Expression of wnt4/5 during reproductive cycle of catfish and wnt5 promoter analysis.

Signaling molecules, Wnt4 and Wnt5, are essential for ovarian growth during developmental stages in mammals. Although these molecules were identified in several teleosts, their precise expression and role in reproductive processes have not yet been explored in any lower vertebrates. In view of this, using catfish, Clarias batrachus as an animal model, cloning and expression analysis of wnt4 and wnt5 were analyzed in different tissues, at various developmental stages, during ovarian reproductive cycle and after gonadotropin induction. These studies indicate a plausible influence of Wnts in ovarian development and recrudescence. Transcript and protein localization revealed their presence in peri-nucleolar, pre-vitellogenic, vitellogenic and follicular layer of post-vitellogenic oocytes. Synchronous expression of pax2 and wnt5 during the ovarian development and recrudescence of catfish led us to analyze the importance of putative binding element of Pax2 in the 5'-promoter motif of wnt5 Promoter activity of wnt5 was analyzed by luciferase assays after transfecting progressive deletion constructs in pGL3 basic vector into the mammalian cell lines (HEK 293 and CHO). The constructs having putative Pax2 motif showed high promoter activity compared with controls. Likewise, the constructs with site-directed mutagenesis showed increased activity after supplementing recombinant Pax2 indicating the prominence of this motif in wnt5 promoter, in vitro Electrophoretic gel mobility shift, supershift and chromatin immunoprecipitation assays confirmed the binding of Pax2 to its corresponding cis-acting element in the upstream of wnt5 This study is the first of its kind to report the critical transcriptional interaction of Pax2 on wnt5 vis-à-vis ovarian development in teleosts.

In vivo induction of human chorionic gonadotropin by osmotic pump advances sexual maturation during pre-spawning phase in adult catfish.

Gonadal maturation is a critical event wherein gonads, under the influence of several hormones and factors, undergo cyclic morphological and physiological changes to produce functional gametes during the spawning phase. However, artificial induction can be effectively used to advance the maturation of gonad vis-à-vis spawning like behavior in seasonal breeders during the off-breeding season. In the present study, osmotic pumps loaded with 5000IU of human chorionic gonadotropin (hCG) or saline as control were implanted intraperitoneally for 21days during the pre-spawning phase (May-June) in catfish Clarias batrachus and C. gariepinus. Significant increase in gonado-somatic index and sperm motility, and in the levels of certain sex steroids were observed in the hCG treated catfish when compared to control while estradiol-17ß (E2) was low. Histological analysis in hCG treated testis revealed densely packed sperm and/or spermatids inside the lumen wherein the control testis displayed normal characteristics of the pre-spawning phase. In females, histological analysis showed a significant increase in post-vitellogenic full-grown immature follicles as seen in the spawning phase. In accordance with this, the steroid hormone profile correlated well with steroidogenic shift from E2 to 17α,20ß-DP indicating oocyte maturation. However, in the control ovaries of C. batrachus, perinucleolar and pre-vitellogenic oocytes were seen to be predominant. In addition, when compared with the control, the hCG treated group displayed a significant increase in the transcripts of several genes associated with gonadal growth. Taken together, artificial induction by slow release of hCG is an effective strategy to advance sexual maturation in catfish in a programmed manner.

Involvement of pax2 in ovarian development and recrudescence of catfish: a role in steroidogenesis.

PAX2, a member of paired box family, is an essential transcription factor for the organ development in vertebrates including teleosts, yet no evidence has been shown for its involvement in reproduction. To study this, partial- and/or full-length cDNA of pax2 was isolated from the ovary of catfish, Clarias batrachus, along with its other Pax family members, pax1 and pax9 Tissue distribution and ontogeny expression analysis indicated the prevalence of pax2 but not pax1 and pax9 in ovary. Varied phase-wise expression during ovarian cycle and elevation of pax2 after human chorionic gonadotropin induction showed probable regulation by gonadotropins. Pax2 could be localized in various stages of oocytes and in follicular layer of vitellogenic and post-vitellogenic oocytes. To assess the functional significance of pax2, transient RNA silencing was performed using primary catfish ovarian follicle culture, in vitro, and in catfish, in vivo, through ovary-targeted injection of PEI-esiRNA. Pax2 siRNA treatment reduced the expression of various transcripts related to ovarian development like signaling molecules such as wnt4 and wnt5, estrogen receptors, several steroidogenic enzymes and transcription factors. These transitions in transcript levels might have been mediated by Pax2 acting upstream of wnt4/5 that may play a role in steroidogenesis and/or ovarian development along with ad4bp/sf-1 or by direct or indirect interaction with steroidogenic enzyme genes, which is evident from the change in the levels of serum estradiol-17ß but not 17α,20ß-dihydroxy-4-pregnen-3-one. Taken together, it seems that pax2 has a plausible role during ovarian development and/or recrudescence of catfish either directly or indirectly through Wnt signaling pathway.