HELQ deficiency impairs the induction of primordial germ cell-like cells.

Helicase POLQ-like (HELQ) is a DNA helicase essential for the maintenance of genome stability. A recent study identified two HELQ missense mutations in some cases of infertile men. However, the functions of HELQ in the process of germline specification are not well known and whether its function is conserved between mouse and human remains unclear. Here, we revealed that Helq knockout (Helq-/-) could significantly reduce the efficiency of mouse primordial germ cell-like cell (PGCLC) induction. In addition, Helq-/- embryonic bodies exhibited a severe apoptotic phenotype on day 6 of mouse PGCLC induction. p53 inhibitor treatment could partially rescue the generation of mouse PGCLCs from Helq mutant mouse embryonic stem cells. Finally, the genetic ablation of HELQ could also significantly impede the induction of human PGCLCs. Collectively, our study sheds light on the involvement of HELQ in the induction of both mouse and human PGCLCs, providing new insights into the mechanisms underlying germline differentiation and the genetic studies of human fertility.

Nitroaromatic Compounds from Secondary Nitrate Formation and Biomass Burning Are Major Proinflammatory Components in Organic Aerosols in Guangzhou: A Bioassay Combining High-Resolution Mass Spectrometry Analysis.

The limited characterization and detection capacity of unknown compounds hinder our understanding of the molecular composition of toxic compounds in PM2.5. The present study applied Fourier transform ion cyclotron resonance mass spectrometry coupled with negative and positive electrospray ionization sources (ESI-/ESI+ FT-ICR-MS) to probe the molecular characteristics and dynamic formation processes of the effective proinflammatory components in organic aerosols (OAs) of PM2.5 in Guangzhou for one year. We detected abundant proinflammatory molecules in OAs, mainly classified as CHON compounds (compounds composed of C, H, O, and N atoms) in elemental and nitroaromatic compounds (NACs) in structures. From the perspective of the formation process, we discovered that these proinflammatory molecules, especially toxic NACs, were largely driven by secondary nitrate formation and biomass burning (in emission source), as well as SO2 (in atmospheric evolution). In addition, our results indicated that the secondary processes had replaced the primary emission as the main contributing source of the toxic proinflammatory compounds in OAs. This study highlights the importance of community measures to control the production of nitroaromatic compounds derived from secondary nitrate formation and biomass burning in urban areas.

Base-Promoted Formal (3 + 2) Cycloaddition of α-Halohydroxamates with Electron-Deficient Alkenyl-iminoindolines To Synthesize Spiro-indolinepyrrolidinones.

Construction of pyrrolidinyl-spiroindoles with easily available starting materials has attracted considerable attention from the synthesis community and is in great demand. Here, we describe a base-promoted formal (3 + 2) cycloaddition of α-halohydroxamates with alkenyl-iminoindolines. The present methodology features mild reaction conditions and a broad substrate scope with up to 99% yield and excellent diastereoselectivity. The versatility of this approach is demonstrated through valuable synthetic transformations. Preliminary mechanistic studies shed light on the mechanism of this cycloaddition process.

Single-cell multi-omics sequencing of human spermatogenesis reveals a DNA demethylation event associated with male meiotic recombination.

Human spermatogenesis is a highly ordered process; however, the roles of DNA methylation and chromatin accessibility in this process remain largely unknown. Here by simultaneously investigating the chromatin accessibility, DNA methylome and transcriptome landscapes using the modified single-cell chromatin overall omic-scale landscape sequencing approach, we revealed that the transcriptional changes throughout human spermatogenesis were correlated with chromatin accessibility changes. In particular, we identified a set of transcription factors and cis elements with potential functions. A round of DNA demethylation was uncovered upon meiosis initiation in human spermatogenesis, which was associated with male meiotic recombination and conserved between human and mouse. Aberrant DNA hypermethylation could be detected in leptotene spermatocytes of certain nonobstructive azoospermia patients. Functionally, the intervention of DNA demethylation affected male meiotic recombination and fertility. Our work provides multi-omics landscapes of human spermatogenesis at single-cell resolution and offers insights into the association between DNA demethylation and male meiotic recombination.

