Chrystelle Le Danvic

Background: Bull fertility is a key parameter in the dairy cattle industry, with significant economic implications. DNA methylation is an epigenetic mark essential for fertility, and recent works have highlighted sperm DNA methylation as a promising source of biomarkers for male fertility, but no operational tool has been developed for predicting bull fertility based on DNA methylation. Here, we leveraged existing methylation data to evaluate the potential of DNA methylation for predicting bull fertility with machine learning models. Results: Our study was conducted on a cohort of 351 Holstein commercial bulls, classified as subfertile, neutral and fertile based on a field fertility index (non-return rate of the inseminated cows after 56 days post-insemination). This dataset includes the largest cohort assembled for bull fertility prediction from epigenetic markers. We developed classification models to discriminate subfertile and fertile individuals using a subset of predictors derived from reduced representation bisulfite sequencing data obtained on frozen semen. Four machine learning algorithms were compared: Least Absolute Shrinkage and Selection Operator (LASSO), Random Forest, Gradient Boosting, and Neural Networks. To optimize performances of each algorithm and conduct an unbiased assessment of classification accuracy, we used a three-fold cross-validation before final testing on an independent test set. To increase the robustness of the results, model performances were averaged over 20 partitions of the data into training and test sets, to account for variability in bull distribution across sets. All models performed better than random classification. The best model was LASSO according to classification accuracy (0.62) and Random Forest according to Area Under Curve (0.69) computed with the independent test set. Conclusions: Even with this moderate performance, our models would lead to a 12.4% point increase of the probability of identifying subfertile individuals. These results suggest that sperm DNA methylation holds potential as a relevant source of predictive markers for bull fertility.

Background: Complex traits are influenced by numerous variants, most of which have regulatory effects on gene expression that can be mediated by DNA methylation. Molecular QTL mapping is an approach that aims to dissect these effects. However, obtaining molecular phenotypes on a large scale is challenging, particularly in livestock species. In cattle, an epigenotyping array called EpiChip has recently been developed in the European RUMIGEN project. The EpiChip, which contains 43,317 CpG sites distributed all over the bovine genome, enables large-scale measurement of DNA methylation. This study aims to characterize the genetic determinism of blood DNA methylation in cows by estimating heritability and mapping cis- and trans- methylation QTLs (meQTLs). Results: Whole blood samples from 4,457 genotyped Holstein cows were epigenotyped. Across all CpG sites, the heritability estimates averaged 24.6%. The local meQTL mapping at sequence-level for variable CpG sites (SD > 2.5%; n = 28,806) detected cis -meQTLs for 80.1% of the CpG sites, with sentinel SNPs located close to their associated CpGs. A two-step analysis was also conducted to identify long-range associations, with a particular focus on trans -meQTL hotspots. First, we identified CpG-SNP trans -associations using medium-density genotypes (50k SNPs) that revealed 31,846 SNPs with significant effects on 1 to 530 trans- CpG sites. Then, regions associated with at least 34 independent trans -CpGs were retained defining 31 hotpots. For each hotspot, a local sequence-level GWAS was conducted using the first principal component derived from the associated trans -CpGs. Out of the 31 detected hotspots, three were located close to transcription factor genes ( RUNX1 , NFIC and FOXA3 ) for which the associated trans -CpGs were enriched for the corresponding binding motif. Two other hotspots were located within KDM5A and KDM5B , and their corresponding trans -CpGs were strongly overrepresented in H3K4me3 narrow peaks in blood as well as in other tissues. Conclusions: By identifying functional candidate genes associated with blood DNA methylation in cattle, these findings provide new insights into the regulatory architecture of DNA methylation in mammals, highlighting the value of large-scale molecular data from livestock populations.

