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 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.