Investigation On Vitrification Of Ovary Tissue Of Mouse

Ovarian tissue cryopreservation is important in reproductive engineering. It provides technical supporting for the investigation which needs to reserve and use egg resource for example fertilization mechanism and somatic cell nuclear transfer. So far, cryoprotection of ovarian tissue has made a series of achievements, but it is still not perfect. Vitrification is a new advancement in tissue preservation, but for ovarian tissue is not yet a perfect system.Vitrification procedure and cryoprotectants are two important factors in vitrification technology. In previous studies, both frozen-thawed oocytes and oocytes obtained from frozen-thawed ovarian tissue can be fertilized and develop into blastocysts or bear offsprings,which shows they maintain developmental potential after frozen-thawed. However,ovarian tissue cryoprotected by the conventional programme method and conventional vitrification method, ethier the recovery rate is not high, or method is of complex, besides the rare investigation of cryopreservation of preantral follicles. In present study , different procedure and formulas of cryoprotectant for vitrification were investigated with separated follicles or ovarian tissue slices. The separated follicles from ICR mice ovarian tissue, were vitrificated using the same cryoprotectants as Dinnyes and compared the recovery rates of conventional vitrification,metal net,- 205℃vitrification and -205℃surface vitrification. The optimal method was then selected from above experiments to test different cryoprotectants. Three formulas of cryoprotectant were chosen to cryoprotect the preantral follicles separated from ovarian tissue, comparing the recovery rates. Using proper procedure and formula, vitrification of ovarian tissue slice was undertaken and the viability and growth were evaluated for the follicles separated from ovarian tissue after frozen-thawed.The results showed as follows:(1) In selection of cryoprotection procedure: in experiment A, the recovery rates of metal net group and -205℃vitrification group are significantly higher than that of conventional vitrification group, and no difference between the two groups. In experiment B, there are significant differences between the recovery rates of -205℃surface vitrification group and -205℃vitrification group, there are differences between the recovery rates of -205℃surface vitrification group and metal net, while there are no differences between metal net and -205℃vitrification. The recovery rates reached 83.02%±4.10% in -205℃surface vitrification.(2) In the investigation of formulas of cryoprotectants: There are significant differences between every two formulas. For formula A, its recovery rates is 18.54%, standard deviation (SD) is 2.65, and variation coefficient (CV) is 14.27; For formula B, the recovery rates is 98.39%, standard deviation (SD) is 1.88, and variation coefficient (CV) is 1.91; For formula C, the recovery rates is 5.83%, standard deviation (SD) is 0.38, and variation coefficient (CV) is 6.57. The effect of formula B is the best in the three.(3)In the vitrification of ovarian tissue slice of mouse after frozen-thawed, when the preincubation time is different, the situation of isolated follicle's rocovery rates and follicular adherent rates are also different. When ovarian tissue slice incubated 48h after recovery, follicular recovery rates and adherent rates are higher.To our investigation, we come to the conclusion :1. -205℃surface vitrification is a good method to cryoprotect preantral follicle of mouse.2. Cryoprotectant B(EFS) is a good cryoprotectant to cryoprotect preantral follicle of mouse.3. Ovarian tissue cryoprotected by the -205℃surface vitrification and cryoprotectant B(EFS), when it is incubated 48h after recovery, the isolated follicle's recovery rates and adherent rates are higher.4. Ovarian tissue vitrificated and thawed needs preincubation for further process.