Palmitoylation of vacuole membrane protein 1 promotes small extracellular vesicle secretion via interaction with ALIX and influences intercellular communication.

BACKGROUND: Small extracellular vesicles (EVs), exemplified by exosomes, mediate intercellular communication by transporting proteins, mRNAs, and miRNAs. Post-translational modifications are involved in controlling small EV secretion process. However, whether palmitoylation regulates small EV secretion, remains largely unexplored. METHODS: Vacuole Membrane Protein 1 (VMP1) was testified to be S-palmitoylated by Palmitoylation assays. VMP1 mutant plasmids were constructed to screen out the exact palmitoylation sites. Small EVs were isolated, identified and compared between wild-type VMP1 or mutant VMP1 transfected cells. Electron microscope and immunofluorescence were used to detect multivesicular body (MVB) number and morphology change when VMP1 was mutated. Immunoprecipitation and Mass spectrum were adopted to identify the protein that interacted with palmitoylated VMP1, while knock down experiment was used to explore the function of targeted protein ALIX. Taking human Sertoli cells (SCs) and human spermatogonial stem cell like cells (SSCLCs) as a model of intercellular communication, SSCLC maintenance was detected by flow cytometry and qPCR at 12 days of differentiation. In vivo, mouse model was established by intraperitoneal injection with palmitoylation inhibitor, 2-bromopalmitate (2BP) for 3 months. RESULTS: VMP1 was identified to be palmitoylated at cysteine 263,278 by ZDHHC3. Specifically, palmitoylation of VMP1 regulated its subcellular location and enhanced the amount of small EV secretion. Mutation of VMP1 palmitoylation sites interfered with the morphology and biogenesis of MVBs through suppressing intraluminal vesicle formation. Furthermore, inhibition of VMP1 palmitoylation impeded small EV secretion by affecting the interaction of VMP1 with ALIX, an accessory protein of the ESCRT machinery. Taking SCs and SSCLCs as a model of intercellular communication, we discovered VMP1 palmitoylation in SCs was vital to the growth status of SSCLCs in a co-culture system. Inhibition of VMP1 palmitoylation caused low self-maintenance, increased apoptosis, and decreased proliferation rate of SSCLCs. In vivo, intraperitoneal injection of 2BP inhibited VMP1 palmitoylation and exosomal marker expression in mouse testes, which were closely associated with the level of spermatogenic cell apoptosis and proliferation. CONCLUSIONS: Our study revealed a novel mechanism for small EV secretion regulated by VMP1 palmitoylation in Sertoli cells, and demonstrated its pivotal role in intercellular communication and SSC niche.

Deficiency of nucleosome-destabilizing factor GLYR1 dampens spermatogenesis in mice.

Aberrant sperm morphology hinders sperm motility and causes male subfertility. Spermatogenesis, a complex process in male germ cell development, necessitates precise regulation of numerous developmental genes. However, the regulatory pathways involved in this process remain partially understood. We have observed the widespread expression of Glyr1, the gene encoding a nucleosome-destabilizing factor, in mouse testicular cells. Our study demonstrates that mice experiencing Glyr1 depletion in spermatogenic cells exhibit subfertility characterized by a diminished count and motility of spermatozoa. Furthermore, the rate of sperm malformation significantly increases in the absence of Glyr1, with a predominant occurrence of head and neck malformation in spermatozoa within the cauda epididymis. Additionally, a reduction in spermatocyte numbers across different meiotic stages is observed, accompanied by diminished histone acetylation in spermatogenic cells upon Glyr1 depletion. Our findings underscore the crucial roles of Glyr1 in mouse spermiogenesis and unveil novel insights into the etiology of male reproductive diseases.

RNA m6A modification regulates L1 retrotransposons in human spermatogonial stem cell differentiation in vitro and in vivo.

