| The development of cryopreservation for mammalian immature oocytes has great significance for the preservation of genetic resources.In spite of some successful reports,the efficiency is still very low at present,because the cryoinjury of structure and function will result in relatively poor oocyte ability during the vitrification process.Therefore,we set up a new method for vitrification of bovine immature oocytes,which treat liquid helium(LHe,-269 °C)as the cold source.The present study indicate that LHe vitrification could increase the cooling rate and reduce the concentration of cryoprotectants at the same time.However,whether the method will affect on the structure and function of oocyte is not clear.This study aimed to investigate the developmental competence,ultrastructure and gene expression of oocytes after LHe OPS vitrification.The bovine immature oocytes are experiment material,and they 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).Experiment 1,evaluation of developmental competence for bovine immature oocyte vitrified by LHe and LN.After LHe vitrification of bovine immature oocytes,oocytes were thawed and cultured,and the variation on the rates of normal morphology,maturation,cleavage,and blastocyst were recorded during in vitro culture,and then the effect of LHe vitrification was analyseds.The viability in 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 viability in the treated groups were lower than those in the control group(100%,73.2%,62%,and 39.8%)(P<0.05).These results show that the developmental competence of freezing oocytes was improved by LHe vitrification.Experiment 2,assessment of vitrification by LHe and LN on the ultrastructure of immature bovine oocytes.Oocytes were thawed and observed the ultrastructure of oocytes by transmission electron microscopy(TEM)after LHe vitrification of bovine immature oocytes,such as zona pellucida,mitochondria,cortical granules and lipid droplets.The ultrastructural damage of oocytes in LHe group was obviously less than LN group,fewer lipid droplets,relatively intact mitochondria,and clustered cortical granules were distributed in the cytoplasm of oocytes in the LHe group.But theultrastructural of vitrified oocytes were severe degeneration than that in control,such as absence of perivitelline space and microvilli,decreased matrix density and the degradation of mitochondria.These results indicate that the damage degree of vitrification was reduced by LHe vitrification.Experiment 3,effect of vitrification by LHe and LN on gene expression in immature bovine oocytes.The mRNA expression levels of P53,CDC20,Eg5 and Npm2 were investigated by real-time quantitative polymerase chain reaction(RTqPCR)after LHe vitrification bovine immature oocytes.The expression levels of gene P53 and Eg5 were higher in the LN group compared with the control and LHe groups(P<0.05),and there was no significant difference between the LHe and control groups for gene P53(P>0.05).CDC20 expression did not differ significantly between the LN and LHe groups(P>0.05),but they both 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).Therefore,the gene expression levels of P53 and Eg5 were improved by LHe vitrification.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 and providing some reference for the exploration of immature oocyte freezing methods. |
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