Mouse spermatogenesis entails the maintenance and self-renewal of spermatogonial stem cells(SSCs),which require a complex web-like signaling network transduced by various cytokines.Although brain-derived neurotrophic ...Mouse spermatogenesis entails the maintenance and self-renewal of spermatogonial stem cells(SSCs),which require a complex web-like signaling network transduced by various cytokines.Although brain-derived neurotrophic factor(BDNF)is expressed in Sertoli cells in the testis,and its receptor tropomyosin receptor kinase B(TrkB)is expressed in the spermatogonial population containing SSCs,potential functions of BDNF for spermatogenesis have not been uncovered.Here,we generate BDNF conditional knockout mice and find that BDNF is dispensable for in vivo spermatogenesis and fertility.However,in vitro,we reveal that BDNF-deficient germline stem cells(GSCs)exhibit growth potential not only in the absence of glial cell line-derived neurotrophic factor(GDNF),a master regulator for GSC proliferation,but also in the absence of other factors,including epidermal growth factor(EGF),basic fibroblast growth factor(bFGF),and insulin.GSCs grown without these factors are prone to differentiation,yet they maintain expression of promyelocytic leukemia zinc finger(Plzf),an undifferentiated spermatogonial marker.Inhibition of phosphoinositide 3-kinase(PI3K),mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase(ERK),and Src pathways all interfere with the growth of BDNF-deficient GSCs.Thus,our findings suggest a role for BDNF in maintaining the undifferentiated state of spermatogonia,particularly in situations where there is a shortage of growth factors.展开更多
Type-A spermatogonia first appear at between 3-7 d postnatally in mice and are the only immortalized diploid cells that reproduce in adulthood in these animals. In our current study, we explored the feasibility of pro...Type-A spermatogonia first appear at between 3-7 d postnatally in mice and are the only immortalized diploid cells that reproduce in adulthood in these animals. In our current study, we explored the feasibility of producing stable transgenic mice using these cells. Enhanced pEGFP-N1 plasmids were suspended in ExGen500 transfection reagent and injected at different angles into the testes of 7-d-old male ICR mice. The resulting type-A spermatogonia-mediated gene transfer （TASMGT） mice were then mated with normal females at different stages of sexual maturity （6, 12, and 24 wk）. The integration and expression of the introduced EGFP gene was evaluated in the F1 transgenic offspring by PCR and Southern blotting analysis. The foreign gene integration rates for a low-dose group （15 μL gene suspension injected into each testis） and a high-dose group （30 μL suspensions injected） at the three stages of female sexual maturity tested were 11.76% （2/17）, 14.29% （3/21）, and 11.11% （2/18）, and 5% （1/20）, 5.56% （1/18）, and 0 （0/17）, respectively. The average integration rates for these two dose groups were 12.5% （7/56） and 3.64% （2/55）, respectively, which was a significant difference （P0.05）. Semi-quantitative RT-PCR analysis further showed that the introduced GFP gene was expressed in 3/9 integration mice. In addition, GFP expression was observed in the sperm cells from the TASMGT mice, and also in the embryos and F2 pups from the F1 generation transgenic mice. Hence, although the foreign gene integration rate for TASMGT is not high and the transgenic offspring show as yet unexplained defects, our results indicate that this method is a potentially feasible and reproducible new approach to creating transgenic mice.展开更多
Background:Spermatogenesis is an intricate developmental process during which undifferentiated spermatogonia,containing spermatogonial stem cells(SSCs),undergo self-renewal and differentiation to generate eventually m...Background:Spermatogenesis is an intricate developmental process during which undifferentiated spermatogonia,containing spermatogonial stem cells(SSCs),undergo self-renewal and differentiation to generate eventually mature spermatozoa.Spermatogenesis occurs in seminiferous tubules within the testis,and the seminiferous tubules harbor Sertoli and germ cells.Sertoli cells are an essential somatic cell type within the microenvironment that support and steer male germ cell development,whereas spermatogonia are the primitive male germ cells at the onset of spermatogenesis.While the developmental progression of Sertoli cells and spermatogonia has been well established in mice,much less is known in other mammalian species including pigs.Results:To acquire knowledge of Sertoli cell and spermatogonial development in pigs,here we collected as many as nine ages of Duroc porcine testes from the neonate to sexual maturity,i.e.,testes from 7-,30-,50-,70-,90-,110-,130-,150-and 210-day-old boars,and performed histological and immunohistochemical analyses on testis sections.We first examined the development of spermatogenic cells and seminiferous tubules in porcine testes.Then,by immunofluorescence staining for marker proteins(AMH,SOX9,DBA,UCHL1,VASA,KIT,Ki67 and/or PCNA),we delved into the proliferative activity and development of Sertoli cells and of spermatogonial subtypes(pro-,undifferentiated and differentiating spermatogonia).Besides,by immunostaining forβ-catenin and ZO-1,we studied the establishment of the blood-testis barrier in porcine testes.Conclusions:In this longitudinal study,we have systematically investigated the elaborate Sertoli cell and spermatogonial developmental patterns in pigs from the neonate to sexual maturity that have so far remained largely unknown.The findings not only extend the knowledge about spermatogenesis and testicular development in pigs,but also lay the theoretical groundwork for porcine breeding and rearing.展开更多
