Col3a1 delivered via extracellular vesicles of Sertoli cells is essential for mice Sertoli cell proliferation.

It is generally believed that Sertoli cells can proliferate only before sexual maturity. In this study, we found that extracellular vesicles of Sertoli cells derived from prepubertal mice (SEVs) have the ability to promote the proliferation of Sertoli cell population. In addition, via proteomic analysis, we compared the functional components of extracellular vesicles derived from Sertoli cells of mice at 12-14 days and 8 weeks. The functional profiling of SEVs suggested important developmental roles, and this was confirmed by analysis comparing the transcriptomic changes in Sertoli cells treated with DMSO and GW4869. The following analysis pointed to Col3a1 as a key factor in SEVs, which was further validated using primary Sertoli cells and TM4 cell line. The present study suggests a possible role for Col3a1 in promoting the proliferation of cultured Sertoli cells and provides a new perspective on the function of extracellular vesicles in Sertoli cell development.

Placental ischemia disrupts DNA methylation patterns in distal regulatory regions in rats.

Preeclampsia (PE) is a leading cause of maternal and fetal morbidity and mortality worldwide. However, the impact of PE on the organization of the functional architecture of the placental methylome remains largely unknown. We performed whole-genome bisulfite sequencing of placental DNA and applied a Hidden Markov Model to investigate epigenome-wide alterations in functional structures, including partially methylated domains (PMDs), low-methylated regions (LMRs), and unmethylated regions (UMRs), in a reduced uterine perfusion pressure (RUPP) rat model of PE. The remarkable similarity we observed between the rat and human placental DNA methylomes suggests that the RUPP rat model is appropriate to elucidate the epigenetic mechanisms underlying human PE. The notable changes in PMDs indicate RUPP-induced perturbation of the stressed placental methylome. This was probably regulated via modulation of the epigenetic modifier expression, including significant downregulation of Dnmt1 and Dnmt3a and upregulation of Tet2. More importantly, changes in RUPP-induced DNA methylation occurred predominately in LMRs (80 %), which represent active enhancers, rather than in canonical UMRs (3 %), which represent promoters, suggesting that placental ischemia disrupts enhancer DNA methylation. Our findings emphasize the role of enhancer methylation in response to PE, corroborating discoveries in human PE studies. We suggest paying more attention to enhancer regions in future studies on PE.

IKBA phosphorylation governs human sperm motility through ACC-mediated fatty acid beta-oxidation.

The nuclear factor-κB (NF-κB) signaling pathway regulates specific immunological responses and controls a wide range of physiological processes. NF-κB inhibitor alpha (IKBA) is an NF-κB inhibitory mediator in the cytoplasm that modulates the nuclear translocation and DNA binding activities of NF-κB proteins. However, whether the upstream cascade of the canonical NF-κB signaling pathway has physiological roles independent of IKBA-mediated transcriptional activation remains unclear. Herein we investigated the function of IKBA in mature sperm in which transcriptional and translational events do not occur. IKBA was highly expressed in human sperm. The repression of IKBA phosphorylation by its inhibitor Bay117082 markedly enhanced sperm motility. On the contrary, lipopolysaccharide-stimulated IKBA phosphorylation significantly decreased sperm motility. Nevertheless, Bay117082 treatment did not affect the motility of IKBA-knockout sperm. Further, untargeted metabolomic analysis and pharmacological blocking assays revealed that the Bay117082-induced increase in sperm motility was attributable to fatty acid ß-oxidation (FAO) enhancement. In addition, we found that IKBA phosphorylation inhibition resulted in a significant reduction of acetyl-CoA carboxylase levels in the FAO metabolic pathway. Our findings indicate that IKBA-mediated signaling orchestrates sperm motility program and improves our understanding of transcription-independent NF-κB signaling pathway in cells.

Targeted bisulfite resequencing of differentially methylated cytosines in pre-eclampsia reveals a skewed dynamic balance in the DNA methylation of enhancers.

