The Molecular Mechanism Of Heat Stress Induced Autophagy In The Boar SCs Through MiR-145-5p Target Arachidonic Acid Generation

Heat stress(HS)is an organism-specific response of mammals to cope with the adverse thermal stimulation,when the ambient temperature is higher than the normal body temperature.HS-induced spermatogenesis in mammals not only affects their reproductive capacity,reduces animal welfare and increases the economic cost of farming,but also influences the male infertility.Sertoli cells(SCs)are essential for spermatogenesis,which provide structural and nutritional support for developing germ cells and promote the formation of the Blood-Testis Barrier(BTB)and the differentiation and proliferation of spermatogenic cells.Therefore,the number of SCs not only determines the ability of germ cell development,but also influences the ultimate testicular size in male adulthood.Previous studies demonstrated that HS leads to the increases of lactate production through upregulation of miRNAs,and also induce the autophagy and apoptosis of SCs through activating the oxidative stress.Do HS affect other metabolites(such as fatty acids)of SCs in testis?Is the underlying mechanism related to miRNA-mediated oxidative stress?What are the phenotype changes of the period?In this study,the influences of HS on the metabolism of SCs and spermatogenesis process were studied through metabonomics sequencing,transcript sequencing,and bioinformatics and molecular biology methods at in vitro and in vivo,respectively.In vitro model,SCs were used from piglets around one month old,and the in vivo model used 6 weeks'male mice.In vitro experiments were conducted to investigate the effects of HS on lipid metabolism and cell stress behavior of SCs and the underlying mechanism of the action.The assay in vivo aimed to verify the mechanism of action of the in vitro assay.And to determine the metabolism is available for spermatogenesis.The results of the studies can be concluded as follows:1.Construction of heat stress model and screening of differential metabolites in SCsThe present study aims to reveal the influence of HS on the metabolic state of SCs.The effect of different HS levels on cell viability and the toxicity of SCs treated at various time and temperature were investigated.The results showed that compared with the control(32°C for 30 min),cell viability increased and then decreased with the increasing temperature,and the proliferative activity of cells treated at 34°C and36°C for 30 min increased significantly(P<0.01),then returning to baseline gradually during 38°C?44°C(P<0.05).Incubated of the SCs at 44°C with 5 min intervals,the cell activity first showed a transient increase and then gradually decreased,the toxicity increases in a zigzag and stair-stepping patterns.The cell activity was the lowest and toxicity was the highest at 44°C and 30 min(P<0.05).The early apoptosis rate of cells was about 2-fold higher than that of the control group(P<0.05),while the late apoptosis rate and normal rate have no significant difference between the two groups(P>0.05).Transmission electron microscopy analysis showed that autophagic vesicles were observed in HS group,and the number of mitochondria decreased,the morphology of the mitochondrial ridge was blurred or even atrophied.The above results indicated that autophagy and apoptosis were induced by exposure cells in HS at 44°C for 30 min.The metabolome sequencing of SCs at this temperature was performed using LC-MS/MS for the purpose of screening for differential metabolites.The results showed that 9352 metabolites were identified in positive ion mode,with 199up-regulated and 184 down-regulated;7304 metabolites were identified in negative ion mode,with 117 up-regulated and 143 down-regulated.20 differential metabolites with molecular characteristics(both in positive and negative ion modes)were obtained upon data exclusion and normalization.It was found that the metabolites are involved in the metabolism of carbohydrates,amino acids,lipids,antibacterial antioxidants and hormones.Among the metabolites which related to energy produce under HS.The most significant changes were found for the sugar-based galactose(FC=7.62,P<0.01)during the analysis of different energy metabolic pathways of in heat stressed SCs,followed by glycerol(FC=4.88,P<0.01).However,the fatty acid metabolite arachidonic acid(AA)was significantly upregulated(FC=2.34,P<0.01)and both of its upstream and downstream derivatives were all detected including linoleic acid,prostaglandins,and leukotrienes(P<0.01).This implies that AA might play a key role in heat stress affecting the function changes of SCs.2.Mechanism of