Takashi Todo

Two subtypes of androgen and progestogen receptors in fish testes.

Two subtypes (alpha and beta) of androgen (AR) and progestogen receptors (PR) are present in the testis of Japanese eel (Anguilla japonica). Amino acid homology of the open reading frames between alpha and beta in AR or PR is approximately 40%, but the DNA- and ligand-binding domains show high homology between subtypes. Judging from these structures, alpha and beta are not isoforms derived from translational initiation at two in-phase ATG codons, alternative splicing, or tetraploidy. In transient transfection assays using a reporter construct containing a steroid-responsive promoter, each subtype showed its corresponding hormone-dependent transactivation. The ligand affinity for transactivation between AR and PR subtypes was similar for physiological ligands. Tissue distribution of both subtype mRNAs was different. Protein interaction between subtypes was demonstrated in vitro by GST pull-down assays. These results clearly indicate that two functional subtypes of AR and PR exist in eel. These findings will advance our understanding of the mechanisms underlying sex steroid signaling.

Molecular Cloning of Estrogen Receptors α and β in the Ovary of A Teleost Fish, the Tilapia (Oreochromis niloticus)

Abstract Estrogen receptors (ER) in mammals have recently been shown to be encoded by two distinct genes, ERα and ERβ. In this study, cDNAs encoding two tilapia ER subtypes, tER5.1 and tER4.3, were cloned from an ovarian cDNA library of a teleost fish, the tilapia Oreochromis niloticus. The tER5.1 and tER4.3 contain complete open reading frames encoding 585 and 557 amino acid residues, respectively. The two receptors share about 12% homology in the A/B domain, 96% in the DNA binding domain (C domain), 12% in the D domain, 57% in the ligand binding domain (E damain), and 20% in the F domain. Phylogenetic analysis of ER proteins from various vertebrate species indicated that vertebrate ERs consist of two major groups (ERα and ERβ); tER5.1 and tER4.3 belong to ERα and ERβ subtypes, respectively. Thus, we consider tER5.1 and tER4.3 to be the tilapia homologs of ERα (tERα) and ERβ (tERβ), respectively. In transient transfection assays using mammalian COS-7 cells, both tERα and tERβ showed estradiol-17β dependent activation of transcription from the estrogen-responsive ERE-Luc promoter. This is the first report of the presence of ERα and ERβ within a single non-mammalian vertebrate species.

Teleost Ovarian Carbonyl Reductase-Like 20β-Hydroxysteroid Dehydrogenase: Potential Role in the Production of Maturation-Inducing Hormone During Final Oocyte Maturation

Abstract 17α,20β-Dihydroxy-4-pregnen-3-one is the major oocyte maturation-inducing hormone of several teleost species. Gonadotropin-induced increase in ovarian 20β-hydroxysteroid dehydrogenase activity is essential for the synthesis of maturation-inducing hormone. Cloning and expression studies suggest that ayu (Plecoglossus altivelis) ovarian carbonyl reductase can function as 20β-hydroxysteroid dehydrogenase. The amino acid sequence deduced from the isolated cDNA had 276 amino acid residues and shared approximately 60% homology with mammalian and teleostean carbonyl reductases. The sequence data search showed that the ayu cDNA clone belongs to the short-chain dehydrogenase/reductase family. The clear lysate prepared from Escherichia coli harboring the cDNA catalyzed the production of maturation-inducing hormone. Its identification was confirmed by two-dimensional, thin-layer chromatography followed by recrystallization. Purification of the E. coli-expressed cDNA product revealed that it possessed both carbonyl reductase and steroid dehydrogenase activities, and 17α-hydroxyprogesterone, the endogenous immediate precursor of maturation-inducing hormone, was one of the preferred substrates. Furthermore, Northern blot analysis denoted that the transcripts are present both in fully grown, immature ovarian follicles and at higher levels in mature ovarian follicles. These results demonstrate that the carbonyl reductase of ayu ovary is involved in the production of maturation-inducing hormone, and they provide evidence for a novel physiological role of this enzyme in the final maturation of oocytes. Based on its functional properties, the enzyme can be referred to as carbonyl reductase-like 20β-hydroxysteroid dehydrogenase.

Isoleucine-15 of rainbow trout carbonyl reductase-like 20β-hydroxysteroid dehydrogenase is critical for coenzyme (NADPH) binding

Carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase (CR/20beta-HSD) is an enzyme that converts 17alpha-hydroxyprogesterone to 17alpha, 20beta-dihydroxy-4-pregnen-3-one (the maturation-inducing hormone of salmonid fish). We have previously isolated two types of CR/20beta-HSD cDNAs from ovarian follicle of rainbow trout (Oncorhynchus mykiss). Recombinant proteins produced by expression in Escherichia coli in vitro showed that one (type A) had CR and 20beta-HSD activity but that the other (type B) did not. Among the three distinct residues between the protein products encoded by the two cDNAs, two residues (positions 15 and 27) are located in the N-terminal Rossmann fold, the coenzyme binding site. To investigate the structure/function relationships of CR/20beta-HSDs, we generated mutants by site-directed mutagenesis at the following positions: MutA/I15T, MutB/T15I, and MutB/Q27K. Enzyme activity of wild-type A was abolished by substitution of Ile-15 by Thr (MutA/I15T). Conversely, enzyme activity was acquired by the replacement of Thr-15 with Ile in type B (MutB/T15I). MutB/T15I mutant showed properties similar to the wild-type A in every aspect tested. Mutation MutB/Q27K had only partial enzyme activity, indicating that Ile-15 plays an important role in enzyme binding of cofactor NADPH.

Transgenic cell lines which stably express progestogen receptors (PRS) α and β and the PR-responsive reporter genes

Transgenic cell lines which stably express progestogen receptors (PRs) and the PR-responsive reporter genes were developed. They are a good system for rapid, sensitive and reproducible screening of various ligands.