Correlation between triglyceride glucose index and collateral circulation formation in patients with chronic total occlusion of coronary arteries in different glucose metabolic states.

BACKGROUND: To investigate the correlation between triglyceride glucose index (TyG) and collateral circulation in patients with chronic total occlusion (CTO) of coronary arteries in different glucose metabolic states. METHODS: A total of 681 patients who underwent coronary angiography between January 2020 and December 2021 to determine the presence of CTO lesions in at least one major coronary artery were retrospectively included in this study. Patients were categorized into a group with poor collateral circulation formation (Rentrop grade 0-1, n = 205) and a group with good collateral circulation formation (Rentrop grade 2-3, n = 476) according to the Rentrop scale. They were also categorized according to their glucose metabolism status: normal glucose regulation (NGR) (n = 139), prediabetes mellitus (Pre-DM) (n = 218), and diabetes mellitus (DM) (n = 324). Correlation between TyG index and collateral circulation formation was analyzed by logistic regression analysis and receiver operating characteristic (ROC) curves. RESULTS: Among patients with CTO, TyG index was significantly higher in the group with poor collateral circulation formation than in the group with good collateral circulation formation. Logistic regression analysis showed that TyG index was an independent risk factor for poor collateral circulation formation (OR 5.104, 95% CI 3.323-7.839, P < 0.001). The accuracy of TyG index in predicting collateral circulation formation was evaluated by the ROC curve, which had an area under the curve of 0.779 (95% CI 0.738-0.820, P < 0.001). The restrictive cubic spline curves showed that the risk of poor collateral circulation formation in the Pre-DM and DM groups was initially flat and finally increased rapidly, except for the NGR group. TyG index was significantly associated with an increased risk of poor collateral circulation formation in the Pre-DM and DM groups. CONCLUSIONS: TyG index was significantly associated with the risk of poor collateral circulation formation in patients with CTO, especially those with Pre-DM and DM.

Unveiling Mechanical Activation: GAIN Domain Unfolding and Dissociation in Adhesion GPCRs.

Adhesion G protein-coupled receptors (aGPCRs) have extracellular regions (ECRs) containing GPCR autoproteolysis-inducing (GAIN) domains. The GAIN domain enables the ECR to self-cleave into N- and C-terminal fragments. However, the impact of force on the GAIN domain's conformation, critical for mechanosensitive aGPCR activation, remains unclear. Our study investigated the mechanical stability of GAIN domains in three aGPCRs (B, G, and L subfamilies) at a loading rate of 1 pN/s. We discovered that forces of a few piconewtons can destabilize the GAIN domains. In autocleaved aGPCRs ADGRG1/GPR56 and ADGRL1/LPHN1, these forces cause the GAIN domain detachment from the membrane-proximal Stachel sequence, preceded by partial unfolding. In noncleavable aGPCR ADGRB3/BAI3 and cleavage-deficient mutant ADGRG1/GPR56-T383G, complex mechanical unfolding of the GAIN domain occurs. Additionally, GAIN domain detachment happens during cell migration. Our findings support the mechanical activation hypothesis of aGPCRs, emphasizing the sensitivity of the GAIN domain structure and detachment to physiological force ranges.

Structural basis for CD97 recognition of the decay-accelerating factor CD55 suggests mechanosensitive activation of adhesion GPCRs.

The adhesion G protein-coupled receptor CD97 and its ligand complement decay-accelerating factor CD55 are important binding partners in the human immune system. Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis, as well as various cancers. Previous literatures have indicated that the CD97 includes 3 to 5 epidermal growth factor (EGF) domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat (SCR) domains of CD55. However, the details of this interaction remain elusive, especially why the CD55 binds with the highest affinity to the shortest isoform of CD97 (EGF1,2,5). Herein, we designed a chimeric expression construct with the EGF1,2,5 domains of CD97 and the SCR1-4 domains of CD55 connected by a flexible linker and determined the complex structure by crystallography. Our data reveal that the two proteins adopt an overall antiparallel binding mode involving the SCR1-3 domains of CD55 and all three EGF domains of CD97. Mutagenesis data confirmed the importance of EGF5 in the interaction and explained the binding specificity between CD55 and CD97. The architecture of CD55-CD97 binding mode together with kinetics suggests a force-resisting shearing stretch geometry when forces applied to the C termini of both proteins in the circulating environment. The potential of the CD55-CD97 complex to withstand tensile force may provide a basis for the mechanosensing mechanism for activation of adhesion G protein-coupled receptors.

