Transcriptionally active and inactive regions of nucleolar chromatin in amplified nucleoli of fully grown oocytes of hibernating frogs, Rana pipiens (Amphibia, Anura). A quantitative electron microscopic study.

Chromatin structures contained in nuclei of full-grown oocytes of hibernating northern leopard frogs, Rana pipiens, kept at 4 degrees C were examined by light and electron microscopy, with special emphasis on the morphology of spread nucleoli. Light microscopic inspection of the in situ arrangement revealed that at this stage of oogenesis the numerous amplified nucleoli, together with the chromosomes, were included in a centrally located aggregate. Characteristically, the nucleoli appeared spheroidal and showed large size differences; many of them revealed a caveolated interior and close association with large dense bodies of granular (GDB) or fibrillar (FDB) substructure. A spreading procedure involving limited dispersal of nucleolar chromatin allowed the analysis of a large number of individual nucleoli from a specific nucleus. Electron microscopy showed that different types of nucleoli which could be distinguished in terms of transcriptional activity occurred in the same nucleus: (i) Nucleoli in which nearly all pre-rRNA gene regions appeared as fully covered by lateral fibrils, indicative of active transcription ('active nucleoli'); (ii) nucleoli that contained exclusively or predominantly chromatin strands free of lateral fibrils and were characterized by a uniformly beaded appearance, interpreted as transcriptionally inactive, nucleosome-packed chromatin ('inactive nucleoli'); and (iii) nucleoli in which a number of typical arrays of lateral fibril gradients occurred besides other regions that were free of such fibrils and apparently non-transcribed ('partly inactive nucleoli'). Quantitative evaluations of the proportions of active and inactive nucleolar chromatin indicated that a minimum of 70% of nucleolar DNA is not transcribed at this stage of oogenesis. Details of the pattern and ultrastructural organization of active and inactive chromatin regions are presented. Lampbrush chromosome structures were observed in the same nuclei and also showed transcriptional structures, though often with reduced packing density of lateral fibrils. The observations show that a number of genes of both kinds, those coding for pre-rRNAs and those coding for proteins, are transcribed in full-grown oocytes of hibernating frogs at 4 degrees C. However, the data also indicate that the number of transcribed genes and the frequency of transcriptional events are greatly reduced at this stage of oogenesis, especially in the nucleolar chromatin.