Identification of multiple dmrt1s in catfish: localization, dimorphic expression pattern, changes during testicular cycle and after methyltestosterone treatment

<jats:p>The double sex and mab-3 related (DM) transcription factor 1 (<jats:italic>dmrt1</jats:italic>) plays an important role in testicular differentiation. Here, we report cloning of multiple <jats:italic>dmrt1</jats:italic>s, a full-length and two alternative spliced forms from adult catfish (<jats:italic>Clarias</jats:italic> <jats:italic>gariepinus</jats:italic>) testis, which encode predicted proteins of 287 (<jats:italic>dmrt1</jats:italic>a), 253 (<jats:italic>dmrt1</jats:italic>b) and 233 (<jats:italic>dmrt1</jats:italic>c) amino acid residues respectively. Interestingly, <jats:italic>dmrt1</jats:italic>c lacks the majority of the DM domain. Multiple <jats:italic>dmrt1</jats:italic>s (<jats:italic>dmrt1</jats:italic>a and <jats:italic>dmrt1</jats:italic>c) were obtained from <jats:italic>Clarias batrachus</jats:italic> as well. Tissue distribution (transcript and protein) of catfish <jats:italic>dmrt1</jats:italic> revealed exclusive expression in testis. Semi-quantitative RT-PCR revealed the presence of multiple <jats:italic>dmrt1</jats:italic>s with high levels of <jats:italic>dmrt1</jats:italic>a in adult testis but not in ovary. Real-time RT-PCR analysis during testicular cycle showed higher levels of <jats:italic>dmrt1</jats:italic> transcripts in preparatory and pre-spawning when compared with spawning and post-spawning phases. Immunocytochemical and immunofluorescence localization revealed the presence of catfish Dmrt1 protein in spermatogonia and spermatocytes, which indicates plausible role in spermatogenesis. Histological analysis indicated initiation of gonadal sex differentiation in catfish around 40–50 days after hatching. The potential role for <jats:italic>dmrt1</jats:italic> in testicular differentiation is evident from its stage-dependent elevated expression in developing testis. Furthermore, dimorphic expressions of <jats:italic>dmrt1</jats:italic>s were evident at different stages of gonadal development or recrudescence in catfish. Treatment of methyl testosterone (MT) during early stages of gonadal sex differentiation resulted in adult males. Interestingly, we also obtained MT-treated fishes having ova-testis gonads. Analysis of <jats:italic>dmrt1</jats:italic>, <jats:italic>sox9a</jats:italic>, <jats:italic>foxl2</jats:italic> and <jats:italic>cyp19a1</jats:italic> expression patterns in MT-treated gonads revealed tissue-specific pattern. These results together suggest that multiple <jats:italic>dmrt1</jats:italic>s are testis-specific markers in catfish.</jats:p>