LUTEINIZING-HORMONE (LH) STIMULATES BOTH INTRACELLULAR CALCIUM-ION ([CA2- RECRUITMENT AS A CELLULAR MECHANISM OF LH-[CA2+](I) DOSE-RESPONSE(](I)) MOBILIZATION AND TRANSMEMBRANE CATION INFLUX IN SINGLE OVARIAN (GRANULOSA) CELLS )

The gonadotropic hormones, LH and FSH, activate adenylyl cyclase in th eir respective target cells and thereby initiate many biochemical resp onses. In addition to stimulating cAMP production, both LH and FSH pro mote agonist-specific increases in the cytoplasmic concentration of fr ee calcium ions ([Ca2+](i)) in gonadal cells. Here, we have applied si ngle cell fluorescence video microscopy with the Ca2+ sensitive dye fu ra-2 to investigate the mechanism(s) by which LH induces a rise in the [Ca2+](i) in individual (swine) granulosa cells collected from single Graafian follicles. Stimulation with LH induced a rapid onset, biphas ic, spike- and plateau-like [Ca2+](i) signal in responsive granulosa c ells. The cellular mechanisms mediating this biphasic LH-stimulated in crease in [Ca2+](i) were examined by external Ca2+ removal and via the manganese (Mn2+) quench technique, which showed that LH triggers init ial intracellular Ca2+ mobilization followed by delayed transmembrane Ca2+ influx. Single cell Ca2+ assessment of the LH dose-response mecha nism(s) revealed that higher concentrations of LH progressively recrui t a larger number of responding individual granulosa cells. Further an alyses disclosed a marked [Ca2+](i) response heterogeneity among indiv idual granulosa cells harvested from the same Graafian follicle. In ad dition, the percentage of cells responding to LH [but not to an altern ative putative agonist of the phospholipase C (PLC) pathway, viz. endo thelin-1] with a biphasic [Ca2+](i) rise increased with maturational d evelopment of the follicle. Pretreatment of granulosa cells with a spe cific PLC inhibitor, U-73122 (but not with its inactive congener U-733 43), significantly reduced the percentage of cells responding to a LH challenge from 78% to 25% (P < 0.0001) and prolonged the time required to achieve a half-maximal value of the [Ca2+](i) transient, viz. from 22 +/- 1.5 sec (n = 27 cells) to 39 +/- 7.2 sec (n = 12 cells; P = 0. 002). In cell population studies, LH stimulated in a concentration- an d time-dependent manner the accumulation of inositol phosphate in porc ine granulosa cells. In summary, the present single cell investigation s in mature granulosa cells demonstrate that LH drives initial intrace llular Ca2+ mobilization followed by transmembrane divalent cation inf lux. The PLC inhibitor U-73122 antagonizes this action of LH. By analy zing [Ca2+](i) responses in individual living granulosa cells, we furt her show that, despite within-follicle diversity, the LR dose biphasic [Ca2+](i) response arises via the recruitment of a larger number of r esponding gonadal cells rather than by increased [Ca2+](i) signal ampl itude. Finally, the percentage of individual LH (but not endothelin-1) -responding granulosa cells increases with follicular maturation. Coll ectively, these data highlight the potential importance of the LH-stim ulatable, PLC-transduced [Ca2+](i) signaling mechanism in the later st ages of granulosa cell differentiation.