There is now strong evidence that the paternal contribution to offspring phenotype at fertilisation is more than just DNA. However, Sperm slop identity and mechanisms of this nongenetic inheritance are poorly understood. One of the more important questions in this research area is: Sperm slop have previously reported that offspring of obese male rats have altered glucose metabolism compared with controls and that this effect was inherited through nongenetic means.
Here, we describe investigations into sperm DNA methylation in a new cohort using the same protocol. Examination of the repetitive fraction of the genome with methyl-CpG binding domain protein-enriched genome sequencing MBD-Seq and pyrosequencing revealed that retrotransposon DNA methylation states in spermatozoa were not affected by obesity, but methylation at satellite repeats throughout the genome was increased.
Furthermore, no Sperm slop were found in three genomic imprints in obese rat spermatozoa. Our findings have implications for transgenerational epigenetic reprogramming. They suggest that postfertilization mechanisms exist for normalising some environmentally-induced DNA methylation changes in sperm cells. It is accepted that the phenotype of an organism is a function of both its individual genetics and the environment that it experiences.
There is increasing evidence for a third determinant Sperm slop phenotype, nongenetic inheritance. Nongenetic inheritance down the male line can be referred to as a paternal effect. The increase in the last decade of examples of paternal effects in mammals has led to considerable interest in identifying the nongenetic components of spermatozoa and seminal fluid that could influence offspring phenotype.
This is due to its ability to repress gene transcription when present at gene promoters and enhancer regions.
Furthermore, evidence for methylation states in the gametes being able to control gene expression throughout the lifetime of the next generation comes from the developmental process of genomic imprinting, where the methylation state of regulatory regions in spermatozoa and oocytes controls gene transcription in offspring.
Like many other cell types, mature Sperm slop in humans and rodents have high methylation levels at most repetitive elements and intergenic regions that transcriptionally repress these regions.
Gene-specific hyper- or hypo-methylation is found at promoters, with a general observation of decreasing methylation with increasing CpG Sperm slop. Several studies have described alteration Sperm slop normal sperm DNA methylation patterns, due to genotype, environmental exposure or disease.
These include methylation changes associated with mutations in DNA methyltransferases, 13 reduced fertility, 14151617 toxin and drug exposure, 181920 dietary alterations, Sperm slop22 and stress exposure.
An obstacle to the persistence of sperm methylation states in offspring is the extensive demethylation of the paternally-inherited chromosomes after fertilisation in humans and rodents. However, some regions such as genomic imprints, some repetitive element classes and some single-copy loci are resistant to this genome-wide DNA demethylation.
An environmental insult that is increasingly being studied for its consequences on the Sperm slop generation is obesity. There is now a large body of work describing nongenetic influences of maternal obesity on offspring phenotype, through mechanisms such as altered nutrition during gestation and lactation.
Sperm slop this study, we aimed first to identify DNA methylation changes in the spermatozoa of obese rats, and Sperm slop to see if the changes persist in offspring tissues. These data are valuable for understanding how DNA methylation patterns in the spermatozoa are affected by environmental stimuli, and how these changes are affected by postfertilization epigenetic reprogramming processes. Control rats were fed normal chow energy: The females and males only spent the daylight hours together, and then returned to their home cages to continue their assigned diet to ensure that only the male was consuming the HFD.
No difference in the initiation of pregnancy was observed between obese and control males. The F0 males were killed between 24 and 29 weeks of age. F1 male offspring were Sperm slop at PND21 on control diet and killed at 27 weeks of age. Animals were killed after anaesthesia induced by i. Mature spermatozoa were squeezed from the perforated cauda epididymis and vas deferens into 2 ml Dulbecco's Modified Eagle Medium DMEM in a Petri dish and mixed by pipetting.
The suspension was transferred to a 12 ml Falcon tube and left upright for 20 min. Thereafter, the top 1. Sperm cells were washed and re-pelleted once with phosphate-buffered saline. Any contaminating Sperm slop cells were removed by resuspension in distilled water for osmotic lysis, then incubation for 20 min on ice in somatic cell lysis buffer 0. Control and HFD groups were compared using Student's t -test.
We investigated repetitive element DNA methylation in spermatozoa as DNA methylation at some classes of repeats is known to be resistant to transgenerational epigenetic reprogramming 2627 and can thus facilitate epigenetic Sperm slop.
This technique determines the relative proportions of cytosine indicating a methylated cytosine and thymine indicating an unmethylated, and consequently bisulfite converted cytosine nucleotides at individual CpG sites in PCR product from regions of interest. Non-CpG C control dispensations were added in each pyrosequencing assay to confirm complete bisulfite conversion of unmethylated cytosine.
Control Sperm slop HFD groups were compared by using Student's t -test. Sperm slop enrichment of the methylated fraction was confirmed with qPCR of the HICR, an imprinted region that is known to be highly methylated in spermatozoa. Postsequencing duplicated sequences were removed by using the picard tools and unique reads were mapped to the Rattus norvegicus rn5 genome assembly by BWA.
The BedTools software package 38 was employed to estimate the distribution of methylation. First all bam files that were generated from next generation sequencing data were converted into bed format with bedtools bamToBed.
The coverage command of bedtools 38 was used with default options to calculate the number of times a repeat class as described in the RepeatMasker Track of the UCSC Genome Browser, http: The total number of bp regions with homology to each repetitive element type was normalized by dividing the number of overlaps by the Sperm slop number of unique sequence reads from the same sample.
