Analyses of inter-individual variations of sperm DNA methylation and their potential implications in cattle

Abstract

Background: DNA methylation has been shown to be involved in many biological processes, including X chromosome inactivation in females, paternal genomic imprinting, and others.

Results: Based on the correlation patterns of methylation levels of neighboring CpG sites among 28 sperm whole genome bisulfite sequencing (WGBS) data (486 × coverage), we obtained 31,272 methylation haplotype blocks (MHBs). Among them, we defined conserved methylated regions (CMRs), variably methylated regions (VMRs) and highly variably methylated regions (HVMRs) among individuals, and showed that HVMRs might play roles in transcriptional regulation and function in complex traits variation and adaptive evolution by integrating evidence from traditional and molecular quantitative trait loci (QTL), and selection signatures. Using a weighted correlation network analysis (WGCNA), we also detected a co-regulated module of HVMRs that was significantly associated with reproduction traits, and enriched for glycosyltransferase genes, which play critical roles in spermatogenesis and fertilization. Additionally, we identified 46 VMRs significantly associated with reproduction traits, nine of which were regulated by cis-SNPs, implying the possible intrinsic relationships among genomic variations, DNA methylation, and phenotypes. These significant VMRs were co-localized (± 10 kb) with genes related to sperm motility and reproduction, including ZFP36L1, CRISP2 and HGF. We provided further evidence that rs109326022 within a predominant QTL on BTA18 might influence the reproduction traits through regulating the methylation level of nearby genes JOSD2 and ASPDH in sperm.

Conclusion: In summary, our results demonstrated associations of sperm DNA methylation with reproduction traits, highlighting the potential of epigenomic information in genomic improvement programs for cattle.

Keywords: Cattle; Methylation haplotype blocks; Reproduction traits; Sperm DNA methylation; Variably methylated regions.

Fig. 1

Schematic overview of the current study. We defined methylation haplotype blocks (MHBs) using whole genome bisulfite sequencing (WGBS) data of 28 sperm samples. We then detected the highly variably methylated regions (HVMRs), conserved hypomethylated regions (Hypo-CMRs) (average methylation level < 20%) and conserved hypermethylated regions (Hyper-CMRs) (average methylation level > 80%) based on the methylation variations among individuals. We next functionally annotated them by integrating DNA methylation, gene expression, GO/KEGG, transcriptional factor binding sites, QTL and WGCNA module-trait relationship. We further detected the variably methylated regions (VMRs) using lenient criteria. We associated the methylation levels of VMRs with 41 complex traits. We also annotated the significant VMRs by examining the functional annotation of their associated genes, and their corresponding expression across 91 tissues. We finally conducted cis-methylation QTL (± 1-Mb) analyses for significant VMRs

Fig. 2

Characterization of sperm methylation haplotype blocks (MHBs) in cattle. a Length distribution of MHBs. b CG density (CG number per base pair) distribution of MHBs. c Co-localization of MHBs with known genomic elements. d Enrichment of MHBs in known genomic features. e Enrichment of MHBs in predicted chromatin core 15-states using chromHMM [25, 63]: 1 TssA: Active Tss; 2 TssAFlnk: Flanking active TSS; 3 TxFlnk: Transcrption at gene 5′ and 3′; 4 EnhA: Active enhancer; 5 EnhAATAC: Active enhancer & ATAC; 6 EnhWk: Weak active enhancer; 7 EnhPois: Poised enhancer; 8 EnhPoisATAC: Poised enhancer & ATAC; 9 EnhWkCTCFATAC: Weak enhancer & CTCF & ATAC; 10 ATAC: ATAC islands; 11 reprWkCTCF: Weak represeed CTCF; 12 BivFlnk: Flanking bivalent TSS/Enhancer; 13 ReprRC: Repressed Polycomb; 14 ReprPCWk: Weak repressed Polycomb; and 15 Quies: Quiescent/Low. f One example of MHB located in the exon 4 of predicted maternal imprinted gene GAREM1. Methylation levels of the MHB were low in sperm but high in oocyte

Fig. 3

Comparison of three categories of regions with extreme methylation variation in sperm. a Standard deviation (SD) vs. mean sperm DNA methylation of all methylation haplotype blocks (MHBs), and distribution of highly variable methylated regions (HVMRs), hypomethylated conserved regions (Hypo-CMRs) and hypermethylated conserved regions (Hyper-CMRs). b Average methylation levels of HVMR, Hypo-CMRs and Hyper-CMRs in 16 somatic tissues. c Average expression levels (FPKM value) of genes associated with HVMR, Hypo-CMRs and Hyper-CMRs in 91 tissues and cell types. d Enrichments of three categories of expression QTL in HVMR, Hypo-CMRs and Hyper-CMRs (eeQTL: exon expression QTLs; geQTL: gene expression QTLs; sQTL: splicing QTLs; these expression QTLs were detected from dairy cattle blood and milk cells, liver and muscle [28]). e Enrichments of selection signatures differentiating dairy and beef cattle breeds [30] in HVMR, Hypo-CMRs and Hyper-CMRs

Fig. 4

Relationship between methylation variations in sperm and complex traits. a Enrichments of six QTL categories (including 232 traits) from Cattle QTL database in HVMRs, Hypo-CMRs and Hyper-CMRs. CE: calving ease; DPR: daughter pregnancy rate; SB: still birth. b Module-trait relationships using a weighted correlation network analysis (WGCNA) (Only reproduction traits were tested). Elements in red dash box were two traits most significantly associated with module 1. HCR: heifer conception rate; CCR: cow conception rate; ST_PL: standard length of productive life; GL: gestation length; SCR: sire conception rate; PL: length of productive life; DPR: daughter pregnancy rate. c Co-methylated regions with 15 MHBs in module 1 enriched for glycosyltransferase genes. M1, M2 and M3 represents three MHBs located with the GALNT2 gene

Fig. 5

Associations between variably methylated regions (VMRs) and reproduction traits. a The -log(P) values of associations of VMRs with four types of complex traits. Red dot line indicates the significant P value after Bonferroni correction (P < 2.89 × 10^-6; 0.05/17323). b Manhattan plots of five traits with significant/suggestively significant VMRs. Red dots indicated the significant VMRs (P < 2.89 × 10⁻⁶; 0.05/17323). Green dots indicated the suggestive significant VMRs (P < 5.77 × 10⁻⁵; 1/17323). CCR: cow conception rate; DPR: daughter pregnancy rate; GL: gestation length; ST_PL: standard length of productive life. c Correlation between predicted transmitting ability (PTA) values of DPR and methylation levels of VMR within the ZFP36L1 gene. d Correlation between PTA values of DPR and methylation levels of VMR located downstream of CRISP2 gene. e Expression levels (FPKM values) of four genes associated with significant VMRs across 91 bovine tissues

Fig. 6

A trait-related variably methylated region (VMR) was associated with a SNP within 1 Mb distance. a UCSC browser of the VMR associated with CCR and DPR as well as the methylation QTL (meQTL) associated with the trait-related VMR (chr18: 57097832–57,097,893). Y axis indicates the –log10(P) from association test. b Association studies between the meQTL (rs109326022) and 35 bovine complex traits in 27, 214 Holstein bulls [44]. PL and SCE were the most significant traits associated with the meQTL. c Methylation levels of the trait-related VMR in three genotypes of rs109326022