Systemic CLIP-seq analysis and game theory approach to model microRNA mode of binding.

microRNAs (miRNAs) associate with Ago proteins to post-transcriptionally silence gene expression by targeting mRNAs. To characterize the modes of miRNA-binding, we developed a novel computational framework, called optiCLIP, which considers the reproducibility of the identified peaks among replicates based on the peak overlap. We identified 98 999 binding sites for mouse and human miRNAs, from eleven Ago2 CLIP-seq datasets. Clustering the binding preferences, we found heterogeneity of the mode of binding for different miRNAs. Finally, we set up a quantitative model, named miRgame, based on an adaptation of the game theory. We have developed a new algorithm to translate the miRgame into a score that corresponds to a miRNA degree of occupancy for each Ago2 peak. The degree of occupancy summarizes the number of miRNA-binding sites and miRNAs targeting each binding site, and binding energy of each miRNA::RNA heteroduplex in each peak. Ago peaks were stratified accordingly to the degree of occupancy. Target repression correlates with higher score of degree of occupancy and number of miRNA-binding sites within each Ago peak. We validated the biological performance of our new method on miR-155-5p. In conclusion, our data demonstrate that miRNA-binding sites within each Ago2 CLIP-seq peak synergistically interplay to enhance target repression.

Recent computational developments on CLIP-seq data analysis and microRNA targeting implications.

Cross-Linking Immunoprecipitation associated to high-throughput sequencing (CLIP-seq) is a technique used to identify RNA directly bound to RNA-binding proteins across the entire transcriptome in cell or tissue samples. Recent technological and computational advances permit the analysis of many CLIP-seq samples simultaneously, allowing us to reveal the comprehensive network of RNA-protein interaction and to integrate it to other genome-wide analyses. Therefore, the design and quality management of the CLIP-seq analyses are of critical importance to extract clean and biological meaningful information from CLIP-seq experiments. The application of CLIP-seq technique to Argonaute 2 (Ago2) protein, the main component of the microRNA (miRNA)-induced silencing complex, reveals the direct binding sites of miRNAs, thus providing insightful information about the role played by miRNA(s). In this review, we summarize and discuss the most recent computational methods for CLIP-seq analysis, and discuss their impact on Ago2/miRNA-binding site identification and prediction with a regard toward human pathologies.

From benchmarking HITS-CLIP peak detection programs to a new method for identification of miRNA-binding sites from Ago2-CLIP data.

Experimental evidence indicates that about 60% of miRNA-binding activity does not follow the canonical rule about the seed matching between miRNA and target mRNAs, but rather a non-canonical miRNA targeting activity outside the seed or with a seed-like motifs. Here, we propose a new unbiased method to identify canonical and non-canonical miRNA-binding sites from peaks identified by Ago2 Cross-Linked ImmunoPrecipitation associated to high-throughput sequencing (CLIP-seq). Since the quality of peaks is of pivotal importance for the final output of the proposed method, we provide a comprehensive benchmarking of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, on four publicly available Ago2-HITS-CLIP datasets and one unpublished in-house Ago2-dataset in stem cells. We measured the sensitivity, the specificity and the position accuracy toward miRNA binding sites identification, and the agreement with TargetScan. Secondly, we developed a new pipeline, called miRBShunter, to identify canonical and non-canonical miRNA-binding sites based on de novo motif identification from Ago2 peaks and prediction of miRNA::RNA heteroduplexes. miRBShunter was tested and experimentally validated on the in-house Ago2-dataset and on an Ago2-PAR-CLIP dataset in human stem cells. Overall, we provide guidelines to choose a suitable peak detection program and a new method for miRNA-target identification.

RNA-mediated epigenetic heredity requires the cytosine methyltransferase Dnmt2.

