Category Archives: Growth Factors

Role of Fibroblast Growth Factors: DISCUSSION(4)

The ability to generate EG cell colonies from PGCs declines beyond 11.5 dpc, with only male EG cells being obtained from 12.5 dpc embryos. Reduced expression of an FGF receptor in female cells provides an explanation for this observation and suggests a mechanism. In both male and female germ cells, c-kit RNA levels decline at 13.5 dpc. It is interesting to speculate that a function of FGFR activation is to maintain c-kit expression in PGCs, thereby contributing to the proliferative stage. This model would predict that in females, FGF receptor expression is reduced by 13.5 dpc, such that the down-regulation of c-kit cannot be prevented by treatment with bFGF. In male PGCs, continuing FGFR expression and subsequent activation may maintain c-kit expression. antibiotics levaquin

Role of Fibroblast Growth Factors: DISCUSSION(3)

DISCUSSION(3)

These areas of FGF4 expression in the preimplantation and postimplantation embryo closely match expected locations for PGCs. Other FGFs, especially FGF-3 and FGF-5, have also been shown to be expressed in tissues immediately adjacent to those expressing FGF-4. These observations suggest that FGFs may be continuously available to premigratory and migratory PGCs. Mice lacking either a functional FGF-3 or FGF-5 gene are fertile, indicating that some PGCs mature in the absence of either factor. Embryos deficient in FGF-4 cease development prior to PGC proliferation, as do embryos lacking functional FGFR-1, preventing an analysis of PGC development in these embryos. Mice lacking a functional FGFR-2 are not presently available. antibiotic levaquin

Role of Fibroblast Growth Factors: DISCUSSION(2)

At present, there are 16 members of the FGF ligand family. The typical FGF receptor has a split intracellular tyrosine kinase domain and an extracellular domain containing up to three ligand-binding immunoglobin-like loops. Alternative splicing within the extracellular domains of FGFR genes can generate great diversity of ligand-binding domains, which can result in different ligand-binding specificities. Additionally, each FGF receptor may transduce a particular mitogenic potential. However, as individual receptors can be activated by several different ligands, specificity may partly depend upon spatial and temporal expression patterns. An exception is FGF7 (keratinocyte growth factor, KGF), which selectively activates a differentially spliced form of FGFR-2. Using RNase protection analysis, we have detected KGF receptor transcripts in urogenital ridge RNA (unpublished results). Neither germ cell nor gonadal defects have been observed in KGF-deficient mice. buy cheap antibiotics

Role of Fibroblast Growth Factors: DISCUSSION(1)

DISCUSSION(1)

The results presented here confirm previous observations that bFGF stimulates PGC growth in culture. This effect is apparent in the absence of a preformed feeder layer or with purified PGCs plated on a feeder layer. Basic FGF stimulates PGC growth on a variety of feeder layers including bone marrow-derived stromal cells (Sl220), primary mouse fibroblasts, and the established mouse fibroblast cell lines STO, NIH3T3, and 10T1/2 cells (unpublished results). The ability of bFGF to stimulate PgC growth independently of the feeder cell type suggests that it may exert its action, in part, directly on the PGCs. birth control yasmin

Role of Fibroblast Growth Factors: RESULTS(3)

PGCs Bound Iodinated bFGF

To more completely distinguish FGF receptor expression in germ cells from that in somatic cells, and to confirm that PGCs express a surface FGF-binding activity, we developed a method in which ex vivo PGCs are incubated with 125I-bFGF, fixed on microscope slides, stained for alkaline phosphatase, and exposed to autoradiographic emulsion. Figure 4 presents a sample photomicrograph from such an experiment performed on 11.5 dpc genital ridges. The presence of autoradiographic grains over alkaline phosphatase expressing PGCs is clearly seen. It is also notable that somatic cells (not expressing alkaline phosphatase) in this preparation bound bFGF, as might be expected for mesodermally derived genital ridge tissue. The level of 125I-bFGF binding by alkaline phosphatase-positive and by alkaline phosphatase-negative cell types appeared comparable. The specificity of this binding was demonstrated by counting grains over stained PGCs and was compared to that of samples in which an excess of unlabeled bFGF had been added (Fig. 5). Binding of 125I-bFGF by most somatic cells was also sensitive to the presence of unlabeled bFGF (not shown). buy flovent inhaler

