Study On Mechanism Of Reactive Free Radical Production By Mouse Early Embryos

Reactive free radicals are involved in many physiological and pathophysiological processes. Under normal conditions, production and clearance of reactive free radicals are in dynamic equilibrium. In this study, unfertilized oocytes and early embryos were obtained by flushing reproductive tracts of superovulated mice; NO concentrations in the conditioned culture medium from mouse embryos were determined indirectly by measurement of nitrites using the method of Griess reagent; reactive oxygen species were assayed by spin-trapping ESR; oxygen exhausting was determined by detecting oxygen concentration in the culture medium with oxygen meter. The results were as follows:1. NO production by mouse embryos Embryos were cultured in Hanks balanced salt solution for 4 hours. Then the culture medium was collected, and equal amount of Griess reagent was added into it. NO concentrations were determined indirectly by spectrophotometry. NO production was not significantly different between unfertilized oocytes and day 1 embryos (P>0.05). However, NO production by day 2, 3, 4 embryos remarkably increased (P<0. 01) and reached the highest level on day 3. Then, it began decreasing (P<0. 05). It seemed that NO was involved in early mouse embryo development. After Vitamin E treatment, NO production by unfertilized oocytes and day 2 embryos decreased significantly (P<0.01) while there was no significant Variation of NO production by day 1, 3, 4 embryos.2. Hydroxyl radical production by mouse embryos DEPMPO is more suitable for studying oxyradicals than DMPO because of its higher signal intensity and longer life of adducts. Therefore, DEPMPO was adopted to trap oxyradicals in our study. Hydroxyl radical was trapped by DEPMPO in embryos for the first time. Hydroxyl radical concentration of unfertilized oocytes was very low,while those of embryos were much higher. With the development of embryos, hydroxyl radical concentration trended to increase and reached its peak in 8-cell embryos. Then, it trended to decrease.3. Oxygen exhausting of mouse embryos There was very low oxygen exhausting in unfertilized oocytes. Meanwhile, oxygen concentration of culture medium containing embryos decreased with time (P<0. 0001). With the development of embryos, oxygen exhausting rate trended to increase. There was certain linear correlation between hydroxyl production and oxygen exhausting (P<0.25).4. Correlation between NO and hydroxyl radical in mouse embryos There was linear correlation between NO production and hydroxyl radical production(P<0.025).In summary, under the present condition, oxygen exhausting rate of mouse embryos is much higher than that of unfertilized oocytes; NO and hydroxyl radical are produced by early embryos; there is linear correlation between NO and hydroxyl radical production; there is certain linear correlation between oxygen exhausting and hydroxyl radicals.