EpigenomicsVol. 5, No. 1 News & ViewsFree AccessResearch Highlights: Highlights from the latest articles in advances in the understanding of sperm epigeneticsDouglas T CarrellDouglas T CarrellAndrology & IVF Laboratories & Departments of Surgery (Urology), Obstetrics & Gynecology, & Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84108, USA. Search for more papers by this authorEmail the corresponding author at douglas.carrell@hsc.utah.eduPublished Online:15 Feb 2013https://doi.org/10.2217/epi.12.79AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Evaluation of: Paradowska AS, Miller D, Spiess AN et al. Genome wide identification of promoter binding sites for H4K12ac in human sperm and its relevance for early embryonic development. Epigenetics 7(9), 1057–1070 (2012).During spermiogenesis, chromatin is uniquely packaged in a multistep process that is initiated, in part, by hyperacetylation of histones and ultimately results in the replacement of the majority of histones, first in a transient replacement with transition proteins, followed by replacement of the transition proteins with protamines. The protamines facilitate a higher order of chromatin compaction, which is necessary for sperm motility and may also protect the sperm DNA from damage during transport through the female reproductive tract. However, protamines are rapidly replaced with maternal histones following fertilization [1]. Although 5–15% of the sperm genome in fertile men remains bound to histones rather than undergoing protamination, abnormal protamination of sperm, either quantitatively or qualitatively (e.g., the ratio of the two protamines expressed) correlates with diminished fertility [2]. These observations have led to the important question of why any histones are retained and if histones are retained for potential epigenetic regulatory control of the sperm genome postfertilization [3].Two relatively recent studies highlighted the potential role of sperm epigenetic programming. Arpanahi et al. investigated the retention of sperm nucleosomes by using endonuclease digestion and found that, generally, nucleosomes were retained in regions associated with regulatory functions, particularly regions recognized by CTCF [4]. Hammoud et al. employed genome-wide sequencing studies and found that nucleosomes were retained in regions of developmental importance, that bivalent histone enrichment (H3K4me3/H3K27me3) was found in a large percentage of developmental genes and that miRNAs, imprinted genes and important transcript factors were marked with activating histone modifications, leading to the hypothesis that some key developmental genes may retain bivalently marked histones, rather than undergoing protamination, to facilitate important early embryonic transcription [5].In the study highlighted herein, Paradowska et al. have further investigated the potential role of sperm chromatin epigenetic marks on embryogenesis by employing chromatin immunoprecipitation of DNA bound to H3K12ac [6], a potentially important histone modification related to gene activation and previously shown to be found in sperm and retained in the male pronucleus [7]. Microarray promoter analysis confirmed the authors’ previous data that generally, H3K12ac is enriched in the promoter regions of developmentally relevant genes and that the enrichment is highest in a 2 kb range around the transcriptional start site. Gene ontogeny analysis identified several gene promoters associated with H3K12ac that potentially have significant roles in embryogenesis, as well as a subset of genes that appear to be important in spermatogenesis. Comparing the H3K12ac data set with previously published data sets, the authors found some overlap between H3K12ac enrichment and H3K9ac enrichment, but little relationship to H3K4me3 and H3K27me3 enrichment.Owing to the extensive and rapid remodeling of the male chromatin during sperm decondensation and pronucleus development, immunofluorescence studies were undertaken to describe the retention of H3K12ac in the sperm nucleus before and after pronuclei development. It was found that in human sperm H3K12ac was primarily localized in the subacrosomal region. In mice, H3K12ac was more strongly observed in the male pronucleus than the female pronucleus, but the female pronucleus progressively became hyperacetylated. Interestingly, in parthenogenetically activated oocytes, both pronuclei were strongly stained for H3K12ac, which may provide clues to the importance of the availability of some hyperacetylated genes in the early embryo.Perhaps the most interesting aspect of the study is the comparison of the H3K12ac-enriched gene sites with levels of retained mRNAs in sperm. Mature sperm are transcriptionally quiescent but retain some mRNAs. Whether these mRNAs are simply a historical record of spermatogenesis or if there is a role for them in facilitating embryonic development is uncertain [8]. Paradowska et al. found a positive relationship between H3K12ac enrichment at specific gene promoters and an elevated level of mRNA transcript of those genes [6]. They have hypothesized that the two mechanisms may jointly result in “a cascade of events resulting in transition of developmentally important mRNA transcripts to the zygote.” While studies have demonstrated a relationship between mRNA levels and fertility, further studies are necessary to determine if the relationship is causative or simply correlational.Delineating the packaging of