Pre-eclampsia (PE) is a major hypertensive disorder of pregnancy. Widespread differentially methylated cytosines (DMCs) with modest changes in methylation level are associated with PE, whereas their cause and biological significance remain unknown. We aimed to clarify DNA methylation patterns around DMCs in 103 placentas using MethylCap targeted bisulfite re-sequencing (MethylCap-seq) assays of 690 selected DMCs. We verified the MethylCap-seq method, then validated 677 (98.1%) of DMCs (vDMCs) in an independent cohort. The validated DMCs were strongly enriched in active placenta-specific enhancers and showed highly dynamic methylation levels. We found high epigenetic heterogeneity between vDMCs and adjacent CpG sites (r2 < 0.2) and a significant decrease in PE in the discovery and replication cohorts (P = 2.00 × 10-24 and 6.43 × 10-9, respectively). We replicated the methylation changes in a hypoxia/reoxygenation cell model. We constructed 112 methylation haplotype blocks and found that the frequencies of unmethylated haplotypes (UMHs) were dynamic with gestational age (GA) and were altered in maternal plasma of patients with PE. Our results uncovered additional DNA methylation features in PE placentas and suggested a model of skewed DNA methylation balance of enhancers in PE.

The association of cesarean section with overweight and neurodevelopment of Chinese children aged 1-5 months.

Objective: The purpose of this survey was to explore the association of delivery mode with overweight and neurodevelopment of Chinese infants aged 1-5 months. Materials and methods: This study was based on a cross-sectional survey. Data for this study were obtained from the Children's Nutrition and Health System Survey in China which was conducted from 2019 to 2020. Characteristics of parents and children and the delivery mode were obtained using interview-administered questionnaires. Body mass index-for age z-score (BMI z) was calculated using World Health Organization (WHO) child growth standards. Children's neurodevelopment was assessed by a trained child health care physician using the Child Psychological Development Scale. The association of delivery mode with infant overweight was analyzed using a multivariable logistic regression model. We conducted a multivariable linear regression model to explore the relationship between delivery modes with neurodevelopment. Results: In total, the present analysis included 1,347 children aged 1-5 months, 35.61% were born via cesarean section, of which 15.21% were overweight. After adjustment for infant characteristics and parental factors, the cesarean section was significantly related with the likehood of being overweight [OR = 1.95; 95% confidence interval (CI): 1.27 to 2.98]. Children born via cesarean section had a 3.41-point decrease in gross motor development (ß = -3.41; 95% CI: -5.77 to -1.05), a 3.65-point decrease in fine motor development (ß = -3.65; 95% CI: -6.03 to -1.28), and a 2.96-point in language development (ß = -2.96; 95% CI: -5.20 to -0.73), a 1.65-point in total development (ß = -1.65; 95% CI: -3.17 to -0.14) compared with those who were vaginal birth. Conclusion: In our study population, cesarean section was associated with overweight and neurodevelopment outcomes. The cesarean section might increase the likehood of infant overweight, and might decrease the developmental scores of gross motor, fine motor and language. Further studies should be conducted to verify the associations and explore the possible mechanisms.

Lysophosphatidic Acid Improves Human Sperm Motility by Enhancing Glycolysis and Activating L-Type Calcium Channels.

Until now, the molecular mechanisms underlining sperm motility defect causing male infertility are still poorly understood. Safe and effective compounds or drugs that can improve sperm motility are also very limited. Lysophosphatidic acid (LPA) is a naturally occurring phospholipid and a bioactive intermediate with multiple biological activities. It has been detected in various body fluids such as serum, plasma, saliva, tears, blister fluids, hen egg white, and ascites from patients with ovarian cancer. LPA is also abundant in seminal plasma and follicular fluid. It enhances follicle stimulation, improves oocyte fertilization, and promotes early embryonic development and embryo implantation. However, the physiological role of LPA in the male reproductive system remains unknown. Here, our study showed that LPA significantly improved the motility parameters of human sperm hyperactivation in a dose-dependent manner. The LPA-induced elevation of sperm motility is dependent on bovine serum albumin (BSA) but independent of the classical BSA-induced sAC/cAMP/PKA signaling pathway. The enhancement of sperm motility by LPA could not be blocked by CCCP, a respiratory inhibitor suppressing mitochondrial ATP production. Moreover, LPA improved the activity of triosephosphate isomerase in glycolysis. Meanwhile, LPA treatment significantly increased ATP and phosphoenolpyruvate levels and decreased ADP content during sperm glycolysis. Notably, none of known or identified LPA receptors was detected in human sperm. Further investigations showed that LPA promoted sperm motility through L-type calcium channels. In summary, this study revealed the involvement of LPA in the regulation for human sperm motility by enhancing glycolysis and activating L-type calcium channels. The current findings may shed new light on the understanding of causes of asthenozoospermia, and indicate that LPA could be used as a novel therapeutic agent to improve sperm function and fertilizing capacity.

