| In mammals, 170-HSDs play essential role in the sex steroid synthesis and metabolism pathway by catalyzing the interconversion between 17-ketosteroids (dehydroepian-drosterone, androstenedione, and estrone) and 17-hydroxysteroids (androstenediol, testosterone, and estradiol-17β). 17β-HSD1 and 17β-HSD3 were regarded as the key enzyme hi specifically catalyzing the synthesis of estrogen and androgen. 17β-HSD1 expressed dominantly in ovary and responsible for the conversion from estrone to estrodiol-17β, while 17β-HSD3 is male specific and it can exclusively catalyze the conversion from androstenedione to testosterone. The expression of mouse 17β-HSD8 is much high in the kidney than that of gonads, and the mutation of 17β-HSD8 led to polycysts kidney. In the Nile tilapia (Oreochromis niloticus), sex steroids play important role in the process of sex differentiation and gametogenesis, more over, the steroidogenesis in tilapia has been well studied comparing to other teleost species. In order to elucidate the role of 17β-HSDs in tilapia steroidogenic pathway, we cloned and characterized it's 17β-HSD1, 17β-HSD3 and 17β-HSD8. The full length cDNAs of 17β-HSD1 and 17β-HSD3, 17β-HSD8 were obtained by library screening and RACE, respectively. Expression patterns (tissue distribution) of three types 17β-HSDs were checked by RT-PCR and Northern blot. The recombinant constructs of 17β-HSDl/pET Blue2 and 17β-HSD8/pcDNA 3.1 were made and subsequently transformed into the corresponding host expression cell of (DE3)pLacI and mammalian HEK 293 cell. Finally, the enzyme activities of 17β-HSD1 and 17β-HSD8 were characterized by thin layer chromatography using recombinant proteins.The full length cDNA of 17β-HSDl was obtained from ovarian library screening, while 17β-HSD3 cloning was performed by RT-PCR followed by RACE. A fragment of 17P-HSD8 was obtained from tilapia EST sequence and 5'- RACE was carried out subsequently. The full length cDNAs of 17P-HSD1, 17β-HSD3, 170-HSD8 are 1054,1963 and 1006bp respectively, and the deduced amino acid sequences of three type 17β-HSDs were 289, 325 and 256AA. All of them share very high homology to the counterparts of human, mouse, puffer fish, zebrafish and medaka, respectively. Tissue distribution analysis by RT-PCR and Northern blot show that 17β-HSD1 was expressed exclusively in the ovary of tilapia while 17β-HSD3 was expressed exclusively in the testis, no signals were detectable in other tissues. On the other hand, 17β-HSD8 was expressed in both ovary and testis, meanwhile, the expressions were also observed in the intestine, gill, heart, liver and kidney.Enzymatic assay were performed using recombinant proteins expressed in E. coli cells. Tilapia 17β-HSD1 is NADP(H) dependent and it can catalyze the interconversion from Estrone to Estradiol efficiently and also from Androstendion to Testosterone but less efficiently. 17β-HSD8/pcDNA3.1 recombinant protein expressed in HEK 293 cells and it can slightly interconvert estrone to estradiol. Moreover, it can catalyze the conversion from Testosterone to Androstenedione. However, 17β-HSD3 didn't show any conversion to all the substrates checked in this study.
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