Ad4bp/sf-1 regulates cyp11b1 and cyp17a1 in the Asian catfish, Clarias batrachus

Abstract

Testicular function in fish is mediated by steroids, with cytochrome P450 11B1, mitochondrial, or 11β-hydroxylase, encoded by cyp11b1, and cytochrome P450 17A1, or 17α-hydroxylase/17,20-lyase, encoded by cyp17a1, being key steroidogenic enzymes. However, the regulation of cyp11b1 and cyp17a1 has not yet been studied in fish. To address this, the 5′ upstream regions of cyp11b1 and cyp17a1 from catfish, Clarias batrachus, were cloned and sequenced. Predicted putative transcription factor binding sites included Ad4bp/sf-1, Foxp1, Pax1, Pax2, Gata1, and Oct1 in both promoter fragments. Luciferase reporter assays in TM3 mouse Leydig cells, with and without human chorionic gonadotropin (hCG) induction, showed significant promoter activity in constructs containing ad4bp/sf-1 and foxp1, but not with the other transcription factors. Site-directed mutagenesis and chromatin immunoprecipitation further confirmed the binding of Ad4bp/sf-1 to the promoters of both cyp11b1 and cyp17a1, while Foxp1 binding was observed only in the cyp11b1 promoter. Immunolocalization of Ad4bp/sf-1 revealed its presence in interstitial/Leydig cells and also in the testicular lumen. The expression of ad4bp/sf-1 increased after hCG induction in vivo and following 11-ketotestosterone (11-KT)/methyl testosterone stimulation in vitro, indicating gonadotropin- and androgen-dependent regulation. Additionally, transient gene silencing of ad4bp/sf1 using small interfering RNA (siRNA) decreased the expression of cyp11b1, cyp17a1, and foxp1, suggesting co-regulation. Finally, decreased serum testosterone and 11-KT levels upon ad4bp/sf-1 siRNA silencing further support its role in regulating male steroidogenesis in males.