Tree shrews(Tupaia belangeri chinensis)share a close relationship to primates and have been widely used in biomedical research.We previously established a spermatogonial stem cell(SSC)-based gene editing platform to g...Tree shrews(Tupaia belangeri chinensis)share a close relationship to primates and have been widely used in biomedical research.We previously established a spermatogonial stem cell(SSC)-based gene editing platform to generate transgenic tree shrews.However,the influences of long-term expansion on tree shrew SSC spermatogenesis potential remain unclear.Here,we examined the in vivo spermatogenesis potential of tree shrew SSCs cultured across different passages.We found that SSCs lost spermatogenesis ability after long-term expansion(>50 passages),as indicated by the failure to colonize the seminiferous epithelium and generate donor spermatogonia(SPG)-derivedspermatocytesor spermatids marking spermatogenesis.RNA sequencing(RNA-seq)analysis of undifferentiated SPGs across different passages revealed significant gene expression changes after sub-culturing primary SPG lines for more than 40 passages on feeder layers.Specifically,DNA damage response and repair genes(e.g.,MRE11,SMC3,BLM,and GEN1)were down-regulated,whereas genes associated with mitochondrial function(e.g.,NDUFA9,NDUFA8,NDUFA13,and NDUFB8)were up-regulated after expansion.The DNA damage accumulation and mitochondrial dysfunction were experimentally validated in high-passage cells.Supplementation with nicotinamide adenine dinucleotide(NAD+)precursor nicotinamide riboside(NR)exhibited beneficial effects by reducing DNA damage accumulation and mitochondrial dysfunction in SPG elicited by long-term culture.Our research presents a comprehensive analysis of the genetic and physiological attributes critical for the sustained expansion of undifferentiated SSCs in tree shrews and proposes an effective strategy for extended in vitro maintenance.展开更多
Objective:To assess the effect of cryopreservation on stemness and proliferation potential of sheep spermatogonial stem cells(SSCs)in vitro.Methods:Sheep testicular cells were isolated and putative SSCs were enriched ...Objective:To assess the effect of cryopreservation on stemness and proliferation potential of sheep spermatogonial stem cells(SSCs)in vitro.Methods:Sheep testicular cells were isolated and putative SSCs were enriched by the laminin-based differential plating method.Putative SSCs were co-cultured with the Sertoli cell feeder prepared by the Datura Stramonium Agglutinin(DSA-lectin)-based method.The cultured putative SSCs were cryopreserved in Dulbecco's Modified Eagle Medium-10%fetal bovine serum mixture(DMEM-10%FBS)media containing 10%dimethyl sulfoxide(DMSO)alone or 10%DMSO plus 200 mM trehalose.Cryopreserved putative SSCs were evaluated for their proliferation potential using in vitro culture and stemness by immunocytochemistry.Finally,the transfection ability of cryopreserved putative SSCs was analyzed.Results:We isolated 91%viable testicular cells from sheep testes.The majority of the laminin enriched cells expressed the SSC related marker,ITGA6.Co-culture of sheep putative SSCs with Sertoli cell feeder resulted in the generation of stable colonies,and the expression of SSC marker was maintained after several passages.A significantly higher number of viable putative SSCs was recovered from SSCs cryopreserved in media containing 10%DMSO and 200 mM trehalose compared to 10%DMSO alone(P<0.01).Cryopreserved putative SSCs formed colonies and showed SSC marker expression similar to the non-cryopreserved putative SSCs.The appearance of green fluorescent colonies over the Sertoli cell feeder indicated that cryopreserved sheep SSCs were successfully transfected.Conclusions:Cryopreserved putative SSCs can retain their stemness,colony forming ability,and transfection efficiency in vitro.Our research may help in the effective preservation of germplasm and the generation of transgenic ovine species.展开更多
Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation of pig spermatogonial stem cells(p SSCs) has not been tested. The aim of this ...Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation of pig spermatogonial stem cells(p SSCs) has not been tested. The aim of this work was to study the effect of sucrose during p SSC cryopreservation and to find the most effective concentration in freezing medium. p SSCs were cryopreserved with freezing media containing different concentrations of sucrose(70, 140, 210, and 280 mmol L^(–1)) and a control group without sucrose. The survival rates, plasma membrane integrity, and mitochondrial membrane potential of thawed cells were detected by trypan blue(TB) staining, SYBR-14/propidium iodide(PI) dual staining, and JC-1 staining, respectively. All the staining results showed an obvious increase in cell survival in the sucrose-treated groups as compared to that in the control group, with the exception of 280 mmol L^(–1) sucrose. Moreover, the 210 mmol L^(–1) sucrose group yielded the highest survival rate among all the groups(P<0.05). The results of SYBR-14/PI dual staining and JC-1 staining were consistent with those of TB staining as above described. Quantitative real-time PCR(q RT-PCR) indicated that the m RNA levels of three apoptosis-promoting genes(BAX, APAF1 and CASPASE9) were significantly higher in thawed cells than in cells before freezing(P<0.05). Moreover, the mR NA level of one anti-apoptotic gene(XIAP) was significantly lower in thawed cells than in cells before freezing(P<0.05). When comparing the m RNA expression of apoptosis-related genes in thawed cells, the m RNA level of the anti-apoptotic genes in the control group was significantly lower than that in the sucrose-treated groups(P<0.05). Western blot analyses showed that the expression levels of cleaved CASPASE9, CASPASE3 and PARP-1 in the sucrose-treated groups were lower than those in the control group and were the lowest in the 210 mmol L^(–1) sucrose group. Both q RT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing. Briefly, sucrose promoted p SSCs survival after freezing and thawing, especially at a concentration of 210 mmol L^(–1), which possibly assisted p SSC dehydration and inhibited cell apoptosis. These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of p SSCs.展开更多
Background:Spermatogonial stem cells(SSCs)are capable of both self-renewal and differentiation to mature functional spermatozoa,being the only adult stem cells in the males that can transmit genetic information to the...Background:Spermatogonial stem cells(SSCs)are capable of both self-renewal and differentiation to mature functional spermatozoa,being the only adult stem cells in the males that can transmit genetic information to the next generation.Porcine SSCs hold great value in transgenic pig production and in establishment of porcine models for regenerative medicine.However,studies and applications of porcine SSCs have been greatly hampered by the low number of SSCs in the testis as well as the lack of an ideal stable long-term culture system to propagate porcine SSCs perpetually.Results:In the present study,by lentiviral transduction of plasmids expressing the simian virus 40(SV40)large T antigen into porcine primary SSCs,we developed two immortalized cell lines with porcine SSC attributes.The established cell lines,with the expression of porcine SSC and germ cell markers UCHL1,PLZF,THY1,VASA and DAZL,could respond to retinoic acid(RA),and could colonize the recipient mouse testis without tumor formation after transplantation.The cell lines displayed infinite proliferation potential,and have now been cultured for more than 7 months and passaged for over 35 times without morphological abnormalities.Conclusions:We have for the first time established porcine SSC lines that could provide abundant cell sources for mechanistic studies on porcine SSC self-renewal and differentiation,thereby facilitating development of an optimal long-term culture system for porcine primary SSCs and their application to animal husbandry and medicine.展开更多
BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stabl...BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stable degradation product widely used in daily life and production and has been proven to affect male fertility.However,the underlying mechanisms therein are unclear.Thus,it is necessary to study the effect and mechanism of NP on spermatogonial stem cells(SSCs).AIM To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway.METHODS SSCs were treated with NP at 0,10,20 or 30μmol.MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs.Flow cytometry was conducted to measure SSC apoptosis.The expression of Bad,Bcl-2,cytochrome-c,pro-Caspase 9,SOX-2,OCT-4,Nanog,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot,and the mRNA expression of SOX-2,OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction.RESULTS Compared with untreated cells(0μmol NP),SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2,Nanog,OCT-4,SOX-2,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,and PLZF(P<0.05),whereas the expression of Bad,cytochrome-c,and pro-Caspase 9 increased significantly(P<0.05).We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K,AKT,mTORC1,and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs(P<0.05).NP exerted the greatest effect at 30μmol among all NP concentrations.CONCLUSION NP attenuated the proliferation,differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress.The associated mechanism may be related to the PI3K/AKT/mTOR pathway.展开更多
BACKGROUND Spermatogonial stem cells(SSCs)are the origin of male spermatogenesis,which can reconstruct germ cell lineage in mice.However,the application of SSCs for male fertility restoration is hindered due to the un...BACKGROUND Spermatogonial stem cells(SSCs)are the origin of male spermatogenesis,which can reconstruct germ cell lineage in mice.However,the application of SSCs for male fertility restoration is hindered due to the unclear mechanisms of proliferation and self-renewal in humans.AIM To investigate the role and mechanism of SPOC domain-containing protein 1(SPOCD1)in human SSC proliferation.METHODS We analyzed publicly available human testis single-cell RNA sequencing(RNAseq)data and found that SPOCD1 is predominantly expressed in SSCs in the early developmental stages.Small interfering RNA was applied to suppress SPOCD1 expression to detect the impacts of SPOCD1 inhibition on SSC proliferation and apoptosis.Subsequently,we explored the target genes of SPOCD1 using RNA-seq and confirmed their role by restoring the expression of the target genes.In addition,we examined SPOCD1 expression in some non-obstructive azoospermia(NOA)patients to explore the correlation between SPOCD1 and NOA.RESULTS The uniform manifold approximation and projection clustering and pseudotime analysis showed that SPOCD1 was highly expressed in the early stages of SSC,and immunohistological results showed that SPOCD1 was mainly localized in glial cell line-derived neurotrophic factor family receptor alpha-1 positive SSCs.SPOCD1 knockdown significantly inhibited cell proliferation and promoted apoptosis.RNA-seq results showed that SPOCD1 knockdown significantly downregulated genes such as adenylate kinase 4(AK4).Overexpression of AK4 in SPOCD1 knockdown cells partially reversed the phenotypic changes,indicating that AK4 is a functional target gene of SPOCD1.In addition,we found a significant downregulation of SPOCD1 expression in some NOA patients,suggesting that the downregulation of SPOCD1 may be relevant for NOA.CONCLUSION Our study broadens the understanding of human SSC fate determination and may offer new theories on the etiology of male infertility.展开更多
BACKGROUND Human spermatogonial stem cells(SSCs)are the basis of spermatogenesis.However,little is known about the developmental regulatory mechanisms of SSC due to sample origin and species differences.AIM To investi...BACKGROUND Human spermatogonial stem cells(SSCs)are the basis of spermatogenesis.However,little is known about the developmental regulatory mechanisms of SSC due to sample origin and species differences.AIM To investigates the mechanisms involved in the proliferation of human SSC.METHODS The expression of mitogen-activated protein kinase kinase 7(MKK7)in human testis was identified using immunohistochemistry and western blotting(WB).MKK7 was knocked down using small interfering RNA,and cell proliferation and apoptosis were detected by WB,EdU,cell counting kit-8 and fluorescenceactivated cell sorting.After bioinformatic analysis,the interaction of MKK7 with c-Jun N-terminal kinases(JNKs)was verified by protein co-immunoprecipitation and WB.The phosphorylation of JNKs was inhibited by SP600125,and the phenotypic changes were detected by WB,cell counting kit-8 and fluorescenceactivated cell sorting.RESULTS MKK7 is mainly expressed in human SSCs,and MKK7 knockdown inhibits SSC proliferation and promotes their apoptosis.MKK7 mediated the phosphorylation of JNKs,and after inhibiting the phosphorylation of JNKs,the phenotypic changes of the cells were similar to those after MKK7 downregulation.The expression of MKK7 was significantly downregulated in patients with abnormal spermatogenesis,suggesting that abnormal MKK7 may be associated with spermatogenesis impairment.CONCLUSION MKK7 regulates the proliferation and apoptosis of human SSC by mediating the phosphorylation of JNKs.展开更多
Objective:To investigate the effects of soft agar on in-vitro proliferation of neonate mouse spermatogonial stem cells co-cultured with Sertoli cells.Methods:Tissues of neonate NMRI male mice testes were used for harv...Objective:To investigate the effects of soft agar on in-vitro proliferation of neonate mouse spermatogonial stem cells co-cultured with Sertoli cells.Methods:Tissues of neonate NMRI male mice testes were used for harvesting spermatogonial stem cells and Sertoli cells.After cell harvest,flow cytometry using promyelocytic leukemia zinc-finger(PLZF)protein antibody was used to assess the purity of the cells.The isolated testicular cells were cultured in the absence(the control group)or presence of soft agar-coated dishes(the experimental group)supplemented with leukemia inhibitory factor and glia cell line–derived neurotrophic factor for two weeks.Alkaline phosphatase activity was assessed in the colonies formed after two weeks of culture by alkaline phosphatase staining.On day 14 of culture,the expression levels of DNA-binding protein inhibitor(ID-4)and PLZF genes in the undifferentiated cells were evaluated by the detection of PLZF protein antibody using real-time PCR and immunocytochemistry techniques.The number and diameter of the colonies of spermatogonial stem cells were assessed by ImageJ software.Results:In the experimental group,the number and the diameter of colonies significantly increased as compared with those in the control group(P<0.05,P<0.01,respectively).In addition,the level of expression of ID-4 and PLZF genes in the undifferentiated cells significantly increased in the experimental group as compared with the control group(P<0.01).However,the expression of tyrosine-protein kinase kit(c-kit)gene in differentiated cells decreased in the experimental group as compared with the control group,but there was no significant difference between the two groups.Conclusions:Spermatogonial stem cells can be efficiently proliferated by culturing the stem cells in soft agar-coated dishes.The new protocol used in this study can be a valuable method for future studies.展开更多
