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Estrogens are steroid hormones produced by the aromatization of androgens by the aromatase enzyme, encoded by theCYP19A1gene. Although generally referred to as “female sex hormones”, estrogen is also produced in the adult testes of many mammals, including humans. To better understand the function of estrogens in the male, we used the rabbit model which is an important biomedical model. First, the expression ofCYP19A1was localized, demonstrating that testicular estrogens are produced by meiotic germ cells inside the seminiferous tubules. Next, the cells expressing ESR1 and ESR2 were identified, showing that estrogens could exert their function on post-meiotic germ cells in the tubules and play a role during sperm maturation, since ESR1 and ESR2 were detected in thecaudaepididymis. Then, CRISPR/Cas9CYP19A1−/−genetically modified rabbits were analyzed.CYP19A1−/−males showed decreased fertility with lower sperm count associated with hypo-spermatogenesis and lower spermatid number. Germ/sperm cell DNA methylation was unchanged, while sperm parameters were affected asCYP19A1−/−males exhibited reduced sperm motility associated with increased flagellar defects. In conclusion, testicular estrogens could be involved in the spermatocyte-spermatid transition in the testis, and in the acquisition of sperm motility in the epididymis.

Background During epididymal transit, spermatozoa go through several functional maturation steps, resulting from interactions with epididymal secretomes specific to each region. In particular, the sperm membrane is under constant remodeling, with sequential attachment and shedding of various molecules provided by the epididymal lumen fluid and epididymosomes, which also deliver sncRNA cargo to sperm. As a result, the payload of sperm sncRNAs changes during the transit from the epididymis caput to the cauda. This work was designed to study the dynamics of cattle sperm sncRNAs from spermatogenesis to final maturation. Results Comprehensive catalogues of sperm sncRNAs were obtained from testicular parenchyma, epididymal caput, corpus and cauda, as well as ejaculated semen from three Holstein bulls. The primary cattle sncRNA sperm content is markedly remodeled as sperm mature along the epididymis. Expression of piRNAs, which are abundant in testis parenchyma, decreases dramatically at epididymis. Conversely, sperm progressively acquires miRNAs, rsRNAs, and tsRNAs along epididymis, with regional specificities. For instance, miRNAs and tsRNAs are enriched in epididymis cauda and ejaculated sperm, while rsRNA expression peaks at epididymis corpus. In addition, epididymis corpus contains mainly 20 nt long piRNAs, instead of 30 nt in all other locations. Beyond the bulk differences in abundance of sncRNAs classes, K-means clustering was performed to study their spatiotemporal expression profile, highlighting differences in specific sncRNAs and providing insights into their putative biological role at each maturation stage. For instance, Gene Ontology analyses using miRNA targets highlighted enriched processes such as cell cycle regulation, response to stress and ubiquitination processes in testicular parenchyma, protein metabolism in epididymal sperm, and embryonic morphogenesis in ejaculated sperm. Conclusions Our findings confirm that the sperm sncRNAome does not simply reflect a legacy of spermatogenesis. Instead, sperm sncRNA expression shows a remarkable level of plasticity resulting probably from the combination of multiple factors such as loss of the cytoplasmic droplet, interaction with epididymosomes, and more surprisingly, the putative in situ production and/or modification of sncRNAs by sperm. Given the suggested role of sncRNA in epigenetic trans-generational inheritance, our detailed spatiotemporal analysis may pave the way for a study of sperm sncRNAs role in embryo development.

