The oral cavity is a complex physiological community encompassing a wide range of microorganisms.Dysbiosis of oral microbiota can lead to various oral infectious diseases,such as periodontitis and tooth decay,and even...The oral cavity is a complex physiological community encompassing a wide range of microorganisms.Dysbiosis of oral microbiota can lead to various oral infectious diseases,such as periodontitis and tooth decay,and even affect systemic health,including brain aging and neurodegenerative diseases.Recent studies have highlighted how oral microbes might be involved in brain aging and neurodegeneration,indicating potential avenues for intervention strategies.In this review,we summarize clinical evidence demonstrating a link between oral microbes/oral infectious diseases and brain aging/neurodegenerative diseases,and dissect potential mechanisms by which oral microbes contribute to brain aging and neurodegeneration.We also highlight advances in therapeutic development grounded in the realm of oral microbes,with the goal of advancing brain health and promoting healthy aging.展开更多
Pigs have emerged as valuable large-animal models for cardiac xenotransplantation;however,the temporal dynamics of myocardial development in this species remains insufficiently defined.This study analyzed gene express...Pigs have emerged as valuable large-animal models for cardiac xenotransplantation;however,the temporal dynamics of myocardial development in this species remains insufficiently defined.This study analyzed gene expression patterns across four key developmental stages(neonatal,juvenile,sexual maturity,and adulthood)to delineate the molecular mechanisms driving porcine myocardial development.Increases in heart weight were accompanied by proportional expansion of myocardial fiber area and chamber size,reflecting coordinated structural development.Transcriptomic profiling of myocardial tissue by RNA sequencing(RNA-seq)identified 2189 differentially expressed genes(DEGs)across stage comparisons.Short time-series expression miner(STEM)analysis classified these DEGs into four major expression clusters enriched in pathways associated with myocardial development,immune responses,cell proliferation,and metabolic processes.Among 359 DEGs conserved across all developmental stages,six candidate genes were strongly associated with myocardial development.Reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR)confirmed a significant correlation between the expression of these candidate genes and myocardial development in porcine tissue.These findings establish a transcriptomic framework for porcine myocardial maturation and provide a molecular basis for advancing cardiac xenotransplantation.展开更多
Sertoli and granulosa cells,the initial differentiated somatic cells in bipotential gonads,play crucial roles in directing male and female gonad development,respectively.The transcription factor Foxo1 is involved in d...Sertoli and granulosa cells,the initial differentiated somatic cells in bipotential gonads,play crucial roles in directing male and female gonad development,respectively.The transcription factor Foxo1 is involved in diverse cellular processes,and its expression in gonadal somatic cells is sex-dependent.While Foxo1 is abundantly expressed in ovarian granulosa cells,it is notably absent in testicular Sertoli cells.Nevertheless,its function in gonadal somatic cell differentiation remains elusive.In this study,we find that ectopic expression of Foxo1 in Sertoli cells leads to defects in testes development.Further study uncovers that the ectopic expression of Foxo1 induces the abundant expression of Foxl2 in Sertoli cells,along with the upregulation of other female-specific genes.In contrast,the expression of male-specific genes is reduced.Mechanistic studies indicate that Foxo1 directly binds to the promoter region of Foxl2,inducing its expression.Our findings highlight that Foxo1 serves as a key regulator for the lineage maintenance of ovarian granulosa cells.This study contributes valuable insights into understanding the regulatory mechanisms governing the lineage maintenance of gonadal somatic cells.展开更多
Pluripotent stem cells(PSCs)are useful for developmental and translational research because they have the potential to differentiate into all cell types of an adult individual.Pigs are one of the most important domest...Pluripotent stem cells(PSCs)are useful for developmental and translational research because they have the potential to differentiate into all cell types of an adult individual.Pigs are one of the most important domestic ungulates,commonly used for food and as bioreactors.Generating stable pluripotent porcine PSC lines remains challenging.So far,the pluripotency gene network of porcine PSCs is poorly understood.Here we found that TBX3-derived induced pluripotent stem cells(iPSCs)closely resemble porcine 4-cell embryos with the capacity of totipotent-like stem cells(TLSCs).Interestingly,our data suggest that TBX3 facilitates the activation of H3K4me3 methyltransferase,specifically MLL1.Subsequent investigations revealed that the porcine 4-cell specific gene,MCL1,is a key downstream effector of the TBX3-MLL1 axis.Together,our study of the TBX3 regulatory network is helpful in the understanding of the totipotency characteristics of pigs.展开更多