Reassessment of the Proteomic Composition and Function of Extracellular Vesicles in the Seminal Plasma.

Seminal plasma contains a high concentration of extracellular vesicles (EVs). The heterogeneity of small EVs or the presence of nonvesicular extracellular matter (NV) pose major obstacles in understanding the composition and function of seminal EVs. In this study, we employed high-resolution density gradient fractionation to accurately characterize the composition and function of seminal EVs and NV. We found that the seminal EVs could be divided into 3 different subtypes-namely, high-density EV (EV-H), medium-density EV (EV-M), and low-density EV (EV-L)-after purification using iodixanol, while NV was successfully isolated. EVs and NV display different features in size, shape, and expression of some classic exosome markers. Both EV-H and NV could markedly promote sperm motility and capacitation compared with EV-M and EV-L, whereas only the NV fraction induced sperm acrosome reaction. Proteomic analysis results showed that EV-H, EV-M, EV-L, and NV had different protein components and were involved in different physiological functions. Further study showed that EV-M might reduce the production of sperm intrinsic reactive oxygen species through glutathione S-transferase mu 2. This study provides novel insights into important aspects of seminal EVs constituents and sounder footing to explore their functional properties in male fertility.

The rate of child restraint system use among children aged under six years in China.

AIMS: The child restraint system (CRS) for vehicles is designed to provide specialized protection for children in the event of a crash. The aim of the study was to investigate the rate of CRS use and analyze the factors associated with CRS use among children aged under six years in China, and to provide further insight into developing strategies for promoting public health education. METHODS: This is a cross-sectional study. The study sites were 36 primary healthcare institutions in 12 provinces across China, and the participants were 34,503 guardians of children aged 0-6 years. Guardians who owned private cars were included and completed surveys about their experience using CRS. Odds ratios and 95% confidence intervals were calculated using multivariate logistic regression models. RESULTS: The overall rate of CRS use among children aged under six years in China was 17.3%. Multivariate logistic regression analysis revealed that living in an urban area, low age of the child, guardians having higher education and being looked after by parents had a significant positive association with CRS use among children aged under six years. CONCLUSIONS: This study confirms that there is a low rate of CRS use among children aged under six years in China, highlighting the considerable need for CRS use education, advocacy and promotion of increasing use.

Anemia in preschool children in China and its association with neuropsychological development / 中国学校卫生

Objective@#To explore the relationship between anemia and neuropsychological development in various domains among preschool children in China.@*Methods@#Data came from the National Nutrition and Health Systematic Survey for children in China, and 3 261 preschool children aged 2-6 years and their parents from 28 sites across 14 provinces were recruited in this study. Parental and child characteristics were obtained by interview administrated questionnaires. Blood hemoglobin(Hb) concentration was determined by Hemocue method. Neuropsychological development quotients were assessed using the Development Scale for Children Aged 0-6 Years(WS/T 580-2017).@*Results@#The average Hb level was (125.23±11.49)g/L and the overall anemia prevalence was 10.30% among preschool children. After adjusting the confounding factors(sex, age, ethnicity, region, feeding mode, maternal status during pregnancy, etc), developmental quotients of gross motor( β=-2.15, 95%CI =-3.89--0.41), fine motor( β=-2.46, 95%CI =-4.12--0.79), adaptive behavior( β=-2.59, 95%CI =-4.42--0.76), language( β=-3.65, 95%CI =-5.53--1.78), personal social behavior( β=-3.11, 95%CI =-4.94--1.28) and full scale( β=-2.79, 95%CI =-4.10--1.49) among children with anemia were significantly lower than non anemic infants( P <0.05).@*Conclusion@#Anemia was negatively associated with developmental quotient, as well as five domains of gross motor, fine motor, adaptive behavior, language, and personal social behavior in preschool children aged 2-6 years. It is suggested to carry out the work of anemia monitoring and intervention in preschool children to further improve their neuropsychological development.