The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m6A) plays an essential role in regulating various biological processes. However, the function of m6A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m6A methyltransferase METTL3 or the m6A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m6A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m6A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m6A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m6A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m6A modification in restricting L1 retrotransposition.

Discovery of (2-(4-Substituted phenyl)quinolin-4-yl)(4-isopropylpiperazin-1-yl)methanone Derivatives as Potent Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitors.

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an increasingly important role in the treatment of hyperlipidemia. In pursuit of potent small molecules that block the PCSK9/low-density lipoprotein receptor (LDLR) protein-protein interaction (PPI), a series of 2-phenylquinoline-4-carboxylic acid derivatives were designed and synthesized based on previously derived molecules. In the in vitro PPI inhibition test, compounds M1, M12, M14, M18 and M27 exhibited potent activities with IC50 values of 6.25 µM, 0.91 µM, 2.81 µM, 4.26 µM and 0.76 µM, respectively, compared with SBC-115337 (IC50 value of 9.24 µM). Molecular docking and molecular dynamics simulations revealed the importance of hydrophobic interactions in the binding of inhibitors to the PPI interface of PCSK9. In LDLR expression and LDL uptake assays, the tested compounds M1, M12 and M14 were found to restore LDLR expression levels and to increase the extracellular LDL uptake capacity of HepG2 cells in the presence of exogenous PCSK9. Collectively, novel small-molecule PCSK9/LDLR PPI inhibitors (especially M12) with in vitro lipid lowering ability, were discovered as lead compounds for further development of hypolipidemic drugs.

A comprehensive meta-analysis to identify susceptibility genetic variants for precocious puberty.

PURPOSE: Currently, several genetic variants in ERα gene (rs2234693 and rs9340799), ERß gene (rs1256049 and rs4986938), KISS1 gene (rs4889, rs1132506 and rs5780218), LIN28B gene (rs314263, rs314276 and rs314280), and MKRN3 gene (rs2239669) have been repeatedly explored for their contribution to precocious puberty (PP) susceptibility. However, the results remain conflicting rather than conclusive. We here performed a meta-analysis to identify the real susceptibility genetic variants for PP. METHODS: After screening by inclusion criteria, 20 related studies were finally included in this meta-analysis. The odds ratios and 95% confidence intervals were calculated to assess the strength of association. Sensitive analysis, publication bias, and trial sequential analysis (TSA) were performed to evaluate the stability and reliability of results. RESULTS: Rs2234693, rs9340799, and rs1256049 were significantly associated with PP susceptibility (p < 0.0084). Stratified analysis according to ethnicity showed that rs2234693 and rs9340799 were significantly associated with PP susceptibility in Asian and Chinese populations. Stratified analysis according to PP subtype showed that rs2234693 and rs9340799 were significantly associated with idiopathic central PP susceptibility in Asian and Chinese populations (p < 0.0084). The results of publication bias, sensitivity analysis, and TSA provided solid evidence for the association between these three variants and PP susceptibility. CONCLUSIONS: Rs2234693 and rs9340799 in ERα gene and rs1256049 in ERß gene may serve as susceptive factors for PP development. The present finding should be confirmed in replication studies and reinforced in functional studies, which will ultimately improve the feasibility of the application of these three PP-susceptible loci in clinical practice.

Hardness and superconductivity in tetragonal LiB4 and NaB4.

Boron-based compounds have triggered substantial attention due to their multifunctional properties, incorporating excellent hardness and superconductivity. While tetragonal metal borides LiB4 and NaB4 with BaAl4-type structure and striking clathrate boron motif have been induced under compression, there is still a lack of deep understanding of their potential properties at ambient pressure. We herein conduct a comprehensive study on I4/mmm-structured LiB4 and NaB4 under ambient pressure via first-principles calculations. Remarkably, both LiB4 and NaB4 are found to possess high Vickers hardness of 39 GPa, which is ascribed to the robust boron framework with strong covalency. Furthermore, their high hardness values together with distinguished stability make them highly potential superhard materials. Meanwhile, electron-phonon coupling analysis reveals that both LiB4 and NaB4 are conventional phonon-mediated superconductors, with critical temperatures of 6 and 8 K at 1 atmosphere pressure (atm), respectively, mainly arising from the coupling of B 2p electronic states and the low-frequency phonon modes associated with Li-, Na-, and B-derived vibrations. This work provides valuable insights into the mechanical and superconducting behaviors of metal borides and will boost further studies of emergent borides with multiple functionalities.

