Generation and Application of Mouse-Rat Allodiploid Embryonic Stem Cells.

Mammalian interspecific hybrids provide unique advantages for mechanistic studies of speciation, gene expression regulation, and X chromosome inactivation (XCI) but are constrained by their limited natural resources. Previous artificially generated mammalian interspecific hybrid cells are usually tetraploids with unstable genomes and limited developmental abilities. Here, we report the generation of mouse-rat allodiploid embryonic stem cells (AdESCs) by fusing haploid ESCs of the two species. The AdESCs have a stable allodiploid genome and are capable of differentiating into all three germ layers and early-stage germ cells. Both the mouse and rat alleles have comparable contributions to the expression of most genes. We have proven AdESCs as a powerful tool to study the mechanisms regulating X chromosome inactivation and to identify X inactivation-escaping genes, as well as to efficiently identify genes regulating phenotypic differences between species. A similar method could be used to create hybrid AdESCs of other distantly related species.

Association between single nucleotide polymorphism of rs1937 in TFAM gene and longevity among the elderly Chinese population: based on the CLHLS study.

BACKGROUND: To investigate whether the mitochondrial transcription factor A (TFAM) rs1937 single nucleotide polymorphism (SNP) is associated with longevity. METHODS: We conducted a case-control study among Chinese long-lived individuals (≥90 years). Data were obtained on 3294 participants who were able to voluntarily provided a saliva sample during 2008-2009 from the Chinese Longitudinal Healthy Longevity Survey (CLHLS). In this study, 1387 young elderly (65-74 years) were allocated to the control group, and 1907 long-lived individuals were recruited as the case group. SNP rs1937 on TFAM were genotyped. Logistic regression models were applied to evaluate the association between rs1937 SNP and longevity. RESULTS: The genotype frequency of the SNP of rs1937 in the two groups had a significant difference (p = 0.003). Binary logistic regression analysis showed that compared to younger elderly, the long-lived individuals with "CC genotype" of rs1937 were more closely related to increased longevity than those with "GG genotype" (OR: 1.989, 95% CI: 1.160-3.411). The positive association between rs1937 SNP and longevity was robust in stratified analyses and sensitivity analyses. CONCLUSIONS: We found the SNP of rs1937 may be a potential biomarker for longer human life span. Further studies are necessary to elucidate the biological mechanism of rs1937 on TFAM with promoting longevity.

Rat embryonic stem cells produce fertile offspring through tetraploid complementation.

Pluripotency of embryonic stem cells (ESCs) can be functionally assessed according to the developmental potency. Tetraploid complementation, through which an entire organism is produced from the pluripotent donor cells, is taken as the most stringent test for pluripotency. It remains unclear whether ESCs of other species besides mice can pass this test. Here we show that the rat ESCs derived under 2i (two small molecule inhibitors) conditions at very early passages are able to produce fertile offspring by tetraploid complementation. However, they lose this capacity rapidly during culture due to a nearly complete loss of genomic imprinting. Our findings support that the naïve ground state pluripotency can be captured in rat ESCs but also point to the species-specific differences in its regulation and maintenance, which have implications for the derivation and application of naïve pluripotent stem cells in other species including human.

Androgenetic haploid embryonic stem cells produce live transgenic mice.

Haploids and double haploids are important resources for studying recessive traits and have large impacts on crop breeding, but natural haploids are rare in animals. Mammalian haploids are restricted to germline cells and are occasionally found in tumours with massive chromosome loss. Recent success in establishing haploid embryonic stem (ES) cells in medaka fish and mice raised the possibility of using engineered mammalian haploid cells in genetic studies. However, the availability and functional characterization of mammalian haploid ES cells are still limited. Here we show that mouse androgenetic haploid ES (ahES) cell lines can be established by transferring sperm into an enucleated oocyte. The ahES cells maintain haploidy and stable growth over 30 passages, express pluripotent markers, possess the ability to differentiate into all three germ layers in vitro and in vivo, and contribute to germlines of chimaeras when injected into blastocysts. Although epigenetically distinct from sperm cells, the ahES cells can produce viable and fertile progenies after intracytoplasmic injection into mature oocytes. The oocyte-injection procedure can also produce viable transgenic mice from genetically engineered ahES cells. Our findings show the developmental pluripotency of androgenentic haploids and provide a new tool to quickly produce genetic models for recessive traits. They may also shed new light on assisted reproduction.

iPS cells produce viable mice through tetraploid complementation.