BMP4 promotes mouse iPS cell differentiation to male germ cells via Smad1/5, Gata4, Id1 and Id2.

Generation of male germ cells from pluripotent cells could provide male gametes for treating male infertility and offer an ideal model for unveiling molecular mechanisms of spermatogenesis. However, the influence and exact molecular mechanisms, especially downstream effectors of BMP4 signaling pathways, in male germ cell differentiation of the induce pluripotent stem (iPS) cells, remain unknown. This study was designed to explore the role and mechanism of BMP4 signaling in the differentiation of mouse iPS cells to male germ cells. Embryoid body (EB) formation and recombinant BMP4 or Noggin were utilized to evaluate the effect of BMP4 on male germ cell generation from mouse iPS cells. Germ cell-specific genes and proteins as well as the downstream effectors of BMP4 signaling pathway were assessed using real-time PCR and Western blots. We found that BMP4 ligand and its multiple receptors, including BMPR1a, BMPR1b and BMPR2, were expressed in mouse iPS cells. Real-time PCR and Western blots revealed that BMP4 could upregulate the levels of genes and proteins for germ cell markers in iPS cells-derived EBs, whereas Noggin decreased their expression in these cells. Moreover, Smad1/5 phosphorylation, Gata4 transcription and the transcripts of Id1 and Id2 were enhanced by BMP4 but decreased when exposed to Noggin. Collectively, these results suggest that BMP4 promotes the generation of male germ cells from iPS cells via Smad1/5 pathway and the activation of Gata4, Id1 and Id2 This study thus offers novel insights into molecular mechanisms underlying male germ cell development.

Establishment and applications of male germ cell and Sertoli cell lines.

Within the seminiferous tubules there are two major cell types, namely male germ cells and Sertoli cells. Recent studies have demonstrated that male germ cells and Sertoli cells can have significant applications in treating male infertility and other diseases. However, primary male germ cells are hard to proliferate in vitro and the number of spermatogonial stem cells is scarce. Therefore, methods that promote the expansion of these cell populations are essential for their use from the bench to the bed side. Notably, a number of cell lines for rodent spermatogonia, spermatocytes and Sertoli cells have been developed, and significantly we have successfully established a human spermatogonial stem cell line with an unlimited proliferation potential and no tumor formation. This newly developed cell line could provide an abundant source of cells for uncovering molecular mechanisms underlying human spermatogenesis and for their utilization in the field of reproductive and regenerative medicine. In this review, we discuss the methods for establishing spermatogonial, spermatocyte and Sertoli cell lines using various kinds of approaches, including spontaneity, transgenic animals with oncogenes, simian virus 40 (SV40) large T antigen, the gene coding for a temperature-sensitive mutant of p53, telomerase reverse gene (Tert), and the specific promoter-based selection strategy. We further highlight the essential applications of these cell lines in basic research and translation medicine.

MicroRNAs and DNA methylation as epigenetic regulators of mitosis, meiosis and spermiogenesis.

Spermatogenesis is composed of three distinctive phases, which include self-renewal of spermatogonia via mitosis, spermatocytes undergoing meiosis I/II and post-meiotic development of haploid spermatids via spermiogenesis. Spermatogenesis also involves condensation of chromatin in the spermatid head before transformation of spermatids to spermatozoa. Epigenetic regulation refers to changes of heritably cellular and physiological traits not caused by modifications in the DNA sequences of the chromatin such as mutations. Major advances have been made in the epigenetic regulation of spermatogenesis. In this review, we address the roles and mechanisms of epigenetic regulators, with a focus on the role of microRNAs and DNA methylation during mitosis, meiosis and spermiogenesis. We also highlight issues that deserve attention for further investigation on the epigenetic regulation of spermatogenesis. More importantly, a thorough understanding of the epigenetic regulation in spermatogenesis will provide insightful information into the etiology of some unexplained infertility, offering new approaches for the treatment of male infertility.