RNA-mediated transmission of phenotypes is an important way to explain non-Mendelian heredity. We have previously shown that small non-coding RNAs can induce hereditary epigenetic variations in mice and act as the transgenerational signalling molecules. Two prominent examples for these paramutations include the epigenetic modulation of the Kit gene, resulting in altered fur coloration, and the modulation of the Sox9 gene, resulting in an overgrowth phenotype. We now report that expression of the Dnmt2 RNA methyltransferase is required for the establishment and hereditary maintenance of both paramutations. Our data show that the Kit paramutant phenotype was not transmitted to the progeny of Dnmt2(-/-) mice and that the Sox9 paramutation was also not established in Dnmt2(-/-) embryos. Similarly, RNA from Dnmt2-negative Kit heterozygotes did not induce the paramutant phenotype when microinjected into Dnmt2-deficient fertilized eggs and microinjection of the miR-124 microRNA failed to induce the characteristic giant phenotype. In agreement with an RNA-mediated mechanism of inheritance, no change was observed in the DNA methylation profiles of the Kit locus between the wild-type and paramutant mice. RNA bisulfite sequencing confirmed Dnmt2-dependent tRNA methylation in mouse sperm and also indicated Dnmt2-dependent cytosine methylation in Kit RNA in paramutant embryos. Together, these findings uncover a novel function of Dnmt2 in RNA-mediated epigenetic heredity.

Preconceptional paternal obesity may increase the risk of congenital urogenital anomalies in offspring: A case-control study.

BACKGROUND: Congenital urogenital anomalies affect 4-60 per 10,000 births. Maternal obesity, along with other risk factors, is well documented as a contributing factor. However, the impact of paternal obesity on risk is unclear. Obesity is prevalent among men of reproductive age, highlighting the need for further research into the potential association between paternal obesity and offspring congenital urogenital anomalies. OBJECTIVES: This study aims to determine the association between paternal obesity and the risk of congenital urogenital malformations in offspring. METHODS: Case-control study conducted on 179 newborns (91 cases, 88 controls) selected from the Notre Dame des Secours-university hospital database. Cases were identified as newborns presenting at least one congenital urogenital abnormality, defined as developmental anomalies that can result in a variety of malformations affecting the kidneys, ureters, bladder, and urethra. Controls were identified as newborns without any congenital abnormalities. The exclusion criteria were maternal obesity, infections during pregnancy, chronic diseases, prematurity, growth retardation, assisted reproductive technologies for conception, substance abuse, down syndrome, and other malformations. Data were collected through phone interviews, medical records, and questionnaires. In this study, the exposure was the preconceptional paternal body mass index (BMI), which was calculated based on self-reported height and weight. According to guidelines from the US Centers for Disease Control and Prevention (CDC), individuals are considered to be in the healthy weight range if their BMI (kg/m2) is between 18.5 and < 25. They are classified as overweight if their BMI is ≥ 25, obese class I if their BMI is between 30 and < 35, obese class II if their BMI is between 35 and < 40, and obese class III if their BMI is 40 or higher. Logistic regression analysis was employed to quantify the association between paternal obesity and urogenital conditions in offspring. RESULTS: Significant differences in median (minimum-maximum) paternal BMI values were noted between the cases and controls at the time of conception (cases: 27.7 (43-20.1), controls: 24.8 (40.7-19.6); p < 0.0001). Logistic regression analysis confirmed that at the time of conception, compared to normal-weight fathers, overweight fathers displayed a heightened risk of offspring congenital malformations, with an odds ratio (OR) of 4.44 (95% CI = 2.1-9.1). Similarly, fathers categorized as obese Class I at conception had approximately eight times higher odds (OR = 8.62, 95% CI = 2.91-25.52) of having offspring with urogenital conditions compared to normal-weight fathers. Additionally, fathers classified as obese Class II at conception exhibited 5.75 times higher odds (OR = 5.75, 95% CI = 0.96-34.44) of having offspring with urogenital conditions in comparison to normal-weight fathers. DISCUSSION AND CONCLUSION: We found that the risk of urogenital malformations increased with paternal BMI during the preconceptional period. The findings suggest the importance of addressing paternal obesity in efforts to reduce the risk of urogenital congenital malformations in offspring.

Paternal peri-conceptional physical activity and the risk of congenital heart disease in offspring: A case-control study.