Since the initial description of induced pluripotent stem (iPS) cells created by forced expression of four transcription factors in mouse fibroblasts, the technique has been used to generate embryonic stem (ES)-cell-like pluripotent cells from a variety of cell types in other species, including primates and rat. It has become a popular means to reprogram somatic genomes into an embryonic-like pluripotent state, and a preferred alternative to somatic-cell nuclear transfer and somatic-cell fusion with ES cells. However, iPS cell reprogramming remains slow and inefficient. Notably, no live animals have been produced by the most stringent tetraploid complementation assay, indicative of a failure to create fully pluripotent cells. Here we report the generation of several iPS cell lines that are capable of generating viable, fertile live-born progeny by tetraploid complementation. These iPS cells maintain a pluripotent potential that is very close to ES cells generated from in vivo or nuclear transfer embryos. We demonstrate the practicality of using iPS cells as useful tools for the characterization of cellular reprogramming and developmental potency, and confirm that iPS cells can attain true pluripotency that is similar to that of ES cells.

Generation of transgenic rats through induced pluripotent stem cells.

The rat is an important animal model for human disease research. Using inhibitors of glycogen synthase kinase 3 and MAPK signaling pathways, rat embryonic stem cells and rat induced pluripotent stem cells (riPSCs) have been derived. However, unlike rat embryonic stem cells, germ line competent riPSCs have only been derived from Wistar rats at low efficiency. Here, we found that an optimized induction medium containing knock-out serum replacement and vitamin C improved the rate and efficiency of riPSCs generation from Dark Agouti rat fibroblasts and Sertoli cells. riPSCs maintained an undifferentiated status for >30 passages and could differentiate into various cells types including germ cells when injected into rat blastocysts. Moreover, transgenic riPSCs could be generated through the PiggyBac transposon, which could be used to generate transgenic rats through germ line transmission. riPSCs can be used as a novel tool in genetic and genomic studies of the rat.

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.

Stem cells and small molecule screening: haploid embryonic stem cells as a new tool.

Stem cells can both self-renew and differentiate into various cell types under certain conditions, which makes them a good model for development and disease studies. Recently, chemical approaches have been widely applied in stem cell biology by promoting stem cell self-renewal, proliferation, differentiation and somatic cell reprogramming using specific small molecules. Conversely, stem cells and their derivatives also provide an efficient and robust platform for small molecule and drug screening. Here, we review the current research and applications of small molecules that modulate stem cell self-renewal and differentiation and improve reprogramming, as well as the applications that use stem cells as a tool for small molecule screening. Moreover, we introduce the recent advance in haploid embryonic stem cells research. Haploid embryonic stem cells maintain haploidy and stable growth over extensive passages, possess the ability to differentiate into all three germ layers in vitro and in vivo, and contribute to the germlines of chimeras when injected into blastocysts. Androgenetic haploid stem cells can also be used in place of sperm to produce fertile progeny after intracytoplasmic injection into mature oocytes. Such characteristics demonstrate that haploid stem cells are a new approach for genetic studies at both the cellular and animal levels and that they are a valuable platform for future small molecule screening.

[Railway Emission Trends in China Based on Fuel Life Cycle Analysis].

Railway transportation is one of the main modes of modern transportation. Under the dual constraints of air quality improvement and carbon neutrality achievement, clarifying the emission trend of CO2 and pollutants in railway transportation is of great significance for pollution and carbon reduction in the transport sector. In this study, the CO2 and pollutant emission characteristics of Chinese railways from 2001 to 2018 were analyzed based on the fuel life cycle method. Then, railway emission trends from 2019-2030 were assessed combined with scenario analysis. The results showed that with the advancement of railway electrification, the use of new diesel locomotives, and the continuous upgrading of fuel standards, the total CO2 and pollutant emissions in the fuel life cycle of railway transportation showed an upward and downward trend, respectively. In 2018, the total emissions of CO2, NOx, CO, BC, and SOx from railway transportation were 3780.29×104t, 11.98×104t, 3.94×104t, 0.20×104t, and 3.08×104t, respectively. Accelerating the improvement of power structure and reducing unit energy consumption were the best single control strategies to reduce railway emissions of CO2, SOx, NOx, BC, and CO, respectively. Under the comprehensive scenario of actively responding to railway pollution and carbon reduction, the emission reduction rates of CO2, NOx, CO, BC, and SOx could reach 35%, 37%, 39%, 32%, and 45%, respectively. The stagnation of power structure reform or the railway electrification process will lead to a significant increase in total emissions of railway transportation. Therefore, the pollution and carbon reduction of railway transportation requires continuous attention.

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.

The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting.

Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.

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.

Single-cell multiomics sequencing reveals the reprogramming defects in embryos generated by round spermatid injection.

