Epigenetic analysis of high and low motile sperm populations reveals methylation variation in satellite regions within the pericentromeric position and in genes functionally related to sperm DNA organization and maintenance in Bos taurus

- Epigenetic analysis of high and low motile sperm populations reveals methylation variation in satellite regions within the pericentromeric position and in genes functionally related to sperm DNA.
- Background: Sperm epigenetics is an emerging area of study supported by observations reporting that abnormal sperm DNA methylation patterns are associated with infertility.
- Here, we explore cytosine-guanine dinucleotides (CpGs) methylation in high (HM) and low motile (LM) Bos taurus sperm populations separated by Percoll gradient..
- HM and LM methylation patterns were investigated by bisulfite sequencing..
- Results: Comparison between HM and LM sperm populations revealed that methylation variation affects genes involved in chromatin organization.
- BTSAT4 was hypomethylated in HM sperm populations..
- Conclusions: The characterization of the epigenome in HM and LM Bos taurus sperm populations provides a first step towards the understanding of the effect of methylation on sperm fertility.
- Methylation variation observed in HM and LM populations in genes associated to DNA structure remodelling as well as in a repetitive element in pericentric regions suggests that maintenance of chromosome structure through epigenetic regulation is probably crucial for correct sperm functionality..
- 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- Full list of author information is available at the end of the article.
- Among the known epigenetic processes in mammalian cells, DNA methylation has been identified as an import- ant regulatory mechanism of genome function in normal embryonic development, X-chromosome inactivation and genomic imprinting [8, 9].
- DNA methylation of the 5-carbon position in cytosine residues was reported to be predominantly present in cytosine-guanine dinucleo- tides (CpG) and especially in GC rich regions called CpG islands (CGIs) [10].
- Sperm epigenetic marks are unique, thus the factors that determine the patterns of DNA methylation differ between male germ cells and somatic cells.
- Although RE are highly methylated in both germ and somatic cells, el- ements from several subfamilies show different levels of methylation in the two cell types [13].
- Most of the epigenetic signatures in germ cells are erased after conception from the morula stage to the blastocyst stage in the inner cell mass (ICM).
- succes- sively, a sharp increase in the level of methylation in the embryo is observed following implantation [16, 17]..
- The level of DNA methylation of round spermatid was reported to be different from that of mature spermatozoa.
- DNA methylation in human spermatozoa was higher in low quality spermatozoa [22].
- Targeted bisulfite sequen- cing also revealed different levels of methylation in the promoter regions between high and low motile human sperm [24]..
- DNA methylation pattern were found to be different between spermatozoa from high-fertile and sub-fertile buffalo bulls [25].
- Recently, assessment of the epigenetic signa- ture of bull spermatozoa using a human DNA methyla- tion microarray [26] and Methyl-Binding Domain (MBD) Sequencing [27] revealed differentially methylated CpG sites and regions associated to bull fertility rate..
- In the present study, the 5-methyl cytosine variations in CpGs were evaluated in high and low motility bull sperm populations following methyl enrichment and bi- sulfite sequencing approach.
- Sperm cells were successfully fractionated into HM and LM populations.
- Bisulfite sequencing was then applied to the methylation- enriched genomic fraction to investigate CpG methylation level at single base resolution in the highly methylated re- gions.
- by unbalanced sequencing between groups of samples due to MBD enrichment, we evaluated cytosine coverage con- sistence between the HM and LM groups.
- After calculating cytosine me- thylation conversion, a high percentage (93.7%) of the cytosines in the CpG enriched regions was methylated in both sperm populations (see Additional file 3 for statis- tics).
- Among these methylated regions (MRs), shared between at least 3 out of 4 for both HM and LM sperm populations, were selected for DNA cytosine methylation profile comparison..
- sperm populations.
- Differentially methylated regions between HM and LM sperm populations.
- A genome-wide analysis of genes and regulatory ele- ments revealed that a small percentage of CpGs shows a significant variation in the methylation level (differen- tially methylated regions (DMRs)/MRs percentage) be- tween HM and LM sperm populations in gene bodies (1.45.