Deletion of Asb15b gene can lead to a significant decrease in zebrafish intermuscular bone.

Intermuscular bones, which are present in numerous economically significant fish species, have a negative impact on the development of aquaculture. The Asb15b gene, primarily expressed in skeletal muscle, plays a crucial role in regulating protein turnover and the development of muscle fibers. It stimulates protein synthesis and controls the differentiation of muscle fibers. In this study, we employed CRISPR/Cas9 technology to generate homozygous zebrafish strains with 7 bp and 49 bp deletions in the Asb15b gene. Subsequent analyses using skeleton staining demonstrated a substantial reduction in the number of intermuscular bones in adult Asb15b-/- -7 bp and Asb15b-/- -49 bp mutants compared to the wild-type zebrafish, with decreases of 30 % (P < 0.001) and 40 % (P < 0.0001), respectively. Histological experiments further revealed that the diameter and number of muscle fibers in adult Asb15b-/- mutants did not exhibit significant changes when compared to wild-type zebrafish. Moreover, qRT-PCR experiments demonstrated significant differences in the expression of bmp6 and runx2b genes, which are key regulators of intermuscular bone development, during different stages of intermuscular bone development in Asb15b-/- mutants. This study strongly suggests that the Asb15b gene plays a crucial role in regulating intermuscular bone development in fish and lays the groundwork for further exploration of the role of the Asb15b gene in zebrafish intermuscular bone development.

Impacts of AlaAT3 transgenic poplar on rhizosphere soil chemical properties, enzyme activity, bacterial community, and metabolites under two nitrogen conditions.

Poplar stands as one of the primary afforestation trees globally. We successfully generated transgenic poplar trees characterized by enhanced biomass under identical nutrient conditions, through the overexpression of the pivotal nitrogen assimilation gene, pxAlaAT3. An environmental risk assessment was conducted for investigate the potential changes in rhizosphere soil associated with these overexpressing lines (OL). The results show that acid phosphatase activity was significantly altered under ammonium in OL compared to the wild-type control (WT), and a similar difference was observed for protease under nitrate. 16SrDNA sequencing indicated no significant divergence in rhizosphere soil microbial community diversity between WT and OL. Metabolomics analysis revealed that the OL caused minimal alterations in the metabolites of the rhizosphere soil, posing no potential harm to the environment. With these findings in mind, we anticipate that overexpressed plants will not adversely impact the surrounding soil environment.

p38 mitogen-activated protein kinase and liver X receptor-α mediate the leptin effect on sterol regulatory element binding protein-1c expression in hepatic stellate cells.

Leptin, a key hormone in regulating energy homeostasis, is mainly produced by adipocytes. Cogent evidence indicates a unique role of leptin in the promotion of liver fibrosis. Hepatic stellate cell (HSC) activation is a pivotal step in the process of liver fibrosis. Sterol regulatory element binding protein (SREBP)-1c, a critical transcription factor for lipid synthesis and adipocyte differentiation, functions as a key transcription factor in inhibition of HSC activation. SREBP-1c is highly expressed in quiescent HSCs and downregulated upon HSC activation. The aim of this study is to examine the effect of leptin on SREBP-1c gene expression in HSCs in vitro and in vivo and elucidate the underlying mechanisms. The results of the present study demonstrated that leptin strongly inhibited SREBP-1c expression in HSCs in vivo and in vitro. p38 MAPK was involved in leptin regulation of SREBP-1c expression in cultured HSCs. Leptin-induced activation of p38 MAPK led to the decreases in liver X receptor (LXR)-α protein level, activity and its binding to the SREBP-1c promoter, which caused the downregulation of SREBP-1c expression. Moreover, leptin inhibition of SREBP-1c expression via p38 MAPK increased the expression of alpha1(I) collagen in HSCs. Our results might provide new insights into the mechanisms of the unique role of leptin in the development of liver fibrosis and might have potential implications for clarifying the molecular mechanisms underlying liver fibrosis in diseases in which circulating leptin levels are elevated such as nonalcoholic steatohepatitis, type 2 diabetes mellitus and alcoholic cirrhosis.

