Effect Of Anti-frozen Protein III On The Freezing Effect Of Rabbit Ovarian Tissue

Objective:Transplantation of cryobanked ovarian tissue is a promising strategy to restore fertility for women. However, follicles damage during frozen-thawed and ischaemia following ovarian tissue grafting can hinder the development of this technology. Many researches had reported that anti-freeze proteins could protect tissues and cells during freezing. The aim of the study was to observe the effect of antifreeze protein Ⅲ (AFP Ⅲ) on rabbit ovarian tissue cryopreservation.Methods:The study takes rabbit ovary as the research object, using vitrification technology to freeze cortex fragment and freezing the whole ovary by slow-freezing and vitrification technology respectively. Through the following ways:analyzing histology characteristics of frozen-thawing ovary though light microscope, observing the ultrastructural changes by transmission electron microscope, detecting the apoptosis of stromal cells, granulose cells and oocytes by TUNEL assays, and testing the survival of follicles by the means of confocal microscope. Results:Though using vitrification technology to freeze cortex fragment,after thawing, normal rate of follicular morphology of the fresh ovarian group is clearly higher than the two frozen groups(P<0.05), the apoptosis rate of oocytes is significantly lower than the two frozen groups (P<0.05).Normal rate of follicular morphology after adding AFP III into the cryoprotectants is significantly higher than conventional vitrification group without adding AFP Ⅲ(P<0.05), the apoptosis rate of oocytes is obviously less than the conventional vitrification group.By using slow-freezing and vitrification technology to freeze the whole ovary of rabbit, after thawing, normal rate of follicular morphology of fresh group, stromal cells density and the survival rate of follicles is distinctly higher than the four freeze-thawing groups (P<0.05);The apoptosis rate of fresh group is visibly lower than the four freeze-thawing groups.When freezing the whole ovary of rabbit by vitrification, normal rate of follicular morphology and stromal cells density after the addition of AFP Ⅲ in the cryoprotectants is definitely higher comparing with the zero-added AFPIII (P<0.05).The apoptosis rate of oocytes is significantly less than that without adding group AFPIII (P<0.05); but for survival rate of follicles, the two groups has no difference (P>0.05. Comparing with slow freezing method, there is no difference in the normal rate of follicular morphology, the survival rate of follicles and the apoptosis rate of oocytes of the whole ovary of rabbit, which are frozen by vitrification (P<0.05). But the stromal cells density is clearly higher than slow-freezing (P<0.05).Conclusions:Using vitrification to freeze ovarian tissue fragment of rabbit, the normal rate of ovarian follicles of the fresh ovarian group is evidently higher than that in the frozen thawed ovarian tissue. The apoptosis rate of oocytes is visibly less than frozen-thawing ovaries, which shows that there is cold injury in the ovarian cortex during the progress of vitrification. However, adding AFP Ⅲ to tryoprotectants can reduce the damage of ovarian cortex and improve the normal rate of follicular morphology, thus which can reduce the rate of oocyte apoptosis.Two methods of cryopreservation of rabbit ovaries:slow freezing and vitrification, whose effects are not so far different, but for the effect of cryopreservation of stromal cells, vitrification is better than slow-freezing. Adding AFP III into the cryoprotectants of slow-freezing and vitrification can improve the normal rate of follicular morphology and increase the stromal cells density, reduce apoptosis of granule cells, stromal cells and oocytes. It also can improve the survival rate of follicles and has a better protective effect on the ultrastructure of the follicle, which is more apparent in the slow-freezing.