Background:Spermatogenesis is a cellular differentiation process that includes three major events:mitosis of spermatogonia,meiosis of spermatocytes and spermiogenesis.Steady-state spermatogenesis relies on functions o...Background:Spermatogenesis is a cellular differentiation process that includes three major events:mitosis of spermatogonia,meiosis of spermatocytes and spermiogenesis.Steady-state spermatogenesis relies on functions of spermatogonial stem cells(SSCs).Establishing and maintaining a foundational SSC pool is essential for continued spermatogenesis in mammals.Currently,our knowledge about SSC and spermatogenesis is severely limited in domestic animals.Results:In the present study,we examined transcriptomes of testes from domestic yaks at four different stages(3,5,8 and 24 months of age)and attempted to identify genes that are associated with key developmental events of spermatogenesis.Histological analyses showed that the most advanced germ cells within seminiferous tubules of testes from 3,5,8 and 24 months old yaks were gonocytes,spermatogonia,spermatocytes and elongated spermatids,respectively.RNA-sequencing(RNA-seq)analyses revealed that 11904,4381 and 2459 genes were differentially expressed during the gonocyte to spermatogonia transition,the mitosis to meiosis transition and the meiosis to post-meiosis transition.Further analyses identified a list of candidate genes than may regulate these important cellular processes.CXCR4,a previously identified SSC niche factor in mouse,was one of the up-regulated genes in the 5 months old yak testis.Results of immunohistochemical staining confirmed that CXCR4 was exclusively expressed in gonocytes and a subpopulation of spermatogonia in the yak testis.Conclusions:Together,these findings demonstrated histological changes of postnatal testis development in the domestic yak.During development of spermatogonial lineage,meiotic and haploid germ cells are supported by dynamic transcriptional regulation of gene expression.Our transcriptomic analyses provided a list of candidate genes that potentially play crucial roles in directing the establishment of SSC and spermatogenesis in yak.展开更多
Col la I （one of the subunit of collagen type I） is a collagen, which belongs to a family of extracellular matrix （ECM） proteins that play an important role in cellular proliferation and differentiation. However, ...Col la I （one of the subunit of collagen type I） is a collagen, which belongs to a family of extracellular matrix （ECM） proteins that play an important role in cellular proliferation and differentiation. However, the role of Col lal in spermatogenesis, especially in the control of proliferation and differentiation of spermatogonial stem cells （SSCs）, remains unknown. In this study, we explored effects of downregulation of Collal on differentiation and proliferation of mouse spermatogonia. Loss-of-function study revealed that Oct4 and Plzf, markers of SSC self-renewal, were significantly decreased, whereas the expression of c-kit and haprin, hallmarks of SSC differentiation, was enhanced after Col la I knockdown. Cell cycle analyses indicated that two-thirds of spermatogonia were arrested in S phase after Collal knockdown. In vivo experiments, DNA injection and electroporation of the testes showed that spermatogonia self-renewal ability was impaired remarkably with the loss-of-function of Collal. Our data suggest that silencing of Collal can suppress spermatogonia self-renewal and promote spermatogonia differentiation.展开更多
Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility.However,the exact mechanisms underlying the behavior of spermatogonia,including spermatogonial stem cell(SSC)self-renewal and s...Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility.However,the exact mechanisms underlying the behavior of spermatogonia,including spermatogonial stem cell(SSC)self-renewal and spermatogonial proliferation and differentiation,are not fully understood.Recent studies demonstrated that the mTOR complex 1(mTORC1)signaling pathway plays a crucial role in spermatogonial development,but whether MTOR itself was also involved in any specific process of spermatogonial development remained undetermined.In this study,we specifically deleted Mtor in male germ cells of mice using Stra8-Cre and assessed its effect on the function of spermatogonia.The Mtor knockout(KO)mice exhibited an age-dependent perturbation of testicular development and progressively lost germ cells and fertility with age.These age-related phenotypes were likely caused by a delayed initiation of Mtor deletion driven by Stra8-Cre.Further examination revealed a reduction of differentiating spermatogonia in Mtor KO mice,suggesting that spermatogonial differentiation was inhibited.Spermatogonial proliferation was also impaired in Mtor KO mice,leading to a diminished spermatogonial pool and total germ cell population.Our results show that MTOR plays a pivotal role in male fertility and is required for spermatogonial proliferation and differentiation.展开更多
The effect of ginsenosides on proliferation of type A spermatogonia was investigated in 7-day-old mice. Spermatogonia were characterized by c-kit expression and cell proliferation was assessed by immunocytochemical de...The effect of ginsenosides on proliferation of type A spermatogonia was investigated in 7-day-old mice. Spermatogonia were characterized by c-kit expression and cell proliferation was assessed by immunocytochemical demonstration of proliferating cell nuclear antigen (PCNA). After 72-h culture, Sertoli cells formed a confluent monolayer to which numerous spermatogonial colonies attached. Spermatogonia were positive for c-kit staining and showed high proliferating activity by PCNA expression. Ginsenosides (1.0～10 μg/ml) significantly stimulated proliferation of spermatogonia. Activation of protein kinase C (PKC) elicited proliferation of spermatogonia at 10-8 to 10-7 mol/L and the PKC inhibitor H7 inhibited this effect. Likewise, ginsenosides-stimulated spermatogonial proliferation was suppressed by combined treatment of H7. These results indicate that the proliferating effect of ginsenosides on mouse type A spermatogonia might be mediated by a mechanism involving the PKC signal transduction pathway.展开更多
Aim： To investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice. Methods： Mice were surgically rendered cryptorchid, then treated with different dose...Aim： To investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice. Methods： Mice were surgically rendered cryptorchid, then treated with different doses of 17β- estradiol （E2） s.c. once a day. Mice were killed at sexual maturity （45 days of age）, and histological analysis and inununofluorescence were performed. Serum follicle stimulating hormone （FSH）, estradiol, testosterone and luteinizing hormone （LH） were measured. Results： Low doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia. Conclusion： E2 has a dose-related mitogenic effect on spermatogonia.展开更多