ß-Catenin pathway is required for TGF-ß1 inhibition of PPARγ expression in cultured hepatic stellate cells.

Hepatic stellate cell (HSC) activation is a key step in process of liver fibrosis. Transforming growth factor-ß1 (TGF-ß1) is the most powerful mediator of HSC activation and plays a central role in liver fibrosis. Peroxisome proliferator-activated receptor-γ (PPARγ) is an important regulator of adipocyte differentiation and has been proposed as a crucial factor for inhibition of HSC activation. The effect of TGF-ß1 on PPARγ in HSCs is largely unknown. This study is aimed to examine whether TGF-ß1 can influence PPARγ expression, focusing on the role of ß-catenin pathway, a key pathway linked to adipogenesis, in TGF-ß1 regulation of PPARγ in cultured HSCs. Our results demonstrated that TGF-ß1 evidently inhibited PPARγ expression and activity in cultured HSCs, which were mediated through ß-catenin pathway. TGF-ß1 promoted ß-catenin expression and also increased the stability of ß-catenin protein through ERK1/2/glycogen synthase kinase-3ß (GSK-3ß) axis in cultured HSCs. Moreover, TGF-ß1 inhibition of PPARγ expression by ß-catenin pathway caused the increase in alpha1(1) collagen and tissue inhibitor of matrix metalloproteinase expression. These results indicated for the first time that TGF-ß1 could down-regulate PPARγ expression through ß-catenin pathway and subsequently contributed to the increase in alpha1(1) collagen level in cultured HSCs.

[Curcumin inhibits iron overload-induced hepatocytic apoptosis and nuclear factor-κB activity].

OBJECTIVE: Iron is an essential micronutrient for human beings but its overload induces various diseases of liver, the main body storage site for iron, such as liver fibrosis. Curcumin is a natural polyphenol derived from turmeric and has been used widely. Its pharmacological action has attracted great attention in recent years. The apoptosis of rat cultured hepatocytes was induced by FeNTA (ferric nitrilotriacetate)-induced Iron overload. The present study was to examine the effect of curcumin at low concentrations on FeNTA-induced apoptosis of hepatocytes and elucidate the underlying mechanisms. METHODS: After the incubation of hepatocytes with 100 µmol/L FeNTA in the presence or absence of 1 - 10 µmmol/L of curcumin, a series of analyses were performed, including the analyses of hepatocytic apoptosis, the expressions of proteins relating with the regulations of cell apoptosis, caspase-3 activity, the production of reactive oxygen species (ROS) and nuclear factor NF-κB activity. RESULTS: Curcumin reduced the FeNTA-induced hepatocytic apoptosis by 46.65% and significantly down-regulated the protein levels of Bcl-2 and Bcl-XL. In contrast, it had no effect on the protein levels of Bax and Bad. The curcumin treatment reduced FeNTA-caused production of ROS and caspase-3 activity by 45.01% and 59.71% respectively. And the NF-κB activity was also inhibited. CONCLUSION: Curcumin at low concentrations reduces iron overload-caused hepatocytic apoptosis and NF-κB activity, the key regulatory transcription factor for the inflammation-related gene expression in cultured hepatocyte.

Identification and Expression Analysis of CAMTA Genes in Tea Plant Reveal Their Complex Regulatory Role in Stress Responses.

