胶质细胞源性神经营养因子引发精原干细胞自我更新的信号通路

胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)是TGF-β超家族的一个相关成员。哺乳动物睾丸曲细精管内支持细胞分泌的GDNF,能促进精原干细胞(spermatogonial stem cells,SSCs)的自我更新与增殖。SSCs去分化诱导产生的多能干细胞已被广泛应用于再生医学领域,且SSCs在制作转基因动物、男性不育治疗和体外实施精子发生过程等方面,具有极大的应用价值。所以,GDNF引发SSCs自我更新的作用机理非常值得探索。通过对GDNF引发SSCs自我更新的信号通路进行系统梳理,我们发现了如下的作用过程:GDNF与GFR-α1特异性结合,活化Ret蛋白酪氨酸激酶,随后激活Ras/ERK1/2、PI3K-Akt和SFK信号通路,促进SSCs的自我更新;同时,在该过程中还存在信号通路间的交联对话现象。     

精原干细胞自我更新和分化的调控

精原干细胞（spermatogonial stem cells,SSCs）是体内自然状态下惟一能将遗传信息传至子代的成体干细胞,它们能通过维持自我更新和分化的稳定从而保证雄性生命过程中精子发生的持续进行。了解SSCs自我更新和分化的调节机制有助于阐明精子发生机理,并为探究其他组织中成体干细胞增殖分化的调节机制提供依据。然而目前对于SSCs自我更新和分化的调控机制所知甚少。SSCs的更新与分化遵循特定模式,受以睾丸支持细胞为主要成分的微环境及各种内分泌因素如胶质细胞源神经营养因子（GDNF）、维生素、Ets转录因子ERM/Etv5等的调控。本文评述了SSCs更新与分化的模式以及上述因素对其更新、分化的调控,探讨了其中可能涉及的信号通路,以期为本领域及其他成体干细胞相关研究提供借鉴。     

神经营养因子与神经干细胞

生长因子在神经干细胞的增殖,分化和存活过程中有重要作用。神经营养因子是其中的一类,它包括神经生长因子（NGF）家族,胶质源性神经营养因子（GDNF）家族和其它神经营养因子。NGF家族包括NGF,BDNF,NT－3,NT－4／5和NT－6。这一家族可促进epidermic growth facter(EGF)反应 海马及前脑室管膜下区神经干细胞的存活和分化。GDNF家族包括GDNF,NTN,PSP和ART。GDNF家族促神经发育的作用主要在外周,它促进肠神经嵴前体细胞的存活和增殖,且对外周感觉神经的发育至关重要。其它生长因子如bFGF和EGF,它们能促进神经干细胞增殖和存活;CNTF和LIF等在神经干细胞的分化中也有重要作用。     

小熊猫精原干细胞的分选与培养

野生动物的保护手段主要包括就地保护、易地保护与离体保护。精原干细胞（SSCs）是雄性动物维持生殖能力的根本,既能通过自我更新产生新细胞,也能通过分化产生精子,在小熊猫（Ailurus fulgens）离体保护方面具有广阔的应用前景。动物睾丸中精原干细胞数量极少,分离纯化与体外培养对于其研究和应用至关重要。本研究选择整合素α6（ITGA6）蛋白作为精原干细胞分子标记,采用免疫磁珠分选（MACS）技术富集了3月龄小熊猫睾丸中的ITGA6阳性细胞。流式细胞术检测发现分选后ITGA6阳性细胞纯度可达74.27% ± 8.73%,显著高于分选前（32.60% ± 3.06%）。将分选后的细胞接种到层粘连蛋白包被的细胞培养板中,用含胶质细胞源性神经营养因子（GDNF）、表皮细胞生长因子（EGF）与成纤维细胞生长因子（bFGF）的培养基进行体外培养。培养10 d后,在显微镜下可观察到典型的精原干细胞集落,结合逆转录PCR（RT-PCR）和细胞免疫荧光染色发现这些细胞集落特异性表达精原干细胞分子标记蛋白ITGA6、早幼粒细胞白血病锌指蛋白（PLZF）和胸腺细胞分化抗原1（THY1）,同时也表达生殖细胞标记蛋白VASA和DAZL。本研究结果证实,ITGA6可作为小熊猫精原干细胞的分子标记用于细胞分选富集,同时初步建立的培养体系也为小熊猫精子发生机制与应用研究提供材料。     

