ABSTRACT: Tobacco-induced microRNAs and mRNAs profile alterations in human spermatozoa: a preliminary study for further knowledge of toxical spermatogenesis impairment
Project description:Tobacco-induced mRNAs profile alterations in human spermatozoa: a preliminary study for further knowledge of toxical spermatogenesis impairment
Project description:Tobacco-induced microRNAs profile alterations in human spermatozoa: a preliminary study for further knowledge of toxical spermatogenesis impairment
Project description:Tobacco smoking generates deleterious effects on human semen quality but mechanisms by which cigarettes smoking can impact spermatogenesis are poorly understood. Recent works have shown that spermatozoa RNAs can be used to understand mechanisms involved in tobacco induced spermatogenesis impairment. We performed a prospective study of 8 smoker and 8 non-smoker patients in an university hospital. All patients were selected according to an occupational exposure standardized questionnaire and the sperm parameters. We performed gene expression and miRNA microarrays using RNA extracts from spermatozoa of 8 smokers and 8 non-smokers. Quantification of selected miRNA was performed using quantitative RT-PCR. We show that 16 genes were differentially expressed between smokers and non-smokers, of which 5 were upregulated and 11 were down regulated in smokers. 23 microRNAs were differentially expressed, of which 16 were upregulated and 7 were down regulated in smokers. Quantitative RT-PCR confirmed the down regulation in smokers for 3 microRNAs. Moreover in smokers, one of the upregulated genes is a putative target for one down regulated microRNA. This is a preliminary and innovating study on spermatozoa RNA extracts in the field of infertility. This preliminary study shows that large scale approaches are non invasive diagnostic tools that may help elucidate the mechanisms that mediate the response to tobacco smoke exposure on human spermatogenesis. This could be used to design biomarkers of human spermatogenetic damage, and help elucidate the fine regulatory mechanisms that mediate responses to environmental agent exposure during human spermatogenesis.
Project description:Tobacco smoking generates deleterious effects on human semen quality but mechanisms by which cigarettes smoking can impact spermatogenesis are poorly understood. Recent works have shown that spermatozoa RNAs can be used to understand mechanisms involved in tobacco induced spermatogenesis impairment. We performed a prospective study of 8 smoker and 8 non-smoker patients in an university hospital. All patients were selected according to an occupational exposure standardized questionnaire and the sperm parameters. We performed gene expression and miRNA microarrays using RNA extracts from spermatozoa of 8 smokers and 8 non-smokers. Quantification of selected miRNA was performed using quantitative RT-PCR. We show that 16 genes were differentially expressed between smokers and non-smokers, of which 5 were upregulated and 11 were down regulated in smokers. 23 microRNAs were differentially expressed, of which 16 were upregulated and 7 were down regulated in smokers. Quantitative RT-PCR confirmed the down regulation in smokers for 3 microRNAs. Moreover in smokers, one of the upregulated genes is a putative target for one down regulated microRNA. This is a preliminary and innovating study on spermatozoa RNA extracts in the field of infertility. This preliminary study shows that large scale approaches are non invasive diagnostic tools that may help elucidate the mechanisms that mediate the response to tobacco smoke exposure on human spermatogenesis. This could be used to design biomarkers of human spermatogenetic damage, and help elucidate the fine regulatory mechanisms that mediate responses to environmental agent exposure during human spermatogenesis.
Project description:MicroRNAs (miRNAs) are involved in nearly every biological process examined to date. Mounting evidence show that some spermatozoa specific miRNAs play important roles in the regulation of spermatogenesis and germ cells development, but little is known of the exact identity and function of miRNA in sperm cells or their potential involvement in spermatogenesis and germ cells development. Here, we investigated the spermatozoa miRNA profiles using illumina deep sequencing combined with bioinformatic analysis using zebrafish as a model system. Deep sequencing of small RNAs yielded 12 million raw reads from zebrafish spermatozoa. Analysis showed that the noncoding RNA of the spermatozoa included tRNA, rRNA, snRNA, snoRNA and miRNA. By mapping to the zebrafish genome, we identified 400 novel and 204 conserved miRNAs which could be grouped into 104 families, including zebrafish specific families, such as mir-731, mir-724, mir-725, mir-729 and mir-2185. We report the first characterization of the miRNAs profiling in zebrafish spermatozoa. The obtained spermatozoa miRNAs profiling will serve as valuable resources to systematically study spermatogenesis in fish and vertebrate. Examination of small RNA populations in zebrafish spermatozoa
Project description:MicroRNAs (miRNAs) are involved in nearly every biological process examined to date. Mounting evidence show that some spermatozoa specific miRNAs play important roles in the regulation of spermatogenesis and germ cells development, but little is known of the exact identity and function of miRNA in sperm cells or their potential involvement in spermatogenesis and germ cells development. Here, we investigated the spermatozoa miRNA profiles using illumina deep sequencing combined with bioinformatic analysis using zebrafish as a model system. Deep sequencing of small RNAs yielded 12 million raw reads from zebrafish spermatozoa. Analysis showed that the noncoding RNA of the spermatozoa included tRNA, rRNA, snRNA, snoRNA and miRNA. By mapping to the zebrafish genome, we identified 400 novel and 204 conserved miRNAs which could be grouped into 104 families, including zebrafish specific families, such as mir-731, mir-724, mir-725, mir-729 and mir-2185. We report the first characterization of the miRNAs profiling in zebrafish spermatozoa. The obtained spermatozoa miRNAs profiling will serve as valuable resources to systematically study spermatogenesis in fish and vertebrate.
