A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific

peer-reviewedBackground: 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.Background: Spermatozoa have a remarkable epigenResults: 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. Conclusions: 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. 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

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Les petits ARN non codants du spermatozoïde bovin : de potentiels biomarqueurs de la fertilité mâle ?

Most dairy cows are inseminated and sub-fertile bulls can have an overall dramatic negative impact on the sustainability of the dairy sector. The bull fertility prediction, through efficient quality control (QC) procedures is thus crucial for the cattle breeding sector. Over the last decades, several biomarkers and quality control procedures have been proposed to guarantee semen fertility, including assessment of key functional parameters using flow cytometry. However, their relevance for routine QC has yet to be ascertained and their predictive value is too limited to confidently discard subfertile bulls. Thus, studies have been conducted by LabCom SeQuaMol to explore to what extent the bull sperm epigenome may be relevant to improve fertility prediction. In this respect, this PhD thesis was focused on small noncoding RNAs (sncRNAs), which have been shown to play a role in spermatogenesis and fertilization. We aimed at improving the scientific knowledge on bull sperm sncRNA content in order to develop a routine tool to predict bull fertility. The work has been organized according to three main themes: providing a comprehensive overview of cattle sperm sncRNA, studying their expression dynamics during sperm maturation along epididymis and identifying biomarkers associated with bull fertility.L’utilisation de taureaux subfertiles entraîne des pertes de production, une augmentation du taux de réforme des vaches pour infertilité et un gaspillage des ressources. C’est pourquoi le contrôle qualité de la semence utilisée en Insémination Animale est une préoccupation majeure des entreprises de sélection. Divers protocoles de contrôle qualité ont été développés au cours des dernières décennies, exploitant des biomarqueurs et des paramètres fonctionnels clés mesurés par cytométrie en flux. Toutefois, la capacité prédictive de ces tests reste limitée et ne permet pas d’identifier les taureaux subfertiles. C’est pourquoi des travaux ont été entrepris dans le cadre du LabCom SeQuaMol, afin d’explorer l’intérêt de l’épigénome spermatique comme biomarqueur de fertilité chez les taureaux. Ainsi, les travaux menés au cours de cette thèse ont portés sur les petits ARNs non codants (sncRNA), dont le rôle au cours de la spermatogénèse et de la fécondation a été suggéré et démontré. L’amélioration des connaissances sur le contenu en sncRNA du spermatozoïde bovin éjaculé avait comme objectif pratique de développer un outil permettant de fiabiliser la prédiction en routine de la fertilité des taureaux. Les travaux ont été organisés selon trois axes : l’établissement du contenu exhaustif en sncRNA du sperme bovin éjaculé, l’étude de la dynamique d’expression de ces sncRNAs au cours de la maturation épididymaire et l'identification in fine de biomarqueurs associés à la fertilité des taureaux

Sperm-borne sncRNAs: potential biomarkers for semen fertility?

International audienceSemen infertility or sub-fertility, whether in humans or livestock species, remains a major concern for clinicians and technicians involved in reproduction. Indeed, they can cause tragedies in human relationships or have a dramatic overall negative impact on the sustainability of livestock breeding. Understanding and predicting semen fertility issues is therefore crucial and quality control procedures as well as biomarkers have been proposed to ensure sperm fertility. However, their predictive values appeared to be too limited and additional relevant biomarkers are still required to diagnose sub-fertility efficiently. During the last decade, the study of molecular mechanisms involved in spermatogenesis and sperm maturation highlighted the regulatory role of a variety of small non-coding RNAs (sncRNAs) and led to the discovery that sperm sncRNAs comprise both remnants from spermatogenesis and post-testicular sncRNAs acquired through interactions with extracellular vesicles along epididymis. This has led to the hypothesis that sncRNAs may be a source of relevant biomarkers, associated either with sperm functionality or embryo development. This review aims at providing a synthetic overview of the current state of knowledge regarding implication of sncRNA in spermatogenesis defects and their putative roles in sperm maturation and embryo development, as well as exploring their use as fertility biomarkers

The epigenome of male germ cells and the programming of phenotypes in cattle

International audience• Bull semen is a commercial product widely used forartificial insemination.• During the differentiation of male germ cells intospermatozoa, there are several windows of epigenomesensitivity to environmental factors.• The epigenome of bull sperm exhibits both conservedfeatures and interindividual variations, some of whichare associated with fertility.• The paternal epigenome contributes to embryo development and to programming the phenotype of offspring

Multi-omics data integration for the identification of biomarkers for bull fertility.

