Effects Of Baicalin On In Vitro Maturation Of Pig Oocytes And In Vitro Development Of Pig Embryos

Baicalin is a traditional Chinese medicine monomer extracted from the root of baicalin plant.Baicalin has a variety of functions including anti-oxidation,anti-apoptosis and anti-inflammation,and is often used to treat diseases caused by a variety of inflammatory factors.In addition,many animal studies reported that baicalin affects the reproductive system including female infertility and miscarriage.In vitro maturation(IVM)of oocyte and in vitro development of embryo will be damaged by high concentration of reactive oxygen species(ROS),resulting in reduction of the efficiency.Therefore,the research on the mechanism of oocyte maturation and embryo development and the application of both in assisted reproductive technology(ART)are greatly restricted.In this study,the addition of baicalin in IVM culture medium of pig oocytes and in vitro development culture medium of pig embryos could significantly improve IVM capacity and quality of pig oocyte,in vitro development ability and quality of pig embryos by inhibiting apoptosis.In addition,in vivo experiments of adult male kunming mice were used to verify the protective effect of baicalin on alcohol-induced sperm injury.1.The effect of baicalin on IVM of pig oocytes and subsequentembryonic development following parthenogenetic activation(PA).Experiment 1.This experiment explored the effect of different baicalin concentrations on the IVM of pig oocytes,and noticed that both 0.1 μg/mL and 0.5 μg/mL baicalin could significantly improve the IVM of pig oocytes(68.1,70.2 vs.59.7,P<0.05).Next,we investigated the effect of these various concentrations of baicalin on reactive oxygen species(ROS)production of pig oocytes.The ROS level was lower in oocytes treated with 0.05,0.1 and 0.5 μg/mL baicalin than control oocytes and those treated with other concentrations of baicalin confirming by fluorescence staining(P<0.05).We decided to treat oocytes with 0.1 μg/mL baicalin in the subsequent experiments.Meanwhile,RT-PCR results showed that mRNA expression of the pro-apoptotic gene BCL2-Associated X(Bax)was significantly lower(P<0.05)in mature oocytes and cumulus cells in the baicalin-treated group than the control group.Experiment 2.To detect the quality of oocytes after baicain treatment,the fluorescent staining was conducted.It showed that mitochondrial potential(△Ψm)and ATP content in baicalin treatment group were significantly higher than that of control group(P<0.05).RT-PCR results showed that mRNA expression of the cumulus cell expansion-related genes(PTGS1,PTX3,TNFAIP6,SHAS2)and nuclear maturation-related genes(BMP15,GDF9,CyclinB,CDKl)was significantly(P<0.05)higher in baicalin-treated oocytes than the control oocytes.Experiment 3.Effect of baicalin treatment during IVM on subsequent development of PA embryos.The development capacity and the number of cells per blastocyst were significantly higher(P<0.05)in the group treated with 0.1 μg/mL baicalin than the control group.At the same time,mRNA expression of the Sox2,Nanog and Bcl2 was significantly higher(P<0.05)while mRNA expression of Bax was lower(P<0.05)in the baicalin-treated group than the control group.In addition,the percentage of apoptotic cells in blastocysts was significantly lower(P<0.05)in the baicalin-treated group than the control group.2.Effects of baicalin on PA,somatic cell nuclear transplantation(SCNT)and in vitro fertilization(IVF)embryo development capacity.Experiment 1.Effects of of baicalin on in vitro development capacity of pig embryos.The percentage of embryos that reached the blastocyst stage and the number of cells per blastocyst were significantly higher(p<0.05)in the group treated with 0.1 μg/mL baicalin(baicalin-treated group)than the control group.Concerning the quality of blastocyst,the percentage of apoptotic cells was also lower(p<0.05)in the baicalin-treated group than the control group.RT-PCR showed that the relative mRNA expression of Oct4,Sox2 and Bcl2 in blastocysts was higher(p<0.05)whereas the relative mRNA expression of Bax was lower(p<0.05)in the baicalin-treated group than the control group.Experiment 2.Effects of baicalin on ROS production,apoptotic-related gene mRNA expression and △Ψm and ATP production in pig PA embryos.Regarding to the quality of embryos,fluorescence staining results showed that the ROS level in embryos at the 2-cell and 4-cell stages was lower(p<0.05)in the baicalin-treated group than the control