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

• Published in: Theriogenology Vol. 87; pp. 91 - 99
• Main Authors: Wu, Hua, Yu, Xue-Li, Guo, Xian-Fei, Zhang, Fan, Pei, Xu-Zhe, Li, Xiao-Xia, Han, Wen-Xia, Li, Ying-Hua
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2017
• Subjects: Animals; Bovine immature oocyte; Cattle - physiology; Cryopreservation - veterinary; Cumulus Cells - physiology; Female; Gene expression; Gene Expression Regulation - physiology; Helium; In Vitro Oocyte Maturation Techniques - veterinary; Liquid helium; Oocytes - physiology; Oocytes - ultrastructure; RNA, Messenger - genetics; RNA, Messenger - metabolism; Ultrastructure; Vitrification; Liquid helium; Bovine immature oocyte; Gene expression; Ultrastructure; Vitrification
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/j.theriogenology.2016.08.010

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.