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.

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.

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.

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.

Stem cell autotomy and niche interaction in different systems

The best known cases of cell autotomy are the formation of erythrocytes and thrombocytes(platelets) from progenitor cells that reside in special niches. Recently, autotomy of stem cells and its enigmatic interaction with the niche has been reported from male germline stem cells(GSCs) in several insect species. First described in lepidopterans, the silkmoth, followed by the gipsy moth and consecutively in hemipterans, foremost the milkweed bug. In both, moths and the milkweed bug, GSCs form finger-like projections toward the niche, the apical cells(homologs of the hub cells in Drosophila). Whereas in the milkweed bug the projection terminals remain at the surface of the niche cells, in the gipsy moth they protrude deeply into the singular niche cell. In both cases, the projections undergo serial retrograde fragmentation with progressing signs of autophagy. In the gipsy moth, the autotomized vesicles are phagocytized and digested by the niche cell. In the milkweed bug the autotomized vesicles accumulate at the niche surface and disintegrate. Autotomy and sprouting of new projections appears to occur continuously. The significance of the GSC-niche interactions, however, remains enigmatic. Our concept on the signaling relationship between stem cell-niche in general and GSC and niche(hub cells and cyst stem cells) in particular has been greatly shaped by Drosophila melanogaster. In comparing the interactions of GSCs with their niche in Drosophila with those in species exhibiting GSC autotomy it is obvious that additional or alternative modes of stem cell-niche communication exist. Thus, essential signaling pathways, including niche-stem cell adhesion(E-cadherin) and the direction of asymmetrical GSC division- as they were found in Drosophila- can hardly be translated into the systems where GSC autotomywas reported. It is shown here that the serial autotomy of GSC projections shows remarkable similarities with Wallerian axonal destruction, developmental axon pruning and dying-back degeneration in neurodegenerative diseases. Especially the hypothesis of an existing evolutionary conserved "autodestruction program" in axons that might also be active in GSC projections appears attractive. Investigations on the underlying signaling pathways have to be carried out. There are two other well known cases of programmed cell autotomy: the enucleation of erythroblasts in the process of erythrocyte maturation and the segregation of thousands of thrombocytes(platelets) from one megakaryocyte. Both progenitor cell types- erythroblasts and megakaryocytes- are associated with a niche in the bone marrow, erythroblasts with a macrophage, which they surround, and the megakaryocytes with the endothelial cells of sinusoids and their extracellular matrix. Although the regulatory mechanisms may be specific in each case, there is one aspect that connects all described processes of programmed cell autotomy and neuronal autodestruction: apoptotic pathways play always a prominent role. Studies on the role of male GSC autotomy in stem cell-niche interaction have just started but are expected to reveal hitherto unknown ways of signal exchange. Spermatogenesis in mammals advance our understanding of insect spermatogenesis. Mammal and insect spermatogenesis share some broad principles, but a comparison of the signaling pathways is difficult. We have intimate knowledge from Drosophila, but of almost no other insect, and we have only limited knowledge from mammals. The discovery of stem cell autotomy as part of the interaction with the niche promises new general insights into the complicated stem cell-niche interdependence.

新生牛雄性生殖干细胞的分离纯化与培养

为建立新生牛雄性生殖干细胞的体外增殖与分化体系,将新生牛睾丸消化成单细胞悬液,分别进行差速贴壁、不连续密度梯度Percoll分选雄性生殖干细胞和支持细胞,对分选生殖干细胞进行体外培养。结果表明,差速贴壁法能有效分选雄性生殖干细胞与支持细胞,2%血清浓度的培养液即可用于雄性生殖干细胞的体外培养,添加100 ng·mL-1的GDNF能显著促进雄性生殖干细胞的增殖,增殖形成的雄性生殖干细胞集落具有一定多能性。

MiR-34c在奶山羊雄性生殖干细胞增殖分化中的作用

microRNA(miRNA)在奶山羊雄性生殖细胞和精子发生过程有重要的调控功能。为研究miR-34c对雄性生殖干细胞增殖与分化中的作用,本文利用视黄酸效应基因8(Stra8)在雄性生殖细胞中随年龄增长,以其表达量上调的表达特征为指针,使用实时定量PCR技术筛选分析miRNAs。结果发现,miR-34c与Stra8的表达规律基本一致。在无精症奶山羊的睾丸组织中,发现miR-34c在无精症奶山羊睾丸组织中表达缺失。利用miR-34c模拟物及抑制剂转染奶山羊雄性生殖干细胞,体外转染miR-34c模拟物及其抑制剂,发现miR-34c能够下调Rarg、Stra8与c-Myc基因的表达,减缓奶山羊雄性生殖干细胞的增殖。结果提示,miR-34c可能具有调控奶山羊雄性生殖干细胞的减数分裂的作用,同时抑制其增殖。