L’œstrus (ou chaleurs) est une période critique dans la mise en place des processus de reproduction au sein des élevages. Or, les techniques de détection actuelles s’avèrent lourdes à mettre en place, onéreuses et n’assurent pas une détection systématique de l’œstrus. Le mâle est capable de détecter l’œstrus par l’intermédiaire de signaux chimiques émis dans les urines de femelle. La connaissance de ces signaux, ainsi que de leurs protéines de liaison dans les tissus olfactifs devrait permettre le développement d’un biosenseur, permettant de détecter de façon automatique et précise les composés volatils émis par les femelles en œstrus. Afin de mettre en évidence les signaux chimiques de l’œstrus chez les bovins (Bos Taurus), des urines de vaches (30) ont été collectées à différents stades de leur cycle œstral. La composition chimique de ces urines a été déterminée par chromatographie en phase gazeuse couplée à la spectrométrie de masse. La comparaison des profils chimiques urinaires au cours de cycle œstral a permis l’identification d’une série de composés présents de façon spécifique aux stades pré-œstrus et/ou œstrus chez certains animaux. Une grande variabilité interindividuelle et entre les collectes a été observée. Aucune protéine impliquée dans la liaison des composés œstrus-spécifiques identifiés n’a pu être mise en évidence dans les urines des femelles émettrices. Chez l’animal détecteur, plusieurs isoformes de la protéine bovine de liaison aux odeurs (OBPb) ont pu être mises en évidence dans les tissus olfactifs. Des études biochimiques ont confirmé la phosphorylation d’un des variants de l’OBPb et la présence d’une chaîne N-glycannique sur la partie N-terminale de la protéine. L’existence d’une diversité d’OBPb suggère une spécificité de liaison de chaque isoforme vis-à-vis des ligands odorants. Leur propriété de liaison a été quantifiée par spectroscopie de fluorescence, ouvrant ainsi la voie à des études de relations structure-fonction de ces OBP.

Background: DNA methylation (DNAm) plays an important functional role and is influenced by genetic variants known as methylation QTLs (meQTLs). The majority of meQTL studies have been conducted in human blood. Despite its unique landscape, the genetic regulation of sperm DNAm remains largely unexplored. In this study, we leveraged DNAm measured in sperm from 405 Holstein bulls using reduced representation bisulfite sequencing (RRBS) and performed sequence-level genome-wide association studies for 166,985 variable CpGs (s.d. >5%). We reported heritability estimates and have mapped both cis-meQTLs and trans-meQTLs. Results: Heritability estimates ranged from 0 to 1 and averaged 0.26 across all selected CpGs, with 76% of estimates above 0.1. The meQTL mapping revealed that 32.9% of the CpGs had a cis-meQTL, 3.6% had a trans-meQTL and 1.0% had both cis- and trans-meQTLs. The cis-CpGs were located on average 261 kb (absolute mean) from their cis-meQTL top SNPs (defined by the most significant association). MeQTLs were enriched in featured genomic annotations, including regions surrounding transcription start sites and ATAC-seq peaks. We also identified spurious trans-associations by analyzing data across multiple genome assemblies, including the construction of a partial pangenome. Additionally, eight trans-meQTL hotspots, defined as variants associated with at least 30 trans-CpGs, were identified and overlapped with genes involved in epigenetic regulation. Using peripheral blood mononuclear cell DNAm from 54 out of the 405 bulls, we did not observe a similar effect of the trans-meQTL hotspots to that one observed in sperm. Conclusions: For the first time, meQTLs have been detected and characterized in bovine sperm, contributing to a better understanding of the transmission of paternally inherited DNAm marks. These findings provide useful information for further research aimed at integrating epigenetic information into the prediction of performance traits.

Despite passing stringent quality control, bulls used in artificial insemination can vary significantly in their fertility, emphasizing the need for reliable markers of sperm quality. This study aimed to identify sperm proteins acting as biomarkers of fertility in 2 different populations of dairy bulls classified based on their field fertility. Semen was collected and cryopreserved from: 54 Holstein bulls located in Ireland, classified according to fertility indexes as low fertility (LF, n = 23), medium fertility (n = 14), or high fertility (HF, n = 17); and 18 Holstein bulls located in Denmark, classified as LF (n = 8) or HF (n = 10). The proteome was measured through liquid chromatography-mass spectrometry and data were analyzed with the R software. Differentially abundant proteins between HF and LF bulls and biomarker proteins were determined through a modified t-test and random forest, respectively, selecting 301 differentially abundant proteins and 34 biomarker proteins. The predictive ability of the 34 biomarkers was evaluated employing support vector machine as the classifier, using their abundance levels in the Irish bulls to train the model and in the Danish bulls for validation. The prediction accuracy was 94.4%, with only one HF bull misclassified, corresponding to the lowest fertility index bull in the HF group. The biomarkers more abundant in sperm of HF bulls enriched axoneme assembly and sperm motility (false discovery rate <0.05), according to functional analysis. In conclusion, a robust model coupled with the application of appropriate bioinformatic tools allowed the identification of functionally relevant sperm proteins predictive of the fertility of Holstein bulls used in artificial insemination.