heat stress-induced autophagy generation in SCs via arachidonic acid stimulationAA and its major metabolites play important roles in pathophysiological processes including cell proliferation and differentiation,signal activation and inflammation,nutrition supply and energy metabolism.In this study,we aimed to reveal the potential mechanisms by which HS is involved in the autophagic response in SCs through the induction of AA production.The results showed that cell viability was reduced with cell atrophy,decreased uniformity of nucleoplasmic distribution,blurred cell contours,decreased expression of Claudin 5 and Claudin 11,and decreased integrity of cell membranes in AAT and HS group,respectively(P<0.05).In addition,the AAT and HS groups promoted intracellular production of ROS and MDA(P<0.05),which could trigger oxidative stress.Excessive oxidative stress leads to the increase of free radicals and ROS accumulation,inhibits the replication of mitochondrial D-loop(P<0.01),activates the expression of respiratory chain protein Complex???and?(P<0.05),resulting in an electron leak,and promotes the production of lactic acid.The injured mitochondrial membrane promotes Ca²⁺leakage from mitochondrial,and flow into the endoplasmic reticulum(ER),then activates the expression of ER stress protein IRE1 and JNK phosphorylation,causes ER stress.In addition,excessive stress causes structural damage to mitochondria and endoplasmic reticulum,which in turn induced the conversion of the cellular autophagy protein LC3I to LC3 II(P<0.05),reduced the activity of P62(P<0.05),promoted the expression of the lysosomal protein LAMP2(P<0.05),enhanced lysosomal activity and induced autophagic vesicle and autophagic lysosomes formation.Cells were treated with the oxidative stress inhibitor NAC,the endoplasmic reticulum stress inhibitor 4-PBA and the autophagy inhibitor Bafilomycin A1 and the arachidonic acid inhibitor Diethylcarbamazine citrate,respectively.It was found that they could alleviate HS-induced oxidative stress and endoplasmic reticulum stress and mitochondrial damage.Additionally,the inhibitors also reduced ROS,MDA and Ca²⁺production(P<0.05),and reversed cellular autophagic behavior(P<0.05).The above results suggest that SCs induced organelle damage and activate intracellular autophagy pathway by enhancing AA metabolism under HS.3.Heat stress mediates autophagy production in SCs through miR-145-5p induced arachidonic acid generationMi RNAs play regulatory roles in cell growth and lipid metabolism.In this study,the upstream target m RNAs and miRNAs for AA production was explored.To study the potential mechanism of miRNA-m RNA crosstalk on regulated autophagy in SCs through mediating AA production.Firstly,RNA-seq and bioinformatics analysis was employed to screening potential miRNAs and m RNAs.The study results demonstrated that a total of 804 differentially altered miRNAs were identified in three biological replicates.Through KEGG pathway enrichment analysis,these miRNAs were found to be enriched in 1)Fatty acid prolongation or catabolic pathway(P<0.05),2)Phosphatidylinositol signaling pathway(P<0.05),3)Cytochrome P450 metabolic pathway(P<0.05).GO functional analysis revealed that they were involved in the regulation of these pathways:1)Transcriptional translation of RNA,DNA,and proteins(P<0.05),2)Mitochondrial,endoplasmic reticulum and Golgi functions(P<0.05),and 3)Cytoskeletal structure and membrane integrity functions(P<0.05),all results indicate that these miRNAs may be involved in intracellular lipid metabolism and affect the processes of cell membrane or organelle damage.The targeting relationship between miR-145-5p and PLA2G4A(PLA2G4A induces the production of AA)was verified through bioinformatics analysis and gene cloning methods and a dual luciferase reporter gene system.The results showed that the fluorescence activity of tool cells(HEK 293T cells)(P<0.05)was significantly reduced by miR-145-5p mimics and psi-CHECK2-WT-PLA2G4A-3'UTR co-transfection group,while miR-145-5p inhibitor and psi-CHECK2-WT-PLA2G4A-3'UTR,on the other hand,enhanced the fluorescence activity of this cell(P<0.05).The mutant psi-CHECK2-MUT-PLA2G4A-3'UTR and mimic/inhibitor co-transfection groups could not influence fluorescence activity.Western Blot assay showed that HS induced down-regulation of miR-145-5p expression(P<0.05)and up-regulation of PLA2G4A expression(P<0.05),their expression variation were correlated negatively.In addition,PLA2G4A expression was significantly inhibited or activated upon transfection with mimic or inhibitor.These results suggest that miR-145-5p is a direct target miRNA of PLA2G4A to generate AA under HS.The