新生牛雄性生殖细胞源类ES的培养及检测

采用贴壁速率差法分离纯化的新生牛雄性生殖干细胞（Male germ stem cell，mGSCs）和支持细胞（Calf sertoli cell，CSCs）；CSCs饲养层细胞一般维持时间不超过8d；新生牛雄性生殖干细胞在CSCs饲养层上1代培养至5～6d可形成类ES细胞集落，采用机械离散集落传代法有利于集落形成或存在，传代培养至少在前2代细胞集落存在；部分典型细胞集落的中心细胞AKP强阳性，周边细胞AKP弱阳性或阴性；免疫组化检测显示，细胞集落SSEA1强阳性、SSEA3弱阳性和Oct-4呈现阳性染色。表明mGSCs具有ES细胞的某些细胞特性。

成年昆明小鼠雄性生殖干细胞的分离培养及其多能性研究

【目的】从成年昆明小鼠睾丸组织中分离具有多能性的雄性生殖干细胞(Male germline stem cells,mGSCs)。【方法】采用机械法和2步酶消化法分离出成年昆明小鼠的精原细胞,经差速贴壁后,培养在小鼠胎儿成纤维细胞(MEF)饲养层上,2周后得到类胚胎干细胞(ESCs)样mGSCs。对得到的mGSCs进行碱性磷酸酶(AP)染色、免疫荧光染色、RT-PCR、体内分化等检测。【结果】mGSCs具有类似ESCs的形态和多能性,AP染色呈阳性,免疫荧光Oct4、Vasa染色呈阳性,RT-PCR检测其表达Oct4、Nanog、Sox2等多能性基因和Vasa、C-kit等生殖细胞的标记基因,在免疫缺陷鼠体内能形成畸胎瘤,组织学检测其可形成包括3个胚层在内的多种类型细胞。【结论】从成年昆明小鼠睾丸分离的精原细胞,在体外培养去分化形成了具有类ESCs生物学特性的mGSCs,mGSCs既具有多能性,也具有生殖细胞的基本生物学特性。

干扰EZH2的表达对奶山羊精原干细胞抗氧化能力的影响

[目的]本试验旨在探究EZH2对奶山羊精原干细胞抗氧化能力的影响。[方法]通过siRNA干扰EZH2在奶山羊精原干细胞中的表达,验证干扰效率,并采用ELISA、qPCR和Western blot检测抗氧化酶活性和抗氧化相关基因与蛋白表达水平。[结果]与对照组相比,干扰EZH2极显著提高奶山羊精原干细胞中过氧化氢酶(CAT)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GPx)的活性(P<0.01),而丙二醛(MDA)含量极显著下降(P<0.01)。在分子水平上,干扰EZH2后,奶山羊精原干细胞中CAT、SOD2蛋白和mRNA表达水平均极显著提高(P<0.01),GPx mRNA表达水平极显著上升(P<0.01)。[结论]在体外培养的奶山羊精原干细胞中,干扰EZH2增强了细胞的抗氧化能力,其分子机制可能与激活CAT和SOD2蛋白并促进其下游抗氧化基因GPx表达相关。

PLZF与哺乳动物雄性生殖干细胞的发育分化

早幼粒细胞白血病锌指蛋白(promyelocytic leukemia zinc finger,PLZF),也被称为ZBTB16(zinc finger and BTB domain containing 16,ZBTB16)或锌指蛋白145(zinc finger protein 145,ZFP145),是我国学者发现与人类疾病相关的蛋白质.人类PLZF的是由673个氨基酸残基组成的转录抑制因子,属于蛋白质超家族.该超家族以N端的BTB/POZ(bric-à-brac,tramtrack,brad complex(BTB)/poxvirus zinc finger(POZ)domain)结构为特征.PLZF蛋白的BTB/POZ结构与个体发育、胚胎发生、染色体的重构等事件相关.近年发现,PLZF在哺乳动物雄性生殖干细胞(male germline stem cells,mGSCs)发育分化过程中也发挥重要作用.探讨PLZF的生物学功能和作用机制,将有助于理解其在mGSCs发育过程中的重要作用.本文就PLZF在维持mGSCs自我更新和在发育分化调控中的作用给予综述.