Surface integrity optimization for ball-end hard milling of AISI D2 steel based on response surface methodology.

This study focuses on systematically revealing how cutting parameters influence the surface integrity of ball-end hard milled surface of AISI D2 steel and proposing optimization scheme from surface integrity, wear resistance and fatigue resistance perspective based on response surface methodology respectively. Results can be summarized into three aspects. Firstly, radial depth of cut with percent contribution ratio (PCR) 62.05% has a decisive influence on surface roughness, followed by spindle speed 13.25% and feed per tooth 6.63%. The work hardening degree was raised from 12.5% to 38.4% when spindle speed changed from 8000 rpm to 2000 rpm. Spindle speed and radial depth of cut are the most significant factor influencing residual stress. The PCR of spindle speed and radial depth of cut reached 73.47% and 18.63% for residual stress in feed direction, 47.11% and 37.51% in step-over direction, respectively. High residual compressive stress can be generated by lowering spindle speed and radial depth of cut benefiting from the aggravated squeeze between ball-end milling cutter and workpiece. Secondly, too small feed per tooth or too small radial depth of cut should be avoided from wear resistance point because though the surface microhardness can be improved, the surface quality will also be deteriorated. The combination of high spindle speed, small feed per tooth together with small radial depth of cut can meet the wear resistance and the machining efficiency requirement. Finally, a medium-sized cutting parameter combination should be adopted to realize satisfying material removal rate and fatigue resistance. This study can be used to guide the selection of cutting parameters during ball-end milling of hardened AISI D2 steel for dies/molds manufacturing industries.

Complex polycyclic aromatic compound mixtures in PM2.5 in a Chinese megacity: Spatio-temporal variations, toxicity, and source apportionment.

Polycyclic aromatic compounds (PACs) are important toxic organic components in fine particulate matter (PM2.5), whereas the links between PM2.5 toxicity and associated PACs in ambient air are poorly understood. This study investigated the spatial-temporal variations of PACs in PM2.5 collected from 11 sampling sites across a Chinese megacity and characterized the reactive oxygen species (ROS) generation and cytotoxicity induced by organic extracts of PM2.5 based on cellular assays. The extra trees regression model based on machine learning and ridge regression were used to identify the key toxicants among complex PAC mixtures. The total concentrations of these PACs varied from 2.12 to 71.7 ng/m3 across the study city, and polycyclic aromatic hydrocarbons (PAHs) are the main PACs. The spatial variations of the toxicological indicators generally resembled those of the PAC concentrations, and the PM2.5 related to waste treatment facilities exhibited the strongest toxic potencies. The ROS generation was highly correlated with high molecular weight PAHs (MW302 PAHs), followed by PAHs with MW<302 amu and oxygenated PAHs, but not with nitrated PAHs and the plastics additives. The cell mortality showed weak correlations with these organic constituents. The associations between the biological endpoints and these PM2.5-bound contaminants were further confirmed by exposure to authentic chemicals. Four primary sources of PACs were identified, among which coal and biomass combustion sources (30.2% of the total PACs) and industrial sources (31.0%) were predominant. PACs emitted from industrial sources were highly associated with ROS generation in this city. Our findings highlight the potent ROS-generating potential of MW302 PAHs and the importance of industrial sources contributing to PM2.5 toxicity in this megacity, raising public concerns and further administration.

Gasdermin D-mediated pyroptosis: mechanisms, diseases, and inhibitors.

