The gonadotropic hormones, LH and FSH, activate adenylyl cyclase in their respective target cells and thereby initiate many biochemical responses. In addition to stimulating cAMP production, both LH and FSH promote agonist-specific increases in the cytoplasmic concentration of free calcium ions ([Ca2+](i)) in gonadal cells. Here, we have applied single cell fluorescence video microscopy with the Ca2+-sensitive dye fura-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, biphasic, spike- and plateau-like [Ca2+](i) signal in responsive granulosa cells. The cellular mechanisms mediating this biphasic LH-stimulated increase in [Ca2+](i) were examined by external Ca2+ removal and via the manganese (Mn2+) quench technique, which showed that LH triggers initial intracellular Ca2+ mobilization followed by delayed transmembrane Ca2+ influx. Single cell Ca2+ assessment of the LH dose-response mechanism(s) revealed that higher concentrations of LH progressively recruit a larger number of responding individual granulosa cells. Further analyses disclosed a marked [Ca2+](i) response heterogeneity among individual granulosa cells harvested from the same Graafian follicle. In addition, the percentage of cells responding to LH [but not to an alternative putative agonist of the phospholipase C (PLC) pathway, viz. endothelin-1] with a biphasic [Ca2+](i) rise increased with maturational development of the follicle. Pretreatment of granulosa cells with a specific PLC inhibitor, U-73122 (but not with its inactive congener U- 73343), 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 see (n = 12 cells; P = 0.002). In cell population studies, LH stimulated in a concentration- and time-dependent manner the accumulation of inositol phosphate in porcine granulosa cells. In summary, the present single cell investigations in mature granulosa cells demonstrate that LH drives initial intracellular Ca2+ mobilization followed by transmembrane divalent cation influx. The PLC inhibitor U-73122 antagonizes this action of LH. By analyzing [Ca2](i) responses in individual living granulosa cells, we further show that, despite within-follicle diversity, the LH dose biphasic [Ca2+](i) response arises via the recruitment of a larger number of responding gonadal cells rather than by increased [Ca2+](i) signal amplitude. Finally, the percentage of individual LH (but not endothelin-1)-responding granulosa cells increases with follicular maturation. Collectively, these data highlight the potential importance of the LH-stimulatable, PLC-transduced [Ca2+](i) signaling mechanism in the later stages of granulosa cell differentiation.
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