Optimization of culture conditions for short-term maintenance, proliferation, and colony formation of porcine gonocytes

Background: Gonocytes give rise to spermatogonial stem cells, and thereby play an essential role in establishing spermatogenesis. Optimized culture conditions for gonocytes provide an opportunity for their study and in vitro manipulation for potential application in reproductive technologies. Using six experiments in a step-wise design, we examined the effects of several culture conditions on the maintenance, proliferation, and colony formation of porcine gonocytes. Testis cells from neonatal piglets were cultured for 7 d in DMEM supplemented with 10% fetal bovine serum. The examined culture conditions included using different cell seeding densities, gonocyte proportions, incubation temperatures, sampling strategies, and medium changing regimens.Results: Confluency of cel s was optimal(>90% by ~6 d) when 3.0 × 104 testis cel s/cm2 containing ~40% gonocytes were used. Incubating the cel s at 35 °C or 37 °C resulted in similar cel number and viability at confluency, but incubation at 35 °C resulted in a delayed confluency. In the first 2 d of culture, gonocytes remained mostly floating in the medium and gradual y settled over the next 5 d. Consequently, not changing the medium for 7 d(as opposed to changing it every 2 d) led to a significant increase in the number of gonocyte colonies by reducing the loss of "floating gonocytes".Conclusion: We found that gonocytes require the presence of a critical minimum number of somatic cel s for settlement, and can proliferate and form growing colonies even in a basic medium. Large numbers of viable gonocytes remain floating in the medium for several days. The optimized culture conditions in the present study included seeding with 3.0 × 104 testis cel s/cm2 containing ~40% gonocytes, incubating at 37 °C, and without changing the medium in the first week, which can result in improved colony formation of porcine gonocytes.

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.

猪雄性生殖干细胞的分离纯化

为了建立猪雄性生殖干细胞的分离纯化方法,以1～2周龄长白猪睾丸组织为材料,采用差异贴壁法和曲细精管培养法对猪雄性生殖干细胞进行分离纯化。结果表明:双酶消化-差异贴壁法和曲细精管培养法均能获得高活力的猪雄性生殖干细胞,但后者的富集效果较好,可以达到40.7%,前者的富集效果为36.9%。差异贴壁法和曲细精管培养法具有操作简单、回收细胞纯度高等优点,可应用于猪雄性生殖干细胞的分离纯化。

牛雄性生殖系干细胞的体外诱导分化

雄性生殖系干细胞(Male germ-line stem cells,mGSCs)是一群具有高度自我更新能力和分化潜能的细胞,是雄性成体内唯一可复制的二倍体永生细胞。转基因技术与雄性生殖系干细胞异体及异种移植技术相结合,将会为克隆动物、转基因动物生产及一些人类遗传性疾病的基因治疗提供新的机遇与途径。本试验采用组合酶消化和选择贴壁法,对5月龄、6月龄牛胎儿及新生牛雄性生殖系干细胞体外培养及分化进行了研究。试验结果显示,睾丸支持细胞对雄性生殖系干细胞体外增殖、分化所必需的,同时对数期睾丸支持细胞对雄性生殖系干细胞贴壁、增殖与分化效果明显;共培养16 d后,牛雄性生殖系干细胞分化为长形精子细胞,试验建立了牛雄性生殖系干细胞体外诱导培养分化体系。

Retinol促进小鼠雄性生殖干细胞的增殖

Retinol(RE)作为维生素A的一种,对生命有机体内多种生理生化和代谢反应均有重要的影响。本试验以雄性生殖干细胞(mGSCs)为对象,用RE培养mGSCs,探讨RE对mGSCs增殖的影响。试验分别设置对照组,RE组,RE+LY(LY294002,PI3K抑制剂)组,RE+PD(PD98059,MAPKK抑制剂)组。选取培养不同时间的mGSCs进行半定量与定量PCR分析,检测基因的表达情况。对培养48h的mGSCs进行Brdu荧光染色。结果显示,RE培养能够显著促进mGSCs的增殖,培养后C-myc等增殖标记基因表达上调,加入LY、PD抑制剂后,增殖效果显著降低;表明RE可能通过PI3K或MAPKK促进mGSCs的增殖。本研究证明了RE具有促进mGSCs增殖的作用,对于探明RE对mGSCs增殖调控的机理具有重要意义。