specific epigenetic marks within sperm chromatin has demonstrated potential epigenetic mechanisms of sperm influences on early embryogenesis. This study has demonstrated another consistent epigenetic pattern that appears to be retained throughout early pronucleus development, and associated with gene-expression profiles through the blastocyst stage. Interesting future studies will include the characterization of these marks in couples with abnormal embryogenesis and the use of genetic models to test epigenetic modifications.References1 Balhorn R. The protamine family of sperm nuclear proteins. Genome Biol.8(9),227 (2007).Crossref, Medline, Google Scholar2 Nanassy L, Liu L, Griffin J, Carrell DT. The clinical utility of the protamine 1/protamine 2 ratio in sperm. Protein Pept. Lett.18(8),772–777 (2011).Crossref, Medline, CAS, Google Scholar3 Carrell DT. Epigenetics of the male gamete. Fertil. Steril.97(2),267–274 (2012).Crossref, Medline, CAS, Google Scholar4 Arpanahi A, Brinkworth M, Iles D et al. Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences. Genome Res.19(8),1338–1349 (2009).Crossref, Medline, CAS, Google Scholar5 Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR. Distinctive chromatin in human sperm packages genes for embryo development. Nature460(7254),473–478 (2009).Crossref, Medline, CAS, Google Scholar6 Paradowska AS, Miller D, Spiess AN et al. Genome wide identification of promoter binding sites for H4K12ac in human sperm and its relevance for early embryonic development. Epigenetics7(9),1057–1070 (2012).Crossref, Medline, CAS, Google Scholar7 Van Der Heijden GW, Derijck AA, Ramos L, Giele M, Van Der Vlag J, De Boer P. Transmission of modified nucleosomes from the mouse male germline to the zygote and subsequent remodeling of paternal chromatin. Dev. Biol.298(2),458–469 (2006).Crossref, Medline, CAS, Google Scholar8 Hamatani T. Human spermatozoal RNAs. Fertil. Steril.97(2),275–281 (2012).Crossref, Medline, CAS, Google ScholarEvaluation of: Rotondo JC, Bosi S, Bazzan E et al. Methylenetetrahydrofolate reductase gene promoter hypermethylation in semen samples of infertile couples correlates with recurrent spontaneous abortion. Hum. Reprod. 27(12), 3632–3638 (2012).MTHFR is a key factor in the ongoing maintenance of DNA methylation, as well as RNA, proteins and lipids, via its role in helping to maintain an endogenous pool of bioavailable methyl groups [1]. Polymorphisms of the MTHFR gene have been shown to be associated with diminished levels of MTHFR enzyme activity, ultimately resulting in global hypomethylation [2]. MTHFR polymorphisms and hypermethylation of the MTHFR gene promoter have both been shown to be associated with reduced sperm concentrations and diminished fertility [3,4].Recurrent spontaneous abortion (RSA) affects approximately 1% of couples and may be due to a myriad of genetic, anatomic, immunologic and biochemical abnormalities in the female. On the male side, genetic abnormalities, including sperm chromosome aneuploidies and elevated levels of DNA damage in the form of single and double strand breaks, have been shown to be associated with RSA [5]. Recently, several reports have evaluated MTHFR polymorphisms and RSA, and a meta-analysis has shown that some polymorphisms of the MTHFR gene are associated with RSA in non-Caucasians [6].Rotondo et al. have evaluated MTHFR gene hypermethylation using methylation-specific PCR in three populations: RSA, non-RSA infertility patients and fertile controls [7]. They report that hypermethylation is seen much more frequently in the RSA population than in the other two populations and the defect is not dependent on sperm quality. The level of hypermethylation was striking, with the methylation status observed in 71% of RSA men with normal semen quality versus 6% of infertile men with normal semen quality and 0% of fertile controls. This report identifies a new potential avenue of evaluating RSA. While the study did not measure global methylation levels in the sperm, nor did it concomitantly evaluate methylation of other specific genes, the data are strongly suggestive of a possible effect on methylation of genes associated with normal embryonic function. The authors have highlighted the need for further studies in this important area. Key questions include understanding the downstream events resulting from hypermethylation and the upstream causes of hypermethylation. While the very high levels of methylation seen in the RSA patients are intriguing, biological relevance must be investigated further.References1 Trimmer EE. Methylenetetrahydrofolate reductase: biochemical characterization and medical significance. Curr. Pharm. Des. doi:10.2174/1381213231163966128 (2012) (Epub ahead of print).Google Scholar2 Matthews RG. Methylenetetrahydrofolate reductase: a common human polymorphism and its biochemical implications. Chem. Rec.2(1),4–12 (2002).Crossref, Medline, CAS, Google Scholar3 Rubes J, Rybar R, Prinosilova P et al. Genetic polymorphisms influence the susceptibility of men to sperm DNA damage associated with exposure to air pollution. Mutat. Res.683(1–2),9–15 (2010).Crossref, Medline, CAS, Google Scholar4 Wu W, Shen O, Qin Y et al. Idiopathic male infertility is strongly associated with aberrant promoter methylation of methylenetetrahydrofolate reductase (MTHFR). PLoS ONE5(11),e13884 (2010).Crossref, Medline, Google Scholar5 Carrell DT, Wilcox AL, Lowy L et al. Elevated sperm chromosome aneuploidy and apoptosis