Influence of Follicular Maturation on Luteinizing Hormone and Guanosine 5′-O-Thiotriphosphate-Promoted Breakdown of Phosphoinositides and Calcium Mobilization in Chicken Granulosa Cells

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Abstract
Previous studies in this laboratory have shown a remarkable increase in capacity of avian granulosa cells to produce progesterone during the last 2-3 days of folliculogenesis, with concomitant increases in the activities of key steroidogeneic enzymes. In view of the proposed involvement of inositol phospholipids and their hydrolytic products in signal transduction in steroidogenic cells, we investigated in this study the influence of follicular maturation on phosphoinositide-specific phospholipase C (PLC) and intracellular Ca2+ release in chicken granulosa cells. Resting concentrations of intracellular calcium ([Ca2+]i) as measured in Fura 2/AM-loaded cells increased significantly during the last 48 h of follicular maturation, from 185 .+-. 9 nM in the third largest follicle (F3) to 355 .+-. 26 in the cells of the largest (F1) follicle. Luteinizing hormone (LH) caused a dose-related rise in [Ca2+]i, but the dose response was left-shifted by more than one order of magnitude in F1 cells compared to F2 cells. In granulosa cells of less developed follicles, LH failed to raise [Ca2+]i. To assess PLC activity, granulosa cells from F1, F2, and F3 follicles were labeled with [3H]inositol for 2 h and then stimulated with LH (0.1 .mu.g/ml). Time course studies showed than within 30 s, phosphatidylinositol-4,5 bisphosphate (PIP2) decreased by 33%, 13% and 11% in F1, F2, and F3 cells, respectively. Similar responses were obtained when permeabilized cells were exposed to guanosine 5''-O-thiotriphosphate, which also caused a corresponding increase in 45Ca efflux from F1 and F2 cells. However, inositol 1,4,5 trisphosphate (IP3) caused the same dose-related efflux of 45Ca efflux in F1, F2and F3 granulosa cells. These results have provided evidence for the existence of an LH-sensitive, PLC-IP3-Ca2+ signalling pathways in chicken granulosa cells. Moreover, the rate-limiting step in this mechanism appears to be PLC, the activity of which significantly increases during the final 2 days of follicualr maturation.