Enhanced pre-pubertal nutrition in Holstein bulls increased reproductive hormone production and sperm production potential with no negative effects on sperm quality. However, recent trends in human epigenetic research have identified pre-pubertal period to be critical for epigenetic reprogramming in males. Our objective was to evaluate the methylation changes in sperm of bulls exposed to different pre-pubertal diets. One-week-old Holstein bull calves (n = 9), randomly allocated to 3 groups, were fed either a high, medium or low diet (20%, 17% or 12.2% crude protein and 67.9%, 66% or 62.9% total digestible nutrients, respectively) from 2 to 32 weeks of age, followed by medium nutrition. Semen collected from bulls at two specific time points, i.e. 55-59 and 69-71 weeks, was diluted, cryopreserved and used for reduced representation bisulfite sequencing. Differential methylation was detected for dietary treatment, but minimal differences were detected with age. The gene ontology term, "regulation of Rho protein signal transduction", implicated in sperm motility and acrosome reaction, was enriched in both low-vs-high and low-vs-medium datasets. Furthermore, several genes implicated in early embryo and foetal development showed differential methylation for diet. Our results therefore suggest that sperm epigenome keeps the memory of diet during pre-pubertal period in genes important for spermatogenesis, sperm function and early embryo development.

Estrogens are steroid hormones produced by the aromatization of androgens by the aromatase enzyme, encoded by the CYP19A1 gene. Although generally referred to as “female sex hormones”, estrogen is also produced in the adult testes of many mammals, including humans. To better understand the function of estrogens in the male, we used the rabbit model which is an important biomedical model. First, the expression of CYP19A1 transcripts was localized mainly in meiotic germ cells. Thus, testicular estrogen appears to be produced inside the seminiferous tubules. Next, the cells expressing ESR1 and ESR2 were identified, showing that estrogens could exert their function on post-meiotic germ cells in the tubules and play a role during sperm maturation, since ESR1 and ESR2 were detected in the cauda epididymis. Then, CRISPR/Cas9 CYP19A1−/− genetically modified rabbits were analyzed. CYP19A1−/− males showed decreased fertility with lower sperm count associated with hypo-spermatogenesis and lower spermatid number. Germ/sperm cell DNA methylation was unchanged, while sperm parameters were affected as CYP19A1−/− males exhibited reduced sperm motility associated with increased flagellar defects. In conclusion, testicular estrogens could be involved in the spermatocyte–spermatid transition in the testis, and in the acquisition of sperm motility in the epididymis.

Semen motility is the most widely recognized semen quality parameter used by Artificial Insemination (AI) centers. With the increasing worldwide export of semen between AI centers there is an increasing need for standardized motility assessment methods. Computer-Assisted Sperm Analysis (CASA) technology is thought to provide an objective motility evaluation; however, results can still vary between laboratories. The aim of present study was to verify the impact of different setting values of the CASA IVOS II on motility, concentration, and morphology of bovine semen samples frozen in an extender with or without egg yolk and then decide on optimal settings for a further validation step across AI centers. Semen straws from 30 different bulls were analyzed using IVOS II with twelve modified settings. No significant changes were observed in semen concentration, percentage of motile sperm or kinetic results for either extender type. However, increasing settings for both STR and VAP progressive (%) from Low, Medium, and High cut-off values significantly (p

Spermatogenesis involves coordinated processes, including meiosis, to produce functional gametes. We previously reportedTopaz1as a germ cell-specific gene highly conserved in vertebrates.Topaz1knockout males are sterile with testes that lack haploid germ cells because of meiotic arrest after prophase I. To better characterizeTopaz1–/–testes, we used RNA-sequencing analyses at two different developmental stages (P16 and P18). The absence of TOPAZ1 disturbed the expression of genes involved in microtubule and/or cilium mobility, biological processes required for spermatogenesis. Moreover, a quarter of P18 dysregulated genes are long non-coding RNAs (lncRNAs), and three of them are testis-specific and located in spermatocytes, their expression starting between P11 and P15. The suppression of one of them,4939463O16Rik, did not alter fertility although sperm parameters were disturbed and sperm concentration fell. The transcriptome of P18-4939463O16Rik–/–testes was altered and the molecular pathways affected included microtubule-based processes, the regulation of cilium movement and spermatogenesis. The absence of TOPAZ1 protein or4930463O16Rikproduced the same enrichment clusters in mutant testes despite a contrasted phenotype on male fertility. In conclusion, althoughTopaz1is essential for the meiosis in male germ cells and regulate the expression of numerous lncRNAs, these studies have identified aTopaz1regulated lncRNA (4930463O16Rik) that is key for both sperm production and motility.

