Testosterone-dependent Porcine MiR-26a-5p And Let-7g-5p Act As Signaling Mediators To Regulate Sperm Apoptosis Via Targeting PTEN And PMAIP1

Recent evidence suggests that prepubertal hemicastration-induced testosterone deficiency can dramatically decrease the sperm quality.Micro RNAs(miRNAs)are conserved mediators of post-transcriptional gene regulation in eukaryotes.However,the systemic regulation and function of miRNAs in sperm quality decline induced by prepubertal hemicastration has not,until now,been studied.Here,we sequenced the miRNA transcriptomes of sperm with prepubertal hemicastration by small RNA-sequencing and compared it to Control libraries.The main results of this study are as follows:(1)We carried out hemicastration in prepubertal Yorkshire boars to establish the testosterone deficiency model.All experimental pigs feed to the body maturation stage(ten-month-old)and several phenotypic index were assessed in HC(n = 15)and CT(n = 15)pigs.These results indicated that there were no obvious alteration in the body weight,body length,and the several organ indexs between HC and CT pigs.However,some items associated with reproduction showed significant difference and embodied that prepubertal hemicastration in Yorkshire boars reduced the serum testosterone,increased the testis index and testis size of the remaining testis,which demonstrated that the testosterone deficiency model in Yorkshire boars were established successfully.(2)To systematically investigate the mechanism of the specific decline of sperm quality in hemicastration pigs at miRNA transcriptome level,the sperm cells were isolated from the semen sample from HC and CT pigs(n = 3,respectively),and then sperm total RNA were extracted and sequenced via Illumina Hiseq platform for miRNA profiling.As a whole,we identified a total of 396 known pre-miRNAs encoding 206 mature miRNAs in the six libraries.Furthermore,16differentially-expressed miRNAs were identified in HC versus CT sperm(defined as those exhibiting a Benjamini-corrected P value ? 0.05 and a fold-change > 2 or < 0.5in HC versus CT sperm).Interestingly,all of them were down-regulated.Using this approach,we identified 16 miRNAs in sperm that were differentially expressed between two libraries,interestingly,all of them were down-regulated.(3)Ranking analysis showed that,although 80%(8/10)miRNAs were shared between HC and CT sperm libraries in the top 10 high-expressed position,the unevenly distribution of miRNA expression were presented in the top 10 miRNAs with the highest expression level,which may be of house-keeping role in the biological function of sperm cells.(4)To explore the potential functions of the DE miRNAs in sperm,we conducted target prediction and functional enrichment analysis using DIANA online software.This result revealed that the target genes of DE miRNAs in sperm were mainly involved in the HIF-1 signaling pathway,the cell cycle,and pathways involved in cell apoptosis,including the m TOR,and P53 signaling pathways.Notably,more than half of the DE miRNAs in sperm(12/16,75.00%)were enriched in the P53 signalling pathway.(5)Furthermore,Gain-and loss-of-function assays were performed by transfection of miRNA mimic(or mimic control),or inhibitor(or inhibitor control)in the treated semen samples.The sperm after transfection for 8h were evaluated the phenotypic parameters.As a result,miR-26a-5p and let-7g-5p significantly improved sperm quality,including the augment of VCL,VSL and VAP,and enhanced the sperm motility.(6)Luciferase reporter assay confirmed that PTEN and PMAIP1 are targets of miR-26a-5p and let-7g-5p,respectively.Overexpression and inhibition of miRNA showed that miR-26a-5p and let-7g-5p could mitigate apoptosis by inhibiting the apoptosis-related target genes PTEN and PMAIP1,and then affected the p53 pathway.(7)Next,spearman correlation between sperm and seminal plasma exosomes indicated they were moderately correlated(CT,r = 0.733,HC,r = 0.719).Meanwhile,hierarchical clustering with the Control-exosomes(C-exosomes)and Hemicastration-exosmes(H-exosomes)showed the same as sperm of separation effect.In conclusion,our results indicate that seminal plasma exosomes maybe can deliver their contents including miRNAs to help sperm to live longer for attaching tooocytes eventually be used for reproductive purposes,thus,act as signaling mediators and exert functional regulation in p53 signal pathways.