Effects And Mechanism Of Antioxidant Capacity On Testis Development Of Hu Sheep

As the reproductive organ of male animals,the testicles mainly produce sperm and testosterone.Testicular tissue lipid metabolism is vigorous because rich in a large amount of polyunsaturated fatty acids（PUFA）.During testicular development and spermatogenesis,it will produce a large amount of reactive oxygen species（ROS）.ROS is cleared by the antioxidant system in the tissue in normal conditions.When the oxidative/antioxidant balance in the tissue is destroyed,excessive ROS can cause oxidative stress,leading to DNA and protein damage in sperm and lipid peroxidation cascade reaction.Oxidative stress affects testicular development and spermatogenesis,leading to a decline in male reproductive capacity.Therefore,in order to explore the impact of oxidative stress levels in testicular tissue on testicular development and its mechanism of action,this study analyzed the oxidative stress levels in testicular tissue of 6-month-old Hu sheep at different developmental levels,and utilized oxidative lipid omics technology to find the oxidative lipid metabolism molecules that affect testicular development.Based on these experiments,a testicular sertoli cell oxidative stress model was constructed in vitro.By adding PUFA to search for its protective effect on cells damaged by oxidative stress,and further analyze its mechanism of action.The main research content and results of this paper are as follows:（1）Study on oxidative stress level of testicular tissue at different development levels:According to the different development levels of testicles,18 6-month-old rams were selected and divided into well developed testicular（L）and poorly developed testicular（S）groups.The average testicular weight of group L and group S were 158.67±5.21 g and 44.58±4.14 g,respectively.The diameter of the seminiferous tubule,the thickness of the seminiferous epithelium,and the sperm density of group L were significantly higher than group S（P<0.05）.Compared with group S,group L has a significant increase in T-AOC（2.69±0.47 vs.1.16±0.22 U/mgprot）and T-SOD（22.35±2.59 vs.992±1.62 U/mgprot）,while MDA（0.72±0.13 vs.34±0.17 n M/mgprot）and mt DNA copy number（P<0.05）.The expression of GPX3 and Cu/Zn SOD m RNA in group L was significantly higher than that in group S（P<0.05）,and the proteins of GPX3 and Cu/Zn SOD were expressed in both Leydig cells and seminiferous tubules,with higher expression levels in group L.The above results indicate that well developed testis has stronger antioxidant capacity and lower oxidative stress level,which is conducive to spermatogenesis.（2）Study on oxylipin of testicular tissues at different developmental levels:The results of oxidized lipid omics analysis showed that a total of 118 oxidized lipid metabolites were detected in testicular tissue,and a total of 20 differential oxidized lipid metabolites were selected.Among them,16 oxylipin had a higher content in underdeveloped testicular tissue.The level of oxylipin ofω-3 PUFAs was higher thanω-6 PUFAs in group S.Differential metabolite KEGG was enriched in the serotonergic synapse and arachidonic acid metabolic pathway.The q PCR results showed that the m RNA expression levels of genes ALOX12,AKR1B1,PTGDS,and PTGER2 involved in regulating oxidative lipid metabolites were higher in the S group than in the L group.The above results indicate that the degree of oxidative stress in underdeveloped testicular tissue is more severe,and the degree of lipid peroxidation is higher.The degree of lipid peroxidation inω-3 PUFAs is higher thanω-6 PUFAs.（3）Study on the protection ofω-3 PUFA（ALA）against oxidative stress damage model of testicular Sertoli cells:Sertoli cells in vitro were isolated and identified.Sertoli cells were treated with different concentrations of H₂O₂（0,100,200,500,800,1000μM）after 12 hours,there was a negative correlation between cell viability and H₂O₂ concentration,with an IC50 concentration of 318μM.Oxidative stress model cells were treated with different concentrations of ALA（0,50,100,200,400μM）for 12 hours,it was found that cell viability of the 200μM ALA treated group was significantly increased（P<0.01）.Compared with the oxidative stress injury model group,the H₂O₂+ALA treatment group showed a significant improvement in cell exfoliation and death,as well as a significant increase in SOD content and m RNA expression of antioxidant gene GPX4 and anti apoptotic gene Bcl2（P<0.05）.The above results indicate that adding ALA in vitro can alleviate the damage of oxidative stress to supporting cells and improve cell viability.In conclusion,PUFAs play an important role in testicular development.Testis with higher development have stronger antioxidant capacity and lower lipid peroxidation levels,which is more conducive to spermatogenesis.ALA of 200μM can alleviate the oxidative damage caused by H₂O₂ in the testicular Sertoli cells of Hu sheep.The above results can be supplemented for productionω-3 PUFAs and provide a theoretical basis for improving the reproductive ability of rams.