While hallmarks of rodent spermatogonia stem cell biomarkers' heterogeneity have recently been identified, their stage and subset distributions remain unclear. Furthermore, it is currently difficult to accurately ...While hallmarks of rodent spermatogonia stem cell biomarkers' heterogeneity have recently been identified, their stage and subset distributions remain unclear. Furthermore, it is currently difficult to accurately identify subset-specific SSC marker distributions due to the poor nuclear morphological characteristics associated with fixation in 4% paraformaldehyde. In the present study, testicular cross-sections and whole-mount samples were Bouin fixed to optimize nuclear resolution and visualized by immunohistochemistry (IHC) and immunofluorescence (IF). The results identified an expression pattern of PLZFhighc-KITpos in A1 spermatogonia, while A2–A4-differentiating spermatogonia were PLZFlowc-KITpos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric Apr and false clones were distinguished based on the presence or absence of TEX14, a molecular maker of intercellular bridges, despite having identical nuclear morphology and intercellular distances that were <25 μm. In conclusion, this optimized Bouin fixation procedure facilitates the accurate identification of spermatogonium subsets based on their molecular profiles and is capable of distinguishing asymmetric and false clones. Therefore, the findings presented herein will facilitate further morphological and functional analysis studies and provide further insight into spermatogonium subtypes.展开更多
In vitro production of functional gametes can revolutionize reproduction by reducing generation intervals and accelerating genetic breeding in aquaculture,especially in fish with relatively long generations.Neverthele...In vitro production of functional gametes can revolutionize reproduction by reducing generation intervals and accelerating genetic breeding in aquaculture,especially in fish with relatively long generations.Nevertheless,functional sperm production from in vitro-cultured spermatogonia remains a challenge in most aquaculture fish.In this study,we isolated and characterized premeiotic spermatogonia from marine four-eyed sleepers(Bostrychus sinensis),which are prone to ovotesticular or sterile testicular development,and induced the differentiation of the spermatogonia into flagellated sperm in a three-dimensional(3D)culture system.Artificial insemination indicated that the in vitro-derived sperm were capable of fertilizing mature oocytes to develop into normal larvae.Furthermore,melatonin significantly promoted spermatogonia proliferation and differentiation through the ERK1/2 signaling pathway,and thus increased the efficiency in functional sperm production.The 3D culture system and resulting functional sperm hold great promise for improving the genetic breeding of aquaculture fish.展开更多
Objective The expression patterns of ribosomal large subunit protein 23 a(RPL23 a)in mouse testes and GC-1 cells were analyzed to investigate the potential relationship between RPL23 a expression and spermatogonia apo...Objective The expression patterns of ribosomal large subunit protein 23 a(RPL23 a)in mouse testes and GC-1 cells were analyzed to investigate the potential relationship between RPL23 a expression and spermatogonia apoptosis upon exposure to X-ray.Methods Male mice and GC-1 cells were irradiated with X-ray,terminal dUTP nick end-labelling(TUNEL)was performed to detect apoptotic spermatogonia in vivo.Apoptotic rate and cell cycle phase of GC-1 cells were analyzed with flow cytometry.Protein interactions were detected by Immunoprecipitation and protein localization as studied by immunofluorescence.Immunoblotting and real-time PCR were applied to analyze to protein and gene expression.Results Ionizing radiation(IR)increased spermatogonia apoptosis,the expression of RPL11,MDM2 and p53,and decreased RPL23 a expression in mice spermatogonia in vivo and in vitro.RPL23 a knockdown weakened the interaction between RPL23 a and RPL11,leading to p53 accumulation.Moreover,knockdown and IR decreased RPL23 a that induces spermatogonia apoptosis via RPL23 a-RPL11-MDM2-p53 pathway in GC-1 cells.Conclusion These results suggested that IR reduced RPL23 a expression,leading to weakened the RPL23 a-RPL11 interactions,which may have activated p53,resulting in spermatogonia apoptosis.These results provide insights into environmental and clinical risks of radiotherapy following exposure to IR in male fertility.The graphical abstract was available in the web of www.besjournal.com.展开更多
The present study was aimed at finding an effective method to isolate and purify the subtype of type A spermatogonial stem cells （SSCs） in juvenile rats. Testes from 9-days-old rats were used to isolate germ cells b...The present study was aimed at finding an effective method to isolate and purify the subtype of type A spermatogonial stem cells （SSCs） in juvenile rats. Testes from 9-days-old rats were used to isolate germ cells by using two-step enzymatic digestion. The expression of c-kit in the testes of the rats was immunohistochemically detected. After isolation, cell suspension was enriched further by discontinuous density gradient centrifugation. Then type A1-A4 spermatogonia was isolated from the purified spermatogonia with c-kit as the marker by using fluorescence-activated cell sorting （FACS）. Electron microscopy was used to observe their ultrastructure. Finally, highly purified and viable subtype of SSCs was obtained. Cells separation with discontinuous density gradient centrifugation significantly increased the concentration of c-kit positive cells [（18.65±1.69）% after the centrifugation versus （3.16±0.84）% before the centrifugation, P〈0.01]. Furthermore, the recovery and viability were also high [（65.9±1.24）% and （85.6±1.14）%]. It is concluded that FACS with c-kit as the marker in combination with discontinuous density gradient centrifugation can well enrich type A1-A4 spermatogonia from the testes of 9-days-old rats.展开更多