Round spermatid injection (ROSI) technique holds great promise for clinical treatment of a proportion of infertile men. However, the compromised developmental potential of ROSI embryos largely limits the clinical application, and the mechanisms are not fully understood. Here, we describe the transcriptome, chromatin accessibility, and DNA methylation landscapes of mouse ROSI embryos derived from early-stage round spermatids using a single-cell multiomics sequencing approach. By interrogating these data, we identify the reprogramming defects in ROSI embryos at the pronuclear stages, which are mainly associated with the misexpression of a cohort of minor zygotic genome activation genes. We screen a small compound, A366, that can significantly increase the developmental potential of ROSI embryos, in which A366 can partially overcome the reprogramming defects by amending the epigenetic and transcriptomic states. Collectively, our study uncovers the reprogramming defects in ROSI embryos for understanding the mechanisms underlying compromised developmental potential and offers an avenue for ROSI technique optimization.

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.

A weakly luminescent Tb-MOF-based "turn-on" sensor for the highly selective and sensitive sensing of an anthrax biomarker.

Bacillus anthracis is an extremely dangerous bacterium that is associated with high morbidity and mortality. 2,6-Pyridine dicarboxylic acid (DPA) is a major biomarker of Bacillus anthracis, and it is of great significance to be able to detect DPA in a rapid, efficient, and sensitive way. Herein, a 3D network metal-organic framework (Tb-MOF) with excellent thermal and water stability was synthesized. Tb-MOF could be used to selectively detect DPA via green fluorescence recovery with a fluorescence intensity enhancement factor of 103. In addition, due to the high detection sensitivity (a detection limit of 2.4 µM) and excellent anti-interference abilities, Tb-MOF was less affected by environmental factors when compared with a "turn-off"-response luminescence sensor; it can be employed as a promising "turn-on" luminescence sensor for DPA in the future. Finally, quantum calculations showed that a large energy difference appeared between the 5D4 level of Tb3+ and the first excited triplet energy level of H2-DHBDC2-, which was the reason that the complex did not show characteristic Tb3+ emission.

A novel 3D coordination polymer constructed by dual-ligand for highly sensitive detection of purine metabolite uric acid.

Uric acid (UA), as the final product of purine metabolism, exists in urine and serum, which plays an important role in human metabolism, immunity and other functions. The sensitive, efficient, and rapid detection of UA has far-reaching significance in clinical diagnosis and disease prevention. Herein, a novel coordination polymer constructed by dual-ligand was successfully prepared, which exhibited excellent thermal and water stability. The polymer was interlaced by coordination bonds and hydrogen bonds to form an infinitely extended three-dimensional framework, which showed a rare and novel topological structure. The complex selectively recognized UA through significant fluorescence quenching response in the presence of various interferences. The excellent detection sensitivity (the limited detection of 1.2 µM), outstanding anti-interference ability and remarkable recyclability marked the complex to be a promising sensor material towards UA. In addition, the detection mechanism of UA by the complex was investigated in detail by combining density functional theory (DFT) and a variety of other analytical methods.

A hydrostable samarium(III)-MOF sensor for the sensitive and selective detection of tryptophan based on a "dual antenna effect".

Tryptophan (Trp) is one of the essential amino acids, which plays important roles in biological systems and the normal growth of human beings, and it is of great significance to be able to detect Trp in a rapid, efficient, and sensitive way. Herein, a 3D network metal-organic framework ([Sm2(BTEC)1.5(H2O)8]·6H2O) with excellent thermal and water stability was synthesized by a hydrothermal method. Interestingly, it could discriminate Trp from other natural amino acids in aqueous solution through a significant fluorescence enhancement effect, and showed high detection sensitivity (LOD = 330 nM) and outstanding anti-interference ability. The sensor system was successfully applied to the detection of Trp in practical samples, so it was expected to be a sensitive and efficient Trp sensor. In addition, the sensing mechanism was explained in detail by a series of characterization methods combined with density functional theory (DFT). There were many coordination water molecules in the crystal structure of the complex. Based on the small steric hindrance and molecular structure of water molecules, it provided the possibility for coordination interaction between Trp and Sm3+. On the other hand, the triplet energy level (T1) of Trp matched with the 4G5/2 vibrational energy level of Sm3+, so Trp could be used as the second "antenna molecule" besides 1,2,4,5-benzenetetracarboxylic acid (H4BTEC). Therefore, it effectively broadened the way for Sm-MOF to absorb excitation light.

Dual functions of pH-sensitive cation Zr-MOF for 5-Fu: large drug-loading capacity and high-sensitivity fluorescence detection.