Analysis of different plant- and animal-based dietary patterns and their relationship with serum uric acid levels in Chinese adults.

BACKGROUND: Dietary patterns play an important role in regulating serum uric acid levels in the body, but evidence for the association between different kinds of plant-based and animal-based dietary patterns and individual serum uric acid levels is scarce and inconsistent. METHODS: We analyzed data from the sixth wave of the China Health and Nutrition Survey. The plant-based diet of 7,806 participants was determined using three consecutive 24-hour dietary recalls, and latent profile analysis was used to identify dietary patterns among participants. Serum uric acid levels were analyzed using the enzymatic colorimetric method. The association between intakes of different types of dietary pattern and individual serum uric acid levels was analyzed using linear regression analysis, after adjusting for confounding variables. RESULTS: We identified three types of plant-based dietary patterns, namely, low tuber starches and vegetable plant-based diet (LTVP), high cereal, tuber starches and vegetable plant-based diet (HCTVP), and high legume and fruit plant-based diet (HLFP). We also identified three types of animal-based dietary patterns, namely, high milk and egg animal-based diet (HMiEA), low egg and fish animal-based diet, and high meat and fish animal-based diet (HMeFA). Significant coefficients for participant serum uric acid levels were observed for the HCTVP diet (ß = -0.022, P = 0.031) and HMeFA diet (ß = 0.061, P < 0.001). The median intake of foods in the HCTVP diet was as follows: cereals and cereal products, 444.83 g/d; tubers and starch products, 166.67 g/d; dried legumes and legume products, 8.33 g/d; vegetables and vegetable products, 333.33 g/d; and fruits and fruit products, 0 g/d. The median intake of foods in the HMeFA diet was as follows: meat and meat products, 73.33 g/d; poultry and poultry products, 0 g/d; milk and milk products, 0 g/d; eggs and egg products, 26.67 g/d; and fish, shellfish, and mollusks, 180.00 g/d. CONCLUSION: We showed that individual serum uric acid levels (1) might decrease under the plant-based HCTVP diet, (2) might increase under the animal-based HMeFA diet, (3) might not decrease under the plant-based HLFP diet, and (4) might not increase under the animal-based HMiEA diet. Further studies are needed to confirm these associations.

[Delivery of epididymis-specific mRNAs from mouse epididymosomes into N2a and TM4 cells].

OBJECTIVE: To investigate whether mouse epididymis-specific mRNAs Adam7 and Crisp1 can be delivered into N2a and TM4 cells, and to provide an experimental basis for exploring the function of epididymal mRNAs. METHODS: Using RT-PCR, we detected the presence of epididymis-specific genes (Adam7, Crisp1, Defb22, Wfdc2, and Wfdc9) in the testis, epididymis, epididymosome and sperm of adult male BALB/c mice as well as in the human testis, seminal vesicles and sperm. We isolated epididymosomes of BALB/c mice by low-speed centrifugation, filtration and ultracentrifugation, fluorescently labeled them by PKH26, co-incubated them for 1 hour with the N2a and TM4 cells after 24 hours of starvation culture, and observed whether they were fused with the N2a and TM4 cells and ingested using the epididymosomes without PKH26 labeling, PKH26 dye without epididymosomes, and non- epididymosome or -PKH26 dye as controls. Then we detected the epididymis-specific genes in the N2a and TM4 cells after 1-hour co-incubation by RT-PCR. RESULTS: Adam7 and Crisp1 were present in the mouse epididymis, epididymosomes and sperm, and in the human seminal vesicles and sperm as well, but not in the testes of either the mice or men. PKH26 and Hoechst33258 fluorescence double-labeling showed that the mouse epididymosomes were fused with the N2a and TM4 cells and ingested; RT-PCR revealed the mRNAs of Adam7 and Crisp1 in the N2a and TM4 cells after 1-hour co-incubation; and Western blot exhibited the CRISP1 protein in the N2a and TM4 cells incubated with epididymosomes. CONCLUSION: Epididymosomes can deliver epididymis-specific mRNAs Adam7 and Crisp1 into N2a and TM4 cells, where Crisp1 may be translated into proteins, though their function and significance need to be further studied.

