- Nevertheless, the process of sperm capacitation remains poorly understood and requires further elucidation. - In the current study, via high throughput sequencing, we identified and explored the differentially expressed microRNAs (miRNAs) and mRNAs involved in boar sperm capacitation.. - Results: We identified a total of 5342 mRNAs and 204 miRNAs that were differentially expressed in fresh and capacitated boar sperms. - From these, 12 miRNAs (8 known and 4 newly identified miRNAs) and their differentially expressed target mRNAs were found to be involved in sperm capacitation-related PI3K-Akt, MAPK, cAMP-PKA and Ca 2. - Our findings provide important insights for the understanding of the RNA profile in boar sperm and future elucidation of the underlying molecular mechanism relevant to mammalian sperm capacitation.. - Multiple physiological and biochemical changes are in- volved in sperm capacitation [3], including protein tyro- sine phosphorylation [4], membrane cholesterol efflux [5], production of reactive oxygen species (ROS) [6], membrane hyper-polarization [5, 7], as well as increase in intracellular pH [8], Ca 2. - Full list of author information is available at the end of the article. - By and large, the process of sperm capacitation and its underlying molecular mechanisms are poorly under- stood and require further elucidation [42]. - In this study, as the first to utilize next generation sequencing for the study of sperm capacitation,we identified and reported differentially expressed mRNA and miRNA profiles in fresh and capacitated boar sperm. - Deep sequencing in- formation was obtained to explore the interaction of miRNA and mRNA and to further understand the underlying mechanism of sperm capacitation.. - In total, Mirdeep2 detected 1092 unique miRNAs in fresh and capacitated sperm. - In brief, we identified 5342 differentially expressed mRNAs. - Results of clustered differentially expressed genes are shown in Fig. - Moreover, 5342 differentially expressed genes (DEGs) were analyzed by KEGG. - These mRNAs were enriched in 283 pathways, and each enriched pathway contained numbers of differentially expressed mRNAs ranging from 1 to 115 (Fig. - From these, 41 mRNAs were enriched in Wnt, MAPK, PI3K-Akt signaling pathways, and energy metabolism and sperm capacitation-related pathways such as Ca 2+ and cAMP signaling pathways (Fig. - We identified a total of 204 differentially expressed miRNAs (DEM) between FS and CS. - Hierarchical cluster analysis of differentially expressed miRNAs in fresh and capaci- tated sperm was shown in Fig. - Protein internetwork of differentially expressed genes Based on the protein interaction network, we observed a distinct interactive relationship between the differentially expressed genes. - The nodes, degrees, aggregation coeffi- cients and edges in the interaction networks reflect the strength of the interactions between differentially expressed genes. - Based on these parameters, we specu- lated that differentially expressed genes, such as MAPK1, PGK1, PPM1B, and PGAM1, may play an important role in the regulation of fresh and capacitated boar sperm (Fig. - Similarly, these pre- dicted target mRNAs are annotated and associated with energy metabolism and sperm capacitation signaling pathways, including phosphatidylinositol-signaling sys- tem, glycolysis, MAPK, calcium, and PI3K-Akt signaling pathway (Fig. - The identified miRNAs and mRNAs ( n = 8 each) were ran- domly selected for verifying their expression level in fresh and capacitated sperm via qRT-PCR. - The commonly shared characteristics of miRNAs and mRNAs in mammals indicate their import- ant roles in regulation, control and guidance of sperm Table 1 Sperm quality parameters of fresh and capacitated boar sperm. - Table 2 Overview of transcriptome and small RNA sequencing in fresh and capacitated boar sperm. - Q30, the percentage of the base that the quality value of clean data is greater than or equal to 30.Mapped reads (single-end), the number of bases for compared to the reference genome’ clean reads and the percentage in clean reads.. - Building on extensive scruitiny of scientific lit- erature, our study reported, for the very first time, the comprehensive and systemic investigation of the miRNA and mRNA profiles in fresh and capacitated boar sperm using high throughput sequencing. - In current investiga- tion, we identified a total of 204 DE miRNAs and 5342 DE mRNAs between fresh and capacitated boar sperm.. - Generally, Ca 2+ and HCO3 − are considered as two important biological ele- ments required for sperm capacitation and are be- lieved to promote protein tyrosine phosphorylation.. - In this study, heparin induces boar sperm capacita- tion and affects the expression of miRNAs and their target mRNAs. - 1 Volcano plot of differentially expressed miRNAs and mRNAs between fresh and capacitated boar sperm. - The red and green dots represent up-regulated and down-regulated differentially expressed genes, respectively. - Blue or black dots represent genes that were not differentially expressed. - 2 Hierarchical cluster analysis of significantly differentially expressed miRNAs and mRNA in fresh and capacitated boar sperm. - In the present study, we analyzed the KEGG pathway of DE miRNAs and their target mRNAs. - The results showed that these DE miRNAs and their target mRNAs were mainly enriched in the PI3K-Akt, MAPK, cAMP-PKA, and calcium signaling pathways, which are thought to be important for protein tyrosine phosphoryl- ation and sperm capacitation. - processes can mimic sperm capacitation and acrosome reaction [21]. - 3 KEGG annotation of differentially expressed target genes of miRNAs and mRNAs. - The vertical axis indicates the name of metabolic pathway of KEGG, and horizontal axis represents the number of genes annotated to the pathway, accounting for the proportion of the total number of genes. - 4 Enrichment analysis of KEGG pathway in differentially expressed target genes of miRNAs and mRNAs. - AKAP3 , as a target of miR-1285 , can combine with PKA and PDE4A to function as skeletal protein in sperm and regulates the concentration of local cAMP and sperm capacitation [45]. - Furthermore, some newly identified miRNAs and their targets may be associated with sperm capacitation. - is a calcium binding tyrosine phosphorylated