Comparison of alkalosis defense mechanism during embryonic stages at conditions of in vivo and in vitro

The regulation of intracellular pH (pHi) is an important factor in order to maintain life of cell in developing embryos. There are three type of pH regulatory-mechanisms: HCO3-/Cl-, which provides defense of alkalosis, Na+/H+ and Na+,HCO3-/Cl-, which protects embryos from the acidosis. The function of these regulatory-mechanisms is to keep the pH value within a certain range for healthy developing embryos. AE activity of embryos in oviduct (tuba uterina) is high but this activity gradually decreases during the embryonic development process. In this study, it is aimed to compare AE activity of preimplantation stage embryos, which were develop in "in vivo" condition and were obtained from "in vitro" condition. It is also aimed to investigate whether there is a difference in terms of the effectiveness of alkalosis defense mechanism of preimplantation stage in vivo and in vitro embryos or not. Superovulation protocol was performed to 5-8 week old mature Balb/c strain mouse. In "in vivo" conditions, two-cell (2-c), four-cell (4-c) and eight-cell (8-c) embryos were obtained as well as morula and blastocyst stage embryos certain time after copulation. On the other hand, PN zygotes were cultured to obtain preimplantation stage embryos in "in vitro" conditions. . KSOM based media were used as culture and recording solutions. pH-sensitive fluorophore 2,7 bis carboxyethyl 5-6 carboxyfluorescein (BCECF) was used to record pHi values by using microspectrofluorometric method. AE activity was determined with Cl- removal method. Recovery of embryos from ammonium induced- alkalosis and effect of DIDS on this recovery has been evaluated to confirm AE function. While AE activity was high at in vivo and in vitro 2-c embryos (0,052±0,01 vs 0,060±0,01 pHU/min), it was gradually decrease as embryos developing 4-c, 8-c, morula and blastocyst stages in both conditions (in pHU/min; 0,040±0,00 vs 0,047±0,00; 0,031±0,00 vs 0,039±0,01; 0,024±0,01 vs 0,021±0,00; 0,017±0,01 vs 0,019±0,00, respectively). The decrease in AE activity was statistically significant at 8-c, morula and blastocyst stages (p<0,05). However, significant difference was not determined between the values of the same stage "in vivo" and "in vitro" embryos. The recovery from induced-alkalosis occurred in all embryonic stages and pHi returned to their initial values. Recovery were suppressed by adding DIDS at early preimplantation stages developed in both conditions . Whereas DIDS did not effect to recovery of embryos at the stages of morula and blastocyst. There was no significantly difference between the responses of in vivo and in vitro developing embryos. AE activity changes and recovery from induced-alkalosis were similar in "in vivo" and "in vitro" developing embryos. Because of there is no difference at embryonic pHi regulation between in vivo and in vitro conditions, long-continued embryo culture seems like no effect on culture success. In the light of this findings, since implantation success is high at proceed stage embryos such as morula or blastocyst, it has been considered that these proceed stage embryos can be used as uterine transfer stage embryos rather than commonly used 8-c stage in "in vitro" fertilization process.