Project description:BACKGROUND: In previous studies using candidate gene approaches, low sperm count (oligospermia) has been associated with altered sperm mRNA content and DNA methylation in both imprinted and non-imprinted genes. We performed a genome-wide analysis of sperm DNA methylation and mRNA content to test for associations with sperm function. METHODS AND RESULTS: Sperm DNA and mRNA were isolated from 21 men with a range of semen parameters presenting to a tertiary male reproductive health clinic. DNA methylation was measured with the Illumina Infinium array at 27,000 CpG loci. Unsupervised clustering of methylation data differentiated the 21 sperm samples by their motility values. Recursively partitioned mixture modeling (RPMM) of methylation data resulted in four distinct methylation profiles that were significantly associated with sperm motility (P=0.01). Linear models of microarray analysis (LIMMA) was performed based on motility and identified 9,189 CpG loci with significantly altered methylation (Q<0.05) in the low motility samples, with many loci located in genes associated with subfertility and epigenetic regulation. In the low motility samples, the majority of disrupted CpG loci (80%) were hypomethylated. Of the aberrantly methylated CpGs, 194 were associated with imprinted genes almost equally distributed into hypermethylated (predominantly paternally expressed) and hypomethylated (predominantly maternally expressed) groups. Sperm mRNA was measured with the Human Gene 1.0 ST Affymetrix GeneChip Array. LIMMA analysis based on motility identified 20 candidate transcripts as differentially expressed in low motility sperm, including HDAC1 (NCBI 3065), SIRT3 (NCBI 23410), and DNMT3A (NCBI 1788). Altered expression of these epigenetic regulatory genes was associated with RPMM DNA methylation class. CONCLUSIONS: Using integrative genome-wide approaches to study epigenetic and gene expression patterns in human sperm we identified CpG methylation profiles and mRNA alterations associated with low sperm motility, and that low motility sperm may have aberrant genome-wide hypomethylation due to excess HDAC1 activity. See summary above
Project description:Nitrogen cavitation of bovine sperm to isolate plasma membrane. FASP digestion using trypsin. Data acquired using 5 workflows: (1) Unfractionated on a 5600 TripleTOF; (2) SCX-ESI-LC-MS/MS on a QSTAR Elite; (3) 1D Gel-LC-ESI-LC-MS/MS on a QSTAR Elite; (4) SCX-LC-MALDI-MS/MS on a UltrafleXtreme; (5) 1D Gel-LC-MALDI-MS/MS on a UltrafleXtreme
Project description:Small noncoding RNAs (sncRNA) are becoming recognized for their participation in a diverse range of cellular functions. In this study, their global characterization in human spermatozoa from donors with proven fertility was undertaken. Reads were analyzed in two classes, those mapping to unique locations and those that could be aligned to up to 10 genomic locations. All libraries showed comparable distribution of reads between intergenic, intronic and exonic genomic regions. Analysis of the sequences revealed the presence of multiple small RNA classes. The miRNAs retained in spermatozoa were found to be associated with promoter regions, suggestive of a role at the transcriptional level. Piwi-interacting sncRNAs as well as repeat-associated small RNAs were identified for the first time in mature spermatozoa from a mammalian species. Human spermatozoa retain a multifaceted population of small non-coding RNAs. Their presence is consistent with the view that they may function to stabilize the genome as part of the confrontation and consolidation of the genomes at fertilization. Examination of sperm samples from 3 individuals, sequenced individually
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:The male gamete is not completely mature after ejaculation and requires further events in the female genital tract to acquire fertilizing ability, including the processes of capacitation and acrosome reaction. In order to shed light on dynamic protein changes experienced by the sperm cell in preparation for fertilization, a comprehensive quantitative proteomic profiling based on isotopic peptide labeling and liquid chromatography followed by tandem mass spectrometry was performed on spermatozoa from three donors of proven fertility under three sequential conditions: purification with density gradient centrifugation, incubation with capacitation medium, and induction of acrosome reaction by the exposure to the calcium ionophore A23187. After applying strict selection criteria for peptide quantification and for statistical analyses, 36 proteins with significant changes in their relative abundance within sperm protein extracts were detected. Moreover, the presence of peptide residues potentially harboring sites for post-translational modification was revealed, suggesting that protein modification may be an important mechanism in sperm maturation. In this regard, increased levels of proteins mainly involved in motility and signaling, both regulated by protein modifiers, were detected in sperm lysates following incubation with capacitation medium. In contrast, less abundant proteins in acrosome-reacted cell lysates did not contain potentially modifiable residues, suggesting the possibility that all those proteins might be relocated or released during the process. Protein-protein interaction analysis revealed a subset of proteins potentially involved in sperm maturation, including ERLIN2, GGH, TMED 10, and ATP synthases. These results contribute to the current knowledge of the molecular basis of human fertilization and propose new candidates to be validated as modulators of male fertility.
