| Testis is the reproductive organ of boars,and the seminiferous tubules are the main site of spermatogenesis.Since the testicles are overhanging outside the body,they are easily affected by environmental temperature changes,especially heat stress caused by high temperature environment,which seriously affects the quality of boar semen.At present,the research of heat stress on boar reproduction mainly focuses on after sexual maturity,but there are few studies on the influence of heat stress on prepubertal boars,and the mechanism is still unclear.The testes of the boars in the prebupertal stage are not fully developed and their ability to regulate temperature is limited,so they are more susceptible to high temperature environment.The antioxidant chlorogenic acid can relieve oxidative stress and reduce tissue oxidative damage.Therefore,this study will explore the alleviating effect of chlorogenic acid on heat stress-induced testicular injury from the perspective of transcriptome,proteome and metabolome by constructing a whole-body heat stress model of prepubertal boars and using multi-omics technology.The aim of this study was to elucidate the omics differences in testis injury caused by heat stress in prepubertal boars,explore the molecular mechanism of heat stress affecting testicular development,and provide data support and new ideas for alleviating the oxidative damage of testis induced by heat stress.The main research contents and results are as follows:1.The effect of heat stress on testes injury of prepubertal boars and the alleviating effect of chlorogenic acid.In this study,diets of prepubertal boars(6-10 weeks of age)were supplemented with 1000 mg/Kg chlorogenic acid at 50%concentration,and it was found that chlorogenic acid could increase the average daily gain and the activity of serum antioxidant indexes such as GSH-Px,SOD,CAT and T-AOC.At the age of 10 weeks,the boars were subjected to short-term heat treatment for 2 hours a day for 7 consecutive days by creating a small unit thermal environment(37-40℃),which resulted in the increase of cortisol content in serum,the decrease of testosterone content,and the damage of the shape of seminiferous tubules in the testis before the first estrous stage.The total antioxidant capacity of testicular tissue,the number of sertoli cells in seminiferous tubules,and the sperm density and motility of boars after sexual maturation were decreased.Compared with the heat stress group,dietary chlorogenic acid significantly increased the T-AOC of testicular tissue,improved the integrity of seminiferous tubules,and increased sperm density after sexual maturation.Heat treatment of sertoli cells in vitro showed that heat stress promoted the increase of intracellular ROS expression,flow cytometry analysis showed that heat stress increased the proportion of sertoli cells apoptosis,and EDU staining showed that the proportion of EDU positive cells decreased.After the addition of chlorogenic acid to the cell medium and heat treatment,the ROS accumulation and cell apoptosis were alleviated,and the number of EDU positive cells was increased.These results indicate that heat stress can damage the morphology of seminiferous tubules in the testis of prepubertal boars,reduce the number of sertoli cells in seminiferous tubules,and reduce sperm density and sperm motility after sexual maturation.Chlorogenic acid can improve the antioxidant function of prepubertal boars,and alleviate the damage of testis heat stress.2.Transcriptome analysis of heat stress and chlorogenic acid on testes of prepubertal boars.The testicular tissues of 3 male pigs were randomly selected in each treatment group.The results of RNA-Seq analysis showed that 1373 DEGs were affected by heat stress compared with the control group.GO analysis showed that DEGs were associated with cyclin-dependent serine/threonine kinase inhibitor activity(GO:0004861),KEGG analysis showed that DEGs were related to apoptosis and tight junctions.A total of 2087 DEGs were affected by chlorogenic acid.GO analysis showed that DEGs were enriched in spermatogenesis and oxidoreductase activity,and KEGG analysis showed that DEGs were significantly enriched in glucagon signaling pathway.Under the condition of heat stress,789 DEGs were affected by chlorogenic acid.GO analysis showed that DEGs were significantly enriched in the antioxidant activity part,and KEGG analysis showed that DEGs were mainly enriched in the glutathione metabolism pathway.The results of in vitro cell experiments showed that heat stress could significantly increase