Expression data from ejaculated spermatozoa of normozoospermic and asthenozoospermic men
ABSTRACT: Analysis of ejaculated spermatozoav from normozoospermic men and asthenozoospermic men. Some of genes were up-regulated or down-regulated in asthenozoospermia, and their abnormal expression were the causes of the impaired sperm motility. Results provide insight into the mechanisms by which asthenozoospermia is controlled. We used microarrays to detail the global programme of gene expression of ejaculated spermatozoa between normozoospermic and asthenozoospermic men and identified distinct classes of genes expressed differentially in two groups. The liquefied semen samples from normozoospermic and asthenozoospermic men were purified by Percoll on a discontinuous density gradients for RNA extraction and hybridization on Affymetrix microarrays. 30 sperm samples of each group were pooled to abtain enough total RNA.
Project description:Analysis of ejaculated spermatozoav from normozoospermic men and asthenozoospermic men. Some of genes were up-regulated or down-regulated in asthenozoospermia, and their abnormal expression were the causes of the impaired sperm motility. Results provide insight into the mechanisms by which asthenozoospermia is controlled. We used microarrays to detail the global programme of gene expression of ejaculated spermatozoa between normozoospermic and asthenozoospermic men and identified distinct classes of genes expressed differentially in two groups. Overall design: The liquefied semen samples from normozoospermic and asthenozoospermic men were purified by Percoll on a discontinuous density gradients for RNA extraction and hybridization on Affymetrix microarrays. 30 sperm samples of each group were pooled to abtain enough total RNA.
Project description:Data about the entire sperm DNA methylome are limited to two sperm donors whereas studies dealing with a greater number of subjects focused only on a few genes or were based on low resolution arrays. This implies that information about what we can consider as a normal sperm DNA methylome and whether it is stable among different normozoospermic individuals is still missing. The definition of the DNA methylation profile of normozoospermic men, the entity of inter-individual variability and the epigenetic characterization of quality-fractioned sperm subpopulations in the same subject (intra-individual variability) are relevant for a better understanding of pathological conditions. We addressed these questions by using the high resolution Infinium 450K methylation array and compared normal sperm DNA methylomes against somatic cells. Our study, based on the largest number of subjects (n = 8) ever considered for such a large number of CpGs (n = 487,517), provided clear evidence for i) a highly conserved DNA methylation profile among normozoospermic subjects; ii) a stable sperm DNA methylation pattern in different quality-fractioned sperm populations of the same individual. The latter finding is particularly relevant if we consider that different quality fractioned sperm subpopulations show differences in their structural features, metabolic and genomic profiles. We demonstrate, for the first time, that DNA methylation in normozoospermic men remains highly uniform regardless the quality of sperm subpopulations. In addition, our analysis provided both confirmatory and novel data concerning the sperm DNA methylome, including its peculiar features in respect to somatic and cancer cells. Our description about a highly polarized sperm DNA methylation profile, the clearly distinct genomic and functional organization of hypo- versus hypermethylated loci as well as the association of histone-enriched hypomethylated loci with embryonic development, which we now extended also to hypomethylated piRNAs-linked genes, provides solid basis for future basic and clinical research. Bisulphite converted DNA from the 26 normal sperm and 2 somatic cell samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip v1.2
Project description:RNA-Seq technique was applied to investigate the effects of two semen collection methods (Pelleted vs Liquefied) and two sperm purification methods (SCLB vs PS) to the integrity of isolated RNAs at different perspectives. The same set of semen samples were applied to investigate the qualitative and quantitative effect of semen collection methods and sperm cell purification methods on sperm transcript profiling.
Project description:The objective of the present study is to investigate if females have the ability to recognise X or Y chromosome bearing spermatozoa and present a different response to different spermatozoa. In order to investigate if females have the ability to recognise X or Y chromosome bearing spermatozoa and present a different response to different spermatozoa. Samples of semen were sorted for chromosomal sex by ﬂow cytometric-sorting. Using laparoscopic insemination one oviduct was inseminated with X spermatozoa and the contralateral oviduct was inseminated with Y spermatozoa.This allowed us to obtain samples from the oviduct in contact with X sperm and Y sperm in the the contrlateral oviduct from the same sow. Oviduct pig samples were collected for RNA isolation and hybridization on Affymetrix microarrays. Four biological replicates were performed (n= 4 sows) and a total of 8 arrays were used for microarrays study (4 arrays for oviduct samples in contact with X spermatozoa and 4 arrays for oviduct samples in contact with Y spermatozoa.
