Dmrt1 regulates the immune response by repressing the TLR4 signaling pathway in goat male germline stem cells

Double sex and mab-3-related transcription factor 1(Dmrt1),which is expressed in goat male germline stem cells(mGSCs)and Sertoli cells,is one of the most conserved transcription factors involved in sex determination.In this study,we highlighted the role of Dmrt1 in balancing the innate immune response in goat mGSCs.Dmrt1 recruited promyelocytic leukemia zinc finger(Plzf),also known as zinc finger and BTB domain-containing protein 16(Zbtb16),to repress the Toll-like receptor 4(TLR4)-dependent inflammatory signaling pathway and nuclear factor(NF)-κB.Knockdown of Dmrt1 in seminiferous tubules resulted in widespread degeneration of germ and somatic cells,while the expression of proinflammatory factors were significantly enhanced.We also demonstrated that Dmrt1 stimulated proliferation of mGSCs,but repressed apoptosis caused by the immune response.Thus,Dmrt1 is sufficient to reduce inflammation in the testes,thereby establishing the stability of spermatogenesis and the testicular microenvironment.

An efficient strategy for generation of transgenic mice by lentiviral transduction of male germline stem cells in vivo

Background： Male germline stem cells（MGSCs） are a subpopulation of germ cells in the testis tissue. MGSCs are capable of differentiation into spermatozoa and thus are perfect targets for genomic manipulation to generate transgenic animals.Method： The present study was to optimize a protocol of production of transgenic mice through transduction of MGSCs in vivo using lentiviral-based vectors. The recombinant lentiviral vectors with either EF-1 or CMV promoter to drive the expression of enhanced green fluorescent protein（e GFP） transgene were injected into seminiferous tubules or inter-tubular space of 7-day-old and 28-day-old mouse testes. At 5 or 6 wk post-surgery, these pre-founders were mated with wild-type C57BL/6J female mice（1.5 to 2.0-month-old）.Results： Sixty-seven percent of F1 generation and 55.56 % of F2 offspring were positive for eG FP transgene under the control of EF-1 promoter via PCR analysis. The transgenic pups were generated in an injection site-and age-independent manner. The expression of transgene was displayed in the progeny derived from lentiviral vector containing CMV promoter to drive transgene, but it was silenced or undetectable in the offspring derived from lentiviral vector with transgene under EF-1 promoter. The methylation level of g DNA in the promoter region of transgene was much higher in the samples derived lentiviral vectors with EF-1 promoter than that with CMV promoter,suggesting e GFP transgene was suppressed by DNA methylation in vivo.Conclusion： This research reported here an effective strategy for generation of transgenic mice through transduction of MGSCs in vivo using lentivirus vectors with specific promoters, and the transgenic offspring were obtained in an injection site-and age-independent manner. This protocol could be applied to other animal species, leading to advancement of animal transgenesis in agricultural and biomedical fields.

GDNF Up-Regulates c-Myc Transcription via the PI3K/Akt Pathway to Promote Dairy Goat Male Germline Stem Cells (mGSC) Proliferation

Studies have demonstrated that regulation of GDNF on male germline stem cells （mGSCs） mainly through Ras/Erk1/2, Src family kinase and PI3K/Akt signaling pathways, but the signaling pathways GDNF-mediated are different when the species and cell lines varied. Whether GDNF regulates self-renewal of mGSCs isolated from livestock has not been reported. Here, we purified mGSCs from dairy goat testis using mixed enzymes and fibronectin. Immunofluoresce staining revealed the cultured dairy mGSCs expressed Vasa, Nanos2, Ngn3, Tert, Dazl, Lin28, Oct4, CD49f, Stra8 and GFRa1, reflecting that these cells were mGSCs phenotype. Then we cultured these dairy goat mGSCs in different concentrations of GDNF （0, 5, 10, or 20 ng mL-1） to optimize the best concentration of GDNF to sustain the dairy goat mGSCs self-renewal, after that the inhibitor of PI3K （LY294002, 10 μmol L-1） was added to the medium which contains the optimal concentration of GDNF we obtained by experiments. The mGSCs cultured in different media were compared through the population doubling time （PDT）, capacity of cell proliferation evaluated by PCNA and BrdU immunofluorescence staining, RT-PCR, QRT-PCR, Western blotting and flow cytometry. Results showed that 10 ng mL-1 was the optimal concentration of GDNF to maintain goat mGSCs self-renewal and GDNF up-regulates c-Myc transcription via the PI3K/Akt pathway to promote goat mGSCs proliferation. This study provides us an efficient model to study the mechanism in mGSCs proliferation and differentiation in goat, and has important implications in unveiling signaling pathways in livestock GSCs.

ALK Family Inhibitor A83-01 Promotes the Proliferation of Mouse Male Germline Stem Cells (mGSCs) Under Serum- and Feeder-Free Conditions

A83-01 is a selective inhibitor of the TGF-β type I receptor ALK,which inhibits the TGF-β-induced epithelial-to-mesenchymal transition（EMT） via the inhibition of Smad2 phosphorylation.Previous studies have showed that A83-01 promoted somatic cellular reprogramming significantly.Male germline stem cells（mGSCs）,as an alternative resource of pluripotent stem cells derived adult testis,have promising valuable in clinic medicine and regeneration,however,the derivation of mGSCs was complex and difficult.What the role A83-01 plays in promoting the proliferation of mGSCs is still unknown.In this study,combined with A83-01 and knockout serum replacement（KSR） medium,we obtained a relatively feeder-and serum-free system for mGSCs culturing in vitro and the optimal concentration of A83-01 was 0.25 μmol L-1.After continuous culturing,the proliferation efficiency of undifferentiated mGSCs and differentiation capacity of mGSC were examined as well.Results showed that,A83-01 dramatically increased the number of mGSCs and AP positive colonies,and the mitosis index according to the BrdU assay.A83-01 could also increase the expression of pluripotent markers including Oct4,Klf4,Nanog and c-Myc,analyzed byreal-time quantative PCR.mGSCs cultured in the optimal feeder-and serum-free system combined with A83-01 could form embryoid bodies（EBs）,which consisted of three embryonic layers detected by immunofluorescence and RT-PCR.Remarkably,the results demonstrated 0.25 μmol L-1A83-01 could promote the proliferation of mouse mGSC colonies and maintain their undifferentiated status under feeder-and serum-free systems.

Establishment and characterization of immortalized bovine male germline stem cell line

Male germline stem cells （mGSCs） are unique adult germ cells with self-renewal potential and spermatogenesis function in the testis. However, further studies are needed to establish a long-term cultural system of mGSCs in vitro, especially for large animals such as bovine mGSCs. In this study, we first established a stable immortalized bovine male germline stem cell line by transducing Simian virus 40 （SV40） large T antigen. The proliferation of these cells was improved significantly. These cells could express spermatogonial stem cell （SSC）-specific markers, such as PLZF, PGP9.5, VASA, LIN28A, and CD49F, both in the mRNA and protein levels. Additionally, these cells could be differentiated into three germ layer cells to enter meiosis, form colonies, and proliferate in the seminiferous tubules of busulfan-induced infertile mice. The immortalized bovine mGSCs maintain the criteria of mGSCs.

Recent advances in isolation,identification,and culture of mammalian spermatogonial stem cells

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.