Research Of Vitrification Protocols For Mouse Ovarian Tissue Cryopreservation

Objective:The aim of this research was to explore the effects of three protocols that different cryoprotectants combination taking aluminum foil as carrier on mouse ovarian tissue vitrification and to supply the animal experiment's data for the human ovarian tissue cryopreservation technique.Methods:Ovarian tissue acquired from twenty female Kun Ming mouse aged 5 to 6 weeks were randomly allocated to fresh, cryoprotectant A group, B group and C group. Follicle morphology, growth phase of fresh and post-cryopreservation tissue were observed by light microscope. Separating follicles from the tissue, and then counting the rate of survival, revival and maturing as followed by observed the distribution of mitochondria through con-focal laser-scanning microscopy.Results:No statistically significant differences were observed between the fresh and cryopreserved groups named A, B, C group (88.71% vs.75.56%,76.00%,73.81%), P>0.05. The normal morphological rate of secondary and preantral follicle in A, B, C group(39.3%,48.57% and 41.94%, respectively) were lower than the fresh group leveled 84.21%, P<0.05). The survival rate of follicle in group A, B, C were significantly lower than fresh group(76.85%,82.81%, 79.07%vs.92.39%),P<0.05. The rate in group B was higher than that in group A and C, P<0.05. There's no difference between group A and C, P>0.05. The revival rate of follicle in group B was higher than that in group A and C (88.92% vs.83.18%,85.59%), P<0.05. There's no difference between group A and C, P>0.05. The fluorescence signal of mitochondria in fresh group appeared stronger and more even than that in cryopreserved groups.Conclusion:1. The success rate of mouse ovarian tissue cryopreservation can be improved when the cryoprotectants were the combination of EG and DMSO.2. The cryoprotectants combination of EG and DMSO added aluminum foil carrier can preserve mouse ovarian tissue morphology well. Also, good survival and revival rate of follicle can be obtained with this protocol. The best concentration of each cryoprotectant was 20% EG,10% DMSO and 0.5mol/L sucrose.3. The research founded the experimental system about ovarian tissue vitrification in Northwest China firstly and laid a foundation for the human ovarian tissue cryopreservation in the future.