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

To analyze the multihormonal control mechanisms of GTH secretion in th e eel, primary culture of pituitary cells from control or estradiol-tr eated female silver eels, a treatment known to stimulate in vivo GTH s ynthesis, was developed. Dispersed eel pituitary cells obtained by enz ymatic (trypsin/DNAse) and mechanical dispersion were cultured in Earl es M199, at 18 degrees. Immunoreactive GTH (ir-GTH) cells were charact erized by the immunoperoxidase method, using antiserum to carp GTH bet a subunit. Ir-GTH cells from control silver eels were small and repres ented 14% of the dispersed pituitary cells. In contrast, ir-GTH cells from estradiol-treated eels were larger (cell area x2.5) and represent ed 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 rele ase was low (less than 2% of cell content) but measurable in cultures from estradiol-treated eels. Subsequent experiments examined effects o f various secretagogues on GTH release from primary cultures of pituit ary cells from estradiol-pretreated eels. GTH release was significantl y increased (x1.5 to x3 basal release) by 10(-6) M GnRH-A as well as b y both native GnRHs in the eel (mammalian GnRH, mGnRH, and chicken GnR H-II, cGnRH-II), at the same concentration. Lower GnRH concentrations had no significant effect, indicating a low sensitivity of gonadotroph s 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 p ituitary GnRH receptor had a low specificity toward various molecular forms, in the eel as in the other nonmammalian species. The protein ki nase C (PKC) activator (phorbol ester: PMA) also stimulated GTH secret ion, with a maximal effect at 10(-8) M, indicating that the PKC pathwa y was functional. In contrast, a depolarizing agent (50 mM KCl) had no significant effect on GTH release, suggesting lack of a functional vo ltage-sensitive calcium channel (VSCC) secretory pathway. Perifusion e xperiments on whole pituitary confirmed the lack of effect of KCl on g onadotrophs from E(2)-pretreated eels and indicated that an additional in vivo treatment with GnRH agonist and dopamine antagonist could ind uce the differentiation of a functional VSCC pathway. These characteri stics of the transduction mechanisms may be related to the immature st ate of the eel gonadotrophs at the silver stage. (C) 1996 Academic Pre ss, Inc.