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.     

Aggregation of Cells from Early Chick Blastoderms

The aggregation of dissociated cells from chick blastoderms at Hamburger and Hamilton stages 1–5 was studied. Aggregation was measured during the first 4 h of culture by determination of the proportion of single cells in the medium. No difference in aggregation was found when cells dissociated by either trypsin or EDTA were studied. Similarly the presence or absence of serum in the medium had no appreciable effect on early phases of aggregation, although at 24 h and thereafter, aggregate size was reduced in serum-free cultures. It was found that at all of the stages studied, cells aggregate and sort out into two groups. One group forms a continuous phase of loosely associated cells while the other segregates into several localised areas of closely associated cells within the aggregate. Examination of aggregates up to 7 days in culture showed progressive differentiation within each phase and several identifiable cell types were observed. Basal laminae were present at the boundary between the compact phase and the loose phase.     

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。     

Improvement of Transfection Efficiency in Cultured Chicken Primordial Germ Cells by Percoll Density Gradient Centrifugation

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifing the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.     

猪原始生殖细胞生物学特性的研究

胚胎生殖细胞（embryonic germ cell,EGC）是由胎儿原始生殖细胞（primordial germ cell,PGC）经体外驯化培养获得的一种多潜能干细胞。研究猪PGC生物学特性对于建立猪EGC及了解猪生殖细胞发育机制具有重要意义。该研究以原代培养的猪PGC为对象,探讨了其生长行为特征及其重编程过程中多能性、生殖系标志基因的表达模式。结果显示,26 d胚胎生殖嵴分离的PGC呈碱性磷酸酶阳性,细胞体积及核质比较大;体外培养初期呈现出较强的增殖及迁移能力,培养第5 d细胞增殖达到平台期,此时克隆高表达Oct4、Sox2、Nanog、c-Myc、Klf4和Ifi tm3（P〈0.05）,低表达Blimp1（P〈0.05）,Nanos1和Stella的表达水平与猪胎儿成纤维细胞无差异;猪PGC形成的原代克隆已经具有多向分化潜能。     

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。     

Ultrastructural Evidence that Chick Primordial Germ Cells Leave the Blood-Vascular System Prior to Migrating to the Gonadal Anlagen

It is known that chick primordial germ cells (PGCs), after separation from the endoderm in early embryonic development, temporarily circulate via the blood-vascular system and eventually migrate to the gonadal anlagen. However, direct evidence that circulating PGCs leave the blood vessels is lacking. The purpose of present study is to describe the ultrastructural features of PGCs as they emerge from the blood vessels. PGCs leaving the blood vessels were first examined with semi-thin sections stained with toluidine blue. Then, some of the sections were re-embedded in Epon 812, and sectioned for electron microscopy. PGCs were observed emerging from the capillaries in the region posterior to the omphalomesenteric arteries of the embryo, between the splanchnic mesoderm and open-gut endoderm, at stages 15–18 (about 2.5 days of incubation). Ultrastructurally, PGCs exhibited the protruding, bulge-like cytoplasmic processes through the endothelial gaps in the capillary walls. Prior to emerging, intravascular PGCs seemed to stick to the endothelium of the blood vessels. Thus, our results offer ultrastructural evidence that the circulating PGCs exit the blood vessels prior to migrating to the gonadal anlagen.     

The Binding of Cations to the Surfaces of Cells from Early Chick Blastoderms

Cells of the early chick blastoderms are either preparing for or undergoing regulated morphogenetic movements which culminate in the formation of a three-layered embryo. Information on the changes in the physical-chemical properties of cell surfaces may help in the understanding of this process. The binding of magnesium, manganese, strontium, barium and lanthanum to surfaces of early embryonic cells was estimated by the changes induced by these cations in the cells' electrophoretic mobilities (EPM). Cells show a positive EPM at concentrations of MgCl₂ and MnCl₂ at 3 × 10⁻² M while SrCl₂, and BaCl₂ were not able to reverse the cells' charge at concentrations up to 6 × 10⁻² M. CaCl₃ reversed the cells' EPM at concentrations as low as 5 × 10⁻³ M.
Our results suggest that the surfaces of early embryonic cells have a high affinity for Mg and Mn. This is indicated by a reversal of polarity which cannot be detected in cells of differentiating or adult tissues at the cation concentrations used in these experiments.     

Primordial Germ Cell-Like Cells Differentiated In Vitro from Skin-Derived Stem Cells

Background

We have previously demonstrated that stem cells isolated from fetal porcine skin have the potential to form oocyte-like cells (OLCs) in vitro. However, primordial germ cells (PGCs), which must also be specified during the stem cell differentiation to give rise to these putative oocytes at more advanced stages of culture, were not systematically characterized. The current study tested the hypothesis that a morphologically distinct population of cells derived from skin stem cells prior to OLC formation corresponds to putative PGCs, which differentiate further into more mature gametes.

