New evidences for the involvement of 20β-hydroxysteroid dehydrogenase in final oocyte maturation of air-breathing catfish

Abstract

20β-hydroxysteroid dehydrogenase (20β-HSD) synthesizes 17α,20β-dihdroxy-4-pregnen-3-one, the steroid required for resumption of prophase-I arrested oocytes in teleosts. Though 20β-HSD cDNAs have been cloned from few fish species, its role in final oocyte maturation (FOM) is still questionable. To study the role of 20β-HSD in FOM more explicitly, we cloned and characterized 20β-HSD from ovary of air-breathing catfish, Clarias gariepinus. Interestingly, Escherichia coli expressed recombinant proteins, both full-length and an N-terminal truncated proteins catalyzed the reduction of steroids and xenobiotics, however there was significant difference between them. Semi-quantitative RT-PCR and Western blots demonstrated the presence of 20β-HSD transcript and protein in various tissues with relatively higher level in gonads, gill, kidney and brain. A positive correlation of 20β-HSD expression was observed in different phases of ovarian cycles. Immunocytochemical/immunofluoroscence analysis with specific antibody identified presence of 20β-HSD in follicular layer of ovary. Real-time RT-PCR and Western blotting showed an induction of 20β-HSD expression during human chorionic gonadotropin (hCG)-induced oocyte maturation, in vitro and in vivo. Concomitantly, a rise in 20β-HSD enzyme activity was also noticed. Specific inhibitors of carbonyl reductase inhibited not only recombinant protein catalytic activity but also hCG-induced oocyte maturation in a dose-dependent manner as evidenced by blocking of germinal vesicle break down. These results together provide new evidences for the involvement of 20β-HSD in the FOM/meiotic maturation. © 2009 Elsevier Inc. All rights reserved.