P-75: The Effect of Hydrostatic Pressure in The Presence of Different Concentrations of Extracellular Calcium, Ethanol and Cytochalasin B on Parthenogenetic Activation of Mouse oocytes

Background: The parthenogenetic of oocytes is important in cloning research, as artificial activation of oocytes is an essential component of nucleus transfer protocols. Hydrostatic pressure can act as a mechanical stimulator that rearranges egg contents. Ethanol promotes a single intracellular Ca2+ increase of greater and longer amplitude than the initial increase observed at fertilization. In this study, we investigated the effect of hydrostatic pressure on parthenogenetic activation of mouse oocytes in presence of different concentration of calcium and ethanol and cytochalasin B. Materials and Methods: 6 to 8-week-old female NMRI mouse were superovulated by an injection of 10 IU of PMSG, followed by 10 IU HCG 48 hours later. Metaphase II oocytes were collected from oviduct 14 hr after HCG injection. Oocytes transferred to T6 medium containing different concentrations of calcium (0, 1.7, 3.4 mM) and 7% ethanol and for 5 minutes, treatments I, II,III ; respectively. They were divided to experiment and control groups. In experiment group, oocytes subjected to 20 mmHg pressure for 20 minutes. Oocytes without exposure to pressure were considered as control. Oocytes from two groups were transferred to T6 medium supplemented with different concentration of calcium and 5 μg/ml cytochalasin B for 4 hours. Oocytes were cultured for 72 hours and embryo development was assessed. Results: After 72 hours, in experiment and control groups treatments I , II, III , the percentage of cleavage was 18.51, 69.63, 45.36 and 9.8, 41.17, 48.03%; respectively. Cleavage rate in experiment group was higher than control group (p<0.05). The highest cleavage rate associated with treatment II which were significantly different from others in experiment and control (p<0.05). Conclusion: The results suggest that exposure to hydrostatic pressure, in the presence of ethanol and cytochalasin B can be considered as an artificial parthenogenic activation protocol in mouse oocyte.