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  • Author: Y. Nagahama x
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W Ge, M Tanaka, M Yoshikuni, Y Eto and Y Nagahama

ABSTRACT

We have cloned a full length cDNA coding for the activin type IIB receptor (GactRIIB) from the goldfish ovary. GactRIIB shares 73 and 70% amino acid identity in the extracellular domain, and 78 and 80% identity in the intracellular domain with the type IIB receptors of the mouse and Xenopus respectively. The intracellular domain of GactRIIB contains two serine kinase consensus sequences, DFKSRN and GTRRYMAPE, in agreement with the reports in other vertebrates that serine/threonine phosphorylation is involved in activin signal transduction. The identity of GactRIIB was confirmed by transient expression in the COS cells followed by activin binding. Iodinated human activin A bound to the GactRIIB-transfected cells and the binding could be completely inhibited by unlabeled activin. Affinity labeling revealed a band of about 85 kDa, which is in agreement with the reported type II receptors in other vertebrates. Together with the fact that activin is expressed in the goldfish ovary, the cloning of activin receptors from the ovary suggests paracrine and autocrine roles for activin in the goldfish ovarian functions.

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T Kitano, K Takamune, T Kobayashi, Y Nagahama and SI Abe

The phenotypic sex of many teleost fishes including flounders can be experimentally altered by treating embryos or larvae with varied temperatures or sex-steroid hormones. To analyse the sex determination mechanism, especially the role of cytochrome P450 aromatase (P450arom), an enzyme that catalyses the conversion of androgens to estrogens, in temperature-dependent gonadal sex differentiation in the Japanese flounder, we generated two populations of larvae, both having XX (genetic females) but each growing up to display all phenotypic females or males, by rearing the larvae at normal (18 degrees C) or high (27 degrees C) water temperatures from days 30 to 100 after hatching respectively. The larvae (XX) were produced artificially by mating normal females (XX) with gynogenetic diploid males (XX) which had been sex-reversed to phenotypic males by 17alpha-methyltestosterone. To study the role of P450arom in sex determination in the flounder, we first isolated a P450arom cDNA containing the complete open reading frame from the ovary. RT-PCR showed that P450arom mRNA was highly expressed in the ovary and spleen but weakly in the testis and brain. Semi-quantitative analyses of P450arom mRNA in gonads during sex differentiation showed that there was no difference in the levels of P450arom mRNA between the female and male groups when the gonad was sexually indifferent (day 50 after hatching). However, after the initiation of sex differentiation (day 60), the mRNA levels increased rapidly in the female group, whereas they decreased slightly in the male group. Similarly, estradiol-17beta levels rose remarkably in the female group, yet remained constant in the male group. These results suggest that induction of sex reversal of genetically female larvae to phenotypic males by rearing them at a high water temperature caused a suppression of P450arom gene expression. Furthermore, we suggest that the maintenance of P450arom mRNA at very low levels is a prerequisite for testicular differentiation, while the increased levels are indispensable for ovarian differentiation.

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L Y Zhou, D S Wang, B Senthilkumaran, M Yoshikuni, Y Shibata, T Kobayashi, C C Sudhakumari and Y Nagahama

In order to elucidate the roles of 17β-HSDs in fish gonadal steroidogenesis, three types of 17β-HSDs (17β-HSD1, 17β-HSD8 and putative 17β-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17β-HSD type 1, 8 and 12 were 1504, 1006 and 1930 bp long, with open reading frames encoding proteins of 289, 256 and 314 aminoacids, respectively. Tissue distribution pattern analyzed by RT-PCR and Northern blot showed that 17β-HSD1 was dominantly expressed in the ovary, while the putative 17β-HSD12, one of the two duplicates found in fish, is a male specific enzyme and expressed exclusively in testis (detected by RT-PCR only). On the other hand, 17β-HSD8 was expressed in the brain, gill, heart, liver, intestine, gonad, kidney and muscle of both male and female. Enzymatic assays of the three types of 17β-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17β-HSD1 expressed in E. coli had the preference for NADP(H) as cofactor and could catalyze the inter-conversion between estrone and estradiol efficiently as well as the inter-conversion between androstenedione and testosterone, but less efficiently. Tilapia 17β-HSD8 recombinant protein expressed in HEK 293 cells could catalyze the conversion of testosterone to androstenedione, as well as the inter-conversion between estrone and estradiol. However, the putative 17β-HSD12 expressed in E. coli or in HEK 293 cells showed no conversion to any of the four substrates tested in this study. Based on enzyme characterization and tissue distribution, it is plausible to attribute crucial roles to 17β-HSDs in the gonadal steroidogenesis of teleosts.

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X T Chang, T Kobayashi, H Kajiura, M Nakamura and Y Nagahama

ABSTRACT

A cDNA clone encoding the complete tilapia (a teleost fish, Oreochromis niloticus) cytochrome P450 aromatase (P450arom) was isolated from an ovarian follicle cDNA library. The deduced amino acid sequence (522 amino acid residues) had 72·2% and 59·5% homology with rainbow trout and catfish P450arom respectively, and about 50% homology with mammalian and avian P450arom. Expression of this cDNA in COS-7 cells produced a protein that converted exogenous testosterone to estrogens. Northern blots using a tilapia P450arom cDNA fragment and Western blots using an antiserum against a tilapia P450arom polypeptide fragment revealed a single P450arom mRNA (2·6 kb) and a single protein (59 kDa) in tilapia ovarian tissue respectively. These analyses also revealed that the levels of both P450arom mRNA and protein were low in early vitellogenic follicles, increased in midvitellogenic follicles, and declined to non-detectable levels in post-vitellogenic follicles. Changes in the ability of follicles to convert exogenous testosterone to estrogens (aromatase activity) were similar to those of P450arom mRNA and protein. These observations indicated that the capacity of tilapia ovarian follicles to synthesize estradiol-17β is closely related to the contents of P450arom mRNA and protein within them.