Relationship between screen time and myopia in children aged 11-14 years in China / 中国学校卫生

Objective@#To explore relationship between screen time and myopia in children aged 11-14 years in China.@*Methods@#The data were extracted from "National Nutrition and Health Systematic Survey and Application for 0-18 Years Old Children". A total of 12 397 children aged 11-14 years old from 14 provinces and 28 districts/counties in seven regions of China were surveyed by using multi stage stratified random sampling method. Daily screen time and visual acuity information were collected through a questionnaire.@*Results@#The myopia rate of 11-14 years old children in China was 45.0%, among which the rate of girls was higher than that of boys, and the rate of urban was higher than that of rural, and it increased with age ( χ 2=178.82,79.25, 495.96 , P <0.01). The daily screen time median of 12 397 children was 40.0 minutes, with boys(40.0 min) longer than girls( 35.0 min ) and urban children(40 min) longer than rural children(33.0 min) ( χ 2=20.86，102.68， P <0.01). The myopia rate of boys ( 42.5 %) with daily screen time greater than or equal to 60 minutes was higher than that of boys (36.4%) with daily screen time less than 60 minutes, and the myopia rate of girls (55.6%) with daily screen time greater than or equal to 60 minutes was higher than that of girls (48.0%)( χ 2=23.62，34.15， P <0.01). After adjusting for age, gender, region, time of medium and high intensity physical activity, intake of sugary food and sugary beverages, daily sleep time, multivariable Logistic regression model showed that girls with daily screen time greater than or equal to 60 minutes ( OR=1.14, 95%CI =1.03-1.27) had a higher risk of myopia than those with less than 60 minutes. After adjusting for confounding factors, there was no correlation between daily screen time and the degree of myopia in boys or girls( P >0.05).@*Conclusion@#Daily screen time greater than or equal to 60 minutes may be a risk factor for myopia in girls aged 11 to 14 years old. Given the complexity of the factors that affect vision, researches are needed to examine the relationship between screen time and myopia.

Sperm epigenetic alterations contribute to inter- and transgenerational effects of paternal exposure to long-term psychological stress via evading offspring embryonic reprogramming.

Paternal life experiences impact offspring health via germline, and epigenetic inheritance provides a potential mechanism. However, global reprogramming during offspring embryogenesis and gametogenesis represents the largest hurdle to conceptualize it. Yet, detailed characterization of how sperm epigenetic alterations carrying "environmental memory" can evade offspring embryonic reprogramming remains elusive. Here, mice exposed to long-term restraint stress were employed to study the mechanisms underlying inter- and transgenerational effects of paternal exposure to a long-term psychological stress. We found that stress could induce paternal inheritance of reproductive, behavioral, and metabolic disorders. Bisulfite methylation profiling of 18 sperm and 12 embryo samples of three consecutive generations identified inter- and transgenerational inheritance of paternal Differential DNA Methylation Regions (DMRs) at frequencies ~11.36% and 0.48%, respectively. These DMRs related to genes with functional implications for psychological stress response, and tissue inheritance of these DMRs passed paternal disorders epigenetically to offspring. More importantly, these DMRs evaded offspring embryonic reprogramming through erasure and subsequent reestablishment, but not via un-erasure way. Nonetheless, their reestablishment proportions in the primitive streak (E7.5) stage were altered. Furthermore, sncRNA-seq revealed that stress-induced tsRNA, miRNA and rsRNA dysregulation in paternal sperm might play important roles in DMRs occurrence and paternal inheritance. These finding implied that sperm epigenetic alterations contribute to inter- and transgenerational effects of paternal exposure to long-term psychological stress, and highlighted the possible underlying molecular mechanism.