Aim： To examine the possible effect of heat treatment on expression of heat shock proteins （Hsps） 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. Methods： Western blot...Aim： To examine the possible effect of heat treatment on expression of heat shock proteins （Hsps） 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. Methods： Western blot analysis, realtime polymerase chain reaction （PCR）, and confocal immunohistochemistry were used to analyze mRNA and protein levels of the Hsps in response to 43~C treatment of Sertoli cells isolated from pubertal monkey testes. Results： Staining with Hoechst 33342 indicated Sertoli cells did not undergo apoptosis after heat treatment. Hspl05 was expressed in cytoplasm of untreated Sertoli cells. Both Hspl05 mRNA and protein levels were increased approximately 20-fold compared to those of the untreated controls at 12 h after heat treatment. Untreated Sertoli cells did not express Hsp70, but heat stress induced its expression in the cell cytoplasm. The time-course of changes in Hsp70 was similar to that of Hsp105. In contrast to Hsp105 and Hsp70, the change in Hsp60 expression was much less obvious. The protein level between 12 h and 48 h after heat treatment was only approximately 1.5-fold that of the untreated control. Extracellular regulated kinase （ERK） 1/2 inhibitor （U0126） or phosphoinositide kinase-3 （PI3K） inhibitor （LY294002） could partially block the response of Hspl05 and Hsp70 induced by heat treatment. Conclusion： These results indicate that the heat-induced expression of the three types of Hsp in monkey Sertoli cells might be regulated by ERK and/or PI3K signal pathways, but the profile of their expression is different, suggesting that they might have different regulatory functions in Sertoli cells.展开更多
Dear Editor, It is now well known that somatic cells can be efficiently reprogrammed into induced pluripotent stem cells （iPSCs） by forced expression of defined factors [1- 3]. These cells, like embryonic stem cel...Dear Editor, It is now well known that somatic cells can be efficiently reprogrammed into induced pluripotent stem cells （iPSCs） by forced expression of defined factors [1- 3]. These cells, like embryonic stem cells （ESCs）, have true pluripotency as shown by the live, fertile mice that can be generated through the tetraploid complementation assay using these iPSCs [4, 5]. So far, iPSCs have been generated from many species including mice, primate,展开更多
High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the importa...High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases （DNMTs）, therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5＇ regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmtl and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones （AF1, AF2 and AF3） showed either hypermethylation or hypomethylation in the 5＇ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized （IVF） fetuses. Besides, we found that the 5＇ regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5＇ regions is probably associated with the high abortion of bovine clones.展开更多
One of the main causes of pregnancy failure and fetus abortion is oocyte aneuploidy,which is increased with maternal aging.Numerous possible causes of oocyte aneuploidy in aged women have been proposed,including cross...One of the main causes of pregnancy failure and fetus abortion is oocyte aneuploidy,which is increased with maternal aging.Numerous possible causes of oocyte aneuploidy in aged women have been proposed,including cross-over formation defect,cohesin loss,spindle deformation,spindle assembly checkpoint malfunction,microtubule-kinetochore attachment failure,kinetochore mis-orientation,mitochondria dysfunction-induced increases in reactive oxygen species,protein over-acetylation,and DNA damage.However,it still needs to be answered if these aneuploidization factors have inherent relations,and how to prevent chromosome aneuploidy in aged oocytes.Epidemiologically,oocyte aneuploidy has been found to be weakly associated with higher homocysteine concentrations,obesity,ionizing radiation and even seasonality.In this review,we summarize the research progress and present an integrated view of oocyte aneuploidization.展开更多
Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in respon...Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.展开更多
The mitochondrion is the major energy provider to power sperm motility. In mammals, aside from the nuclear genome, mitochondrial DNA （mtDNA） also contributes to oxidative phosphorylation to impact production of ATP ...The mitochondrion is the major energy provider to power sperm motility. In mammals, aside from the nuclear genome, mitochondrial DNA （mtDNA） also contributes to oxidative phosphorylation to impact production of ATP by coding 13 polypeptides. However, the role of sperm mitochondria in fertilization and its final fate after fertilization are still controversial. The viewpoints that sperm bearing more mtDNA will have a better fertilizing capability and that sperm mtDNA is actively eliminated during early embryogenesis are widely accepted. However, this may be not true for several mammalian species, including mice and humans. Here, we review the sperm mitochondria and their mtDNA in sperm functions, and the mechanisms of maternal mitochondrial inheritance in mammals.展开更多
Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells （ESCs）, which may be an important source ofhistocompatible cells and tissues for cell therapy. Here we describe the derivat...Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells （ESCs）, which may be an important source ofhistocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines （hPES-1 and hPES-2） from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA- 1-60, and TRA- 1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies （EBs） of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers （hPES-2 did not）. Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat （STR） results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.展开更多
The enrichment and identification of human epidermal stem cells （EpSCs） are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been...The enrichment and identification of human epidermal stem cells （EpSCs） are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rap- idly adherent keratinocytes into three populations that range in size from 5-7 μm （population A）, to 7-9 μm （population B）, to ≥9μm （population C） in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of plintegrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency （CFE）. When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to pl-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.展开更多
Recent data from several laboratories have provided evidence that the newly fertilized oocyte inherits epigenetic signals from the sperm chromatin that are required for proper embryonic development. For the purposes o...Recent data from several laboratories have provided evidence that the newly fertilized oocyte inherits epigenetic signals from the sperm chromatin that are required for proper embryonic development. For the purposes of this review, the term epigenetic is used to describe all types of molecular information that are transmitted from the sperm cell to the embryo. There are at least six different forms of epigenetic information that have already been established as being required for proper embryogenesis in mammals or for which there is evidence that it may do so. These are （i） DNA methylation; （ii） sperm-specific histones, （iii） other chromatin-associated proteins; （iv） the perinuclear theca proteins; （v） sperm-born RNAs and, the focus of this review; and （vi） the DNA loop domain organization by the sperm nuclear matrix. These epigenetic signals should he considered when designing protocols for the manipulation and cryopreservation of spermatozoa for assisted reproductive technology as necessary components for effective fertilization and subsequent embryo development.展开更多
Knockout Brown Norway （BN） rat could be a useful disease model for human disorders,however,a failure to derive embryonic stem （ES） cells disturbs the further development of the model.In this study,we reported a ca...Knockout Brown Norway （BN） rat could be a useful disease model for human disorders,however,a failure to derive embryonic stem （ES） cells disturbs the further development of the model.In this study,we reported a case of successful derivation of the BN rat ES cells with the derivation efficiency comparable to that of Sprague Dawley （SD） rats.The BN rat ES cells expressed the key transcription factors,and were able to form embryonic bodies （EBs） when being differentiated in vitro.After injecting the BN rat ES cells into the SD rat blastocysts,high-contribution chimeric rats were generated and could survive to their adulthood.Our success in generating pluripotent rat ES cells will benefit the generation of the knockout rats in the future.展开更多
Mouse and human somatic cells can be induced to become pluripotent stem （iPS） cells by retroviral transduction with defined transcription factors . Reprogrammed pluripotent stem cells have great potential for regene...Mouse and human somatic cells can be induced to become pluripotent stem （iPS） cells by retroviral transduction with defined transcription factors . Reprogrammed pluripotent stem cells have great potential for regenerative medicine and also provide a good experi- mental system for studying epigenetic reprogramming and differentiation. However, at present, the reprogram- ming process is still somewhat slow and ineffective.展开更多
Maintaining metabolic homeostasis is essential for cellular and organismal health throughout life.Multiple signaling pathways that regulate metabolism also play critical roles in aging,such as PI3K/AKT,mTOR,AMPK,and s...Maintaining metabolic homeostasis is essential for cellular and organismal health throughout life.Multiple signaling pathways that regulate metabolism also play critical roles in aging,such as PI3K/AKT,mTOR,AMPK,and sirtuins(SIRTs).Among them,sirtuins are known as a protein family with versatile functions,such as metabolic control,epigenetic modification and lifespan extension.Therefore,by understanding how sirtuins regulate metabolic processes,we can start to understand how they slow down or accelerate biological aging from the perspectives of metabolic regulation.Here,we review the biology of SIRT3,SIRT4,and SIRT5,known as the mitochondrial sirtuins due to their localization in the mitochondrial matrix.First,we will discuss canonical pathways that regulate metabolism more broadly and how these are integrated with aging regulation.Then,we will summarize the current knowledge about functional differences between SIRT3,SIRT4,and SIRT5 in metabolic control and integration in signaling networks.Finally,we will discuss how mitochondrial sirtuins regulate processes associated with aging and aging-related diseases.展开更多
