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-relatedHMGbox 9) is unlikely to initiateAMHin 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 assayswhencoexpressed in Xenopus kidney-derivedA6cells. However, Sox9 expression showed no sexual dimorphism when AMH expression was up-regulated in the developing testis. These results, taken together, suggest thatAMHis 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.
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