Molecular characterization and expression of FSHbeta, LHbeta, and common alpha-subunit in male Atlantic halibut (hippoglossus hippoglossus)

Summary

To elucidate the role of the gonadotropins in the multiple spawner Atlantic halibut (Hippoglossus hippoglossus) full length cDNAs encoding FSHβ, LHβ, and the common α-subunit were cloned from pituitary glands by RACE-PCR. The three cDNAs consisted of 614, 595, and 666 nucleotides encoding peptides of 131, 146, and 124 amino acids, respectively. Halibut FSHβ and LHβ showed unique structural features among the vertebrate glycoprotein hormones. First, in contrast to all known FSHβ, which contain either one or two conserved N-glycosylation sites, no potential binding site was found in Atlantic halibut FSHβ. Second, the conserved glycosylation site in the N-terminus of all vertebrate LHβ has been substituted with a unique C-terminal binding site in Atlantic halibut LHβ. Furthermore, a specific cysteine residue of importance for the folding and heterodimerization of mammalian FSH is lacking in the FSHβ from Atlantic halibut as well as many other teleosts. However, teleost FSHβ is characterized by an additional N-terminal cysteine, which has likely replaced the missing residue, implicating a modified folding pattern of this subunit. In situ hybridization of mature male pituitaries revealed that FSHβ and LHβ mRNA were expressed in distinct cell types throughout the proximal pars distalis of the adenohypophysis, while α-subunit mRNA was identified in all parts of the proximal pars distalis, and also along the periphery of pars intermedia. Consistently, Northern blot analysis of pituitary RNA from mature males showed that FSHβ, LHβ, and α-subunit mRNAs were highly expressed. In juvenile male pituitaries very few cells containing FSHβ, LHβ, and α-subunit mRNA were identified by in situ hybridization. Low mRNA levels encoding LHβ and the α-subunit were also demonstrated by Northern blot analysis of the juvenile pituitaries, while no FSHβ mRNA was detected using this less sensitive technique. The results suggest that both FSH and LH play a role during both the very early and the final reproductive stages in Atlantic halibut males.