The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous...The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous epithlium cells were isolated and purified, the cells were cryopreserved after identification, and the effects of different purification and cyopreservation methods on bovine testicular cells were studied. The results showed that there were spermatogonial stem cells and sertoli cells in the neonatal bovine seminiferous tubules, differential adherent selection methods could effectively separate these two cell types. Spermatogonial stem cells were positive after AKP, C-kit, and OCT-4 identification; sertoli cells were positive after oil red O and vimentin identification. Frozen stock solution supplemented with 10% DMSO had the best effect in spermatogonial stem cell cryopreservation, while frozen stock solution supplemented with 10% of ethylene glycol and 0.1 mmol·L-1 trehalose had the best effect in sertoli cells cryopreservation.展开更多
With the decline in male fertility in recent years,strategies for male fertility preservation have received increasing attention.In this study,by reviewing current treatments and recent publications,we describe resear...With the decline in male fertility in recent years,strategies for male fertility preservation have received increasing attention.In this study,by reviewing current treatments and recent publications,we describe research progress in and the future directions of stem cell-based therapies for male fertility preservation,focusing on the use of spermatogonial stem cells(SSCs),SSC niches,SSC-based testicular organoids,other stem cell types such as mesenchymal stem cells,and stem cell-derived extracellular vesicles.In conclusion,a more comprehensive understanding of the germ cell microenvironment,stem cell-derived extracellular vesicles,and testicular organoids will play an important role in achieving male fertility preservation.展开更多
Continuous self-renewal and differentiation of spermatogonial stem cells(SSCs)is vital for maintenance of adult spermatogenesis.Although several spermatogonial stem cell regulators have been extensively investigated i...Continuous self-renewal and differentiation of spermatogonial stem cells(SSCs)is vital for maintenance of adult spermatogenesis.Although several spermatogonial stem cell regulators have been extensively investigated in rodents,regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established.We analyzed single-cell sequencing data from the human testis and found that forkhead box P4(FOXP4)expression gradually increased with development of SSCs.Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential.Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis.FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis.These findings imply that FOXP4 is involved in human SSC proliferation,which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.展开更多
The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as te...The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.展开更多
The surrogate reproduction technique,such as inter-specific spermatogonial stem cells(SSCs)transplantation(SSCT),provides a powerful tool for production of gametes derived from endangered species or those with desirab...The surrogate reproduction technique,such as inter-specific spermatogonial stem cells(SSCs)transplantation(SSCT),provides a powerful tool for production of gametes derived from endangered species or those with desirable traits.However,generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily,by SSCT,has not succeeded.Here,using two small cyprinid fishes from different subfamilies,Chinese rare minnow(gobiocypris rarus,for brief:Gr)and zebrafish(danio rerio),we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure.The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis,resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells.Interestingly,the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior.When pou5f3 and chd knockout Gr SSCs were transplanted,Gr-derived genome-edited sperm was successfully produced in zebrafish.This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT,and surrogate production of genome-edited gametes from another species as well.This method is feasible to be applied to future breeding of commercial fish and livestock.展开更多
Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells(SSCs).However,such therapy for infertility has n...Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells(SSCs).However,such therapy for infertility has not been experimentally investigated yet.In this study,a mouse model with an X-linked testis-expressed 11(TEX11)mutation(Tex11PM/Y)identified in azoospermia patients exhibited meiotic arrest due to aberrant chromosome segregation.Tex11PM/Y SSCs could be isolated and expanded in vitro normally,and the mutation was corrected by clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated endonuclease 9(Cas9),leading to the generation of repaired SSC lines.Whole-genome sequencing demonstrated that the mutation rate in repaired SSCs is comparable with that of autonomous mutation in untreated Tex11PM/Y SSCs,and no predicted off-target sites are modified.Repaired SSCs could restore spermatogenesis in infertile males and give rise to fertile offspring at a high efficiency.In summary,our study establishes a paradigm for the treatment of male azoospermia by combining in vitro expansion of SSCs and gene therapy.展开更多
Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells(SSCs).SSCs,the only male reproductive stem cells that transmit genetic material to subsequent generations,possess...Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells(SSCs).SSCs,the only male reproductive stem cells that transmit genetic material to subsequent generations,possess an inherent self-renewal ability,which allows the maintenance of a steady stem cell pool.SSCs eventually differentiate to produce sperm.However,in an in vitro culture system,SSCs can be induced to differentiate into various types of germ cells.Rodent SSCs are well defined,and a culture system has been successfully established for them.In contrast,available information on the biomolecular markers and a culture system for livestock SSCs is limited.This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process,the biology and niche of SSCs,the isolation and culture systems of SSCs,and the biomolecular markers and identification of SSCs.This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals,enhancement of human male fertility,reproductive medicine,and protection of endangered species.展开更多