in patients with unexplained recurrent pregnancy loss. Obstet. Gynecol.101(6),1229–1235 (2003).Crossref, Medline, Google Scholar6 Wu X, Zhao L, Zhu H, He D, Tang W, Luo Y. Association between the MTHFR C677T polymorphism and recurrent pregnancy loss: a meta-analysis. Genet. Test. Mol. Biomarkers16(7),806–811 (2012).Crossref, Medline, CAS, Google Scholar7 Rotondo JC, Bosi S, Bazzan E et al. Methylenetetrahydrofolate reductase gene promoter hypermethylation in semen samples of infertile couples correlates with recurrent spontaneous abortion. Hum. Reprod.27(12),3632–3638 (2012).Crossref, Medline, CAS, Google ScholarEvaluation of: Ankolkar M, Patil A, Warke H et al. Methylation analysis of idiopathic recurrent spontaneous miscarriage cases reveals aberrant imprinting at H19 ICR in normozoospermic individuals. Fertil. Steril. 98(5), 1186–1192 (2012).Nuclear transplantation studies have been instructive in highlighting the necessity of both paternal and maternal chromatin for normal embryogenesis in mammals. A major reason for the necessity of a mixed genotype is the phenomenon of imprinting, in which certain genes are selectively expressed from one parental allele and repressed in the other parental allele through selective imprinting [1]. The IGF2/H19 gene cluster on human chromosome 11 is one such example of an obligatory imprinting in humans. Insulin-like growth factor is expressed from the paternally-derived allele, while H19 is expressed from the maternally-derived allele. Proper regulation of expression of the two genes is essential for normal development. The H19 imprinting control region (ICR) is thought to be primarily regulated by methylation of a specific CTCF binding site that affects downstream regulatory events.It has been demonstrated that couples undergoing treatment for infertility have a low, but significantly elevated risk of having children with syndromes that result from imprinting errors, and that these defects are usually the result of abnormal methylation of imprinted genes derived from the sperm [2]. Subsequent studies have evaluated methylation of numerous genes and found that elevated errors of methylation are seen in various subfertile phenotypes, including oligozoospermia and abnormal chromatin packaging [3]. An attractive hypothesis is that methylation defects in sperm, along with alterations in chromatin packaging, may be associated with abnormal embryogenesis, including failed embryo development, as well as spontaneous abortion [4].Ankolkar et al. have analyzed the H19 ICR in sperm from men with recurrent spontaneous abortion (RSA), as defined by three or more miscarriages [5]. Compared with fertile controls, the sperm from men with RSA had hypomethylation of the H19 ICR, as reported by the percentage of patients with complete H19 methylation, the percentage of clones methylated and the percentage of CpG sites methylated. Although the differences were statistically significant, it is important to note that the actual differences were not large (i.e., 97.05 vs 98.31% of complete ICR methylation). The methylation of CTCF site 6, thought to be the most biologically relevant, was also decreased in the RSA sperm. The authors propose that hypomethylation at this site may lead to CTCF binding that affects other genes, including the silencing of the IGF2 gene, which would likely result in abortion.Spermatogenesis is intriguing in that defects are seldom isolated. Defects of spermatogenesis in infertile men are often temporally and mechanistically very diverse. Therefore, it is important to consider the results of this study broadly, as well as mechanistically. Disregulation of the H19 ICR may reflect broad methylation defects that are cumulatively, or in specific combinations, responsible for pathologies. Whether this report has identified a specific defect responsible for miscarriage can only be ascertained by broader studies. Genetic and environmental studies will also be necessary to ascertain the underlying etiologies.Financial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.References1 Morcos L, Ge B, Koka V et al. Genome-wide assessment of imprinted expression in human cells. Genome Biol.12(3),R25 (2011).Crossref, Medline, CAS, Google Scholar2 Kobayashi H, Hiura H, John RM et al. DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm. Eur. J. Hum. Genet.17(12),1582–1591 (2009).Crossref, Medline, CAS, Google Scholar3 Hammoud SS, Nix DA, Hammoud AO, Gibson M, Cairns BR, Carrell DT. Genome-wide analysis identifies changes in histone retention and epigenetic modifications at developmental and imprinted gene loci in the sperm of infertile men. Hum. Reprod.26(9),2558–2569 (2011).Crossref, Medline, CAS, Google Scholar4 Nanassy L, Carrell DT. Paternal effects on early embryogenesis. J. Exp. Clin. Assist. Reprod.5,2 (2008).Crossref, Medline, Google Scholar5 Ankolkar M, Patil A, Warke H et al. Methylation analysis of idiopathic recurrent spontaneous miscarriage cases reveals aberrant imprinting at H19 ICR in normozoospermic individuals. Fertil. Steril.98(5),1186–1192 (2012).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByGenome wide methylation analysis to uncover genes related to recurrent pregnancy loss8 February 2021 | Genes & Genomics, Vol. 43, No. 4 Vol. 5, No. 1 Follow us on social media for the latest updates Metrics Downloaded 591 times History Published online 15 February 2013 Published in print February 2013 Information© Future Medicine LtdPDF download