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Artificial fertilization of cultured fish is essential for seed production using breeding techniques. However, in tuna species, the success rate of artificial fertilization is tremendously low. In this study, it was reported that the adequate procedure for ovulated egg collection and storage for artificial fertilization in kawakawa Euthynnus affinis. The collection of ovulated eggs was attempted using new techniques that disrupt only spawning activity without discontinuing ovulation. The available time to use ovulated eggs was also examined by assessing the optimal preservation process and temperature. As a result, artificial fertilization was effectively executed by assessing spawning time and thoroughly extracting ovulated eggs immediately after ovulation, with a success rate of 70% and an ovulation rate of 51.7%. Ovulated eggs could be stored with small quantities of ovarian fluid to sustain fertility. However, fertility was better preserved with Hanks' solution. Ovulated eggs with high productivity were achieved 3 h after egg extraction when maintained in Hanks' solution at 20 °C, leading to a supply of one-cell stage embryo for microinjection treatment constantly by continuously executing artificial fertilization. This systematic procedure permitted selective breeding by 1:1 mating between top-quality parental fish and applying several developmental engineering techniques to kawakawa breeding. [ABSTRACT FROM AUTHOR]

Transcription of vitellogenin ( vtg ) genes are initiated when estradiol-17β (E 2 )-estrogen receptor (ER) complexes bind estrogen response elements (ERE) located in the gene promoter region. Transcriptional regulation of dual vtg subtypes (major salmonid A-type vtg : vtgAs ; minor C-type vtg : vtgC ) by E 2 was investigated under co-expression of a potential major transcriptional factor, erα1 , in cutthroat trout. Two forms of trout vtgAs promoters (1 and 2) and one vtgC promoter were sequenced. These promoters structurally differ based on the number of EREs present. The vtgAs promoter 1 exhibited the highest maximal transcriptional activity by in vitro gene reporter assays. The concentration of E 2 that induces 50% of gene reporter activity (half-maximal effective concentrations, EC 50 ) was similar among all vtg promoters and also to the EC 50 of E 2 administered to induce vtg transcription in vivo . This study revealed a difference in transcriptional properties of multiple vtg promoters for the first time in a salmonid species, providing the basis to understand mechanisms underlying regulation of vitellogenesis via dual vtg gene expression. [ABSTRACT FROM AUTHOR]

One or more distinct forms of the nuclear estrogen receptor (ER) have been isolated from many vertebrates to date. To better understand the molecular evolution of ERs, we cloned and characterized er cDNAs from the inshore hagfish, Eptatretus burgeri , a modern representative of the most primitive vertebrates, the agnathans. Two er cDNAs, er1 and er2 , were isolated from the liver of a reproductive female hagfish. A phylogenetic analysis placed hagfish ER1 into a position prior to the divergence of vertebrate ERs. Conversely, hagfish ER2 was placed at the base of the vertebrate ERβ clade. The tissue distribution patterns of both ER subtype mRNAs appeared to be different, suggesting that each subtype has different physiological roles associated with estrogen actions. An estrogen responsive-luciferase reporter assay using mammalian HEK293 cells was used to functionally characterize these hagfish ERs. Both ER proteins displayed estrogen-dependent activation of transcription. These results clearly demonstrate that the hagfish has two functional ER subtypes. [ABSTRACT FROM AUTHOR]

