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
PGCs Demonstrated Developmental- and Sex-Specific Changes in bFGF Binding
We also investigated potential developmental regulation of this FGF-binding activity by performing this procedure on germ cells obtained from 8.5 and 11.5 dpc embryos as well as samples from sex-segregated 12.5 and 13.5 dpc embryos, the earliest stages at which gender is morphologically distinguishable. The results from this experiment, also presented in Figure 5, illustrate that at all stages tested, male germ cells exhibited specific binding of 125I-bFGF Surprisingly, while germ cells obtained from 12.5 dpc female embryos also bound bFGF, binding was reduced to nonspecific levels by 13.5 dpc. At this stage most oogonia are entering meiotic prophase I.
FIG. 4. Basic FGF binding by PGCs. Single-cell suspensions of genital ridges from 11.5 dpc embryos were incubated in the presence of 125I-bFGF as described in Materials and Methods. PGCs were identified by alkaline phosphatase staining, which is seen as a red precipitate surrounding the cell (large arrow). Cells not staining for alkaline phosphatase are considered to be somatic cells (open arrowheads). Grains (small arrow) observed in areas lacking cells are considered background.
FIG. 5. Analysis of 125I-bFGF binding by PGCs. PGCs from embryos of the indicated age and sex were analyzed for binding of 125I-bFGF. The average number and SD of autoradiographic grains over an alkaline phosphatase-expressing PGC was determined (solid bars). In each case, nonspecific binding was monitored by a control incubation containing 0.5 ^g unlabeled bFGF competitor (gray bars). At least 10 cells were counted to determine an average number of grains. The mean levels of grains per PGC under the Experimental condition (total binding) significantly exceeded (p = 0.0001) those under the Plus Competitor condition (nonspecific binding) at every time except for females at 13.5 dpc (p = 0.1085).