The precise distribution of DNA and RNA within the human Sertoli cell nucleolus

On the basis of autoradiographic studies, it used to be accepted that rDNA transcription takes place in the dense fibrillar component of the nucleolus (reviewed by Goessens, 1984; Fakan, 1986). Over the past few years, however, data based on immunolocating procedures at the electron microscope level have strongly suggested that rDNA transcription occurs in the fibrillar centres (Scheer and Benavente, 1990; Thiry et al., 1991). A variety of methods, applied to various cell-type nucleoli, have detected DNA exclusively in the fibrillar centres, preferentially in their peripheral regions, but not in the dense fibrillar component (Scheer et al., 1987; Thiry, 1988, 1992; Thiry et al., 1988, 1991, 1992). Recent in situ hybridization studies have, however, yielded apparently contradictory results concerning the location of rRNA genes in the nucleolar fibrillar components. In Ehrlich tumour cells (Thiry and Thiry-Blaise, 1989, 1991), HeLa cells, and mouse 3T3 cells (Puvion-Dutilleul et al., 1991), rDNA was detected only in the fibrillar centres and not in the dense fibrillar component, while the reverse was observed for human Sertoli cells (Wachtler et al., 1992) and human lymphocytes (Wachtler et al., 1990). Although the functional organization of the nucleoli may be different in different cells or species, we have put forward the hypothesis that the divergent conclusions might arise from difficulties in distinguishing the various nucleolar components under in situ hybridization conditions (Thiry and Goessens, 1992). In this respect, we have recently shown that the rDNA signal obtained after in situ hybridization on human lymphocytes should be attributed to the condensed chromatin crossing through the dense fibrillar component rather than to the dense fibrillar component itself (Vandelaer et al., 1992). To shed light on the nucleolus of human Sertoli cells, we have used the in situ terminal deoxynucleotidyl transferase/immunogold (TdT/immunogold) technique to investigate, in great detail, the precise distribution of DNA within the nucleolus. This method was applied to acetylated material, conditions that provide an excellent distinction between the various nucleolar components and make it possible to visualize condensed chromatin with high contrast. To obtain additional information concerning the morphofunctional organization of the nucleolus, the distribution of RNA was further investigated by means of the in situ polyadenylate nucleotidyl transferase/immunogold (PnT/immunogold) technique. The results reveal DNA in the chromatin enclosed in the nucleolar interstices, especially those in contact with the fibrillar centres, as well as in the fibrillar centres, but not in the dense fibrillar component of the human Sertoli cell nucleolus. Moreover, our results indicate that the fibrillar centre is the only site where DNA and RNA are visualized together. 33 Journal of Cell Science 105, 33-39 (1993) Printed in Great Britain © The Company of Biologists Limited 1993