Calmodulin-binding transcription activators (CAMTAs) are evolutionarily conserved transcription factors and have multi-functions in plant development and stress response. However, identification and functional analysis of tea plant (Camellia sinensis) CAMTA genes (CsCAMTAs) are still lacking. Here, five CsCAMTAs were identified from tea plant genomic database. Their gene structures were similar except CsCAMTA2, and protein domains were conserved. Phylogenetic relationship classified the CsCAMTAs into three groups, CsCAMTA2 was in group I, and CsCAMTA1, 3 and CsCAMTA4, 5 were, respectively, in groups II and III. Analysis showed that stress and phytohormone response-related cis-elements were distributed in the promoters of CsCAMTA genes. Expression analysis showed that CsCAMTAs were differentially expressed in different organs and under various stress treatments of tea plants. Three-hundred and four hundred-one positive co-expressed genes of CsCAMTAs were identified under cold and drought, respectively. CsCAMTAs and their co-expressed genes constituted five independent co-expression networks. KEGG enrichment analysis of CsCAMTAs and the co-expressed genes revealed that hormone regulation, transcriptional regulation, and protein processing-related pathways were enriched under cold treatment, while pathways like hormone metabolism, lipid metabolism, and carbon metabolism were enriched under drought treatment. Protein interaction network analysis suggested that CsCAMTAs could bind (G/A/C)CGCG(C/G/T) or (A/C)CGTGT cis element in the target gene promoters, and transcriptional regulation might be the main way of CsCAMTA-mediated functional regulation. The study establishes a foundation for further function studies of CsCAMTA genes in stress response.

The relationship between mean platelet volume lymphocyte ratio and collateral circulation in patients with chronic total coronary occlusion.

Objective: To correlate mean platelet volume lymphocyte ratio (MPVLR) and coronary collateral circulation (CCC) in patients with chronic total occlusion (CTO). Materials and methods: A total of 643 patients who were hospitalized at a single large academic medical center from January 2020 to October 2021 and had CTO lesions in at least one major coronary artery confirmed by coronary angiography were retrospectively analyzed. Patients were divided according to the Rentrop criteria into poorly formed CCC (Rentrop grade 0-1, n = 235) and well-formed CCC (Rentrop grade 2-3, n = 408) groups. Mean platelet volume lymphocyte ratio (MPVLR) was calculated from routine laboratory data (MPV divided by lymphocyte count). The clinical data of the two groups were compared, and relationships between MPVLR and CCC formation were analyzed. Results: The MPVLR of patients with poorly formed CCC was significantly higher than that of patients with well-formed CCC (7.82 ± 3.80 vs. 4.84 ± 1.42, P < 0.01). The prevalence of diabetes mellitus and C-reactive protein levels were significantly higher in the poor CCC group than in the good CCC group (P < 0.01), while the proportions of patients with CTO or multivessel lesions in the right coronary artery were significantly lower in the poor CCC group than in the good CCC group (P < 0.01). Multivariate logistic regression analysis identified MPVLR (OR: 2.101, 95% CI: 1.840-2.399, P < 0.01), C-reactive protein level (OR: 1.036, 95% CI: 1.008-1.064, P < 0.05), a history of diabetes mellitus (OR: 2.355, 95% CI: 1.532-3.621, P < 0.01), and right coronary CTO ratio (OR: 0.313, 95% CI: 0.202-0.485, P < 0.01) as independent risk factors for CCC formation. The area under the ROC curve of MPVLR for predicting poorly formed CCC was 0.82 (95% CI: 0.784-0.855, P < 0.01), the best cut-off point was 6.02 and the sensitivity and specificity of MPVLR for predicting poorly formed CCC were 72.3 and 82.4%, respectively. Conclusion: In patients with coronary CTO, MPVLR was negatively correlated with CCC and a high MPVLR level was an independent predictor of poorly formed CCC.

A Comparative Study of the Clinical Benefits of Rivaroxaban and Warfarin in Patients With Non-valvular Atrial Fibrillation With High Bleeding Risk.

