Effects Of Adding Zearalenone And Melatonin On Pig Spermatogonial Stem Cells

The sensitivity of swine Spermatogonial Stem Cells makes them susceptible to external substances.So it is a new way to improve boar performance by clarifying the damage mechanism of foreign substances to SSCs and identifying beneficial substances,which can promote the proliferation and differentiation of SSCs.In order to explore the influence mechanism of different additives on pig SSCs,the SSCs in the testicles of 7-day-old large white pigs were isolated by differential adhesion method and magnetic activated cell sorting.After morphological observation,alkaline phosphatase staining,marker gene detection and immunofluorescence staining identification,SSCs were used as experimental materials to study the effect mechanism of exogenous additives on pig SSCs.The main findings are as follows:1.Set different concentrations of Zearalenone to treat SSCs.The result of CCK8 detection suggested that the activity of SSCs can be significantly reduced when ZEA concentration and treatment time are greater than 25 ?mol/m L and 24.With the increasing of ZEA concentration,the level of SSCs Reactive oxygen species and Malonydialdehyde content increased significantly,and the mitochondrial membrane potential,total Superoxide Dismutase activity and Glutathione content decreased significantly.2.The result of Quantitative Real-time PCR detection showed that ZEA could increase the m RNA levels of apoptosis genes,such as Bax,Caspase3,Fas,P53,simultaneously decrease the m RNA levels of Bcl-2.The result of Western Blot detection showed that Caspase3 protein expression and Bax/Bcl-2 increased,simultaneously SOD2 protein expression decreased with the increase ZEA concentration.3.Treated SSCs by Proantho cyanidins,Folic acid,Melatonin and only when MT concentration and treatment time were greater than 50 ?mol/m L and 48 h,treatment time greater than 48 h,the vitality of SSCs could be promoted.The results of the combined treatment showed that MT and PC can significantly reduce the apoptosiseffect of ZEA on SSCs.4.With the increase of MT concentration,the content of MDA in SSCs decreased significantly,and the total SOD activity and GSH content was not changed obviously.5.The result of Quantitative Real-time PCR detection showed that MT could decrease the m RNA expression of the apoptotic genes Bax,Caspase3 and P53.Western Blot detection showed that Caspase3 protein expression and the proportion of Bax/Bcl-2decreased with the increase of MT concentration,and the protein expression of marker protein UCHL1 increased with the addition of MT.These results indicated that ZEA can cause oxidative damage to pig SSCs,activate apoptosis pathways to reduce cell viability.Among PC,FA and MT,only MT can promote the vitality of SSCs pigs,and PC and MT have the function of reducing the apoptosis effect of ZEA.MT has the effect of eliminating intracellular ROS,inhibiting the expression of apoptotic genes,and promoting the vitality of pig SSCs.