Gasdermin D (GSDMD)-mediated pyroptosis and downstream inflammation are important self-protection mechanisms against stimuli and infections. Hosts can defend against intracellular bacterial infections by inducing cell pyroptosis, which triggers the clearance of pathogens. However, pyroptosis is a double-edged sword. Numerous studies have revealed the relationship between abnormal GSDMD activation and various inflammatory diseases, including sepsis, coronavirus disease 2019 (COVID-19), neurodegenerative diseases, nonalcoholic steatohepatitis (NASH), inflammatory bowel disease (IBD), and malignant tumors. GSDMD, a key pyroptosis-executing protein, is linked to inflammatory signal transduction, activation of various inflammasomes, and the release of downstream inflammatory cytokines. Thus, inhibiting GSDMD activation is considered an effective strategy for treating related inflammatory diseases. The study of the mechanism of GSDMD activation, the formation of GSDMD membrane pores, and the regulatory strategy of GSDMD-mediated pyroptosis is currently a hot topic. Moreover, studies of the structure of caspase-GSDMD complexes and more in-depth molecular mechanisms provide multiple strategies for the development of GSDMD inhibitors. This review will mainly discuss the structures of GSDMD and GSDMD pores, activation pathways, GSDMD-mediated diseases, and the development of GSDMD inhibitors.

Lovastatin promotes the self-renewal of murine and primate spermatogonial stem cells.

The spermatogonial stem cell (SSC) niche is critical for SSC maintenance and subsequent spermatogenesis. Numerous reproductive hazards impair the SSC niche, thereby resulting in aberrant SSC self-renewal and male infertility. However, promising agents targeting the impaired SSC niche to promote SSC self-renewal are still limited. Here, we screen out and assess the effects of Lovastatin on the self-renewal of mouse SSCs (mSSCs). Mechanistically, Lovastatin promotes the self-renewal of mSSCs and inhibits its inflammation and apoptosis through the regulation of isoprenoid intermediates. Remarkably, treatment by Lovastatin could promote the proliferation of undifferentiated spermatogonia in the male gonadotoxicity model generated by busulfan injection. Of note, we demonstrate that Lovastatin could enhance the proliferation of primate undifferentiated spermatogonia. Collectively, our findings uncover that lovastatin could promote the self-renewal of both murine and primate SSCs and have implications for the treatment of certain types of male infertility using small compounds.

Pollution source and chemicals structure of the water-soluble fractions in PM2.5 that induce apoptosis in China.

Identify risk drivers is the key condition in air pollution control, and biological effect-directed analysis is the most commented method for combing chemical identify and human health. The water-soluble organic matter contained in PM2.5 plays an important role in human health, while it is also the most difficult to identify its chemical information. Exploring the structural characteristics and pollution sources of its key toxic components is the optimized strategy to meet this question. In this study, the induction of apoptosis by the water-soluble fractions (WSF) of PM2.5 samples collected in 10 major cities in China over a period of 1 year was observed in vitro in Beas-2b cells. Organic carbon structures were examined using nuclear magnetic resonance; air potential sources were identified using δ13C and 14C isotopic markers. Apoptosis induction by WSF in PM2.5 was generally stronger in northern cities than in southern cities, and in winter than in summer. Organic compounds with aromatic and double-bond carbon structures from secondary products of motor vehicle exhausts, coal-derived emissions, and emissions derived from the burning of core residues may be primarily responsible for apoptosis induction by PM2.5. Our results will contribute to understanding the toxic substances contained in WSF and provide basic data for accurate pollution control.

Targeting APLN/APJ restores blood-testis barrier and improves spermatogenesis in murine and human diabetic models.