BACKGROUND: Genetic and environmental factors have been shown to contribute to the development of congenital heart disease (CHD). To date, the focus of scientific articles has primarily centered on genetics and maternal environmental factors, with comparatively less attention given to paternal risk factors. OBJECTIVES: This study aims to investigate the potential association between paternal pre-conceptional physical activity levels (PA), along with paternal peri-conceptional smoking and alcohol consumption, and the risk of CHD in offspring. MATERIALS AND METHODS: An observational case-control study was conducted in Lebanon, with 279 participants, aiming to investigate potential risk factors for CHD. We included children with confirmed CHD, born between 2012 and 2022. Controls born in the same timeframe were selected randomly from the general population using online questionnaire forms. Mean age of children included was 6 years old (0-10). The pre-conceptional PA was assessed using the Global Physical Activity Questionnaire validated in Arabic. In addition, paternal smoking, alcohol consumption, and maternal risk factors were collected. RESULTS: The study included 128 CHD cases (45.9%) and their parents, as well as 151 healthy infants (54.1%) and their parents. There were no statistically significant variations in the alcohol consumption noted between the fathers in the case and control groups (p = 0.18). The paternal involvement in recreational-related PA during the peri-conception period was associated with a reduced risk of the CHD development in offspring by 46.9% (OR = 0.531, 95% CI: 0.301-0.936, p = 0.029). Additionally, increasing paternal total sitting time by 1 h above the average, which was approximately 260 min (4 h), increased the risk of CHD in offspring by 0.4% (p = 0.001). Moreover, paternal smoking exhibited an apparent association with a 56% increased risk of offspring developing CHD, notwithstanding that the confidence intervals included the null (OR = 1.56, 95% CI: 0.86-2.8, p = 0.136). DISCUSSION AND CONCLUSION: This observational study is the first to report a potential association between paternal PA, and CHD in offspring. This study aligns with previous reports, advocating for the paternal engagement in PA and the adoption of healthy lifestyle habits, especially during the critical stages of conception. Such practices are strongly recommended to enhance fertility and promote optimal health for offspring. However, due to the subjectivity in reporting PA and lack of molecular proof, additional prospective and molecular studies are required to validate these findings.

Impact of testicular cancer on sperm small non-coding RNA signature: a pilot study.

Testicular germ cell tumours (TGCTs) are the most common tumours in young adults of European ancestry. The high heritability and the constantly increased incidence, which has doubled over the last 20 years, strongly suggest that both genetic and environmental factors are likely to shape the TGCT susceptibility. While genome-wide association studies have identified loci associated with TGCT susceptibility, the role played by environmental molecular vectors in TGCT susceptibility remains unclear. Evidence shows that sperm non-coding RNAs provide a good vision of the environmental stresses experienced by men. Here, to determine whether TGCT impacts the abundance of specific non-coding RNAs in sperm, small RNA deep sequencing analysis of sperm of 25 men aged between 19 and 42 years, diagnosed with (n = 16) or without (n = 9) TGCT was performed. The primary analysis showed no statistical significance in the sncRNA population between the TGCT and non-TGCT groups. However, when sperm physiological parameters were considered to look for differentially expressed sncRNA, we evidenced 11 differentially expressed sncRNA between patients and control which allow a clear discrimination between control and TGCT samples after Hierarchical Clustering analysis. Together, these findings indicate that sperm small non-coding RNAs abundance may have the potential for diagnosing men with TGCT. However, specific care should be taken regarding sperm physiological parameters of the TGCT patients. Hence, larger studies are needed to confirm our findings and to determine whether such a signature associates with the risks to develop TGCT.

The miR-124-Sox9 paramutation: RNA-mediated epigenetic control of embryonic and adult growth.

The size of the mammalian body is determined by genetic and environmental factors differentially modulating pre- and postnatal growth. We now report a control of growth acting in the mouse from the first cleavages to the postnatal stages. It was evidenced by a hereditary epigenetic modification (paramutation) created by injection of a miR-124 microRNA into fertilized eggs. From the blastocyst to the adult, mouse pups born after microinjection of this miRNA showed a 30% increase in size. At the blastocyst stage, frequent duplication of the inner cell mass resulted in twin pregnancies. A role of sperm RNA as a transgenerational signal was confirmed by the giant phenotype of the progeny of transgenic males expressing miR-124 during spermiogenesis. In E2.5 to E8.5 embryos, increased levels of several transcripts with sequence homology to the microRNA were noted, including those of Sox9, a gene known for its crucial role in the progenitors of several adult tissues. A role in embryonic growth was confirmed by the large size of embryos expressing a Sox9 DNA transgene. Increased expression in the paramutants was not related to a change in miR-124 expression, but to the establishment of a distinct, heritable chromatin structure in the promoter region of Sox9. While the heritability of body size is not readily accounted for by Mendelian genetics, our results suggest the alternate model of RNA-mediated heritable epigenetic modifications.