Background: Conflicting results regarding alterations to sperm DNA methylation in cases of spermatogenesis defects, male infertility and poor developmental outcomes have been reported in humans. Bulls used for artificial insemination represent a relevant model in this field, as the broad dissemination of bull semen considerably alleviates confounding factors and enables the precise assessment of male fertility. This study was therefore designed to assess the potential for sperm DNA methylation to predict bull fertility. Results: A unique collection of 100 sperm samples was constituted by pooling 2–5 ejaculates per bull from 100 Montbéliarde bulls of comparable ages, assessed as fertile (n = 57) or subfertile (n = 43) based on non-return rates 56 days after insemination. The DNA methylation profiles of these samples were obtained using reduced representation bisulfite sequencing. After excluding putative sequence polymorphisms, 490 fertility-related differentially methylated cytosines (DMCs) were identified, most of which were hypermethylated in subfertile bulls. Interestingly, 46 genes targeted by DMCs are involved in embryonic and fetal development, sperm function and maturation, or have been related to fertility in genome-wide association studies; five of these were further analyzed by pyrosequencing. In order to evaluate the prognostic value of fertility-related DMCs, the sperm samples were split between training (n = 67) and testing (n = 33) sets. Using a Random Forest approach, a predictive model was built from the methylation values obtained on the training set. The predictive accuracy of this model was 72% on the testing set and 72% on individual ejaculates collected from an independent cohort of 20 bulls. Conclusion: This study, conducted on the largest set of bull sperm samples so far examined in epigenetic analyses, demonstrated that the sperm methylome is a valuable source of male fertility biomarkers. The next challenge is to combine these results with other data on the same sperm samples in order to improve the quality of the model and better understand the interplay between DNA methylation and other molecular features in the regulation of fertility. This research may have potential applications in human medicine, where infertility affects the interaction between a male and a female, thus making it difficult to isolate the male factor.

The water vole Arvicola terrestris is endemic to Europe where its outbreak generates severe economic losses for farmers. Our project aimed at characterising putative chemical signals used by this species, to develop new sustainable methods for population control that could also be used for this species protection in Great Britain. The water vole, as well as other rodents, uses specific urination sites as territorial and sex pheromone markers, still unidentified. Lateral scent glands and urine samples were collected from wild males and females caught in the field, at different periods of the year. Their volatile composition was analysed for each individual and not on pooled samples, revealing a specific profile of flank glands in October and a specific profile of urinary volatiles in July. The urinary protein content appeared more contrasted as males secrete higher levels of a lipocalin than females, whenever the trapping period. We named this protein arvicolin. Male and female liver transcript sequencing did not identify any expression of other odorant-binding protein sequence. This work demonstrates that even in absence of genome, identification of chemical signals from wild animals is possible and could be helpful in strategies of species control and protection.

Background: Small ungulates (sheep and goat) display a seasonal breeding, characterised by two successive periods, sexual activity (SA) and sexual rest (SR). Odours emitted by a sexually active male can reactivate the ovulatory cycle of anoestrus females. The plasticity of the olfactory system under these hormonal changes has never been explored at the peripheral level of odours reception. As it was shown in pig that the olfactory secretome (proteins secreted in the nasal mucus) could be modified under hormonal control, we monitored its composition in females of both species through several reproductive seasons, thanks to a non-invasive sampling of olfactory mucus. For this purpose, two-dimensional gel electrophoresis (2D-E), western-blot with specific antibodies, MALDI-TOF and high-resolution (nano-LC-MS/MS) mass spectrometry, RACE-PCR and molecular modelling were used. Results: In both species the olfactory secretome is composed of isoforms of OBP-like proteins, generated by post-translational modifications, as phosphorylation, N-glycosylation and O-GlcNAcylation. Important changes were observed in the olfactory secretome between the sexual rest and the sexual activity periods, characterised in ewe by the specific expression of SAL-like proteins and the emergence of OBPs O-GlcNAcylation. In goat, the differences between SA and SR did not come from new proteins expression, but from different post-translational modifications, the main difference between the SA and SR secretome being the number of isoforms of each protein. Proteomics data are available via ProteomeXchange with identifier PXD014833. Conclusion: Despite common behaviour, seasonal breeding, and genetic resources, the two species seem to adapt their olfactory equipment in SA by different modalities: the variation of olfactory secretome in ewe could correspond to a specialization to detect male odours only in SA, whereas in goat the stability of the olfactory secretome could indicate a constant capacity of odours detection suggesting that the hallmark of SA in goat might be the emission of specific odours by the sexually active male. In both species, the olfactory secretome is a phenotype reflecting the physiological status of females, and could be used by breeders to monitor their receptivity to the male effect.