Project description:Increasingly, studies have shown that mature spermatozoa contain many transcripts including mRNAs and miRNAs. However, the expression profile of long noncoding RNAs (lncRNAs) in mammalian sperm has not been systematically investigated. Here, we used highly purified RNA to investigate lncRNA expression profiles in mouse mature sperm by stranded-specific RNA-seq. We identified 20907 known and 4088 novel lncRNAs transcripts, and the existence of intact lncRNAs was confirmed by RT-PCR and fluorescence in situ hybridization on two representative lncRNAs. Compared with testes, 2873 upregulated and 6629 downregulated lncRNAs and 1237 upregulated and 2513 downregulated mRNAs were identified in sperm. Based on the “Cis and Trans” RNA-RNA interaction principle, we found 1793 targeted coding genes of differently expressed lncRNAs. In terms of GO analysis, differentially expressed lncRNAs targeted genes mainly related to nucleic acid metabolic, protein modification, chromatin and histone modification, sperm function, and spermatogenesis. In contrast, differentially expressed transcripts of mRNAs were highly enriched for nucleic acid metabolic, spermatogenesis, sperm function, and chromatin organization. Furthermore, KEGG pathway analysis showed that the differentially expressed lncRNAs were involved in mRNA surveillance and AMPK signaling pathways. Besides these two pathways, RNA transport splicesome was another pathway incorporated with the differentially expressed mRNA. In summary, this study provides a preliminary database valuable for identifying lncRNAs critical in the late stage of spermatogenesis or important for sperm function regulation.
Project description:Malnad Gidda is one among the 43 registered cattle breeds of India with unique traits and spread over Western Ghats and coastal regions of Karnataka state in India. Selection of highly elite fertile bulls for the breeding purpose is a critical control point in animal breeding programmes. Therefore, to characterize the semen proteome and to understand the semen biology of this breed, a comprehensive proteomic analysis of spermatozoa and seminal plasma has been carried out by employing SCX and bRPLC fractionation strategies in a mass-spectrometry platform. The semen samples from three Malnad Gidda bulls maintained at Southern Regional Station of ICAR-NDRI under standard managemental conditions were used in the study. The proteomic characterisation of semen from Malnad Gidda breed resulted in the identification of 5, 84,520 PSMs, from 24,467 peptides from 2,815 proteins in spermatozoa and identification of 2, 77,583 PSMs from 12,047 peptides, which resulted in 1,974 proteins from seminal plasma. Out of 2,815 proteins in spermatozoa and 1,974 proteins from seminal plasma, 969 proteins were common to both seminal plasma and spermatozoa. The biological processes and cellular localization of spermatozoa proteins were studied using DAVID tool and were further enriched the identical GO terms using REVIGO online tool. The functional enrichment analysis of identified proteins indicated their roles in the biological processes like sperm motility, spermatid development, spermatogenesis, and so on. GO studies showed the commonalities and differences in the molecular functions of the proteins exclusively identified in spermatozoa, seminal plasma and common proteins. This is the first proteomic investigation conducted on the semen samples of an Indian indigenous breed; therefore, we believe that our preliminary data should significantly advance our understanding of semen proteome of Indian cattle.
Project description:In higher eukaryotes, histone methylation is involved in the maintenance of cellular identity during somatic development. During spermatogenesis, Since most nucleosomes are replaced by protamines during spermatogenesis . Iit is therefore unclear whether if histone modifications function in paternal transmission of epigenetic information. Here we show that H3K4 di-methylation (H3K4me2) and H3K27 tri-methylation (H3K27me3), two modifications important for Trithorax and Polycomb-mediated gene regulation, display methylation-specific distributions at regulatory regions in human spermatozoa. H3K4 dimethylation H3K4me2-marksed promoters of genes relevant control gene functions in spermatogenesis and cellular homeostasis suggesting that this mark reflects germline transcription. In contrast, H3K27 trimethylation (H3K27me3) marks promoters of key developmental regulators in sperm like in somatic cells. Promoters of orthologous genes are similarly modified in mouse spermatozoa. Further, particularly genes with extensive H3K27me3 coverage around transcriptional start sites are never expressed during male and female gametogenesis, nor in pre-implantation embryos. These data are compatible with a function for Polycomb in repressing somatic determinants across generations. Importantly, however, we observe only modest selective retention of nucleosomes at regulatory regions in human sperm suggesting that paternal transmission of H3K27me3-encoded epigenetic information may be subjected to variegation. Identification of nucleosome containing regions in 6 human sperm samples
Project description:<p>There is a clear need to develop biomarkers for Parkinson disease (PD) diagnosis and monitoring disease progression. In this study we evaluated cerebrospinal fluid (CSF) proteins, which are known to be critically involved in PD or identified in our preliminary profiling studies, aptamers, and RNAs as potential PD biomarkers. Access to subjects for this study was via the Pacific Northwest Udall Center (PANUC) and the Alzheimer's Disease Research Center (ADRC) at the University of Washington and Oregon Health and Sciences University (OHSU). Using CSF samples from 30 well-characterized patients with PD and 30 age-, sex-matched healthy controls, we prepared RNA seq libraries and performed deep sequencing of all RNA species, including small and long RNA, mRNAs, noncoding RNAs and differentially spliced transcripts. We then tried several methods for RNAseq data analysis to optimize our analysis pipeline. We identified a total of 3381 transcripts corresponding to 182 long intergenic RNAs (LincRNAs), 11 microRNAs (miRNAs), 2861 protein-coding transcripts, 200 pseudogenes and 127 antisense RNAs; some of them were differentially expressed between PD and control groups. Selected differentially expressed RNAs have been validated in the same set of CSF samples using real-time PCR (RT-PCR). Further validations in independent, larger cohorts of samples are still ongoing. Our results obtained so far suggested that CSF proteins and RNAs could be used as good indexes for PD diagnosis and disease severity/progression. This study is a part of the NIDDS-funded Parkinson's Disease Biomarkers Program (PDBP).</p>