- Considering CGIs, a higher proportion of the methylome (9.77%) was remodelled in HM vs LM sperm populations.
- Hierarchical analysis of the 20 most hyper and hypo methylated DMRs found in CGIs, in gene bodies, 5’UTR and 3’UTR well discriminated HM from LM samples (Fig.
- Methylated regions (MRs) were stratified based on the average methylation level of CpGs (ranging from 0 to 100%).
- HM and LM sperm populations were compared and 20 most hyper and 20 most hypo methylated DMRs from each comparison were used for clustering samples.
- In addition, GO terms related to hindbrain function, epithelia and endo- thelia migration metabolic processes were also observed to differ between HM and LM sperm populations.
- Out of 23,431 CGIs annotated in the bovine genome, 3869 were detected (in at least 3 out of 4 samples for HM and LM) in our dataset.
- and dis- tribution of CGIs length was calculated in each class in HM and LM sperm populations (Fig.
- Out of 2434 BTSAT4 elements annotated in the bovine genome 720 were detected (in at least 3 out of 4 samples for HM and LM) in our dataset..
- Analysis of CpG methylation outlined an overall low level of BTSAT4 methylation in the HM sperm popula- tion.
- Considering 159 DMRs in the BTSAT4 regions, 122 were more methylated in LM sperm populations (Additional file 12) (Fig.
- The overall CGIs covered by MBD sequencing represent about 1% of the total cow genome, while sequenced BTSAT4 regions represent 0.2%.
- 3 Distribution of CGIs length for Methylated regions (MRs) grouped based on CpG methylation level for HM and LM sperm populations..
- shows that 70,6% of the BTSAT4 base pairs fall within CGIs, 80% of the BTSAT4 repeats being completely or partially located within CGIs (Additional file 13)..
- In this work, the pattern of methylation in high and low motile bull sperm populations was determined using an enrichment step of methyl-CpG sequences combined with bisulfite sequencing..
- The comparison of different genomic features in HM and LM sperm populations revealed several differentially methylated regions flanking genes with a role in chroma- tin organization and maintenance.
- Interestingly, HM and LM sperm populations showed variation in methylation of telomerase reverse transcriptase (TERT) and telomerase- associated protein 1 (TEP1).
- Previous studies reported a strict association between sperm DNA methylation levels and both sperm chromatin condensation and DNA integrity, suggesting that the formation of a compact chro- matin and proper DNA methylation are closely related events during spermatogenesis [21]..
- methylation and 80 – 100% methylation) in high motile (HM) and low motile (LM) sperm populations.
- KDM1 is required for spermatogonial differentiation, as well as germ cell survival, in the developing testis [40]..
- An evolutionarily conserved pathway between histone H3-K9 methylation and DNA methylation exists in mammals, that is likely to be important to reinforce het- erochromatic subdomains stability and to protect genome integrity.
- located within genomic satellite repeats and in particular BTSAT4 Bovine satellite I [29] was observed to be less methylated in HM sperm populations.
- In the bovine genome BTSAT4 is likely to be the counterpart.
- Methylated regions (MRs), left panel, and differentially methylated region (DMRs), right panel, were stratified based on the average methylation level of CpGs (ranging from 0 to 100%) for HM and LM sperm populations.
- Methylation profiling in bovine semen revealed differen- tial methylation of the BTSAT4 repetitive element in pericentric regions between HM and LM sperm popula- tions.
- DNA extraction, library preparation and sequencing Four HM and four LM sperm samples obtained in previ- ous step were used for DNA extraction.
- The General Linear Model procedure (PROC GLM) was used to evaluate the efficiency of the sperm separation comparing semen qual- ity parameters at thawing and in the HM population.
- The model included the fixed effect of the sperm population, and bull as random.
- Data are available in the Sequence Reads Archive (SRA), (Accession Number SRP119411)..
- Seq- monk software (version 0.34.1) was used for visualization and analysis of the Bismark output (http://www.bioinfor- matics.babraham.ac.uk/projects/seqmonk.