effects on the function of SCs were evaluated through overexpression miR-145-5p and knockdown of miR-145-5p,or downregulated the expression of PLA2G4A by si RNA.The results showed that oxidative stress was alleviated by miR-145-5p mimic and PLA2G4A si RNA following by the reduced expression of Complexes I,II,and V,(P<0.05)and decreased the activity of ROS and MDA(P<0.05).In addition,the expression of autophagy-related protein LC3 was decreased(P<0.05),while the expression of P62 was increased(P<0.05),which suppressed the formation of autophagic vesicles and lysosomes and inhibited cellular autophagy.These results indicated that HS decreased AA generation and inhibited SCs autophagy by increasing miR-145-5p expression,and vice versa.4.Effects of heat stress-induced AA metabolism in SCs on male spermatogenic capacityTo investigate the effect of the AA induced autophagy of SCs on spermatogenesis process.We used 6 weeks male mice as a model and incubate them in a smart incubation chamber at 40°C and relative humidity of 60.00%±2.31 for 3 d or endowed mice intraperitoneal injections of AAT,AAY and AAT+NAC for 7?10 d,and then their effects on testicular morphology and male fertility were examined.The morphological results showed that the testes in the AAT and HS groups were filled with partial congestion,and the ratio of testes weight/body weight was higher in the AAT group than that in all other groups(P<0.05).It was observed that the testicular germinal tubules in the AAT and HS groups exhibited severe atrophy,the inner lumen of some of the germinal tubules and the polarity of the streamline cells of the entire germinal tubules were expanded,the basement membrane was incomplete,and the germinal cells are distributed in different levels of spermatogenic epithelium.The expression of SCs marker protein GATA-4 in the germinal epithelium of testicular tissue was detected by immunohistochemistry(IHC).The results showed that the expression of the protein did not follow its regularity upon AAT and HS treatment,which was scattered throughout the germinal tubules with a tendency to shift toward the middle luminal area,in which reflect a decrement in SCs.However,the cell morphology and GATA-4 protein distribution were significantly improved after adding AAY or NAC groups.Additionally,AAT and HS group induced an increased MDA(P<0.05)but decreased T-AOC level(P<0.01),and led to the enhancement of ROS fluorescence activity in male mice testis tissues.AA and HS also elevated the mitochondrial respiratory chain proteins Complex?and?expression,respectively(P<0.05).The variations in the localization and expression of GFP-LC3 in testicular tissues were subsequently evaluated by labeling the autophagic sites with GFP immunofluorescence staining.In the AAT and HS groups,germ cell autophagy levels were stronger than that inside the varicocele,which in turn were stronger than that in the basal lamina,and the fluorescence in the middle of the germinal epithelium varied from a clear punctate distribution to a diffuse distribution,indicating that the formation of autophagosomes was induced with a higher degree of autophagy.On the other hand,Western Blot results showed that the conversion of LC3-I to LC3-II was increased and a degradation of P62 were present in both AAT and HS groups(P<0.05).However,the autophagy level first enhanced and then gradually fell back with the remediation of NAC and AAY.Sperm viability assay showed that the sperm were deeply stained and some body types appeared abnormal although a large number of sperm could be found in the epididymis of the AAT and HS groups,indicating that sperm damage was induced by AA.Reproductive ability assays showed that females mated with males in the AAT group were unable to acquire fertility with the failure of pregnancies,and females mated with the HS group had prolonged prodromal time and low litter size and low offspring first birth weight(P<0.05).All of the results suggested that HS-induced AA production causes reduced reproductive ability in males.In summary,HS enhances the expression of PLA2G4A and the level of free fatty acid AA in SCs by decreasing the expression of miR-145-5p,and leads to the increase of lipid peroxides,activation of oxidative stress,damaged of the mitochondrial function and endoplasmic reticulum stress,which induces autophagy and impedes sperm production.Inhibition of AA production or addition of antioxidant NAC could reverse the spermatogenic decline induced by HS.This study not only identified the molecular etiology of HS-induced reduction in male fertility,but also provided for novel small molecules or metabolites to improve heat tolerance in boars.