the proportion of cells in G0/G1 and S phases,increase the apoptosis rate,the expression of Caspase 3 and Bcl-2proteins,and significantly promote the expression of glutathione S transferase(GSTO1)in the glutathione metabolism pathway.The activity of glutathione peroxidase(GSH-Px)was significantly inhibited by chlorogenic acid,and the expression of GSTO1 was significantly down-regulated and the activity of GSH-Px was significantly up-regulated after heat stress.These results confirmed that heat stress could affect testicular development by affecting cell cycle progression and cell apoptosis.Chlorogenic acid can regulate the expression of GSTO1 mRNA and the activity of GSH-Px in the glutathione metabolism pathway to play an antioxidant function,and then alleviate the damage of testis tissue caused by heat stress.3.Effects of heat stress and chlorogenic acid on proteome of testes of prepubertal boars.The proteomic sequencing analysis of testicular tissue samples showed that there were 41differentially expressed proteins(DEPs)affected by heat stress,which were mainly enriched in the oxidative phosphorylation pathway.208 DEPs were affected by chlorogenic acid,which were mainly enriched in steroid hormone biosynthesis and glutathione metabolism pathways.Under the condition of heat stress,34 DEPs were affected by chlorogenic acid,and most of them were enriched in metabolic pathways.Through testicular tissue transcriptome and proteome data integration analysis found that under the influence of heat stress,the mRNA and protein expression trends of seven genes were consistent,among which Ezrin(EZR)gene was enriched in the tight junction pathway and its expression was down-regulated,while biliverdin reductase A(BLVRA)gene was enriched in the porphyrin and chlorophyll metabolic pathway and its expression was up-regulated;Under the premise of heat stress,the mRNA and protein expression trends of 3 genes affected by chlorogenic acid were consistent,among which only the expression of BLVRA was affected by chlorogenic acid to change the heat-induced expression level.suggesting that BLVRA plays a role in the alleviation of heat stress by chlorogenic acid.Combined analysis confirmed that heat stress could damage the testicular tissue by affecting the tight junction pathway,and chlorogenic acid could inhibit the testicular tissue damage induced by heat stress by regulating glutathione metabolism and metabolic pathway.4.Effects of heat stress and chlorogenic acid on the metabolome of prepubertal boars’testes.~1H-NMR technology was used to analyze the metabolites of testicular tissues,and 28metabolites including branching-chain amino acid,lactic acid and alanine were identified.15 metabolites including choline,phosphatidylcholine,lactic acid,taurine,glutathione,glutamic acid,valine,glycine,ethanolamine,acetate,tyrosine,isocitrate,glutamine,creatine and alanine were important metabolites affected by heat stress and chlorogenic acid in porcine testis before estrus.The content of lactate and the activity of lactate dehydrogenase(LDH)increased after heat stress,and the expression of LDHA and LDHD mRNA increased after heat stress.Under the condition of heat stress,chlorogenic acid can increase the activity of LDH and decrease the content of intracellular lactic acid.These results indicated that heat stress caused the increase of lactate content and accelerated energy metabolism,and chlorogenic acid increased the resistance of cells to heat stress and maintained the energy stability of the body.In conclusion,the whole body heat stress model of prepubertal boars and the in vitro heat stress model of testis sertoli cells were established in this study,through the analysis of the multiple omics testicular tissue of differentially expressed genes,proteins,and metabolites,indicates that thermal stress mainly make prepubertal boar testicular tissues glutathione peroxidase activity is impaired,cause oxidative stress,furthermore,chlorogenic acid can further induce cell cycle arrest in G0/G1 and S phases and apoptosis,increase lactate secretion,damage cell tight junctions,and affect sperm number and sperm motility after sexual maturation.Chlorogenic acid can inhibit cell apoptosis caused by heat stress and alleviate heat stress injury by maintaining the balance of tissue antioxidant system.This study preliminarily analyzed the mechanism of heat stress-induced oxidative damage in prepubertal boars,and provided a theoretical basis for chlorogenic acid to alleviate heat stress injury. |
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