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First published online 11 January 2006
doi: 10.1242/dev.02246

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Granulosa cells regulate intracellular pH of the murine growing oocyte via gap junctions: development of independent homeostasis during oocyte growth

¹ Hormones, Growth and Development Program, Ottawa Health Research Institute, Ottawa, ON K1Y 4E9, Canada.
² Department of Obstetrics and Gynecology (Division of Reproductive Medicine), University of Ottawa, Ottawa, ON K1Y 4E9, Canada.
³ Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON K1Y 4E9, Canada.

View larger version (39K): [in a new window]   Fig. 1. The follicle sets oocyte pHi during growth. (A) An example of one experiment in which follicle-enclosed and denuded oocytes were microinjected with SNARF-dex to determine pHi. Brightfield (left) and fluorescence (right) images are shown. Note that fluorescence is restricted to the oocyte. Scale bar 50 µm. (B) Oocytes within each experiment were grouped in 5 µm increments. pHi increased significantly with increasing size in denuded oocytes (P<0.0001), whereas there is no significant difference in pHi between size groupings in follicle-enclosed oocytes (P>0.25). Each point comprises between four and 17 oocytes from two to eight replicates.

View larger version (19K): [in a new window]   Fig. 2. The follicle affords the growing oocyte HCO3-/Cl- activity. (A) pHi was monitored in denuded (top row) and follicle-enclosed (bottom row) oocytes simultaneously. Cl- was removed from the bathing medium at 10 minutes in each experiment. Traces shown are the mean of all experiments performed. Note that Cl- removal causes sharp increases in pHi in all follicle-enclosed oocytes, but only in larger denuded oocytes. (B) Plot of the rate of pHi increase, which provides an indication of HCO3-/Cl- activity. HCO3-/Cl- exchange rate increases significantly with increasing oocyte size in denuded oocytes (P<0.0001), whereas there is no difference between sizes in follicle-enclosed oocytes (P>0.8). DIDS inhibited Cl--induced pHi increases in follicle-enclosed oocytes (A, bottom row, far right). Each point comprises between four and 17 oocytes from two to eight replicates.

View larger version (22K): [in a new window]   Fig. 3. Granulosa cells influence oocyte pHi in cumulus-oocyte complexes. (A) Example of an experiment in which cumulus-enclosed and denuded oocytes were compared with SNARF-dextran. Brightfield (left) and fluorescence (right) images are shown. (B) Cl- was removed from the bath at 10 minutes. (C,D) Cumulus-enclosed oocytes exhibited a significantly higher resting pHi (C; P<0.05) and rate of pHi increase (D; P<0.01) than did denuded oocytes. Data are from four replicates. Total oocytes: denuded, n=25; cumulus-enclosed, n=23.

View larger version (20K): [in a new window]   Fig. 4. Gap-junction inhibitors prevent Cl- removal-induced pHi 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 pHi increase upon Cl- removal in follicle-enclosed oocytes was quantified. Note that AGA and octanol both significantly inhibit Cl- removal-induced pHi 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.

View larger version (15K): [in a new window]   Fig. 5. Gap junction inhibitors significantly inhibit the recovery from alkalosis in follicle-enclosed oocytes. Follicle-enclosed oocytes (45-60 µm diameter) from mice aged 10-12 days were microinjected with SNARF. NH4Cl (35 mM) was added to the bathing media 10 minutes after initiation of pHi measurements at t=0. Drug treatments are the same as in Fig. 4. Three replicates were performed for each treatment, with a total of 18, 18, and 21 follicles for DMSO, AGA and octanol, respectively. The records shown are the average of all three replicates for each treatment.

View larger version (45K): [in a new window]   Fig. 6. Cl- removal triggers rapid pHi increases in oocytectomised (OOX) cumulus-oocyte complexes. (A) Examples of OOX complexes produced by aspirating the oocyte from within cumulus-oocyte complexes (see Materials and methods). (B) pHi was recorded during removal of Cl- (at 10 minutes) in SNARF-AM loaded complexes. Cl- removal caused a very rapid increase in pHi (0.91±0.02 pH units/minute). Data shown is the average of three replicates, from a total of 11 complexes. Inset shows an example of fluorescence images of OOX complexes.

View larger version (29K): [in a new window]   Fig. 7. Cl- removal-induced pHi changes in partly denuded cumulus-oocyte complexes. Cumulus-oocyte complexes were partly denuded by careful pipetting with a narrow-bore pipette, and subsequently loaded with SNARF-AM. This allowed simultaneous visualization of the oocyte and attached granulosa cells (see inset). Cl- was removed from the bathing media at 10 minutes. Fluorescence was measured separately in regions within the oocyte and within granulosa cell areas peripheral to the oocyte in order to monitor pHi in the two compartments. Notice that Cl- removal causes pHi increases within oocyte and granulosa cells that are indistinguishable. Trace shown is the average of eight complexes in one of two similar experiments.