Effect of liquid helium vitrification on cytoskeleton of immature cattle oocytes.

The developmental potential and the changes in cytoskeleton structures in immature oocytes of cattle resulting from liquid helium (LHe) vitrification was evaluated in this study. Immature oocytes were randomly divided into three groups: fresh oocytes (negative control), oocytes vitrified in liquid nitrogen (LN group, positive control), and oocytes vitrified in LHe (LHe group). In Experiment 1, the proportions for normal morphology, maturation, cleavage, and blastocyst were greater in the LHe group than in the LN group (88.3% compared with 79.1%, 51.7% compared with 43.3%, 42.6% compared with 33.0%, and 11.0% compared with 4.7%, respectively; P<0.05), and the rates of oocyte development were greater in the control group (100%, 72.8%, 64.3%, and 40.3%) than in the vitrified groups (P<0.05). In Experiment 2, the effect of vitrification by LHe and LN on cytoskeleton of cattle oocytes was examined. The cytoskeleton had varying degrees of damage, and the negative influence of LHe vitrification on the cytoskeleton was less than that of LN vitrification (P<0.05), and the vitrified group had greater cytoskeleton degeneration than the control group (P<0.05). In conclusion, LHe vitrification reduced the negative effect of cryoinjury on cytoskeleton structure and improved the viability of immature oocytes of cattle compared with LN vitrification.

Effect of liquid helium vitrification on the ultrastructure and related gene expression of mature bovine oocytes after vitrifying at immature stage.

This study aimed to investigate the developmental potential of and the ultrastructural changes and gene expression differences resulting from liquid helium (LHe; -269 °C) vitrification in immature bovine oocytes. Immature oocytes were randomly divided into three groups: fresh oocytes (control, negative control), oocytes vitrified in liquid nitrogen (LN group, positive control), and oocytes vitrified in LHe (LHe group). In experiment 1, the rates of normal morphology, maturation, cleavage, and blastocyst in the LHe group were higher than those in the LN group (87.1% vs. 80.5%, 51% vs. 48%, 41.7% vs. 36.8%, and 13% vs. 8.5%, respectively; P < 0.05), and the rates of development in the control group (100%, 73.2%, 62%, and 39.8%) were higher than those in the treated groups (P < 0.05). In experiment 2, oocytes displayed various degrees of injury at the ultrastructural level after vitrification, but more severe degeneration was observed in the LN group, such as formation of several lipid droplets, swelling of mitochondria, and absence of cortical granules. Compared with the LN group, fewer lipid droplets, relatively intact mitochondria, and clustered cortical granules were distributed in the cytoplasm of oocytes in the LHe group. In experiment 3, the mRNA expression levels of p53, CDC20, Eg5, and Npm2 were investigated by real-time quantitative polymerase chain reaction. Expression levels of the kinesin Eg5 and the apoptotic gene p53 expression levels were higher in the LN group compared with the control and LHe groups (P < 0.05). CDC20 and Npm2 expression did not differ significantly between the LN and LHe groups (P > 0.05), the CDC20 expression in the LN and LHe groups were lower than control group (P < 0.05), the Npm2 expression in LHe group was lower than control group (P < 0.05), but there was no significant difference between the LN and control groups (P > 0.05). In conclusion, LHe vitrification decreased the negative effect of cryoinjury on the ultrastructure of some organelles and the expression of some related genes, thereby improving the viability of immature bovine oocytes compared to LN vitrification.