Intrauterine insemination with donor sperm(IUI-D)is an assisted reproductive technology(ART)offered to couples with definitive male infertility or risk of genetic disease transmission.Here,we sought to evaluate our pr...Intrauterine insemination with donor sperm(IUI-D)is an assisted reproductive technology(ART)offered to couples with definitive male infertility or risk of genetic disease transmission.Here,we sought to evaluate our practice in IUI-D and identify factors that influenced the success rate.We performed a retrospective,single-center study of all IUI-D procedures performed at Lille University Medical Center(Lille,France)between January 1,2007,and December 31,2017.Single and multivariate analyses with a mixed logistic model were used to identify factors associated with clinical pregnancies and live births.We included 322 couples and 1179 IUI-D procedures.The clinical pregnancy rate was 23.5%,and the live birth rate was 18.9%per IUI-D.In a multivariate analysis,the women’s age was negatively associated with the live birth rate.The number of motile spermatozoa inseminated was the only factor associated with both clinical pregnancies and live births,with a chosen threshold of 0.75 million.The clinical pregnancy and live birth rates were,respectively,17.3%and 13.0%below the number of motile spermatozoa inseminated threshold and 25.9%and 21.0%at or above the threshold(all P=0.005).The number of motile spermatozoa inseminated was the only factor that significantly influenced both pregnancies and live-birth rates after IUI-D.Indeed,below a threshold of 0.75 million motile spermatozoa inseminated,those rates were significantly lower.Application of this number of motile spermatozoa inseminated threshold may help centers to allocate donations more effectively while maintaining reasonable waiting times for patients.展开更多
Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chro...Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.展开更多
Rat is a valuable model for pharmacological and physiological studies. Germline-competent rat embryonic stem （rES） cell lines have been successfully established and the molecular networks maintaining the self-renewi...Rat is a valuable model for pharmacological and physiological studies. Germline-competent rat embryonic stem （rES） cell lines have been successfully established and the molecular networks maintaining the self-renewing, undifferentiated state of rES cells have also been well uncovered. However, little is known about the differentiation strategies and the underlying mechanisms of how these authentic rat pluripotent stem ceils give rise to specific cell types. The aim of this study is to investigate the neural differentiation capacity of rES cells. By means of a modified procedure based on previous publications - combination of mitogen-activated protein kinase （MAPK） and glycogen synthase kinase 3 （GSK3） inhibitors （two inhibitors, ＂2i＂） with feeder-conditioned medium, we successfully obtained high- quality rat embryoid bodies （rEBs） from rES cells and then differentiated them to tripotent neural progenitors. These rES cell-derived neural progenitor cells （rNPCs） were capable of self-renewing and giving rise to all three neural lineages, including astrocytes, oligo- dendrocytes, and neurons. Besides, these rES cell-derived neurons stained positive for y-aminobutyric acid （GABA） and tyrosine hydroxylase （TH）. In summary, we develop an experimental system for differentiating rES cells to tripotent neural progenitors, which may provide a powerful tool for pharmacological test and a valuable platform for studying the pathogenesis of many neurodegenerative disorders such as Parkinson＇s disease and the development of rat nervous system.展开更多
Transcription activator-like effectors （TALEs） that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TA...Transcription activator-like effectors （TALEs） that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent （USER）, an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases （TALENs） generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tetl locus, and proved that all can specifically target Tetl gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.展开更多
基金supported by the National Natural Science Foundation of China,No.81921006(to GHL)。
文摘The oral cavity is a complex physiological community encompassing a wide range of microorganisms.Dysbiosis of oral microbiota can lead to various oral infectious diseases,such as periodontitis and tooth decay,and even affect systemic health,including brain aging and neurodegenerative diseases.Recent studies have highlighted how oral microbes might be involved in brain aging and neurodegeneration,indicating potential avenues for intervention strategies.In this review,we summarize clinical evidence demonstrating a link between oral microbes/oral infectious diseases and brain aging/neurodegenerative diseases,and dissect potential mechanisms by which oral microbes contribute to brain aging and neurodegeneration.We also highlight advances in therapeutic development grounded in the realm of oral microbes,with the goal of advancing brain health and promoting healthy aging.