Nanomaterials, as carriers of small molecular drugs, have been a focal point in recent years. In this work, a carbazolyl functionalized metal-organic framework, UiO-67-CDC, was successfully synthesized employing the ligand 9H-carbazole-2,7-dicarboxylic acid (9H-2,7-CDC). Postsynthetic approaches targeted the cationization and replacement of the Lewis base carbazole site with two methyl groups, resulting in the positively charged skeleton, which has proven to be a promising carrier for the anticancer drug 5-fluorouracil (5-Fu). The prepared cationic framework UiO-67-CDC-(CH3)2 showed moderately high surface area, hierarchical pore structures, and positive surface characteristics, which effectively and selectivity encapsulated the electron-rich 5-Fu molecules through electrostatic attraction, with a relatively high loading of up to 56.5% (wt%). The drug delivery in simulated blood environment (pH = 7.4) exhibited a more effective release, demonstrating a physiological pH-responsive sustained release. Significantly, the electron-deficient Zr-MOF itself, as a kind of high-sensitivity fluorescence detector, has a unique fluorescence "turn-on" effect with 5-Fu. These results pave the way towards designing surface-engineered MOF materials of interest in drug delivery and fluorescent sensing applications.

Deciphering the autophagy regulatory network via single-cell transcriptome analysis reveals a requirement for autophagy homeostasis in spermatogenesis.

Background: Autophagy has been implicated as a crucial component in spermatogenesis, and autophagy dysfunction can lead to reproductive disorders in animal models, including yeast, C. elegans and mice. However, the sophisticated transcriptional networks of autophagic genes throughout human spermatogenesis and their biological significance remain largely uncharacterized. Methods: We profiled the transcriptional signatures of autophagy-related genes during human spermatogenesis by assessing specimens from nine fertile controls (including two normal persons and seven obstructive azoospermia (OA) patients) and one nonobstructive azoospermia (NOA) patient using single-cell RNA sequencing (scRNA-seq) analysis. Dysregulation of autophagy was confirmed in two additional NOA patients by immunofluorescence staining. Gene knockdown was used to identify the role of Cst3 in autophagy during spermatogenesis. Results: Our data uncovered a unique, global stage-specific enrichment of autophagy-related genes. Human-mouse comparison analysis revealed that the stage-specific expression pattern of autophagy-related genes was highly conserved in mammals. More importantly, dysregulation of some clusters of autophagy-related genes was observed in NOA patients, suggesting the association of autophagy with male infertility. Cst3, a human-mouse conserved and autophagy-related gene that is actively expressed in spermatogonia and early spermatocytes, was found to regulate spermatogonial stem cell (SSC) maintenance and subsequent male germ cell development. Knockdown of Cst3 increased autophagic activity in mouse SSCs and subsequently suppressed the transcription of SSC core factors such as Oct4, Id1, and Nanos3, which could be efficiently rescued by manipulating autophagic activity. Conclusions: Our study provides comprehensive insights into the global transcriptional signatures of autophagy-related genes and confirms the importance of autophagy homeostasis in SSC maintenance and normal spermatogenesis, opening new avenues for further dissecting the significance of the autophagy regulatory network in spermatogenesis as well as male infertility.

The histone demethylase KDM2B regulates human primordial germ cell-like cells specification.

Germline specification is a fundamental step for human reproduction and this biological phenomenon possesses technical challenges to study in vivo as it occurs immediately after blastocyst implantation. The establishment of in vitro human primordial germ cell-like cells (hPGCLCs) induction system allows sophisticated characterization of human primordial germ cells (hPGCs) development. However, the underlying molecular mechanisms of hPGCLC specification are not fully elucidated. Here, we observed particularly high expression of the histone demethylase KDM2B in male fetal germ cells (FGCs) but not in male somatic cells. Besides, KDM2B shared similar expression pattern with hPGC marker genes in hPGCLCs, suggesting an important role of KDM2B in germ cell development. Although deletion of KDM2B had no significant effects on human embryonic stem cell (hESC)'s pluripotency, loss of KDM2B dramatically impaired hPGCLCs differentiation whereas ectopically expressed KDM2B could efficiently rescue such defect, indicating this defect was due to KDM2B's loss in hPGCLC specification. Mechanistically, as revealed by the transcriptional profiling, KDM2B suppressed the expression of somatic genes thus inhibited somatic differentiation during hPGCLC specification. These data collectively indicate that KDM2B is an indispensable epigenetic regulator for hPGCLC specification, shedding lights on how epigenetic regulations orchestrate transcriptional events in hPGC development for future investigation.