Sperm chromatin packaging and DNA methylation: Relevance to ART
Systems Biology in Reproductive Medicine, 2009
Research Article Identification of Genetic Variation in the 5 0 and 3 0 Non-coding Regions of the Protamine Genes in Patients with Protamine Deregulation
Deregulation of sperm nuclear protamine ratio (P1=P2) has been shown to correlate with male facto... more Deregulation of sperm nuclear protamine ratio (P1=P2) has been shown to correlate with male factor infertility in humans, but the cause of this abnormal protein expression has yet to be identified. Recent studies have shown that there is little genetic variability in the coding regions of either of the protamine gene sequences. However, these studies did not investigate the 5 0 or 3 0 non-coding regions of these genes for mutations that might account for changes in the transcriptional or translational regulation of the protamines. In an effort to determine if genetic variation in these non-coding regions may account for aberrant protamine expression, we have sequenced the 5 0 and 3 0 untranslated regions (UTRs) of both protamine 1 (P1) and protamine 2 (P2) genes in a population of infertile men with protamine deregulation, men presenting for infertility work-up with normal protamine ratios, and a population of unrelated, fertile men from the Utah Genetic Reference Project (UGRP). Th...
Sessile serrated polyps (SSPs) have emerged as important precursors for a large number of sporadi... more Sessile serrated polyps (SSPs) have emerged as important precursors for a large number of sporadic colorectal cancers. They are difficult to detect during colonoscopy due to their flat shape and the excessive amounts of secreted mucin that cover the polyps. The underlying genetic and epigenetic basis for the emergence of SSPs is largely unknown with existing genetic studies confined to a limited number of oncogenes and tumor suppressors. A full characterization of the genetic and epigenetic landscape of SSPs would provide insight into their origin and potentially offer new biomarkers useful for detection of SSPs in stool samples. We used a combination of genome-wide mutation detection, exome sequencing and DNA methylation profiling (via methyl-array and whole-genome bisulfite sequencing) to analyze multiple samples of sessile serrated polyps and compared these to familial adenomatous polyps. Our analysis revealed BRAF-V600E as the sole recurring somatic mutation in SSPs with no addi...
DNA methylation on cytosine in vertebrates such as zebrafish serves to silence gene expression by... more DNA methylation on cytosine in vertebrates such as zebrafish serves to silence gene expression by interfering with the binding of certain transcription factors and through the recruitment of repressive chromatin machinery. Cytosine DNA methylation is chemically stable and heritable through the germline - but also reversible through many modes, making it a useful and dynamic epigenetic modification. Virtually all of the enzymes and factors involved in the deposition, binding, and removal of cytosine methylation are conserved in zebrafish, and therefore the organism an excellent model for understanding the use of DNA methylation in the control of gene regulation and other processes. Here, we discuss the main approaches to quantifying DNA methylation levels genome-wide in zebrafish: one is an established method for revealing regional methylation (methylated DNA immunoprecipitation (MeDIP)), and the other is an emerging method that reveals DNA methylation at base-pair resolution (shotgu...
Deregulation of sperm nuclear protamine ratio (P1=P2) has been shown to correlate with male facto... more Deregulation of sperm nuclear protamine ratio (P1=P2) has been shown to correlate with male factor infertility in humans, but the cause of this abnormal protein expression has yet to be identified. Recent studies have shown that there is little genetic variability in the coding regions of either of the protamine gene sequences. However, these studies did not investigate the 5 0 or 3 0 non-coding regions of these genes for mutations that might account for changes in the transcriptional or translational regulation of the protamines. In an effort to determine if genetic variation in these non-coding regions may account for aberrant protamine expression, we have sequenced the 5 0 and 3 0 untranslated regions (UTRs) of both protamine 1 (P1) and protamine 2 (P2) genes in a population of infertile men with protamine deregulation, men presenting for infertility work-up with normal protamine ratios, and a population of unrelated, fertile men from the Utah Genetic Reference Project (UGRP). This analysis has identified 14 single nucleotide polymorphisms (SNPs), of which 13 were novel SNPs in the UTRs of P1 and P2, and verified the existence of a variable length repeat (VLR), GA n , in the P2 5 0 region. The SNP frequencies and VLR allelic frequencies did not achieve statistical significance between the populations, however, one of the SNPs identified in the 3 0 UTR of protamine 2 was found at a low frequency in the abnormal protamine patients, but was completely absent in men with verified normal protamine ratio and donors of known fertility. In conclusion, a number of SNPs have been reported in the protamine genes and the untranslated regions, however, these gene variants do not appear to be responsible for protamine deficiency. Hence, the underlying cause for aberrant protamine expression may possibly be due to abnormalities in candidate spermatogenic transcriptional=translational regulators, post-translational modifiers, or as-of-yet unidentified factors affecting the testicular environment.
Along with many of the genome-wide transitions in chromatin composition throughout spermatogenesi... more Along with many of the genome-wide transitions in chromatin composition throughout spermatogenesis, epigenetic modifications on histone tails and DNA are continuously modified to ensure stage specific gene expression in the maturing spermatid. Recent findings have suggested that the repertoire of epigenetic modifications in the mature sperm may have a potential role in the developing embryo and alterations in the epigenetic profile have been associated with infertility. These changes include DNA demethylation and the retention of modified histones at important developmental, signaling and micro-RNA genes, which resemble the epigenetic state of an embryonic stem cell. This review assesses the significance of epigenetic changes during spermatogenesis, and provides insight on recent associations made between altered epigenetic profiles in the mature sperm and its relationship to infertility.