Role of Fibroblast Growth Factors: RESULTS(2)

RESULTS(2)

FGFR-1 and FGFR-2 Were Expressed in the Fmbryonic Gonad and Were Readily Detectable in Highly Purified PGC RNA

To further explore whether bFGF acts directly on PGCs, we sought to determine whether PGCs express an FGF receptor. Four murine high-affinity tyrosine kinase receptor genes for FGFs have been identified to date. To investigate whether PGCs might express one of these receptors, 11.5 dpc genital ridge RNA was tested for expression for all four genes by RNase protection assays. FGFR-1 and FGFR-2 RNA were readily detected in the genital ridge RNA (Fig. 2). buy yasmin online

Role of Fibroblast Growth Factors: RESULTS(1)

Basic FGF Stimulated the Growth of Premigratory and Postm igratory PGCs

To more thoroughly define the functions of bFGF in the in vitro PGC culture system, we plated 11.5 dpc PGCs in the presence of bFGF under a variety of conditions. As previously shown, bFGF stimulated the accumulation of PGCs plated on mitotically inactivated STO fibroblast feeder layers (Fig. 1A). We have previously shown that the optimal stimulatory effect of bFGF in this system occurs at a bFGF concentration of 1 ng/ml. Given the important role that LIF plays in PGC culture, and as bFGF can rapidly increase the accumulation of LIF RNA by STO feeder layers (unpublished results), we questioned whether the effect of bFGF on PGCs was indirect, acting primarily through the feeder layer. buy ventolin inhalers

Role of Fibroblast Growth Factors: MATERIALS AND METHODS(4)

MATERIALS AND METHODS(4)

Purified populations of PGCs were isolated as described above. RNA probes labeled with 32P were prepared and RNase protection assays performed as described previously and analyzed on 6% acrylamide-urea sequencing gels. A p-actin internal control (Ambion, Austin, TX) was included in each assay. The protected fragments used were the 277-base pair (bp) PvuII to SphI fragment of the extracellular domain of FGF receptor (FGFR)-1, the 326-bp EcoRV to EcoRI fragment in the extracellular domain of FGFR-2, the DdeI fragment in the extracellular domain of FgFr-3 , and the 387-bp EcoRV to BamHI fragment of FGFR-4. All fragments were subcloned in p-Bluescript SK or KS and transcribed with T7 or T3 polymerase (Stratagene, La Jolla, CA) as appropriate. buy cipro

Role of Fibroblast Growth Factors: MATERIALS AND METHODS(3)

Slides were immersed twice in a 1:1 mixture of Kodak NTB-2 (Eastman Kodak, Rochester, NY) autoradiographic emulsion:water at room temperature and stored in the dark at 4°C for 26 days prior to development. Factor binding to the cell surface was visualized by the appearance of grains caused by 125I exposure of the photographic emulsion. flovent inhaler

Differences between Experimental and Plus Competitor means, as well as trend hypotheses, were determined with the use of /-tests and Wilcoxon rank sum tests. To control the type I family error rate, we used the Bonferroni criterion and set the alpha level at 0.01 to be required for significance. As outcomes from /-tests and Wilcoxon tests gave identical interpretations, probability values reported are from /-tests.

Role of Fibroblast Growth Factors: MATERIALS AND METHODS(2)

MATERIALS AND METHODS(2)

FGF-Binding Assays

PGC cell suspensions were obtained as described above, but trypsinization was terminated after 5 min at 37°C by addition of one drop of FBS. The cell suspensions were quickly washed twice in 1 ml binding buffer (a 1:1 mixture of DMEM and Ham’s F-12 supplemented with insulin and transferrin [Sigma Chemical Co., St. Louis, MO] and 0.1% BSA). Cell suspensions were diluted to 6 X 106 total cells/ ml. Cell suspension (100 ^l) was incubated with 750 000 cpm of 125I-bFGF (800-1200 Ci/mmol; Amersham, Arlington Heights, IL) with agitation for 45 min at 4°C. buy birth control online

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