Purification of Mouse Primordial Germ Cells by MiniMACS Magnetic Separation System

During migration toward gonadal ridges, primordial germ cells (PGCs; the earliest identifiable germ cells in the embryo) are very few in number, move along different tissues, and are not identifiable by morphological criteria alone. Here we report the use of the magnetic cell sorter MiniMACS as a tool for the isolation of such rare cells from 10.5- to 13.5-days post coitum mouse embryos. Cells stained sequentially by TG-1 (a monoclonal IgM antibody known to bind to the surface of PGCs) and superparamagnetic microbeads coated with secondary anti-mouse IgM antibody were separated on a magnetic column. Unlabeled cells (somatic cells) pass through the column, while labeled cells (germ cells) are retained. The retained cells can be eventually easily eluted and immediately used for biochemical studies or grown in suitable in vitro culture systems.     

Implication of DNA Demethylation and Bivalent Histone Modification for Selective Gene Regulation in Mouse Primordial Germ Cells

Primordial germ cells (PGCs) sequentially induce specific genes required for their development. We focused on epigenetic changes that regulate PGC-specific gene expression. mil-1, Blimp1, and Stella are preferentially expressed in PGCs, and their expression is upregulated during PGC differentiation. Here, we first determined DNA methylation status of mil-1, Blimp1, and Stella regulatory regions in epiblast and in PGCs, and found that they were hypomethylated in differentiating PGCs after E9.0, in which those genes were highly expressed. We used siRNA to inhibit a maintenance DNA methyltransferase, Dnmt1, in embryonic stem (ES) cells and found that the flanking regions of all three genes became hypomethylated and that expression of each gene increased 1.5- to 3-fold. In addition, we also found 1.5- to 5-fold increase of the PGC genes in the PGCLCs (PGC-like cells) induced form ES cells by knockdown of Dnmt1. We also obtained evidence showing that methylation of the regulatory region of mil-1 resulted in 2.5-fold decrease in expression in a reporter assay. Together, these results suggested that DNA demethylation does not play a major role on initial activation of the PGC genes in the nascent PGCs but contributed to enhancement of their expression in PGCs after E9.0. However, we also found that repression of representative somatic genes, Hoxa1 and Hoxb1, and a tissue-specific gene, Gfap, in PGCs was not dependent on DNA methylation; their flanking regions were hypomethylated, but their expression was not observed in PGCs at E13.5. Their promoter regions showed the bivalent histone modification in PGCs, that may be involved in repression of their expression. Our results indicated that epigenetic status of PGC genes and of somatic genes in PGCs were distinct, and suggested contribution of epigenetic mechanisms in regulation of the expression of a specific gene set in PGCs.     

Clonal Variation In Proliferation Rate of Cultures of Gpk Cells

Pedigrees of twenty-six clones of a line of keratocytes derived from guinea-pig ear epidermis (GPK cells) were analysed from time-lapse film. the mean interdivision time (IDT) for the culture was 1143 φ 215 (SD) min. the mean generation rates (mean reciprocal interdivision times) of clones varied over a range of 3.93–10.2 × 10^-4/min and the standard deviation of the clonal mean generation rates was 16.8% of the average value. Transient intraclonal variations in IDT due to mitoses in a plane perpendicular to the substratum were observed. the data were also analysed on the basis of cell location in sixteen equal zones (quadrats) of the filmed area. the mean generation rate of quadrats was 8.73 x 10^-4/min (SD = 4.9%). the spatial distribution showed some clustering of cells. the mean local density of the clones (2.25 φ 0.62 cells/10^-4 cm²) was significantly higher than the quadrat density (1.76 φ 0.8 cells/10^-4 cm²). There was no significant correlation between clonal density and mean generation rates, whereas for quadrats a significant negative correlation was found (P= 2.7%). the results support the proposition that cell lineage is the major determinant of the proliferation rate of subconfluent cultures.     

从原始生殖细胞分离克隆鸡胚胎生殖细胞的研究

从孵化 5 5天的鸡胚生殖腺中分离得到大量原始生殖细胞 (PGCs)集落 ,这些集落的细胞经多次克隆传代具有胚胎生殖细胞 (EG)的诸多特征 ,如有连续传代的能力 (传至第 9代 ) ,细胞集落有典型鸟巢状结构 ,PAS染色阳性 ,AKP染色阳性 ,在无饲养层无分化抑制因子LIF时可以自发分化成几种细胞类型 ,包括成纤维细胞、神经细胞、自律细胞等 ,悬浮培养时具有形成类胚体的能力。上述发现表明该细胞具EG细胞的诸多特性 ,为类EG细胞     