Spermatogenesis comprises a coordinated process, including meiosis, to produce fertile male gametes. Previous study reported that Topaz1 is a germ cell specific gene highly conserved in vertebrates. Topaz1 knockout male mice are sterile. The mutant testes lack haploid germ cells and meiosis arrests at the first-division prophase-metaphase transition. Here, in order to better characterize the testicular phenotype of Topaz1-/- mice, we used RNA-seq analyses at two different developmental stages. At postnatal days 16 (P16), 205 genes were differentially expressed genes (DEGs) in Topaz1-/- testes. They suggest stress conditions in mutant testes. At P18, the number of DEGs was increased 10-fold and 90% were down-regulated. The absence of Topaz1 seems to disturb the expression of genes involved in microtubule and/or cilium mobility, spermatogenesis and first meiotic division during the transition from prophase to metaphase. This is consistent with the Topaz1-/- testis phenotype where microtubule networks and centrosomes are disrupted. Moreover, a quarter of P18-DEGs are long non-coding RNAs (lncRNAs). Three of them, down-regulated at P16 and P18, were studied. They are testis-specific, located in spermatocytes and their expression starts between P11 and P15. We report here the effects of the suppression of one of these lncRNAs, 4939463O16Rik. The mouse fertility is not affected although the sperm parameters are disturbed. Transcriptome of P18-4939463O16Rik-/- testes is altered and the affected molecular pathways include microtubule-based process, regulation of cilium movement, spermatogenesis, male gamete differentiation. The absence of TOPAZ1 protein or of 4930463O16Rik lncRNA showed the same enrichment clusters in mutant testes despite a contrasted phenotype on the male fertility. In conclusion, Topaz1 is an essential gene for male fertility in mice and seems to stabilize the expression of many lncRNAs. Its absence leads to meiotic arrest. Suppression of one lncRNA is dispensable for mouse fertility but is necessary during terminal differentiation of male gametes. Author SummaryThe Topaz1 gene was initially characterized during the meiotic initiation in the sheep fetal ovary. In order to determine its function, the KO of the murine gene was performed. In this species, only males are sterile and spermatogenesis is blocked before the first meiotic division. Here, we show that cytoskeletal elements are strongly disturbed in mutant testes, a sign that these elements take an important function in spermatogenesis. While the mitotic spindle of spermatogonia is normal, the meiotic spindle of spermatocytes is a hemispindle-shaped and the homologous chromosome pairs cannot position themselves on the equatorial plate. In addition, lncRNAs represent 25% of genes whose testis expression varies significantly due to the absence of Topaz1. This suggests a key role of these factors in spermatogenesis. Largely testis specific, they could be involved in spermatogenesis with a more or less critical role on the mouse fertility, probably also depending of their redundancies.

Background Mature sperm carry thousands of RNAs, including mRNAs, lncRNAs, tRNAs, rRNAs and sncRNAs, though their functional significance is still a matter of debate. Growing evidence suggests that sperm RNAs, especially sncRNAs, are selectively retained during spermiogenesis or specifically transferred during epididymis maturation, and are thus delivered to the oocyte at fertilization, providing resources for embryo development. However , a deep characterization of the sncRNA content of bull sperm and its expression profile across breeds is currently lacking. To fill this gap, we optimized a guanidinium–Trizol total RNA extraction protocol to prepare high-quality RNA from frozen bull sperm collected from 40 representative bulls from six breeds. Deep sequencing was performed (40 M single 50-bp reads per sample) to establish a comprehensive repertoire of cattle sperm sncRNA. Results Our study showed that it comprises mostly piRNAs (26%), rRNA fragments (25%), miRNAs (20%) and tRNA fragments (tsRNA, 14%). We identified 5p-halves as the predominant tsRNA subgroup in bull sperm, originating mostly from Gly and Glu isoacceptors. Our study also increased by ~ 50% the sperm repertoire of known miRNAs and identified 2022 predicted miRNAs. About 20% of sperm miRNAs were located within genomic clusters, expanding the list of known polycistronic pri-miRNA clusters and defining several networks of co-expressed miRNAs. Strikingly, our study highlighted the great diversity of isomiRs, resulting mainly from deletions and non-templated additions (A and U) at the 3p end. Substitutions within miRNA sequence accounted for 40% of isomiRs, with G>A, U>C and C>U substitutions being the most frequent variations. In addition, many sncRNAs were found to be differentially expressed across breeds. Conclusions Our study provides a comprehensive overview of cattle sperm sncRNA, and these findings will pave the way for future work on the role of sncRNAs in embryo development and their relevance as biomarkers of semen fertility.

