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.