A quantitative assay for concanavalin A- and Ricinus communis agglutinin-mediated agglutinations of rat ascites hepatoma cells: Relationship between concanavalin a binding and cell agglutination

A simple quantitative assay method was developed for the agglutination of rat ascites hepatoma cells mediated by Concanavalin A or Ricinus communis agglutinin. This method was based on the principle that the turbidity of a cell suspension is proportional to the sum of the cross-sectional area of cells and aggregates. As predicted by the theoretical consideration, the turbidity decreased when cells were aggregated and the decrease was a function of the average number of the cells in aggregates.The agglutinability of the cells, judged by this method, showed a maximum value at a certain concentration of the agglutinin. By further addition of the agglutinin, the agglutinability slightly decreased from the maximum. These phenomena were observed both for Concanavalin A and Ricinus communis agglutinin. The binding and the agglutination experiments using [³H] concanavalin A revealed that the binding to approx. 20% of the total receptors caused a maximal agglutination. This suggested that the receptors responsible for the agglutination constitute only a small part of the total receptors on the surface.     

A quantitative assay for concanavalin A- and Ricinus communis agglutinin-mediated agglutinations of rat ascites hepatoma cells. Relationship between concanavalin A binding and cell agglutination.

A simple quantitative assay method was developed for the agglutination of rat ascites hepatoma cells mediated by Concanavalin A or Ricinus communis agglutinin. This method was based on the principle that the turbidity of a cell suspension is proportional to the sum of the cross-sectional area of cells and aggregatesmas predicted by the theoretical consideration, the turbidity decreased when cells were aggregated and the decrease was a function of the average number of the cells in aggregates. The agglutinability of the cells, judged by this method, showed a maximum value at a certain concentration of the agglutinin. By further addition of the agglutinin, the agglutinability slightly decreased from the maximum. These phenomena were observed both for Concanavalin A and Ricinus communis agglutinin. The binding and the agglutination experiments using [3-H]concanavalin A revealed that the binding to approx;0% of the total receptors caused a maximal agglutination. This suggested that the receptors responsible for the agglutination constitute only a small part of the total receptors on the surface.     

Germ line chimera produced by transfer of cultured chick primordial germ cells.

Intrinsic primordial germ cells (PGCs) from stage 27 (5-day-old) chick embryonic germinal ridges were cultured in vitro for a further 5 days, and shown to proliferate on stroma cells derived from the germinal ridge. To determine whether these cultured PGCs could colonize and contribute to the germ-line, PGCs were isolated by gentle pipetting, labeled with PKH26 fluorescent dye and injected into the blood stream of stage 17 (2.5-day-old) chick embryos. The recipient embryos were incubated until they reached stage 28. Thin sections of these embryos were analysed by fluorescent confocal laser microscopy. These analyses showed that the labeled donor PGCs had migrated into the germinal ridges of the recipient embryos, and transplanted PGCs had undergone at least 3-7 divisions. These results suggest that PGCs that had passed far beyond the migration stage in vivo were still able to migrate, colonize and proliferate in recipient chick embryonic gonads.     

不同时期鸡胚原始生殖细胞分离的研究

采用Ficoll密度梯度离心,酶解离两种方法在鸡胚孵化的第14期、19期、28期,分离、培养鸡胚中的原始生殖细胞(PGCs)。探索PGCs分离、培养的适宜时期及方法,以期获得较多数量,较高活力的PGCs作介导生产转基因鸡。结果表明：1．提取、分离PGCs的最佳时期依次为19期、28期。2．两种分离方法均能分离到一定数量的PGCs细胞。但在19期和28期,酶解离法分离到的PGCs的相对数量较多,存活时间较长,是一种较适宜的分离方法。     

Lectin-mediated agglutination of murine lymphoma cells. Cell surface deformability and reversibility of agglutination by saccharides.