Methodology/Principal Findings

When induced to differentiate in an appropriate microenvironment, a subpopulation of morphologically distinct cells, some of which are alkaline phosphatase (AP)-positive, also express Oct4, Fragilis, Stella, Dazl, and Vasa, which are markers indicative of germ cell formation. A known differentially methylated region (DMR) within the H19 gene locus, which is demethylated in oocytes after establishment of the maternal imprint, is hypomethylated in PGC-like cells compared to undifferentiated skin-derived stem cells, suggesting that the putative germ cell population undergoes imprint erasure. Additional evidence supporting the germ cell identity of in vitro-generated PGC-like cells is that, when labeled with a Dazl-GFP reporter, these cells further differentiate into GFP-positive OLCs.

Significance

The ability to generate germ cell precursors from somatic stem cells may provide an in vitro model to study some of the unanswered questions surrounding early germ cell formation.     

Purification of Primordial Germ Cells from TNAPMouse Embryos Using FACS-gal

Tissue nonspecific alkaline phosphatase (TNAP), the product of theAkp2locus, is expressed in mouse primordial germ cells (PGC) for an extensive period during embryogenesis. Mice with theAkp2^tm1Sormutant allele of TNAP expresslacZ(β-galactosidase; β-gal) under control of theAkp2locus. PGCs were purified fromAkp2^tm1Sorembryos using fluorescence activated cell sorting of β-gal expressing cells (FACS-gal). Analysis of the purified cells by alkaline phosphatase staining and immunocytochemistry with anti-c-kitantibody demonstrated that highly (98%) purified PGCs can be isolated using this method. This technique will facilitate experiments that require highly purified preparations of PGCs including cell culture and gene expression analyses.     

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.     

Derivation of Intermediate Pluripotent Stem Cells Amenable to Primordial Germ Cell Specification

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Indispensability of Iron for the Growth of Cultured Chick Cells

In order to clarify the role of iron in the growth promoting effect of transferrin (Tf), the effects of the following substances were examined in cultured chick skeletal myogenic cells: transition metal ions (Fe²⁺, Fe³⁺, Cr³⁺, Cu²⁺, Mn²⁺, Co²⁺, Cd²⁺, Zn²⁺ and Ni²⁺), Tf complexes with these metals and metal-free apoTf.
The cells did not grow well when incubated in a culture medium composed of Eagle's minimum essential medium and horse serum. But they grew well in the presence of Fe²⁺ or Fe³⁺ (10–100 μM) or iron-bound Tf (10–500 nM) in the medium. None of the transition metal ions other than iron was effective. Neither apoTf nor Tf complexes with these metals showed the growth promoting effect. The generality of the requirement of iron for cell growth was ascertained in the primary culture of other types of chick embryonic cells: fibroblasts, cardiac myocytes, retinal pigment cells and spinal nerve cells.
The results show that iron is one of the indispensable substances for cell growth and suggest that Tf protein plays a role in facilitating the transport of iron into the cells.     

Licensing of Primordial Germ Cells for Gametogenesis Depends on Genital Ridge Signaling

In mouse embryos at mid-gestation, primordial germ cells (PGCs) undergo licensing to become gametogenesis-competent cells (GCCs), gaining the capacity for meiotic initiation and sexual differentiation. GCCs then initiate either oogenesis or spermatogenesis in response to gonadal cues. Germ cell licensing has been considered to be a cell-autonomous and gonad-independent event, based on observations that some PGCs, having migrated not to the gonad but to the adrenal gland, nonetheless enter meiosis in a time frame parallel to ovarian germ cells -- and do so regardless of the sex of the embryo. Here we test the hypothesis that germ cell licensing is cell-autonomous by examining the fate of PGCs in Gata4 conditional mutant (Gata4 cKO) mouse embryos. Gata4, which is expressed only in somatic cells, is known to be required for genital ridge initiation. PGCs in Gata4 cKO mutants migrated to the area where the genital ridge, the precursor of the gonad, would ordinarily be formed. However, these germ cells did not undergo licensing and instead retained characteristics of PGCs. Our results indicate that licensing is not purely cell-autonomous but is induced by the somatic genital ridge.     

Specific Insulin-Mediated Regulation of Glutamine Synthetase in Cultured Chick Astroglial Cells

The expression of glutamine synthetase (GS; L-glutamate ammonia ligase; EC 6.3.1.2) in primary cultures of chick astroglial cells and neurons grown in a chemically defined medium, with and without insulin added, was investigated. An inhibitory effect of insulin toward GS activity, and specific to chick astroglial cells, was observed. Neurons in culture were not sensitive to the hormone effect. Modulation of the activating effect of hydrocortisone on glial GS by insulin was also observed. The data suggest that insulin contributes to the regulation of the metabolism of amino acid neurotransmitters via its effect on GS.