| Oocyte cryopreservation has become an important assisted reproductive technique and played a valuable role in many fields of society.But,cryopreservation can induce a decline of oocyte quality,which results in decline of embryo developmental competence and abnormal fetus.Melatonin can improve the developmental competence of vitrified-warmed oocyte,but its main stage promoting oocyte development potential is not clear.Therefore,this experiment aimed to study the effect of melatonin on the zygotic cell cycle in parthenogenetic development of vitrified-warmed mouse matured（metaphaseⅡ,MⅡ）oocyte,which could identify the main stage and the possible mechanism.Firstly,fresh MⅡoocytes were randomly divided into three groups:Control group,Vitrification group,Vitrification+MT(10-9mol/L)group.Then,we detected the developmental potential of vitrified-warmed oocyte with whole stage supplementation of melatonin and the phased supplementation of melatonin,which is used to explore the effect of melatonin on blastocyst rate and the first zygotic cleavage rate of vitrified-warmed oocyte.Finally,we analysed the pronuclear formation,transition of S phase and events associated with them,which contains spindle recovery,chromosome separation,mitochondrial function and ROS levels.The results are as follows:1)Melatonin promotes the developmental competence of vitrified-warmed oocyteParthenogenetic blastocyst development rate of mouse MⅡoocytes in vitrification group was significantly reduced（33.61±2.92%vs 66.67±0.59%,P<0.05）.However,when10-9 mol/L melatonin was added to the vitrification group in whole stage,the parthenogenetic blastocyst development rate was significantly increased（57.14±7.47%vs.33.61±2.92%,P<0.05）.Similarly,when oocytes parthenogenetic developed to 2-cell embryo in vitrification+MT group,and then those 2-cell embryos were transferred to melatonin-free medium,the blastocyst development rate of oocytes was also significantly increased（76.47±0.60%vs.53.02±3.15%,P<0.05）.Those results indicated that melatonin certainly increased the developmental competence of vitrified-warmed oocyte and the main stage may be the zygote stage.2)Melatonin could mediate the cell cycle process of zygoteCompared to the control group,the time required for the first cleavage of 50%parthenogenetic zygotes in vitrification group was significantly increased（14.76±0.22vs.15.47±0.14,P<0.05）.However,the vitrification+MT group was significantly shortened（14.77±0.18 vs.15.47±0.14,P<0.05）.In addition,At 6 h and 7 h after parthenogenetic activation,the proportion of G1/S transition of vitrification group was significantly lower than control group（14.56±3.81%and 53.11±3.30%vs.43.71±2.18%and 78.92±7.89%,P<0.05）.When melatonin was added to the vitrification group,the rates of zygote G1/S transition were significantly higher than vitrification group（24.31±3.34%and 80.63±8.28%vs.14.56±3.81%and 53.11±3.30%,P<0.05）.At 11 h after parthenogenetic activation,the rate of zygotes in vitrification group stayed in S phase was significantly higher than control group（49.46±5.89%vs.10.58±4.19%,P<0.05）.In vitrification+MT group,the proportion of the zygote stayed in S phase was significantly lower than vitrification group（20.34±1.61%vs.10.58±4.19%,P<0.05）.This indicated that melatonin could alleviate the delay phenomenon of S phase caused by vitrification.Further findings showed that zygote at the end of G1（5 h after parthenogenetic activation）,the mitochondrial membrane potential level（1.67±0.02 vs.2.26±0.08,P<0.05）and membrane potential-related ATP level（0.448±0.015 vs.0.536±0.021,P<0.05）in the vitrification group were significantly decreased.When melatonin was added to vitrification group,the mitochondrial membrane potential level and ATP level of zygote increased significantly（1.67±0.02 and 0.448±0.015 vs.2.10±0.06 and 0.495±0.007,P<0.05）.Similarly,zygote at S phase（9h after parthenogenetic activation）,the ROS level in vitrification group was significantly increased（0.01128±0.00032 vs.0.0149±0.00128,P<0.05）.After melatonin was added to the vitrification group,the ROS level significantly decreased（0.0149±0.00128 vs.0.00895±0.00034,P<0.05）.These results showed that melatonin may improve the cell cycle process of zygote from vitrified-warmed oocyte by mediating mitochondrial function and ROS level.3)Melatonin promotes the parthenogenetic pronuclear formation of vitrified-warmed oocyteAfter 3-6 h parthenogenetic activation of oocytes,the rate of pronuclear formation in vitrification group was significantly lower than control group（13.39-97.54%vs.4.14-75.74%,P<0.05）.When melatonin was added to the vitrification group,the pronuclear formation rate of zygotes were significantly higher than vitrification group（14.60-94.07%vs.4.14-75.74%,P<0.05）.After oocyte parthenogenetic activation,the time required for 50%zygotes to form pronuclear was significantly increased in the vitrification group（3.99±0.056 vs.4.89±0.17,P<0.05）.When melatonin was added to vitrification group,the time required for 50%zygotes to form pronuclear was significantly reduced（4.89±0.17 vs.4.09±0.033,P<0.05）.when meiosis process was further analysed,we found that grade of spindle and chromosome morphology of oocytes recovered 1 h after vitrified-warmed treatment was significantly decreased when compared to control group（3.24±0.09 vs.2.67±0.11,P<0.05）.And after 1 hour post activation,the chromosome separation rate of oocytes in vitrification group was significantly decreased（83.33±6.25%vs.43.63±5.27%,P<0.05）.After 2 hours post activation,the situation was similar to 1 hour post activation.After melatonin was added to vitrification group,grade of spindle and chromosome morphology significantly increased（2.67±0.11 vs.3.17±0.11,P<0.05）.And after 1 hour post activation,the chromosome separation rate of oocytes increased significantly（43.63±5.27%vs.62.63±3.79%,P<0.05）,and the chromosome separation rate of vitrification+MT group recovered to control group（77.93±4.03%vs.85.70±4.81%,P>0.05）after 2 hours post activiation.These results illustrated that the state of spindle recovery and chromosome separation in vitrification group may affect the pronuclear information,which induced an abnormal cell cycle.But,supplementation of melatonin could alleviate this phenomenon.In summary,melatonin can promote the recovery of spindle morphology and chromosome separation,pronuclear formation and the first cleavage of zygotes,improve mitochondrial function and reduce ROS levels,improving zygote quality and promote later embryonic development.And for low developmental competence of vitrified-warmed oocyte of livstocks,our conclusion of this experiment could provide a proof for melatonin which promotes the developmental competence of vitrified-warmed oocyte and in vitro production of embryo. |
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