| During the in vitro maturation of oocytes and the in vitro culture of early embryos,environmental stress is inevitable.Oxidative stress is one of the important factors hindering the in vitro development of oocytes and early embryos.The imbalance of the intracellular redox system can lead to excessive production of reactive oxygen species(ROS)in cells,and the excessive accumulation of this ROS can reduce the efficiency of oocyte maturation in vitro and embryo production in vitro.As a traditional Chinese medicine in my country,Dendrobium has been widely used for many years and has various effects such as anti-inflammatory and analgesic,and has attracted much attention in recent years.Dendrobium contains a natural alkaloid(Dendrobine,DNE)that can scavenge free radicals,reduce ROS generation and inhibit apoptosis.However,so far,no research on DNE in the field of female mammalian oocyte and early embryonic development has been reported.The purpose of this experiment is to explore the role of DNE in the maturation and early embryonic development of mouse oocytes cultured in vitro.This experiment is divided into the following two parts:1.The effect of dendrobine on the maturation of mouse oocytesThe purpose of this experiment was to investigate the effect of DNE on the in vitro maturation of mouse oocytes.By adding different concentrations(0,5,10,20,50 μM)of DNE to the in vitro maturation medium of Kunming mouse oocytes for 12 h in vitro,the optimal concentration of DNE to promote oocyte maturation was verified.The results showed that the first polar body(PBI)excretion rate of mouse oocytes treated with 10 μM and 20 μM DNE was significantly increased compared with the 0 μM group(P< 0.05).Immunofluorescence staining,fluorescence intensity measurement,real-time quantitative PCR(q RT-PCR)and other methods were used to detect the relevant indicators during the maturation of mouse oocytes.The results showed that the addition of 10 μM and 20 μM DNE could significantly reduce the level of ROS in mouse oocytes(P< 0.05),significantly increase the level of GSH(P<0.05),and significantly increase the level of MMP(P< 0.05).In addition,the m RNA expression levels of antioxidant-related genes STRT1,STRT2,and SOD2 in mouse oocytes treated with 10 μM and 20 μM DNE were significantly higher than those in the 0 μM group(P< 0.05).2.The effect of dendrobine on the early embryonic developmental potential of miceThe purpose of the experiment was to investigate the potential effect of DNE on early embryonic development in mice.First,different concentrations(0,5,10,20,50μM)of DNE were added to the early embryo medium,the fertilized eggs were cultured for 24 h or 96 h,and the early embryo division rate and blastocyst rate were calculated by cell morphology observation..The results showed that the blastocyst rate was significantly increased in the 10 μM and 20 μM DNE treatment groups(P<0.05).Then,immunofluorescence staining,fluorescence intensity measurement,q RT-PCR and other methods were used to detect the relevant indicators of the mouse two-cell stage,that is,the zygotic genome activation(ZGA)stage.The results showed that the ROS levels in the 10 μM and 20 μM DNE treatment groups were significantly lower than those in the 0 μM group(P< 0.05),and the GSH levels were significantly increased(P< 0.05).MMP levels in the 10 μM DNE-treated group were significantly higher than those in the 0 μM group(P< 0.05).The m RNA transcription levels of ZGA activation-related genes DUX,YAP1 and TRC in mouse two cells treated with 10 μM and 20 μM DNE were significantly higher than those in the 0 μM group(P< 0.05).Finally,the relevant indicators affecting the developmental potential of mouse blastocysts were detected by immunofluorescence staining,fluorescence intensity measurement,q RT-PCR and other methods.The results showed that the total number of blastocyst cells in the 10 μM and 20 μM DNE treatment groups was significantly higher than that in the 0 μM group(P< 0.05),the blastocyst apoptotic cell rate was significantly decreased(P< 0.05),and the ROS level was significantly lower than that in the 0 μM group.(P<0.05).The GSH level in the 20 μM DNE-treated group was significantly higher than that in the 0 μM group(P< 0.05).And CB levels were significantly decreased after 10 μM and 20 μM DNE treatment(P< 0.05).The m RNA expression levels of mouse early embryo antioxidant-related genes SIRT1,SIRT2,and SOD2 in the 10 μM and 20 μM DNE groups were significantly higher than those in the 0 μM group(P< 0.05).The expression levels of apoptosis-related genes Caspase-3 and BAX m RNA in the 10 μM DNE treated group were significantly lower than those in the 0 μM group(P< 0.05).The expression levels of anti-apoptosis-related gene BCL-2 in the 10 μM and 20 μM DNE treatment groups were significantly higher than those in the 0 μM group(P<0.05).In conclusion,this study shows that DNE can effectively promote the maturation of mouse oocytes and improve the early embryonic development potential.Specifically,DNE can promote the discharge of the first polar body of mouse oocytes and increase the blastocyst rate of early embryos in mice.DNE can balance the redox system and improve the function of mitochondria,thereby promoting oocyte maturation;DNE can also increase the number of blastocyst cells in mice,reduce the rate of apoptotic cells,and balance the redox system,thereby improving the early embryonic development of mice potential. |
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