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.

JOHN CHANG; HARRY COOK; JAMES WIGGS; GUSTAVO M SOMOZA; RENATO DE LEEUW; RICHARD E PETER; GUSTAVO M SOMOZA

GENERAL AND COMPARATIVE ENDOCRINOLOGY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Año: 1990 vol. 77 p. 256 - 273

0016-6480

Two cell dispersion methods for excised goldfish pituitary glands were tested, and acultured dispersed cell system based on trypsin enzymatic tissue digestion was developedand characterized. Controlled trypsin/DNase treatment of goldfish pituitary gland yieldeddispersed cells of high viability (trypsin blue exclusion test) that responded to gonadotropin(GTH)-releasing hormone (GnRH) challenges with GTH secretion in a time- and dosedependentmanner following overnight culture. Electron microscopy revealed that cell preparationsproduced by the trypsin dispersion were free of cell debris and nerve terminals. Thedispersed pituitary cells also retained distinct morphological and immunological identities.Under static incubation conditions, 2-hr treatments with 0.1 nM to 1 t&l [Trp’, Let?]-GnRH(sGnRH) and [D-Arg6, Pro’-N-ethylamide]-sGnRH (sGnRHa) stimulated GTH release withsimilar efficacy, but with ED,,+ of 1.92 f 0.48 and 0.19 2 0.08 nM, respectively. [His’, Trp’,Tyr*]-GnRH (cGnRH-II) stimulated GTH release in a nonsigmoidal, but dose-dependentmanner, and with a higher efficacy than sGnRH. In contrast, sGnRH, sGnRHa, andcGnRH-II were equipotent in inducing growth hormone (GH) secretion in static culturestudies and with ED,,s of 0.29 ? 0.13,0.18 f 0.11, and 0.19 2 0.17 n&f, respectively. Whentrypsin/DNase-dispersed cells cultured overnight with cytodex beads were tested in a cellcolumn perifusion system, dose-related increase in GTH secretion, as well as GH release,were also observed with 0.5 to 50 ml4 sGnRH. These results suggest that trypsin-dispersedgoldfish pituitary cells can be used effectively to study the actions of GnRH on teleostpituitary either in short-term static incubation or column perifusion studies. Differences inthe GTH and GH responses to the two native GnRH forms, sGnRH and cGnRH-II, are alsoindicated.