Gonad differentiation is a crucial step conditioning the future fertility of individuals and most of the master genes involved in this process have been investigated in detail. However, transcriptomic analyses of developing gonads from different animal models have revealed that hundreds of genes present sexually dimorphic expression patterns. DMXL2 was one of these genes and its function in mammalian gonads was unknown. We therefore investigated the phenotypes of total and gonad-specific Dmxl2 knockout mouse lines. The total loss-of-function of Dmxl2 was lethal in neonates, with death occurring within 12 hours of birth. Dmxl2-knockout neonates were weak and did not feed. They also presented defects of olfactory information transmission and severe hypoglycemia, suggesting that their premature death might be due to global neuronal and/or metabolic deficiencies. Dmxl2 expression in the gonads increased after birth, during follicle formation in females and spermatogenesis in males. DMXL2 was detected in both the supporting and germinal cells of both sexes. As Dmxl2 loss-of-function was lethal, only limited investigations of the gonads of Dmxl2 KO pups were possible. They revealed no major defects at birth. The gonadal function of Dmxl2 was then assessed by conditional deletions of the gene in gonadal supporting cells, germinal cells, or both. Conditional Dmxl2 ablation in the gonads did not impair fertility in males or females. By contrast, male mice with Dmxl2 deletions, either throughout the testes or exclusively in germ cells, presented a subtle testicular phenotype during the first wave of spermatogenesis that was clearly detectable at puberty. Indeed, Dmxl2 loss-of-function throughout the testes or in germ cells only, led to sperm counts more than 60% lower than normal and defective seminiferous tubule architecture. Transcriptomic and immunohistochemichal analyses on these abnormal testes revealed a deregulation of Sertoli cell phagocytic activity related to germ cell apoptosis augmentation. In conclusion, we show that Dmxl2 exerts its principal function in the testes at the onset of puberty, although its absence does not compromise male fertility in mice., Author summary DMXL2 gene dysfunction underlies various human diseases, including breast cancer, non-syndromic hearing loss, and polyendocrine-polyneuropathy syndrome, demonstrating the large range of potential actions of DMXL2. We show here that Dmxl2 expression is crucial for survival in mice, as neonates die within hours of birth when this gene is inactivated. The transmission of olfactory information is affected, leading to an absence of suckling and impaired feeding. Severe hypoglycemia is also observed in male neonates. We observed Dmxl2 expression in several organs, including the brain, heart and adrenal glands, potentially corresponding to some of the phenotypes observed in Dmxl2-deficient pups. We also described Dmxl2 expression in the reproductive tracts and gonads and showed that Dmxl2 inactivation specifically in the testes has a significant effect on the initial waves of spermatogenesis, resulting in very low levels of sperm production at puberty. Our results suggest that DMXL2 deficits should be considered in men with impaired fertility, as pathogenic variants of this gene may be associated with male infertility in humans.