Odorant-binding proteins (OBP) are secreted in the nasal mucus at the vicinity of olfactory receptors (ORs). They act, at least, as an interface between hydrophobic and volatile odorant molecules and the hydrophilic medium bathing the ORs. They have also been hypothesized to be part of the molecular coding of odors and pheromones, by forming specific complexes with odorant molecules that could ultimately stimulate ORs to trigger the olfactory transduction cascade. In a previous study, we have evidenced that pig olfactory secretome was composed of numerous olfactory binding protein isoforms, generated by O-GlcNAcylation and phosphorylation. In addition, we have shown that recombinant OBP (stricto sensu) produced in yeast is made up of a mixture of isoforms that differ in their phosphorylation pattern, which in turn determines binding specificity. Taking advantage of the high amount of OBP secreted by a single animal, we performed a similar study, under exactly the same experimental conditions, on native isoforms isolated from pig, Sus scrofa, nasal tissue. Four fractions were obtained by using strong anion exchange HPLC. Mapping of phosphorylation and O-GlcNAcylation sites by CID-nanoLC-MS/MS allowed unambiguous localization of phosphosites at S13 and T122 and HexNAc sites at S13 and S19. T112 or T115 could also be phosphorylated. BEMAD analysis suggested extra phosphosites located at S23, S24, S41, S49, S57, S67, and T71. Due to the very low stoichiometry of GlcNAc-peptides and phosphopeptides, these sites were identified on total mixture of OBP isoforms instead of HPLC-purified OBP isoforms. Nevertheless, binding properties of native OBP isoforms to specific ligands in S. scrofa were monitored by fluorescence spectroscopy. Recombinant phosphorylated OBP-Pichia isoforms bind steroids and fatty acids with slight differences. Native isoforms, that are phosphorylated but also O-GlcNAcylated show radically different binding affinities for the same compounds, which strongly suggests that O-GlcNAcylation increases the binding specificity of OBP isoforms. These findings extend the role of O-GlcNAc in regulating the function of proteins involved in many mechanisms of metabolic homeostasis, including extracellular signaling in olfaction. Data is available via ProteomeXChange with identifier PXD011371.

Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. Accordingly, perturbations in the establishment of DNA methylation patterns during male germ cell differentiation have been associated with infertility in several species. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bull spermatozoa is still scarce. The purpose of this study was therefore to characterize the bull sperm methylome relative to both bovine somatic cells and the sperm of other mammals through a multiscale analysis. The quantification of DNA methylation at CCGG sites using luminometric methylation assay (LUMA) highlighted the undermethylation of bull sperm compared to the sperm of rams, stallions, mice, goats and men. Total blood cells displayed a similarly high level of methylation in bulls and rams, suggesting that undermethylation of the bovine genome was specific to sperm. Annotation of CCGG sites in different species revealed no striking bias in the distribution of genome features targeted by LUMA that could explain undermethylation of bull sperm. To map DNA methylation at a genome-wide scale, bull sperm was compared with bovine liver, fibroblasts and monocytes using reduced representation bisulfite sequencing (RRBS) and immunoprecipitation of methylated DNA followed by microarray hybridization (MeDIP-chip). These two methods exhibited differences in terms of genome coverage, and consistently, two independent sets of sequences differentially methylated in sperm and somatic cells were identified for RRBS and MeDIP-chip. Remarkably, in the two sets most of the differentially methylated sequences were hypomethylated in sperm. In agreement with previous studies in other species, the sequences that were specifically hypomethylated in bull sperm targeted processes relevant to the germline differentiation program (piRNA metabolism, meiosis, spermatogenesis) and sperm functions (cell adhesion, fertilization), as well as satellites and rDNA repeats. These results highlight the undermethylation of bull spermatozoa when compared with both bovine somatic cells and the sperm of other mammals, and raise questions regarding the dynamics of DNA methylation in bovine male germline. Whether sperm undermethylation has potential interactions with structural variation in the cattle genome may deserve further attention.