KnockoutTM SR无血清培养基支持小鼠精原干细胞的短期存活(简报)

精原干细胞(spermatogonial stem cells,SSCs)是睾丸内具有自我复制和分化为精子潜能的干细胞,它的体外培养是精子发生机理研究和制作转基因动物等的新途径[1,2].近几年的研究表明,SSCs在体外的自我增殖需要GDNF(glial cell line-derived neu-rotrophie factor)因子和饲养层细胞等的支持[3-10].并且睾丸支持细胞(Sertoli's cells)和血清都导致培养的SSCs分化[1,6].因此,使用无血清培养基培养高度纯化的SSCs是培养成败的关键之一.     

精原干细胞niche的研究进展

精原干细胞(spermatogonial stem cells,SSCs)是指睾丸内位于曲精细管基膜上既能自我更新维持自身适量恒定,又能定向分化产生精母细胞的一类原始精原细胞。随着干细胞深入的研究,人们发现了一种控制着干细胞可塑性与命运的微环境,此微环境被称为干细胞niche,干细胞niche由niche细胞、细胞外基质、细胞因子等构成。精原干细胞niche是由黏附因子、生长因子、支持细胞、间质细胞以及小管周肌肉细胞组成。大量的研究表明支持细胞在睾丸中是主要的成体细胞,通过分泌可溶性的因子来影响精原干细胞niche的结构与功能,同时支持细胞还能够间接的影响其他的成体细胞。随着年龄的增长使得精原干细胞niche的功能下降。精原干细胞数量以及精原干细胞niche为我们研究组织特异性干细胞生物学以及保持再生组织平衡提供了很宝贵的线索,精原干细胞对于保持组织的自我更新具有很重要的作用,并且受到人们大量的关注,然而精原干细胞niche也起到很重要的作用,它为治疗一些疾病提供新途径.本文将综述精原干细胞niche及其变化对精原干细胞功能调节的相关研究进展。     

Niche调控精原干细胞命运

<正>常的精子发生是一个由多种因子参与的精密、有序调控的生理过程。精原干细胞的自我更新维持了精原干细胞本身数量的稳定,而其分化成为精原祖细胞的节奏保障了形成精子的规模。睾丸微环境(niche)在精原干细胞自我更新和分化过程中起着举足轻重的作用。该文简要描述了精子发生过程中精原干细胞及其微环境形成过程中所涉及的主要细胞因子及其调控机制,并探讨了该研究过程中所遇到的问题,最后展望了相关基础研究在临床医疗和科学研究领域中的应用前景。     

精原干细胞与睾丸生殖衰老的研究进展

雄性睾丸内精子的生成及其质量随年龄增长逐渐降低。精原干细胞是精子生成的起点,其数量和质量决定了精子的生成,而精原干细胞niche是调节精原干细胞自我更新与分化的重要因素。在衰老过程中,干细胞微环境退化,精原干细胞自我更新和分化失衡,被认为是衰老导致睾丸生殖功能衰退的的主要因素。本文将综述衰老引起的精原干细胞与niche变化及其对生殖的影响相关研究进展。     

小鼠精原干细胞与胚胎干细胞样细胞

近年来,通过培养小鼠精原干细胞(spermatogonial stem cells,SSCs)获得了胚胎干细胞样细胞(,embryonic stem cell-like cells,ES样细胞).这些研究表明小鼠精原干细胞不仅具备特异分化为精子的干细胞潜能,而且具备胚胎干细胞(embryonic stem cell,ES)分化为三胚层的多向分化潜能.因此.这将有助于研究干细胞的分化调控机制,并且这些研究成果延伸至人类精原干细胞,也将为再生医学获取特殊的胚胎干细胞样细胞或特异分化的精子细胞开辟了蹊径.     