OBJECTIVE: To compare the clinical benefits of rivaroxaban and warfarin in patients with non-valvular atrial fibrillation (NVAF) with high bleeding risk. METHODS: A retrospective study was conducted on patients with high bleeding risk NVAF who were hospitalized at the First Affiliated Hospital of Zhengzhou University between May 31, 2016 and May 31, 2019 and took at least rivaroxaban and warfarin. The clinical benefits of both drugs were assessed by efficacy benefit and safety risk. The primary efficacy benefit was a composite end point for stroke (both ischemic and hemorrhagic) and systemic embolism. The secondary efficacy end points were death and myocardial infarction (MI). The principal safety end point was the composite end point of fatal bleeding and critical organ bleeding. RESULTS: A total of 1,246 patients with high bleeding risk were enrolled, including 787 patients in the rivaroxaban group and 459 patients in the warfarin group. Results of the primary efficacy benefit endpoint were obtained from 104 patients (13.2%) in the rivaroxaban group and 88 (19.2%) patients in the warfarin group (hazard ratio [HR]: 0.681; 95% confidence interval [CI]: 0.512-0.906; P < 0.001 for non-inferiority). The principal safety end points were observed in 49 (6.23%) patients in the rivaroxaban group and in 55 (11.98%) patients in the warfarin group (HR: 0.469 in the rivaroxaban group; 95% CI: 0.314-0.702; P < 0.001). With respect to secondary efficacy and benefit endpoints, 28 (3.56%) patients in the rivaroxaban group and 22 (4.79%) patients in the warfarin group died, with an HR of 0.760 (95% CI: 0.435-1.329; P = 0.336); 32 (4.07%) patients in the rivaroxaban group; and 26 (5.66%) patients in the warfarin group had MI, with an HR of 1.940 (95% CI: 0.495-1.069, P = 0.254) in the rivaroxaban group. CONCLUSIONS: Rivaroxaban is non-inferior to warfarin in the prevention of stroke and systemic embolism in patients with high blood NVAF. Rivaroxaban is superior to warfarin in reducing fatal bleeding and bleeding in critical organs. CLINICAL TRIAL REGISTRATION: Chinese Clinical Trials Registry, identifier ChiCTR2100052454.

Cold Acclimation for Enhancing the Cold Tolerance of Zebrafish Cells.

Cold stress is an important threat in the life history of fish. However, current research on the tolerance mechanisms of fish to cold stress is incomplete. To explore the relevant molecular mechanisms enabling cold stress tolerance in fish, here we studied ZF4 cells subjected to short-term (4 days) low temperature stress and long-term (3 months) low temperature acclimation. The results showed that cell viability decreased and the cytoskeleton shrank under short-term (4 days) low temperature stress, while the cell viability and the cytoskeleton became normal after cold acclimation at 18°C for 3 months. Further, when the cells were transferred to the lower temperature (13°C), the survival rate was higher in the acclimated than non-acclimated group. By investigating the oxidative stress pathway, we found that the ROS (reactive oxygen species) content increased under short-term (4 days) cold stress, coupled with changes in glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) enzyme activity levels. In addition, overproduction of ROS disrupted physiological cellular homeostasis that generated apoptosis via the activation of the mitochondrial pathway. However, when compared with the non-domesticated group, both ROS levels and apoptosis were lowered in the long-term (3 months) domesticated cells. Taken together, these findings suggest that cold acclimation can improve the low temperature tolerance of the cells. This exploration of the mechanism by which zebrafish cells tolerate cold stress, thus contributes to laying the foundation for future study of the molecular mechanism of cold adaptation in fish.

Characterization, isolation, and culture of spermatogonial stem cells in Macaca fascicularis.