- Cytosines count for each position (n count>5X, present at least in 3 out of 4 in HM and LM samples) was determined and the Edge-R package was used to evaluate if over or under-representation occurs in our dataset between HM and LM groups..
- in both HM and LM sperm populations.
- Reads count in BTSAT4 REs was determined and the Edge-R package was used to evaluate if over or under-representation occurs in our dataset between HM and LM groups.
- Differentially meth- ylated regions (DMRs) between HM and LM populations were calculated using the logistic regression filter in R to assess differential methylation (FDR <.
- GO classification of the DMRs was performed according to canonical GO categories, using the Cytoscape plug-in ClueGO which in- tegrates GO [52] and enhances biological interpretation of large lists of genes.
- Evaluation of REs in CGIs was per- formed by intersecting genomic positions of both features by Bedtools intersect (http://bedtools.readthedocs.io), thus frequencies for each RE category were calculated for low/intermediate methylation CGIs (20–60% methyla- tion) and high methylation CGIs (80–100% methyla- tion), in both HM and LM sperm populations and in Bos taurus genome..
- Edge-R smear plot representing the Average Log Count Per Millions (CPM) and the abundance differences (logFC = log Fold Change) for the cytosine counts between HM and LM groups.
- Methylated Regions (MRs) found at least in three samples in both high motile (HM) and low motile (LM) sperm populations overlapping gene bodies.
- Methylated Regions (MRs) found at least in three samples in both high motile (HM) and low motile (LM) sperm populations upstream of genes (5 ’ UTR).
- Methylated Regions (MRs) found at least in three samples in both high motile (HM) and low motile (LM) sperm populations downstream of genes (3 ’ UTR).
- Methylated Regions (MRs) found at least in three samples in both high motile (HM) and low motile (LM) sperm.
- Differentially Methylated Regions (DMRs) found at least in three samples in both high motile (HM) and low motile (LM) sperm populations overlapping gene bodies.
- Differentially Methylated Regions (DMRs) found at least in three samples in both high motile (HM) and low motile (LM) sperm populations upstream of genes (5 ’ UTR).
- Differentially Methylated Regions (DMRs) found at least in three samples in both high motile (HM) and low motile (LM) sperm populations downstream of genes (3 ’ UTR).
- Differentially Methylated Regions (DMRs) found at least in three samples in both high motile (HM) and low motile (LM) sperm populations overlapping CpG islands (CGIs).
- Differentially Methylated Regions (DMRs) found at least in three samples in both high motile (HM) and low motile (LM) sperm populations overlapping BTSAT4 satellite.
- The funding bodies did not play any roles in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript..
- Influence of the male on embryo quality.
- DNA methylation in early development.
- Functions of DNA methylation: islands, start sites, gene bodies and beyond.
- Role of the Dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse.
- DNA methylation in mouse gametogenesis.
- Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells.
- A unique regulatory phase of DNA methylation in the early mammalian embryo.
- The DNA methylation landscape of human early embryos.
- Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids.
- Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm.
- Sperm global DNA methylation level:.
- Different levels of DNA methylation detected in human sperms after morphological selection using high magnification microscopy.
- Age-associated sperm DNA methylation alterations: possible implications in offspring disease susceptibility.
- Promoter targeted bisulfite sequencing reveals DNA methylation profiles associated with low sperm motility in asthenozoospermia.
- Genome-wide profiling of sperm DNA methylation in relation to buffalo ( Bubalus bubalis ) bull fertility.
- Differentially methylated CpG sites in bull spermatozoa revealed by human DNA methylation arrays and bisulfite analysis.
- Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos.
- Profiling genome-wide DNA methylation..
- Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin.
- DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.
- Aberrant DNA methylation reprogramming in bovine SCNT preimplantation embryos.
- Obesity-induced sperm DNA methylation changes at satellite repeats are reprogrammed in rat offspring.
- Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between high- and low-motile sperm populations.
- Development of preimplantation embryos of the golden hamster in a defined culture medium

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