International audience

International audience; Nonsteroidal antiinflammatory drugs and analgesic drugs, such as N-acetyl- p-aminophenol (APAP; acetaminophen, paracetamol), are widely used by pregnant women. Accumulating evidence has indicated that these molecules can favor genital malformations in newborn boys and reproductive disorders in adults. However, the consequences on postnatal testis development and adult reproductive health after exposure during early embryogenesis are still unknown. Using the mouse model, we show that in utero exposure to therapeutic doses of the widely used APAP-ibuprofen combination during the sex determination period leads to early differentiation and decreased proliferation of male embryonic germ cells, and early 5-methylcytosine and extracellular matrix protein deposition in 13.5 d postcoitum exposed testes. Consequently, in postnatal testes, Sertoli-cell maturation is delayed, the Leydig-cell compartment is hyperplasic, and the spermatogonia A pool is decreased. This results in a reduced production of testosterone and in epididymal sperm parameter defects. We observed a reduced sperm count (19%) in utero-exposed (F0) adult males and also a reduced sperm motility (40%) in their offspring (F1) when both parents were exposed, which leads to subfertility among the 6 mo old F1 animals. Our study suggests that the use of these drugs during the critical period of sex determination affects the germ-line development and leads to adverse effects that could be passed to the offspring.-Rossitto, M., Marchive, C., Pruvost, A., Sellem, E., Ghettas, A., Badiou, S., Sutra, T., Poulat, F., Philibert, P., Boizet-Bonhoure, B. Intergenerational effects on mouse sperm quality after in utero exposure to acetaminophen and ibuprofen.

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La présence de taureaux subfertiles sur le marché de l’insémination artificielle influence l’efficacité et la durabilité des élevages, en entraînant des pertes de production, une augmentation du taux de réforme des génisses et un gaspillage des ressources. Aujourd’hui, l’évaluation de la fertilité des taureaux, basée sur l’analyse combinée de marqueurs génétiques et de marqueurs morphologiques, cinétiques et métaboliques de la semence, n’est pas suffisante pour identifier les taureaux subfertiles avant leur entrée en production. Notre objectif est d’utiliser l’épigénome de la semence bovine pour identifier de nouveaux marqueurs fiables et précoces de la fertilité mâle.Nous avons mené une analyse pan-génomique de la méthylation de l’ADN spermatique par Reduced Representation Bisulfite Sequencing (RRBS). La préparation des librairies ainsi que l‘analyse bioinformatique et statistique des données ont été automatisées au laboratoire, rendant possible l’étude d’une cohorte de 58 taureaux de races Holstein et Montbéliard. Ces taureaux ont été répartis en trois catégories selon l’adéquation entre une prédiction de fertilité basée sur l’analyse de leur génotype, et leur fertilité réelle sur le terrain : fertiles, subfertiles et déviants (prédits comme fertiles mais subfertiles sur le terrain). Des analyses descriptives réalisées sur l’ensemble des CpG couverts (> 1 million) montrent que les déviants peuvent être distingués des autres animaux chez les Holstein uniquement. En accord avec cette observation, davantage de CpG différentiellement méthylés (DMC) ont été identifiés entre les catégories de fertilité chez les taureaux Holstein que chez les Montbéliard (8884 et 4823, respectivement). De plus, ces DMC présentent une claire tendance à l’hypométhylation chez les déviants Holstein, et sont situés à proximité de gènes impliqués dans le développement embryonnaire précoce. Dans les deux races, les DMC identifiés permettent de prédire de façon robuste le statut de fertilité. Des exemples de validation par pyroséquençage pourront être présentés, soulignant l’importance des paramètres utilisés pour l’analyse statistique des résultats de RRBS. Enfin, nous aborderons la problématique de l’intégration avec le génotype, qui illustre l’effet direct et indirect de polymorphismes de séquence sur la méthylation de l’ADN.En conclusion, cette étude montre que des profils de méthylation de l’ADN spermatique peuvent être associés à la fertilité chez le bovin. Ces résultats seront complétés par l’analyse de microARN (voir Sellem et al.) et de modifications post-traductionnelles d’histones. L’ensemble des biomarqueurs identifiés serviront de base au développement d’outils technologiques et statistiques pouvant être utilisés en routine pour améliorer la prédiction de la fertilité mâle.

Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bovine spermatozoa is still scarce. In the context of genomic selection, more information on the epigenetic features transferred to the embryo alongside the paternal genetic heritage is necessary, in order to improve semen quality control procedures and bull fertility. We characterized the bull sperm methylome relative to bovine fibroblasts and monocytes using reduced restriction bisulfite sequencing (RRBS). A wide majority of differentially methylated cytosines (DMCs) were less methylated in sperm than in somatic cells. Consistent with previous studies in other species, these hypomethylated DMCs were enriched for genes relevant to the germline differentiation program and sperm functions. The most remarkable observation was a dramatic enrichment for repeats and particularly for satellites, which may partly account for the lower global methylation we observed in bull sperm relative to sperm from other species (LUminometric Methylation Assay). We next investigated whether fertility biomarkers could be identified in the sperm methylome. Based on field fertility indicators, we compared fertile ejaculates, subfertile ejaculates and ejaculates from bulls with a disappointing career in contradiction with genomic predictions (Montbéliarde breed, n=10/group). The median age at production was consistent among groups and the phenotypic characterization revealed no significant differences except for the proportion of motile sperm, which was reduced in disappointing bulls. We identified 4823 DMCs targeting genes involved in signaling pathways, angiogenesis and the response to growth factors. Since DNA methylation is affected by the CpG content of the genome and its alteration by DNA polymorphism, an integrative approach was conducted to identify CpGs and SNPs in correlation, which revealed both direct and indirect interactions between the genome and the epigenome.

International audience

Deficiencies in bull mating behaviour have implications for bovine artificial insemination activities. The aim of this study was to identify the compounds present in fluids emitted by cows during oestrus, which could enhance bull libido. Chemical analysis of urine samples from cows led to the characterization of molecules varying specifically at the pre-oestrous and oestrous stages. The synthetic counterpart molecules (1,2-dichloroethylene, squalene, coumarin, 2-butanone, oleic acid) were used to investigate the biological effects on male sexual behaviour and sperm production. When presented to males 2-butanone and oleic acid synthetic molecules significantly lowered mounting reaction time and ejaculation time (-33% and 21% following 2-butanone inhalation, respectively p

Herd fertility is a key factor for the sustainability of cattle farming. However, declining fertility has become one of the main causes of culling and replacement of cows in developed countries. Omics technologies have been successfully developed in cattle, refining our knowledge of molecular mechanisms governing cattle reproduction. However, most studies have failed to establish clear links between the genome and final phenotypes. Further studies will be needed to integrate all Omics data and develop a systems biology modeling approach to tackle this complex biological function.

International audience

National audience; L’épigénome du spermatozoïde mature est unique. En effet, il joue un rôle fondamental dans l’acquisition des propriétés morphologiques et fonctionnelles du spermatozoïde sans être en lien direct avec une activité transcriptionnelle. D’autre part, alors qu’il est le fruit de remaniements épigénétiques ayant démarré dès la vie fœtale, sa destinée est d’être reprogrammé rapidement après la fécondation. Malgré la large diffusion de semence bovine sur le marché de l’insémination artificielle, il existe peu de données sur la méthylation de l’ADN dans le spermatozoïde bovin. Nous avons d’abord caractérisé le méthylome spermatique bovin en comparaison avec des cellules somatiques adultes, et montré que certaines familles de séquences répétées (satellites, rDNA) présentaient un taux de méthylation très faible dans les spermatozoïdes. La surreprésentation de ces séquences dans le génome bovin, ainsi que leur faible méthylation, pourraient expliquer pourquoi les spermatozoïdes bovins présentent un taux de méthylation global plus bas que les spermatozoïdes d’autres espèces de mammifères. Nous avons ensuite étudié les paramètres intrinsèque (race du taureau), physiologique (âge à la production de semence) et pathologique (anomalies morphologiques des spermatozoïdes) associés à des variations du méthylome spermatique. La race, qui se confond avec l’origine géographique des taureaux étudiés (Holstein, Montbéliarde, Normande, Charolais, Blanc Bleu Belge et Abondance), semble modeler le méthylome spermatique au-delà de la présence de polymorphismes de séquence inter-races. Des taureaux de races différentes pourraient donc transmettre à leurs descendants non seulement des patrimoines génétiques différents, mais également des patrimoines épigénétiques différents. De manière plus surprenante, nous avons mis en évidence un effet majeur de l’âge du taureau au moment de la collecte de semence dans toutes les races étudiées. Les variations du méthylome liées à l’âge semblent quantitativement plus importantes que les perturbations associées à des anomalies morphologiques causant la réforme des taureaux, ce qui pose question sur la collecte de semence à un âge de plus en plus précoce dans les races laitières. Ces données montrent que dans leur ensemble, les pratiques de sélection et d’élevage des reproducteurs, ainsi que de collecte de semence, ont un impact majeur sur le patrimoine épigénétique véhiculé par le spermatozoïde. Il semble légitime de s’interroger sur les répercussions potentielles que pourraient avoir ces variations du méthylome spermatique sur le phénotype de la descendance. La mise en place d’un « contrôle qualité épigénétique » systématique de la semence bovine devient plus que jamais une nécessité, en cette période d’intense restructuration du secteur et de forte évolution des pratiques.