Spermatogonial stem cells (SSCs) have great applications in both reproductive and regenerative medicine. Primates including monkeys are very similar to humans with regard to physiology and pathology. Nevertheless, little is known about the isolation, the characteristics, and the culture of primate SSCs. This study was designed to identify, isolate, and culture monkey SSCs. Immunocytochemistry was used to identify markers for monkey SSCs. Glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRA1)-enriched spermatogonia were isolated from monkeys, namely Macaca fascicularis (M. fascicularis), by two-step enzymatic digestion and magnetic-activated cell sorting, and they were cultured on precoated plates in the conditioned medium. Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and RNA sequencing were used to compare phenotype and transcriptomes in GFRA1-enriched spermatogonia between 0 day and 14 days of culture, and xenotransplantation was performed to evaluate the function of GFRA1-enriched spermatogonia. SSCs shared some phenotypes with rodent and human SSCs. GFRA1-enriched spermatogonia with high purity and viability were isolated from M. fascicularis testes. The freshly isolated cells expressed numerous markers for rodent SSCs, and they were cultured for 14 days. The expression of numerous SSC markers was maintained during the cultivation of GFRA1-enriched spermatogonia. RNA sequencing reflected a 97.3% similarity in global gene profiles between 0 day and 14 days of culture. The xenotransplantation assay indicated that the GFRA1-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-KitW/W (W) mutant mice. Collectively, GFRA1-enriched spermatogonia are monkey SSCs phenotypically both in vitro and in vivo. This study suggests that monkey might provide an alternative to human SSCs for basic research and application in human diseases.

Derivation and propagation of spermatogonial stem cells from human pluripotent cells.

OBJECTIVES: This study is designed to generate and propagate human spermatogonial stem cells (SSCs) derived from human pluripotent stem cells (hPSCs). METHODS: hPSCs were differentiated into SSC-like cells (SSCLCs) by a three-step strategy. The biological characteristics of SSCLCs were detected by immunostaining with antibodies against SSC markers. The ability of self-renewal was measured by propagating for a long time and still maintaining SSCs morphological property. The differentiation potential of SSCLCs was determined by the generation of spermatocytes and haploid cells, which were identified by immunostaining and flow cytometry. The transcriptome analysis of SSCLCs was performed by RNA sequencing. The biological function of SSCLCs was assessed by xeno-transplantation into busulfan-treated mouse testes. RESULTS: SSCLCs were efficiently generated by a 3-step strategy. The SSCLCs displayed a grape-like morphology and expressed SSC markers. Moreover, SSCLCs could be propagated for approximately 4 months and still maintained their morphological properties. Furthermore, SSCLCs could differentiate into spermatocytes and haploid cells. In addition, SSCLCs displayed a similar gene expression pattern as human GPR125+ spermatogonia derived from human testicular tissues. And more, SSCLCs could survive and home at the base membrane of seminiferous tubules. CONCLUSION: SSCLCs were successfully derived from hPSCs and propagated for a long time. The SSCLCs resembled their counterpart human GPR125+ spermatogonia, as evidenced by the grape-like morphology, transcriptome, homing, and functional characteristics. Therefore, hPSC-derived SSCLCs may provide a reliable cell source for studying human SSCs biological properties, disease modeling, and drug toxicity screening.

Gene Silencing of Human Sertoli Cells Utilizing Small Interfering RNAs.

Sertoli cells, as the unique somatic cells within the seminiferous tubules, play essential roles in regulating normal spermatogenesis. In addition, recent studies have demonstrated that Sertoli cells could have significant applications in regenerative medicine due to their great plasticity. However, the roles of genes in controlling the fate determinations of human Sertoli cells remain largely unknown. Silencing genes of human Sertoli cells utilizing small interfering RNAs (siRNAs) is an important method to explore their functions and mechanisms in human Sertoli cells. We isolated and identified human Sertoli cells. RNA interference (RNAi) was employed to probe the roles and signaling pathways of BMP6 and BMP4 in mediating the proliferation and apoptosis of human Sertoli cells. Specifically, siRNAs against BMP6 and BMP4 were used to knock down the expression levels of BMP6 and BMP4 and examine the function and mechanism in controlling the fate decisions of human Sertoli cells. In this chapter, we provided the detailed methods of RNAi in silencing BMP6 gene of human Sertoli cells. Quantitative real-time PCR demonstrated that the designed BMP6 siRNAs apparently silenced BMP6 mRNA in human Sertoli cells at 24 h after transfection. Western blots showed that the siRNAs silenced the expression of BMP6 protein effectively at 48 h after transfection. In summary, siRNAs can effectively and specifically knock down targeting genes at both transcriptional and translational levels utilizing RNAi in human Sertoli cells.