The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis.The single-cell RNA sequencing(scRNA-seq)analysis of the testis was performed to identify genes upr...The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis.The single-cell RNA sequencing(scRNA-seq)analysis of the testis was performed to identify genes upregulated in spermatogonia.Using scRNAseq analysis,we identified the spermatogonia upregulated gene origin recognition complex subunit 6(Orc6),which is involved in DNA replication and cell cycle regulation;its protein expression in the human and mouse testis was detected by western blot and immunofluorescence.To explore the potential function of Orc6 in spermatogonia,the C18-4 cell line was transfected with control or Orc6 siRNA.Subsequently,5-ethynyl-2-deoxyuridine(EdU)and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assays,flow cytometry,and western blot were used to evaluate its effects on proliferation and apoptosis.It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells.Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated(Wnt)/β-catenin signaling.Western blot revealed that the expression ofβ-catenin protein and its phosphorylation(Ser675)were significantly decreased when silencing the expression of ORC6.Our findings indicated that Orc6 was upregulated in spermatogonia,whereby it regulated proliferation and apoptosis by activating Wnt/β-catenin signaling.展开更多
The black rockfish (Sebastes schlegelii) is a marine species that is economically important in aquaculture, and the efficiency of its spermatogenesis is vital for its success in the aquaculture industry. Spermatogonia...The black rockfish (Sebastes schlegelii) is a marine species that is economically important in aquaculture, and the efficiency of its spermatogenesis is vital for its success in the aquaculture industry. Spermatogonia serve as the foundation of spermatogenesis in fish, possessing the ability for continuous self-renewal and progressive differentiation into mature spermatozoa. Moreover, Sertoli cells are crucial in modulating the proliferation and differentiation of spermatogonia. This study focused on the regulation of glycolysis by PLIN2a in Sertoli cells of the black rockfish and examined how the inhibition of glycolysis in these cells impacted the proliferation and differentiation processes of spermatogonia. We found that effective regulation of glycolysis was crucial for the metabolic activity and functional maintenance of Sertoli cells in black rockfish. Overexpression of plin2a in vitro enhanced glycolysis in Sertoli cells, whereas inhibition of glycolysis impaired their normal metabolic activity. In vivo inhibition of glycolysis in black rockfish testes lead to apoptosis of Sertoli cells and significantly suppressed the proliferation and differentiation of spermatogonia. These results underscore the essential role of glycolysis in the development and metabolic activity of Sertoli cells and highlight the critical regulatory role of glycolysis in determining the fate of spermatogonia. This study emphasizes the importance of regulating energy metabolism pathways, particularly glycolysis, in Sertoli cells to indirectly influence the development of spermatogonia, offering significant insights into the reproductive mechanisms of black rockfish and other teleost species.展开更多
Black rockfish(Sebastes schlegelii)is one of the most important marine economic viviparous fishes.Recently,germplasm degradation and genetic diversity reduction have occurred due to overfishing and long-term artificia...Black rockfish(Sebastes schlegelii)is one of the most important marine economic viviparous fishes.Recently,germplasm degradation and genetic diversity reduction have occurred due to overfishing and long-term artificial breeding.Germ cell transplantation combined with cryopreservation may be an alternative way to protect genetic resources.However,in viviparous fish that undertake fertilization and embryo development in vivo,transplantation is more difficult than in oviparous fish,including selection of transplantation stage,isolation of germ stem cells,and preparation of sterile recipients.This seriously restricts the development of viviparous transplantation.Therefore,in this study,we aimed to explore a transplantation method suitable for these species.Donor cells were isolated from cryopreserved whole testes of 300–400g male Sebastes schlegelii in May,labeled by PKH26,and intra-peritoneally transplanted into allogeneic larvae at 5–10 days post-birth.Subsequently,the development of donor-derived cells in recipients were continuously detected by fluorescence labeling,histology,microsatellite markers,and fecundity tests.The results showed that donors were rich in spermatogonia(75%)and recipients maintained a high survival rate after transplantation,with a rate of>20%at sexual maturity.Further,donor-derived cells successfully migrated(100%),colonized,and incorporated into the developing recipient gonad(93.33%).Finally,transplanted recipients could normally develop and differentiate into male and female individuals,with donor-derived gametes found in 65.38%of mature recipients.In the present study,we first establish a simple and suitable transplantation method for Sebastes schlegelii using immature males and specific larvae,which will serve as a promising tool in the protection of germplasm resources for this transplantation-restricted marine viviparous species.展开更多
Dear Editor,I would like to congratulate Mamsen et al.i on their extensive and scientifically valuable work.I analyzed their raw data presented in Table 1 of the original article from a different perspective and disco...Dear Editor,I would like to congratulate Mamsen et al.i on their extensive and scientifically valuable work.I analyzed their raw data presented in Table 1 of the original article from a different perspective and discovered an effect not mentioned in the article.My analysis showed that luteinizing hormone(LH)levels are significantly lower in patients at high infertility risk(HIR),whose testes lack A dark(Ad)spermatogonia and display an abnormal ratio of germ cells per crosssectional tubule(G/T).展开更多
基金partly supported by Grant-in-Aid for Scientific Research on Innovative Areas funding from the Ministry of Education,Culture,Sports,Science,and Technology(MEXT)KAKENHI(No.22H04677 to KO)a grant from the Japan Society for the Promotion of Science KAKENHI(No.20K09543 to ST,No.20K07228 to KK,and No.19KK0183 to KO)the Takeda Science Foundation(2021 to KK).
