Investigation of activity changes of anion exchanger against intracellular alkalosis during early preimplantation embryonic stages

Intracellular pH (pHi) regulation is an important component of mammalian cell homeostasis. pHi is maintained within a narrow range by three major regulatory-mechanisms: the HCO3-/Cl- exchanger (AE), which alleviates alkalosis, and the Na+/H+ (NHE) and Na+-dependent HCO3-/Cl- (NDCBE) exchangers, both of which alleviate acidosis. pHi should be kept within narrow range by these regulators for healthy developing embryos, as well. Findings of studies performed showed that although AE activity of oocyte at prophase I stage high, this activity is inhibited during meiotic maturation. In this study, we aimed to investigate AE activity during early preimplantation embryonic developmental process, from PN zygote to 8-c stage. In this study, PN zygotes, two-cell (2-c), four-cell (4-c), eight-cell (8-c) stage embryos were obtained by classical procedure from 5-8 week old mature female Balb/c strain mouse following superovulation protocol. KSOM based solutions were used as culture and recording solutions. Intracellular pH (pHi) was recorded by using microspectrofluorometric technique from zygotes/embryos which were loaded with pH-sensitive florophore, BCECF. AE activity was determined by Cl- removal assay. Function of AE was investigated using recovery response from induced-alkalosis and confirmed by DIDS, specific inhibitor of AE. While AE activity was high at PN zygotes (0,068±0,01 pHU/min), this activity gradually decreased as embryos develop to 2-c, 4-c and 8-c stages (in pHU/min; 0,058±0,01, 0,042±0,01 and 0,033±0,01, respectively). These activity changes were statistically significant at PN zygote and 2-c embryo when compared to 8-c stage (p<0,05). Complete recovery from induced-alkalosis was determined at all-stages of embryos; and, DIDS inhibited these recovery responses at all stages except 8-c embryo. In the light of these findings, it is possible to conclude that embryos, which are developing and changing their metabolisms, constitute appropriate defense mechanisms in accordance with the environment, as they move along from alkaline oviduct to acidic uterus. Keywords: Anion exchanger, mouse, intracellular pH regulation, zygote, embryo