Cloning, expression and enzyme activity analysis of testicular 11β-hydroxysteroid dehydrogenase during seasonal cycle and after hCG induction in air-breathing catfish Clarias gariepinus

Abstract

A full-length cDNA encoding 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) was cloned from testis of air-breathing catfish, Clarias gariepinus which showed high sequence homology to zebrafish and eel. The open reading frame of 11β-HSD2 was then transfected to COS-7 cells, which converted 11β-hydroxytestosterone (11-OHT) to 11-ketotestosterone (11-KT). Using NAD+, 11β-HSD2 from testicular microsomes oxidized 11-OHT with apparent Km 56±4nM and Vmax 55±6pmol/h/mgprotein values. Tissue distribution analysis revealed prominent expression in testis, anterior kidney, liver and gills. Expression of 11β-HSD2 in testis and serum levels of 11-KT were high in the prespawning phase. Administration of human chorionic gonadotropin (hCG) during prespawning and resting phases revealed initial rise in 11β-HSD2 transcript at 4h followed by gradual increase at 8h, 12h and peaking at 24h, only in testis of prespawning phase. Rate of conversion of 11-OHT to 11-KT by testicular microsomes during different testicular phases and after hCG administration corroborated well with the expression of 11β-HSD2. Ontogeny study indicated that this enzyme is expressed during testicular development. Thus the spatio-temporal expression supported with putative dehydrogenase activity and circulating 11-KT levels clearly suggest a major role for 11β-HSD2 during testicular differentiation and seasonal testicular cycle in catfish. © 2010 Elsevier Ltd.