Inherited variation at the epigenetic level: paramutation from the plant to the mouse.

In contrast with a wide definition of the 'epigenetic variation', including all changes in gene expression that do not result from the alteration of the gene structure, a more restricted class had been defined, initially in plants, under the name 'paramutation'. It corresponds to epigenetic modifications distinct from the regulatory interactions of the cell differentiation pathways, mitotically stable and sexually transmitted with non-Mendelian patterns. This class of epigenetic changes appeared for some time restricted to the plant world, but examples progressively accumulated of epigenetic inheritance in organisms ranging from mice to humans. Occurrence of paramutation in the mouse and possible mechanisms were then established in the paradigmatic case of a mutant phenotype maintained and hereditarily transmitted by wild-type homozygotes. Together with the recent findings in plants indicative of a necessary step of RNA amplification in the reference maize paramutation, the mouse studies point to a new role of RNA, as an inducer and hereditary determinant of epigenetic variation. Given the known presence of a wide range of RNAs in human spermatozoa, as well as a number of unexplained cases of familial disease predisposition and transgenerational maintenance, speculations can be extended to possible roles of RNA-mediated inheritance in human biology and pathology.

RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse.

Paramutation is a heritable epigenetic modification induced in plants by cross-talk between allelic loci. Here we report a similar modification of the mouse Kit gene in the progeny of heterozygotes with the null mutant Kit(tm1Alf) (a lacZ insertion). In spite of a homozygous wild-type genotype, their offspring maintain, to a variable extent, the white spots characteristic of Kit mutant animals. Efficiently inherited from either male or female parents, the modified phenotype results from a decrease in Kit messenger RNA levels with the accumulation of non-polyadenylated RNA molecules of abnormal sizes. Sustained transcriptional activity at the postmeiotic stages--at which time the gene is normally silent--leads to the accumulation of RNA in spermatozoa. Microinjection into fertilized eggs either of total RNA from Kit(tm1Alf/+) heterozygotes or of Kit-specific microRNAs induced a heritable white tail phenotype. Our results identify an unexpected mode of epigenetic inheritance associated with the zygotic transfer of RNA molecules.

Association between metabolic disorders and seminal plasma miRNA levels: a pilot study.

BACKGROUND: Excess weight and metabolic disorders have a negative impact on male reproductive functions. The mechanisms involved are numerous and complex and epigenetic mechanisms may also be involved, notably through the small non-coding RNAs. Among them, microRNAs (miRNAs) are of particular interest. This preliminary study aimed to identify the miRNAs differentially enriched in seminal plasma related to metabolic disorders and if some are also associated with spermatic parameters alterations. One hundred and sixty men between 18 to 45 years, partners of infertile couple, were included in this cohort. The miRNAs associated with metabolism were selected from the literature and assayed by quantitative real-time PCR using TaqMan gene expression assays. A subset of those with an interesting profile in seminal plasma were secondarily tested in blood. RESULTS: Among the 11 selected miRNAs, seven were detected in seminal plasma (miR10b, miR19a, miR19b, miR34b, miR34c, miR133b, miRlet7c). A negative correlation was observed between seminal miR19a levels and metabolic syndrome, blood glucose and C-peptide. Seminal miR19b levels were also negatively correlated with metabolic syndrome. Seminal miR34c levels were negatively correlated with body mass index (BMI) and waist circumference. Seminal miR133b levels were positively correlated with BMI, waist circumference and leptin levels. Interestingly, modifications of miRNAs in seminal plasma seem specific since highlighted above correlations were not retrieved in the blood plasma for the miR19a, 19b, 10b, 34c. CONCLUSION: Few metabolic and anthropometric disorders are correlated with the level of specific miRNAs in seminal plasma. Further studies will be required to decipher if other small non-coding RNAs may also be correlated with metabolic and anthropometric disorders and to assess their potential implication in the alteration of reproductive functions in men with obesity or metabolic disorders. CLINICAL STUDY: Metabolic Syndrome and Male Infertility (Metasperme): Trial registration:  NCT01974947 . Registered 18 July 2013.