基金supported by the National Natural Science Foundation of China(32222079 to L.J.and 32202621 to D.D.W.)。
文摘Pigs have emerged as valuable large-animal models for cardiac xenotransplantation;however,the temporal dynamics of myocardial development in this species remains insufficiently defined.This study analyzed gene expression patterns across four key developmental stages(neonatal,juvenile,sexual maturity,and adulthood)to delineate the molecular mechanisms driving porcine myocardial development.Increases in heart weight were accompanied by proportional expansion of myocardial fiber area and chamber size,reflecting coordinated structural development.Transcriptomic profiling of myocardial tissue by RNA sequencing(RNA-seq)identified 2189 differentially expressed genes(DEGs)across stage comparisons.Short time-series expression miner(STEM)analysis classified these DEGs into four major expression clusters enriched in pathways associated with myocardial development,immune responses,cell proliferation,and metabolic processes.Among 359 DEGs conserved across all developmental stages,six candidate genes were strongly associated with myocardial development.Reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR)confirmed a significant correlation between the expression of these candidate genes and myocardial development in porcine tissue.These findings establish a transcriptomic framework for porcine myocardial maturation and provide a molecular basis for advancing cardiac xenotransplantation.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB0820000)the National Natural Science Foundation of China(82421003,32270902,32170855)+1 种基金the Faculty Resources Project of the College of Life Sciences,Inner Mongolia University(2022-104)Initiative Scientific Research Program,Institute of Zoology,Chinese Academy of Sciences(20231OZ0102).
文摘Sertoli and granulosa cells,the initial differentiated somatic cells in bipotential gonads,play crucial roles in directing male and female gonad development,respectively.The transcription factor Foxo1 is involved in diverse cellular processes,and its expression in gonadal somatic cells is sex-dependent.While Foxo1 is abundantly expressed in ovarian granulosa cells,it is notably absent in testicular Sertoli cells.Nevertheless,its function in gonadal somatic cell differentiation remains elusive.In this study,we find that ectopic expression of Foxo1 in Sertoli cells leads to defects in testes development.Further study uncovers that the ectopic expression of Foxo1 induces the abundant expression of Foxl2 in Sertoli cells,along with the upregulation of other female-specific genes.In contrast,the expression of male-specific genes is reduced.Mechanistic studies indicate that Foxo1 directly binds to the promoter region of Foxl2,inducing its expression.Our findings highlight that Foxo1 serves as a key regulator for the lineage maintenance of ovarian granulosa cells.This study contributes valuable insights into understanding the regulatory mechanisms governing the lineage maintenance of gonadal somatic cells.
基金funded by the National Basic Research Program of China(2022YFD1302200,2023YFF1000904,2023ZD0404303 and 2021YFD1200301)the National Natural Science Foundation of China(32072806,32372970 and 32002246)+2 种基金the Program of Shaanxi Province Science and Technology Innovation Team,China(2019TD-036)the Key Technologies Demonstration of Animal Husbandry in Shaanxi Province,China(20221086 and 20230978)the Inner Mongolia Autonomous Region Open Competition Project,China(2022JBGS0025).
文摘Pluripotent stem cells(PSCs)are useful for developmental and translational research because they have the potential to differentiate into all cell types of an adult individual.Pigs are one of the most important domestic ungulates,commonly used for food and as bioreactors.Generating stable pluripotent porcine PSC lines remains challenging.So far,the pluripotency gene network of porcine PSCs is poorly understood.Here we found that TBX3-derived induced pluripotent stem cells(iPSCs)closely resemble porcine 4-cell embryos with the capacity of totipotent-like stem cells(TLSCs).Interestingly,our data suggest that TBX3 facilitates the activation of H3K4me3 methyltransferase,specifically MLL1.Subsequent investigations revealed that the porcine 4-cell specific gene,MCL1,is a key downstream effector of the TBX3-MLL1 axis.Together,our study of the TBX3 regulatory network is helpful in the understanding of the totipotency characteristics of pigs.