文摘Mouse spermatogenesis entails the maintenance and self-renewal of spermatogonial stem cells(SSCs),which require a complex web-like signaling network transduced by various cytokines.Although brain-derived neurotrophic factor(BDNF)is expressed in Sertoli cells in the testis,and its receptor tropomyosin receptor kinase B(TrkB)is expressed in the spermatogonial population containing SSCs,potential functions of BDNF for spermatogenesis have not been uncovered.Here,we generate BDNF conditional knockout mice and find that BDNF is dispensable for in vivo spermatogenesis and fertility.However,in vitro,we reveal that BDNF-deficient germline stem cells(GSCs)exhibit growth potential not only in the absence of glial cell line-derived neurotrophic factor(GDNF),a master regulator for GSC proliferation,but also in the absence of other factors,including epidermal growth factor(EGF),basic fibroblast growth factor(bFGF),and insulin.GSCs grown without these factors are prone to differentiation,yet they maintain expression of promyelocytic leukemia zinc finger(Plzf),an undifferentiated spermatogonial marker.Inhibition of phosphoinositide 3-kinase(PI3K),mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase(ERK),and Src pathways all interfere with the growth of BDNF-deficient GSCs.Thus,our findings suggest a role for BDNF in maintaining the undifferentiated state of spermatogonia,particularly in situations where there is a shortage of growth factors.
基金supported by the National Transgenic Breeding Project (2008ZX08010-004)the National Natural Science Foundation of China (30830080)+1 种基金the National 973 Program of China (G2006CB102105,2009CB941604)the National 863 Program of China (20060110Z1039, 2008AA10Z143)
文摘Type-A spermatogonia first appear at between 3-7 d postnatally in mice and are the only immortalized diploid cells that reproduce in adulthood in these animals. In our current study, we explored the feasibility of producing stable transgenic mice using these cells. Enhanced pEGFP-N1 plasmids were suspended in ExGen500 transfection reagent and injected at different angles into the testes of 7-d-old male ICR mice. The resulting type-A spermatogonia-mediated gene transfer （TASMGT） mice were then mated with normal females at different stages of sexual maturity （6, 12, and 24 wk）. The integration and expression of the introduced EGFP gene was evaluated in the F1 transgenic offspring by PCR and Southern blotting analysis. The foreign gene integration rates for a low-dose group （15 μL gene suspension injected into each testis） and a high-dose group （30 μL suspensions injected） at the three stages of female sexual maturity tested were 11.76% （2/17）, 14.29% （3/21）, and 11.11% （2/18）, and 5% （1/20）, 5.56% （1/18）, and 0 （0/17）, respectively. The average integration rates for these two dose groups were 12.5% （7/56） and 3.64% （2/55）, respectively, which was a significant difference （P0.05）. Semi-quantitative RT-PCR analysis further showed that the introduced GFP gene was expressed in 3/9 integration mice. In addition, GFP expression was observed in the sperm cells from the TASMGT mice, and also in the embryos and F2 pups from the F1 generation transgenic mice. Hence, although the foreign gene integration rate for TASMGT is not high and the transgenic offspring show as yet unexplained defects, our results indicate that this method is a potentially feasible and reproducible new approach to creating transgenic mice.
基金supported by the National Natural Science Foundation of China(Grant No.32002178 and 31772605)the Undergraduate Training Program for Innovation and Entrepreneurship(X202110712185).
文摘Background:Spermatogenesis is an intricate developmental process during which undifferentiated spermatogonia,containing spermatogonial stem cells(SSCs),undergo self-renewal and differentiation to generate eventually mature spermatozoa.Spermatogenesis occurs in seminiferous tubules within the testis,and the seminiferous tubules harbor Sertoli and germ cells.Sertoli cells are an essential somatic cell type within the microenvironment that support and steer male germ cell development,whereas spermatogonia are the primitive male germ cells at the onset of spermatogenesis.While the developmental progression of Sertoli cells and spermatogonia has been well established in mice,much less is known in other mammalian species including pigs.Results:To acquire knowledge of Sertoli cell and spermatogonial development in pigs,here we collected as many as nine ages of Duroc porcine testes from the neonate to sexual maturity,i.e.,testes from 7-,30-,50-,70-,90-,110-,130-,150-and 210-day-old boars,and performed histological and immunohistochemical analyses on testis sections.We first examined the development of spermatogenic cells and seminiferous tubules in porcine testes.Then,by immunofluorescence staining for marker proteins(AMH,SOX9,DBA,UCHL1,VASA,KIT,Ki67 and/or PCNA),we delved into the proliferative activity and development of Sertoli cells and of spermatogonial subtypes(pro-,undifferentiated and differentiating spermatogonia).Besides,by immunostaining forβ-catenin and ZO-1,we studied the establishment of the blood-testis barrier in porcine testes.Conclusions:In this longitudinal study,we have systematically investigated the elaborate Sertoli cell and spermatogonial developmental patterns in pigs from the neonate to sexual maturity that have so far remained largely unknown.The findings not only extend the knowledge about spermatogenesis and testicular development in pigs,but also lay the theoretical groundwork for porcine breeding and rearing.
