Sperm DNA Methylation Analysis in Swine Reveals Conserved and Species-Specific Methylation Patterns and Highlights an Altered Methylation at the GNAS Locus in Infertile Boars1

Abstract

Male infertility is an increasing health issue in today's society for both human and livestock populations. In livestock, male infertility slows the improvement of animal selection programs and agricultural productivity. There is increasing evidence that epigenetic marks play an important role in the production of good-quality sperm. We therefore screened for specific or common epigenetic signatures of livestock infertility. To do so, we compared DNA methylation level in sperm DNA from fertile and infertile boars. We evaluated first the global level of sperm DNA methylation and found no difference between the two groups of boars. We then selected 42 loci of interest, most of them known to be imprinted in human or mice, and assessed the imprinting status of five of them not previously described in swine tissues: WT1, CNTN3, IMPACT, QPCT, and GRB10. DNA methylation level was then quantified in fertile and infertile boars at these 42 loci. Results from fertile boars indicated that the methylation level of the selected loci is highly conserved between pig, human, and mice, with a few exceptions, including the POU5F1 (OCT4) promoter and RTL1. Comparison between fertile and infertile boars revealed that one imprinted region, the GNAS locus, shows an increase in sperm DNA methylation in three out of eight infertile boars with low semen quality. This increase in DNA methylation is associated with an altered expression of the genes belonging to the GNAS locus, suggesting a new role for GNAS in the proper formation of functional gametes.

Meiotic pairing and gene expression disturbance in germ cells from an infertile boar with a balanced reciprocal autosome-autosome translocation

“…Total RNAs were extracted from testis samples from three fertile control boars with normal semen parameters (Supplementary Table 1) and from the t(1;14) boar using the method described in Congras et al (2014). Three independent extractions from three samples were performed for each animal.…”

In vitro exposure to CPF affects bovine sperm epigenetic gene methylation pattern and the ability of sperm to support fertilization and embryo development

“…NESP55‐GNAS locus, which was found to be imprinted in cattle (Khatib et al, ), mice (Fröhlich et al, ), humans (Grybek et al, ), and pigs (Oczkowicz et al, ), covers more than 11 Kb on bovine chromosome 13. Congras et al () have previously described an altered methylation at this locus in infertile boars. A high sequence homology was found between pig and bovine sequences, so we opted to analyze the same region.…”

“…However, in contrast, genome-wide DNA methylation profiles of spermatozoa DNA, as well as methylation analysis on the GNAS promoter region of sperm treated with the pesticide, did not show evident alterations at the concentrations used for the experimental design. Nonetheless, our interest for GNAS was still justified by evidence reported in previous studies; higher methylation levels of GNAS promoter were found in sterile boars compared to fertile boars (Congras et al, 2014).…”

Teratozoospermia and asthenozoospermia are associated with specific epigenetic signatures

“…Specifically, we identified 1080 alterations associated with low viability, 164 altered regions associated with low motility, and 12 alterations associated with low count (Table S2). Interestingly, in these analyses PEG10, MEST, and GNAS (commonly cited imprinted genes that are reportedly associated with male infertility in multiple mammalian species) were genes that had commonly altered methylation profiles (Poplinski et al, 2010;Congras et al, 2014). We assessed the distance from the transcription start site (TSS) of the regions identified to be associated with both low motility and low viability (Fig.…”