Foxl2 cDNA from the ovary of the frog Rana rugosa and Xenopus laevis and examined its expression during gonadal sex differentiation in R. rugosa. Alignment of known Foxl2 sequences from vertebrates showed high identity of the Foxl2 open reading frame and protein sequences, in particular the forkhead domain and C-terminal region, with other vertebrate sequences. Among different adult tissues, Foxl2 was expressed at its highest level in the ovary. Real-time RT-PCR analysis showed that Foxl2 expression was sexually dimorphic during gonadal sex differentiation in R. rugosa. In addition, Foxl2, which was detected immunochemically in somatic cells surrounding oocytes in the ovary, promoted R. rugosa CYP19 transcription in luciferase promoter assays conducted in A6 cells. We also found by FISH analysis that Foxl2 was an autosomal gene. Altogether, these results suggest that Foxl2 probably plays a very important role in ovarian differentiation of R. rugosa by possibly regulating CYP19 expression. The factor that up-regulates Foxl2 expression in female gonads still remains to be identified. [Copyright &y& Elsevier]

Abstract: 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 17βHSD types 8 (17βHSD8) and 12 (17βHSD12), 5α-reductase type 1 (5αRed1), 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, 3βHSD, 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 17βHSD8 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, 3βHSD, 17βHSD12, 5αRed1 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. [Copyright &y& Elsevier]

Abstract: Sex steroids play pivotal roles in gonadal differentiation in many species of vertebrates. The sex can be reversed from female to male by testosterone in the Japanese wrinkled frog Rana rugosa, but it is still unclear what genes are up- or down-regulated during the XX sex-reversal in this species. To search the genes for the female-to-male sex-reversal, we employed differential display and 5′/3′-RACE. Consequently, we isolated from the gonads at day 8 after testosterone injection 24 different cDNA fragments showing a testosterone treatment-related change and then obtained three full-length cDNAs, which we termed Zfp64, Zfp112, and Rrp54. The former two cDNAs encoded different proteins with zinc-finger domains, whereas the latter cDNA encoded an unknown protein. Transcripts of the three genes were hardly detectable in the sex-reversing gonads at day 24 after the injection; at this time few growing oocytes were observed in the sex-reversing gonad. Besides, in situ hybridization analysis showed positive signals of the three genes in the cytoplasm of growing oocytes of an ovary when testosterone was injected into a tadpole. Thus, the decrease in expression of these three genes was probably due to the disappearance of growing oocytes and not to their direct involvement in the testis formation. To find the key-gene for testis formation, it will be necessary to analyze, by the differential display method, more genes showing a change in expression pattern during sex reversal. [Copyright &y& Elsevier]

Abstract: The objective of this investigation was to ascertain whether bisphenol A (BPA), which has a structural resemblance to thyroid hormone (TH), acts as a TH agonist or antagonist in terms of affecting the release of thyrotropin (TSH). To this end, we exposed adult bullfrog (Rana catesbeiana) pituitary cells to BPA and/or TH in the presence or absence of corticotropin-releasing factor (CRF), which is known to have a potent TSH-releasing activity in amphibians. BPA (10−9–10−4 M) did not affect the basal release of TSH. However, it suppressed CRF-inducible TSH release at 10−4 M, but not at 10−5 M. Triiodothyronine (T3) at 10−7 M and l-thyroxine (T4) at 10−6 M also suppressed the CRF-inducible release of TSH. The combination of T3 (10−7 M) or T4 (10−6 M) with BPA (10−4 M) had an additive effect in suppressing TSH release. A comparison of the suppressive effects of BPA and T3 on the release of TSH following the addition of actinomycin D or cycloheximide to the culture medium revealed that both of the latter compounds blocked T3-inducible but not BPA-inducible suppression of TSH release. The results indicate that the mechanism of action of BPA is different from that of T3 in that T3 action involves RNA and protein synthesis, whereas BPA action does not involve either of these processes. Furthermore, BPA was found to suppress the thyrotropin-releasing hormone-inducible release of both prolactin (PRL) and TSH. Our results suggest that BPA acts not only as a blocker of TSH secretagogues but also as a blocker of a PRL secretagogue at the pituitary level. Estradiol affected neither the release of TSH nor the release of PRL in the presence or absence of their secretagogues, suggesting that the suppression of the release of TSH and PRL caused by BPA may not be derived from its estrogenic activity. [Copyright &y& Elsevier]

Abstract: Sex steroids play decisive roles in gonadal differentiation in many species of vertebrates. The sex can be changed by sex steroids in some species of amphibians, but the mechanism of the sex-reversal is largely unknown. In this study, we cloned and characterized 3 cDNAs encoding sex steroid-synthesizing enzymes, i.e., CYP11A1, CYP17 and 3β-HSD from the frog Rana rugosa. RT-PCR analysis showed that the CYP17 expression was much higher in male gonads than in female ones during sex determination in R. rugosa, whereas CYP11A1 and 3β-HSD showed no sexually dimorphic expression. When testosterone was injected into tadpoles for female-to-male sex reversal, CYP17 expression appeared to be very strong in the gonad at days 16 and 24 after injection of testosterone. CYP11A1 was also transcribed higher at day 16, but its expression was weaker when compared with that of CYP17. The expression of 3β-HSD did not change during the sex reversal. In addition, in situ hybridization analysis revealed that CYP17 was expressed in somatic cells of the indifferent male gonad and in those of the testis. Positive signals of CYP17 were also produced in somatic cells of a female-to-male sex-reversed gonad (testis) at days 16 and 24 post testosterone injection, but not in the ovary. Taken together, the results suggest that CYP17 is very involved in testicular differentiation of the gonad in R. rugosa. [Copyright &y& Elsevier]

Abstract: Due to the lack of a radioimmunoassay (RIA) system for amphibian thyrotropin (TSH), no direct evidence that thyroid hormone suppresses the release of TSH from the amphibian pituitary has been obtained. However, we recently developed an RIA for bullfrog (Rana catesbeiana) TSH and thus were able to study the effect of thyroid hormone on the release of TSH from the bullfrog pituitary. Enzymatically dispersed pituitary cells of larval, juvenile, and adult bullfrogs were cultured in the absence or presence of 100nM corticotropin-releasing factor of bullfrog origin (fCRF), which is known to be a potent stimulator of the release of TSH. The amount of spontaneously released TSH was higher in late prometamorphic and climactic tadpoles than in early prometamorphic larvae and juvenile and adult frogs. Pituitary cells from tadpoles at metamorphic climax responded to fCRF to release much more TSH than those from early and late prometamorphic tadpoles and juvenile and adult frogs. In all cases, the fCRF (100nM)-induced, but not the basal, release of TSH was significantly suppressed by 1nM triiodothyronine (T3) and 1000nM thyroxine (T4), when examined using adult pituitary cells. The suppressive effect of thyroid hormones was revealed to be dependent on their concentrations. [Copyright &y& Elsevier]