Luteinizing Hormone (LH) Stimulates Both Intracellular Calcium Ion ([Ca]i) Mobilization and Transmembrane Cation Influx in Single Ovarian (Granulosa) Cells: Recruitment as a Cellular Mechanism of LH-[Ca]i Dose Response*

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 ([Ca]i) in gonadal cells. Here, we have applied single cell fluorescence video microscopy with the Casensitive dye fura-2 to investigate the mechanism(s) by which LH induces a rise in the [Ca]i in individual (swine) granulosa cells collected from single Graafian follicles. Stimulation with LH induced a rapid onset, biphasic, spikeand plateau-like [Ca]i signal in responsive granulosa cells. The cellular mechanisms mediating this biphasic LH-stimulated increase in [Ca]i were examined by external Ca removal and via the manganese (Mn) quench technique, which showed that LH triggers initial intracellular Ca mobilization followed by delayed transmembrane Ca influx. Single cell Ca 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 [Ca]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 [Ca]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 [Ca]i transient, viz. from 22 6 1.5 sec (n 5 27 cells) to 39 6 7.2 sec (n 5 12 cells; P 5 0.002). In cell population studies, LH stimulated in a concentrationand timedependent 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 Ca mobilization followed by transmembrane divalent cation influx. The PLC inhibitor U-73122 antagonizes this action of LH. By analyzing [Ca]i responses in individual living granulosa cells, we further show that, despite within-follicle diversity, the LH dose biphasic [Ca]i response arises via the recruitment of a larger number of responding gonadal cells rather than by increased [Ca]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 [Ca]i signaling mechanism in the later stages of granulosa cell differentiation. (Endocrinology 139: 3606–3612, 1998) G transfection experiments have recently documented the ability of a single receptor to activate dual intracellular signaling pathways after interacting with its cognate ligand. For example, the human TSH, hCG/LH, and PTH/PTH-related peptide receptors expressed in monkey kidney (COS) cells all mediate single ligand stimulation of both adenylyl cyclase and phospholipase C (PLC) (1, 2). Intracellular cAMP and soluble inositol phosphates are also generated by activation of the porcine calcitonin receptor (3), the murine LH receptor (4), and the rat LH receptor expressed in heterologous host cells (5). However, gene transfection experiments in nonnative cell populations do not establish whether and how specific signaling mechanisms are implemented by native receptors within the physiological milieu of the individual homologous (native) cell. Indeed, in the particular case of LH, reports documenting PLC activation by this gonadotropin in untransformed ovarian cells are limited and controversial. Although some investigations have reported that LH does not affect inositol phosphate accumulation in rat and pig granulosa (6) and luteal cells (7), other studies have described LH-stimulated inositol phosphate accumulation in rat granulosa cells (8, 9), and bovine (10) and porcine luteal cells (11). Here we implement monitoring of cytoplasmic concentration of free calcium ions ([Ca]i) with high temporal resolution by semiquantitative fluorescence video microscopy in single (swine) granulosa cells to examine the mechReceived January 28, 1998. Address all correspondence and requests for reprints to: Dr. Jorge A. Flores, Biology Department, West Virginia University, Brooks Hall, P.O. Box 6057, Morgantown, West Virginia 26506-6057. * This work was supported in part by NIH Grant R01-HD-16806 (to J.D.V.), a supplement to this NICHHD grant (to J.A.F.), NIH P-30 Reproduction Center Grant 1-P30-HD-28934, and the National Science Foundation Center for Biological Timing (to J.A.F., C.A., J.D.V., and O.S.), and the Hospital Clinico de la Universidad de Chile (to C.A.). † Present address: Organon, Inc., West Orange, New Jersey 07052. 0013-7227/98/$03.00/0 Vol. 139, No. 8 Endocrinology Printed in U.S.A. Copyright © 1998 by The Endocrine Society