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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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Vitellogenesis has been extensively studied in oviparous vertebrates, including teleost fishes, while not much is known with regard to jawless hagfishes, modern representatives of the most primitive vertebrate class. This study aimed to characterize vitellogenin (Vtg) and yolk protein (YP) in the inshore hagfish (Eptatretus burgeri) as an initial step to understand vitellogenesis in this species. A putative Vtg fraction was purified from the serum of female hagfish by combinations of hydroxylapatite and ion-exchange chromatography, followed by gel filtration. The purified fraction appeared to contain two distinct Vtgs (Vtg1 and Vtg2) and exhibited biochemical properties resembling those previously reported for teleost Vtgs; these appeared to be female-specific serum proteins and high-molecular-weight proteins in gel filtration (˜505 kDa as the mixture fraction of both Vtgs) and in SDS-PAGE (Vtg1 and Vtg2; ˜210 kDa and ˜195 kDa, respectively). A major YP was also purified from hagfish eggs by combinations of hydroxylapatite chromatography and gel filtration; the apparent native mass of the purified YP was unusually large (> 669 kDa). The purified YP consisted of four polypeptides in SDS-PAGE; the peptide pattern indicated that it consisted of two lipovitellins (Lv1 and Lv2) giving rise to two sets of heavy chains (˜116 kDa and ˜106 kDa, respectively) and two light chains (˜32 kDa and ˜28 kDa, respectively). Additional immunological analysis, Nterminal amino acid sequencing and cDNA cloning firmly confirmed the precursor-product relationship between hagfish Vtgs and Lvs.