Type 2 diabetes mellitus is one of the most prevalent metabolic diseases presenting with systemic pathologies, including reproductive disorders in male diabetic patients. However, the molecular mechanisms that contributing to spermatogenesis dysfunction in diabetic patients have not yet been fully elucidated. Here, we perform STRT-seq to examine the transcriptome of diabetic patients' testes at single-cell resolution including all major cell types of the testis. Intriguingly, whereas spermatogenesis appears largely preserved, the gene expression profiles of Sertoli cells and the blood-testis barrier (BTB) structure are dramatically impaired. Among these deregulate pathways, the Apelin (APLN) peptide/Apelin-receptor (APJ) axis is hyper-activated in diabetic patients' testes. Mechanistically, APLN is produced locally by Sertoli cells upon high glucose treatment, which subsequently suppress the production of carnitine and repress the expression of cell adhesion genes in Sertoli cells. Together, these effects culminate in BTB structural dysfunction. Finally, using the small molecule APLN receptor antagonist, ML221, we show that blocking APLN/APJ significantly ameliorate the BTB damage and, importantly, improve functional spermatogenesis in diabetic db/db mice. We also translate and validate these findings in cultured human testes. Our findings identify the APLN/APJ axis as a promising therapeutic target to improve reproduction capacity in male diabetic patients.

Coordination compound-derived Fe4N/Fe3N/Fe/CNT for efficient electrocatalytic oxygen evolution: a facile one-step synthesis in the absence of extra nitrogen source.

By annealing an Fe(III)-coordination compound (Fe-CC), [FeCl3(Hbta)2] (Hbta = benzotriazole) in the presence of a carbon nanotube precursor (PCNT) template, an Fe4N/Fe3N/Fe/CNT heterostructure was successfully synthesized without an extra nitrogen source. The decomposition of the Hbta in Fe-CC under high-temperature annealing can produce carbon sheets and reduced graphene oxide (rGO), and the presence of CNTs can alleviate the stacking of thein situ-generated carbon materials. Meanwhile, iron nitride nanoparticles (NPs) can be anchored on the carbon sheets, and the anchoring effect efficiently prevents the agglomeration of NPs and increases the amount of active catalytic sites for the oxygen evolution reaction (OER). Fe4N/Fe3N/Fe/CNT shows an excellent OER activity with a Tafel slope of 63 mV dec-1as well as overpotentials of 121 (η10) and 275 mV (η100) at 10 and 100 mA cm-2, respectively - far exceeding commercial RuO2and other catalysts. Density functional theory calculations show that the excellent OER performance of Fe4N/Fe3N/Fe/CNT is associated with the Fe4N/Fe3N heterojunction, which can improve the electron conductivity and boost the electron transfer from N to Fe. The Fe4N/Fe3N/Fe/CNT catalyst exhibits long-term OER activity during 100 h of electrolysis at 20 mA cm-2. This is related to the dual coatings of thein situ-generated thin carbon shell and few-layered rGO on the surface of the iron nitride NPs, which can protect the fast leaching of iron nitride during the OER process. Furthermore, the effects of the annealing temperature, the PCNT template and the heating rate on the calcined products were investigated.

Cell-fate transition and determination analysis of mouse male germ cells throughout development.

Mammalian male germ cell development is a stepwise cell-fate transition process; however, the full-term developmental profile of male germ cells remains undefined. Here, by interrogating the high-precision transcriptome atlas of 11,598 cells covering 28 critical time-points, we demonstrate that cell-fate transition from mitotic to post-mitotic primordial germ cells is accompanied by transcriptome-scale reconfiguration and a transitional cell state. Notch signaling pathway is essential for initiating mitotic arrest and the maintenance of male germ cells' identities. Ablation of HELQ induces developmental arrest and abnormal transcriptome reprogramming of male germ cells, indicating the importance of cell cycle regulation for proper cell-fate transition. Finally, systematic human-mouse comparison reveals potential regulators whose deficiency contributed to human male infertility via mitotic arrest regulation. Collectively, our study provides an accurate and comprehensive transcriptome atlas of the male germline cycle and allows for an in-depth understanding of the cell-fate transition and determination underlying male germ cell development.

Coordination Polymer-Derived Fe3N Nanoparticles for Efficient Electrocatalytic Oxygen Evolution.