基金Acknowledgment This study was supported by the ＂973＂ project （No. 2006CB504001）, the Major Research Plan （No. 2006CB944001）, the CAS Innovation Project （KSCA2- YW-R-55）, the National Natural Science Foundation of China （No. 3061800530230190 30600311）, and the Beijing Natural Science Foundation （No. 5073032）.
文摘Aim： To examine the possible effect of heat treatment on expression of heat shock proteins （Hsps） 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. Methods： Western blot analysis, realtime polymerase chain reaction （PCR）, and confocal immunohistochemistry were used to analyze mRNA and protein levels of the Hsps in response to 43~C treatment of Sertoli cells isolated from pubertal monkey testes. Results： Staining with Hoechst 33342 indicated Sertoli cells did not undergo apoptosis after heat treatment. Hspl05 was expressed in cytoplasm of untreated Sertoli cells. Both Hspl05 mRNA and protein levels were increased approximately 20-fold compared to those of the untreated controls at 12 h after heat treatment. Untreated Sertoli cells did not express Hsp70, but heat stress induced its expression in the cell cytoplasm. The time-course of changes in Hsp70 was similar to that of Hsp105. In contrast to Hsp105 and Hsp70, the change in Hsp60 expression was much less obvious. The protein level between 12 h and 48 h after heat treatment was only approximately 1.5-fold that of the untreated control. Extracellular regulated kinase （ERK） 1/2 inhibitor （U0126） or phosphoinositide kinase-3 （PI3K） inhibitor （LY294002） could partially block the response of Hspl05 and Hsp70 induced by heat treatment. Conclusion： These results indicate that the heat-induced expression of the three types of Hsp in monkey Sertoli cells might be regulated by ERK and/or PI3K signal pathways, but the profile of their expression is different, suggesting that they might have different regulatory functions in Sertoli cells.
文摘Dear Editor, It is now well known that somatic cells can be efficiently reprogrammed into induced pluripotent stem cells （iPSCs） by forced expression of defined factors [1- 3]. These cells, like embryonic stem cells （ESCs）, have true pluripotency as shown by the live, fertile mice that can be generated through the tetraploid complementation assay using these iPSCs [4, 5]. So far, iPSCs have been generated from many species including mice, primate,
基金the National Basic Re-search Program of China (973 Program) (No. 2006CB504004 and 2006CB944004)the National Natural Science Foundation of China (No. 30430530)the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KSCX2-YW-N-017).
文摘High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases （DNMTs）, therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5＇ regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmtl and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones （AF1, AF2 and AF3） showed either hypermethylation or hypomethylation in the 5＇ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized （IVF） fetuses. Besides, we found that the 5＇ regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5＇ regions is probably associated with the high abortion of bovine clones.
基金supported by the National Natural Science Foundation of China(31801245,81671425 and 81971357)Key Research&Development Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory(2019GZR110104001)
文摘One of the main causes of pregnancy failure and fetus abortion is oocyte aneuploidy,which is increased with maternal aging.Numerous possible causes of oocyte aneuploidy in aged women have been proposed,including cross-over formation defect,cohesin loss,spindle deformation,spindle assembly checkpoint malfunction,microtubule-kinetochore attachment failure,kinetochore mis-orientation,mitochondria dysfunction-induced increases in reactive oxygen species,protein over-acetylation,and DNA damage.However,it still needs to be answered if these aneuploidization factors have inherent relations,and how to prevent chromosome aneuploidy in aged oocytes.Epidemiologically,oocyte aneuploidy has been found to be weakly associated with higher homocysteine concentrations,obesity,ionizing radiation and even seasonality.In this review,we summarize the research progress and present an integrated view of oocyte aneuploidization.
基金Acknowledgment This study was supported by the National Natural Science Foundation of China （30230190）, the National Basic Science Research and Development Project （973） （G1999055901） and the Chinese Academy of Sciences （CAS） Knowledge Innovation Program （KSCX-2-SW-201）.
文摘Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.