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M. Tanaka, T. M. Telecky, S. Fukada, S. Adachi, S. Chen and Y. Nagahama

ABSTRACT

The enzyme aromatase P-450 (P450arom) catalyses the conversion of androgen to oestrogen. A cDNA insert encoding P450arom was isolated from a rainbow trout (Oncorhynchus mykiss) ovary cDNA library. The insert was sequenced and found to contain an open-reading frame predicted to encode a protein of 522 amino acid residues. The deduced polypeptide is 52% homologous with human, mouse and rat P450arom and 53% homologous with that of chicken. The insert was confirmed to encode P450arom by introducing it into COS-1 monkey kidney tumour cells (COS-1 cells) and detecting the conversion of testosterone to oestradiol-17β by radioimmunoassay. The N-terminal region of the deduced polypeptide was 19 amino acids longer than that of the other four species, and was found by hydropathy plotting to be very hydrophobic.

Northern blot analysis revealed 2.6kb RNA transcripts which were present in the trout ovary during vitellogenesis and hybridized to the cDNA insert. In preparations from subsequent stages of ovarian development, no RNA transcripts hybridized to the probe. Since the RNA transcripts are present only during the stage of oestradiol-β production by the ovarian follicles, oestradiol-17β production may be regulated, in part, by the amount of P450arom mRNA present.

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W Ge, T Miura, H Kobayashi, R E Peter and Y Nagahama

ABSTRACT

We have cloned a full length cDNA coding for activin βB subunit from the goldfish ovary. Sequence analysis of the goldfish activin βB shows that this peptide is extremely conserved across vertebrates. The mature region of goldfish activin βB has 93 and 98% amino acid identity with that of human and zebrafish βB subunit respectively. The identity of the cloned goldfish activin βB was further confirmed by expressing the protein in the Chinese hamster ovary (CHO) cells followed by detection of the specific activity of activin in the culture medium using F5-5 cell assay. mRNA of goldfish activin βB is expressed in a variety of goldfish tissues including ovary, testis, brain, pituitary, kidney and liver, suggesting a wide range of physiological roles for activin in the goldfish.

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C Mittelholzer, E Andersson, D Consten, T Hirai, Y Nagahama and B Norberg

Abstract

In order to better quantify the molecular mechanisms regulating final oocyte maturation and spawning, complete coding sequences with partially or fully untranslated regions for the steroidogenic enzymes, cytochrome P450 aromatase and 20β-hydroxysteroid dehydrogenase, were cloned from ovaries of Atlantic cod (Gadus morhua). The nucleotide and amino acid sequences showed high homologies with the corresponding sequences of other fish species, and conserved features important for functionality were identified in both predicted proteins. The sequences of the corresponding genomic loci were also determined, allowing the design of mRNA-specific quantitative PCR assays. As a reference gene for the real-time RT-PCR assays, eukaryotic elongation factor 1α was chosen, and the mRNA as well as the genomic sequence was determined. In addition, a real-time quantitative PCR assay for the 18S rRNA was adapted to be used in cod. Analysis of immature and maturing female cod from July to January respectively showed that the enzyme genes showed the expected quantitative changes associated with physiological regulation. However, mRNA for eukaryotic elongation factor 1α, and to a lesser extent even 18S rRNA, showed variable expression in these samples as well. To find accurate standards for real-time PCR in such a dynamic organ as the cod ovary is not an easy task, and several possible solutions are discussed.

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JQ Jiang, DS Wang, B Senthilkumaran, T Kobayashi, HK Kobayashi, A Yamaguchi, W Ge, G Young and Y Nagahama

The Japanese eel (Anguilla japonica) and Nile tilapia (Oreochromis niloticus) 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) cDNAs were isolated from their respective testes cDNA libraries. The cDNAs predict two peptides of 436 and 406 amino acid residues that share about 42% homology with mammalian 11beta-HSD type 2 proteins. Analysis of the tissue distribution pattern by RT-PCR reveals that 11beta-HSD2 is expressed in a wide variety of tissues in tilapia, with higher expression in kidney and gill of both sexes, and with the highest expression in testis. 11beta-Dehydrogenase activity of the eel 11beta-HSD2 was confirmed by demonstrating the conversion of cortisol to cortisone by the recombinant protein after transient expression of this cDNA clone in COS-1 cells. Bands of approximately 2.7 and approximately 3.8 Kb were detected in Northern blot of eel and tilapia testes respectively, which is consistent with the cloned cDNA sizes of the two species. Northern blot analysis also revealed that the expression of the eel testis 11beta-HSD2 gene could be induced by human chorionic gonadotropin (hCG) injection, implying a role of 11beta-HSD2 in hCG-induced 11-ketotestosterone production and spermatogenesis in the Japanese eel.