Intracellular pH changes during the following porcine oocyte activation

During fertilization in the sea urchin and toad, there is an important intracellular pH increase that follows the transient rise in intracellular free calcium levels in the oocyte. The rise in intracellular pH triggers what are known as the late events of oocyte activation, and is critical for complete activation of the oocyte. Recent studies in the mouse and rat have indicated that intracellular pH does not increase during fertilization. The objective of the present set of experiments was to determine if intracellular pH changes occur during parthenogenetic activation or the fertilization process of the pig. Intracellular pH was measured during treatment of porcine IVM oocytes with 7% ethanol, 200 muM thimerosal or 50 and 100 muM of the calcium ionophore A23187. All four treatments resulted in an intracellular pH increase, although thimerosal treatment only resulted in an increase in intracellular pH beginning after approximately 12 minutes. Bovine oocytes were found to produce an increase in intracellular pH increase when using calcium ionophore, but not ethanol or thimerosal treatments. Murine oocytes produced an increase in intracellular pH upon treatment with all three methods, although response was of a differing magnitude to that of the porcine oocyte. Inhibitors were used to show that the rise in intracellular pH in porcine oocytes when using the different activation treatments had distinct underlying mechanisms. Studies were undertaken to determine mRNA transcript levels of the Na+/H+ exchange genes NHE1--NHE5. Transcripts of the Na+/H + exchange genes, NHE1, 3, 4 and 5, were found during preimplantation development of the pig, with NHE1 found in the oocyte and four-cell stage embryo, but not the blastocyst stage embryo. Transcripts of NHE2 were absent during preimplantation development. Transcripts of NHE3 were found only in the oocyte. Transcripts of NHE4 were found in the oocyte and blastocyst stage embryo and NHE5 transcripts were found in the oocyte, four-cell and blastocyst stage embryos. By using calcium-free medium and BAPTA-AM loaded oocytes in calcium-free medium, it was determined that calcium is not necessary for increasing the intracellular pH during parthenogenetic activation, but is necessary for pronuclear formation when activating with thimerosal or calcium ionophore. Ethanol activation produced pronuclei at an equal level in calcium-free and calcium-containing media. Finally, fusion of the sperm and oocyte plasma membranes were observed to occur within 30 minutes of subzonal sperm injection (SUZI). There were no significant changes in intracellular pH when oocytes were monitored for 30 minutes following SUZI, although pronuclei formed after six hours of culture.