Luteinizing Hormone (LH) Drives Diverse Intracellular Calcium Second Messenger Signals in Isolated Porcine Ovarian Thecal Cells: Preferential Recruitment of Intracellular Ca2+ Oscillatory Cells by Higher Concentrations of LH*

Abstract

Abstract The present study examines Ca2+ second messenger signaling driven by LH in isolated porcine thecal cells. To this end, we implemented semiquantitative fluorescent (fura-2) videomicroscopic imaging of single thecal cells in vitro. Stimulation of 388 cells with LH (5 μg/ml) elicited an intracellular Ca2+ ([Ca2+]i) signal in 85± 5.3% of individual thecal cells (n = 11 experiments). Among 337 LH-responsive cells, we identified four predominant temporal modes of [Ca2+]i signaling: 1)[ Ca2+]i oscillations with periodicities of 0.5 to 4.5 min−1 (63 ± 4.5%), 2) a[ Ca2+]i spike followed by a sustained plateau (17 ± 2.6%), 3) a [Ca2+]i spike only (5.8 ± 2.6%); and 4) a [Ca2+]i plateau only (3.8 ± 1.5%). The prevalence, but not the amplitude or frequency, of LH-induced [Ca2+]i oscillations in thecal cells was dependent on the agonist concentration. Reduced availability of extracellular Ca2+ induced by treatment with EGTA or cobaltous chloride did not block the initiation, but reversibly abolished ongoing [Ca2+]i oscillations (72% of cells) or increased the mean[ Ca2+]i interspike periodicity from 1.09± 0.16 to 0.59 ± 0.07 min−1 (P< 0.05). Putative phospholipase C inhibition with U-73122 (10μ m) also abolished or frequency-damped LH-driven[ Ca2+]i oscillations in 95 ± 4.7% of cells. [Ca2+]i oscillations in thecal cells were not abrogated by overnight pretreatment with pertussis toxin. We conclude that 1) thecal cells (unlike earlier findings in granulosa cells) manifest a diverse array of [Ca2+]i signaling responses to LH at the single cell level; 2) LH can dose dependently recruit an increasing number of individually[ Ca2+]i oscillating thecal cells; 3) extracellular Ca2+ is required for LH to sustain (but not initiate) frequent and high amplitude [Ca2+] oscillations in thecal cells; and 4) these signaling actions of LH are mediated via phospholipase C, but not a pertussis-toxin sensitive mechanism. Accordingly, the present data extend the apparent complexity of LH-induced [Ca2+]i second messenger signaling and identify at the single cell level LH’s dose-responsive drive of[ Ca2+]i oscillations in gonadal cells.