Transcriptional regulation of P63 on the apoptosis of male germ cells and three stages of spermatogenesis in mice.

Infertility affects 10-15% of couples worldwide, and male factors account for 50%. Spermatogenesis is precisely regulated by genetic factors, and the mutations of genes result in abnormal spermatogenesis and eventual male infertility. The aim of this study was to explore the role and transcriptional regulation of P63 in the apoptosis and mouse spermatogenesis. P63 protein was decreased in male germ cells of P63(+/-) mice compared with wild-type mice. There was no obvious difference in testis weight, sperm motility, and fecundity between P63(+/-) and wild-type mice. However, abnormal germ cells were frequently observed in P63(+/-) mice at 2 months old. Notably, apoptotic male germ cells and the percentage of abnormal sperm were significantly enhanced in P63(+/-) mice compared to wild-type mice. Spermatogonia, pachytene spermatocytes and round spermatids were isolated from P63(+/-) and wild-type mice using STA-PUT velocity sedimentation, and they were identified phenotypically with high purities. RNA sequencing demonstrated distinct transcription profiles in spermatogonia, pachytene spermatocytes, and round spermatids between P63(+/-) mice and wild-type mice. In total, there were 645 differentially expressed genes (DEGs) in spermatogonia, 106 DEGs in pachytene spermatocytes, and 1152 in round spermatids between P63(+/-) mice and wild-type mice. Real time PCR verified a number of DEGs identified by RNA sequencing. Gene ontology annotation and pathway analyzes further indicated that certain key genes, e.g., Ccnd2, Tgfa, Hes5, Insl3, Kit, Lef1, and Jun were involved in apoptosis, while Dazl, Kit, Pld6, Cdkn2d, Stra8, and Ubr2 were associated with regulating spermatogenesis. Collectively, these results implicate that P63 mediates the apoptosis of male germ cells and regulates three stages of spermatogenesis transcriptionally. This study could provide novel targets for the diagnosis and treatment of male infertility.

MiR-663a Stimulates Proliferation and Suppresses Early Apoptosis of Human Spermatogonial Stem Cells by Targeting NFIX and Regulating Cell Cycle.

Human spermatogonial stem cells (SSCs) could have significant applications in reproductive medicine and regenerative medicine because of their great plasticity. The fate determinations of human SSCs are mediated by epigenetic factors. However, nothing is known about the regulation of non-coding RNA on human SSCs. Here we have explored for the first time the expression, function, and target of miR-663a in human SSCs. MiR-663a was upregulated in human spermatogonia compared with pachytene spermatocytes, as indicated by microRNA microarray and real-time PCR. CCK-8 and 5-Ethynyl-2'-deoxyuridine (EDU) assays revealed that miR-663a stimulated cell proliferation and DNA synthesis of human SSCs. Annexin V and propidium iodide (PI) staining and flow cytometry demonstrated that miR-663a inhibited early and late apoptosis of human SSCs. Furthermore, NFIX was predicted and verified as a direct target of miR-663a. NFIX silencing led to an enhancement of cell proliferation and DNA synthesis and a reduction of the early apoptosis of human SSCs. NFIX silencing neutralized the influence of miR-663a inhibitor on the proliferation and apoptosis of human SSCs. Finally, both miR-663a mimics and NFIX silencing upregulated the levels of cell cycle regulators, including Cyclin A2, Cyclin B1, and Cyclin E1, whereas miR-663a inhibitor had an adverse effect. Knockdown of Cyclin A2, Cyclin B1, and Cyclin E1 led to the decrease in the proliferation of human SSCs. Collectively, miR-663a has been identified as the first microRNA that promotes the proliferation and DNA synthesis and suppresses the early apoptosis of human SSCs by targeting NFIX via cell cycle regulators Cyclin A2, Cyclin B1, and Cyclin E1. This study thus provides novel insights into the molecular mechanisms underlying human spermatogenesis, and it could offer novel targets for treating male infertility and other human diseases.