| At present,artificial insemination is still the most widely used reproductive biotechnology in pig industry.Semen quality is the most important factor to determine the number of offspring and the success rate of artificial insemination.Boar semen is usually stored in liquid,however,during liquid storage,sperm cells continue to metabolize,resulting in the depletion of available nutrients and the accumulation of metabolic end products.Reactive oxygen species(ROS)is a by-product of oxygen metabolism in sperm mitochondria and one of the main determinants of sperm quality and effective survival time during liquid storage.However,excessive reactive oxygen species induced oxidative stress causes damage to a certain proportion of sperm cells,eventually leading to cell function impairment and cell death.Therefore,with the extension of storage time,the quality of semen becomes worse and the pregnancy rate decreases.It is known that seminal plasma has antioxidant capacity and can remove ROS to protect sperm from oxidative stress.Exosomes in seminal plasma contain lipids,proteins,micro RNA(miRNA)and messenger RNA(m RNA),will have a positive impact on key processes related to sperm function.Nowadays,exosomes have become a research hotspot in various fields,miRNA and metabonomics can effectively predict target factors.Therefore,in order to explore the protective effect of seminal plasma exosomes on pig sperm during liquid storage at 17℃,10 Landrace boars with reproductive ability and an average age of 22.4 months were selected in this experiment.The sperm quality was evaluated through computer assisted sperm analysis system(CASA),and 6 samples meeting the standard were selected.Each sample was collected and homogenized with the same amount of semen(10 m L).The specific research results are as follows :1、The exosomes of boar seminal plasma were separated by ultracentrifugation.The obvious cup-shaped structure was observed by transmission electron microscope(TEM),and nano particle tracking analysis(NTA)showed that the average diameter was 107 nm,western blot showed that the presence of CD81,CD63 and CD9 markers,which were consistent with the characteristics of exosomes.2、The exosomes labeled with Dil dye were added to the sperm dilution,and the fusion of exosomes and sperm was verified by immunofluorescence.The results showed that the fusion of exosomes and sperm increased with the increase of time and exosome concentration.3、Different concentrations of seminal plasma exosomes(Exo-0,Exo-1,Exo-4,Exo-8,Exo-16)were added to the diluent and stored at 17℃ for 0,2,4 and 6 days to detect the quality parameters of sperm.The results showed that,with the prolongation of preservation time,the DNA integrity,total antioxidant capacity,the expression levels of antioxidant genes(SOD1,CAT,GPX1),anti-apoptotic genes(BCL2),viability and mitochondrial membrane potential,and the expression levels of MT-ND1,MT-ND6,PRDX3 and PRDX5 of sperm in each group decreased gradually;The levels of lipid oxidation,expression of apoptotic genes Bax and Caspase3,mitochondrial reactive oxygen species(mt ROS),ADP/ATP ratio and MTCH1 increased gradually;Compared with Exo-0 group,the DNA integrity,total antioxidant capacity,anti-apoptotic capacity and mitochondrial function of sperm cells in(Exo-1,Exo-4,Exo-8,Exo-16)groups were increased,and there was no significant difference between Exo-8 and Exo-16 groups(P < 0.05);The expression of AWN and AQN-1 in sperm of Exo-8 group and cleavage rate after in vitro fertilization were significantly higher than those of Exo-0 group(P < 0.05).4、High throughput sequencing was used to compare the differences of sperm miRNA transcripts stored at 17℃ for the fourth day with Exo-0 and Exo-8.In the Exo-8 group,31 miRNAs were up-regulated and 21 miRNAs were down regulated,q RT-PCR confirmed the sequencing results of down regulation of ssc-miR-6516,up regulation of ssc-miR-128,ssc-miR-296-3p and ssc-let-7a,luciferase reporter gene experiment verified that SETD7 and MTCH1 were target genes of ssc-miR-128 and ssc-miR-296-3p,respectively.5、Non targeted metabolomics(LC-MS / MS)was used to analyze the metabolic changes of sperm cells stored at 17℃ for the fourth day with Exo-0 and Exo-8.There are many metabolites related to “lipid and lipid molecules” in sperm incubated with Exo-0 and Exo-8,KEGG pathway is mainly related to “metabolic pathway”,the content of lysophosphatides in sperm incubated with Exo-8 was lower than that without exosomes,and nearly half of them were lysophosphatidylcholine(Lyso PC).In conclusion,seminal plasma exosomes were successfully extracted in this study,and the use of Exo-8 and Exo-16 concentrations of exosomes can effectively improve semen quality parameters.The function of seminal plasma exosomes during 17℃preservation of boar semen was analyzed by miRNA expression profile and metabolomics.It was found that during the storage at 17℃,exosomes in boar seminal plasma may decreased the production of mt ROS and sperm apoptosis by down regulating SETD7 by ssc–miR–128,down regulating MTCH1(PSAP)by ssc–miR–296–3p and reducing the metabolism of Lyso PC,so as to protect spermatozoa from oxidative stress,prolong storage time and improve the cleavage rate.The completion of this project will provide an important theoretical basis for the study of the function of porcine seminal plasma exosomes in sperm physiology,enhance the understanding of oxidative stress in 17℃ liquid phase preservation of boar semen,prolong the preservation time of semen,improve reproductive efficiency and economic benefits of pig industry. |
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