New evidences for the involvement of 20beta-hydroxysteroid dehydrogenase in final oocyte maturation of air-breathing catfish.
20beta-hydroxysteroid dehydrogenase (20beta-HSD) synthesizes 17alpha,20beta-dihdroxy-4-pregnen-3-one, the steroid required for resumption of prophase-I arrested oocytes in teleosts. Though 20beta-HSD cDNAs have been cloned from few fish species, its role in final oocyte maturation (FOM) is still questionable. To study the role of 20beta-HSD in FOM more explicitly, we cloned and characterized 20beta-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 20beta-HSD transcript and protein in various tissues with relatively higher level in gonads, gill, kidney and brain. A positive correlation of 20beta-HSD expression was observed in different phases of ovarian cycles. Immunocytochemical/immunofluoroscence analysis with specific antibody identified presence of 20beta-HSD in follicular layer of ovary. Real-time RT-PCR and Western blotting showed an induction of 20beta-HSD expression during human chorionic gonadotropin (hCG)-induced oocyte maturation, in vitro and in vivo. Concomitantly, a rise in 20beta-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 20beta-HSD in the FOM/meiotic maturation.
Sreenivasulu
G
G
Department of Animal Sciences, School of Life Sciences, University of Hyderabad, P.O. Central University, Hyderabad 500 046, Andhra Pradesh, India.
Senthilkumaran
B
B
D015089:20-Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002397:Catfishes / Q000254:growth & development / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology* / Q000254:growth & development*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D012333:RNA, Messenger; D015089:20-Hydroxysteroid Dehydrogenases