RéSUMé: CONTEXTE: L'excès de poids et les troubles métaboliques ont un impact négatif sur les fonctions de reproduction masculine. Les mécanismes impliqués sont nombreux et complexes, et des mécanismes épigénétiques peuvent également intervenir, notamment par le biais des petits ARN non codants. Parmi eux, les microRNAs (miRNAs) présentent un intérêt particulier. Cette étude préliminaire visait à identifier les miRNAs différentiellement enrichis dans le plasma séminal en relation avec des troubles métaboliques et si certains étaient également associés à des altérations des paramètres spermatiques. Cent soixante hommes âgés de 18 à 45 ans, partenaires de couple infertile, ont été inclus dans cette cohorte. Les miRNAs associés au métabolisme ont été sélectionnés dans la littérature et analysés par PCR quantitative en temps réel à l'aide de tests d'expression génique TaqMan. Un sous-ensemble de ceux présentant un profil intéressant dans le plasma séminal ont été secondairement testés dans le sang. RéSULTATS: Parmi les 11 miRNAs sélectionnés, sept ont été détectés dans le plasma séminal (miR10b, miR19a, miR19b, miR34b, miR34c, miR133b, miRlet7c). Une corrélation négative a été observée entre les niveaux du miR19a séminal et le syndrome métabolique, la glycémie et le C-peptide. Les niveaux de miR19b séminaux étaient également corrélés négativement avec le syndrome métabolique. Les niveaux de miR34c séminaux étaient négativement corrélés avec l'IMC et le tour de taille. Les niveaux de miR133b séminaux étaient positivement corrélés avec l'IMC, le tour de taille et les niveaux de leptine. Il est intéressant de noter que les modifications des miRNA dans le plasma séminal semblent spécifiques puisque les corrélations mises en évidence ci-dessus n'ont pas été retrouvées dans le plasma sanguin pour les miR19a, 19b, 10b, 34c. CONCLUSION: Quelques désordres métaboliques et anthropométriques ont été observés corrélés avec le niveau de certains miRNAs dans le plasma séminal. Des études complémentaires sont nécessaires pour déterminer si d'autres petits ARN non codants sont corrélés aux troubles métaboliques et anthropométriques et pour évaluer leur implication potentielle dans l'altération des fonctions de reproduction chez les hommes souffrant d'obésité ou de troubles métaboliques.

Paternal High-Protein Diet Programs Offspring Insulin Sensitivity in a Sex-Specific Manner.

The impact of maternal nutrition on offspring is well documented. However, the implication of pre-conceptional paternal nutrition on the metabolic health of the progeny remains underexplored. Here, we investigated the impact of paternal high-protein diet (HPD, 43.2% protein) consumption on the endocrine pancreas and the metabolic phenotype of offspring. Male Wistar rats were given HPD or standard diet (SD, 18.9% protein) for two months. The progenies (F1) were studied at fetal stage and in adulthood. Body weight, glycemia, glucose tolerance (GT), glucose-induced insulin secretion in vivo (GIIS) and whole-body insulin sensitivity were assessed in male and female F1 offspring. Insulin sensitivity, GT and GIIS were similar between F1 females from HPD (HPD/F1) and SD fathers (SD/F1). Conversely, male HPD/F1 exhibited increased insulin sensitivity (p < 0.05) and decreased GIIS (p < 0.05) compared to male SD/F1. The improvement of insulin sensitivity in HPD/F1 was sustained even after 2 months of high-fat feeding. In male HPD/F1, the ß cell mass was preserved and the ß cell plasticity, following metabolic challenge, was enhanced compared to SD/F1. In conclusion, we provide the first evidence of a sex-specific impact of paternal HPD on the insulin sensitivity and GIIS of their descendants, demonstrating that changes in paternal nutrition alter the metabolic status of their progeny in adulthood.