chemical fiber sheath protein involved in sperm capacitation [28]. - promotes the maturation and tyrosine phosphorylation of acrosinthat are closely related to sperm capacitation [53]. - One previ- ous study has also demonstrated that the bovine sperm capacitation process requires AKAP3 -degrada- tion. - In our investigation, we found that a number of mRNAs re- lated to sperm capacitation in other species were also differentially expressed in boar sperm. - 5 Protein interaction network analysis for the differentially expressed genes between fresh and capacitated sperm. - 6 The pie chart of annotated target genes associated with energy metabolism and sperm capacitation signaling pathways. - 7 qRT-PCR validation of differentially expressed miRNAs and mRNAs. - (a) and (c): qRT-PCR results of eight differentially expressed mRNAs and miRNAs between fresh and capacitated boar sperm. - (b) and (d): The small RNA and transcriptome sequencing results of eight differentially expressed mRNAs and miRNAs between fresh and capacitated boar sperm. - miR PDE4A – cAMP-PKA Regulating the concentration of local cAMP and sperm capacitation [45]. - miR-133a-3p − 26.7016 PRDX Preventing oxidative stress during human sperm capacitation [73]. - conservative PSPI Participating in the regulation of reproductive immunity, and the process of fertilization. - unconservative CABYR Calcium binding tyrosine phosphoric acid fiber vitamin sheathing protein, participates in sperm capacitation [28]. - unconservative ACRBP Promotion of acrosin maturation and sperm capacitation [53]. - We also generated valuable data on novel boar miRNAs and genomic clusters expressed in fresh and ca- pacitated boar sperm. - Our findings, along with existing evidences, support the notion that differentially expressed miRNAs and their target mRNAs in fresh and capacitated boar sperm play considerable role in the regulation of sperm apoptosis, mitochondrial membrane potential and spermatogenesis alteration. - These discov- eries indicate a functional redundancy of these genes in controlling sperm capacitation and thereby, fertility. - Our findings provide important insights for the understand- ing of the RNA profile in boar sperm and future elucida- tion of the underlying molecular mechanism relevant to mammalian sperm capacitation.. - Sperm collection and induction of sperm capacitation Fresh ejaculates were harvested from 11 sexually mature Landrace boars using manual collection method as pre- viously described [55]. - The samples were incubated for 10 min on ice for lysis of the somatic cells. - 1 were assigned as differentially expressed.. - For prediction of the potential targets of differentially expressed miRNAs, miRnada [66] and RNAhybrid [67]. - GO enrichment analysis was performed on target genes of miRNAs and differentially expressed mRNA usingthe GOseqR package [68]. - In addition, the differentially expressed protein coding genes were also analyzed using GO. - Statistical analyses of the expression levels of the miRNA and mRNA were performed using unpaired t test (SPSS software version 18.0, IBM). - Differential expression of mRNAs between fresh and capacitated boar sperm. - Differential expression of miRNAs between fresh and capacitated boar sperm.. - 69 specifically expressed mRNAs in capacitated boar sperm. - 4554 specifically expressed mRNAs in fresh boar sperm. - 434 co-expressed mRNAs in fresh and capacitated boar sperm. - DEGs: Differentially expressed genes. - DEM: Differentially expressed miRNAs;. - PTP: Protein tyrosine phosphorylation. - The authors thank Chongzhou pig farm of the Sichuan Giastar Group for generously allowing semen collection.. - This work was supported partly by a grant from the National Natural Science Foundation of China (No.31570533).The funding bodies do not have any role in the design of the study, the collection, analysis and interpretation of data and the authoring of the manuscript.. - Part of the data generated or analyzed during this study are included in this published article and additional files.. - CJZ granted, concept, designed the experiment and revised, given final approval version of the manuscript to be published. - All procedures for boar semen collection from Chongzhou pig farm of the Sichuan Giastar Group were approved by the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, revised in June 2004) and the Institutional Animal Care and Use Committee in the College of Animal Science and Technology, Sichuan Agricultural University, Sichuan, China, under permit No. - Chongzhou pig farm of the Sichuan Giastar Group also understood and granted permission for Prof. - The capacitation of the mammalian sperm. - Sodium and epithelial sodium channels participate in the regulation of the capacitation-associated hyperpolarization in mouse sperm. - Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation. - Effect of progesterone on bovine sperm capacitation and acrosome reaction. - A seminal vesicle autoantigen of mouse is able to suppress sperm capacitation-related events stimulated by serum albumin. - Ca(2+)-regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis. - Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation. - Kinases, phosphatases and proteases during sperm capacitation. - Role and regulation of PI3K in sperm capacitation and the acrosome reaction. - Identification of the major tyrosine phosphorylated protein of capacitated hamster spermatozoa as a homologue of mammalian sperm a kinase anchoring protein. - Presence of RNA in the sperm nucleus. - Signalling pathways involved in sperm capacitation. - Expression and tyrosine phosphorylation of sp32 regulate the activation of the boar proacrosin/acrosin system. - Initial Comparison of CTC Staining among Different Porcine Sperm Capacitation Impacts. - Effects of Heparin on in vitro Sperm Capacitation in Liaoning Cashmere Goats. - AKAP3 degradation in sperm capacitation is regulated by its tyrosine phosphorylation[J]. - Peroxiredoxins prevent oxidative stress during human sperm capacitation[J]. - Characterization of a novel role for the dynamin mechanoenzymes in the regulation of human sperm acrosomal exocytosis[J]. - Clusterin in the mouse epididymis: possible roles in sperm maturation and capacitation[J]
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