Fundamental knowledge on the regulation of reproduction by gonadotropins (Gths) is quite limited in viviparous fishes. In the present study, we performed molecular cloning and characterization of cDNAs encoding two Gth subunits (fshb and lhb) from the pituitaries of viviparous white-edged rockfish, Sebastes taczanowskii ; expression profiles of both gene transcripts were elucidated in the pituitaries of reproductive males and females which were kept in a captive environment. The cloned fshb and lhb fragments exhibited high sequence identities with corresponding β-subunit sequences from black rockfish, S. schlegelii. Notably, the fshb of white-edged rockfish appeared to lack a putative N-glycosylation site, whereas lhb conserved it. Expression of fshb and lhb transcripts in the rockfish pituitaries largely changed in synchrony but for minor exceptions. In males, levels of both transcripts increased with progression of spermatogenesis, although the peak for fshb (October) appeared slightly earlier than that for lhb (November). In females, both gene transcripts exhibited synchronous bimodal changes. High expression of fshb and lhb transcripts in the female pituitary during the gestation period, followed by the drastic decrease at parturition, suggest their possible involvement in regulation of gestation of this species. The knowledge gained for Sebastes in this study superimposes fundamental information necessary for further physiological understanding of viviparity in teleost fish. [Display omitted] • cDNAs encoding fshb and lhb were cloned in viviparous white-edged rockfish. • The β-subunit sequences were remarkably identical with those in black rockfish. • Reproductive changes in the β-subunits were tracked in the pituitary gland. • The β-subunit mRNAs were expressed synchronously. • The β-subunit mRNA levels were high in the gestating females. [ABSTRACT FROM AUTHOR]

• Choriogenin, vitellogenin and estrogen receptor-1a genes were estrogen-responsive. • These estrogen responsive genes responded differentially to estradiol-17β (E2). • E2 transactivated these gene promoters in the presence of four estrogen receptors. Estradiol-17β (E2) regulates transcription of estrogen-responsive genes via estrogen receptors (Esr). In many teleost species, choriogenin (chg), vitellogenin (vtg) and esr genes are transactivated by E2 in the liver. This study aimed i) to compare expression properties of all subtypes of these genes (chg : chgHα , chgHβ , chgL ; vtg : vtgAs , vtgC ; esr : esr1a , esr1b , esr2a , esr2b) in response to estrogen stimulation, and ii) to confirm how each of four Esr subtypes is involved in the transcriptional regulation of these estrogen-responsive genes in cutthroat trout hepatocytes. In hepatocytes in primary culture, all chg and vtg subtype mRNA levels, and those of esr1a , were increased by E2 treatment (10−6 M) at 24 and 72 h post initiation (hpi), but esr1b , esr2a and esr2b mRNA levels were not. Treatment of hepatocytes with various concentrations of E2 (10−11–10−6 M) induced dose-dependent increases in the levels of all chg and vtg subtype mRNAs at 24 and 72 hpi. At both time points, the lowest dose that induced a significant increase in the expression levels of mRNAs (LOEC) for E2 differed among the genes; LOECs were estimated as 10−11 M for chgHα at 24 hpi, as 10−9 M for vtgC at 72 hpi, and as 10−10 M for other mRNAs at both 24 and 72 hpi. Meanwhile, the levels of esr1a mRNA exhibited a dose-dependent increase at 24 and 72 hpi, but the LOEC shifted from 10−9 M at 24 hpi to 10−7 M at 72 hpi because of a decrease in mRNA levels at treatment groups exposed to high concentrations of E2. All Esr subtypes transactivated chg , vtg and esr1a promoters in the presence of E2 in vitro. The activation levels indicated that promoter activity of chgHα ≥ vtgAs > chgHβ > chgL ≥ vtgC ≥ esr1a when mediated by Esr1a, chgHβ > chgHα > chgHL > vtgAs ≥ vtgC ≥ esr1a by Esr1b, chgHβ ≥ chgL > chgHα ≥ vtgAs > vtgC > esr1a by Esr2a, and chgHβ ≥ chgHα ≥ vtgAs > chgL ≥ vtgC > esr1a by Esr2b. Collectively, different Esr subtypes were distinctly different in their ability to transactivate estrogen-responsive target genes, resulting in differential expression of chg , vtg and esr1a genes in the estrogen-exposed hepatocytes. [ABSTRACT FROM AUTHOR]

Fish egg yolk is largely derived from vitellogenins, which are synthesized in the liver, taken up from the maternal circulation by growing oocytes via receptor-mediated endocytosis and enzymatically processed into yolk proteins that are stored in the ooplasm. Lipid droplets are another major component of fish egg yolk, and these are mainly composed of neutral lipids that may originate from maternal plasma lipoproteins. This review aims to briefly summarize our current understanding of the molecular mechanisms underlying yolk formation in fishes. A hypothetical model of oocyte growth is proposed based on recent advances in our knowledge of fish yolk formation. [ABSTRACT FROM AUTHOR]