Characterization of long-term ex vivo expansion of tree shrew spermatogonial stem cells

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.

Cryopreserved ovine spermatogonial stem cells maintain stemness and colony forming ability in vitro

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.

Optimal dose of busulfan for depleting testicular germ cells of recipient mice before spermatogonial transplantation

Successful spermatogonial transplantation requires depletion of the host germ cells to allow efficient colonization of the donor spermatogonial stem cells. Although a sterilizing drug,busulfan （Myleran）,is commonly used for preparing a recipient mouse before transplantation,the optimal dose of this drug has not yet been defined.The present study investigated the effects of different doses of busulfan （10-50 mg per kg body weight） on survival rate,testicular mass and histomorphology,and on the haploid spermatids and spermatozoa of male BALB/c mice.The results suggest that a dosage of 30 mg kg^-1 is optimal for the ablative treatment withbusulfan used to prepare the recipient mice. This dose results in an adequate depletion of the host germ cells for colonization of donorderived spermatogonial stem cells and causes the lowest death rate of the animals.

Meclofenamic acid represses spermatogonial proliferation through modulating m^6A RNA modification

Background: N6-Methyladenosine(m^6A), the most prevalent modification in mammalian m RNA, plays important roles in numerous biological processes. Several m^6A associated proteins such as methyltransferase like 3(METTL3),methyltransferase like 14(METTL14), α-ketoglutarate-dependent dioxygenase Alk B homolog 5(ALKBH5) and YTH domain containing 2(YTHDC2) are involved in the regulation of spermatogenesis and oogenesis. However, the role of the first detected m^6A demethylase, fat mass and obesity associate protein(FTO), in germ cells remains elusive.Elucidation of FTO roles in the regulation of germ cell fate will provide novel insights into the mammalian reproduction.Methods: Mouse GC-1 spg cells were treated with the ester form of meclofenamic acid(MA2) to inhibit the demethylase activity of FTO. The cellular m^6A and m^6Amlevel were analyzed through high performance liquid chromatography combined with tandem mass spectrometry(HPLC/MS-MS). The cell apoptosis was detected via TUNEL and flow cytometry. The cell proliferation was detected through Ed U and western blot. The m RNA level of core cyclin dependent kinases(CDKs) was quantified via q-PCR. RNA decay assay were performed to detect RNA stability. Dual fluorescence assay was conducted to study whether MA2 affects the expression of CDK2 dependent on the m^6A modification at 3’UTR.Results: MA2 significantly increased the cellular m^6A level and down-regulated the expression of CDK1, CDK2, CDK6 and Cd C25 a, resulting in arrest of G1/S transition and decrease of cell proliferation. MA2 downregulated CDK2 m RNA stability. Additionally, mutation of the predicted m^6A sites in the Cdk2–3’UTR could mitigated the degradation of CDK2 m RNA after MA2 treatment.Conclusion: MA2 affected CDKs expression through the m^6A-dependent m RNA degradation pathway, and thus repressed spermatogonial proliferation.

Manipulation of spermatogonial stem cells in livestock species

We are entering an exciting epoch in livestock biotechnology during which the fundamental approaches(such as transgenesis, spermatozoa cryopreservation and artificial insemination) will be enhanced based on the modern understanding of the biology of spermatogonial stem cells(SSCs) combined with the outstanding recent advances in genomic editing technologies and in vitro cell culture systems. The general aim of this review is to outline comprehensively the promising applications of SSC manipulation that could in the nearest future find practical application in livestock breeding. Here, we will focus on 1) the basics of mammalian SSC biology;2) the approaches for SSC isolation and purification;3) the available in vitro systems for the stable expansion of isolated SSCs;4) a discussion of how the manipulation of SSCs can accelerate livestock transgenesis;5) a thorough overview of the techniques of SSC transplantation in livestock species(including the preparation of recipients for SSC transplantation,the ultrasonographic-guided SSC transplantation technique in large farm animals, and the perspectives to improve further the SSC transplantation efficiency), and finally, 6) why SSC transplantation is valuable to extend the techniques of spermatozoa cryopreservation and/or artificial insemination. For situations where no reliable data have yet been obtained for a particular livestock species, we will rely on the data obtained from studies conducted in rodents because the knowledge gained from rodent research is translatable to livestock species to a great extent. On the other hand, we will draw special attention to situations where such translation is not possible.

