Hepatic estrogen-responsive genes relating to oogenesis in cutthroat trout (Oncorhynchus clarki): The transcriptional induction in primary cultured hepatocytes and the in vitro promoter transactivation in responses to estradiol-17β

Abstract

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.