Evaluation of intracellular pH regulation and alkalosis defense mechanisms in preimplantation embryos

摘要

Intracellular pH (pH(i)) regulation is an important homeostatic function of cells. There are three major pH(i)-regulatory mechanisms: HCO3-/C1(-) exchanger (anion exchanger [AEI), which alleviates alkalosis, and the Na+/H+ and Na+,HCO3-/C1(-) exchangers, both of which alleviate acidosis. We hypothesized that there would be developmental changes in pH,regulatory activity in preimplantation embryos as conditions in the oviduct are alkaline but acidic in the uterus. This study focused on the AE mechanism in pronuclear (PN) zygotes, two-cell (2-c), four-cell (4-c), morula, and blastocyst stage embryos from Balb/c mice. Microspectrofluorometry was used to monitor changes in pH; in embryos subjected to Cl--free media in presence or absence of an AE inhibitor, DIDS, and in embryos recovering from NH(4)C1-induced alkalosis. Real-time polymerase chain reaction was used to identify AE isoforms. The pH; changes were greatest in PN zygotes (0.086 +/- 007 pHU/min) but fell as embryos developed to the 2-c, 4-c, morula, and blastocyst stages (0.063 +/- 006; 0.035 +/- 007; 0.024 +/- 004, and 0.014 +/- 004 pHU/min, respectively). DIDS significantly reduced the rise in pH; caused by Cl- removal in all embryos; the finding pointed out that this pH; changes are due to AE activity. But DIDS only inhibited the recovery responses of PN zygote, 2-c and 4-c embryos but not morula or blastocyst stage embryos. In bicarbonate-containing medium, all embryos recovered from induced alkalosis but only the morula and blastocyst stages could fully compensate from ammonium induced-alkalosis in bicarbonate-free medium. The finding showed that commonly used ammonium pulse method to investigate AE function against alkalosis is not suitable for morula and blastocyst embryonic stages. All embryos expressed SLC4A2 and SLC4A4 coding for AE-2 and AE-4, but none expressed either AE-1 or AE-3. The gradual change in the response to alkalosis in preimplantation embryos may be adaptations to their normal in vivo environment, where the early embryos are located in the alkaline oviduct, whereas the morula and blastocyst move into the acidic uterus. (C) 2015 Elsevier Inc. All rights reserved.