Establishment of cell lines with porcine spermatogonial stem cell properties

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.

Spermatogonial stem cells:Current biotechnological advances in reproduction and regenerative medicine

Spermatogonial stem cells(SSCs) are the germ stem cells of the seminiferous epithelium in the testis. Through the process of spermatogenesis, they produce sperm while concomitantly keeping their cellular pool constant through self-renewal. SSC biology offers important applications for animal reproduction and overcoming human disease through regenerative therapies. To this end, several techniques involving SSCs have been developed and will be covered in this article. SSCs convey genetic information to the next generation, a property that can be exploited forgene targeting. Additionally, SSCs can be induced to become embryonic stem cell-like pluripotent cells in vitro. Updates on SSC transplantation techniques with related applications, such as fertility restoration and preservation of endangered species, are also covered on this article. SSC suspensions can be transplanted to the testis of an animal and this has given the basis for SSC functional assays. This procedure has proven technically demanding in large animals and men. In parallel, testis tissue xenografting, another transplantation technique, was developed and resulted in sperm production in testis explants grafted into ectopical locations in foreign species. Since SSC culture holds a pivotal role in SSC biotechnologies, current advances are overviewed. Finally, spermatogenesis in vitro, already demonstrated in mice, offers great promises to cope with reproductive issues in the farm animal industry and human clinical applications.

Cytotoxicity of nonylphenol on spermatogonial stem cells via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway

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.

Effect of sucrose on cryopreservation of pig spermatogonial stem cells

Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation ofpig spermatogonial stem cells （pSSCs） has not been tested. The aim of this work was to study the effect of sucrose during pSSC cryopreservation and to find the most effective concentration in freezing medium, pSSCs 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 （qRT-PCR） indicated that the mRNA 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 mRNA level of one anti-apoptotic gene （XIAP） was significantly lower in thawed cells than in cells before freezing （P〈0.05）. When comparing the mRNA expression of apoptosis-related genes in thawed cells, the mRNA 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 qRT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing. Briefly, sucrose promoted pSSCs survival after freezing and thawing, especially at a concentration of 210 mmol L^-1, which possibly assisted pSSC dehydration and inhibited cell apoptosis. These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of pSSCs.

Xeno-free culture of human spermatogonial stem cells supported by human embryonic stem cell-derived fibroblast-like cells

Spermatogonial stem cells （SSCs） divide continuously to support spermatogenesis throughout postnatal life and transmit genetic information to the next generation. Here, we report the successful establishment of the method for the isolation and identification of human SSCs from testicular tissue, and to determine the culture conditions required to expand SSCs on human embryonic stem cell-derived fibroblast-like cells （hdFs）. Large-scale cultures of SSCs were maintained on hdF feeder layers and expanded in the presence of a combination of cytokines and glial cell line-derived neurotrophic factor for at least 2 months. Cell surface marker analysis showed that SSCs retained high levels of alkaline phosphatase activity and stained strongly for anti-stage-specific embryonic antigen （SSEA）-1, OCT4 and CD49f. They also expressed the genes OCT4, SOX3 and STRA8 as detected by reverse transcription polymerase chain reaction （RT-PCR） analysis. These data clearly illustrate a novel approach for the growth of human SSCs using hdFs as feeder cells, potentially eliminating xenogeneic contaminants. This system provides a new opportunity for the study of the regulatory mechanism of the ‘niche＇ that governs SSC self-renewal, and will be a valuable source of SSCs for potential clinical applications.

