ABSTRACT
Sperm chromatin packaging is a very complex and highly regulated phenomenon. While most of the sperm chromatin is replaced by protamines, some are retained in nucleosomes. It is recently being recognised that these nucleosomes are intentionally retained and could be contributing to the expression of genes in the very early stages of embryogenesis. Endocrine disruption has been previously shown to affect reproductive outcome and sperm DNA methylation. This study aims to decipher the possibility of changes in nucleosome occupancy in sperm chromatin, induced by tamoxifen (selective oestrogen receptor modulator) and cyproterone acetate (androgen antagonist). We used next-generation sequencing approach (MNase-Seq) to identify changes in the nucleosome landscape of the spermatozoa. We demonstrated that endocrine disruption affects nucleosome occupancy at critical regions of the genome and many of them harbour genes relevant for embryogenesis. This study emphasises that environmental factors could affect embryo development by way of modulating male epigenetic factors.
Subject(s)
Endocrine Disruptors/toxicity , Nucleosomes/drug effects , Spermatozoa/drug effects , Animals , Cyproterone Acetate , Genomic Imprinting , Male , Nucleosomes/metabolism , Rats, Sprague-Dawley , Spermatozoa/metabolism , Tamoxifen , Transcription Initiation SiteABSTRACT
Endocrine control of reproduction is very well known and has been echoed by many research groups. However, recent developments point to the ability of toxic endocrine disrupting chemicals (EDC) to alter epigenetic information of the gametes which gets transferred to the developing embryo and affects the immediate reproductive outcome or even persists transgenerationally. These epigenetic aberrations contribute to the ensuing pathophysiology of reproductive disorders. Investigations of the female in cases of poor reproductive outcome have been the main strategy towards diagnosis. However, despite the male partner contributing half of his genome to the progeny, thorough investigations in the male have been ignored. Environmental pollutants are all pervading and are encountered in our day-to-day life. Many of these pollutants have potential to disrupt the endocrine system. Here, we discuss how the male gametes (spermatozoa) are susceptible to a myriad of epigenetic insults inflicted by exposure to endocrine disruptors and how important is the contribution of the epigenetic marks of the spermatozoa in healthy reproduction. We advocate that sperm epigenetics should be considered as a significant contributor to reproductive health and should be researched further and be subsequently included in routine diagnostic workup in cases of poor reproductive outcome.
Subject(s)
Endocrine Disruptors/adverse effects , Environmental Pollutants/adverse effects , Epigenesis, Genetic/drug effects , Reproduction/drug effects , Spermatozoa/drug effects , Animals , Female , Humans , Infertility, Male/chemically induced , Infertility, Male/genetics , Infertility, Male/pathology , Male , Spermatozoa/metabolism , Spermatozoa/pathologyABSTRACT
Genomic imprinting is an epigenetic phenomenon known to regulate fetal growth and development. Studies from our laboratory have demonstrated that treatment of adult male rats with tamoxifen increased postimplantation loss around mid gestation. Further studies demonstrated the aberrant expression of transcripts of several imprinted genes in the resorbing embryos at days 11 and 13 of gestation including IGF2. In addition, decreased methylation at the Igf2-H19 imprint control region was observed in spermatozoa and in resorbing embryos sired by tamoxifen-treated males. In this study, methylation analysis of the imprinted genes, which were found to be differentially expressed, was done using EpiTYPER in the spermatozoa of tamoxifen-treated rats and in postimplantation embryos sired by tamoxifen-treated rats. Differentially methylated regions (DMRs) for most imprinted genes have not been identified in the rats. Hence, initial experiments were performed to identify the putative DMRs in the genes selected for the study. Increased methylation at CpG islands present in the putative DMRs of a number of imprinted genes was observed in the resorbing embryos sired by tamoxifen-treated male rats. This increase in methylation is associated with the downregulation of most of these genes at the transcript level in resorbing embryos. No change in the methylation status of these genes was observed in spermatozoa. These observations suggest that a deregulation of mechanisms protecting unmethylated alleles from a wave of de novo methylation occurs following implantation.
