👤 Shigeho Ijiri

17α, 20β-Dihydroxy-4-pregnen-3-one (DHP) is a maturation-inducing steroid in many teleost fish. Carbonyl reductase-like 20β-hydroxysteroid dehydrogenase (CR/20β-HSD) is a candidate enzyme responsible for DHP production during oocyte maturation in various fish, including Nile tilapia. However, a novel type of 17β-hydroxysteroid dehydrogenase, type 12-like (17β-HSD12L), is responsible for DHP production during oocyte maturation in masu salmon. 17β-HSD12 (presumably orthologous to salmon 17β-HSD12L) has been detected in Nile tilapia; however, its enzymatic activity and specific ability to convert the DHP substrate 17α-hydroxyprogesterone (17OHP) have not been examined. This study aimed to determine whether CR/20β-HSD or 17β-HSD12 is responsible for DHP production during oocyte maturation in the Nile tilapia. Mammalian expression vectors containing tilapia hsd17b12 or CR/20bhsd were transfected into HEK293T cells, followed by incubation with 17OHP. HEK293T cells transfected with hsd17b12 exhibited a strong ability to convert exogenous 17OHP to DHP (73.8% yield). Cells transfected with CR/20bhsd or the control vector converted only 7.4% and 7.5% of 17OHP to DHP, respectively. In addition, based on LC-MS/MS analyses, 17β-HSD12 did not convert any substrates other than 17OHP, including DHP, adrenosterone, androstenedione, estrone, testosterone, 11-ketotestosterone, and estradiol-17β. CR/20β-HSD showed strong 17β-HSD oxidoreductase activity especially with adrenosterone and androstenedione. Tissue-specific hsd17b12 expression analyzed by RT-PCR showed that hsd17b12 mRNA was strongest amplification in full-grown follicles. Finally, full-grown ovarian follicles were incubated with salmon pituitary extract (SPE, 100 µg/mL) or human chorionic gonadotropin (HCG, 100 IU/mL) to induce 20β-HSD activity in vitro, and enzyme activity was assessed by co-incubation with 100 ng/mL 17OHP for 2, 4, 8, and 16 h. Conversion of 17OHP to DHP by ovarian follicles incubated with SPE and HCG peaked at 16 h, subsequent with increased follicular hsd17b12 mRNA levels, which were significantly higher than those in control incubations. However, the levels of CR/20bhsd mRNA remained low and did not differ among time points. The present study strongly suggests that 17β-HSD12, and not CR/20β-HSD, is the 20β-HSD responsible for DHP production by ovarian follicles during oocyte maturation in Nile tilapia. Show less

The production of 11-ketotestosterone (11KT), an important steroid hormone in piscine spermatogenesis, is regulated by the pituitary gonadotropins [Gths: follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh)] and it is synthesized by catalytic reactions involving several steroidogenic enzymes. Among these enzymes, the role of 17β-hydroxysteroid dehydrogenases (Hsd17bs) that exhibited 17-ketosteroid reducing activity (17KSR activity) responsible for 11KT synthesis is still poorly understood. In the present study, for the deeper understanding of testicular 11KT biosynthesis, we first investigated the steroidogenic pathway to produce 11KT in Japanese eel testis. In vitro incubation of the testis with androstenedione (A4) and the subsequent analysis of the metabolites by thin-layer chromatography indicated that 11KT was synthesized from A4 via 11β-hydroxyandrostenedione (11OHA4) and 11-ketoandrostenedione (11KA4), which indicated that the steroidogenic enzyme exhibiting the 17KSR activity responsible for converting 11KA4 to 11KT is crucial for 11KT production. Subsequently, cDNAs encoding three candidate enzymes, Hsd17b type3 (Hsd17b3), Hsd17b type12a (Hsd17b12a), and 20β-hydroxysteroid dehydrogenase type2 (Hsd20b2), potentially with the 17KSR activity were isolated and characterized in the Japanese eel. The isolated hsd17b3, hsd17b12a, and hsd20b2 cDNAs putatively encoded 308, 314, and 327 amino acid residues with high homology to those of other vertebrate counterparts, respectively. The Hsd17b3, Hsd17b12a, and Hsd20b2 expressed either in HEK293T or in Hepa-E1 converted 11KA4 to 11KT. Tissue-distribution analysis by quantitative real time PCR revealed that hsd17b12a and hsd20b2 mRNAs were detected in the testis, while hsd17b3 mRNA was not detectable. Furthermore, we examined the effects of Gths on the 17KSR activity and the expression of the candidate genes in the immature testis. The 17KSR activity was upregulated by administration of Gths. Furthermore, only expression of hsd17b12a among three candidates was upregulated by Gths as well as the 17KSR activity. These findings strongly suggested that Hsd17b12a is one of the enzymes with 17KSR activity responsible for 11KT synthesis in the testis of Japanese eel. Show less