小鼠gdnf基因在真核细胞NIH-3T3中的表达

为了得到超量表达胶质细胞源神经营养因子'(glial cell line-derived neuotrophic factor,GDNF)的NIH-3T3细胞株,用于制作精原干(spermatogonial stem cells,SSCs)培养的饲养层,通过RT-PCR方法成功地从幼年小鼠睾丸中克隆了gdnf矿基因,构建了真核表达载体pcDNA3.1-gdnf,并用其转染NIH-3T3细胞.对筛选出的阳性细胞克隆进行的免疫荧光染色、RT-PCR和Western blotting的结果表明,获得了超量表达gdnf基因的NIH-3T3细胞株,这为精原干细胞的培养奠定了基础.     

Role of Src family kinases and N-Myc in spermatogonial stem cell proliferation

Spermatogonial stem cells are required for the initiation of spermatogenesis and the continuous production of sperm. In addition, they can acquire pluripotency and differentiate into derivatives of the three embryonic germ layers when cultured in the appropriate conditions. Therefore, understanding the signaling pathways that lead to self-renewal or differentiation of these cells is of paramount importance for the treatment of infertility, the development of male contraceptives, the treatment of testicular cancers, and ultimately for tissue regeneration. In this report, we studied some of the signaling pathways triggered by glial cell line-derived neurotrophic factor (GDNF), a component of the spermatogonial stem cell niche produced by the somatic Sertoli cells. As model systems, we used primary cultures of mouse spermatogonial stem cells, a mouse spermatogonial stem cell line and freshly isolated testicular tubules. We report here that GDNF promotes spermatogonial stem cell proliferation through activation of members of the Src kinase family, and that these kinases exert their action through a PI3K/Akt-dependent pathway to up-regulate N-myc expression. Thus, to proliferate, spermatogonial stem cells activate mechanisms that are similar to the processes observed in brain stem cells and lung progenitors.     

Akt mediates self-renewal division of mouse spermatogonial stem cells

Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, we assessed the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells.     

Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture

Functional roles of spermatogonial stem cells in spermatogenesis are self-renewing proliferation and production of differentiated daughter progeny. The ability to recapitulate these actions in vitro is important for investigating their biology and inducing genetic modification that could potentially lead to an alternative means of generating transgenic animals. The objective of this study was to evaluate the survival and proliferation of frozen-thawed bovine spermatogonial stem cells in vitro and investigate the effects of exogenous glial cell line-derived neurotrophic factor (GDNF). In order to accomplish this objective we developed a bovine embryonic fibroblast feeder cell line, termed BEF, to serve as feeder cells in a coculture system with bovine germ cells. Bovine spermatogonial stem cell survival and proliferation in vitro were evaluated by xenogeneic transplantation into the seminiferous tubules of immunodeficient mice. Bovine germ cells cocultured for 1 wk resulted in significantly more round cell donor colonies in recipient mouse testes compared to donor cells transplanted just after thawing. Bovine germ cells cocultured for 2 wk had fewer colony-forming cells than the freshly thawed cell suspensions or cells cultured for 1 wk. Characterization of the feeder cell line revealed endogenous expression of Gdnf mRNA and protein. Addition of exogenous GDNF to the culture medium decreased the number of stem cells present at 1 wk of coculture, but enhanced stem cell maintenance at 2 wk compared to cultures without added GDNF. These data indicate that frozen-thawed bovine spermatogonial stem cells survive cryopreservation and can be maintained during coculture with a feeder cell line in which the maintenance is influenced by GDNF.     

Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor

Spermatogonial stem cells (SSC) are a small self-renewing subpopulation of type A spermatogonia, which for the rest are composed of differentiating cells with a very similar morphology. We studied the development of primary co-cultures of prepubertal bovine Sertoli cells and A spermatogonia and the effect of glial cell line-derived neurotropic factor (GDNF) on the numbers and types of spermatogonia, the formation of spermatogonial colonies and the capacity of the cultured SSC to colonize a recipient mouse testis. During the first week of culture many, probably differentiating, A spermatogonia entered apoptosis while others formed pairs and chains of A spermatogonia. After 1 week colonies started to appear that increased in size with time. Numbers of single (A(s)) and paired (A(pr)) spermatogonia were significantly higher in GDNF treated cultures at Days 15 and 25 (P < 0.01 and 0.05, respectively), and the ratio of A(s) to A(pr) and spermatogonial chains (A(al)) was also higher indicating enhanced self-renewal of the SSC. Furthermore, spermatogonial outgrowths in the periphery of the colonies showed a significantly higher number of A spermatogonia with a more primitive morphology under the influence of GDNF (P < 0.05). Spermatogonial stem cell transplantation experiments revealed a 2-fold increase in stem cell activity in GDNF treated spermatogonial cultures (P < 0.01). We conclude that GDNF rather than inducing proliferation, enhances self-renewal and increases survival rates of SSC in the bovine spermatogonial culture system.     

Genetic and epigenetic properties of mouse male germline stem cells during long-term culture

Although stem cells are believed to divide infinitely by self-renewal division, there is little evidence that demonstrates their infinite replicative potential. Spermatogonial stem cells are the founder cell population for spermatogenesis. Recently, in vitro culture of spermatogonial stem cells was described. Spermatogonial stem cells can be expanded in vitro in the presence of glial cell line-derived neurotrophic factor (GDNF), maintaining the capacity to produce spermatogenesis after transplantation into testis. Here, we examined the stability and proliferative capacity of spermatogonial stem cells using cultured cells. Spermatogonial stem cells were cultured over 2 years and achieved approximately 10(85)-fold expansion. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, spermatogonial stem cells retained the euploid karyotype and androgenetic imprint during the 2-year experimental period, and produced normal spermatogenesis and fertile offspring. However, the telomeres in spermatogonial stem cells gradually shortened during culture, suggesting that they are not immortal. Nevertheless, the remarkable stability and proliferative potential of spermatogonial stem cells suggest that they have a unique machinery to prevent transmission of genetic and epigenetic damages to the offspring, and these characteristics make them an attractive target for germline modification.     

Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro

Spermatogonial stem cells (SSCs) support life-long spermatogenesis by self-renewing and producing spermatogonia committed to differentiation. In vitro, SSCs form three-dimensional spermatogonial aggregates (clusters) when cultured with glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2); serial passaging of clusters results in long-term SSC maintenance and expansion. However, the role of these growth factors in controlling patterns of SSC division and fate decision has not been understood thoroughly. We report here that in a short-term culture, GDNF and FGF2 increase the number of dividing SSCs, but not the total SSC number, compared to a no-growth-factor condition. Since the total germ cell number increases with growth factors, these results suggest that GDNF and FGF2 promote a SSC division pattern that sustains the size of the stem cell pool while generating committed progenitors. Our data also show that SSC numbers increase when the cluster structure is disintegrated and cell–cell interaction in clusters is disrupted. Collectively, these results suggest that in this culture system, GDNF and FGF2 stimulate SSC divisions that promote self-renewal and differentiation in the SSC population, and imply that the destruction of the cluster structure, a potential in vitro niche, may contribute to SSC expansion.     

The Niche-Derived Glial Cell Line-Derived Neurotrophic Factor (GDNF) Induces Migration of Mouse Spermatogonial Stem/Progenitor Cells

In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF), a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.