基金supported by National Key Research&Development Project grant(2016YFC0501805)Qinghai Department of Science and Technology grants(2017-NK-154 and 2016-ZJ-917Q)+2 种基金a STS grant from Chinese Academy of Sciences(KFJ-STS-QYZD-113)supported by the CAS“100 Talents” and Qinghai “1000 Talents” programsfunded by CAS “Light of West China Foundation”
文摘Background:Spermatogenesis is a cellular differentiation process that includes three major events:mitosis of spermatogonia,meiosis of spermatocytes and spermiogenesis.Steady-state spermatogenesis relies on functions of spermatogonial stem cells(SSCs).Establishing and maintaining a foundational SSC pool is essential for continued spermatogenesis in mammals.Currently,our knowledge about SSC and spermatogenesis is severely limited in domestic animals.Results:In the present study,we examined transcriptomes of testes from domestic yaks at four different stages(3,5,8 and 24 months of age)and attempted to identify genes that are associated with key developmental events of spermatogenesis.Histological analyses showed that the most advanced germ cells within seminiferous tubules of testes from 3,5,8 and 24 months old yaks were gonocytes,spermatogonia,spermatocytes and elongated spermatids,respectively.RNA-sequencing(RNA-seq)analyses revealed that 11904,4381 and 2459 genes were differentially expressed during the gonocyte to spermatogonia transition,the mitosis to meiosis transition and the meiosis to post-meiosis transition.Further analyses identified a list of candidate genes than may regulate these important cellular processes.CXCR4,a previously identified SSC niche factor in mouse,was one of the up-regulated genes in the 5 months old yak testis.Results of immunohistochemical staining confirmed that CXCR4 was exclusively expressed in gonocytes and a subpopulation of spermatogonia in the yak testis.Conclusions:Together,these findings demonstrated histological changes of postnatal testis development in the domestic yak.During development of spermatogonial lineage,meiotic and haploid germ cells are supported by dynamic transcriptional regulation of gene expression.Our transcriptomic analyses provided a list of candidate genes that potentially play crucial roles in directing the establishment of SSC and spermatogenesis in yak.
文摘Col la I （one of the subunit of collagen type I） is a collagen, which belongs to a family of extracellular matrix （ECM） proteins that play an important role in cellular proliferation and differentiation. However, the role of Col lal in spermatogenesis, especially in the control of proliferation and differentiation of spermatogonial stem cells （SSCs）, remains unknown. In this study, we explored effects of downregulation of Collal on differentiation and proliferation of mouse spermatogonia. Loss-of-function study revealed that Oct4 and Plzf, markers of SSC self-renewal, were significantly decreased, whereas the expression of c-kit and haprin, hallmarks of SSC differentiation, was enhanced after Col la I knockdown. Cell cycle analyses indicated that two-thirds of spermatogonia were arrested in S phase after Collal knockdown. In vivo experiments, DNA injection and electroporation of the testes showed that spermatogonia self-renewal ability was impaired remarkably with the loss-of-function of Collal. Our data suggest that silencing of Collal can suppress spermatogonia self-renewal and promote spermatogonia differentiation.
基金This work was supported by the Ministry of Science and Technology of the People’s Republic of China(NO.2014CB943103)the National Natural Science Foundation of China(No.31671203 and NO.31471104)the Science and Technology Commission of Shanghai Municipality(NO.17JC1420100).
文摘Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility.However,the exact mechanisms underlying the behavior of spermatogonia,including spermatogonial stem cell(SSC)self-renewal and spermatogonial proliferation and differentiation,are not fully understood.Recent studies demonstrated that the mTOR complex 1(mTORC1)signaling pathway plays a crucial role in spermatogonial development,but whether MTOR itself was also involved in any specific process of spermatogonial development remained undetermined.In this study,we specifically deleted Mtor in male germ cells of mice using Stra8-Cre and assessed its effect on the function of spermatogonia.The Mtor knockout(KO)mice exhibited an age-dependent perturbation of testicular development and progressively lost germ cells and fertility with age.These age-related phenotypes were likely caused by a delayed initiation of Mtor deletion driven by Stra8-Cre.Further examination revealed a reduction of differentiating spermatogonia in Mtor KO mice,suggesting that spermatogonial differentiation was inhibited.Spermatogonial proliferation was also impaired in Mtor KO mice,leading to a diminished spermatogonial pool and total germ cell population.Our results show that MTOR plays a pivotal role in male fertility and is required for spermatogonial proliferation and differentiation.
基金Project supported by the Program for New Century Excellent Talents in University of the Ministry of Education of China (No. NCET-05-0514)the Science and Technology Department of Zhejiang Province, China (No. 2008C22040)
文摘The effect of ginsenosides on proliferation of type A spermatogonia was investigated in 7-day-old mice. Spermatogonia were characterized by c-kit expression and cell proliferation was assessed by immunocytochemical demonstration of proliferating cell nuclear antigen (PCNA). After 72-h culture, Sertoli cells formed a confluent monolayer to which numerous spermatogonial colonies attached. Spermatogonia were positive for c-kit staining and showed high proliferating activity by PCNA expression. Ginsenosides (1.0～10 μg/ml) significantly stimulated proliferation of spermatogonia. Activation of protein kinase C (PKC) elicited proliferation of spermatogonia at 10-8 to 10-7 mol/L and the PKC inhibitor H7 inhibited this effect. Likewise, ginsenosides-stimulated spermatogonial proliferation was suppressed by combined treatment of H7. These results indicate that the proliferating effect of ginsenosides on mouse type A spermatogonia might be mediated by a mechanism involving the PKC signal transduction pathway.
基金Acknowledgment This work was supported by the National Natural Science Foundation of China （No. 30200195）. We thank Dr Hai-Bin Wang for taking photographs and Dr Su-Hui Wu （Henan Normal University, China） for statistical analysis. We thank the faculty of Huanghuai University for supporting Dr En-Zhong Li.
