ABSTRACT
Objective To explore the effects of hypoxia on the growth,mitochondria distribution and function of mouse embryonic fibroblasts(MEFs).Methods MEFs were sub-cultured in the hypoxia group containing 5％ oxygen and normal oxygen group containing 20％ oxygen,every 24 hours,living MEFs were counted by using trypan blue staining.Mito-Tracker Green was used to stain mitochondria,then cells were observed by using laser confocal microscope.The ATP kit was used to detect ATP synthesis.Results During the logarithmic phase,the numbers of living cells in the hypoxia group were higher than those in the normal oxygen group,the differences were statistically significant (P＜0.05).The percentages of perinuclear mitochondrial in the hypoxia group were higher than those in the normal oxygen group,the differences were statistically significant (P＜0.05).Meanwhile,the significant difference was found in the ATP level between the two groups (P＜0.05).Conclusion The distribution of mitochondria in MEFs and energy synthesis are influenced by the hypoxic culture condition,which could be better for promoting cell growth compared with normal oxygen culture condition.
ABSTRACT
OBJECTIVE: The present study was undertaken to examine the effects of magnesium ion in the culture medium on the development of mouse fertilized oocytes either before or after pronuclear formation, and to investigate whether the effect of magnesium ion is related with the redistributional change of mitochondria. METHODS: Fertilized oocytes obtained from the oviducts of mice at 15 hr after hCG injection before pronuclear formation (pre-PN) or 21 hr after hCG injection after pronuclear formation (post-PN) were used. The embryos were cultured for 3 days with basic T6 medium-magnesium free and various concentrations of magnesium ion, 0.0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM, respectively. After culture, the developmental stages of embryos and the number of nuclei were evaluated. To observe the effects of magnesium ion on the mitochondrial distribution, fertilized oocytes were collected at 21 hr after hCG injection and cultured for 6 hr with various concentration of magnesium ion. As a control, fertilized oocytes with pronuclei at 27 hr after hCG injection were used. RESULTS: The concentration of magnesium ion to accelerate the in vitro development of mouse fertilized oocytes appeared to be at 2.0 mM for the pre-PN and the post-PN stage embryos. In the mitochondrial redistribution patterns, the embryos cultured in 2.0 mM concentration of magnesium ion showed the highest percentage (22.6%) of distinct perinuclear clustering pattern comparing to other experimental group. CONCLUSION: The effect of magnesium ion may be related to the cytoplasmic redistribution of mitochondria. This relationship seems to connect the developmental competence of preimplantation mouse embryos in vitro. These results can suggest that higher concentration of magnesium ion (2.0 mM) than those of conventional culture medium (0.2~1.2 mM) is more suitable for in vitro culture of preimplantation mouse embryos.