Based on a coordination polymer, FeCl2(4,4'-bpy) (4,4'-bpy = 4,4'-bipyridine) and the carbon nanotube (CNT)/NaCl dual template, Fe3N nanoparticles (NPs) were synthesized via chemical thermolysis in the absence of an extra nitrogen source. The decomposition of 4,4'-bpy under high temperature produces thin carbon coating for Fe3N NPs. Also, the CNT template anchors the Fe3N NPs to avoid aggregation. The sample (denoted as Fe3N-C N) exhibits excellent electrocatalytic oxygen evolution reaction (OER) behavior even with a small molar ratio of Fe3N (Fe: 4.9 at. %), which can deliver a current density of 10 mA cm-2 at an overpotential of 218 mV with a Tafel slope of 84 mV dec-1 and long-term OER activity during 60 h electrolysis at 20 mA cm-2. Furthermore, the sample after 20 h electrolysis, denoted as Post-Fe3N-C N (20 h), displays enhanced OER activity with a smaller Tafel slope of 41 mV dec-1 and overpotentials of 195 and 327 mV at 10 and 100 mA cm-2, respectively, which is mainly due to the partial transformation of Fe3N into FeOOH. The OER mechanism is investigated by density functional theory calculations, and it is found that the surface partial oxidation of Fe3N leads to the effective OER electrolysis, which changes the electron density of the superficial atoms and induces the moderate adsorption for the intermediates.

[Effects of drought stress on physiological and biochemical and chemical components of Cinnamomum cassia seedlings].

Six month old Cinnamomum cassia seedlings were used to simulate drought stress with polyethylene glycol(PEG 6000). The physiological indicators(osmotic substances, antioxidant enzymes, etc.) and chemical components of seedlings under different drought levels and the correlation between the two were studied. The results showed that the chlorophyll content and relative water content decreased gradually with the increase of PGE 6000(0, 5%, 10%, 15%) concentration and time(3, 5, 7 d), while the soluble protein content, soluble sugar content and catalase(CAT) activity increased, but the rising rate slowed down with the time. The activities of peroxidase(POD), superoxide dismutase(SOD), malondialdehyde(MDA) and proline content increased at first and then decreased. The content of coumarin, cinnamaldehyde, cinnamic acid and dimethoxycinnamaldehyde decreased, while the content of cinnamyl alcohol continued to increase.Under drought stress, the fluorescence signals of reactive oxygen species and no contents in roots of C. cassia seedlings were significantly stronger than those of the control.Further correlation analysis showed that coumarin content, di-methoxycinnamaldehyde content and osmoregulation substance content were significantly negatively correlated(P<0.05), cinnamic acid content was significantly negatively correlated with POD and SOD activities(P<0.01).It was found that C. cassia seedlings showed a certain degree of drought tolerance under short-term or mild drought stress, but if the drought exceeded a certain degree, the physiological metabolism of the seedlings would be unbalanced.

The chromatin accessibility landscape reveals distinct transcriptional regulation in the induction of human primordial germ cell-like cells from pluripotent stem cells.

In vitro induction of human primordial germ cell-like cells (hPGCLCs) provides an ideal platform to recapitulate hPGC development. However, the detailed molecular mechanisms regulating the induction of hPGCLCs remain largely uncharacterized. Here, we profiled the chromatin accessibility and transcriptome dynamics throughout the process of hPGCLC induction. Genetic ablation of SOX15 indicated the crucial roles of SOX15 in the maintenance of hPGCLCs. Mechanistically, SOX15 exerted its roles via suppressing somatic gene expression and sustaining latent pluripotency. Notably, ETV5, a downstream regulator of SOX15, was also uncovered to be essential for hPGCLC maintenance. Finally, a stepwise switch of OCT4/SOX2, OCT4/SOX17, and OCT4/SOX15 binding motifs were found to be enriched in closed-to-open regions of human embryonic stem cells, and early- and late-stage hPGCLCs, respectively. Collectively, our data characterized the chromatin accessibility and transcriptome landscapes throughout hPGCLC induction and defined the SOX15-mediated regulatory networks underlying this process.