Paternal multigenerational exposure to an obesogenic diet drives epigenetic predisposition to metabolic diseases in mice.

Obesity is a growing societal scourge. Recent studies have uncovered that paternal excessive weight induced by an unbalanced diet affects the metabolic health of offspring. These reports mainly employed single-generation male exposure. However, the consequences of multigenerational unbalanced diet feeding on the metabolic health of progeny remain largely unknown. Here, we show that maintaining paternal Western diet feeding for five consecutive generations in mice induces an enhancement in fat mass and related metabolic diseases over generations. Strikingly, chow-diet-fed progenies from these multigenerational Western-diet-fed males develop a 'healthy' overweight phenotype characterized by normal glucose metabolism and without fatty liver that persists for four subsequent generations. Mechanistically, sperm RNA microinjection experiments into zygotes suggest that sperm RNAs are sufficient for establishment but not for long-term maintenance of epigenetic inheritance of metabolic pathologies. Progressive and permanent metabolic deregulation induced by successive paternal Western-diet-fed generations may contribute to the worldwide epidemic of metabolic diseases.

Prediction of coronary heart disease incidence in a general male population by circulating non-coding small RNA sRNY1-5p in a nested case-control study.

During the development of atherosclerotic lesion, s-RNYs (small RNAs of about 24/34 nucleotides) are derived by the processing of long Ro-associated non-coding RNAs (RNYs) in macrophages. The levels of serum s-RNYs have been found significantly upregulated in patients with coronary heart disease (CHD) compared to age-matched CHD-free individuals. The present study aimed to examine the predictive value of serum s-RNYs for CHD events in the general male population. Within the frame of nested-case-control study, the GENES study, we measured the absolute expression of a RNY-derived small RNA, the s-RNY1-5p, in the serum of individuals (without CHD at baseline) who encountered a CHD event within 12 years of follow-up (n = 30) (Cases) and compared them to individuals who remained event-free (Controls) (n = 30). The expression of s-RNY1-5p in serum was significantly upregulated in Cases compared to Controls (p = 0.027). The proportion of CHD event-free was significantly higher among individuals with serum s-RNY1-5p below the median value (631 molecules/mL). In a multivariable model adjusted for age, smoking, hypertension, diabetes and dyslipidemia, the risk of CHD events increased more than fourfold in individuals with serum s-RNY1-5p above the median value (HR, 4.36; 95% CI 1.22-15.60). A positive association with CHD events was also observed when considering s-RNY1-5p as a continuous variable (p = 0.022). Based on our results, we conclude that serum s-RNY1-5p is an independent predictor of CHD events in a general male population and might be a relevant biomarker for early detection of cardiovascular diseases.

Mother or Father: Who Is in the Front Line? Mechanisms Underlying the Non-Genomic Transmission of Obesity/Diabetes via the Maternal or the Paternal Line.

Extensive epidemiological and experimental evidence have shown that exposure to an adverse intrauterine environment as observed in offspring of pregnancies complicated by obesity or diabetes, can program susceptibility to metabolic, endocrine and cardiovascular disorders later in life. Although most studies have concentrated on the maternal environment, it is also becoming evident that paternal exposure to obesity or diabetes can result in the later development of metabolic disorders in the offspring. Such programmed effects might not be limited to the first directly exposed generation, but could be transmitted to subsequent generations. This suggests the existence of mechanisms by which metabolic changes in parental phenotype are transmissible to offspring. The mechanisms which underpin the transmission of the programmed effects across generations are still unclear. However, epigenetic regulation of transcription has emerged as a strong candidate for mediating the heritability of metabolic diseases. Here, we review the most relevant evidence from human and animal studies showing transmission of programming effects of obesity or diabetes across generations, and the current mechanisms underlying either maternal or paternal influences on the metabolic status of offspring.

Role of miRNA in the Transmission of Metabolic Diseases Associated With Paternal Diet-Induced Obesity.

