Preliminary Study On The Influence Of Ereezing-thawing Damage On Oocytes Mediated By Calcium Ions

The recovery rate,fertilization rate,embryo developmental rate and suvival rate of oocyte after cryopreserved resuscitation have not been ideal,the reasons for this gap are complex.The increse of intracellular calcium concentration may be an important reason for the decrease of developmental rate.To investigate the effect of intracellular calcium changes on oocytes,the ultrastructure,sperm penetration,hyaluronic digestion,developmental rate,calcium concentration and fluctuation of oocytes in each group before and after freezing were studied by TEM(Transmission electron microscopy)and LSCM(Laser scanning confocal microscopy).It is therefore discovered that:(1)Under the TEM,oocytes in the fresh control group,TG toxic group,2-APB toxic group have complete zonas and mitochondrial structures,while oocytes in the freezing group and TG freezing group were severely damaged,with broadened zonas,damaged edges,widened ovum gaps and unclear mitochondrial structures;oocytes in 2-APB freezing group were slightly injured,with part of the organelle structures like zonas and mitochondrial structures being damaged.(2)Digestion time of zonas and number of sperms combined with zonas:in 2-APB freezing group,the figures were(230.5 ± 7.14,4.17 ± 1.11);in TG freezing group,(172.5±5.91 and 1.17±1.03);in the freezing group,(221.22±9.46 and 2.75±1.29)and in the fresh control group,(300.14±7.45 and 8.00±2.45).Compared with the digestion time of zonas and number of sperms combined with zonas in the fresh control group,those figures in the former three groups in 2-APB freezing group were distinctly lower(P<0.05);compared with the digestion time and number of sperms combined with zonas in TG freezing group and in the freezing group,figures in 2-APB freezing group were substantially higher(P<0.05).(3)In terms of the development rate of each group,compared with the cleavage rate of the fresh control group,cleavage rates of the freezing group(59.39%),TG toxic group(71.91%),TG freezing group(52.29%),2-APB toxic group(85.71%)and 2-APB freezing group(66.39%)were significantly different(P<0.05);cleavage rate and blastocyst rate in TG freezing group(52.29%and 25.44%)were substantially lower(P<0.05)than those in TG toxic group(71.91%and 35.95%);cleavage rate in 2-APB freezing group(66.39%)was distinctly lower(P<0.05)than that in 2-APB toxic group(86.03%).(4)It was discovered through laser confocal detection that,intracellular calcium concentrations of all the groups were significantly differents(P<0.05),among which the figure in 2-APB freezing group(122.21±4.33)is apparently lower(P<0.05)than those in TG freezing group(146.87±7.49)and in freezing group(139.70±6.00).It was discovered through the detection of intracellular calcium concentration that,calcium ion in the control group remains at a dynamically stable state,while the calcium ion in toxic group,TG group and 2-APB group fluctuates remarkably,and the peak of fluctuation in 2-APB group was significantly lower than those of the rest two groups.Besides,the duration of fluctuation in 2-APB group was lower than those in the other two.The results indicated that TG was capable of raising intracellular calcium concentration,worsening zona sclerosis and damage,leading to low development rate;oocytes processed by 2-APB cryopreservation had the capacity to lower intracellular calcium concentration,relieved the the damage of oocytes and thus leaded to a high development rate.Therefore,2-APB could be applied to lower the damage on the oocytes caused by cryopreservation during cell cryopreservation.