Modeling a cataract disorder in mice with prime editing.

Prime editing enables efficient introduction of targeted transversions, insertions, and deletions in mammalian cells and several organisms. However, genetic disease models with base deletions by prime editing have not yet been reported in mice. Here, we successfully generate a mouse model with a cataract disorder through microinjection of prime editor 3 (PE3) plasmids to efficiently induce targeted single-base deletion. Notably, a generated mouse with a high G-deletion rate (38.2%) displays a nuclear cataract phenotype; the PE3-induced deletions in mutant mice achieve high rates of germline transmission to their progenies, with phenotypic inheritance of cataract. Our data propose that modeling a genetic disease with a single nucleotide deletion in mice can be achieved with prime genome editing in vivo.

Effect of Superabsorbent Polymer on the Mechanical Performance and Microstructure of Concrete.

The internally cured material known as superabsorbent polymer (SAP) is an important innovation in concrete engineering technology. This paper investigates the effect of adding a polymer with superabsorbent capabilities on the physical and mechanical performance of concrete. The microstructure of the new hybrid concrete was also studied, and the influence of the polymer particle size and volume on the mechanical durability was evaluated. The mechanical properties of the new hybrid concrete, such as compressive strength, flexural strength, elastic modulus, and splitting tensile strength, were measured through laboratory experiments. The microstructure characteristics of the concrete were also investigated by scanning electron microscopy (SEM). The results show that shrinkage was reduced, while the volume stability of the concrete improved. Moreover, we found that cracking was reduced, while issues such as chloride penetration and freeze-thaw resistance were also improved. In addition, the SAP could effectively improve the microstructure of the concrete and refine the pore structure, as seen in the microscopic test. This paper helps to promote the development of internally cured material and improve technology for the prevention of concrete construction cracks.

The Alteration of Salivary Immunoglobulin A in Autism Spectrum Disorders.

Objectives: Autism spectrum disorders (ASD) are neurodevelopmental disorders with changes in the gut and oral microbiota. Based on the intimate relationship between the oral microbiota and oral mucosal immunity, this study aimed to investigate changes in salivary immunoglobulin A (IgA) level in ASD and the underlying mechanism for any such changes. Methods: We recruited 36 children diagnosed with ASD and 35 normally developing children and measured their salivary IgA content using enzyme-linked immunosorbent assay (ELISA). The valproate (VPA) -treated ASD mouse model was established by prenatal exposure to valproate and mouse salivary IgA content was also quantified by ELISA. The submandibular glands of VPA and control mice were isolated and analyzed using qRT-PCR, immunofluorescence staining, and flow cytometry. ASD-related Streptococci were co-incubated with the human salivary gland (HSG) cell line, and western blotting was used to detect the levels of relevant proteins. Results: We found that salivary IgA content was significantly decreased in patients with ASD and had a significant ASD diagnostic value. The salivary IgA content also decreased in VPA mice and was significantly correlated with autistic-like behaviors among them. The mRNA and protein levels of the polymeric immunoglobulin receptor (Pigr) were downregulated in the submandibular glands of VPA mice and the Pigr mRNA level was positively correlated with mouse salivary IgA content. HSG cells treated with ASD-related Streptococci had reduced PIGR protein level. Conclusion: Therefore, protective IgA levels were reduced in the saliva of individuals with ASD, which correlated with the bacteria-induced downregulation of Pigr in salivary glands. This study suggests a new direction for ASD diagnosis and prevention of oral diseases in ASD cohorts and provides evidence for the ASD mucosal immunophenotype in the oral cavity.

The Awareness and Attitude of Contracted Service Among General Medical Practitioners in Community Health Service Centers in Urban China: A Cross-Sectional Study.

