Research On The Effects Of Aging On Male/male Reproduction During Childbearing Age And Related Mechanism

Part one:Study of the effect of aging on fertility in male rats during reproductive period and its mechanismObjective:By exploring the effects of aging on fertility in male rats during reproductive period and its mechanisms,we provide a theoretical basis for further improvement of male fertility at advanced ages.Methods:The rats were divided into three age groups:low（6 months old）,medium（9.5 months old）and high（12.5 months old）,with 6 rats in each group.After anesthesia and execution,the rats were weighed and sampled,and their body weight,testicular and epididymal mass were recorded.The sperm parameters of the epididymis were measured by a computer-aided sperm analyzer after 20-fold dilution,and the reactive oxygen species（ROS）and apoptosis of the spermatozoa of the rats were detected by flow cytometry analysis,and the differences in the indicators among the groups were statistically analyzed.Flow cytometric analysis was applied to detect lipid peroxidase,malondialdehyde,divalent iron ion content and reduced/oxidized glutathione in rat testicular tissue cells,and PCR array was applied to detect differentially expressed genes related to iron death in rat testicular tissue;the regulation of miRNAs on genes related to iron death was studied by constructing cloning vectors.Results:The testis and epididymis mass and testis/body weight ratio of the rats in the senior group were significantly lower than those in the junior and middle age groups（P<0.05）.There were no significant difference in sperm concentration,total sperm viability and forward-moving sperm among the three groups（P>0.05）.The results of flow cytometry showed that the ROS fluorescence intensity of epididymal spermatozoa in the three groups tended to decrease with age growth,with the senior group being significantly lower than the lower and middle age groups（P<0.05）;the percentage of apoptotic epididymal spermatozoa increased gradually with age growth,with the senior group being significantly higher than the lower and middle age groups（P<0.05）.The lipid peroxidase content of rat testicular tissue cells tended to increase with age growth,but there were no significant differences（P>0.05）.In addition,no significant differences were found in malondialdehyde,divalent iron ion content and reduced/oxidized glutathione values among the age groups（P>0.05）,which were not statistically significant.The genes related to the iron death signalling pathway that were differentially expressed between the testis tissues of the junior and senior age groups were Nox3,Slc7a11 and Steap3,respectively,and all of them increased in expression with age.Dual luciferase reporter gene assay showed that Hsa-miR-127-3p had a regulatory effect on Steap3.Conclusion:Aging causes a gradual decrease in fertility in male rats during reproductive period and increases the susceptibility of testicular tissue cells to ferroptosis as age growth.miRNAs may play a regulatory role in ferroptosis-related genes for age growth during reproductive period.Part two:Expression profile of miRNAs in spermatozoa of normal fertile men with agingObjective:To detect and analyze the miRNAs expression profiles of spermatozoa of men with normal fertility at different ages,to screen for sperm miRNAs associated with male fertility and early embryonic development and offspring health,to scientifically guide the fertility of senior men,and to provide molecular markers and targets for human fertility regulation.Methods:Normal fertility men were divided into three groups according to age（Group A,n=8,aged 20-30 years;Group B,n=10,aged 31-40 years;Group C,n=9,aged 41-55 years）,and sperm miRNAs profiles were detected by high-throughput sequencing,differentially expressed miRNAs between groups were analysed,target gene prediction was performed for differentially expressed miRNAs,and target genes obtained by prediction GO and KEGG analyses were performed.Besides,sperm from an additional 65 men with normal fertility（22 cases,22 cases and 21 cases from groups A,B and C respectively）were used for qRT-PCR to validate the differentially expressed miRNAs confirmed by sequencing.Results:A total of 2160 miRNAs were detected:1223 were known,937 were newly identified and unnamed,and 191 were expressed in all donors.Seven,five and 17 differentially expressed miRNAs were identified in the comparison of group A with group B,group B with group C and group A with group C,respectively.Twenty-two miRNAs were statistically associated with aging.Combining the results of differentially expressed miRNAs and statistical analysis,a total of 12 miRNAs were screened and identified as age-associated,including hsa-miR-127-3p,mmu-miR-5100_L+2R-1,efu-miR-9226_L-2₁ss22GA,cgr-miR-1260_L+1,hsa-miR-652-3p_R+1.pal-miR-9993 a-3p_L+2R-1,hsa-miR-7977₁ss6AG,hsa-miR-106b-3p_R-1,hsa-miR-186-5p,PC-3p₅9611₁11,hsa-miR-93-3p_R+1 and aecamir-8986a-p5₁ss1GA.9165 target genes for age-associated miRNAs were predicted.GO analysis of the target genes revealed significant enrichment in molecular functions,cellular components and biological processes in protein binding,cell membrane,and cell cycle,respectively;KEGG enrichment analysis had 139 signalling pathways,such as signalling pathways regulating stem cell pluripotency,substance metabolism pathways,and Hippo signalling pathways.Expanded sample size qRT-PCR validation of miR-127-3p,miR-106b-3p and miR-1260 showed the same expression trends as sequencing.Conclusion:This study shows that the expression profile of intra-sperm miRNAs changes with age,suggesting that intra-sperm miRNAs may play an important role in regulating male fertility in the opposite direction,providing a new basis for studying the mechanism of age-related male fertility decline.