Comparison of in vivo and in vitro acidosis defense mechanisms in preimplantation embryonic development

Aim:Intracellular pH (pHi) regulation is crutial for all cell types including preimplantiton embryos. There are three mechanisms that are used for pHi regulation in preimplantation embryos. HCO3⁻/Cl- exchanger alleviates alkalosis while Na+/H+ and Na+, HCO₃⁻ /Cl- exchangers alleviate acidosis. In this study, we aimed to compare acidosis defence mechanisms provided by Na+/H+ exchanger activity alone and together with Na+,HCO₃⁻/Cl- exchanger activity in in vivo and in vitro embryos. Materials and Methods: Embryos were collected from 5-8 week old Balb/c strain female mice. In vivo and in vitro embryos loaded with 2,7 bis carboxyethyl 5-6 carboxy fluorescein (BCECF) and recovery rates from induced acidosis were recorded by microspectrofluorometric method. Specific inhibitors (Cariporide, S3226) were used to determine the contribution of Na+/H+ exchanger isoforms (NHE1 and 3) to recovery from acidosis. In a group of embryos Na+/H+ and Na+, HCO₃⁻/Cl- exchanger proteins visualised through indirect immunofluorescence protocol. Results: Na+/H+ exchanger activity was not significantly different between in vivo and in vitro same stage embryos. NHE1 and 3 isoforms contributed to recovery from acidosis. We found that the major mechanism for defence against acidosis is the Na+/H+ exchanger and the Na+, HCO₃-/Cl- exchanger plays a complementary role, and embryos developed in vitro also showed similar results. Conclusion: The exchanger activities were high at the 2-cell stage of the embryos and there was no significant decrease during the developmental stages of the embryos. These results suggest that there are effective acidosis defence mechanisms in accordance with the increased metabolic activity during embryonic development and the acidic environment of the uterus. The absence of significant changes in acidosis defence mechanisms between in vivo and in vitro embryos at all developmental stages may indicate that cultured advanced stage embryos can also be used for IVF treatment. Key Words:Intracellular pH regulation, Na+/H+ exchanger, Na+ ,HCO₃⁻/Cl- exchanger, preimplantation embryo development, immunofluorescence staining