Molecular characterization and expression patterns of Nanog gene validating its involvement in the embryonic development and maintenance of spermatogonial stem cells of farmed carp,Labeo rohita

Background: The homeobox containing transcription factor Nanog plays crucial roles in embryonic development/proliferation and/or maintenance of spermatogonial stem cells(SSCs) via interacting with transcription factors such as Oct4 and Sox2 in mammals. However, knowledge of its exact mechanistic pathways remains unexploited. Very little is known about teleost Nanog. Information on the Nanog gene of farmed rohu carp(Labeo rohita) is lacking. We cloned and characterized the Nanog gene of rohu carp to understand the expression pattern in early developmental stages and also deduced the genomic organization including promoter elements.Results: Rohu Nanog(LrNanog) cDNA comprised an open reading frame of 1,161 nucleotides bearing a structural homeodomain; whereas, the genomic structure contained four exons and three introns suggesting that it is homologous to mammalian counterparts. Phylogenetical y, it was closely related to freshwater counterparts. Protein sequence(386 AA of42.65 kDa) comparison revealed its low similarity with other vertebrate counterparts except that of the conserved homeodomain. Tissue distribution analysis revealed the existence of LrNanog transcripts only in adult gonads. The heightened abundances in the ovary and proliferating spermatogonia suggested its participations in maternal inheritance and male germ cell development. The potentiating abundances from fertilized egg onwards peaking at blastula stage vis-à-vis decreasing levels from gastrula stage onwards demonstrated its role in embryonic stem cell development. We also provided evidence of its presence in SSCs by western blotting analysis. Further, the promoter region was characterized, predicting a basal core promoter and other consensus elements.Conclusion: The molecular characterization of LrNanog and its documented expression profiling at transcript and protein levels are indicative of its functional linkage with embryonic/spermatogonial stem cell maintenance. This is the first report of LrNanog genomic organization including its promoter sequence information with predicted regulatory elements of a large-bodied carp species. This will be useful for elucidating its mechanism expression in future. Nanog could be used as a potential biomarker for proliferating carp SSCs.

Production of Transgenic Animals Using Spermatogonial Stem Cells

Spermatogonial stem cells （SSCs） are a type of adult stem cell found in male mammals.These cells have the capacity for self renewal and are capable of differentiating in the niche of testis.They are also the only adult stem cells in a normal postnatal body that undergo self-renewal throughout life,transferring genetic information to the offspring.Since a technique for transplanting SSCs was first described by Brinster and his colleagues in 1994,more and more researchers have become interested in exploring the possibility of utilizing adult SSCs to generate transgenic animals.In this mini-review,we attempt to summarize the current research progress in the area of spermatogonial stem cells including the source,types and differentiation of the SSCs,and the application on transgenic animals,with a particular focus on the strategy of SSCs delivery including seminiferous tubule injection and spermatogonial stem cell transplantation.

SPOC domain-containing protein 1 regulates the proliferation and apoptosis of human spermatogonial stem cells through adenylate kinase 4

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.

Study on the role of JAK/STAT signaling pathway during chicken spermatogonial stem cells generation based on RNA-Seq

Spermatogonia! stem cells（SSCs） form the foundation for spermatogenesis and sustain male fertility.To explore the regulatory mechanisms of chicken SSCs generation,we obtained highly purified chicken embryonic stem cells（ESCs）,primordial germ cells（PGCs） and SSCs by fluorescence-activated cell sorting（FACS）.High-throughput analysis methods（RNA-Seq） were used to sequence the transcriptome level of these cells.Gene ontology and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment were used to analyze RNA-Seq results.BMP4 was used to induce chicken ESCs differentiation to SSCs-like cells in vitro.The quantitative real-time（qRT）-PCR was used to detect the expression changes of the key genes.The results showed that 22 relevant critical pathways were found by RNA-Seq,one of them was the Janus kinase/signal transducer and activator of transcription（JAK/STAT） signaling pathway.Total of 103 related genes were detected in this pathway.Protein-protein interactions analysis found that 87 proteins were significantly related to 19 key proteins in this pathway.These 87 proteins were enriched in 21 biological processes and 18 signaling pathways.Moreover,during the differentiation of chicken ESCs to SSCs-like cells induced by BMP4 in vitro,JAK2 and STAT3 were activated.The qRT-PCR results showed that the expression trends of JAK2 and STAT3 were basically the same as in vivo.We concluded that JAK/STAT signaling pathway plays an important role in the process of chicken SSCs generation both in vivo and in vitro;it may achieve its function through multiple biological processes and other related pathways.

