Useful Technique for the Study of Intracellular Calcium Fluxes in Single Porcine Granulosa Cells in Culture

Abstract

ContentsA new experimental model was applied to study calcium involvement in the mechanisms of action of the pituitary hormones on porcine granulosa cells (GC) on single cells in culture and in a time‐dependent manner. The model involved laser cytometry with calcium‐sensitive dye fluo‐3AM. This study describes pituitary hormone‐mediated changes of calcium (Ca2+) concentrations in cultured porcine GC, at the single cell level. Cells were isolated from medium sized follicles of cycling mature gilts (day 16–19 post‐oestrus) and were cultured at a concentration of 50–100 × 103 cells/Petri dish in 2 ml of enriched M199 medium for 2–3 days. Image scans were performed on individual cells that had been loaded with calcium‐sensitive dye, acetoxymethyl ester of fluo‐3 (fluo‐3 AM; 488ex/526em nm). In Experiment A (nexp = 6), cell response was measured after treatment with porcine luteinizing hormone (pLH) or prolactin (pPrl) (doses of 0, 1, 10 and 100 ng/ml in pH indicator‐free HBSS containing 2 m m of Ca2+). In cultures treated with 1, 10 and 100 ng of pLH (Ncells = 329), increases in relative fluorescence were observed in 37.1, 80.0 and 72.9% of cells scanned, respectively. In cultures treated with similar dosages of pPrl (Ncells = 259), responses were noted in 41.8, 57.3 and 47.2% of cells scanned, respectively. In control cultures treated with medium alone (Ncells = 85) the fluorescence increases were not observed. Fluorescence intensity was increased in the hormone‐treated cells only (p < 0.05). Seven‐fold and five‐fold relative fluorescence increases were observed for cells exposed to 100 ng of pLH or 10 ng of pPrl, respectively. In Experiment B, the effects of the Ca2+ ionophore (A23187) and Ca2+ chelator (EGTA) on the changes of calcium concentrations in GC‐treated with both pLH (100 ng/ml) and pPrl (10 ng/ml) were examined. In the presence of A23187 (10 μm), fluorescence of the cells increased two‐fold (p < 0.05) within 400 s in cultures treated with combination of both pLH and pPrl. However, EGTA (2.5 m m) completely abolished the responses of GC to both pLH and pPrl (Ncells = 168, nexp = 16) and the fluorescence decreased to levels similar to the control group (Ncells = 159, nexp = 5). The studies clearly demonstrated that intracellular free calcium changes occur in porcine GC when they are exposed to pLH or pPrl. The present study has provided a useful 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 time‐dependent manner.