Effect Of Melatonin On In Vitro Maturation Of Vitrified-warmed Mouse Germinal Vesicle Oocytes

Melatonin（MT）can promote the in vitro development of oocytes.However,the exact role of MT in modulating the putative mechanisms relevant to in vitro development of cryopreserved oocytes remains largely unclear,particularly,it is not fully understood at what concentration MT might improve the developmental potential of vitrified-warmed oocytes at GV-stage.Therefore,this study was designed to investigate the effect of MT supplementation on in vitro maturation and developmental potential of germinal vesicle（GV）stage mouse oocytes.Initially,GV-stage oocytes were vitrified by open-pulled straw（OPS）method.In first phase of this research,in order to obtain an optimal concentration of MT for subsequent experiments,following warming,different concentrations of MT(0,10^-9,10^-7,10^-5,10^-3mol/L)were added to the vitrification,thawing and in vitro maturation media.Group receiving no MT supplementation served as control.The development rate of oocytes was evaluated at this stage.The concentration of MT at which highest devolvement rate was observed was then adopted for further experiments.We envisage that these findings will lend reasonable basis for future research aimed at improving the in vitro development of frozen-thawed mammalian oocytes,and at large,will also form the theoretical basis for fertility preservation and strengthening of application of assisted reproductive biotechnology.Briefly,following cryopreservation,the in vitro developmental rate of GV-stage oocytes（ratio of Metaphase I and Metaphase II oocytes）was significantly decreased（P<0.05）in vitrified group compared to the MT-treated(0,10^-9,10^-7,10^-5,10^-3mol/L)groups.Besides,it was observed that germinal vesicle breakdown（GVBD）,MI,and MII development rates were significantly higher in 10^-7mol/L MT-treated group compared to the vitrified group with no added melatonin.Therefore,based on foregoing observations,we envisaged that addition of 10^-7mol/L MT might ameliorate the in vitro developmental potential of vitrified GV-stage mouse oocytes.Whereas in the next phase of this research,the effects of 10^-7mol/L MT on biomarkers such as ROS and GSH levels,mitochondrial membrane potential and ATP levels,spindle morphology,and expression of spindle assembly checkpoint-related genes（Mps1,Bub R1,Mad1,Mad2）were studied in vitrified-warmed oocytes matured in vitro.Results revealed that,when GV-stage oocytes were in vitro cultured and matured（to MI and MII oocytes）following vitrification-warming,ROS levels were significantly lower（P<0.05）in MT-treated(10^-7mol/L)group compared to the vitrified group with no added MT.Besides,the GSH levels,mitochondrial membrane potential,and ATP levels were all significantly higher（P<0.05）in MT-treated(10^-7mol/L)group compared to the vitrified group with no added MT.The results of spindle morphology evaluation showed that,following vitrification-warming,when GV-stage oocytes were in vitro cultured and matured to MII-stage oocytes,the average grade of spindle morphology of oocytes in MT-treated(10^-7mol/L)group was significantly higher（P<0.05）compared to the vitrified group.In addition,the results of quantitative PCR indicated that relative m RNA expression of Mps1,Bub R1,Mad1,Mad2 genes in in vitro matured MII-stage oocytes in vitrified group was significantly different（P<0.05）compared to the fresh group.However,the m RNA expression of Mps1,Mad1 genes was significantly higher（P<0.05）in MT-treated group(10^-7mol/L)compared to the vitrified group.To sum up,cryopreservation of mouse GV-stage oocytes results in oxidative damage,decreased energy（ATP）levels,abnormal spindle morphology,abnormal gene expression,and decreased the development potential of oocytes cultured and matured in vitro.Intriguingly,the addition of 10^-7mol/L MT could significantly ameliorate the balance between the redox reactions and antioxidant system in oocytes exposed to the vitrification-warming.Melatonin supplementation also improved the mitochondrial membrane potential and ATP levels,and showed positive implication on expression of spindle assembly checkpoint-related genes,thus enabling the normal assembly of spindles,and further improving the maturation and developmental competence oocytes in vitro.