The concept of Developmental Origins of Health and Diseases (DOHaD) recognizes that an unfavorable maternal environment alters the developmental trajectory of the fetus and can lead to long-term risk of developing chronic noncommunicable diseases. More recently, the concept of a paternal transmission [Paternal Origins of Health and Diseases (POHaD)] has emerged stressing the impact of paternal overweight or obesity on offspring's health and development. While very few examples of paternal epigenetic inheritance of metabolic disorders have been evidenced in human, many experimental mouse models based on high-fat diet (HFD)-induced paternal obesity have been developed to breakdown molecular mechanisms involved in the process. Besides DNA methylation and chromatin structure, sperm short noncoding RNAs have been considered as the main epigenetic vector of inheritance of paternally environmentally induced changes. Among them, sperm miRNAs are one particular subspecies sensitive to environmental changes and obesity can modify the sperm miRNA profile. Once delivered into the zygote, these molecules might induce epigenetic modifications in the embryo, thereby leading to consequences for fetus development and offspring physical and metabolic health later on in life. Furthermore, some data also suggest that metabolic pathologies may be intergenerationally or transgenerationally transmitted.

Adverse effects of paternal obesity on the motile spermatozoa quality.

Growing evidence suggests that paternal obesity may decrease male fertility potential. During infertility treatment with intra-cytoplasmic sperm injection (ICSI), a morphologically normal motile spermatozoon is injected into a mature egg, when possible. However, sperm motility and morphology per se do not reflect the sperm molecular composition. In this study, we aimed to assess the quality of motile spermatozoa in the context of obesity by analysing their conventional and molecular characteristics as well as their ability to promote early embryonic development. A prospective study was conducted on 128 infertile men divided into three groups: 40 lean, 42 overweight, and 46 obese men. Conventional sperm parameters (concentration, motility and morphology) and sperm molecular status (chromatin composition and integrity, 5-methycytosine (5-mC) and 5-hydroxycytosine (5-hmC) contents and oxidative stress level) were analysed on raw semen and/or on motile spermatozoa selected by density gradient or swim-up techniques. Morphokinetic analysis of the embryos derived from ICSI was performed using the Embryoviewer software. Our results showed that the motile sperm-enriched fraction from obese men exhibited higher levels of retained histones (p<0.001), elevated percentage of altered chromatin integrity (p<0.001), and decreased contents of 5-hmC (p<0.001), and 5-mC (p<0.05) levels as compared to that from lean men. Importantly, there were no statistically significant correlations between these molecular parameters and the percentages of morphologically normal motile spermatozoa. Regarding embryo morphokinetics, the CC1 (p<0.05) and CC3 (p<0.05) embryonic cell cycles were significantly delayed in the cleavage embryos of the obese group as compared to the embryos of the lean group. Our data is of particular interest because, besides demonstrating the negative impacts of obesity on motile spermatozoa molecular composition, it also highlights the possible risk of disturbing early embryonic cell cycles kinetics in the context of paternal obesity.

RNY (YRNA)-derived small RNAs regulate cell death and inflammation in monocytes/macrophages.

The recent discovery of new classes of small RNAs has opened unknown territories to explore new regulations of physiopathological events. We have recently demonstrated that RNY (or Y RNA)-derived small RNAs (referred to as s-RNYs) are an independent class of clinical biomarkers to detect coronary artery lesions and are associated with atherosclerosis burden. Here, we have studied the role of s-RNYs in human and mouse monocytes/macrophages and have shown that in lipid-laden monocytes/macrophages s-RNY expression is timely correlated to the activation of both NF-κB and caspase 3-dependent cell death pathways. Loss- or gain-of-function experiments demonstrated that s-RNYs activate caspase 3 and NF-κB signaling pathways ultimately promoting cell death and inflammatory responses. As, in atherosclerosis, Ro60-associated s-RNYs generated by apoptotic macrophages are released in the blood of patients, we have investigated the extracellular function of the s-RNY/Ro60 complex. Our data demonstrated that s-RNY/Ro60 complex induces caspase 3-dependent cell death and NF-κB-dependent inflammation, when added to the medium of cultured monocytes/macrophages. Finally, we have shown that s-RNY function is mediated by Toll-like receptor 7 (TLR7). Indeed using chloroquine, which disrupts signaling of endosome-localized TLRs 3, 7, 8 and 9 or the more specific TLR7/9 antagonist, the phosphorothioated oligonucleotide IRS954, we blocked the effect of either intracellular or extracellular s-RNYs. These results position s-RNYs as relevant novel functional molecules that impacts on macrophage physiopathology, indicating their potential role as mediators of inflammatory diseases, such as atherosclerosis.