Agglutination of S49 mouse lymphoma cells by Ricinus communis I agglutinin can be reversed by the competing haptenic saccharide, lactose, soon after agglutination, but after further incubation in the absence of lectin the agglutination reaction could not be reversed by lactose and the cells remained as multicell aggregates. The irreversibility of S49 cell agglutination was time, temperature and lectin concentration dependent and its onset correlated with ultrastructurally observed deformation of adjacent cell surfaces and an increase in the proportion of adjacent cell surface areas in close apposition within multicell aggregates. Pretreatment of S49 cells with cytochalasin B or cytochalasin B plus vinblastine enhanced R. communis I agglutinin-mediated agglutination, while vinblastine alone and fluoride plus azide had essentially no effect. When drug-treated cells were agglutinated and then incubated in lectin-free drug-containing media for various times prior to lactose addition, the drug effects were more pronounced. Cytochalasin B alone or with vinblastine inhibited lactose reversal of S49 cell agglutination compared to the drug-free controls, while fluoride plus azide enhanced hapten reversibility. Electron microscopic analysis revealed that the onset of agglutination irreversibility correlated with cell surface deformation in the drug-treated cells. Cell aggregates that were more readily reversible by lactose (fluoride plus azide) were unchanged or less deformed, while S49 aggregates treated with cytochalasin B plus vinblastine were more deformed compared to controls without drugs. These experiments suggest a role for cell surface deformability as an important secondary effect during lectin-mediated cell agglutination of S49 lymphoma cells.     

Production of germ line chimera by transfer of primordial germ cells in the domestic chicken (Gallus domesticus)

Blood was collected from Stage 13 to 14 (1) chick embryos. Primordial germ cells (PGCs) were separated from blood cells by Ficoll density gradient centrifugation. One hundred Rhode Island Red PGCs per embryo were transferred to the blood stream of Stage 14 to 15 White Leghorn embryos. Also, one hundred White Leghorn PGCs per embryo were transferred to the blood stream of Stage 14 to 15 Rhode Island Red embryos. Hatched male and female chicks were raised until sexual maturity, and progeny tests were performed by mating these PGC recipients with Rhode Island Red chickens of the opposite sex. Chicks apparently derived from the transferred PGCs, based on the feather color of the chicks, were produced from all 4 possible mating combinations. The present results indicate that the germ line of PGC recipient chickens consists of 2 distinct populations of germ cells.     

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12–15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.     

Isolation of teleost primordial germ cells using flow cytometry

Primordial germ cells (PGCs) generate gametes, the only cells that can transmit genetic information to the next generation. A previous report demonstrated that a fusion construct of green fluorescent protein (gfp) and zebrafish nos 1 3UTR mRNA could be used to label PGCs in a number of fish species. Here, we sought to exploit this labeling strategy to isolate teleost PGCs by flow cytometry (FCM), and to use these isolated PGCs to examine germ cell migration to the gonadal region. In zebrafish, medaka and goldfish, the PGCs were labeled by injecting the gfp-nos1 3UTR mRNA into 1- 4 cell embryos. When the embryos had developed to the somitogenesis or later stages, they were enzymatically disaggregated and GFP positive cells isolated using FCM. PGCs in the different species clustered in the same segments of the FCM scatter diagrams for total embryonic cells produced by plotting the forward scatter intensity against GFP intensity. In situ hybridization showed that the sorted zebrafish cells expressed vasa RNA in their cytoplasm, suggesting that they were PGCs. When the migration ability of the sorted cells from zebrafish was examined in an in vivo transplantation experiment, approximately 30% moved to the gonadal region of host embryos. These observations demonstrate that PGCs can be isolated without use of transgenic fishes and that the isolated PGCs retain the ability to migrate. Our data indicate that this technique will be of value for isolating PGCs from a range of fish species.     

Lectin-mediated agglutination of murine lymphoma cells. Cell surface deformability and reversibility of agglutination by saccharides

Agglutination of S49 mouse lymphoma cells by Ricinus communis I agglutinin can be reversed by the competing haptenic saccharide, lactose, soon after agglutination, but after further incubation in the absence of lectin the agglutination reaction could not be reversed by lactose and the cells remained as multicell aggregates. The irreversibility of S49 cell agglutination was time, temperature and lectin concentration dependent and its onset correlated with ultrastructurally observed deformation of adjacent cell surfaces and an increase in the proportion of adjacent cell surface areas in close apposition within multicell aggregates. Pretreatment of S49 cells with cytochalasin B or cytochalasin B plus vinblastine enhanced R. communis I agglutinin-mediated agglutination, while vinblastine alone and fluoride plus azide had essentially no effect. When drug-treated cells were agglutinated and then incubated in lectin-free drug-containing media for various times prior to lactose addition, the drug effects were more pronounced. Cytochalasin B alone or with vinblastine inhibited lactose reversal of S49 cell agglutination compared to the drug-free controls, while fluoride plus azide enhanced hapten reversibility. Electron microscopic analysis revealed that the onset of agglutination irreversibility correlated with cell surface deformation in the drug-treated cells. Cell aggregates that were more readily reversible by lactose (fluoride plus azide) were unchanged or less deformed, while S49 aggregates treated with cytochalasin B plus vinblastine were more deformed compared to controls without drugs. These experiments suggest a role for cell surface deformability as an important secondary effect during lectin-mediated cell agglutination of 849 lymphoma cells.     