基金supported by the Major Basic Research Program(Nos.2012CB944404 and 2011CB944501)the National Natural Science Foundation of China(No.30930065)to Q.Y.S
文摘The mitochondrion is the major energy provider to power sperm motility. In mammals, aside from the nuclear genome, mitochondrial DNA （mtDNA） also contributes to oxidative phosphorylation to impact production of ATP by coding 13 polypeptides. However, the role of sperm mitochondria in fertilization and its final fate after fertilization are still controversial. The viewpoints that sperm bearing more mtDNA will have a better fertilizing capability and that sperm mtDNA is actively eliminated during early embryogenesis are widely accepted. However, this may be not true for several mammalian species, including mice and humans. Here, we review the sperm mitochondria and their mtDNA in sperm functions, and the mechanisms of maternal mitochondrial inheritance in mammals.
文摘Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells （ESCs）, which may be an important source ofhistocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines （hPES-1 and hPES-2） from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA- 1-60, and TRA- 1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies （EBs） of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers （hPES-2 did not）. Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat （STR） results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.
文摘The enrichment and identification of human epidermal stem cells （EpSCs） are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rap- idly adherent keratinocytes into three populations that range in size from 5-7 μm （population A）, to 7-9 μm （population B）, to ≥9μm （population C） in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of plintegrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency （CFE）. When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to pl-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.
文摘Recent data from several laboratories have provided evidence that the newly fertilized oocyte inherits epigenetic signals from the sperm chromatin that are required for proper embryonic development. For the purposes of this review, the term epigenetic is used to describe all types of molecular information that are transmitted from the sperm cell to the embryo. There are at least six different forms of epigenetic information that have already been established as being required for proper embryogenesis in mammals or for which there is evidence that it may do so. These are （i） DNA methylation; （ii） sperm-specific histones, （iii） other chromatin-associated proteins; （iv） the perinuclear theca proteins; （v） sperm-born RNAs and, the focus of this review; and （vi） the DNA loop domain organization by the sperm nuclear matrix. These epigenetic signals should he considered when designing protocols for the manipulation and cryopreservation of spermatozoa for assisted reproductive technology as necessary components for effective fertilization and subsequent embryo development.
基金supported in part by the grants from the Chinese National Basic Research Program (No 2007CB948001)the National High Technology R&D Program of China (No 2006AA02A101)Integrated Project funded by the Sixth Framework Programme (FP6) of the European Union (Contract:LSHG-CT-2005-019015)
文摘Knockout Brown Norway （BN） rat could be a useful disease model for human disorders,however,a failure to derive embryonic stem （ES） cells disturbs the further development of the model.In this study,we reported a case of successful derivation of the BN rat ES cells with the derivation efficiency comparable to that of Sprague Dawley （SD） rats.The BN rat ES cells expressed the key transcription factors,and were able to form embryonic bodies （EBs） when being differentiated in vitro.After injecting the BN rat ES cells into the SD rat blastocysts,high-contribution chimeric rats were generated and could survive to their adulthood.Our success in generating pluripotent rat ES cells will benefit the generation of the knockout rats in the future.
基金These three authors contributed equally to this work. This study was supported in part by grants from the Hi-Tech Research and Development Program of China （2006AA02A101 to QZ）, the National Natural Science Foundation of China （30670229 to QZ）, China National Basic Research Program （2006CB701500, 2007CB947700 and 2007CB947800）, the Shanghai Leading Academic Discipline Project （$30201）, and STCSM Project （08dj 1400502）.
文摘Mouse and human somatic cells can be induced to become pluripotent stem （iPS） cells by retroviral transduction with defined transcription factors . Reprogrammed pluripotent stem cells have great potential for regenerative medicine and also provide a good experi- mental system for studying epigenetic reprogramming and differentiation. However, at present, the reprogram- ming process is still somewhat slow and ineffective.