Enhancing T cell anti-tumor efficacy with a PD1-TIGIT chimeric immune-checkpoint switch receptor.

Chimeric antigen receptor (CAR) T cell immunotherapy has demonstrated success in the treatment of hematological malignancies; however, its efficacy and applications in solid tumors remain limited. Immunosuppressive factors, particularly inhibitory checkpoint molecules, restrict CAR T cell activity inside solid tumors. The modulation of checkpoint pathways has emerged as a promising approach to promote anti-tumor responses in CAR T cells. Programmed cell death protein 1 (PD1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) are two critical immune-checkpoint molecules that suppress anti-tumor activity in T cells. Simultaneous targeting of these two inhibitory molecules could be an efficient checkpoint modulation strategy. Here, we developed a PD1-TIGIT chimeric immune-checkpoint switch receptor (CISR) that enhances the efficacy of CAR T cell immunotherapy by reversing the inhibitory checkpoint signals of PD1/PDL1 and/or TIGIT/CD155. In addition to neutralizing PDL1 and CD155, this chimeric receptor is engineered with the transmembrane region and intracellular domain of CD28, thereby effectively enhancing T cell survival and tumor-targeting functions. Notably, under simultaneous stimulation of PDL1 and CD155, CISR-CAR T cells demonstrate superior performance in terms of cell survival, proliferation, cytokine release, and cytotoxicity in vitro, compared with conventional CAR T cells. Experiments utilizing both cell line- and patient-derived xenotransplantation tumor models showed that CISR-CAR T cells exhibit robust infiltration and anti-tumor efficiency in vivo. Our results highlight the potential for the CISR strategy to enhance T cell anti-tumor efficacy and provide an alternative approach for T cell-based immunotherapies.

Effectiveness of Nutritional Therapies in Male Factor Infertility Treatment: A Systematic Review and Network Meta-analysis.

BACKGROUND: Nutritional therapies are effective alternative treatments for male infertility or subfertility. These are cost-effective and easily implementable, unlike other advanced invasive treatments. Even moderate improvements in sperm quality could improve spontaneous pregnancy. OBJECTIVE: We aimed to compare the effectiveness of all nutritional therapies in male infertility/subfertility treatment and ranked their efficacy based on type and etiology. We intend to aid clinicians with an evidence-based approach to affordable and safer initial infertility treatment for those who mainly do not wish to have other advanced invasive treatments or could not afford or have access to them. METHODS: We included 69 studies with 94 individual study arms identified from bibliographic databases and registries. We included studies in adult men with proven infertility or subfertility that investigated nutritional or dietary supplement therapies compared with control or placebo and at least reported on a sperm parameter. We undertook a network meta-analysis and performed a pairwise meta-analysis on all sperm parameter outcomes and meta-regression. No language or date restriction was imposed. A systematic article search was concluded on August 29, 2022. RESULTS: Our network meta-analysis is the first to compare all dietary interventions in a single analysis, sub-grouped by intervention type and type of infertility. L-Carnitine with micronutrients, antioxidants, and several traditional herbal supplements showed statistically and clinically significant improvement in sperm quality. Meta-regression identified that improvement in the sperm count, motility and morphology translated into increased pregnancy rates (p < 0.001; p < 0.001; p < 0.002, respectively). In particular, L-carnitine with micronutrient therapy (risk ratio [RR]: 3.60, 95% CI 1.86, 6.98, p = 0.0002), followed by zinc (RR 5.39, 95% CI 1.26, 23.04, p = 0.02), significantly improved pregnancy rates. Men with oligozoospermia (RR 4.89), followed by oligoasthenozoospermia (RR 4.20) and asthenoteratozoospermia (RR 3.53), showed a significant increase in pregnancy rates. CONCLUSION: We ranked nutritional therapies for their ability to improve sperm quality in men with infertility. Nutritional therapies, particularly L-carnitine alone or combined with micronutrients, significantly improved sperm parameters and pregnancy rates even under severe conditions. We believe these affordable solutions may be valuable for people without access to or who do not wish to undergo more invasive and costly fertility treatments.

