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

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 Nephrops norvegicus 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 Homarus americanus. 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 Escherichia coli fused to glutathione-S-transferase. Immunocytochemical studies were carried out by using this antibody in N. norvegicus and in other crustaceans, Munida rugosa and Squilla mantis; 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.