This study aims to explore the attitude, willingness, and satisfaction with contracted service (CS) among staff in community health service (CHS) centers in urban China and to explore the associated factors of satisfaction with CS. From August 2016 to July 2017, five CHS centers in three provinces of China were selected. Setting-level information was collected by official document review; and personal information on demographic characteristics, awareness, willingness, and attitude of CS among staff was collected by questionnaire survey. Univariate and multivariable logistic regression models were fitted to explore the associated factors of satisfaction with CS. Multiple correspondence analysis (MCA) was used to visually demonstrate the correlations among category data related with satisfaction with CS. The CS signing rates were 30.78, 12.72, 22.20, 14.32, and 21.19% in the five CHS centers. A total of 286 staff included family doctors (40.91%), nurses (31.12%), and others (27.97%) completed the survey. For the sense of self-worth, 86.01% (246/286) participants hold a positive attitude. The predominant barrier of CS signing was caused by the work pressure due to CS performance assessment (48.60%, 139/286). About 30% of family doctors and nurses reported a heavy work pressure, and more than 30% of doctors had great feeling of fatigue. Notably, 51.69% family doctors would like to change their job in the future. Compared with other staff, family doctors were more likely to be unsatisfied with CS (OR: 2.793, 95% CI: 1.155-6.754, p = 0.022). Participants in Sichuan province have lower satisfaction than other places. The MCA yielded similar factors consistent with multivariable results of clustering with different levels of CS satisfaction. Our study revealed that the CS coverage and satisfaction among staff from the primary healthcare system varied geographically and are associated with professional field, workload, and pressure. Measures that aim to promote the stability of primary care human resource should be considered in the future.

Dietary restriction and/or exercise training impairs spermatogenesis in normal rats.

Dietary restriction and/or exercise has been shown to have multiple benefits for health. However, its effects on reproductive health and the mechanisms by which it regulates reproductive function remain unclear. Here, to evaluate its effects on spermatogenesis and sperm function, rats were divided into 4 groups: ad libitum-fed sedentary control, dietary restriction (DR), exercise training (ET), and dietary restriction plus exercise training (DR+ET) groups. Results indicated that body weight, epididymal fat pad weight, and sperm counts were significantly reduced in the DR, ET, and DR+ET groups. Moreover, sperm motility and capacitation-associated protein tyrosine phosphorylation were suppressed in the DR and DR+ET groups, but not the ET group. Microarray analysis revealed that the number of downregulated genes was higher than that of upregulated genes in the DR and/or ET groups. About half of the downregulated genes are common after exercise training and/or diet restriction. Gene ontology analysis showed that downregulated genes in the DR, ET, and DR+ET groups affected spermatogenesis through overlapping pathways, including glucocorticoid, corticosteroid, extracellular structure organization, and estradiol responses. Our findings suggest that diet restriction and/or exercise training may present potential risks to male reproductive dysfunction by disrupting normal gene expression patterns in the testis. Novelty: Dietary restriction and/or exercise can lead to the damage of spermatogenesis as well as sperm maturation. Sperm functional changes are more sensitive to dietary restriction than exercise training. Dietary restriction and exercise impair spermatogenesis through overlapping biological pathways in the testis.

Distinct mRNA and long non-coding RNA expression profiles of decidual natural killer cells in patients with early missed abortion.

Early non-chromosome-related missed abortion (MA) is commonly associated with an altered immunological environment during pregnancy. Human decidual natural killer (dNK) cells, the most abundant lymphocyte population within the first-trimester maternal-fetal interface, are vital maternal regulators of immune tolerance mediating successful embryo implantation and placentation. Previous studies have shown that dNK cells may play a role in MA. However, the gene expression status and specific altered manifestations of dNK cells in patients with early MA remain largely unknown. Here, we show that MA dNK cells have distinct mRNA and lncRNA expression profiles through RNA sequencing, with a total of 276 mRNAs and 67 lncRNAs being differentially expressed compared with controls. Protein-protein interaction analysis of differentially expressed mRNAs was performed to identify hub genes and key modules. An lncRNA-mRNA regulatory network characterized by the small-world property was constructed to reveal the regulation of mRNA transcription by differential hub lncRNAs. Functional annotation of differentially expressed mRNAs and lncRNAs was performed to disclose their potential roles in MA pathogenesis. Our data highlight several enriched biological processes (immune response, inflammatory response, cell adhesion, and extracellular matrix [ECM] organization) and signaling pathways (cytokine-cytokine receptor interaction, ECM-receptor interaction, Toll-like receptor signaling pathway, and phosphatidylinositol signaling system) that may influence MA. This study is the first to demonstrate the involvement of altered mRNA and lncRNA expression profiles in the dNK cell pathogenesis of early MA, facilitating a better understanding of the underlying molecular mechanisms and the development of novel MA therapeutic strategies targeting key mRNAs and lncRNAs.

