Fertilization-mediated Protein Juno Freeze-thaw Damage On Mouse Oocyte Membrane

With the development of embryonic biotechnology,frozen oocytes have become an emerging alternative for female fertility preservation.Clinical data also show that cryopreservation of mature oocytes is a safe and effective method.However,some scholars have found that vitrification of oocytes has an effect on the fertilization process and embryo dynamics,and the fertilization rate is lower than that of fresh oocytes.The sperm membrane protein of Izumo1 and the receptor Juno on the corresponding oocyte are considered to be necessary factors for the interaction and fusion of sperm and oocyte.Therefore,this study uses experimental techniques such as immunofluorescence labeling to observe the distribution of Juno protein before and after freezing,the change in quantity and the repair after injury,and to observe and study the influence of anti-juno protein on the fertilization and development of oocytes at various stages before and after freezing,and on the zona pellucida.To explore the effect of vitrification on the fertilization-mediated protein Juno on the oocyte membrane.The results revealed that:(1)In the effect of vitrification freezing and anti-Juno protein on oocyte development,the oocyte oocyte cleavage rate after in vitro fertilization was significantly lower(P<0.05)in the frozen group compared with the control group at all periods.The oocyte in vitro fertilization rate of oocytes treated with anti-Juno protein was significantly lower(P<0.05)than that of the control and frozen groups in both the unfrozen and pre-and post-freezing anti-Juno protein treated oocytes.The results showed that vitrification and freezing reduced the oocytic rate of IVF in oocytes,and that the addition of anti-Juno protein before and after vitrification and freezing could effectively prevent the fertilization process.(2)In the effect of vitrification freezing and anti-Juno protein on the digestion time of oocyte zona pellucida,the time taken for oocytes to digest ZP was significantly lower in the frozen group compared to the control group at all periods(P<0.05).There was no significant difference in the time taken to digest the ZP in the non-frozen group treated with anti-Juno protein compared with the control group.The time taken to digest the ZP in the anti-Juno protein-treated oocytes before and after freezing was not significantly different from that in the frozen group,but was significantly lower than that in the control group and the anti-Juno protein-treated unfrozen group(P<0.05).The results showed that the damage to the zona pellucida was mainly caused by the vitrification freezing process,and the effect of anti-Juno protein treatment on the zona pellucida was not significant.(3)In the effect of vitrification freezing and anti-Juno protein on the number of bound spermatozoa in oocytes,the number of bound spermatozoa was significantly lower(P <0.05)in the frozen oocytes in each group compared to the control oocytes.The number of bound spermatozoa in the unfrozen group treated with anti-Juno protein was significantly lower compared with the control group(P<0.05).The anti-Juno protein group before and after freezing was not significantly different from the frozen and anti-Juno groups and was significantly lower than the control group(P<0.05).The results suggest that vitrification freezing may lead to sclerosis of the zona pellucida,which decreases the number of oocyte-bound sperm;the addition of monoclonal antibody to Juno protein also significantly decreased the number of oocyte-bound sperm.(4)The fluorescence intensity of Juno protein on the surface of mouse oocytes matured in vitro at 0,12,24,36 hours and in vivo was significantly lower in the frozen group compared with the control group at all times(P<0.05);after thermal repair with Tris-EDTA antigen repair solution at p H 9,the fluorescence intensity of Juno protein on the surface of oocytes was significantly higher compared with the frozen group(P<0.05).The fluorescence intensity of Juno protein on the surface of oocytes was significantly higher(P<0.05)compared with the frozen group,but not significantly different compared with the control group.In summary,vitrification freezing damaged the fertilization-mediated protein Juno protein on the surface of oocytes,and the reduced expression resulted in a weakened ability of mouse oocyte membranes to bind sperm and a reduced fertilization rate.