Male infertility has evolved from a common reproductive system disease to a major social issue.Youjing granule(YG)is a Chinese medicinal material used as a therapy method for tonifying the kidneys and removing dampnes...Male infertility has evolved from a common reproductive system disease to a major social issue.Youjing granule(YG)is a Chinese medicinal material used as a therapy method for tonifying the kidneys and removing dampness due to its pathogenic characteristics.YG has been shown to regulate sperm quality in clinical trials,but the underlying mechanism is not fully understood.The present study was aimed to explore the protective effects and mechanism of action of YG on male reproductive system damage caused by methyl methane sulfonate(MMS).We first established an infertility model of rats through oral administration of MMS and then treated with YG.To determine the effect of YG,spermatogenesis,microvascular density,and secretory function of Leydig cells and Sertoli cells in rats were assessed.Spermatogonial stem cells(SSCs)were co-cultured with mouse embryo fibroblast(MEF)cells as an in vitro cell model before exposure to serum containing YG.Furthermore,the proliferation and apoptosis of SSCs were measured.Results indicated that YG increased the expression of self-renewal and proliferation-related molecules such as glial cell line derived neurotrophic factor(GDNF)and fibroblast growth factor-2(FGF2),and improved the quality of sperm and the proliferation of SSCs.In conclusion,YG may protect spermatogenetic function of rats through regulating the proliferation and self-renewal of SSCs.展开更多
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 i...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(GFRAl)-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 GFRAl-enriched spermatogonia between 0 day and 14 days of culture,and xenotransplantation was performed to evaluate the function of GFRAl-enriched spermatogonia.SSCs shared some phenotypes with rodent and human SSCs.GFRAl-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 GFRAl-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 GFRAl-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-Kitw/w(W)mutant mice.Collectively,GFRAl-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.展开更多
Mammalian spermatogenesis is a complicated and precisely controlled process that requires spermatogonial stem cells(SSCs).SSCs maintain the stem cell pool,balance self-renewal–commitment with differentiation,and prod...Mammalian spermatogenesis is a complicated and precisely controlled process that requires spermatogonial stem cells(SSCs).SSCs maintain the stem cell pool,balance self-renewal–commitment with differentiation,and produce millions of sperm daily.Self-renewal and differentiation are controlled by intrinsic factors within SSCs and extrinsic factors from the"niche."In this review,we discuss the biology of SSCs and the factors regulating their self-renewal and differentiation.展开更多
Although somatic cells can be reprogrammed to pluripotent stem cells(PsCs)with pure chemicals,authentic pluripotency of chemically induced pluripotent stem celis(CipsCs)has never been achieved through tetraploid compl...Although somatic cells can be reprogrammed to pluripotent stem cells(PsCs)with pure chemicals,authentic pluripotency of chemically induced pluripotent stem celis(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 effciency 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 ceil 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 ight on the unique regulatory network underpinning SsC reprogramming,providing insights to understand generic mechanisms for cell-fate decision and epigenetic-relateddisorders in regenerative medicine.展开更多
Objective:Testosterone plays an essential role in maintaining spermatogenesis and male fertility,and the primary known source of testosterone is testicular Leydig cells,which are regulated by luteinizing hormone(LH).H...Objective:Testosterone plays an essential role in maintaining spermatogenesis and male fertility,and the primary known source of testosterone is testicular Leydig cells,which are regulated by luteinizing hormone(LH).However,whether any other ways of testosterone secretion exist still remains unknown.Methods:Transmission electron microscopy was used to detect testicular extracellular vesicles(EVs),which were isolated by an ultracentrifuge process.Separately,the concentrations of follicle-stimulating hormone(FSH),LH,and testosterone were measured by enzyme-linked immunosorbent assay.Results:Some EVs were found by tail vein injection to be present in mouse testes that elevate the circulating testosterone and LH levels in the blood,but do not affect FSH.Separately,they also promote testosterone production in the TM3 Leydig cell line in vitro.To determine whether the EVs from spermatogonia were involved in the secretion of testosterone,we used spermatogonial stem/progenitor cell line C18-4 cells and revealed that C18-4 cells promote production of testosterone in the TM3 Leydig cell line using the EVs.Conclusions:EVs in mouse testes likely originate from spermatogonia and involved in the regulation of the serum testosterone.Our results provide a new mechanism for the regulation of testosterone production.展开更多
基金supported by the Ministry of Science and Technology of China (2021YFF0702700,STI2030-Major Project2021ZD0200900)National Natural Science Foundation of China (U2102202,U1702284)Yunnan Province (202305AH340006)。
文摘Tree shrews(Tupaia belangeri chinensis)share a close relationship to primates and have been widely used in biomedical research.We previously established a spermatogonial stem cell(SSC)-based gene editing platform to generate transgenic tree shrews.However,the influences of long-term expansion on tree shrew SSC spermatogenesis potential remain unclear.Here,we examined the in vivo spermatogenesis potential of tree shrew SSCs cultured across different passages.We found that SSCs lost spermatogenesis ability after long-term expansion(>50 passages),as indicated by the failure to colonize the seminiferous epithelium and generate donor spermatogonia(SPG)-derivedspermatocytesor spermatids marking spermatogenesis.RNA sequencing(RNA-seq)analysis of undifferentiated SPGs across different passages revealed significant gene expression changes after sub-culturing primary SPG lines for more than 40 passages on feeder layers.Specifically,DNA damage response and repair genes(e.g.,MRE11,SMC3,BLM,and GEN1)were down-regulated,whereas genes associated with mitochondrial function(e.g.,NDUFA9,NDUFA8,NDUFA13,and NDUFB8)were up-regulated after expansion.The DNA damage accumulation and mitochondrial dysfunction were experimentally validated in high-passage cells.Supplementation with nicotinamide adenine dinucleotide(NAD+)precursor nicotinamide riboside(NR)exhibited beneficial effects by reducing DNA damage accumulation and mitochondrial dysfunction in SPG elicited by long-term culture.Our research presents a comprehensive analysis of the genetic and physiological attributes critical for the sustained expansion of undifferentiated SSCs in tree shrews and proposes an effective strategy for extended in vitro maintenance.
