Measurement of low concentrations of bovine thyrotrophin by iodide uptake and organification in porcine thyrocytes

Abstract

ABSTRACT Thyrocytes isolated from porcine thyroids by mechanical and enzymatic dispersion and cultured in Eagle's minimal essential medium, supplemented with 5% (v/v) fetal calf serum, glutamine and cortisol, formed a continuous monolayer within 48 h. This monolayer was without cytochemical peroxidase and diaphorase (NADPH reoxidation) activity. In the presence of bovine thyrotrophin (bTSH; 50 mu./l) the cells developed a follicular-like architecture which was maximal at 4 days before reverting back to a uniform monolayer at 6 days. There were no detectable changes in the total DNA content over this period. The follicular structures had marked diaphorase and peroxidase activity, the latter being apically distributed. Concomitant with follicle formation bTSH induced uptake and organification of iodide presented to the cells during the last 6 h of culture. The extent of this process depended on the dose of bTSH and the duration of stimulation. The most sensitive effects for both iodide uptake and organification occurred with 1 mu. bTSH/1 and were maximal with 100 mu./l. Uptake and organification were increased 20±8-fold and 9·6±2- fold (n = 10) respectively over the control with 100 mu./l and the doses of bTSH at which a half maximal response was seen (ED50) were 15±2 and 7±1 (s.d) mu./l (n = 10) respectively. On changing the culture medium to a serum-free system using HB101 culture medium the stimulation time for the most sensitive bTSH effect was reduced to 2·5 days. Moreover the sensitivity of iodide uptake to bTSH was dramatically increased being significantly stimulated by 0·1 mu./l, saturated with 10 mu./l, and having an ED50 of 7 ± 0·2 mu. bTSH/1 (n = 7). Over the dose range 0·1–10 mu. bTSH/l intra- and interassay coefficients of variation were 7%±1·5 (n = 15) and 11% ± 2·5 (n = 15) respectively. Cyclic AMP production in cultures incubated under similar conditions (i.e. after chronic TSH stimulation) was also stimulated by bTSH doses in the range 15·6–125 mu./l. Cells stimulated for 2·5 days with dibutyryl cyclic AMP (range 1 μmol/1–2 mmol/l) also actively concentrated iodide in a dose-dependent fashion. The presence of normal human serum in the medium yielded a progressive and dose-related decrease in TSH-stimulated iodide uptake. This inhibitory effect occurred with serum concentrations as low as 0·1%. In conclusion, a porcine cell culture system is described in which iodide uptake is significantly stimulated by 0·1 mu. bTSH/l, a sensitivity inferior only to that reported with the cytochemical bioassay. J. Endocr. (1985) 106, 13–20