Gonad-inhibiting hormone of the Norway lobster (Nephropsnorvegicus): cDNA cloning, expression, recombinant protein production, and immunolocalization

Abstract

The gonad-inhibiting hormone (GIH) belongs to a neuropeptide family synthesized and released in a neurohemal complex of crustacean eyestalks. The GIH is involved in gonad maturation and plays a more complex role in the control of reproduction and molting. With a combination of reverse transcription–polymerase chain reaction (RT–PCR) and rapid amplification of cDNA ends approaches we determined the cDNA sequence of the Norway lobster Nephropsnorvegicus prepro GIH. The open reading frame of 339 bp codes for a polypeptide of 112 amino acids showing 96% identity with the other known GIH of Homarusamericanus. The precursor peptide consists of a putative signal peptide of 31 amino acids and a putative mature peptide region of 81. RT–PCR analysis shows that GIH mRNA is expressed mainly in eyestalks, both in female and male; the expression of GIH mRNA also in supraesophageal ganglia suggests the existence of additional GIH-producing neurons besides those of eyestalks. A specific polyclonal antibody was raised against a portion of the mature peptide region obtained through expression in Escherichiacoli fused to glutathione-S-transferase. Immunocytochemical studies were carried out by using this antibody in N. norvegicus and in other crustaceans, Munidarugosa and Squillamantis; these locate GIH in superficial axon terminals of the releasing organ, the sinus gland. The identification of a second GIH sequence in crustaceans allows to hypothesize the occurrence, within the neuropeptide family, of three subfamilies probably involved in different functions: crustacean hyperglycemic hormones, GIHs and molt-inhibiting hormones/mandibular organ-inhibiting hormones.