文摘Aim： To investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice. Methods： Mice were surgically rendered cryptorchid, then treated with different doses of 17β- estradiol （E2） s.c. once a day. Mice were killed at sexual maturity （45 days of age）, and histological analysis and inununofluorescence were performed. Serum follicle stimulating hormone （FSH）, estradiol, testosterone and luteinizing hormone （LH） were measured. Results： Low doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia. Conclusion： E2 has a dose-related mitogenic effect on spermatogonia.
基金the grants from the National Key Research and Development Program of China (Project No. 2016YFC1000200)the National Natural Science Foundation of China (Project No. 31472054).
文摘While hallmarks of rodent spermatogonia stem cell biomarkers' heterogeneity have recently been identified, their stage and subset distributions remain unclear. Furthermore, it is currently difficult to accurately identify subset-specific SSC marker distributions due to the poor nuclear morphological characteristics associated with fixation in 4% paraformaldehyde. In the present study, testicular cross-sections and whole-mount samples were Bouin fixed to optimize nuclear resolution and visualized by immunohistochemistry (IHC) and immunofluorescence (IF). The results identified an expression pattern of PLZFhighc-KITpos in A1 spermatogonia, while A2–A4-differentiating spermatogonia were PLZFlowc-KITpos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric Apr and false clones were distinguished based on the presence or absence of TEX14, a molecular maker of intercellular bridges, despite having identical nuclear morphology and intercellular distances that were <25 μm. In conclusion, this optimized Bouin fixation procedure facilitates the accurate identification of spermatogonium subsets based on their molecular profiles and is capable of distinguishing asymmetric and false clones. Therefore, the findings presented herein will facilitate further morphological and functional analysis studies and provide further insight into spermatogonium subtypes.
基金supported by the National Key R&D Program of China(2018YFD0901205)National Natural Science Foundation of China(31771587,31970535)Guangdong Basic and Applied Basic Research Foundation(2020A1515010358)。
文摘In vitro production of functional gametes can revolutionize reproduction by reducing generation intervals and accelerating genetic breeding in aquaculture,especially in fish with relatively long generations.Nevertheless,functional sperm production from in vitro-cultured spermatogonia remains a challenge in most aquaculture fish.In this study,we isolated and characterized premeiotic spermatogonia from marine four-eyed sleepers(Bostrychus sinensis),which are prone to ovotesticular or sterile testicular development,and induced the differentiation of the spermatogonia into flagellated sperm in a three-dimensional(3D)culture system.Artificial insemination indicated that the in vitro-derived sperm were capable of fertilizing mature oocytes to develop into normal larvae.Furthermore,melatonin significantly promoted spermatogonia proliferation and differentiation through the ERK1/2 signaling pathway,and thus increased the efficiency in functional sperm production.The 3D culture system and resulting functional sperm hold great promise for improving the genetic breeding of aquaculture fish.
基金the National Natural Science Foundation of China[31902339]the Foundation for Youth Doctor of Gansu Province[2021QB-026]+2 种基金the Youth Talent Program of“Fuxi”[Gaufx-03Y02]the Scientific Research Start-up Funds for Openly-Recruited Doctors of Gansu Agricultural University[2017RCZX-13]the Special Funds for Discipline Construction of Gansu Agricultural University[GAU-XKJS-2018-067]。
文摘Objective The expression patterns of ribosomal large subunit protein 23 a(RPL23 a)in mouse testes and GC-1 cells were analyzed to investigate the potential relationship between RPL23 a expression and spermatogonia apoptosis upon exposure to X-ray.Methods Male mice and GC-1 cells were irradiated with X-ray,terminal dUTP nick end-labelling(TUNEL)was performed to detect apoptotic spermatogonia in vivo.Apoptotic rate and cell cycle phase of GC-1 cells were analyzed with flow cytometry.Protein interactions were detected by Immunoprecipitation and protein localization as studied by immunofluorescence.Immunoblotting and real-time PCR were applied to analyze to protein and gene expression.Results Ionizing radiation(IR)increased spermatogonia apoptosis,the expression of RPL11,MDM2 and p53,and decreased RPL23 a expression in mice spermatogonia in vivo and in vitro.RPL23 a knockdown weakened the interaction between RPL23 a and RPL11,leading to p53 accumulation.Moreover,knockdown and IR decreased RPL23 a that induces spermatogonia apoptosis via RPL23 a-RPL11-MDM2-p53 pathway in GC-1 cells.Conclusion These results suggested that IR reduced RPL23 a expression,leading to weakened the RPL23 a-RPL11 interactions,which may have activated p53,resulting in spermatogonia apoptosis.These results provide insights into environmental and clinical risks of radiotherapy following exposure to IR in male fertility.The graphical abstract was available in the web of www.besjournal.com.
基金the National Natural Sciences Foundation of China (No. 30371424)
文摘The present study was aimed at finding an effective method to isolate and purify the subtype of type A spermatogonial stem cells （SSCs） in juvenile rats. Testes from 9-days-old rats were used to isolate germ cells by using two-step enzymatic digestion. The expression of c-kit in the testes of the rats was immunohistochemically detected. After isolation, cell suspension was enriched further by discontinuous density gradient centrifugation. Then type A1-A4 spermatogonia was isolated from the purified spermatogonia with c-kit as the marker by using fluorescence-activated cell sorting （FACS）. Electron microscopy was used to observe their ultrastructure. Finally, highly purified and viable subtype of SSCs was obtained. Cells separation with discontinuous density gradient centrifugation significantly increased the concentration of c-kit positive cells [（18.65±1.69）% after the centrifugation versus （3.16±0.84）% before the centrifugation, P〈0.01]. Furthermore, the recovery and viability were also high [（65.9±1.24）% and （85.6±1.14）%]. It is concluded that FACS with c-kit as the marker in combination with discontinuous density gradient centrifugation can well enrich type A1-A4 spermatogonia from the testes of 9-days-old rats.