CDH1, a Novel Surface Marker of Spermatogonial Stem Cells in Sheep Testis

Spermatogonial stem cells（SSCs） are unique stem cells in adult body that can transmit genetic information to the next generation. They have self-renewal potential and can continuously support spermatogenesis throughout life of a male animal. However, the SSC population is extremely small, isolation and purification of the SSCs is challenging, especially for livestock animals. It has been confirmed that CDH1（cadherin-1, also known as E-cadherin） can be expressed in undifferentiated SSCs of mouse and rats, but it has not been verified in sheep. Here, CDH1 was found as a novel surface marker for sheep SSCs. In this paper, sheep antiCDH1 polyclonal antibodies were prepared and its activity was checked. Using the obtained antibodies and immunohistochemistry analysis, we confirmed that CDH1 can be expressed by SSCs in sheep testis.

Antioxidant effects of quercetin in freeze-thawing process of mouse spermatogonialstem cells

Objective: To evaluate the antioxidant effect of quercetin on cell viability, reactive oxygen species (ROS) contents and apoptosis of cryopreserved mouse spermatogonial stem cells (mSSCs). Methods: mSSCs were isolated from neonate mice and cultivated in culture medium containing 30 μM quercetin for 48 h and then frozen for 2 weeks. After thawing, MTT assay was carried out to analyze the cell viability. Moreover, intracellular ROS levels were measured by flow cytometery and apoptosis was evaluated by detection of phosphatidylserine externalization assay and also real-time polymerase chain reaction. Results: Pre-treatment of mSSCs by 30 μM quercetin significantly decreased intracellular ROS content and apoptotic cell numbers and improved viability of mSSCs. Moreover, the gene expression of Bcl-2 and Bax significantly increased and decreased respectively after the freeze-thawing process. Conclusions: Pre-treatment of mSSCs with quercetin can improve cell viability and reduce apoptosis during freeze-thawing process. It can be a promising way to improve the quality and efficiency of cryopreservation protocols used in fertility preservation strategies.

MKK7-mediated phosphorylation of JNKs regulates the proliferation and apoptosis of human spermatogonial stem cells

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.

Colonization of neonate mouse spermatogonial stem cells co-culture with Sertoli cells in the presence and absence soft agar

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.

Preparation, cryopreservation and in vitro culture of spermatogonial stem cells of new born calves

The experiment was designed to study the preparation, cryopreservation and culture of calf spermatogonial stem cells in order to provide some data for theoretical research and practical use of calf spermatogonial stem cells. As for enzymolysis of testis tissue, two digestive method A and B. The cryopreservation cooling procedure was that spermatogonial stem cells was equilibrated at 4℃ for lh, at -20℃ for lh, at -40℃ for 2h, at -80℃ overnight and then stored in liquid nitrogen. The different concentration of three eryoprotectants were compared. The results indicated that both method A and method B achieved the same high motility rates of cells with method B needing time longer, calf spermatogonial stem cells could be well cryopreserved by the stage cooling procedure. Satisfactory cryopreservative results were gotten by adding 10% DMSO or 10% EG to cryopreservative medium, the former was better and adding sucrose could improved cryopreservative effect. The culture behaviors of spermatogonial stem cells kept the same before and after cryopreservation. It was concluded that an effective method was verified for preparation, cryopreservation and culture of new calf spermatogonial stem cells.

FOXP4 promotes proliferation of human spermatogonial stem cells

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.