Bull fertility is an important economic trait, and the use of subfertile semen for artificial insemination decreases the global efficiency of the breeding sector. Although the analysis of semen functional parameters can help to identify infertile bulls, no tools are currently available to enable precise predictions and prevent the commercialization of subfertile semen. Because male fertility is a multifactorial phenotype that is dependent on genetic, epigenetic, physiological and environmental factors, we hypothesized that an integrative analysis might help to refine our knowledge and understanding of bull fertility. We combined -omics data (genotypes, sperm DNA methylation at CpGs and sperm small non-coding RNAs) and semen parameters measured on a large cohort of 98 Montbéliarde bulls with contrasting fertility levels. Multiple Factor Analysis was conducted to study the links between the datasets and fertility. Four methodologies were then considered to identify the features linked to bull fertility variation: Logistic Lasso, Random Forest, Gradient Boosting and Neural Networks. Finally, the features selected by these methods were annotated in terms of genes, to conduct functional enrichment analyses. The less relevant features in -omics data were filtered out, and MFA was run on the remaining 12,006 features, including the 11 semen parameters and a balanced proportion of each type of-omics data. The results showed that unlike the semen parameters studied the-omics datasets were related to fertility. Biomarkers related to bull fertility were selected using the four methodologies mentioned above. The most contributory CpGs, SNPs and miRNAs targeted genes were all found to be involved in development. Interestingly, fragments derived from ribosomal RNAs were overrepresented among the selected features, suggesting roles in male fertility. These markers could be used in the future to identify subfertile bulls in order to increase the global efficiency of the breeding sector

The effect of adjusting settings within a Computer-Assisted Sperm Analysis (CASA) system on bovine sperm motility and morphology results

International audienceSemen 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 cutoff values significantly (p<0.05) reduced the percentage of detected progressive spermatozoa, in egg yolk extender from 49.5±15.2, 37.2±11.9 to 11.9±5.3%, and in clear extender from 51.9±9.1, 35.8±7.3 to 10.0±2.4%, respectively. In clear extender only, the modification of droplet proximal head length significantly affected the detection of normal sperm percentages (88.0± 4.7 to 95.0±0.6 and 96.0±0.6%) and of the percentage of detected proximal droplets (12.2±4.7, 2.5±2.7 to 0.6±0.2%) for Low, Medium and High values respectively (p<0.05). The identification of sensitivity within the CASA system to changes in set parameters then led to the determination of an optimal IVOS II setting. The existing variability among centers for these phenotypes was reduced when the standardized settings were applied across different CASA units. The results clearly show the importance of applied settings for the final CASA results and emphasize the need for standardized settings to obtain comparable data

Structural and Functional Characterization of a Testicular Long Non-coding RNA (4930463O16Rik) Identified in the Meiotic Arrest of the Mouse Topaz1–/– Testes

International audienceSpermatogenesis involves coordinated processes, including meiosis, to produce functional gametes. We previously reported Topaz1 as a germ cell-specific gene highly conserved in vertebrates. Topaz1 knockout males are sterile with testes that lack haploid germ cells because of meiotic arrest after prophase I. To better characterize Topaz1 –/– 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 or 4930463O16Rik produced the same enrichment clusters in mutant testes despite a contrasted phenotype on male fertility. In conclusion, although Topaz1 is essential for the meiosis in male germ cells and regulate the expression of numerous lncRNAs, these studies have identified a Topaz1 regulated lncRNA ( 4930463O16Rik ) that is key for both sperm production and motility