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-a-vis ovarian development in teleosts.