文摘Objective:To assess the effect of cryopreservation on stemness and proliferation potential of sheep spermatogonial stem cells(SSCs)in vitro.Methods:Sheep testicular cells were isolated and putative SSCs were enriched by the laminin-based differential plating method.Putative SSCs were co-cultured with the Sertoli cell feeder prepared by the Datura Stramonium Agglutinin(DSA-lectin)-based method.The cultured putative SSCs were cryopreserved in Dulbecco's Modified Eagle Medium-10%fetal bovine serum mixture(DMEM-10%FBS)media containing 10%dimethyl sulfoxide(DMSO)alone or 10%DMSO plus 200 mM trehalose.Cryopreserved putative SSCs were evaluated for their proliferation potential using in vitro culture and stemness by immunocytochemistry.Finally,the transfection ability of cryopreserved putative SSCs was analyzed.Results:We isolated 91%viable testicular cells from sheep testes.The majority of the laminin enriched cells expressed the SSC related marker,ITGA6.Co-culture of sheep putative SSCs with Sertoli cell feeder resulted in the generation of stable colonies,and the expression of SSC marker was maintained after several passages.A significantly higher number of viable putative SSCs was recovered from SSCs cryopreserved in media containing 10%DMSO and 200 mM trehalose compared to 10%DMSO alone(P<0.01).Cryopreserved putative SSCs formed colonies and showed SSC marker expression similar to the non-cryopreserved putative SSCs.The appearance of green fluorescent colonies over the Sertoli cell feeder indicated that cryopreserved sheep SSCs were successfully transfected.Conclusions:Cryopreserved putative SSCs can retain their stemness,colony forming ability,and transfection efficiency in vitro.Our research may help in the effective preservation of germplasm and the generation of transgenic ovine species.
基金supported by the China Postdoctoral Science Foundation(2014M560809)the Shaanxi Province Postdoctoral Science Foundation,China+1 种基金the Fundamental Research Funds for the Central Universities,China(NWSUAF,2452015145)the National Basic Research Program of China(2014CB943100)
文摘Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation of pig spermatogonial stem cells(p SSCs) has not been tested. The aim of this work was to study the effect of sucrose during p SSC cryopreservation and to find the most effective concentration in freezing medium. p SSCs were cryopreserved with freezing media containing different concentrations of sucrose(70, 140, 210, and 280 mmol L^(–1)) and a control group without sucrose. The survival rates, plasma membrane integrity, and mitochondrial membrane potential of thawed cells were detected by trypan blue(TB) staining, SYBR-14/propidium iodide(PI) dual staining, and JC-1 staining, respectively. All the staining results showed an obvious increase in cell survival in the sucrose-treated groups as compared to that in the control group, with the exception of 280 mmol L^(–1) sucrose. Moreover, the 210 mmol L^(–1) sucrose group yielded the highest survival rate among all the groups(P<0.05). The results of SYBR-14/PI dual staining and JC-1 staining were consistent with those of TB staining as above described. Quantitative real-time PCR(q RT-PCR) indicated that the m RNA levels of three apoptosis-promoting genes(BAX, APAF1 and CASPASE9) were significantly higher in thawed cells than in cells before freezing(P<0.05). Moreover, the mR NA level of one anti-apoptotic gene(XIAP) was significantly lower in thawed cells than in cells before freezing(P<0.05). When comparing the m RNA expression of apoptosis-related genes in thawed cells, the m RNA level of the anti-apoptotic genes in the control group was significantly lower than that in the sucrose-treated groups(P<0.05). Western blot analyses showed that the expression levels of cleaved CASPASE9, CASPASE3 and PARP-1 in the sucrose-treated groups were lower than those in the control group and were the lowest in the 210 mmol L^(–1) sucrose group. Both q RT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing. Briefly, sucrose promoted p SSCs survival after freezing and thawing, especially at a concentration of 210 mmol L^(–1), which possibly assisted p SSC dehydration and inhibited cell apoptosis. These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of p SSCs.
基金This study was supported by the National Natural Science Foundation of China(Grant No.31572401,31772605)to W.Z.the Open Fund of Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province(Grant No.SNDK-KF-201804)Young Talent fund of University Association for Science and Technology in Shaanxi,China(Grant No.20180204)and a startup fund from Northwest A&F University(Grant No.2452018037)to Y.Z.
文摘Background:Spermatogonial stem cells(SSCs)are capable of both self-renewal and differentiation to mature functional spermatozoa,being the only adult stem cells in the males that can transmit genetic information to the next generation.Porcine SSCs hold great value in transgenic pig production and in establishment of porcine models for regenerative medicine.However,studies and applications of porcine SSCs have been greatly hampered by the low number of SSCs in the testis as well as the lack of an ideal stable long-term culture system to propagate porcine SSCs perpetually.Results:In the present study,by lentiviral transduction of plasmids expressing the simian virus 40(SV40)large T antigen into porcine primary SSCs,we developed two immortalized cell lines with porcine SSC attributes.The established cell lines,with the expression of porcine SSC and germ cell markers UCHL1,PLZF,THY1,VASA and DAZL,could respond to retinoic acid(RA),and could colonize the recipient mouse testis without tumor formation after transplantation.The cell lines displayed infinite proliferation potential,and have now been cultured for more than 7 months and passaged for over 35 times without morphological abnormalities.Conclusions:We have for the first time established porcine SSC lines that could provide abundant cell sources for mechanistic studies on porcine SSC self-renewal and differentiation,thereby facilitating development of an optimal long-term culture system for porcine primary SSCs and their application to animal husbandry and medicine.
