Study Of Mouse Ovarian Tissues Vitrification

Background: Cryopreservation of follicles in ovarian cortical tissue has been suggested as a method for preserving fertility for young women who need to undergo cytotoxic therapy. Vitrification is assumed to be a promising method to cryopreserve human ovarian tissue but still needs optimization. In this study, mouse ovarian tissues were vitrified without carrier, and the effect of different cryopreservation protocols on morphology ,survival rates and spindle configuration was assessed.Objectives:To study the survival rates and the variance of DNA of the follicles in the mouse ovarian tissues with different exposure time of 1.5 M EG by vitrification. To campare the survival rates of oocytes in the ovarian tissues by vitrification with different compositions of cryoprotectant solutions. To observe the ultra-structure change of the ovarian tissues after vitrification. To evaluate the effect of CPAs containing EG and DMSO on the spindle configuration.Methods : 1. Mouse ovaries were cut into pieces of 1–1.5 mm3 ,then ovarian tissues were vitrified by being treated with different time of pre-equilibration:5min,10min and 15min, being exposed in the vitrification solution with the same time, then being directely plunged into the liquid nitrogen ,with the method of two-steps,and using ethylene glycol(EG) and sucrose as cryoprectant.After thawing,the vitrified-warmed ovarian tissues and fresh ovarian tissues were stained with hematoxylin and eosin. DNA was observed using agarose gel.2. mouse ovarian tissues were randomly assigned to one of three groups: (i) EG5.5 [5.5M ethylene glycol (EG)+1.0M sucrose]; (ii)DMSO [15% EG + 15% dimethylsulphoxide (DMSO)+0.5M sucrose]; and (iii) GC(25% EG+25% GC). After warming, one part of each group was isolated by mechanical dissection with 26G needles and was stained with PI to examine the survival rates of oocytes.3.After vitrification/warming , the ovarian tissue was analysed using transmission electron microscopy (TEM);After isolation, some GV oocytes was matured in vitro, and were immunostained for tubulin and chromatin before visualization using confocal microscopy.Results: 1.After warming,the rates of the integrate follicles in the ovarian tissues were 11.90,31.82and 17.39%,respectively. The group pre-equaled with ten minutes remains better morphology than other groups. The DNA in the agarose gel of all research groups was similar as the fresh-control group.2. The survival rates after warming were 26.5, 35.6 and 26.1%,respectively.The rate of DMSO group was higher than the others', and both have significant difference(P<0.05),but no significant differences were observed between results in solutions containing EG5.5 versus GC .3. TEM showed apparent Vacuolization and swollen mitochondria in the primordial follicles,The nuclear membrane and basal membrane are intact, but the cell membrane of the oocyte has lost attachment from the granulosa cells in large areas. After mechanical isolation and IVM, the spindles configuration was poor and not good compared with the control.The normal morphology rates of spindle in vitrification and the control group were 35.0 and 75.0%,respectively. There was significant difference between them(P<0.05).Conclusions: 1.The ovarian tissues should be pre-treated about ten minutes. Vitrification may not affect DNA of cells in the ovarian tissues. 2.In vitrification, DMSO is more adaptable to ovarian tissues than EG or GC.3.The fastest cooling rate in combination with EG and DMSO as cryoprotectants injured the ultra-structure, but the oocytes isolated from vitrified ovarian tissues could mature in vitro, and may form normal spindle. This technology is a feasible method for ovarian tissue cryopreservation, but needs more research before being applyed in the clinic.