Sperm Methylome Profiling Can Discern Fertility Levels in the Porcine Biomedical Model

Abstract

A combined Genotyping By Sequencing (GBS) and methylated DNA immunoprecipitation (MeDIP) protocol was used to identify—in parallel—genetic variation (Genomic-Wide Association Studies (GWAS) and epigenetic differences of Differentially Methylated Regions (DMR) in the genome of spermatozoa from the porcine animal model. Breeding boars with good semen quality (n = 11) and specific and well-documented differences in fertility (farrowing rate, FR) and prolificacy (litter size, LS) (n = 7) in artificial insemination programs, using combined FR and LS, were categorized as High Fertile (HF, n = 4) or Low Fertile (LF, n = 3), and boars with Unknown Fertility (UF, n = 4) were tested for eventual epigenetical similarity with those fertility-proven. We identified 165,944 Single Nucleotide Polymorphisms (SNPs) that explained 14–15% of variance among selection lines. Between HF and LF individuals (n = 7, 4 HF and 3 LF), we identified 169 SNPs with p ≤ 0.00015, which explained 58% of the variance. For the epigenetic analyses, we considered fertility and period of ejaculate collection (late-summer and mid-autumn). Approximately three times more DMRs were observed in HF than in LF boars across these periods. Interestingly, UF boars were clearly clustered with one of the other HF or LF groups. The highest differences in DMRs between HF and LF experimental groups across the pig genome were located in the chr 3, 9, 13, and 16, with most DMRs being hypermethylated in LF boars. In both HF and LF boars, DMRs were mostly hypermethylated in late-summer compared to mid-autumn. Three overlaps were detected between SNPs (p ≤ 0.0005, n = 1318) and CpG sites within DMRs. In conclusion, fertility levels in breeding males including FR and LS can be discerned using methylome analyses. The findings in this biomedical animal model ought to be applied besides sire selection for andrological diagnosis of idiopathic sub/infertility.

Seminal Plasma: Relevant for Fertility?

“…Differential analyses of the pig sperm transcriptome have shown the relevant associations with sperm quality [189] and fertility [27,190], confirming data in other species [182,[191][192][193][194]. The SP EVs also contain diverse smallRNAs, among which the majority are messenger RNA fragments (mRNA, about 25% of the total reads) followed by piRNAs (15%, almost 20,000), miRNAs (9%, almost 300) and very small amounts of tsRNA (0.01%) [140].…”

“…Such double location reinforces the need of considering the role of EVs as adding material to the spermatozoa yet reminding us of the relevance of proper isolation of the components of semen, so that they can sequenced separately but compared at the same time. Unfortunately, sample sizes are uneven, and semen from boars with well-studied and established fertility must be primarily studied [ 190 ], a shortage nowadays. However, how are these miRNAs affecting fertility?…”

“…Responses from the female can be issued by spermatozoa alone [ 198 ] or even by the act of copulation [ 199 ], but the evidence is that these changes are rather modest, compared to what the SP can elicit, either alone or as semen (i.e., including the suspended spermatozoa). Many of the top miRNAs detected in pig spermatozoa [ 27 ] and SP EVs [ 140 ] can impact TLR4 signaling [ 200 ], and promote the production of cytokines that modulates immune responses [ 27 , 180 , 201 ] to the extent of modulating the fertility of the males considered [ 27 , 190 ].…”

“…Molecules showing association with (in)fertility are innumerable, but association between variables is not definitory of function in a general population. Markers for sperm dysfunction [ 218 ] and (in)fertility have been detected in spermatozoa [ 27 , 190 , 219 , 220 ] and in the SP [ 221 , 222 ] of many species, including humans [ 218 , 222 , 223 , 224 , 225 ] and livestock [ 70 , 226 ]. They vary from gene presence [ 227 ], markers of Sertoli cell function [ 228 ], proteins [ 36 , 229 , 230 , 231 ], anti-oxidative enzymes [ 223 , 232 , 233 ] and metabolites [ 234 , 235 , 236 ] to exosome-associated molecules [ 237 , 238 ] and microRNAs [ 181 , 239 , 240 , 241 ].…”

Non-coding RNAs and chromatin: key epigenetic factors from spermatogenesis to transgenerational inheritance

“…In a similar biomedical model, boars with high quality semen parameters but lower fertility success have shown that most of the DMRs were hypermethylated on genes related to spermatogenesis, sperm function, fertilization and fertility/prolificacy. Additionally, they showed the same disregulation of sperm miR-153 that occurs in humans, affecting fertilization and embryo rates after IVF [ 129 ].…”