How the extra X chromosome impairs the development of male fetal germ cells.

The dosage of X-linked genes is accurately regulated with the development of fetal germ cells (FGCs)1,2. How aberrant dosage of X-linked genes impairs FGC development in humans remains poorly understood. FGCs of patients with Klinefelter syndrome (KS), who have an extra X chromosome, provide natural models for addressing this issue3. Here we demonstrate that most human FGCs in KS are arrested at an early stage, characterized by the upregulation of genes related to pluripotency, the WNT pathway and the TGF-ß pathway, along with the downregulation of genes involved in FGC differentiation. The limited KS FGCs that are capable of reaching the late stage remain relatively naive. X chromosomes are not inactivated and the dosage of X-linked genes is excessive in KS FGCs. X-linked genes dominate the differentially expressed genes and are enriched in critical biological processes associated with the developmental delay of KS FGCs. Moreover, aberrant interactions between Sertoli cells and FGCs disrupt the migration of late FGCs to the basement membrane in KS. Notably, inhibition of the TGF-ß pathway improves the differentiation of KS FGCs. Our findings elucidate how the extra X chromosome impairs the development of male FGCs and reveal the initial molecular events preceding germ cell loss in KS.

Sphingomyelin Synthase 2 Promotes Endothelial Dysfunction by Inducing Endoplasmic Reticulum Stress.

Endothelial dysfunction (ED) is an important contributor to atherosclerotic cardiovascular disease. Our previous study demonstrated that sphingomyelin synthase 2 (SMS2) promotes ED. Moreover, endoplasmic reticulum (ER) stress can lead to ED. However, whether there is a correlation between SMS2 and ER stress is unclear. To examine their correlation and determine the detailed mechanism of this process, we constructed a human umbilical vein endothelial cell (HUVEC) model with SMS2 overexpression. These cells were treated with 4-PBA or simvastatin and with LiCl and salinomycin alone. The results showed that SMS2 can promote the phosphorylation of lipoprotein receptor-related protein 6 (LRP6) and activate the Wnt/ß-catenin pathway and that activation or inhibition of the Wnt/ß-catenin pathway can induce or block ER stress, respectively. However, inhibition of ER stress by 4-PBA can decrease ER stress and ED. Furthermore, when the biosynthesis of cholesterol is inhibited by simvastatin, the reduction in intracellular cholesterol coincides with a decrease in ER stress and ED. Collectively, our results demonstrate that SMS2 can activate the Wnt/ß-catenin pathway and promote intracellular cholesterol accumulation, both of which can contribute to the induction of ER stress and finally lead to ED.

Simvastatin Attenuates H2O2-Induced Endothelial Cell Dysfunction by Reducing Endoplasmic Reticulum Stress.

Atherosclerosis is the pathological basis of cardiovascular disease, whilst endothelial dysfunction (ED) plays a primary role in the occurrence and development of atherosclerosis. Simvastatin has been shown to possess significant anti-atherosclerosis activity. In this study, we evaluated the protective effect of simvastatin on endothelial cells under oxidative stress and elucidated its underlying mechanisms. Simvastatin was found to attenuate H2O2-induced human umbilical vein endothelial cells (HUVECs) dysfunction and inhibit the Wnt/ß-catenin pathway; however, when this pathway was activated by lithium chloride, endothelial dysfunction was clearly enhanced. Further investigation revealed that simvastatin did not alter the expression or phosphorylation of LRP6, but reduced intracellular cholesterol deposition and inhibited endoplasmic reticulum (ER) stress. Inducing ER stress with tunicamycin activated the Wnt/ß-catenin pathway, whereas reducing ER stress with 4-phenylbutyric acid inhibited it. We hypothesize that simvastatin does not affect transmembrane signal transduction in the Wnt/ß-catenin pathway, but inhibits ER stress by reducing intracellular cholesterol accumulation, which blocks intracellular signal transduction in the Wnt/ß-catenin pathway and ameliorates endothelial dysfunction.

DNA methylation abnormalities induced by advanced maternal age in villi prime a high-risk state for spontaneous abortion.

BACKGROUND: Advanced maternal age (AMA) has increased in many high-income countries in recent decades. AMA is generally associated with a higher risk of various pregnancy complications, and the underlying molecular mechanisms are largely unknown. In the current study, we profiled the DNA methylome of 24 human chorionic villi samples (CVSs) from early pregnancies in AMA and young maternal age (YMA), 11 CVSs from early spontaneous abortion (SA) cases using reduced representation bisulfite sequencing (RRBS), and the transcriptome of 10 CVSs from AMA and YMA pregnancies with mRNA sequencing(mRNA-seq). Single-cell villous transcriptional atlas presented expression patterns of targeted AMA-/SA-related genes. Trophoblast cellular impairment was investigated through the knockdown of GNE expression in HTR8-S/Vneo cells. RESULTS: AMA-induced local DNA methylation changes, defined as AMA-related differentially methylated regions (DMRs), may be derived from the abnormal expression of genes involved in DNA demethylation, such as GADD45B. These DNA methylation changes were significantly enriched in the processes involved in NOTCH signaling and extracellular matrix organization and were reflected in the transcriptional alterations in the corresponding biological processes and specific genes. Furthermore, the DNA methylation level of special AMA-related DMRs not only significantly changed in AMA but also showed more excessive defects in CVS from spontaneous abortion (SA), including four AMA-related DMRs whose nearby genes overlapped with AMA-related differentially expressed genes (DEGs) (CDK11A, C19orf71, COL5A1, and GNE). The decreased DNA methylation level of DMR near GNE was positively correlated with the downregulated expression of GNE in AMA. Single-cell atlas further revealed comparatively high expression of GNE in the trophoblast lineage, and knockdown of GNE in HTR8-S/Vneo cells significantly impaired cellular proliferation and migration. CONCLUSION: Our study provides valuable resources for investigating AMA-induced epigenetic abnormalities and provides new insights for explaining the increased risks of pregnancy complications in AMA pregnancies.