基金Health and Family Planning Committee Joint Fund Project of Hubei Province,No.WJ2018H0020Fundamental Research Funds for the Central Universities,No.2042016kf0187 and No.2042017kf0068Zhongnan Hospital of Wuhan University Science,Technology and Innovation Seed Fund,No.znpy2016022.
文摘BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stable degradation product widely used in daily life and production and has been proven to affect male fertility.However,the underlying mechanisms therein are unclear.Thus,it is necessary to study the effect and mechanism of NP on spermatogonial stem cells(SSCs).AIM To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway.METHODS SSCs were treated with NP at 0,10,20 or 30μmol.MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs.Flow cytometry was conducted to measure SSC apoptosis.The expression of Bad,Bcl-2,cytochrome-c,pro-Caspase 9,SOX-2,OCT-4,Nanog,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot,and the mRNA expression of SOX-2,OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction.RESULTS Compared with untreated cells(0μmol NP),SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2,Nanog,OCT-4,SOX-2,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,and PLZF(P<0.05),whereas the expression of Bad,cytochrome-c,and pro-Caspase 9 increased significantly(P<0.05).We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K,AKT,mTORC1,and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs(P<0.05).NP exerted the greatest effect at 30μmol among all NP concentrations.CONCLUSION NP attenuated the proliferation,differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress.The associated mechanism may be related to the PI3K/AKT/mTOR pathway.
基金the National Natural Science Foundation for Young Scholars of China,No.82201771National Natural Science Foundation of China,No.32270912+2 种基金Natural Science Foundation of Changsha,No.kq2202491Research Grant of CITIC-Xiangya,No.YNXM202109 and No.YNXM202115Hunan Provincial Grant for Innovative Province Construction,No.2019SK4012。
文摘BACKGROUND Spermatogonial stem cells(SSCs)are the origin of male spermatogenesis,which can reconstruct germ cell lineage in mice.However,the application of SSCs for male fertility restoration is hindered due to the unclear mechanisms of proliferation and self-renewal in humans.AIM To investigate the role and mechanism of SPOC domain-containing protein 1(SPOCD1)in human SSC proliferation.METHODS We analyzed publicly available human testis single-cell RNA sequencing(RNAseq)data and found that SPOCD1 is predominantly expressed in SSCs in the early developmental stages.Small interfering RNA was applied to suppress SPOCD1 expression to detect the impacts of SPOCD1 inhibition on SSC proliferation and apoptosis.Subsequently,we explored the target genes of SPOCD1 using RNA-seq and confirmed their role by restoring the expression of the target genes.In addition,we examined SPOCD1 expression in some non-obstructive azoospermia(NOA)patients to explore the correlation between SPOCD1 and NOA.RESULTS The uniform manifold approximation and projection clustering and pseudotime analysis showed that SPOCD1 was highly expressed in the early stages of SSC,and immunohistological results showed that SPOCD1 was mainly localized in glial cell line-derived neurotrophic factor family receptor alpha-1 positive SSCs.SPOCD1 knockdown significantly inhibited cell proliferation and promoted apoptosis.RNA-seq results showed that SPOCD1 knockdown significantly downregulated genes such as adenylate kinase 4(AK4).Overexpression of AK4 in SPOCD1 knockdown cells partially reversed the phenotypic changes,indicating that AK4 is a functional target gene of SPOCD1.In addition,we found a significant downregulation of SPOCD1 expression in some NOA patients,suggesting that the downregulation of SPOCD1 may be relevant for NOA.CONCLUSION Our study broadens the understanding of human SSC fate determination and may offer new theories on the etiology of male infertility.
基金Supported by China Postdoctoral Science Foundation,No.2019M661521and National Natural Science Foundation of China,No.82001634.
文摘BACKGROUND Human spermatogonial stem cells(SSCs)are the basis of spermatogenesis.However,little is known about the developmental regulatory mechanisms of SSC due to sample origin and species differences.AIM To investigates the mechanisms involved in the proliferation of human SSC.METHODS The expression of mitogen-activated protein kinase kinase 7(MKK7)in human testis was identified using immunohistochemistry and western blotting(WB).MKK7 was knocked down using small interfering RNA,and cell proliferation and apoptosis were detected by WB,EdU,cell counting kit-8 and fluorescenceactivated cell sorting.After bioinformatic analysis,the interaction of MKK7 with c-Jun N-terminal kinases(JNKs)was verified by protein co-immunoprecipitation and WB.The phosphorylation of JNKs was inhibited by SP600125,and the phenotypic changes were detected by WB,cell counting kit-8 and fluorescenceactivated cell sorting.RESULTS MKK7 is mainly expressed in human SSCs,and MKK7 knockdown inhibits SSC proliferation and promotes their apoptosis.MKK7 mediated the phosphorylation of JNKs,and after inhibiting the phosphorylation of JNKs,the phenotypic changes of the cells were similar to those after MKK7 downregulation.The expression of MKK7 was significantly downregulated in patients with abnormal spermatogenesis,suggesting that abnormal MKK7 may be associated with spermatogenesis impairment.CONCLUSION MKK7 regulates the proliferation and apoptosis of human SSC by mediating the phosphorylation of JNKs.
基金This study was supported by Shahroud University of Medical Sciences(Grant No.98131).