Sertoli cell-only syndrome: advances, challenges, and perspectives in genetics and mechanisms.

Male infertility can be caused by quantitative and/or qualitative abnormalities in spermatogenesis, which affects men's physical and mental health. Sertoli cell-only syndrome (SCOS) is the most severe histological phenotype of male infertility characterized by the depletion of germ cells with only Sertoli cells remaining in the seminiferous tubules. Most SCOS cases cannot be explained by the already known genetic causes including karyotype abnormalities and microdeletions of the Y chromosome. With the development of sequencing technology, studies on screening new genetic causes for SCOS are growing in recent years. Directly sequencing of target genes in sporadic cases and whole-exome sequencing applied in familial cases have identified several genes associated with SCOS. Analyses of the testicular transcriptome, proteome, and epigenetics in SCOS patients provide explanations regarding the molecular mechanisms of SCOS. In this review, we discuss the possible relationship between defective germline development and SCOS based on mouse models with SCO phenotype. We also summarize the advances and challenges in the exploration of genetic causes and mechanisms of SCOS. Knowing the genetic factors of SCOS offers a better understanding of SCO and human spermatogenesis, and it also has practical significance for improving diagnosis, making appropriate medical decisions, and genetic counseling. For therapeutic implications, SCOS research, along with the achievements in stem cell technologies and gene therapy, build the foundation to develop novel therapies for SCOS patients to produce functional spermatozoa, giving them hope to father children.

MicroRNA profiling reveals the role of miR-133b-3p in promoting apoptosis and inhibiting cell proliferation and testosterone synthesis in mouse TM3 cells.

Late-onset hypogonadism (LOH) is an age-related clinical and biological syndrome in which serum testosterone deficiency is an important characteristic and diagnostic indicator. In this study, we firstly analyzed the difference in the expression level of three miR-133 s (including miR-133a-3p, miR-133a-5p, and miR-133b-3p) in rat testis samples, blood samples from mice before and 1 wk after testis removal, and mouse TM3 cells. Secondly, the mimics and inhibitors corresponding to the three miR-133 s of mouse were transfected into TM3 cells separately to determine the correlation between the three miRNAs. Finally, using mouse TM3 cells to analyze the effect of miR-133b overexpression or inhibition on the proliferation and apoptosis of mouse testicular Leydig cells, the effect on genes related to testosterone synthesis, and the effect on the level of testosterone in the culture medium. We found that, compared with the testis tissue of newborn rats, miR-133a-5p was increased in adult rats, and miR-133a-3p and miR-133b-3p were decreased. In addition, 1 wk after the testis was removed, the expression levels of these three miRNAs in the blood of adult mice decreased. The correlation of the three miRNAs was summarized, and it was found that miR-133b-3p played an important role in it. In TM3 cells, overexpression of miR-133b-3p suppressed the proliferation and promotes apoptosis of cells, suppressed the expression level of most genes related to cell proliferation and testosterone synthesis, and the concentration of testosterone in the culture medium decreased while these phenomena can be reversed by the inhibition of miR-133b-3p expression. It was found that miR-133b-3p can regulate testosterone production in TM3 cells at least by targeting FSCN1. The above results suggest that miR-133b-3p plays an important role in regulating testosterone synthesis. These findings also provide new candidate diagnostic indicators for late-onset hypogonadism in men and provide new clues for the further study of pathogenesis.