基金supported by the National Natural Science Foundation of China (91949209, 91749202, 92149301, 92168201)the National Key Research and Development Program of China (2018YFC2000100, 2020YFA0804000)+5 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010000)the National Natural Science Foundation of China (81921006, 81625009, 82125011)the Key Research Program of the Chinese Academy of Sciences (KFZD-SW-221)the 14th Five-year Network Security and Informatization Plan of Chinese Academy of Sciences (WX145XQ0718)Informatization Plan of Chinese Academy of Sciences (CASWX2021SF-0301)the Milky Way Research Foundation (MWRF)
文摘Maintaining metabolic homeostasis is essential for cellular and organismal health throughout life.Multiple signaling pathways that regulate metabolism also play critical roles in aging,such as PI3K/AKT,mTOR,AMPK,and sirtuins(SIRTs).Among them,sirtuins are known as a protein family with versatile functions,such as metabolic control,epigenetic modification and lifespan extension.Therefore,by understanding how sirtuins regulate metabolic processes,we can start to understand how they slow down or accelerate biological aging from the perspectives of metabolic regulation.Here,we review the biology of SIRT3,SIRT4,and SIRT5,known as the mitochondrial sirtuins due to their localization in the mitochondrial matrix.First,we will discuss canonical pathways that regulate metabolism more broadly and how these are integrated with aging regulation.Then,we will summarize the current knowledge about functional differences between SIRT3,SIRT4,and SIRT5 in metabolic control and integration in signaling networks.Finally,we will discuss how mitochondrial sirtuins regulate processes associated with aging and aging-related diseases.
文摘Intrauterine insemination with donor sperm(IUI-D)is an assisted reproductive technology(ART)offered to couples with definitive male infertility or risk of genetic disease transmission.Here,we sought to evaluate our practice in IUI-D and identify factors that influenced the success rate.We performed a retrospective,single-center study of all IUI-D procedures performed at Lille University Medical Center(Lille,France)between January 1,2007,and December 31,2017.Single and multivariate analyses with a mixed logistic model were used to identify factors associated with clinical pregnancies and live births.We included 322 couples and 1179 IUI-D procedures.The clinical pregnancy rate was 23.5%,and the live birth rate was 18.9%per IUI-D.In a multivariate analysis,the women’s age was negatively associated with the live birth rate.The number of motile spermatozoa inseminated was the only factor associated with both clinical pregnancies and live births,with a chosen threshold of 0.75 million.The clinical pregnancy and live birth rates were,respectively,17.3%and 13.0%below the number of motile spermatozoa inseminated threshold and 25.9%and 21.0%at or above the threshold(all P=0.005).The number of motile spermatozoa inseminated was the only factor that significantly influenced both pregnancies and live-birth rates after IUI-D.Indeed,below a threshold of 0.75 million motile spermatozoa inseminated,those rates were significantly lower.Application of this number of motile spermatozoa inseminated threshold may help centers to allocate donations more effectively while maintaining reasonable waiting times for patients.
基金supported by the National Natural Science Foundation of China(No.30930065 and No.31271605)
文摘Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.
基金supported in part by the grants from the National Basic Research Program of China(No.2012CB966501 to X.Z.and 2011CB965300 to L.W.)the"Strategic Priority Research Program"of the Chinese Academy of Sciences(No. XDA01020100 to Q.Z.)
文摘Rat is a valuable model for pharmacological and physiological studies. Germline-competent rat embryonic stem （rES） cell lines have been successfully established and the molecular networks maintaining the self-renewing, undifferentiated state of rES cells have also been well uncovered. However, little is known about the differentiation strategies and the underlying mechanisms of how these authentic rat pluripotent stem ceils give rise to specific cell types. The aim of this study is to investigate the neural differentiation capacity of rES cells. By means of a modified procedure based on previous publications - combination of mitogen-activated protein kinase （MAPK） and glycogen synthase kinase 3 （GSK3） inhibitors （two inhibitors, ＂2i＂） with feeder-conditioned medium, we successfully obtained high- quality rat embryoid bodies （rEBs） from rES cells and then differentiated them to tripotent neural progenitors. These rES cell-derived neural progenitor cells （rNPCs） were capable of self-renewing and giving rise to all three neural lineages, including astrocytes, oligo- dendrocytes, and neurons. Besides, these rES cell-derived neurons stained positive for y-aminobutyric acid （GABA） and tyrosine hydroxylase （TH）. In summary, we develop an experimental system for differentiating rES cells to tripotent neural progenitors, which may provide a powerful tool for pharmacological test and a valuable platform for studying the pathogenesis of many neurodegenerative disorders such as Parkinson＇s disease and the development of rat nervous system.
基金supported by the National Natural Science Foundation of China (No. Y211291131)the "Strategic Priority Research Program" of the Chinese Academy of Sciences (No. XDA01040109)
文摘Transcription activator-like effectors （TALEs） that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent （USER）, an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases （TALENs） generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tetl locus, and proved that all can specifically target Tetl gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.