文摘Objective:To investigate the effects of soft agar on in-vitro proliferation of neonate mouse spermatogonial stem cells co-cultured with Sertoli cells.Methods:Tissues of neonate NMRI male mice testes were used for harvesting spermatogonial stem cells and Sertoli cells.After cell harvest,flow cytometry using promyelocytic leukemia zinc-finger(PLZF)protein antibody was used to assess the purity of the cells.The isolated testicular cells were cultured in the absence(the control group)or presence of soft agar-coated dishes(the experimental group)supplemented with leukemia inhibitory factor and glia cell line–derived neurotrophic factor for two weeks.Alkaline phosphatase activity was assessed in the colonies formed after two weeks of culture by alkaline phosphatase staining.On day 14 of culture,the expression levels of DNA-binding protein inhibitor(ID-4)and PLZF genes in the undifferentiated cells were evaluated by the detection of PLZF protein antibody using real-time PCR and immunocytochemistry techniques.The number and diameter of the colonies of spermatogonial stem cells were assessed by ImageJ software.Results:In the experimental group,the number and the diameter of colonies significantly increased as compared with those in the control group(P<0.05,P<0.01,respectively).In addition,the level of expression of ID-4 and PLZF genes in the undifferentiated cells significantly increased in the experimental group as compared with the control group(P<0.01).However,the expression of tyrosine-protein kinase kit(c-kit)gene in differentiated cells decreased in the experimental group as compared with the control group,but there was no significant difference between the two groups.Conclusions:Spermatogonial stem cells can be efficiently proliferated by culturing the stem cells in soft agar-coated dishes.The new protocol used in this study can be a valuable method for future studies.
基金Supported by Scientific Research Foundation for Doctors of Northeast Agricultural University (2012RCB27)Postdoctoral Fund of Heilongjiang Provincial Academy of Agricultural Sciences (LRB04-185)
文摘The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous epithlium cells were isolated and purified, the cells were cryopreserved after identification, and the effects of different purification and cyopreservation methods on bovine testicular cells were studied. The results showed that there were spermatogonial stem cells and sertoli cells in the neonatal bovine seminiferous tubules, differential adherent selection methods could effectively separate these two cell types. Spermatogonial stem cells were positive after AKP, C-kit, and OCT-4 identification; sertoli cells were positive after oil red O and vimentin identification. Frozen stock solution supplemented with 10% DMSO had the best effect in spermatogonial stem cell cryopreservation, while frozen stock solution supplemented with 10% of ethylene glycol and 0.1 mmol·L-1 trehalose had the best effect in sertoli cells cryopreservation.
文摘With the decline in male fertility in recent years,strategies for male fertility preservation have received increasing attention.In this study,by reviewing current treatments and recent publications,we describe research progress in and the future directions of stem cell-based therapies for male fertility preservation,focusing on the use of spermatogonial stem cells(SSCs),SSC niches,SSC-based testicular organoids,other stem cell types such as mesenchymal stem cells,and stem cell-derived extracellular vesicles.In conclusion,a more comprehensive understanding of the germ cell microenvironment,stem cell-derived extracellular vesicles,and testicular organoids will play an important role in achieving male fertility preservation.
基金This study was supported by the grants from the Scientific Research Planning Project of Hunan Provincial Health and Family Planning Commission(B2017143)the Natural Science Foundation of Changsha(kq2202491)the Research Grant of CITIC-Xiangya(YNXM 202109 and YNXM 202115。
文摘Continuous self-renewal and differentiation of spermatogonial stem cells(SSCs)is vital for maintenance of adult spermatogenesis.Although several spermatogonial stem cell regulators have been extensively investigated in rodents,regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established.We analyzed single-cell sequencing data from the human testis and found that forkhead box P4(FOXP4)expression gradually increased with development of SSCs.Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential.Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis.FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis.These findings imply that FOXP4 is involved in human SSC proliferation,which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.
基金Supported by the National Natural Science Foundation of China(Grant No.30430030)Specialized Research Fund for the Doctoral Program of Higher Education(Grant No.20061117004)
文摘The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.
基金supported by the National Natural Science Foundation of China(32025037 and 31721005)the National Key R&D Project of China(2018YFA0801000 and 2018YFD0901205)+1 种基金Chinese Academy of Sciences(XDA24010108)State Key Laboratory of Freshwater Ecology and Biotechnology(2019FBZ05)。
文摘The surrogate reproduction technique,such as inter-specific spermatogonial stem cells(SSCs)transplantation(SSCT),provides a powerful tool for production of gametes derived from endangered species or those with desirable traits.However,generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily,by SSCT,has not succeeded.Here,using two small cyprinid fishes from different subfamilies,Chinese rare minnow(gobiocypris rarus,for brief:Gr)and zebrafish(danio rerio),we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure.The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis,resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells.Interestingly,the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior.When pou5f3 and chd knockout Gr SSCs were transplanted,Gr-derived genome-edited sperm was successfully produced in zebrafish.This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT,and surrogate production of genome-edited gametes from another species as well.This method is feasible to be applied to future breeding of commercial fish and livestock.
基金This study was supported by Genome Tagging Project and grants from the Chinese Academy of Sciences,the National Key Research and Development Program of China,Shanghai Municipal Commission for Science and Technology,and the National Natural Science Foundation of China(XDB19010204,2019YFA0109900,OYZDJ-SSW-SMC023Facility-based Open Research Program,19411951800,17JC1420102,31821004,32030029,31730062,31530048,and 81672117)The research is partly supported by the Fountain-Valley Life Sciences Fund of University of Chinese Academy of Sciences Education Foundation。
文摘Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells(SSCs).However,such therapy for infertility has not been experimentally investigated yet.In this study,a mouse model with an X-linked testis-expressed 11(TEX11)mutation(Tex11PM/Y)identified in azoospermia patients exhibited meiotic arrest due to aberrant chromosome segregation.Tex11PM/Y SSCs could be isolated and expanded in vitro normally,and the mutation was corrected by clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated endonuclease 9(Cas9),leading to the generation of repaired SSC lines.Whole-genome sequencing demonstrated that the mutation rate in repaired SSCs is comparable with that of autonomous mutation in untreated Tex11PM/Y SSCs,and no predicted off-target sites are modified.Repaired SSCs could restore spermatogenesis in infertile males and give rise to fertile offspring at a high efficiency.In summary,our study establishes a paradigm for the treatment of male azoospermia by combining in vitro expansion of SSCs and gene therapy.