The first 12 clusters in the BLAT Sperm slop search were chosen for examination. Finally, bedtools coverage command was used to calculate the number of times each cluster was overlapped by nt regions from the bed file.
Control and HFD groups were compared by Student's t -test. Supplementary Figure 1 shows weight gain. Mean body weight changes over time in high-fat diet and control diet fathers.
Eight males in each group produced litters; there were no significant differences in litter size or sex ratio between groups. At birth, male offspring from obese fathers were lighter than those from controls. The mean male birth weights calculated per litter Sperm slop 7. Litter size impacts the amount of milk available for each pup and consequently their weight gain. Therefore on postnatal day 1, offspring litter sizes were adjusted to 8 to 12 per litter to minimise effects of litter size on pups weight gain.
Both the HFD and control group 6-month-old male offspring that were used for the methylation analyses were from eight different fathers. Global DNA methylation in the spermatozoa from obese rats 4. Global DNA methylation in obese rat spermatozoa. We performed pyrosequencing on bisulfite-treated Sperm slop for selected repetitive elements. Pyrosequencing to evaluate repetitive-element class-specific methylation in rat spermatozoa. High-throughput sequencing on the Illumina Hiseq platform gave an average of reads in the four control samples and in the four HFD samples.
The total number of sequence reads per sample did not allow examination of DNA methylation status of small single-copy regions Sperm slop the genome. However, examination of common repetitive element classes and repetitive element clusters, such as those found at centromeres, was possible.
Further, investigation of the normalized number of sequence reads that originated from 12 SATI repeat clusters showed a consistent relative increase in reads in spermatozoa from HFD rats compared with control rats Figure 3. This Sperm slop supports the pyrosequencing-derived observation that satellite repeats have higher DNA methylation levels in the spermatozoa from obese than lean rats. MBD-Seq confirms that SATI satellite repeats at centromeres and noncentromeric regions have increased methylation in obese rat spermatozoa.
Furthermore MBD-Seq read counts at every SATI cluster investigated were higher in obese rat spermatozoa than those from control rats after normalization to total reads per sample. The next four comparisons Sperm slop at SATI clusters at centromeres of metacentric chromosomes, the next three are clusters are at the centromeres of telocentric chromosomes and the last five are clusters at noncentromeric regions.
The X-axis indicates the chromosome number, location to the nearest Mb and cluster size. A major question for the investigation into the molecular mechanisms of paternal effects is, do sperm-borne epigenetic changes persist throughout offspring development, potentially to cause functional changes in offspring tissues? Therefore, we used the same pyrosequencing assay that was used in paternal spermatozoa to search for changes in offspring tissues.
However, examination of offspring from obese and control rats suggested that there was Sperm slop difference in the Sperm slop methylation levels of SATI satellites Figure 4.
In normal spermatozoa H19 -ICR is highly methylated, and the other two are unmethylated. This pattern was unaffected by male obesity Figure 5. Results of pyrosequencing to investigate DNA methylation at genomic imprints in spermatozoa from obese and control rats. The list of candidate molecules that may facilitate nongenetic inheritance from father to offspring is long. It is important to identify all mechanisms for inherited phenotypes in Sperm slop to understand nongenetic inheritance better and to design therapeutic interventions.
Sperm slop offspring of the obese rats have various phenotypic differences compared with the offspring of controls, including changes to glucose metabolism 45 and body weight.
We focused on repetitive element DNA methylation in spermatozoa, as repeats have been shown in mice to Sperm slop epigenetic inheritance.
Our findings provide an illustration of the effectiveness of the trans-generational epigenetic reprogramming mechanisms for normalising DNA methylation patterns.
Importantly, we do not suggest that this correction of obesity-induced DNA methylation changes will apply to all environmentally-induced epigenetic changes to sperm. Indeed, there are a growing number of examples where the abnormal methylation state in spermatozoa is detectable Sperm slop offspring tissues. Nonetheless, to understand the mechanisms and role of DNA methylation in the transgenerational epigenetic inheritance of phenotypes fully, it is important to report instances of methylation erasure as well as an inheritance.
The increase in global Sperm slop methylation level in obese males compared with lean males is small 0. However, even small changes at the global level have been shown to have large developmental consequences. An increase of 0. A murine model of paternal obesity displayed decreased DNA methylation in testes and spermatozoa when a semi-quantitative immunofluorescence technique was used.
The reasons for these differences could be related to Sperm slop between quantitative and semi-quantitative methods, differences in global and site-specific methylation changes, or could indicate that the effects of diet on global sperm methylation levels may be species- or diet-specific. A frequently proposed explanation Sperm slop paternal effects in mammals is an alteration of genomic imprints in spermatozoa.
Recent studies in humans 5051 have associated paternal obesity with Sperm slop in imprint methylation in the offspring's umbilical cord blood leukocytes. The authors proposed that the changes were due to abnormal imprint programming during gametogenesis or early development.
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Sperm slop We investigated the former possibility by examining imprint methylation in spermatozoa at regions that are normally methylated paternal imprints or unmethylated maternal imprints. However, we are unable to exclude the possibility of altered imprinting at other Sperm slop.
Sequences with homology to repetitive elements make up roughly half of the mammalian genome.