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, sox9 is a transcription factor that plays a crucial role in testicular development and chondrogenesis. Here, we report cloning of isoforms of sox9 (sox9a and sox9b) from air-breathing catfish Clarias gariepinus, which undergoes an annual reproductive cycle. Tissue distribution pattern showed differential expression of sox9 duplicates, wherein both forms were highly expressed in brain and gonads. Furthermore, we observed a dimorphic expression pattern of sox9a and sox9b in both adult and developing gonads using RT-PCR, indicating that sox9a retained its function in testis while sox9b might have a new role to play in ovary. Changes in sox9 mRNA levels using real-time quantitative PCR (qRT-PCR) during the seasonal reproductive cycle revealed that sox9a transcript 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-ketotestosterone in vitro up-regulated sox9a mRNA levels in the testicular slices at 12 and 24 h time points, suggesting that gonadotropins might stimulate sox9 expression. These results suggest that sox9 might have a plausible role in the entrainment of the testicular cycle. In contrast, during the ovarian cycle, sox9b mRNA levels gradually declined from preparatory to post-spawning phases. Immunohistochemical (IHC) data showed that, in testis, sox9 is 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.

Expression of SOX3 and SOX9 Genes in Gonads of Blue Gourami

“…These results suggest that SOX24 plays a role as a transcriptional regulator during oogenesis [35]. Our results regarding the change of mRNA in PV compared to VT and FOM as was found in blue gourami are in agreement with the result for SOX9 transcription in oogensis in catfish [34].…”

“…A high level of sperm was found in the testis in both these stages [32], and spermatogenesis was controlled by gonadotropins (FSH and LH) and androgens hormones [20] [33]. Raghuveer and Senthilkumaran [34] observed that in breathing catfish Clarias gariepinus, which underwent an annual reproductive cycle, a dimorphic expression pattern of SOX9a and SOX9b was found in both adult and developing gonads using RT-PCR, indicating that SOX9a retained its function in testis while SOX9b might play a new role in ovary, as was revealed in the current study in blue gourami using real-time PCR.…”

Dimorphic Expression of Various Transcription Factor and Steroidogenic Enzyme Genes during Gonadal Ontogeny in the Air-Breathing Catfish, <i>Clarias gariepinus</i>

“…On the other hand, dmrt1 expression was negligible or undetectable in female gonads during gonadal ontogeny. Previously, we isolated 2 duplicate isoforms of sox9 which were named sox9a and sox9b [Raghuveer and Senthilkumaran, 2010b]. In this study, sox9a and sox9b transcripts were first noticed at low levels in indifferent gonads at 30 dph and at moderately high levels in bipotential gonads around 40 dph ( fig.…”

“…Sections were then incubated overnight at 4 ° C either with Dmrt1, Sox9, Cyp19a1, or Foxl2 antibodies. The specificity of Dmrt1 [Raghuveer and Senthilkumaran, 2009], Sox9 [Raghuveer and Senthilkumaran, 2010b] and Foxl2 [Sridevi and Senthilkumaran, unpublished data] antibodies used for IHC were evaluated previously in our laboratory. In brief, we used catfish-specific antibodies that were raised in our laboratory for the localization of Dmrt1 and Foxl2 proteins.…”

“…For the detection of Cyp19a1, we used a heterologous antibody specific to tilapia Cyp19a1 protein. Human SOX9-specific antibody (heterologous) was used for detection of Sox9a in catfish [Raghuveer and Senthilkumaran, 2010b]. Following incubation with primary antibody, sections were washed in PBS, 0.1% Tween 20 (PBST) for 10 min and then incubated with 1: 500 dilution of biotinylated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, Calif., USA) for the horseradish peroxidase detection.…”