Differences between normal and scaleless (sc/sc) chicken epidermal cell surfaces detected with wheat germ agglutinin

Dissociated epidermal cells derived from the backskin of scaleless chick embryos (stage 34 or 35) form larger agglutinates with wheat germ agglutinin (WGA) than epidermal cells from normal embryonic skin. [³H]Acetyl WGA binding to the scaleless cells is twice as great as to normal epidermal cells. Treatment of these cells with concanavalin A (conA) results in equivalent agglutination of both mutant and normal epidermal cells, whereas neither scaleless nor normal epidermal cells are agglutinated by Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) or Ulex europeus agglutinin (UEA). This alteration in cell surface carbohydrates may be related to the failure of the scaleless mutant embryonic epidermis to undergo normal morphogenesis.     

Changing levels of uv light and carcinogen-induced unscheduled DNA synthesis in mouse oocytes during meiotic maturation.

The effects of cytochalasin B (CB) and colchicine on the lectin-mediated agglutination of dissociated cells from chick embryos at the early primitive streak stage were studied. Cells incubated in the absence of the above-mentioned drugs were agglutinable with concanavalin A (ConA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA). A pre-incubation with neuraminidase was required to render the cells agglutinable with soybean agglutinin (SBA). This treatment had no appreciable effect on the agglutinability of the cells with the other three lectins. Treatment with the drug colchicine had no appreciable effect on the extent of agglutination with any of the above-mentioned lectins. Cells treated with CB dissolved in dimethylsulfoxide (DMSO) in saline, exhibited a reduced lectin-mediated agglutinability. However, a similar decline in agglutinability was observed in controls incubated in saline containing DMSO alone. It is suggested that structures sensitive to colchicine and CB do not play a major role in the control of surface lectin receptors in early embryonic cells.     

Production of germline chimeric chickens following the administration of a busulfan emulsion

Busulfan (1,4-butanediol dimethanesulfonate) was used to deplete endogenous germ cells for the enhanced production of chicken germline chimeras. Utilizing immunohistochemical identification of primordial gem cells (PGCs) in Stage 27 chicken embryos, two delivery formulations were compared relative to the degree of endogenous PGC depletion, a busulfan suspension (BS) and a solublized busulfan emulsion (SBE). Both busulfan treatments resulted in a significant reduction in PGCs when compared to controls. However, the SBE resulted in a more consistent and extensive depletion of PGCs than that observed with the BS treatment. Repopulation of SBE-treated embryos with exogenous PGCs resulted in a threefold increase of PGCs in Stage 27 embryos. Subsequently, germline chimeras were produced by the transfer of male gonadal PGCs from Barred Plymouth Rock embryos into untreated and SBE-treated White Leghorn embryos. Progeny testing of the presumptive chimeras with adult Barred Plymouth Rock chickens was performed to evaluate the efficiency of germline chimera production. The frequency of germline chimerism in SBE-treated recipients increased fivefold when compared to untreated recipients. The number of donor-derived offspring from the germline chimeras also increased eightfold following SBE-treatment of the recipient embryos. These results demonstrated that the administration of a busulfan emulsion into the egg yolk of unincubated eggs improved the depletion of endogenous PGCs in the embryo and enhanced the efficiency of germline chimera production.     

