Useful technique for the study of intracellular calcium fluxes in single porcine granulosa cells in culture

A new experimental model was applied to study calcium involvement in the me chanisms of action of the pituitary hormones on porcine granulosa cells (GC ) on single cells in culture and in a time-dependent manner. The model invo lved laser cytometry with calcium-sensitive dye fluo-3AM. This study descri bes pituitary hormone-mediated changes of calcium (Ca2+) concentrations in cultured porcine GC, at the single cell level. Cells were isolated from med ium sized follicles of cycling mature gilts (day 16-19 post-oestrus) and we re cultured at a concentration of 50-100 x 10(3) cells/Petri dish in 2 ml o f enriched M199 medium for 2-3 days. Image scans were performed on individu al cells that had been loaded with calcium-sensitive dye, acetoxymethyl est er of fluo-3 (fluo-3 AM; 488(ex)/526(em) nm). In Experiment A (n(exp) = 6), cell response was measured after treatment with porcine luteinizing hormon e (pLH) or prolactin (pPrl) (doses of 0, 1, 10 and 100 ng/ml in pH indicato r-free HBSS containing 2 mM of Ca2+). In cultures treated with 1, 10 and 10 0 ng of pLH (N-cells = 329), increases in relative fluorescence were observ ed in 37.1, 80.0 and 72.9% of cells scanned, respectively. In cultures trea ted with similar dosages of pPrl (N-cells = 259), responses were noted in 4 1.8, 57.3 and 47.2% of cells scanned, respectively. In control cultures tre ated with medium alone (N-cells = 85) the fluorescence increases were not o bserved. Fluorescence intensity was increased in the hormone-treated cells only (p < 0.05). Sevenfold and five-fold relative fluorescence increases we re observed for cells exposed to 100 ng of pLH or 10 ng of pPrl, respective ly. In Experiment B, the effects of the Ca2+ ionophore (A23187) and Ca2+ ch elator (EGTA) on the changes of calcium concentrations in GC-treated with b oth pLH (100 ng/ml) and pPrl (10 ng/ml) were examined. In the presence of A 23187 (10 mu M), fluorescence of the cells increased two-fold (p < 0.05) wi thin 400 s in cultures treated with combination of both pLH and pPrl. Howev er, EGTA (2.5 mM) completely abolished the responses of GC to both pLH and pPrl (N-cells = 168, n(exp) = 16) and the fluorescence decreased to levels similar to the control group (N-cells = 159, n(exp) = 5). The studies clear ly demonstrated that intracellular free calcium changes occur in porcine GC when they are exposed to pLH or pPrl. The present study has provided a use ful technique for studying the role of calcium in regulation of porcine GC by different factors at the single porcine cell level in culture and in a t ime-dependent manner.