title: Primary cultures of dispersed pituitary cells from estradiol-pretreated female silver eels (Anguilla anguilla L.): immunocytochemical characterization of gonadotropic cells and stimulation of gonadotropin release.
abstract: To analyze the multihormonal control mechanisms of GTH secretion in the eel, primary culture of pituitary cells from control or estradiol-treated female silver eels, a treatment known to stimulate in vivo GTH synthesis, was developed. Dispersed eel pituitary cells obtained by enzymatic (trypsin/DNAse) and mechanical dispersion were cultured in Earles M199, at 18 degrees. Immunoreactive GTH (ir-GTH) cells were characterized by the immunoperoxidase method, using antiserum to carp GTH beta subunit. Ir-GTH cells from control silver eels were small and represented 14% of the dispersed pituitary cells. In contrast, ir-GTH cells from estradiol-treated eels were larger (cell area x 2.5) and represented a higher proportion (21%) of the pituitary cells. Intracellular and medium contents of GTH were measured by radioimmunoassay for the GTH beta subunit. In vivo estradiol-treatment increased more than 100-fold the GTH content of cell cultures. GTH release, studied over 1 to 4 hr, was undetectable in cultures from normal eels. In contrast, GTH release was low (less than 2% of cell content) but measurable in cultures from estradiol-treated eels. Subsequent experiments examined effects of various secretagogues on GTH release from primary cultures of pituitary cells from estradiol-pretreated eels. GTH release was significantly increased (x1.5 to x3 basal release) by 10(-6) M GnRH-A as well as by both native GnRHs in the eel (mammalian GnRH, mGnRH, and chicken GnRH-II, cGnRH-II), at the same concentration. Lower GnRH concentrations had no significant effect, indicating a low sensitivity of gonadotrophs to GnRH, likely to be related to their immature state at the silver stage. The similar efficacy of mGnRH and cGnRH-II suggested that the pituitary GnRH receptor had a low specificity toward various molecular forms, in the eel as in the other nonmammalian species. The protein kinase C (PKC) activator (phorbol ester: PMA) also stimulated GTH secretion, with a maximal effect at 10(-8) M, indicating that the PKC pathway was functional. In contrast, a depolarizing agent (50 mM KCl) had no significant effect on GTH release, suggesting lack of a functional voltagesensitive calcium channel (VSCC) secretory pathway. Perifusion experiments on whole pituitary confirmed the lack of effect of KCl on gonadotrophs from E2-pretreated eels and indicated that an additional in vivo treatment with GnRH agonist and dopamine antagonist could induce the differentiation of a functional VSCC pathway. These characteristics of the transduction mechanisms may be related to the immature state of the eel gonadotrophs at the silver stage.
authors.affiliation: Laboratoire de Physiologie Générale et Comparée, Muséum National d'Historie Naturelle, URA 90 CNRS, Paris, France.
authors.forename: M
authors.initials: M
authors.lastname: Montero
authors.forename: N
authors.initials: N
authors.lastname: Le Belle
authors.forename: B
authors.initials: B
authors.lastname: Vidal
authors.forename: S
authors.initials: S
authors.lastname: Dufour
mesh_terms: D000811:Anguilla / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000494:pharmacology; D006065:Gonadotropins, Pituitary / Q000096:biosynthesis*; D007150:Immunohistochemistry; D010477:Perfusion; D010703:Phorbol Esters / Q000494:pharmacology; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011863:Radioimmunoassay
chemical_list: D006065:Gonadotropins, Pituitary; D010703:Phorbol Esters; D007987:Gonadotropin-Releasing Hormone; D004958:Estradiol; D011189:Potassium Chloride; C047717:LHRH, His(5)-Trp(7)-Tyr(8)-