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First published online 31 October 2007
doi: 10.1242/dev.005272

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Granulosa cells regulate oocyte intracellular pH against acidosis in preantral follicles by multiple mechanisms

¹ Ottawa Health Research Institute, University of Ottawa, 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 (17K): [in this window] [in a new window]   Fig. 1. Activation of Na+/H+ exchange during oocyte growth. Intracellular pH was monitored in growing oocytes using epifluorescence microscopy. (A) Each panel shows a typical replicate of a given experiment, each individual trace representing a single oocyte. Acidosis was induced in mid-growth phase (left panel) and fully grown (middle panel) oocytes using a 10-minute pulse of NH4Cl (black bar). Note that neither population of oocytes mounts a substantial recovery from acidosis in media devoid of Na+ (gray bar), but resting pH is rapidly restored in fully grown oocytes following Na+ replacement. Insets show examplar fluorescence micrographs from each respective experiment. Scale bar: 50 µm. Where used, amiloride (1 mM) was added at t=20 minutes, and remained in the bath thereafter. Note that amiloride retards recovery from acidosis in fully grown oocytes (right panel). (B) Analysis of rate of recovery from acidosis following Na+ replacement. Different letters above the bars indicate significant differences P<0.01 (ANOVA). Three replicates were performed of each experiment - a total of 52, 32 and 39 oocytes, respectively.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Identification of NHE isoforms that correct acidosis in fully grown oocytes. (Ai) RT-PCR of NHE isoforms in fully grown oocytes from 20-day-old mice. Amplicons formed by reverse transcription and PCR of positive control tissues (+), three oocyte equivalents (O), or the final oocyte wash drop (-) are shown for each isoform, as indicated. For further details see Materials and methods. DNA ladder is in 100 bp increments. Distinct amplicons were always generated from oocyte samples by NHE1, NHE3 and NHE4 primers, but were never by NHE5 primers, and a very faint band was produced by NHE2 primers using 40 cycles of PCR in four of nine replicates. (Aii) RT-PCR of denuded oocytes from day-10 mice. Note that distinct amplicons were generated by primers for NHE1, NHE3 and NHE4. Amplicons were not generated by primers for NHE2 or NHE5 (not shown). (B) Recovery from acidosis in mid-growth phase and fully grown oocytes in bicarbonate-free medium. Note that, as was also the case in the presence of bicarbonate (see Fig. 1), only fully grown oocytes recover form acidosis. (C) Typical examples of NH4Cl pulse experiments on fully grown oocytes in bicarbonate-free media in the presence of cariporide and S3226, as indicated. In each case, the drug was added at t=20 minutes, and remained throughout the experiment unless otherwise indicated. Note that 1 µM cariporide completely and reversibly inhibited acidosis recovery. (D) Summary of all experiments performed in this series plotted on a logarithmic scale. Drugs were diluted in DMSO such that the final concentration of DMSO was 0.1% throughout. Note the break in the x-axis to allow inclusion of a drug-free group (DMSO only). DMSO alone did not influence the rate of recovery (P>0.5). Each data point represents mean±s.d. of the initial rate of pH recovery from three separate replicates, comprising between 41 and 26 oocytes.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Recovery from acidosis in isolated granulosa cells. Granulosa shells were prepared by removing the oocyte from preantral follicles recovered from 10-day-old mice, and then loaded with SNARF-AM. (A) Left, a series of three photomicrographs illustrating the procedure by which the oocyte (arrowheads) is removed from a preantral follicle. A higher resolution exemplar photomicrograph of the resulting granulosa shell is also shown (center) and a fluorescence micrograph of SNARF-loaded shells (right). Scale bar: 50 µm. (B) Recovery from acidosis in SNARF-loaded granulosa shells following a NH4Cl pulse. Note the rapid increase in pH when Na+ was returned to the bath (experiments were performed in triplicate, n=23 in total). Note also that amiloride retarded Na+-dependent acidosis correction (experiments were performed in triplicate, n=23 in total). Amiloride was added at t=10 minutes. (C) Average rate of recovery from acidosis following Na+ replacement. *, P<0.05.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Identification of NHE isoforms that correct acidosis in granulosa shells. (A) RT-PCR of NHE isoforms in granulosa shells isolated from 10-day-old mice. For each isoform, products formed by reverse transcription and PCR of positive control tissues (+), one follicle-equivalent of granulosa cells (G), or oocyte bathing media (-) are shown. For further details see Materials and methods. (B) Acidosis recovery in granulosa cells in bicarbonate-free medium. Note that amiloride retards the Na+-dependent pHi increase that occurs under control conditions. (C) Examples of NH4Cl pulse experiments of granulosa shells in bicarbonate-free medium in the presence of cariporide and S3226 (as indicated in each panel). In each case, the drug was added at t=10 minutes, and remained throughout the experiment. (D) Summary of all experiments performed in this series. DMSO (vehicle) was 0.1% throughout. DMSO alone had no effect upon acidosis recovery. Since some recovery occurs during the Na+-free period in granulosa cells in these experiments, the rate of pHi increase during the Na+-free period was subtracted to obtain the Na+-dependent component of recovery. Each data point represents mean±s.d. of the Na+-dependent pHi recovery from three to five separate replicates, comprising between 18 and 32 granulosa shells. The results of the amiloride and the 10 µM cariporide + 1 µM S3226 co-treatment experiments have been added as a bar graph for ease of comparison.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Investigation of Na+-independent pH increases in granulosa cells. Granulosa cells were acidified by NH4Cl pulse in bicarbonate-free medium and Na+-independent recovery examined. (A) Typical examples of each treatment group. Note that in control experiments, acidosis is almost fully relieved despite the absence of Na+. Drugs or vehicles were added to the bath at t=10 minutes, and remained throughout the experiment. NEM (100 µM) and bafilomycin (20 nM or 200 nM) retarded the Na+-independent alkalinization that occurs following NH4Cl removal in controls. A lower level of NEM (10 µM) partially inhibited recovery, whereas a higher level (1000 µM) did not further inhibit recovery (not shown). Bafilomycin was maximally effective at both 20 and 200 nM, and these data were combined (three replicates: one of 200 nM and two of 20 nM; due to the high cost of bafilomycin and the large volumes required for these experiments, the data for 20 and 200 nM were combined to yield three replicates). Concanamycin (10 nM) caused partial inhibition, which was not increased at 100 nM (not shown). (B) Summary of Na+-independent acidosis correction. Each bar represents mean±s.d. of the initial rate of pH recovery from three replicates. Different letters indicate P<0.01 (ANOVA).