Alors que la semence bovine est un produit largement diffusé sur le marché de l’insémination artificielle, il existe peu de données sur la méthylation de l’ADN dans le spermatozoïde bovin. Le méthylome spermatique est pourtant le fruit d’une reprogrammation ayant démarré dès la vie fœtale, et joue un rôle essentiel dans la compaction de la chromatine et la protection du patrimoine génétique paternel. Nous avons donc mené une étude multi-échelle de la méthylation de l’ADN dans le spermatozoïde bovin comparativement à d’autres tissus et espèces.Une analyse globale par LUMA (LUminometric Methylation Assay, voir Prézelin et al.) a d’abord révélé un taux de méthylation extrêmement faible dans les spermatozoïdes bovins (autour de 45%, contre 60-80% pour les tissus somatiques examinés). L’analyse des spermatozoïdes d’autres espèces (béliers, boucs, étalons, verrats, souris mâles et Homme) a montré que cette hypométhylation était une spécificité du spermatozoïde bovin, et qu’elle était indépendante de la race et de l’état frais ou congelé de la semence. Les sites ciblés par LUMA ont été annotés par rapport à différents éléments géniques, aux îlots CpG et aux éléments répétés dans les espèces étudiées, ne révélant aucune particularité du génome bovin qui pourrait fournir des éléments de compréhension. Pour déterminer les séquences hypométhylées, nous avons ensuite comparé le méthylome spermatique avec celui d’autres tissus bovins (fibroblastes, foie, monocytes) en utilisant deux approches pan-génomiques couvrant des régions différentes du génome : RRBS (Reduced Representation Bisulfite Sequencing, voir Perrier et al.) et MeDIP-chip (immunoprécipitation de l’ADN méthylé et hybridation sur une puce de promoteur). Dans les deux cas, un nombre important de régions différenciellement méthylées (DMR, MeDIP) ou de CpG différenciellement méthylés (DMC, RRBS) ont été mis en évidence, dont la grande majorité se trouve être hypométhylée spécifiquement dans les spermatozoïdes (1144 sur 1678 DMR et 137861 sur 174103 DMC). Ces DMR/DMC hypométhylés sont enrichis en gènes de la spermatogenèse et du spliceosome ainsi qu’en séquences répétées de type satellite. Les résultats obtenus ont été validés par pyroséquençage. Si l’hypométhylation du spermatozoïde a occasionnellement été décrite chez l’Homme ou la souris, ou même chez le bovin pour les séquences satellites, l’originalité de ce travail est de montrer que cette hypométhylation est largement plus importante chez le bovin, et d’identifier les séquences hypométhylées en utilisant des approches complémentaires. Ces résultats suggèrent que des mécanismes spécifiques à l’espèce bovine sont à l’œuvre pour la reprogrammation ou le maintien de la méthylation dans la lignée germinale mâle.