Search

Recently, more evidences of epigenetic impact on the male fertility, particularly on sperm DNA methylation have been reported. Data related to this issue in livestock males is still limited. The present study analyzed the DNA methylation status of the important gene for spermatogenesis, SIRT1, in ram sperm and its correspondence with semen quality and fertilizing ability. The ejaculates of 10 rams (5 rams - 1.5 years old, and 5 rams - 4 years old) from Synthetic Population Bulgarian Milk breed were evaluated and used for the artificial insemination of 174 ewes in breeding season. Two semen samples from each animal were used for DNA extraction followed by bisulfite conversion. The DNA methylation status of SIRT1 was detected through quantitative methylation-specific PCR using two sets of primers designed specifically for bisulfite-converted DNA sequences to attach methylated and unmethylated sites. On the base of age and conception rate the rams were divided in different groups. Data of semen quality, DNA methylation status of SIRT1 and reproductive performances of each group were statistically processed. Results showed a high average value of DNA methylation of SIRT1 in ram sperm (78.5±23.9%) and wide individual variability among investigated animals, with a coefficient of variation of 34.4%. The 1.5 years old animals tended to have a higher level of SIRT1 methylation than 4 years old animals. The rams in group with high fertilizing ability had significantly higher DNA methylation of SIRT1 in sperm than those with low fertilizing ability. In conclusion, results of this study provided evidence that the alteration of sperm SIRT1 methylation is associated with fertility performances of the rams and, probably, with their age.

In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age‐related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S rDNA, and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human α‐satellite and interspersed LINE1 repeats, marmoset α‐satellite, bovine alpha‐ and testis satellite I, mouse minor and major satellite, and LINE1‐T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also depend on the region and sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (>50%) methylated alleles, which are thought to be epigenetically silenced, increase with donor's age. All analyzed CpGs in the sperm rDNA transcription unit show comparable age‐related methylation changes. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo., Sperm methylation of the rDNA transcription unit increases with donor's age in mammals with widely different life spans. Scatter plots show positive correlations between donor's age (x‐axis in percentage of life span) and mean methylation (y‐axis in %) of 18S rDNA in 80 mouse (purple dots), 36 bull (yellow), 16 marmoset (red), and 295 human (green) sperm samples. An orthologous region covering 8 CpGs was analyzed by bisulfite pyrosequencing. The black regression line applies to all 427 analyzed samples.

BackgroundDNA methylation has been shown to be involved in many biological processes, including X chromosome inactivation in females, paternal genomic imprinting, and others.ResultsBased on the correlation patterns of methylation levels of neighboring CpG sites among 28 sperm whole genome bisulfite sequencing (WGBS) data (486 × coverage), we obtained 31,272 methylation haplotype blocks (MHBs). Among them, we defined conserved methylated regions (CMRs), variably methylated regions (VMRs) and highly variably methylated regions (HVMRs) among individuals, and showed that HVMRs might play roles in transcriptional regulation and function in complex traits variation and adaptive evolution by integrating evidence from traditional and molecular quantitative trait loci (QTL), and selection signatures. Using a weighted correlation network analysis (WGCNA), we also detected a co-regulated module of HVMRs that was significantly associated with reproduction traits, and enriched for glycosyltransferase genes, which play critical roles in spermatogenesis and fertilization. Additionally, we identified 46 VMRs significantly associated with reproduction traits, nine of which were regulated by cis-SNPs, implying the possible intrinsic relationships among genomic variations, DNA methylation, and phenotypes. These significant VMRs were co-localized (± 10 kb) with genes related to sperm motility and reproduction, includingZFP36L1,CRISP2andHGF. We provided further evidence that rs109326022 within a predominant QTL on BTA18 might influence the reproduction traits through regulating the methylation level of nearby genesJOSD2andASPDHin sperm.ConclusionIn summary, our results demonstrated associations of sperm DNA methylation with reproduction traits, highlighting the potential of epigenomic information in genomic improvement programs for cattle.

Summary Considering the widespread use of assisted reproductive techniques (ART), DNA methylation of specific genes involved in spermatogenesis achieves increasingly clinical relevance, representing a possible explanation of increased incidence of syndromes related to genomic imprinting in medically assisted pregnancies. Several trials suggested a relationship between male sub-fertility and sperm DNA methylation, although its weight on seminal parameters alteration is still a matter of debate. To evaluate whether aberrant sperm DNA methylation of imprinted genes is associated with impaired sperm parameters. Meta-analysis of controlled clinical trials evaluating imprinted genes sperm DNA methylation comparing men with idiopathic infertility to fertile controls. Twenty-four studies were included, allowing a meta-analytic evaluation for H19, MEST, SNRPN, and LINE-1. When a high heterogeneity of the results was demonstrated, the random effect model was used. H19 methylation levels resulted significantly lower in 879 infertile compared with 562 fertile men (7.53%, 95% CI: 5.14–9.93%, p