View larger version (31K): [in this window] [in a new window]   Fig. 6. Gap junctions provide follicle-enclosed oocytes with access to mechanisms that correct acidosis. SNARF-dextran was microinjected into denuded and follicle-enclosed oocytes from day-10 mice. (A) Recovery from acidosis was examined simultaneously in denuded and follicle-enclosed oocytes from day-10 mice using SNARF-dextran in bicarbonate-free medium. The graph shows a representative replicate of this experiment, in which six follicle-enclosed (black traces) and five denuded oocytes were examined. The follicle-enclosed oocytes recover from acidosis when Na+ is replaced. The average rate of recovery following Na+-replacement is significantly greater in the follicle-enclosed oocytes over the course of three replicates (P<0.05). Note that the SNARF-dextran remains restricted to the oocyte within the follicle (inset). (B) Recovery from acidosis was examined in follicle-enclosed oocytes in the presence of DMSO (left panel) or 150 µM AGA (centre), which were added during the experiment at t=20 minutes. A representative replicate of each treatment group is shown. (C) Analyses of the rate of pH recovery during the Na+-free period and following Na+ replacement are shown. Each bar represents the mean of three separate replicates, a total of 19-29 follicle-enclosed oocytes. Different letters above bars indicate significant differences (ANOVA, P<0.01). Note that gap-junction inhibition abrogates both Na+-dependent and Na+-independent phases of acidosis recovery in follicle-enclosed oocytes. Note also that AGA does not inhibit acidosis recovery in fully grown oocytes, or shells of granulosa cells (B, right, one of two similar experiments is shown for each).

View larger version (23K): [in this window] [in a new window]   Fig. 7. Synchronous recovery from acidosis in oocytes and accompanying granulosa cells. Cumulus oocyte complexes from the ovaries of PMSG-primed adult mice were carefully cleaned so as to produce denuded oocytes, and oocytes with a part-covering of granulosa cells. Following loading with SNARF-AM, both oocyte and granulosa cell regions are clearly visible, such that regions of interest (ROI) for analysis can be positioned to measure pH independently in the different compartments (see inset; ROI examples outlined in white). The graph shown is one of four independent replicates, which comprised 26 denuded and 30 part-enclosed oocytes in total. The average curve for each cell type is shown. Note that pHi of part-enclosed oocytes closely reflects that of the accompanying granulosa cells, including a steady pHi increase which occurs during the Na+-free period, which is not evident in denuded oocytes. Over the course of three experiments, 26 oocyte-granulosa complexes and 30 denuded oocytes were examined.

View larger version (10K): [in this window] [in a new window]   Fig. 8. Model summarizing regulation of ooplasmic pHi by granulosa cell transport mechanisms. The small growing oocyte is incapable of regulating its own pHi, but the gap junctions, which couple the oocyte and granulosa, allow exchangers on the granulosa cell surface to regulate the ooplasm. Granulosa cell exchangers that participate are indicated: Na+/H+ exchanger isoform 1 (light gray circles), isoform 3 (dark gray), and V-ATPase (open circle). The molecular identity of the HCO3-/Cl- exchangers that regulate against alkalosis is unknown (black circles). The broad arrows indicate diffusion of proton equivalents between oocyte and granulosa (see text).

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