Project description:Alterations in the presence of sperm RNAs have been identified using microarrays in teratozoospermic (abnormal morphology) or other types of infertile patients. However, so far no studies had been reported on the sperm RNA content using microarrays in asthenozoospermic patients (low motility). We started the present project to with the goal to characterize the RNA expression in asthenozoospermic infertile patients as compared to normozoospermic fertile controls. We selected four normal fertile donors and four severe asthenozoospermic infertile patients. Equal amounts of RNA were extracted from the sperm samples, subjected to different quality controls and hybridized to the Affymetrix U133 Plus version 2 arrays.
Project description:A greater understanding of the proteins involved in reproduction can benefit animal production. New advances in proteomics are having a major impact on our understanding of how spermatozoa acquire their capacity for fertilization . Sperm proteomics aims at the identification of the proteins that compose the sperm cell and the study of their function . The sperm cell is one of the most highly differentiated cells and is composed of a head with a highly compacted chromatin structure and a large flagellum with midpiece that contains the required machinery for movement and therefore to deliver the paternal genetic and epigenetic content to the oocyte . By being so highly differentiated, spermatozoa are advantageous cells to study proteomics of specific compartments such as the membrane, which basically is the area of major importance for its role in interacting with the surroundings and the oocyte . The fusion of a sperm and an oocyte is a sophisticated process that must be preceded by suitable changes in the sperm's membrane composition . Recent studies of spermatozoa from the proteomic point of view have allowed the identification of different proteins in spermatozoa that are responsible for the regulation of normal/defective sperm functions . While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics . Using this method, detailed proteomic data are now available for human , macaque [9,10], mouse , rat , bull [13-15], stallion , fruit fly , Caenorhabditis elegans , carp , rainbow trout , mussel , ram , honeybee  and rooster  sperm membrane proteins. Rabbit (Oryctolagus cuniculus) is an important mammalian species worldwide, being at the same time of commercial interest and a research model animal. European rabbit meat production is approximately 500 thousand tons, corresponding to a 30% share of world production . Besides, rabbits account for the seventh highest number of animals slaughtered per year in the European Union-27, with 347,603 × 1000 head in 2014 . In a previous work, we identified and quantified rabbit seminal plasma proteins between two different genotypes , concluding the clear effect of genotype in the abundance of certain seminal plasma proteins. However, it is unknown at present whether these differences also exist at sperm proteome level. Therefore, the aim of the present study was to characterise rabbit sperm membrane proteins through NanoLC-MS/MS analysis focusing on the influence of the genetic origin.
Project description:Herein, we constructed a human lectin microarray with 60 human lectin and lectin-like proteins. All of the lectins and lectin-like proteins were purified from yeast, and most showed binding to human glycans. To demonstrate the applicability of the human lectin microarray, human sperm were probed on the microarray and strong bindings were observed for several lectins, including galectin-1, 7, 8, GalNAc-T6, and ERGIC-53 (LMAN1). These bindings were validated by flow cytometry and fluorescence immunostaining. Further, mass spectrometry analysis showed that galectin-1 binds several membrane proteins including HSP90. Finally, functional assays showed that binding of galectin-8 could significantly enhance the acrosome reaction within human sperms. To our knowledge, this is the first construction of a human lectin microarray, and we anticipate it will find wide use for a range of human or mammalian studies, alone or in combination with plant lectin microarrays.
Project description:Artificial insemination in small ruminants is most commonly performed using fresh semen due to the low fertility rates typically achieved with frozen spermatozoa. Usually, when developing and applying assisted reproductive technologies, sheep and goats are often lumped together as one specie. In order to optimize sperm cryopreservation protocols in sheep and goat, differences in sperm proteomics between ram and buck are necessary to detect, which may contribute to differences in sperm function and fertility.