group.In addition,RT-PCR results showed that the relative mRNA expression of the Bax in embryos at the 2-cell and 4-cell stages was lower(p<0.05)in the baicalin-treated group than the control group.By contrast,the relative mRNA expression of Bcl2 gene in embryos at the 4-cell stage was significantly higher(p<0.05)in the baicalin-treated group than the control group.Meanwhile,△Ψm and the ATP level in PA embryos at the 2-cell stage were higher(p<0.05)in the baicalin-treated group than the control group.Experiment 3.Effects of baicalin on mRNA and protein expression of genes related to sonic hedgehog(SHH)signaling in pig PA embryos.RT-PCR and immunostaining results showed that mRNA and protein expression of genes related to this pathway in pig PA embryos at the 2-cell stage.The mRNA expression of SHH signaling pathway-related genes(SHH,SMO,PTCH1,and GLI1)was significantly higher(p<0.05)in the baicalin-treated group than the control group.To confirm that SHH signaling is involved in the mechanism via which baicalin improves embryonic development,we investigated the effects of baicalin in the absence or presence of 2 μM cyclopamine(Cy)on ROS production,△Ψm,and the ATP level in pig PA embryos at the 2-cell stage.The inhibitory effect of baicalin on ROS production was abolished in the presence of Cy.Moreover,the baicalin-induced increases in △Ψm and the ATP level were abrogated in the presence of Cy.At the same time,the baicalin-induced improvements in the development capacity,the number of cells per blastocyst and expression of pluripotency and apoptosis-related genes in blastocysts were abolished in the presence of Cy.Experiment 4.Effects of baicalin on development of pig SCNT and IVF embryos.The results showed that baicalin also can significantly improve the development capacity of SCNT and IVF pig embryos.2.Protective effects of Baicalin on sperm damage in adult male miceinduced by alcoholExperiment 1:This study established a model of alcohol-induced sperm damage in male kunming mice.Ten mice with similar characteristics were randomly divided into experimental group(five)and control group(five).Experiment group were subjected to alcohol daily intragastric administration for consecutive four weeks(2 g/kg per day:first week;4 g/kg per day:second week,6 g/kg per day:third week,and 8 g/kg per day:fourth week,g/kg-ratio of alcohol mass to mouse weight)and the control group was administrated the same amount of normal saline.These mice were euthanized after four weeks.The results showed that the weight and the mortality of alcohol group was decreased and increased compared with control group,respectively.In addition,the sperm density detected in the cell counting plate showed that the sperm content in the alcohol group was significantly lower(p<0.05)than that in the control group.Experiment 2:To detect the protective effect of different concentrations baicalin on alcohol-induced sperm damage in mice,this experiment was divided into four groups:control group(normal saline),alcohol group,alcohol with 25mg/kg baicalin treatment group,alcohol with 50mg/kg baicalin treatment group(mg/kg-ratio of baicalin mass to mouse weight).The administration amount of normal saline and alcohol is according to EXP1 method.The results showed that 50 mg/kg of biacalin has a partial protective effect on the decrease of sperm density and the increase of sperm malformation rate induced by alcohol in mice.Experiment 3:The effects of different concentrations baicalin were evaluated by measuring the mRNA expression of genes related to inflammatory factors and apoptosis of testicular tissue.The results showed that alcohol treatment significantly increased the mRNA expression of caspase-3,Bax,IL-6,TNF-a and decreased the expression of Bcl2 in testicular tissues(P<0.05).However,compared with the alcohol group,50 mg/kg of biacalin treatment group has significantly decreased the mRNA expression of caspase-3,Bax,IL-6,TNF-a genes(p<0.05)and increased the expression of Bcl2(P<0.05).Conclusions:This study confirmed that 0.1 μg/mL baicalin significantly improved in vitro maturation of pig oocytes and in vitro developmental capacity of PA,IVF and SCNT embryos,especially lowing ROS level and improving △Ψm and ATP content.It also proved that baicalin affected the development capacity of pig embryo via the activation of SHH signaling pathway.Furthermore,this study demonstrated that baicalin showed the protective effects on male kunming mice with alcohol-induced sperm damage through in vivo experiments.