Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin-releasing hormone action in the goldfish, Carassius auratus I. Initial morphological, static, and cell column perifusion studies

Abstract

Two cell dispersion methods for excised goldfish pituitary glands were tested, and a cultured dispersed cell system based on trypsin enzymatic tissue digestion was developed and characterized. Controlled trypsin/DNase treatment of goldfish pituitary gland yielded dispersed cells of high viability (trypsin blue exclusion test) that responded to gonadotropin (GTH)-releasing hormone (GnRH) challenges with GTH secretion in a time- and dose-dependent manner following overnight culture. Electron microscopy revealed that cell preparations produced by the trypsin dispersion were free of cell debris and nerve terminals. The dispersed pitiitary cells also retained distinct morphological and immunological identities. Under static incubation conditions, 2-hr treatments with 0.1 n M to 1 μ M [Trp 7, Leu 8]-GnRH (sGnRH) and [ d-Arg 6, Pro 9- N-ethylamide]-sGnRH (sGnRHa) stimulated GTH release with similar efficacy, but with ED 50s of 1.92 ± 0.48 and 0.19 ± 0.08 n M, respectively. [His 5, Trp 7, Tyr 8]-GnRH (cGnRH-II) stimulated GTH release in a nonsigmoidal, but dose-dependent manner, and with a higher efficacy than sGnRH. In contrast, sGnRH, sGnRHa, and cGnRH-II were equipotent in inducing growth hormone (GH) secretion in static culture studies and with ED 50s of 0.29 ± 0.13, 0.18 ± 0.11, and 0.19 ± 0.17 n M, respectively. When trypsin/DNase-dispersed cells cultured overnight with cytodex beads were tested in a cell column perifusion system, dose-related increase in GTH secretion, as well as GH release, were also observed with 0.5 to 50 n M sGnRH. These results suggest that trypsin-dispersed goldfish pituitary cells can be used effectively to study the actions of GnRH on teleost pituitary either in short-term static incubation or column perifusion studies. Differences in the GTH and GH responses to the two native GnRH forms, sGnRH and cGnRH-II, are also indicated.