Promising novel biomarkers and therapy targets: The application of cell-free seminal nucleotides in male reproduction research.

Liquid biopsy has the advantage of diagnosing diseases in a non-invasive manner. Seminal plasma contains secretions from the bilateral testes, epididymides, seminal vesicles, bulbourethral glands, and the prostate. These organs are relatively small and contain delicate tubes that are prone to damage by invasive diagnosis. Cell-free seminal nucleic acids test is a newly emerged item in liquid biopsy. Here, we present a comprehensive overview of all known cell-free DNA and cell-free RNAs (mRNA, miRNA, lncRNA, circRNA, piRNA, YRNA, tsRNA, etc.) and discuss their roles as biomarker candidates in liquid biopsy. With great advantages, including high stability, sensitivity, representability, and non-invasiveness, cell-free DNA/RNAs may be developed as promising biomarkers for the screening, diagnosis, prognosis, and follow-up of diseases in semen-secreting organs. Moreover, RNAs in semen may participate in important processes, including sperm maturation, early embryo development, and transgenerational disease inheritance, which may be developed as potential treatment targets for future clinical use.

Preconception paternal mental disorders and child health: Mechanisms and interventions.

Mental illness is a significant global health issue with a steady prevalence. High heritability is suspected, but genome-wide association studies only identified a small number of risk genes associated with mental disorders. This 'missing inheritance' can be partially explained by epigenetic heredity. Evidence from numerous animal models and human studies supports the possibility that preconception paternal mental health influences their offspring's mental health via nongenetic means. Here, we review two potential pathways, including sperm epigenetics and seminal plasma components. The current review highlights the role of sperm epigenetics and explores epigenetic message origination and susceptibility to chronic stress. Meanwhile, possible spatiotemporal windows and events that induce sexually dimorphic modes and effects of paternal stress transmission are inferred in this review. Additionally, we discuss emerging interventions that could potentially block the intergenerational transmission of paternal psychiatric disorders and reduce the incidence of mental illness. Understanding the underlying mechanisms by which preconception paternal stress impacts offspring health is critical for identifying strategies supporting healthy development and successfully controlling the prevalence of mental illness.

[Molecular markers for predicting the success of micro-TESE in non-obstructive azoospermia].

Non-obstract azoospermia (NOA) is a serious male infertility disease. At present, testicular sperm extraction (micro-TESE) is performed in combination with intracytoplasmic sperm injection (ICSI) technology, NOA patients can have their own consanguine offspring. However, due to the invasiveness and uncertainty of micro-TESE surgery, it is difficult for patients to accept it. Therefore, finding an accurate method to predict the possibility of micro-TESE successful sperm retrival would be beneficial to azoospermia patients. Many genes are transcribed and expressed during spermatogenesis, and molecular assays have irreplaceable sensitivity and specificity in predicting the success sperm retrivel of micro-TESE. This article reviews the methods to predict the success sperm retrivel of micro-TESE including mRNA, non-coding RNA (piRNA, microRNA, cirRNA, tFRNAs) and some protein so far, to provide certain reference value for clinical and subsequent research.

The developmental miR-17-92 cluster and the Sfmbt2 miRNA cluster cannot rescue the abnormal embryonic development generated using obstructive epididymal environment-producing sperm in C57BL/6 J mice.