Decreased male fertility is a big concern in both human society and the livestock industry. Sperm DNA methylation is commonly believed to be associated with male fertility. However, due to the lack of accurate male fertility records (i.e., limited mating times), few studies have investigated the comprehensive impacts of sperm DNA methylation on male fertility in mammals. In this study, we generated 10 sperm DNA methylomes and performed a preliminary correlation analysis between signals from sperm DNA methylation and signals from large-scale (n = 27,214) genome-wide association studies (GWAS) of 35 complex traits (including 12 male fertility-related traits). We detected genomic regions, which experienced DNA methylation alterations in sperm and were associated with aging and extreme fertility phenotypes (e.g., sire-conception rate or SCR). In dynamic hypomethylated regions (HMRs) and partially methylated domains (PMDs), we found genes (e.g., HOX gene clusters and microRNAs) that were involved in the embryonic development. We demonstrated that genomic regions, which gained rather than lost methylations during aging, and in animals with low SCR were significantly and selectively enriched for GWAS signals of male fertility traits. Our study discovered 16 genes as the potential candidate markers for male fertility, including SAMD5 and PDE5A. Collectively, this initial effort supported a hypothesis that sperm DNA methylation may contribute to male fertility in cattle and revealed the usefulness of functional annotations in enhancing biological interpretation and genomic prediction for complex traits and diseases.

Aberrant sperm DNA methylation patterns, mainly in imprinted genes, have been associated with male subfertility and oligospermia. Here, we performed a genome-wide methylation analysis in sperm samples representing a wide range of semen parameters. Sperm DNA samples of 38 males attending a fertility centre were analysed with Illumina HumanMethylation27 BeadChips, which quantify methylation of >27 000 CpG sites in cis-regulatory regions of almost 15 000 genes. In an unsupervised analysis of methylation of all analysed sites, the patient samples clustered into a major and a minor group. The major group clustered with samples from normozoospermic healthy volunteers and, thus, may more closely resemble the normal situation. When correlating the clusters with semen and clinical parameters, the sperm counts were significantly different between groups with the minor group exhibiting sperm counts in the low normal range. A linear model identified almost 3000 CpGs with significant methylation differences between groups. Functional analysis revealed a broad gain of methylation in spermatogenesis-related genes and a loss of methylation in inflammation- and immune response-related genes. Quantitative bisulfite pyrosequencing validated differential methylation in three of five significant candidate genes on the array. Collectively, we identified a subgroup of sperm samples for assisted reproduction with sperm counts in the low normal range and broad methylation changes (affecting approximately 10% of analysed CpG sites) in specific pathways, most importantly spermatogenesis-related genes. We propose that epigenetic analysis can supplement traditional semen parameters and has the potential to provide new insights into the aetiology of male subfertility.

STUDY QUESTION: Which are the main determinants, if any, of sperm DNA methylation levels?SUMMARY ANSWER: Geographical region resulted associated with the sperm methylation status assessed on genome-wide repetitive sequences.WHAT IS KNOWN ALREADY: DNA methylation level, assessed on repetitive sequences from peripheral blood lymphocyte, can vary with age, gender, alcohol consumption and white blood cell counts.STUDY DESIGN, SIZE, DURATION: A cross-sectional study. Individual data were collected from 269 young healthy men of proven fertility living in three geographical regions: Inuits from Greenland, Caucasians from Warsaw (Poland) and Kharkiv (Ukraine). Semen samples were collected between May 2002 and February 2004 and aliquots were immediately frozen.PARTICIPANTS/MATERIALS, SETTING, METHODS: We estimated sperm DNA global methylation level (DGML) in two ways. First DNA methylation in repetitive DNA sequences (LINE-1, Satα and Alu) was quantified by PCR pyrosequencing after bisulfite conversion and second by flow cytometry (FCM) using fluorescently labeled monoclonal antibodies anti-5-methylcytosine. We analyzed whether personal characteristics and habits, body mass index, semen quality parameters, sperm chromatin integrity, biomarkers of accessory gland function and the plasma concentration of reproductive hormones were associated with sperm DNA methylation levels in men. Associations were evaluated by analysis of variance and linear regression analyses.MAIN RESULTS AND THE ROLE OF CHANCE: The geographical location emerged as the main determinant when using the methylation level in repetitive sequences. FCM DGML results were not associated with those from repetitive sequence analysis. No other consistent associations between methylation markers and the assessed variables were identified across countries.LIMITATIONS, REASONS FOR CAUTION: The methods used are only surrogates of the actual sperm methylome and the methylation levels at individual specific loci were not explored.WIDER IMPLICATIONS OF THE FINDINGS: Sperm DGML is relatively independent from semen quality parameters and is a new candidate biomarker for epidemiological studies of the impact of environmental contaminants on male fertility.STUDY FUNDING/COMPETING INTERESTS: The study is part of the project CLEAR (Climate change, Environmental contaminants and Reproductive health) supported by the European Commission 7th framework program, contract no: FP7-ENV-2008-1-226217. No competing interest is declared.