During the elongating spermatid stage of spermatogenesis, there is a step-wise replacement of nuc... more During the elongating spermatid stage of spermatogenesis, there is a step-wise replacement of nuclear histones with protamines 1 and 2. In fertile men, the ratio of protamine 1 ⁄ protamine 2 (P1 ⁄ P2) is within the narrow range of 0.8-1.2. Ratios above or below that range are associated with infertility, exhibiting a wide range of defects including decreased sperm counts, morphology, fertilization ability, and embryo implantation capacity. In this review, we highlight studies evaluating potential causes of abnormal protamine expression, including the sequencing of genes relevant to protamine expression in both affected patients and controls. While the variants of the protamine genes themselves do not appear to be responsible for most observed defects, variants of the Contrin gene, a transcription factor and translation repressor, appear to be contributory to some cases of abnormal expression. Additionally, we explore the potential effects of abnormal protamine replacement on the epigenome of human sperm. Ongoing studies are evaluating the role of retained histones and DNA methylation in sperm, which may be affected in sperm with aberrant protamine replacement. This important area of epigenetic research has profound clinical implications.
The sperm chromatin of fertile men retains a small number of nucleosomes that are enriched at dev... more The sperm chromatin of fertile men retains a small number of nucleosomes that are enriched at developmental gene promoters and imprinted gene loci. This unique chromatin packaging at certain gene promoters provides these genomic loci the ability to convey instructive epigenetic information to the zygote, potentially expanding the role and significance of the sperm epigenome in embryogenesis. We hypothesize that changes in chromatin packaging may be associated with poor reproductive outcome. methods: Seven patients with reproductive dysfunction were recruited: three had unexplained poor embryogenesis during IVF and four were diagnosed with male infertility and previously shown to have altered protamination. Genome-wide analysis of the location of histones and histone modifications was analyzed by isolation and purification of DNA bound to histones and protamines. The histone-bound fraction of DNA was analyzed using high-throughput sequencing, both initially and following chromatin immunoprecipitation. The protamine-bound fraction was hybridized to agilent arrays. DNA methylation was examined using bisulfite sequencing. results: Unlike fertile men, five of seven infertile men had non-programmatic (randomly distributed) histone retention genome-wide. Interestingly, in contrast to the total histone pool, the localization of H3 Lysine 4 methylation (H3K4me) or H3 Lysine 27 methylation (H3K27me) was highly similar in the gametes of infertile men compared with fertile men. However, there was a reduction in the amount of H3K4me or H3K27me retained at developmental transcription factors and certain imprinted genes. Finally, the methylation status of candidate developmental promoters and imprinted loci were altered in a subset of the infertile men. conclusions: This initial genome-wide analysis of epigenetic markings in the sperm of infertile men demonstrates differences in composition and epigenetic markings compared with fertile men, especially at certain imprinted and developmental loci. Although no single locus displays a complete change in chromatin packaging or DNA modification, the data suggest that moderate changes throughout the genome exist and may have a cumulative detrimental effect on fecundity.
Main Outcome Measure(s): The DNA methylation patterns were analyzed using bisulfite sequencing. T... more Main Outcome Measure(s): The DNA methylation patterns were analyzed using bisulfite sequencing. The percentage of methylation was compared between fertile and infertile patients displaying abnormal protamination. Result(s): At six of the seven imprinted genes, the overall DNA methylation patterns at their respective differentially methylated regions were significantly altered in both infertile patient populations. When comparing the severity of methylation alterations among infertile patients, the oligozoospermic patients were significantly affected at mesoderm-specific transcript (MEST), whereas abnormal protamine patients were affected at KCNQ1, overlapping transcript 1 (LIT1), and at small nuclear ribonucleoprotein polypeptide N (SNRPN). Patients with male factor infertility had significantly increased methylation alteration at six of seven imprinted loci tested, with differences in significance observed between oligozoospermic and abnormal protamine patients. This could suggest that risk of transmission of epigenetic alterations may be different with diagnoses. However, this study does not provide a causal link for epigenetic inheritance of imprinting diseases, but does show significant association between male factor infertility and alterations in sperm DNA methylation at imprinted loci. (Fertil Steril Ò 2010;94:1728-33. Ó2010 by American Society for Reproductive Medicine.
cytogenetic results to either normal or aneuploid. The donor's demographics, cycle parameters and... more cytogenetic results to either normal or aneuploid. The donor's demographics, cycle parameters and semen parameters were similar for both groups. Aneuploidy was found in 30.0% of abortuses among patients undergoing oocyte donation. No significant increase in fetal aneuploidy rate was noted with increasing paternal age (<40 years ¼ 25.0%, 40-50 years ¼ 38.8%, >50 years ¼ 25.0%). A higher rate of aneuploidy though not statistically significant was seen in the ICSI vs. conventional IVF group (39.1% vs. 22.2% (P 0.20, RR 0.78, 95% CI 0.53-1.15)). We compared the cytogenetic results of the 50 oocyte recipient couples to a group of 23 female patients (mean age 28.7 AE 1.1) who had a missed abortion following IVF using autologous oocytes. There was no statistical difference in sperm characteristics between the egg recipient and IVF patients. There was a significant difference in the male partner age between these two groups (33.7 AE 7.6 vs. 41.5 AE 6.8) (P<0.05). No statistically significant difference in aneuploidy rate was observed between egg recipients and IVF patients younger then 30 years using autologous oocytes (30.0% vs. 26.1% (P 0.66, RR 0.93, 95% CI 0.68, 1.27)). CONCLUSIONS: These data support that paternal age has little effect on aneuploidy rates. The increase in aneuploidy noted with ICSI requires further evaluation.
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