The distribution of Ag-NORs proteins and ribosomal RNA genes in human spermatogonia: an original procedure associating silver-staining, fish and confocal microscopy.

It is generally accepted that Ag-NORs proteins are associated with nucleolar components engaged in rRNA synthesis. Since conflicting results have been reported concerning the intranucleolar sites of active ribosomal genes, the relationships between the silver-stained areas and the distribution of rDNA were analyzed in human spermatogonia using confocal microscopy of fluorescent in situ hybridized preparations previously stained with silver. The fluorescent signal strictly corresponded to the silver-stained area, identified at the ultrastructural level as the fibrillar center and the surrounding dense fibrillar component. A quantitative evaluation of nucleolar activity solely based on silver-staining may be misleading, taking into account that only Ag-positive proteins located in the dense fibrillar component can be associated with transcriptional activity, while those stored in the fibrillar center are associated with non-transcribed rRNA genes.