GSE134362
Changes in rat sperm DNA methylation induced by age and by perinatal exposure to brominated flame retardant flame retardants 2,2',4,4'-tetrabromodiphenyl ether (BDE-47)
Advanced paternal age at fertilization has been suggested to be a risk factor for neurodevelopmental, psychiatric and other disorders in offspring. One emerging hypothesis suggests that altered offspring phenotype is linked with age-related accumulation of epigenetic changes in the sperm of fathers. Given that paternal age is increasing in the developed world, understanding aging-related epigenetic changes in sperm is needed as well as environmental factors that modify such changes. In this study, we characterize age-dependent changes in sperm DNA methylation profiles between young pubertal (postnatal day (PNDs) 65) and mature (PND120) Wistar rats. We also analyze these changes in rats exposed perinatally to 0.2 mg/kg of ubiquitous environmental xenobiotic 2,2',4,4'-tetrabromodiphenyl ether (BDE-47). Reduced representation bisulfite sequencing (RRBS) libraries were prepared from caudal epididymal sperm DNA and differentially methylated regions (DMRs; = 10x coverage depth, = 3 CpGs per cluster, = 5% methylation change, q < 0.05) were identified via MethPipe package. We identified 21 and 9 exposure-related DMRs in sperm collected on PND65 and PND120, respectively. Two DMRs overlapped between the two time-points. This is the first study to demonstrate that environmentally-relevant perinatal exposure to PBDE results in long-lasting changes in sperm DNA methylation. In control animals, 5,319 age-dependent DMRs were identified, with 99.3% DMRs hypermethylated in mature animals compared to young pubertal rats. These age-related DMRs were enriched for functional categories essential for embryonic development, such as pattern specification, forebrain and sensory organ development, and the Wnt pathway. In BDE-47 exposed rats, sperm DNA methylation was higher in young pubertal and lower in mature animals when compared to controls, which resulted in a significant attenuation in the number of age-dependent DMRs (N = 189) identified in the exposed group. In conclusion, our results indicate that the natural aging process has profound effects on sperm methylation levels and this effect may be modified by environmental exposures. Moreover, our results further support the role of sperm DNA methylation as a likely mechanism by which advanced paternal age is associated with adverse offspring health and development. Overall design: Seven-week-old Wistar rats were purchased from Charles River Laboratories (Kingston, NY, USA) on the sixth day of pregnancy, housed in a temperature- and humidity-controlled room with a 12-h light cycle and maintained at 23±2?°C. All rats were fed ad libitum with a rodent chow (Prolab Isopro RMH 3000, Cat. # 5P75, LabDiet, St. Louis, MO). Between pregnancy day 8 and postnatal day 21 (PND21) dams were fed daily from the tip of a pipette 0.2 µl/gram body weight of vehicle (tocopherol stripped corn oil, MP Biomedicals, Solon, OH) or same volume of 1 mg/ml solution of BDE-47 (AccuStandard, Inc., New Haven, CT; 100% purity) daily (n=6 per exposure group). In the latter group it resulted in exposure level of 0.2 mg/kg body weight BDE-47 per day.  Pups were weaned on PND21. On PND65 and PND120 one male pup was randomly  selected from each litter fasted for 2 hours and euthanized using cervical dislocation.  At each euthanasia both distal cauda epididymis were collected, incised longitudinally and incubated at 37°C for 30 min in 1 ml of sperm wash buffer (Cat. # ART1006, Origio, Denmark) to collect motile spermatozoa via the swim-up procedure. For sperm DNA-methylation analysis we used sperm collected from 6 animals per time-point per exposure group. To remove somatic cells contamination 1.5 ml sperm samples were loaded on top of 1 ml density gradient (40% Isolate, Irvine Scientific, USA) in 15 ml conical tubes and centrifuge for 25 min at 500 g. Pelleted spermatozoa were used to extract DNA. Bisulfite converted libraries were prepared from 100 ng of sperm DNA using Ovation RRBS Methyl-Seq System (Cat. # 0353, NuGEN) and EpiTect Fast DNA Bisulfite Kit (Cat. # 59824, Qiagen) following manufacturers' protocols. Sequencing of libraries was done on HiSeq 2500 (Illumina) in Deep Sequencing Core Facility of the University of Massachusetts School of Medicine (Shrewsbury, MA).  Differentially methylated regions were analyzed using a MethPipe pipeline. In short, raw reads were processed in accordance with recommended protocol for libraries prepared with Ovation RRBS Methyl-Seq System (NuGEN) and then mapped to rn6 Rattus norvegicus reference genome using Bismark (version 0.16.1) and bowtie-2 (version 2.2.9).  PCR-duplicates were removed using nudup.py (version 2.2). Subsequent analyses were restricted to CpGs with = 10x coverage to comply with ENCODE recommendations and recommendations of MethPipe developers. Differentially methylated regions (DMRs) were identified using MethPipe. All DMR spatial annotations with regard to various genome structures were produced using closest-features (v. 2.4.25) tool from bedOps package.