The combination of DNA methylome and transcriptome revealed the intergenerational inheritance on the influence of advanced maternal age.

BACKGROUND: The number of women delivering at advanced maternal age (AMA; > = 35) continuously increases in developed and high-income countries. Large cohort studies have associated AMA with increased risks of various pregnancy complications and adverse pregnancy outcomes, which raises great concerns about the adverse effect of AMA on the long-term health of offspring. Specific acquired characteristics of parents can be passed on to descendants through certain molecular mechanisms, yet the underlying connection between AMA-related alterations in parents and that in offspring remains largely uncharted. METHODS: We profiled the DNA methylomes of paired parental peripheral bloods and cord bloods from 20 nuclear families, including 10 AMA and 10 Young, and additional transcriptomes of 10 paired maternal peripheral bloods and cord bloods. RESULTS: We revealed that AMA induced aging-like changes in DNA methylome and gene expression in both parents and offspring. The expression changes in several genes, such as SLC28A3, were highly relevant to the disorder in DNA methylation. In addition, AMA-related differentially methylated regions (DMRs) identified in mother and offspring groups showed remarkable similarities in both genomic locations and biological functions, mainly involving neuron differentiation, metabolism, and histone modification pathways. AMA-related differentially expressed genes (DEGs) shared by mother and offspring groups were highly enriched in the processes of immune cell activation and mitotic nuclear division. We further uncovered developmental-dependent dynamics for the DNA methylation of intergenerationally correlated DMRs during pre-implantation embryonic development, as well as diverse gene expression patterns during gametogenesis and early embryonic development for those common AMA-related DEGs presenting intergenerational correlation, such as CD24. Moreover, some intergenerational DEGs, typified by HTRA3, also showed the same significant alterations in AMA MII oocyte or blastocyst. CONCLUSIONS: Our results reveal potential intergenerational inheritance of both AMA-related DNA methylome and transcriptome and provide new insights to understand health problems in AMA offspring.

Simvastatin promotes endothelial dysfunction by activating the Wnt/ß­catenin pathway under oxidative stress.

Atherosclerosis is a major pathogenic factor in patients with cardiovascular diseases, and endothelial dysfunction (ED) plays a primary role in its occurrence and development. Simvastatin is a lipid­lowering drug, which is commonly used to prevent or treat risk factors of cardiovascular diseases with a significant anti­atherogenic effect. However, its impact on endothelial cells under conditions of oxidative stress and broader mechanisms of action remain unclear. The present study evaluated the effect of simvastatin on human umbilical vein endothelial cells (HUVECs) under oxidative stress with H2O2, and the associated mechanisms. At a high dose (1 µM), simvastatin exacerbated H2O2­induced endothelial cell dysfunction. Moreover, inhibition of the Wnt/ß­catenin pathway by salinomycin significantly suppressed the simvastatin­associated HUVEC dysfunction. Western blot analysis further demonstrated that simvastatin promoted the phosphorylation of low­density lipoprotein receptor­related protein 6 (LRP6) and activated the Wnt/ß­catenin pathway. Simvastatin also activated endoplasmic reticulum (ER) stress, which was reversed by salinomycin treatment. Based on these results, it was hypothesized that simvastatin may promote ER stress by facilitating LRP6 phosphorylation and the subsequent activation of the Wnt/ß­catenin pathway, thereby enhancing H2O2­induced ED. Therefore, high­dose simvastatin treatment could have potential toxic side effects, indicating the need for close clinical management, monitoring and patient selection.

Sphingomyelin synthase 2 promotes H2O2-induced endothelial dysfunction by activating the Wnt/ß-catenin signaling pathway.

Atherosclerosis (AS) is the primary cause of various cardiovascular and cerebrovascular diseases and has high morbidity and mortality rates. Oxidative stress­induced endothelial cells (ECs) dysfunction is the pathological basis of AS. In addition, sphingomyelin (SM) and the Wnt/ß­catenin signaling pathway are considered to be closely associated with AS; however, the specific mechanism is not clear. Therefore, the present study investigated whether SM may induce ECs dysfunction through the Wnt/ß­catenin signaling pathway. Firstly, a sphingomyelin synthase 2 (SMS2) overexpression cell model was constructed. It was identified that the expression of SMS2 was increased when ECs were treated with H2O2. In addition, these results demonstrated that SMS2 overexpression promoted apoptosis and macrophage adhesion of H2O2­induced ECs, thereby increasing the expression of ß­catenin. Furthermore, SMS activity was inhibited with Dy105, combined with simultaneous treatment with LiCl or H2O2. This additionally confirmed that Dy105 significantly inhibited SMS activity and decreased the level of ECs dysfunction and ß­catenin content; however, LiCl served a key role in activating the Wnt/ß­catenin signaling pathway to promote ECs dysfunction. Collectively, these results suggested that SMS2 overexpression may promote ECs dysfunction by activating the Wnt/ß­catenin signaling pathway, while Dy105 may inhibit the evolution of oxidative stress­induced dysfunction.