Subject(s)
DNA Methylation/drug effects , Embryo, Mammalian/drug effects , Embryonic Development/drug effects , Genomic Imprinting/drug effects , Insulin-Like Growth Factor II/metabolism , Paternal Exposure/adverse effects , Selective Estrogen Receptor Modulators/adverse effects , Animals , Calcium-Binding Proteins , CpG Islands/drug effects , Down-Regulation/drug effects , Embryo Loss/chemically induced , Embryo Loss/metabolism , Embryo, Mammalian/metabolism , Female , Insulin-Like Growth Factor II/genetics , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Male , Pregnancy , Rats , Rats, Sprague-Dawley , Spermatozoa/drug effects , Spermatozoa/metabolism , Tamoxifen/adverse effects , Up-Regulation/drug effects , ras-GRF1/genetics , ras-GRF1/metabolismABSTRACT
OBJECTIVE: To study methylation aberrations in spermatozoa at developmentally important imprinted regions to ascertain their role in early embryo loss in idiopathic recurrent spontaneous miscarriages (RSM). DESIGN: Case-control study. SETTING: Academic research setting at National Institute for Research in Reproductive Health, Parel, Mumbai. PATIENT(S): Male partners of couples with a history of RSM and male partners of couples with proven fertility (control group). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): DNA methylation levels at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) by Epityper Massarray and global methylation levels as measured by LINE-1 methylation and anti-5-methyl cytosine antibody in spermatozoa of 23 men in control group and 23 men in RSM group. RESULT(S): We did not observe any aberration in the total methylation levels in any of the imprinted genes or global methylation analyzed. CONCLUSION(S): Our results indicate that paternal methylation aberrations at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) and global methylation levels are not associated with idiopathic RSM and may not be good epigenetic markers (unlike the H-19 imprinting control region) for diagnosis of idiopathic RSM.
Subject(s)
Abortion, Habitual/genetics , DNA Methylation/genetics , Genetic Markers/genetics , Genomic Imprinting/genetics , Spermatozoa/physiology , Abortion, Habitual/epidemiology , Abortion, Habitual/metabolism , Calcium-Binding Proteins , Case-Control Studies , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Female , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Long Interspersed Nucleotide Elements/genetics , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Pregnancy , Proteins/genetics , Proteins/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
OBJECTIVE: To study H19 ICR methylation levels in association with sperm parameters routinely analyzed in idiopathic recurrent spontaneous miscarriage cases. DESIGN: Case-control study. SETTING: Academic research setting. PATIENT(S): Male partners of couples with a history of idiopathic recurrent spontaneous miscarriage (RSM group) and male partners of couples with proven fertility (control group). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Paternal age, sperm concentration, motility, chromatin compaction status, morphology, and H19 ICR methylation were assessed in control and idiopathic RSM group participants. RESULT(S): Paternal age and basic semen parameters analyzed did not show any significant difference between the two groups; however H19 ICR methylation levels were reduced significantly in the idiopathic RSM group compared with the control group. CONCLUSION(S): Significant reduction in the H19 ICR methylation without significant difference in the sperm parameters demonstrates aberrant imprinting to be associated with idiopathic RSM.
Subject(s)
Abortion, Habitual/genetics , DNA Methylation , Genomic Imprinting , Insulin-Like Growth Factor II/genetics , RNA, Long Noncoding/genetics , Spermatozoa/metabolism , Age Factors , Case-Control Studies , Cell Shape , Chromatin Assembly and Disassembly , Down-Regulation , Female , Genetic Predisposition to Disease , Humans , India , Male , Pregnancy , Semen Analysis/methods , Sperm Count , Sperm Motility , Spermatozoa/pathologyABSTRACT
The selective estrogen receptor modulator, tamoxifen, has been shown to reduce DNA methylation at Insulin-like growth factor 2/H19 differentially methylated region (Igf2/H19 DMR) in the spermatozoa of the Holtzman rats. Since imprint at this locus is acquired during spermatogenesis in the male germ-line, we hypothesized role for estrogen signaling in the methylation dynamics in the testis. The present study was designed to identify putative estrogen response elements (ERE) at Igf2/H19 DMR and their interaction with DNA methylation pathway. Here, we demonstrate presence of functional ERE at 2637/2655 base pair on Igf2/H19 DMR in testicular germ cells, which was found to bind to estrogen receptor beta (ER beta) in the chromatin immunoprecipitation assay. Tamoxifen attenuated ER beta-ERE association thereby acting as an estrogen antagonist at this locus. Further mechanistic study involving colocalization and immunoprecipitation assay revealed interaction of ER beta and Dnmt1 in the testis. The study provides evidence for the role for estrogen in acquisition of imprint at Igf2/H19 DMR in testis and help in understanding molecular mechanism of environmental estrogens impacting male fertility.