DNA Methylation Biomarkers of IQ Reduction are Associated with Long-term Lead Exposure in School Aged Children in Southern China.

Although lead associated with intelligence decline in children has long been reported, studies combining intelligence determination, molecular mechanisms exploration, and biomarker screen are quite rare. In this study, based on 333 children aged 9-11, we determined the role of DNA methylation (DNAm) in the relationship of lead exposure with children's intelligence. DNAm was measured from children's blood DNA specimens, and mediation analysis was performed to identify DNAm biomarkers mediating the lead-intelligence relationship. We identified forty-three differentially methylated regions (DMRs), and two fragments (FAM50B1 and PTCHD3) significantly mediated the lead-intelligence relationship, with contribution rates of 30.36% (p = 0.010) and 60.36% (p < 0.001), respectively. In addition, blood lead levels (BLLs) lower than 100 µg/L still adversely affected children's IQs and DNAm of the two fragments. Our data suggests that DNAm mediates lead-associated cognitive delay in children and blood lead reference value for school-aged children (100 µg/L) should be revised, and the candidate biomarkers can be used in related neurological diseases in future.

Effects of drought stress on physiological and biochemical and chemical components of Cinnamomum cassia seedlings / 中国中药杂志

Six month old Cinnamomum cassia seedlings were used to simulate drought stress with polyethylene glycol(PEG 6000). The physiological indicators(osmotic substances, antioxidant enzymes, etc.) and chemical components of seedlings under different drought levels and the correlation between the two were studied. The results showed that the chlorophyll content and relative water content decreased gradually with the increase of PGE 6000(0, 5%, 10%, 15%) concentration and time(3, 5, 7 d), while the soluble protein content, soluble sugar content and catalase(CAT) activity increased, but the rising rate slowed down with the time. The activities of peroxidase(POD), superoxide dismutase(SOD), malondialdehyde(MDA) and proline content increased at first and then decreased. The content of coumarin, cinnamaldehyde, cinnamic acid and dimethoxycinnamaldehyde decreased, while the content of cinnamyl alcohol continued to increase.Under drought stress, the fluorescence signals of reactive oxygen species and no contents in roots of C. cassia seedlings were significantly stronger than those of the control.Further correlation analysis showed that coumarin content, di-methoxycinnamaldehyde content and osmoregulation substance content were significantly negatively correlated(P<0.05), cinnamic acid content was significantly negatively correlated with POD and SOD activities(P<0.01).It was found that C. cassia seedlings showed a certain degree of drought tolerance under short-term or mild drought stress, but if the drought exceeded a certain degree, the physiological metabolism of the seedlings would be unbalanced.

MicroRNA 182 promotes T helper 1 cell by repressing hypoxia induced factor 1 alpha in experimental autoimmune encephalomyelitis.

MicroRNA 182 is important for the clonal expansion of CD4+ T cells (Th) following IL-2 stimulation and is a potential therapeutic target for autoimmune diseases. In the present study, we investigated the role of microRNA 182 in the differentiation of pro-inflammatory CD4+ T helper cell by overexpressing or silencing microRNA 182 expression both in in vivo and in vitro settings. We report that in the studied Chinese cohort, microRNA 182 is upregulated in patients with relapse and remitting multiple sclerosis (RRMS) and this upregulation is associated with increased IFN-γ producing CD4+ Th1 cells in the circulation. In the murine experimental autoimmune encephalomyelitis (EAE) model, global microRNA 182 overexpression exacerbates clinical symptoms and results in augmented CD4+ IFN-γ+ Th1 and CD4+ IL-17+ Th17 differentiation in vivo. Addition of microRNA 182 mimics in vitro represses both the protein expression and transcriptional activity of hypoxia induced factor 1α (HIF-1α) but increases the level of IFN-γ transcripts in sorted murine CD4+ T cells. Together, our results provide evidence that microRNA 182 may be one of the transitional hubs contribution to regulate Th cells expansion in response to self-antigens and differentiation of antigen specific Th cells during the progression of autoimmune inflammations.