Detection and characterization of primordial germ cells in pheasant (Phasianus colchicus) embryos

The developmental similarity between the chicken and pheasant (Phasianus colchicus) allows the novel biotechnologies developed in the chicken to be applied to the production of transgenic pheasants and interspecies germline chimeras. To detect pheasant primordial germ cells (PGCs) efficiently, which is important for inducing germline transmission, the ultrastructure of PGCs and their reactivity to several antibodies (2C9, QB2, anti-SSEA-1, and QCR1) and periodic acid-Schiff's solution (PAS) were examined. To obtain PGCs, blood was taken from embryos incubated for 62-72 h or from gonads from embryos incubated for 156-216 h. The PGCs collected from both sources had the typical ultrastructure of pluripotent cells: a large nucleus with a distinct nucleolus, a high ratio of nuclear to cytoplasmic volume, and a distinct cytoplasmic membrane. In comparing the morphology of PGCs collected from different sites, more mitochondria and better-developed membrane microvilli were found in gonadal PGCs than in circulating PGCs. The nucleus of gonadal PGCs was flattened and had a large eccentrically positioned nucleolus. Of the antibodies tested, only QCR1 antibody reacted with an epitope in pheasant PGCs, and no specific signal was detected to other antibodies. The temporal change in the PGC populations in the blood and gonads of embryos was examined. In blood, the population was greater (P < 0.0001) in embryos incubated for 64 h than in embryos incubated for 62 or 66-72 h (31.4 versus 5.6-16.2 microL(-1)). In embryonic gonads, the number of PGCs increased continuously from 156 to 216 h of incubation (193-2,718 cells/embryo), although the ratio of PGCs to total gonadal cells did not change significantly (0.50-0.61%). In conclusion, pheasant PGCs have typical germ cell morphology and possess the QCR1 epitope. Circulating blood and the gonads of embryos incubated for 64 and 216 h, respectively, are good sources of PGCs.     

Inter-species transplantation and migration of primordial germ cells in cyprinid fish

Primordial germ cells (PGCs) are the only cells in developing embryos that can transmit genetic information to the next generation. PGCs therefore have considerable potential value for gene banking and cryopreservation, particularly via production of donor gametes using germ-line chimeras. In some animal species, including teleost fish, the feasibility of using PGC transplantation to obtain donor-derived offspring, within and between species, has been demonstrated. Successful use of PGC transplantation to produce germ-line chimeras is absolutely dependent on the migration of the transplanted cells from the site of transplantation to the host gonadal region. Here, we induced germ-line chimeras between teleost species using two different protocols: blastomere transplantation and single PGC transplantation. We evaluated the methods using the rate of successful migration of transplanted PGCs to the gonadal region of the host embryo. First, we transplanted blastomeres from zebrafish, pearl danio, goldfish, or loach into blastula-stage zebrafish embryos. Some somatic cells, derived from donor blastomeres, were co-transplanted with the PGCs and formed aggregates in the host embryos; a low efficiency of PGC transfer was achieved. Second, a single PGC from the donor species was transplanted into a zebrafish embryo. In all inter-species combinations, the donor PGC migrated toward the gonadal region of the host embryo at a comparatively high rate, regardless of the phylogenetic relationship of the donor and host species. These transplantation experiments showed that the mechanism of PGC migration is highly conserved beyond the family barrier in fish and that transplantation of a single PGC is an efficient method for producing inter-species germ-line chimeras.     

Xenogenic oogenesis of chicken (Gallus domesticus) female primordial germ cells in germline chimeric quail (Coturnix japonica) ovary

In present study, chicken primordial germ cells (PGCs) were transferred into quail embryos to investigate the development of these germ cells in quail ovary. Briefly, 2 microl of chicken embryonic blood (stage 14) or about 100 purified circulating PGCs were transferred into quail embryo. Contribution of chicken PGCs were detected in gonads of chimeric quail embryos (stage 28) by immunocytochemical staining of cell surface antigen SSEA-1, and by in situ hybridization (ISH) with female chicken specific DNA probe. As a result, 52.0+/-43.2 (n=18) and 42.7+/-27.3 (n=17) chicken PGCs were found in the gonads of chimeric quail embryo that was injected with chicken embryonic blood (stage 14) and about 100 purified circulating PGCs, respectively. Furthermore, the ovaries of 81.8% (9/11) 12 days post incubation (dpi) chimeric quail embryos were observed with a mean of 457.6+/-237.1 female chicken PGCs-derived oogonia scattered in ovarian cortex area. In 9 out of 12 newly hatched and one week old chimeric quail chicks, on average of 2883.0+/-1924.1 primary oocytes and 3 follicles derived from chicken PGCs were found, respectively. The present results suggest that chicken female PGCs are able to migrate, colonize, proliferate and differentiate into oogonia, primary oocytes in chimeric quail ovary.     

