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A cDNA encoding a putative gonadoliberin receptor was cloned from the pituitary of the African catfish. Conceptual translation predicts a protein of 379 amino acids which shows typical characteristics of GTP-binding-protein-coupled receptors. The isolated cDNA was stable expressed in human embryonic kidney (HEK) 293 cells which were used for studies on gonadoliberin-activated second messenger systems (inositol phosphate production; increase in CAMP andor intracellular Ca2' ). The isolated cDNA encoded a functional receptor, designated catfish gonadoliberin receptor (cfGnRH-R), which had an amino acid sequence similarity of 38 % with mammalian gonadoliberin receptors. In contrast to its mammalian counterparts which lack an intracellular carboxy-terminal domain, the cfGnRH-R contains an additional 49 amino acid residues. From the two endogenous gonadoliberins in African catfish, chicken gonadoliberin-11 had a several hundredfold higher potency than catfish gonadoliberin to activate cfGnRH-R-associated second messenger systems in transfected HEK 293 cells. This is in line with the previously determined higher gonadotropin-release capacity of chicken gonadoliberin-I1 in catfish. Stimulation of second messenger systems with chicken gonadoliberin-11, but not with catfish gonadoliberin, resulted in a biphasic effect and chicken gonadoliberin-I1 led to a higher maximum stimulation than catfish gonadoliberin. Challenging cfGnRH-R simultaneously with chicken gonadoliberin-I1 and catfish gonadoliberin did not lead to additive effects. In contrast, two types of mutual inhibitory effects were recorded. These data indicate that a single cognate cfGnRH-R couples with distinct efficacies to signal transduction systems upon stimulation by the two endogenous gonadoliberins which, in addition, may interact negatively.Keywords: GTP-binding-protein-coupled receptor; gonadoliberin receptor; cDNA ; pituitary ; transfection.Gonadoliberin is the central regulator of reproduction in vertebrates. It is produced by neuroendocrine neurons in the ventral forebrain which release the hormone into the hypophyseal portal blood system or, in the case of teleost fish, directly in the vicinity of pituitary gonadotrophs. Nine different molecular forms of this decapeptide have been characterized in vertebrate brains so far. In highly evolved eutherian mammals only mammalian gonadoliberin (Glp-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH,) Note. The novel nucleotide sequence published here has been submitted to the EMBL data bank and is available under accession number x97497. which all show a high degree of sequence similarity and display the seven transmembrane structure characteristic for GTP-binding-protein-(G-protein)-coupled receptors [3 -91. lnteraction of gonadoliberin with its receptor results in the activation of a G protein that catalyzes phosphatidylinositol turnover. This stimulates the pituitary gonadotrophs to release gonadotropins, which in turn regulate the activity of the gonads. No information is available on the structure of gonadoliberi...

Primordial germ cell (PGC) development in the zebrafish is poorly understood. The expression of vasa RNA, the only molecular marker so far found to be expressed in fish PGCs, suggests its function in the establishment of the germline. The protein product of vasa is present throughout the life cycle in the germline of Drosophila, Caenorhabditis and Xenopus. The expression pattern of the Vasa protein in zebrafish, is still unresolved. We generated an anti-Vasa polyclonal antibody and show that it is maternally expressed initially throughout the embryo. Interestingly, from the two-cell- to the 1000-cell stage the protein is highly concentrated in two 'dots' near the center of the blastomeres and as such remains longest detectable in the animal pole blastomeres. The first distinct cell-specific expression occurs at 60% epiboly on one side of the blastoderm margin. The Vasa protein in the PGCs is organized in a subcellular granular-like conformation which is dynamic throughout development.

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The expression of prepro-catfish GnRH mRNA and prepro-chicken GnRH-II mRNA was investigated by means of in situ hybridization. The differential distribution of cells expressing the respective mRNAs was compared with the distribution of cells immunoreactive for (1) catfish (cf) GnRH and chicken (c) GnRH-II and (2) both GnRH-associated peptides (GAPs). It was found that the prepro-cfGnRH mRNA expressing cells were located in the ventral forebrain, with a similar distribution of the cfGnRH- and cfGAP-immunoreactive perikarya. The prepro-cGnRH-II mRNA expressing cells were exclusively located in the midbrain tegmentum, at the same position as a group of large cGnRH-II- and CIIGAP-immunoreactive perikarya. It was concluded that the peptidergic neurons in the ventral forebrain contain cfGnRH, whereas cGnRH-II perikarya are restricted to the midbrain. The proximal pars distalis of the pituitary, containing the gonadotropin cells, is innervated by fibers immunoreactive for both cfGnRH and cfGAP and originating from the cfGnRH neurons in the ventral forebrain. We could, however, not detect fibers innervating the pituitary that were immunoreactive for cIIGAP.

The cDNAs encoding the catfish prepro-gonadotropin-releasing hormone and the chicken prepro-gonadotropin-releasing hormone I1 of the African catfish (Clarias gariepinus) have been isolated and sequenced. The catfish gonadotropin-releasing-hormone precursor and the chicken gonadotropin-releasing-hormone-I1 precursor have the same overall architecture as other gonadotropinreleasing-hormone precursors identified so far ; each is composed of a signal peptide, gonadotropinreleasing hormone and a gonadotropin-releasing-hormone-associated peptide which is connected to gonadotropin-releasing hormone by a Gly-Lys-Arg sequence. The amino acid sequences of catfish gonadotropin-releasing hormone and chicken gonadotropin-releasing hormone 11, in combination with the Gly-Lys-Arg sequence, are highly conserved during evolution when compared with the corresponding regions of mammalian, avian (chicken gonadotropin-releasing hormone I) and other fish gonadotropin-releasing-hormone precursors. However, the gonadotropin-releasing-hormone-associated peptide regions are markedly divergent. Northem-blot analysis revealed the presence of a single catfish gonadotropin-releasing-hormone mRNA species of about 470 bases, and the presence of a single chicken gonadotropin-releasing-hormone-I1 mRNA species of about 650 bases in the African catfish brain. In situ hybridization revealed catfish gonadotropin-releasing-hormone cell bodies rostro-caudally scattered in the olfactory nerve, along both sides of the midline of the telencephalon, in the preoptic area of the ventral hypothalamus, and in the infundibular stalk close to the pituitary. Chicken gonadotropin-releasing-hormone-I1 cell bodies, however, were exclusively found in the midbrain tegmentum.Gonadotropin-releasing hormone plays a central role in the development and in the maintenance of reproductive functions in vertebrates. The secretion of gonadotropin-releasing hormone by neurosecretory cells regulates the synthesis and release of pituitary gonadotropins, which stimulate gametogenesis and steroidogenesis in the gonads. In turn, gonadal steroids feedback to the hypothalamus and to the pituitary, thus establishing the hypothalamus-pituitary-gonadal axis.Since the elucidation of the structure of mammalian gonadotropin-releasing hormone [ 1, 21, seven other forms of gonadotropin-releasing hormone with distinct primary structures have been reported [3]. The gonadotropin-releasing hormones are named after the species from which they were first isolated by peptide chemistry, but their distribution is not limited to the namesake species. In brain extracts of most submammalian vertebrates, including different fish species, more than one form of gonadotropin-releasing hormone has Correspondence to J. Bogerd,