Inflammation Response of Water-Soluble Fractions in Atmospheric Fine Particulates: A Seasonal Observation in 10 Large Chinese Cities.

Spatiotemporal trends in pro-inflammatory (interleukin (IL)-6 and IL-8) release after exposure to the water-soluble fractions of PM2.5 sampled in 10 large Chinese cities over 1 year were investigated. Chemical components (water-soluble ions, metal(loid) elements, water-soluble organic carbon (WSOC), humic-like substances (HULIS), and endotoxins) in PM2.5 samples were measured, and the molecular structure of WSOC was also analyzed by nuclear magnetic resonance. Changes in DNA methylation and gene expression of candidate genes were also evaluated to explore the potential mechanisms. PM2.5 from southern cities induced lower pro-inflammatory responses compared to those from northern cities. Seasonal differences in toxicity were noted among the cities. IL-6 was significantly correlated with HULIS (as the main fraction of WSOC with oxygenated carbohydrate structures characteristic), Pb, and endotoxin. Furthermore, DNA methylation and gene expression changes in RASSF2 and CYP1B1 were related to pro-inflammatory secretion. Certain components of PM2.5, rather than PM2.5 mass itself, determine the pro-inflammatory release. In particular, HULIS, which originated from primary biomass burning and residual coal combustion, and secondary organic aerosols, appear to be the key component in PM2.5 to induce human health risk.

Efficient generation of human primordial germ cell-like cells from pluripotent stem cells in a methylcellulose-based 3D system at large scale.

BACKGROUND: The mechanisms underlying human germ cell development and infertility remain largely unknown due to bioethical issues and the shortage of experimental materials. Therefore, an effective in vitro induction system of human primordial germ-like cells (hPGCLCs) from human pluripotent stem cells (hPSC) is in high demand. The current strategies used for the generation of hPGCLCs are not only costly but also difficult to perform at a large scale, thereby posing barriers to further research. In this study, we attempted to solve these problems by providing a new 3D culture system for hPGCLC differentiation. METHODS: The efficiency and relative yield of a methylcellulose (MC)-based 3D hPGCLC induction system were first compared with that of a conventional U96 system. Then, we examined the gene expression of germ cell marker genes and the key epigenetic modifications of the EpCAM-/INTEGRINα6-high cells from the 3D MC induction system and the U96 system via quantitative PCR and immunofluorescence. Finally, the reliability of the MC-based 3D hPGCLC induction system was evaluated via the generation of induced pluripotent stem cells (iPSCs) from the testicular cells of one patient with obstructive azoospermia (OA) and followed by the subsequent differentiation of iPSCs into the germ cell lineage. RESULTS: In the present study, we demonstrated that the 3D MC induction system combined with low-cell attachment plates facilitated the generation of hPGCLCs at a large scale. We found that the hPGCLCs generated via the MC system shared similar characteristics to that via the U96 system in terms of the gene expression profiles, germ cell-specific markers, epigenetic modification states and cellular states. In addition, hPGCLCs from iPSCs derived from one OA patient were generated with high efficiency via the present 3D MC induction system. DISCUSSION: The in vitro induction of hPGCLCs from human embryonic stem cells (hESCs)/human induced pluripotent stem cells (hiPSCs) has significant implications in exploring the underlying mechanisms of the origin and specification of hPGCs and the epigenetic programming of the human germ line as well as treating male infertility. Here, we developed a simple and efficient 3D induction system to generate hPGCLCs from hESCs/hiPSCs at a large scale, which facilitated the study of human germ cell development and stem cell-based reproductive medicine.