Project description:Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genomewide investigations of transcriptome complexities in major mammalian organs and their underlying cellular sources, transcriptional mechanisms, and functional relevance have been scarce. Here we first show, using extensive RNAseq data, that transcription of both functional and nonfunctional genomic elements is substantially more widespread in the testis than in other organs across representative mammals. By scrutinizing the transcriptomes of all main testicular cell types in the mouse, we then reveal that meiotic spermatocytes and especially postmeiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein coding genes and long noncoding RNA genes but also poorly conserved intergenic sequences, suggesting that much of it is not of immediate functional relevance. Rather, our analyses of genomewide epigenetic data show that this prevalent transcription, which apparently promoted the birth of new genes during evolution, results from a highly permissive chromatin state during and after meiosis that may ultimately facilitate the replacement of histones by protamines during late spermatogenesis. To study the cellular source and mechanisms of high transcriptome complexity in the mammalian testis, we generated strand-specific deep coverage RNASeq data for purified sertoli cells, spermatogonia, spermatocytes, spermatids and spermatozoa as well as for brain, liver and the whole testis from the mouse. We prepared 8 sequencing libraries for the polyadenylated RNA fraction of each sample and sequenced each library in 3 lanes of the Illumina Genome Analyser IIx platform, yielding a total of >60 millions strand-specific reads of 76 base pairs per sample. In addition, we generated ChIP-Seq data for the H3K4me2 modification as well as RRBS data for brain, liver, testis, spermatocytes and spermatids. RNA-seq, ChIP-seq and RRBS data were generated from the same individual or pool of individuals, in the case of purified cells. RNASeq data for purified sertoli cells, spermatogonia, spermatocytes, spermatids and spermatozoa
Project description:In higher eukaryotes, histone methylation is involved in the maintenance of cellular identity during somatic development. During spermatogenesis, most nucleosomes are replaced by protamines. Therefore, it is unclear if histone modifications function in paternal transmission of epigenetic information. Here we show that active H3K4 di-methylation (H3K4me2) and repressive H3K27 tri-methylation (H3K27me3), two modifications important for Trithorax and Polycomb-mediated gene regulation, are present in chromatin of human spermatozoa and show methylation-specific distributions at regulatory regions. H3K4me2-marked promoters control gene functions in spermatogenesis and cellular homeostasis suggesting that this mark reflects germline transcription. In contrast, H3K27me3 marks promoters of key developmental regulators in sperm as in soma. Many H3K27me3-marked genes are never expressed in the male and female germline, and in early “totipotent” embryos, suggesting a function for Polycomb in repressing somatic determinants across generations. Targets of H3K4me2 and H3K27me3 are also modified in mouse spermatozoa, implicating an evolutionary conserved role for histone methylation in chromatin inheritance via the male germline. Chromatin immuno precipitation (ChIP) was performed on sperm samples obtained from 9 normospermic donors. DNA associated with H3K4me2 or H3K27me3 was precipitated using specific antibodies. Input and precipitated DNA were amplified and hybridized to a tiling microarray (NimbleGen Systems Inc.) representing 18029 promoter regions (2200bp upstream to 500bp downstream of transcription start sites) of all RefSeq annotated human genes. For each modification 3 independent ChIP experiments were performed of which one was hybridized in a dye swap configuration.
Project description:H4K12 acetylation mapped by chromatin Chromatin immunoprecipitation (ChIP) in human sperm. ChIP mapping of chromatin modification. Three biological replicates hybridized to each of two chips.
Project description:Scarcely understood defects lead to asthenozoospermia which results in poor fertility outcomes. Incomplete knowledge of these defects hinders the development of new therapies and reliance on interventional therapies, such as in vitro fertilization, increases. Sperm cells, being transcriptionally and translationally silent, necessitate the proteomic approach to study the sperm funnction. We have performed a differential proteomics analysis of human sperm and seminal fluid and identified over 1,700 proteins. We have included 667 proteins in sperm and 430 proteins in seminal plasma dataset for further analysis. Statistical and mathematical analysis combined with pathway analysis and self organizing maps clustering and correlation was performed on the dataset. It was found that sperm proteomic signature combined with statistical analysis as opposed to the seminal plasma proteomic signature can differentiate the normozoospermic versus the asthenozoospermic sperm samples.
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:Using a combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography - electrospray ionization tandem mass spectrometry, we identified 348 proteins in carp spermatozoa, most of which were for the first time identified in fish. Dynein, tubulin, HSP90, HSP70, HSP60, adenosylhomocysteinase, NKEF-B, brain type creatine kinase, mitochondrial ATP synthase, and valosin containing enzyme represent high abundance proteins in carp spermatozoa. These proteins are functionally related to sperm motility and energy production as well as the protection of sperm against oxidative injury and stress. Moreover, carp sperm is equipped with functionally diverse proteins involved in signal transduction, transcription, translation, protein turnover and transport. About 15% of carp sperm proteins identified here were also detected in seminal plasma which may be a result of leakage from spermatozoa into seminal plasma, adsorption of seminal plasma proteins on spermatozoa surface, and expression in both spermatozoa and cells secreting seminal plasma proteins. The availability of a catalogue of carp sperm proteins provides substantial advances for an understanding of sperm function and for future development of molecular diagnostic tests of carp sperm quality, the evaluation of which is currently limited to certain parameters such as sperm count, morphology and motility or viability.