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.
Henan Provincial Key Laboratory for Grass-Feeding Animal, Henan University of Science and Technology, Luoyang, China.
Hua
H
Wu
Henan Provincial Key Laboratory for Grass-Feeding Animal, Henan University of Science and Technology, Luoyang, China. Electronic address: yxl4282333@126.com.
Xue-Li
XL
Yu
Henan Provincial Key Laboratory for Grass-Feeding Animal, Henan University of Science and Technology, Luoyang, China.
Xian-Fei
XF
Guo
Henan Provincial Key Laboratory for Grass-Feeding Animal, Henan University of Science and Technology, Luoyang, China.
Fan
F
Zhang
Henan Provincial Key Laboratory for Grass-Feeding Animal, Henan University of Science and Technology, Luoyang, China.
Xu-Zhe
XZ
Pei
Henan Provincial Key Laboratory for Grass-Feeding Animal, Henan University of Science and Technology, Luoyang, China.
Xiao-Xia
XX
Li
Henan Provincial Key Laboratory for Grass-Feeding Animal, Henan University of Science and Technology, Luoyang, China.
Wen-Xia
WX
Han
Henan Provincial Key Laboratory for Grass-Feeding Animal, Henan University of Science and Technology, Luoyang, China.
Ying-Hua
YH
Li
D000818:Animals; D002417:Cattle / Q000502:physiology*; D015925:Cryopreservation / Q000662:veterinary*; D054885:Cumulus Cells / Q000502:physiology; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D006371:Helium; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary*; D009865:Oocytes / Q000502:physiology* / Q000648:ultrastructure*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D058989:Vitrification
D012333:RNA, Messenger; D006371:Helium
Bovine immature oocyte; Gene expression; Liquid helium; Ultrastructure; Vitrification