Simple method for isolation of primordial germ cells from chick embryos.

A simple one step centrifugation method was developed for purification of primordial germ cells (PGCs) of chick embryos. PGCs, constituting less than 0.1% of the total blood cells of stage 13-14 embryos that contained a microliter amount of blood, were concentrated at the interface of a 6.3% (w/v) and 14.4% (w/v) Ficoll bilayer by centrifugation at 800 x g for 30 min. the purity of these PGCs was 86%, which was 22 times that obtained previously.     

大鼠原生殖细胞培养和分化的研究

研究大鼠胚胎原生殖细胞（primordial germ cells,PGCs)的培养及分化,取受精后11-12.5天大鼠PGCs进行原代培养,光、电镜观察PGCs及其分化细胞的微细结构,碱性磷酸酶染色检测细胞的分化程度,结果显然显示大鼠PGCs大而圆,散在分布,或多个聚集成团,胞质中含有椭圆形的线粒体和丰富的核糖体,在鼠胚成纤维细胞饲养层存在的情况下,PGCs保持未分化状态,碱性磷酸酶反应呈强阳性,在缺乏饲养层的条件下PGCs很快分化,形态不规则,有伪足,碱性磷酸酶反应减弱,进一步分化可形成具有细长突起的神经元样细胞,胞质中含有细丝束的表皮细胞,可见节律性跳动的心肌细胞,具有分泌颗粒的分泌细胞及似血管,心脏形状的管腔结构等,由PGCs分化来的细胞碱性磷酸酶反应均呈阴性,结果表明大鼠PGCs能够分化形成三个胚层的衍生物,生殖嵴来源的PGCsp是一种具有发育全能性的胚胎多能干细胞,本研究同时证明鼠胚饲养层能抑制大鼠PGCs的分化。     

Loss of heterochromatin protein 1 gamma reduces the number of primordial germ cells via impaired cell cycle progression in mice

Signals from extraembryonic tissues in mice determine which proximal epiblast cells become primordial germ cells (PGCs). After their specification, approximately 40 PGCs appear at the base of the allantoic bud and migrate to the genital ridges, where they expand to about 25?000 cells by Embryonic Day (E)13.5. The heterochromatin protein 1 (HP1) family members HP1alpha, HP1beta, and HP1gamma (CBX5, CBX1, and CBX3, respectively) are thought to induce heterochromatin structure and to regulate gene expression by binding methylated histone H3 lysine 9. We found a dramatic loss of germ cells before meiosis in HP1gamma mutant (HP1gamma(-/-)) mice that we generated previously. The reduction in PGCs in HP1gamma(-/-) embryos was detectable from the early bud stage (E7.25), and the number of HP1gamma(-/-) PGCs was gradually reduced thereafter. Bromodeoxyuridine incorporation into PGCs was significantly reduced in E7.25 and E12.5 HP1gamma(-/-) embryos. Furthermore, a lower proportion of HP1gamma(-/-) PGCs than wild-type PGCs was in S phase, and a higher proportion, respectively, was in G1 phase at E12.5. Moreover, the proportion of p21 (Cip, official symbol CDKN1A)-positive HP1gamma(-/-) PGCs was increased, suggesting that the G1/S phase transition was inhibited. However, no differences were detected between fate determination, migration, apoptosis, or histone modification of PGCs of control embryos and those of HP1gamma(-/-) embryos. Therefore, the reduction in PGCs in HP1gamma(-/-) embryos could be caused by impaired cell cycle in PGCs. These results suggest that HP1gamma plays an important role in keeping enough germ cells by regulating the PGC cell cycle.     

Lectin-mediated agglutination of preimplantation mouse embryos

Plant lectins were used to monitor qualitative changes in carbohydrate-containing receptors during preimplantation mouse development. Beginning at the morula stage, an age-related decline was observed in agglutination of early mouse embryos by concanavalin A (ConA). In contrast, wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA) agglutinated embryos strongly throughout preimplantation development.