基金This work was supported by the National Key Research and Development Program of China(No.2022YFC2702700)the National Natural Science Foundation of China(No.82171597)Clinical Research Plan of Shanghai Hospital Development Center(No.SHDC2020CR3077B).
文摘The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis.The single-cell RNA sequencing(scRNA-seq)analysis of the testis was performed to identify genes upregulated in spermatogonia.Using scRNAseq analysis,we identified the spermatogonia upregulated gene origin recognition complex subunit 6(Orc6),which is involved in DNA replication and cell cycle regulation;its protein expression in the human and mouse testis was detected by western blot and immunofluorescence.To explore the potential function of Orc6 in spermatogonia,the C18-4 cell line was transfected with control or Orc6 siRNA.Subsequently,5-ethynyl-2-deoxyuridine(EdU)and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assays,flow cytometry,and western blot were used to evaluate its effects on proliferation and apoptosis.It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells.Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated(Wnt)/β-catenin signaling.Western blot revealed that the expression ofβ-catenin protein and its phosphorylation(Ser675)were significantly decreased when silencing the expression of ORC6.Our findings indicated that Orc6 was upregulated in spermatogonia,whereby it regulated proliferation and apoptosis by activating Wnt/β-catenin signaling.
基金supported by the National Natural Science Foundation of China[No.32273133 and No.31970492]High-performance Computing Platform of YZBSTCACC.
文摘The black rockfish (Sebastes schlegelii) is a marine species that is economically important in aquaculture, and the efficiency of its spermatogenesis is vital for its success in the aquaculture industry. Spermatogonia serve as the foundation of spermatogenesis in fish, possessing the ability for continuous self-renewal and progressive differentiation into mature spermatozoa. Moreover, Sertoli cells are crucial in modulating the proliferation and differentiation of spermatogonia. This study focused on the regulation of glycolysis by PLIN2a in Sertoli cells of the black rockfish and examined how the inhibition of glycolysis in these cells impacted the proliferation and differentiation processes of spermatogonia. We found that effective regulation of glycolysis was crucial for the metabolic activity and functional maintenance of Sertoli cells in black rockfish. Overexpression of plin2a in vitro enhanced glycolysis in Sertoli cells, whereas inhibition of glycolysis impaired their normal metabolic activity. In vivo inhibition of glycolysis in black rockfish testes lead to apoptosis of Sertoli cells and significantly suppressed the proliferation and differentiation of spermatogonia. These results underscore the essential role of glycolysis in the development and metabolic activity of Sertoli cells and highlight the critical regulatory role of glycolysis in determining the fate of spermatogonia. This study emphasizes the importance of regulating energy metabolism pathways, particularly glycolysis, in Sertoli cells to indirectly influence the development of spermatogonia, offering significant insights into the reproductive mechanisms of black rockfish and other teleost species.
基金This work was supported by the Shandong Province Natural Science Foundation(ZR2020KC038)Research and Development Program of Shandong Province(2021LZGC029)+1 种基金National Key Research and Development Program(2018YFD0901205,2018YFD0901204)China Agriculture Research System(CARS-47).
文摘Black rockfish(Sebastes schlegelii)is one of the most important marine economic viviparous fishes.Recently,germplasm degradation and genetic diversity reduction have occurred due to overfishing and long-term artificial breeding.Germ cell transplantation combined with cryopreservation may be an alternative way to protect genetic resources.However,in viviparous fish that undertake fertilization and embryo development in vivo,transplantation is more difficult than in oviparous fish,including selection of transplantation stage,isolation of germ stem cells,and preparation of sterile recipients.This seriously restricts the development of viviparous transplantation.Therefore,in this study,we aimed to explore a transplantation method suitable for these species.Donor cells were isolated from cryopreserved whole testes of 300–400g male Sebastes schlegelii in May,labeled by PKH26,and intra-peritoneally transplanted into allogeneic larvae at 5–10 days post-birth.Subsequently,the development of donor-derived cells in recipients were continuously detected by fluorescence labeling,histology,microsatellite markers,and fecundity tests.The results showed that donors were rich in spermatogonia(75%)and recipients maintained a high survival rate after transplantation,with a rate of>20%at sexual maturity.Further,donor-derived cells successfully migrated(100%),colonized,and incorporated into the developing recipient gonad(93.33%).Finally,transplanted recipients could normally develop and differentiate into male and female individuals,with donor-derived gametes found in 65.38%of mature recipients.In the present study,we first establish a simple and suitable transplantation method for Sebastes schlegelii using immature males and specific larvae,which will serve as a promising tool in the protection of germplasm resources for this transplantation-restricted marine viviparous species.
文摘Dear Editor,I would like to congratulate Mamsen et al.i on their extensive and scientifically valuable work.I analyzed their raw data presented in Table 1 of the original article from a different perspective and discovered an effect not mentioned in the article.My analysis showed that luteinizing hormone(LH)levels are significantly lower in patients at high infertility risk(HIR),whose testes lack A dark(Ad)spermatogonia and display an abnormal ratio of germ cells per crosssectional tubule(G/T).