BACKGROUND: Sperm, during epididymal transit, acquires microRNAs(miRNAs), which are crucial for embryonic development. However, whether sperm miRNAs influenced by an obstructive epididymal environment affect embryonic development remains unknown. METHOD: The sham operation and vasectomy were performed in C57BL/6 J mice to create the control group (CON) and the obstructive epididymal environment group(OEE) group, respectively. The morphology of the testis and epididymis was observed using hematoxylin and eosin staining (HE staining) to establish the OEE mice model. The sperm quality test, intracytoplasmic sperm injection (ICSI), and epididymosomes fusion were employed to observe the effect of the obstructive epididymal environment on sperm and resultant embryonic development. The alteration of the sperm small RNA (sRNA) profile was analyzed by sRNA sequencing. RT-qPCR and DNA methylation were applied to observe the effect of obstructive epididymis on the expression of sperm miRNAs. The miRNAs microinjection was used to explore the impacts of sperm miRNAs on embryonic development. RESULTS: We confirmed postoperative 8-week mice as the OEE mice model by examining the morphology of the testis and epididymis. In the OEE group, we observed that sperm quality degraded and the development potential of embryos was reduced, which can be saved by the normal epididymal environment. The sperm sRNA sequencing revealed that the expression of the developmental miR-17-92 cluster and the Sfmbt2 miRNA cluster was downregulated in the OEE group. The expression of these two miRNA clusters in epididymis was also downregulated and regulated by DNA methylation. However, the downregulation of either the miR-17-92 cluster or the Sfmbt2 miRNA cluster in normal zygotes did not impair embryonic development. CONCLUSION: The obstructive epididymal environment influences sperm quality and resultant embryonic development, as well as the abundance of the developmental miR-17-92 cluster and the Sfmbt2 miRNA cluster in sperm, but these miRNA clusters are not the cause of abnormal embryonic development. It implies that epididymis is important in early embryonic development and may play a potential role in sperm epigenome.

The balance between NANOG and SOX17 mediated by TET proteins regulates specification of human primordial germ cell fate.

BACKGROUND: Human primordial germ cells (hPGCs) initiate from the early post-implantation embryo at week 2-3 and undergo epigenetic reprogramming during development. However, the regulatory mechanism of DNA methylation during hPGC specification is still largely unknown due to the difficulties in analyzing early human embryos. Using an in vitro model of hPGC induction, we found a novel function of TET proteins and NANOG in the hPGC specification which was different from that discovered in mice. METHODS: Using the CRISPR-Cas9 system, we generated a set of TET1, TET2 and TET3 knockout H1 human embryonic stem cell (hESC) lines bearing a BLIMP1-2A-mKate2 reporter. We determined the global mRNA transcription and DNA methylation profiles of pluripotent cells and induced hPGC-like cells (hPGCLCs) by RNA-seq and whole-genome bisulfite sequencing (WGBS) to reveal the involved signaling pathways after TET proteins knockout. ChIP-qPCR was performed to verify the binding of TET and NANOG proteins in the SOX17 promoter. Real-time quantitative PCR, western blot and immunofluorescence were performed to measure gene expression at mRNA and protein levels. The efficiency of hPGC induction was evaluated by FACS. RESULTS: In humans, TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) impaired the NODAL signaling pathway and impeded hPGC specification in vitro, while the hyperactivated NODAL signaling pathway led to gastrulation failure when Tet proteins were inactivated in mouse. Specifically, TET proteins stimulated SOX17 through the NODAL signaling pathway and directly regulates NANOG expression at the onset of hPGCLCs induction. Notably, NANOG could bind to SOX17 promoter to regulate its expression in hPGCLCs specification. Furthermore, in TKO hESCs, DNMT3B-mediated hypermethylation of the NODAL signaling-related genes and NANOG/SOX17 promoters repressed their activation and inhibited hPGCLC induction. Knockout of DNMT3B in TKO hESCs partially restored NODAL signaling and NANOG/SOX17 expression, and rescued hPGCLC induction. CONCLUSION: Our results show that TETs-mediated oxidation of 5-methylcytosine modulates the NODAL signaling pathway and its downstream genes, NANOG and SOX17, by promoting demethylation in opposition to DNMT3B-mediated methylation, suggesting that the epigenetic balance of DNA methylation and demethylation in key genes plays a fundamental role in early hPGC specification.