Whole-exome sequencing of a large Chinese azoospermia and severe oligospermia cohort identifies novel infertility causative variants and genes.

Rare coding variants have been proven to be one of the significant factors contributing to spermatogenic failure in patients with non-obstructive azoospermia (NOA) and severe oligospermia (SO). To delineate the molecular characteristics of idiopathic NOA and SO, we performed whole-exome sequencing of 314 unrelated patients of Chinese Han origin and verified our findings by comparing to 400 fertile controls. We detected six pathogenic/likely pathogenic variants and four variants of unknown significance, in genes known to cause NOA/SO, and 9 of which had not been earlier reported. Additionally, we identified 20 novel NOA candidate genes affecting 25 patients. Among them, five (BRDT, CHD5, MCM9, MLH3 and ZFX) were considered as strong candidates based on the evidence obtained from murine functional studies and human single-cell (sc)RNA-sequencing data. These genetic findings provide insight into the aetiology of human NOA/SO and pave the way for further functional analysis and molecular diagnosis of male infertility.

Overexpression of human-derived DNMT3A induced intergenerational inheritance of DNA methylation and gene expression variations in rat brain and testis.

In mammals, DNA methylation patterns are established by various types of DNA methyltransferases and can be stably passed on during cell division, thus creating a paradigm for epigenetic regulation that can mediate long-lasting changes in gene expression even when the initial triggering signal has disappeared. Although functional deficiency of DNMT3A, one of the methyltransferases, leads to abnormal DNA methylation patterns that result in developmental deficits in mammals, the impacts of its overexpression on tissue gene expression and DNA methylation patterns remain unclear. Here, our previously established hDNMT3A transgenic rat model and mRNA sequencing and bisulphite sequencing PCR were used to analyse the impact of hDNMT3A overexpression on tissue transcriptome and methylome, and whether the impact could be inherited intergenerationally was subsequently investigated. Our results revealed that the overexpression of hDNMT3A could induce notable gene expression variations in rat testis and brain. More importantly, 36.02% and 38.89% of these variations could be intergenerationally inherited to offspring without the transmission of the initial endogenic trigger in the brain and testis, respectively. Furthermore, we found that intergenerationally inherited DNA methylation variations in their promoters and exons could be the underlying mechanism. Compared with inheritable variations that were passively induced by environmental factors, these variations were actively induced by endogenous epigenetic modifiers. This study provided evidence for the epigenetic inheritance of endogenous factors that actively induce gene expression and DNA methylation variations; however, more studies are needed to determine the number of generations that these variations can be stably inherited.

Locus-specific DNA methylation of Mecp2 promoter leads to autism-like phenotypes in mice.

Autism spectrum disorder (ASD) is a neurodevelopmental disease with a strong heritability, but recent evidence suggests that epigenetic dysregulation may also contribute to the pathogenesis of ASD. Especially, increased methylation at the MECP2 promoter and decreased MECP2 expression were observed in the brains of ASD patients. However, the causative relationship of MECP2 promoter methylation and ASD has not been established. In this study, we achieved locus-specific methylation at the transcription start site (TSS) of Mecp2 in Neuro-2a cells and in mice, using nuclease-deactivated Cas9 (dCas9) fused with DNA methyltransferase catalytic domains, together with five locus-targeting sgRNAs. This locus-specific epigenetic modification led to a reduced Mecp2 expression and a series of behavioral alterations in mice, including reduced social interaction, increased grooming, enhanced anxiety/depression, and poor performance in memory tasks. We further found that specifically increasing the Mecp2 promoter methylation in the hippocampus was sufficient to induce most of the behavioral changes. Our finding therefore demonstrated for the first time the casual relationship between locus-specific DNA methylation and diseases symptoms in vivo, warranting potential therapeutic application of epigenetic editing.