鸡胚胎原始生殖细胞体外培养

以14-15期鸡胚血液为材料,采用Ficoll密度梯度离心方法,提取鸡胚胎原始生殖细胞(primordial germ cells,PGCs),在无基质细胞和基质细胞上分别进行体外培养。从实验结果可以看出：在含有胎牛血清(fetal bovine serum,FBS)、鸡血清(chicken serum,CS)、碱性成纤维细胞生长因子(bFGF)、人胰岛素样生长因子(hIGF-1)、小鼠白血病抑制因子(mLIF)和青,链霉素双抗的M199培养液中培养时,鸡PGCs最多能够存活4天：当采用细胞因子和5天鸡胚胎性腺基质细胞共培养时能存活23代且每代细胞增殖可达近10倍。提纯后的PGCs细胞冻存复苏后,经台盼蓝染色鉴定存活率可达80％左右。     

果蝇原生殖细胞特化的分子机制

原生殖细胞在许多有性生殖动物的胚胎发育早期就已特化出来,并进一步分化为生殖细胞以产生新的子代。动物原生殖细胞的特化主要有生殖质决定和诱导两种模式,果蝇原生殖细胞的特化模式属于前者。研究表明,果蝇原生殖细胞特化过程中生殖质组装的关键基因是osk,其调控下游基因转录产物的定位和翻译,如vas和tud。此外,基因转录沉默是原生殖细胞特化过程的一个重要特征,其与生殖质中的成分如基因nos、gcl、pgc的表达产物密切相关。现对果蝇原生殖细胞特化分子机制进行综述。     

原始生殖细胞体外培养及应用研究

原始生殖细胞是来源于胚胎生殖嵴的一类具有多向分化潜能的干细胞,其形态、细胞表面标志、分化潜能均与来源于囊胚内细胞团的干细胞相似.在饲养细胞层和多种生长因子的共同作用下,可保持原生殖细胞在体外不断增殖而不分化,最终建立EG细胞系.本文就原始生殖细胞体外培养,建立EG细胞系及其应用前景作一综述.     

PAR6, A Potential Marker for the Germ Cells Selected to Form Primordial Follicles in Mouse Ovary

Partitioning-defective proteins (PAR) are detected to express mainly in the cytoplast, and play an important role in cell polarity. However, we showed here that PAR6, one kind of PAR protein, was localized in the nuclei of mouse oocytes that formed primordial follicles during the perinatal period, suggesting a new role of PAR protein. It is the first time we found that, in mouse fetal ovaries, PAR6 appeared in somatic cell cytoplasm and fell weak when somatic cells invaded germ cell cysts at 17.5 days post coitus (dpc). Meanwhile, the expression of PAR6 was observed in cysts, and became strong in the nuclei of some germ cells at 19.5 dpc and all primordial follicular oocytes at 3 day post parturition (dpp), and then obviously declined when the primordial follicles entered the folliculogenic growth phase. During the primordial follicle pool foundation, the number of PAR6 positive germ cells remained steady and was consistent with that of formed follicles at 3 dpp. There were no TUNEL (apoptosis examination) positive germ cells stained with PAR6 at any time studied. The number of follicles significantly declined when 15.5 dpc ovaries were treated with the anti-PAR6 antibody and PAR6 RNA interference. Carbenoxolone (CBX, a known blocker of gap junctions) inhibited the expression of PAR6 in germ cells and the formation of follicles. Our results suggest that PAR6 could be used as a potential marker of germ cells for the primordial follicle formation, and the expression of PAR6 by a gap junction-dependent process may contribute to the formation of primordial follicles and the maintenance of oocytes at the diplotene stage.     

哺乳动物原始生殖细胞体内特化和体外培养

原始生殖细胞(primordial germ cells, PGCs)是胚胎中最先出现的生殖细胞。PGCs来源于上胚层,最早出现在后肠,随后向生殖嵴迁移。这一过程伴随一系列复杂的分子调控机制,以及DNA甲基化重编程和组蛋白修饰等表观遗传过程。PGCs经过不断的分裂、发育及分化,最终形成配子。为了更好地研究PGCs发育与分化的调控和表观遗传过程,体外培养的研究变得越来越重要。本文以小鼠和人为例,介绍了哺乳动物PGCs的特化过程、PGCs特化过程中的表观遗传过程和PGCs的体外培养研究进展。     

On the Origin of Primordial Germ Cells in the Chick Embryo

An attempt was made to re-examine the location of the primordial germ cells (PGCs) in very young chick embryos. Freshly laid blastoderms, prior to hypoblast formation, of a known anterio-posterior axis, were transversely bisected and each half was separately grown in vitro. Both anterior and posterior halves were shown to be fertile and each was shown to contain roughly the same amount of PGCs as a normal control embryo. It has been concluded that in the chick as well as in the duck there is no concentration of cells containing germinal plasm in the posterior part of the blastoderm.
Two other possibilities should be investigated:
1. A concentric arrangement of cells containing germinal plasm. 2. The absence of a germinal plasm and a relatively late appearance of PGCs as a result of induction.     

Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.     

Dullard/Ctdnep1 Modulates WNT Signalling Activity for the Formation of Primordial Germ Cells in the Mouse Embryo

Dullard/Ctdnep1 is a member of the serine/threonine phosphatase family of the C-terminal domain of eukaryotic RNA polymerase II. Embryos lacking Dullard activity fail to form primordial germ cells (PGCs). In the mouse, the formation of PGCs is influenced by BMP4 and WNT3 activity. Although Dullard is reputed to negatively regulate BMP receptor function, in this study we found mutations in Dullard had no detectable effect on BMP4 and p-Smad activity. Furthermore Dullard mutations did not influence the dosage-dependent inductive effect of Bmp4 in PGC formation. However, Dullard may function as a positive regulator of WNT signalling. Combined loss of one copy each of Dullard and Wnt3 had a synergistic effect on the reduction of PGC numbers in the compound heterozygous embryo. In addition, loss of Dullard function was accompanied by down-regulation of WNT/β-catenin signalling activity and a reduction in the level of Dishevelled 2 (Dvl2). Therefore, Dullard may play a role in the fine-tuning of WNT signalling activity by modulating the expression of ligands/antagonists and the availability of Dvl2 protein during specification of the germ cell lineage.     

Regulation of Prolactin and Growth Hormone Production by Clonal Strains of Functional Pituitary Cells in Culture

This report describes the use of clonal strains of rat pituitarytumor cells to study the regulation of prolactin and growthhormone production. Emphasis is placed on the effects on prolactinproduction of the hypothalamic tripeptide pGlu-His-ProNH₂, alsocalled thyrotropin releasing hormone (TRH). TRH binds to specificcellular receptors and stimulates initially the release of previouslysynthesized prolactin; several hours later it stimulates thesynthesis of prolactin. Several kinds of experimental resultsare consistent with the hypothesis that cyclic AMP mediatesthe TRH-stimuIated release of prolactin. However, all of theeffects of TRH on these cells in culture are not mimicked bycyclic AMP analogs, for TRH decreases growth hormone productionwhile it increases the synthesis of prolactin, and the analogdibutyryl cyclic AMP increases the production of both hormones.     

影响牛胚胎干细胞分离克隆因素的研究

采用与同源胎儿成纤维细胞共同培养及传统饲养层培养方式,以高糖DMEM,添加0.1mM2-巯基乙醇,犊牛血清,细胞因子为培养基,以4-13周龄屠宰牛胎儿为实验材料,探讨影响牛原始生殖细胞分离克隆胚胎干细胞的相关因素,结果发现：当犊牛血清为15%时效果最好;细胞因子添加与否对胚胎干细胞的分离及同源牛胎儿成纤维细胞的贴壁与生长影响并不显著,而在传代过程中中有一定影响;以0.2%胰酶 0.04?TA为细胞消化液效果最佳;以同源胎儿成纤维细胞共培养的方式分离克隆牛胚胎干细胞,本研究观察到效果最好。     

Appearance of Primordial Germ Cells in Young Chick Blastoderms Cultured in vitro

Appearance of primordial germ cells (PGCs) in young chick blastoderms was investigated by the cultivation of only the epiblast or hypoblast. Presumptive PGCs exist in the epiblast before primitive-streak formation. They translocate gradually to the lower layer during early stages of primitive-streak formation, though substantial number of presumptive PGCs remain in the upper layer. The existing primary hypoblast under the epiblast is dispensable for the further differentiation of the PGCs.     

鸡胚血液中原始生殖细胞的分离及其培养的研究

从孵化４８～５５小时鸡胚中抽取血液,每只胚胎可获血液２～６μｌ左右。一步法离心分离原始生殖细胞,可使其浓度由０．１％以下提高到５０％以上。将多余血细胞用微量吸管移走后,加入添加１０％胎牛血清的ＴＣＭ—１９９做为培养基,３７．５℃,５％ＣＯ２,９５％空气,饱和湿度下培养,原始生殖细胞可成活２４小时左右。