基金This work was supported by the National Natural Science Foundation of China(No.31672425).
文摘Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells(SSCs).SSCs,the only male reproductive stem cells that transmit genetic material to subsequent generations,possess an inherent self-renewal ability,which allows the maintenance of a steady stem cell pool.SSCs eventually differentiate to produce sperm.However,in an in vitro culture system,SSCs can be induced to differentiate into various types of germ cells.Rodent SSCs are well defined,and a culture system has been successfully established for them.In contrast,available information on the biomolecular markers and a culture system for livestock SSCs is limited.This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process,the biology and niche of SSCs,the isolation and culture systems of SSCs,and the biomolecular markers and identification of SSCs.This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals,enhancement of human male fertility,reproductive medicine,and protection of endangered species.
基金supported by the National Natural Science Foundation of China(No.81774314)the Foundation of Clinical Science and Technology of Wuxi(No.Q202059)+1 种基金the Top Talent Support Program for Young and Middle-Aged People of Wuxi Health Committee(Nos.BJ2020105 and HB2020108)the Clinical Medical Science and Technology Development Foundation of Jiangsu University(No.JYL20180068)。
文摘Male infertility has evolved from a common reproductive system disease to a major social issue.Youjing granule(YG)is a Chinese medicinal material used as a therapy method for tonifying the kidneys and removing dampness due to its pathogenic characteristics.YG has been shown to regulate sperm quality in clinical trials,but the underlying mechanism is not fully understood.The present study was aimed to explore the protective effects and mechanism of action of YG on male reproductive system damage caused by methyl methane sulfonate(MMS).We first established an infertility model of rats through oral administration of MMS and then treated with YG.To determine the effect of YG,spermatogenesis,microvascular density,and secretory function of Leydig cells and Sertoli cells in rats were assessed.Spermatogonial stem cells(SSCs)were co-cultured with mouse embryo fibroblast(MEF)cells as an in vitro cell model before exposure to serum containing YG.Furthermore,the proliferation and apoptosis of SSCs were measured.Results indicated that YG increased the expression of self-renewal and proliferation-related molecules such as glial cell line derived neurotrophic factor(GDNF)and fibroblast growth factor-2(FGF2),and improved the quality of sperm and the proliferation of SSCs.In conclusion,YG may protect spermatogenetic function of rats through regulating the proliferation and self-renewal of SSCs.
基金the National Natural Science Foundation of China(31671550,31872845)National Key R&D Project(2016YFC1000606)+3 种基金High Level Talent Gathering Project in Hunan Province(2018RS3066)Major Scientific and Technological Projects for Collaborative Prevention and Control of Birth Defect in Hunan Province(2019SK1012)Key Grant of Research and Development in Hunan Province(2020DK2002)The Open Fund of the NHC Key Laboratory of Male Reproduction and Genetics(KF201802).
文摘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(GFRAl)-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 GFRAl-enriched spermatogonia between 0 day and 14 days of culture,and xenotransplantation was performed to evaluate the function of GFRAl-enriched spermatogonia.SSCs shared some phenotypes with rodent and human SSCs.GFRAl-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 GFRAl-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 GFRAl-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-Kitw/w(W)mutant mice.Collectively,GFRAl-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.
文摘Mammalian spermatogenesis is a complicated and precisely controlled process that requires spermatogonial stem cells(SSCs).SSCs maintain the stem cell pool,balance self-renewal–commitment with differentiation,and produce millions of sperm daily.Self-renewal and differentiation are controlled by intrinsic factors within SSCs and extrinsic factors from the"niche."In this review,we discuss the biology of SSCs and the factors regulating their self-renewal and differentiation.
基金supported by grants from the National Key R&D Program of China(2020YFA0113300 to M.W.,2018YFA0107601 to F.T.,2019YFA0801802 to M.W.,2022YFA0806300 to X.-Y.Z.)the National Natural Science Foundation of China(82071711 to X.-Y.Z.,32170866 to M.W.,U22A20278 to X.-Y.Z.)+2 种基金Key Research&Development Program of Bioland Laboratory(Guangzhou Regenerative Medicine and Health Guangdong Laboratory)(2018GZR110104002 to X.-Y.Z.)Guangdong Basic and Applied Basic Research Foundation(2021A1515010802 to M.W.)National Demonstration Center for Experimental Education of Basic Medical Sciences(Southerm Medical University).
文摘Although somatic cells can be reprogrammed to pluripotent stem cells(PsCs)with pure chemicals,authentic pluripotency of chemically induced pluripotent stem celis(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 effciency 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 ceil 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 ight on the unique regulatory network underpinning SsC reprogramming,providing insights to understand generic mechanisms for cell-fate decision and epigenetic-relateddisorders in regenerative medicine.
基金Financial support was received from the National Key Research and Development Program of China(No.2018YFC1003500 to F.S)the National Natural Science Foundation of China(Nos.81430027 and 81671510 to F.S).
文摘Objective:Testosterone plays an essential role in maintaining spermatogenesis and male fertility,and the primary known source of testosterone is testicular Leydig cells,which are regulated by luteinizing hormone(LH).However,whether any other ways of testosterone secretion exist still remains unknown.Methods:Transmission electron microscopy was used to detect testicular extracellular vesicles(EVs),which were isolated by an ultracentrifuge process.Separately,the concentrations of follicle-stimulating hormone(FSH),LH,and testosterone were measured by enzyme-linked immunosorbent assay.Results:Some EVs were found by tail vein injection to be present in mouse testes that elevate the circulating testosterone and LH levels in the blood,but do not affect FSH.Separately,they also promote testosterone production in the TM3 Leydig cell line in vitro.To determine whether the EVs from spermatogonia were involved in the secretion of testosterone,we used spermatogonial stem/progenitor cell line C18-4 cells and revealed that C18-4 cells promote production of testosterone in the TM3 Leydig cell line using the EVs.Conclusions:EVs in mouse testes likely originate from spermatogonia and involved in the regulation of the serum testosterone.Our results provide a new mechanism for the regulation of testosterone production.