The dose-effect relationships of cigarette and alcohol consumption with depressive symptoms: a multiple-center, cross-sectional study in 5965 Chinese middle-aged and elderly men.

BACKGROUND: Although association of depressive symptoms with cigarette or alcohol is well documented, the dose-response relationship between them is rarely studied. This study aims to evaluate dose-response relationships of cigarette and alcohol consumption with depressive symptoms in Chinese middle-aged and elderly men, providing evidence to guide cigarette and alcohol control. METHODS: This multiple-center, cross-sectional study including 5965 Chinese men aged 40-79 years was conducted in 2013-2016 in China. Depressive symptoms were evaluated by Beck Depression Inventory-Short Form. History of cigarette smoking and alcohol drinking were collected with a structured questionnaire. Prevalence of depressive symptoms was compared depending on cigarette and alcohol consumption. Adjusted odds ratios (OR) and 95% confidence interval (CI) were estimated by binary logistic regression. Interpolation analysis was applied to test dose-effect relationships. RESULTS: A parabolic-shaped relationship was observed between cigarette consumption and depressive symptoms. Compared to never smokers, 59.0% (OR = 1.59, 95% CI 1.30-1.94) and 29.0% (OR = 1.29, 95% CI 1.08-1.54) higher odds of depressive symptoms were observed in men smoking < 10 cigarettes/day and 10-20 cigarettes/day, whereas, similar odds of depressive symptoms among men smoking > 20 cigarettes/day (P = 0.092). An inverted J-shaped relationship was observed between alcohol consumption and depressive symptoms. Compared to never drinkers, a tendency of higher prevalence of depressive symptoms (OR = 1.16, 95% CI 0.99-1.36) was observed in men drinking < 140 g/week, and similar prevalence was observed in those drinking 140-280 g/week (P = 0.920), whereas, 29.4% (OR = 0.71, 95% CI 0.57-0.88) lower odds in men drinking > 280 g/week. CONCLUSIONS: Associations of cigarette smoking and alcohol drinking with depressive symptoms differ with consumption in middle-aged and elderly men. Health-care providers should exercise great caution on depressive symptoms in conducting cigarette and alcohol control.

Transcriptome and DNA methylome analysis of peripheral blood samples reveals incomplete restoration and transposable element activation after 3-months recovery of COVID-19.

Comprehensive analyses showed that SARS-CoV-2 infection caused COVID-19 and induced strong immune responses and sometimes severe illnesses. However, cellular features of recovered patients and long-term health consequences remain largely unexplored. In this study, we collected peripheral blood samples from nine recovered COVID-19 patients (median age of 36 years old) from Hubei province, China, 3 months after discharge as well as 5 age- and gender-matched healthy controls; and carried out RNA-seq and whole-genome bisulfite sequencing to identify hallmarks of recovered COVID-19 patients. Our analyses showed significant changes both in transcript abundance and DNA methylation of genes and transposable elements (TEs) in recovered COVID-19 patients. We identified 425 upregulated genes, 214 downregulated genes, and 18,516 differentially methylated regions (DMRs) in total. Aberrantly expressed genes and DMRs were found to be associated with immune responses and other related biological processes, implicating prolonged overreaction of the immune system in response to SARS-CoV-2 infection. Notably, a significant amount of TEs was aberrantly activated and their activation was positively correlated with COVID-19 severity. Moreover, differentially methylated TEs may regulate adjacent gene expression as regulatory elements. Those identified transcriptomic and epigenomic signatures define and drive the features of recovered COVID-19 patients, helping determine the risks of long COVID-19, and guiding clinical intervention.