Gonadotrophs but not somatotrophs carry gonadotrophin-releasing hormone receptors: receptor localisation, intracellular calcium, and gonadotrophin and GH release.

Gonadotrophs are the primary target cells for GnRH in the pituitary. However, during a limited period of neonatal life in the rat, lactotrophs and somatotrophs respond to GnRH as well. Also, in the adults of a number of teleost fishes (e.g. carp, goldfish, and tilapia but not trout), GnRH is a potent GH secretagogue. In studying hypophysiotrophic actions of the two forms of GnRH present in the African catfish (Clarias gariepinus), chicken GnRH-II ([His5,Trp7,Tyr8]GnRH; cGnRH-II) and catfish GnRH ([His5,Asn8]GnRH; cfGnRH), we have investigated the effects of GnRH on catfish gonadotrophs and somatotrophs. GnRH binding was examined by incubating dispersed pituitary cells attached to coverslips with 125I-labelled [D-Arg6,Trp7,Leu8,Pro9-Net]GnRH (sGnRHa), a salmon GnRH analogue with high affinity for the GnRH receptor. Following fixation and immunohistochemistry using antisera against catfish LH and GH, 125I-labelled sGnRHa was localised autoradiographically and silver grains were quantified on gonadotrophs and somatotrophs. Specific binding of 125I-labelled sGnRHa was restricted to gonadotrophs. Both cfGnRH and cGnRH-II dose-dependently inhibited 125I-labelled sGnRHa binding to gonadotrophs. To substantiate the localisation of functional GnRH receptors, the effects of cfGnRH and cGnRH-II on the cytosolic free calcium concentration ([Ca2+]i) were examined in Fura-2-loaded somatotrophs and gonadotrophs. GnRH-induced increases in [Ca2+]i appeared to be confined to gonadotrophs, in which both endogenous GnRHs caused a single and transient increase in [Ca2+]i. The amplitude of this [Ca2+]i transient depended on the GnRH dose and correlated well with the GnRHs' effect on LH release. In vivo experiments demonstrated that GnRH treatments which markedly elevated plasma LH levels had no effect on plasma GH levels, while a dopamine agonist (apomorphine) significantly elevated plasma GH levels. We conclude that the two endogenous forms of GnRH in the African catfish are not directly involved in the regulation of the release of GH, suggesting that GnRHs cannot be considered as GH secretagogues in teleosts in general.

Effect of route and frequency of administration of apomorphine on growth hormone release in African catfish (Clarias gariepinus).

Apomorphine is known to stimulate growth hormone release in African catfish following an intraperitoneal (IP) injection. In the present study the effect of apomorphine (5 or 20 mg/kg body weight) on plasma GH levels was evaluated after gastro-intestinal or parenteral delivery. Apomorphine increased the plasma GH concentration regardless of the route of administration, indicating that apomorphine can be absorbed from the intestinal tract. The effect of repeated administration of apomorphine differed clearly between the tested doses. Although a single IP injection with 20 mg apomorphine/kg body weight resulted in a clear increase in plasma GH levels, a second injection given 12 hours later was ineffective. In contrast the last of 4 consecutive injections with 5 mg apomorphine/kg body weight given at intervals of 12 hours stimulated the plasma GH levels in a similar way to a single IP injection with the same dose.

A radioimmunoassay for African catfish growth hormone: validation and effects of substances modulating the release of growth hormone.

A highly sensitive radioimmunoassay has been developed for measuring plasma growth hormone (GH) concentrations in the African catfish (Clarias gariepinus). The lower detection limit of the assay was 0.1 ng/ml and the standard curve had an ED50 value of 0.5 ng/ml. The validity of the assay was established and the effects of several neurotransmitters on the release of GH were examined. In vitro experiments, using a static culture system for dispersed pituitary cells, demonstrated that the GH release in African catfish was affected by growth hormone-releasing hormone and somatostatin. Single intraperitoneal injections with a dopamine agonist, apomorphine, produced significant and dose-dependent increases in plasma GH levels. Unlike carp, goldfish, and tilapia, a super-active analogue of salmon gonadotrophin-releasing hormone did not alter plasma GH levels in African catfish.

One-step immunoaffinity purification and partial characterization of hypophyseal growth hormone from the African catfish, Clarias gariepinus (Burchell).

Growth hormone (GH) was purified from African catfish (Clarias gariepinus) pituitary extracts in a single step by use of immunoaffinity chromatography. A monoclonal antibody to chicken GH, which labels the catfish hypophyseal somatotropes in immunocytochemistry, was coupled to CNBr-activated Sepharose, and crude alkaline pituitary extracts were run over the immunoadsorbent. Reversed-phase high-performance liquid chromatography analysis of the eluted material suggested heterogeneity, whereas silver staining upon SDS-polyacrylamide gel electrophoresis showed one single band with an estimated molecular weight between 22,000 and 23,000 Da. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the same preparation revealed the presence of several components with molecular weights ranging from 20,170 to 20,900 Da. The amino terminus of the protein was homogeneous, and the first 50 residues matched the proposed sequence of GH from two other siluran species (Ictalurus punctatus and Pangasius pangasius), except for one substitution at position 3. These data unequivocally confirm the identity of the purified molecule as suggested by immunochemical evidence. The bioactivity of the GH preparation was demonstrated by the short-term effect of GH on T3 plasma levels in juvenile catfish.