Fig. 4. Gap-junction inhibitors prevent Cl^- removal-induced pH_i increases in follicle-enclosed oocytes. (A) Follicle-enclosed oocytes were recovered from mice aged 10-12 days and microinjected with SNARF-dex. Cl^- removal was performed after 20 minutes in the presence of DMSO (vehicle; 0.3%), AGA (150 µM, 0.3% DMSO) or 1-octanol (1 mM, 0.1% DMSO). Only oocytes sized 45-60 µm were selected for analysis. (B) The rate of pH_i increase upon Cl^- removal in follicle-enclosed oocytes was quantified. Note that AGA and octanol both significantly inhibit Cl^- removal-induced pH_i increases in follicle-enclosed oocytes compared with control (P<0.01). Three replicates were performed for each treatment, a total of 17, 12 and 25 oocytes for DMSO, AGA and octanol, respectively. Insets in A show the effect of similar treatments upon fully-grown denuded oocytes (insets: AGA, n=28; octanol, n=24; both of three replicates). (C) Fluorescein was injected into follicle-enclosed oocytes following similar pre-incubation periods in DMSO, AGA or octanol. Images were obtained 30-60 minutes after microinjection. Note that the granulosa cells surrounding the oocyte can be clearly visualized in DMSO-treated (control) follicles, indicating that fluorescein can pass easily between the oocyte and its granulosa cells. Similar results were obtained for untreated controls in the absence of DMSO (not shown). Note also that AGA and octanol both inhibited fluorescein from entering the granulosa cells. Images presented are unprocessed, and similar microscope settings were used in each acquisition. Controls were carried out on the same days as drug treatments. Bar chart shows the analysis of relative intensity of fluorescence in the oocyte and granulosa regions of follicles from each group. The relative fluorescence intensity in granulosa cells compared with the oocyte is significantly reduced by AGA or octanol (P<0.01). A relative fluorescence value of approximately 0.1 in octanol- and AGA-treated oocytes may reflect a small amount of residual dye transfer from the oocyte to the granulosa cells, or light scattering from the oocyte. n=22, 9, 16 and 14 for untreated, DMSO, AGA and octanol treatments, respectively.