Paternal obesity: how bad is it for sperm quality and progeny health?

There is substantial evidence that paternal obesity is associated not only with an increased incidence of infertility, but also with an increased risk of metabolic disturbance in adult offspring. Apparently, several mechanisms may contribute to the sperm quality alterations associated with paternal obesity, such as physiological/hormonal alterations, oxidative stress, and epigenetic alterations. Along these lines, modifications of hormonal profiles namely reduced androgen levels and elevated estrogen levels, were found associated with lower sperm concentration and seminal volume. Additionally, oxidative stress in testis may induce an increase of the percentage of sperm with DNA fragmentation. The latter, relate to other peculiarities such as alteration of the embryonic development, increased risk of miscarriage, and development of chronic morbidity in the offspring, including childhood cancers. Undoubtedly, epigenetic alterations (ie, DNA methylation, chromatin modifications, and small RNA deregulation) of sperm related to paternal obesity and their consequences on the progeny are poorly understood determinants of paternal obesity-induced transmission. In this review, we summarize and discuss the data available in the literature regarding the biological, physiological, and molecular consequences of paternal obesity on male fertility potential and ultimately progeny health.

De plus en plus de données tendent à montrer que l'obésité paternelle a non seulement des effets néfastes sur la santé métabolique et reproductive de l'individu mais également sur celle de sa descendance. Les mécanismes mis en jeu dans ce processus incluraient des altérations physiologiques et hormonales des fonctions reproductives de l'homme obèse ainsi que des altérations épigénétiques au niveau du génome spermatique. Les modifications hormonales associées à l'obésité et qui se caractérisent principalement par une réduction du taux d'androgènes et une augmentation du niveau d'estrogène induiraient une altération des paramètres spermatiques, une diminution de la concentration ou de la numération totale en spermatozoïde et du volume séminal. Le stress oxydatif dans le testicule induirait une augmentation de la fragmentation de l'ADN spermatique et pourrait rendre compte de l'augmentation des risques de fausses-couches, des problèmes de développement embryonnaire ainsi que de l'augmentation des risques de mortalité chez la descendance, problèmes fréquemment rencontrés lorsque le père est. obèse. Les modifications épigénétiques (altérations des profils de méthylation de l'ADN, de la structure de la chromatine ou/et des profils d'expression des ARN spermatiques) induites par l'obésité sont, quant à elles, loin d'être comprises, même si elles sont, surement, les vecteurs clés de la transmission épigénétique paternelle des maladies métaboliques. L'objet de cette revue est. de résumer puis de discuter les différentes études expérimentales et épidémiologiques publiés à ce jour sur les conséquences physiologiques et moléculaire de l'obésité paternelle sur la santé de l'individu et sur celle de sa descendance.

Post-transcriptional gene silencing mediated by microRNAs is controlled by nucleoplasmic Sfpq.

There is a growing body of evidence about the presence and the activity of the miRISC in the nucleus of mammalian cells. Here, we show by quantitative proteomic analysis that Ago2 interacts with the nucleoplasmic protein Sfpq in an RNA-dependent fashion. By a combination of HITS-CLIP and transcriptomic analyses, we demonstrate that Sfpq directly controls the miRNA targeting of a subset of binding sites by local binding. Sfpq modulates miRNA targeting in both nucleoplasm and cytoplasm, indicating a nucleoplasmic commitment of Sfpq-target mRNAs that globally influences miRNA modes of action. Mechanistically, Sfpq binds to a sizeable set of long 3'UTRs forming aggregates to optimize miRNA positioning/recruitment at selected binding sites, including let-7a binding to Lin28A 3'UTR. Our results extend the miRNA-mediated post-transcriptional gene silencing into the nucleoplasm and indicate that an Sfpq-dependent strategy for controlling miRNA activity takes place in cells, contributing to the complexity of miRNA-dependent gene expression control.