Yasutoshi Yoshiura

Synergistic Expression of Ad4BP/SF-1 and Cytochrome P-450 Aromatase (Ovarian Type) in the Ovary of Nile Tilapia, Oreochromis niloticus, During Vitellogenesis Suggests Transcriptional Interaction

Abstract Involvement of Ad4BP/SF-1 in the ovarian cytochrome P-450 aromatase (oP450arom) gene expression was investigated using ovarian follicles of the Nile tilapia, possessing an average 14-day spawning cycle. The promoter region (5′ flanking region) of oP450arom gene cloned from tilapia contains two Ad4 binding sites. Subsequently, a cDNA encoding Ad4BP/SF-1 was cloned from the ovarian follicles. It is expressed in gonadal tissues, brain, and kidney. Oligonucleotide probes containing putative orphan nuclear receptor binding motifs (derived from promoter region of the aromatase gene) formed complexes with in vitro-translated Ad4BP/SF-1 and nuclear extracts of tilapia ovarian (midvitellogenic) follicles, indicating that Ad4BP/SF-1 is one of the transcriptional regulators for aromatase gene expression. Northern blot analysis revealed that the expression of both oP450arom and Ad4BP/SF-1 increased in parallel with ovarian growth from Day 0 to Day 5 after spawning and declined sharply from Day 8 to Day 11. On the day of spawning (Day 14), the expression of both correlates became undetectable. In vitro incubation of post vitellogenic full-grown immature follicles (corresponding to Day 11 after spawning) with hCG purged both oP450arom and Ad4BP/SF-1 messenger RNA transcripts at 18 h. Conversely, in vitro incubation of late vitellogenic follicles (corresponding to Day 8 after spawning) with hCG retained Ad4BP/SF-1 messenger RNA transcripts more or less steadily and up-regulated oP450arom. Ad4BP/SF-1 probably acts as a transcriptional modulator to implement the paradoxical actions of gonadotropins on oP450arom gene.

Medaka (Oryzias latipes) FTZ-F1 potentially regulates the transcription of P-450 aromatase in ovarian follicles: cDNA cloning and functional characterization.

Our previous findings suggest the activity of cytochrome P-450 aromatase (P-450arom), the enzyme which converts testosterone to estradiol-17beta, in the ovarian follicle of medaka (Oryzias latipes) is regulated at the transcriptional level. In this study, we cloned a cDNA encoding a FTZ-F1-like protein (mdFtz-F1) from ovarian follicles of medaka. In vitro translated mdFTZ-F1, and nuclear extract from medaka ovarian follicles, formed complexes with oligonucleotide probes containing putative orphan nuclear receptor binding motifs, which are present in the promoter region of the medaka P-450arom gene. The expression pattern of mdFtz-F1 transcripts during oogenesis coincides with that of P-450arom transcripts. Transfection assays further suggest a potential transcriptional regulatory activity of mdFTZ-F1 upon the medaka P-450arom promoter. Taken together, these results suggest a potential role of mdFTZ-F1 in the transcriptional regulation of P-450arom in the ovarian follicle of medaka.

Fish gonadotropin and thyrotropin receptors: the evolution of glycoprotein hormone receptors in vertebrates.

We have cloned and characterized, for the first time in fish, two different gonadotropin receptors (GTHR) and a single thyrotropin receptor (TSHR) from amago salmon (Oncorhynchus rhodurus) and Nile tilapia (Oreochromis niloticus). Phylogenetic analyses and intron/exon structure suggest that the two GTHRs in fish are comparable to tetrapod follicle stimulating hormone and luteinizing hormone receptors. Temporal and spatial expression patterns, examined by Northern blot analysis and in situ hybridization, paralleled those seen in mammals and birds. Consequently, genetic and functional divergence of two GTHRs and TSHR probably occurred before the teleost and tetrapod split.

Molecular Cloning of cDNA Encoding a 20S Proteasome α2 Subunit from Goldfish (Carassius auratus) and Its Expression Analysis

Abstract Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most regulated intracellular protein breakdown in eukaryotic cells. The core proteinase of this complex, the 20S proteasome, is comprised of four stacked rings with seven subunits each. The outer two rings are made up of seven, distinct α-type subunits, while the two inner rings are composed of seven, different β-type subunits. Here we present the cloning, sequencing and expression analysis of Carassius auratus, α2_ca, which encodes one of the proteasome α subunits from goldfish ovary. The cloned cDNA is 838 bp long and encodes 234 amino acids. The deduced amino acid sequence is highly homologous to α2 subunits from other vertebrates. The expression of mRNA for α2_ca occurs at very high levels in ovary and muscle and moderately high levels in testis, brain and gill. It was also shown that protein content was different from mRNA expression levels.