Figure 6.

The G_q-family G-protein inhibitor YM-254 890 prevents Ca²⁺ oscillations in response to LH, and delays ovulation. Experiments were performed using follicles 290–360 μm in diameter that had been cultured for ∼24 h in the presence of 1 nM FSH. (A, B) A follicle expressing GCaMP6s was pre-incubated with 10 μM YM-254 890 for 60 min before adding 10 nM LH. The yellow box outlines an area for which enlarged images are shown at four time points: before LH (image 1), after perfusion of 10 nM LH (images 2 and 3), and after perfusion of 300 nM LH (image 4); scale bar = 25 μm. The yellow circle (10 μm in diameter) indicates a mural granulosa cell for which the fluorescence intensity is graphed in B. Supplementary movie S6 shows the entire time series from which these images were selected. Representative of 3 follicles.(C, D) Inhibition of G_q-family G-proteins by preincubation of follicles with 10 μM YM-254 890 for 60 min before addition of 10 nM LH does not inhibit NEBD, but does delay ovulation. Graphs in C and D show the mean ± s.e.m of 3 and 4 experiments, respectively, with 10–18 follicles each. Data in (D) were analyzed by repeated measures two-way ANOVA, with the Holm-Sidak correction for multiple comparisons applied to the t-tests between the DMSO and YM-254 890 groups at each time point; ** indicates P < 0.01 and **** indicates P < 0.0001.