| To explore the effects of vitrification on mitochondrion of oocytes,investigate the mechanism of vitrification of mice oocytes and the factors of reduction of fertilization rate of the vitrified-thawed oocytes,provide research thoughts and theoretical foundations for improving the vitrification methods and the fertilization rate of the vitrified-thawed oocytes,oocytes were devided into three groups:the fresh-contrast group、the poisonous-exposed group and the vitrified-thawed group.Ultrastructural changes were observed of mice oocytes by transmission electron microscopy,ATP content was measured by biological chemiluminescence method,mitochondrial membrane potential Δ Ψm)、distribution of mitochondrion and lysosomes and ROS content were measured by enzyme linked immunosorbent assay(ELISA).The results showed that:1.Ultrastructural damages were found on oocytes of vitrified-thawed group,including:disintegration of the intermediate filaments in the ooplasm;some microvillis shortened,even disappeared;mitochondrion became coarse and vague;the number of cortical granules decreased;the zona pellucida was vague,and some places cracked;plenty of vacuoles occured in the ooplasm,coupling with mitochondria-vacuole complex.2.△ Ψm of the vitrified-thawed group(0.2443±0.03819)was significantly lower than the fresh-contrast group(03977±0.04329)and the poisonous-exposed group(0.3297±0.02967)(P<0.05),but there was no significant difference between the fresh-contrast group and the poisonous-exposed group through One-Way ANOVA.3.The distribution of mitochondrion contained 4 types:Ⅰ Homogeneous、Ⅱ Polarization.ⅢClustered and IVDeficient.Among the 4 types,Ⅰ and Ⅱ are normal distribution,Ⅲ and Ⅳ are abnormal distribution.The distribution in the fresh-contrast group were Homogeneous(59.54%)and Polarization(36.42%);the majority of distribution in the poisonous-exposed group were Homogeneous(19.63%)and Clustered(69.33%);while the mainly distribution of mitochondrion in the vitrified-thawed group were Clustered(56.25%)and Deficient(24.38%).There was significant difference between each other in normal distribution(P<0.05).4、ATP content of the poisonous-exposed group(0.248±0.006pmol/cell)and the vitrified-thawed group(0.212±0.013 pmol/cell)were lower than the fresh-contrast group(0.298±0.020pmol/cell)significantly(P<0.05)through One-Way ANOVA.5.The ROS content of the fresh-contrast group、the poisonous-exposed group and the vitrified-thawed group were 33.75±0.59cps/cell、37.79±1.19cps/cell and 63.81±0.94cps/cell respectively;There was a significant difference between each other(P<0.05)through One-Way ANOVA.6、There were fewer lysosomes in the fresh-contrast oocyte and the poisonous-exposed oocyte,locating in the center of oocyte,besides,spaces were not observed around the lysosomes.While the lysosomes in the vitrified-thawed oocyte were much more,bigger and brighter,dispersing in the cytoplasm,and with many spaces around the lysosomes.Conclusions:Vitrification can make mitochondrion coarse and vague,lead to the reduction of mitochondrial membrane potential(ΔΨm)、the normal distribution of mitochondrion and the ATP content,result in the increase of the ROS content,besides,the distribution of lysosomes became diffusion. |
