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Unveiling the hormonal sex-differentiation mechanisms in Hyla eximia treefrog elucidated from the AR/NR3C4 and ESR1/NR3A1

developmental-biology
evolutionary-studies
genetics
molecular-biology
zoology

Abstract

Sex-differentiation mechanisms in vertebrates, particularly amphibians, are governed by a wide array of sex-linked genes. Genetic sex-differentiation mechanisms are often tied to steroidogenic enzymes and receptors. However, the role of the androgen receptor (AR/NR3C4) and estrogen receptor-alpha (ESR1/NR3A1) has not been thoroughly explored. To examine this issue, we utilized the adult Hyla eximia treefrog as a model. The molecular characterization revealed that AR/NR3C4 has an ORF of 2367 bp, encoding 788 amino acids with a molecular weight of about 87.5 kDa. Molecular cloning results indicated the ESR1/NR3A1-cDNA was a 2161-bp sequence with a 1758-bp ORF encoding 586 amino acids. Phylogenetic analysis showed that AR/NR3C4 and ESR1/NR3A1 had higher identities with the amphibian group than with other vertebrate groups. We found that AR/NR3C4 was expressed in all tested tissues, and its expression level was higher in the testis during the breeding season than in the non-breeding season. During the breeding season, transcripts were expressed at higher levels in the male vocal sac than in the female vocal sac. However, the expression of AR/NR3C4 in female anterior muscle was higher in the non-breeding season than in male anterior muscle. mRNA expression assays suggested a sexually dimorphic expression of the ESR1/NR3A1 transcript in the brain and gonads between males and females throughout both non-breeding and breeding seasons, with expression significantly higher in males than in females. The highest expression levels were recorded in the testis during the breeding season, followed by the brain and ovary. The gene expression pattern indicated in the breeding season suggests that AR/NR3C4 in the testis, vocal sac, and other tissues may play a negligible role or take part in maintaining male reproductive functionality and secondary sex characteristics. This indicates that the gene actively participates in cerebral and testicular function during breeding. Furthermore, it implies that the treefrog ESR1/NR3A1 could play a pivotal role in regulating male sex-differentiation mechanisms.
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