Isolation of sox9 duplicates in catfish: localization, differential expression pattern during gonadal development and recrudescence, and hCG-induced up-regulation of sox9 in testicular slices

Abstract

In vertebrates,sox9is a transcription factor that plays a crucial role in testicular development and chondrogenesis. Here, we report cloning of isoforms ofsox9(sox9aandsox9b) from air-breathing catfishClarias gariepinus, which undergoes an annual reproductive cycle. Tissue distribution pattern showed differential expression ofsox9duplicates, wherein both forms were highly expressed in brain and gonads. Furthermore, we observed a dimorphic expression pattern ofsox9aandsox9bin both adult and developing gonads using RT-PCR, indicating thatsox9aretained its function in testis whilesox9bmight have a new role to play in ovary. Changes insox9mRNA levels using real-time quantitative PCR (qRT-PCR) during the seasonal reproductive cycle revealed thatsox9atranscript in testis was abundant during testicular recrudescence (during spermatogenesis), and its expression significantly decreased during spawning and post-spawning phases. Furthermore, treatments of human chorionic gonadotropin and 11-ketotestosteronein vitroup-regulatedsox9amRNA levels in the testicular slices at 12 and 24 h time points, suggesting that gonadotropins might stimulatesox9expression. These results suggest thatsox9might have a plausible role in the entrainment of the testicular cycle. In contrast, during the ovarian cycle,sox9bmRNA levels gradually declined from preparatory to post-spawning phases. Immunohistochemical (IHC) data showed that, in testis,sox9is detectable in Sertoli and spermatogonial cell types except spermatid/spermatozoa. In the ovary, it is localized in the ooplasm of primary and pre-vitellogenic oocytes. These results were further confirmed by whole-mount IHC and qRT-PCR.