Figure 3

Transcription of rDNA in spermatogenic cells. A) Nuclear run-on transcription in nuclei of different spermatogenic cells in the presence (top 3 panels) and absence of α-amanitin (bottom 3 panels). Labelled transcripts were hybridised with each of the domains of cold pre-rRNA and nucleolin. 2n, diploid cells; 4n, meiotic spermatocytes; and n, spermatid cells. Lane 1-nucleolin; lane 2-beta actin; lane 3-External transcribed spacer (ETS); lane 4–28S rRNA; lane 5–18S rRNA; lane 6-Internal transcribed spacer (ITS). B) Rate of rDNA transcription assayed by nuclear run-on transcription in diploid, meiotic spermatocytes and spermatid cells. RNA isolated at different time periods were hybridised to ETS. Each value represents an average of two independent experiments. Line with blue icon, gametic diploid cells; green, meiotic spermatocytes and yellow, spermatid cells. C) In situ run on transcription assayed in the presence of α-amanitin. BrUTP labelled transcripts were visualized using mouse monoclonal anti-BrdU. Nucleus is stained with DAPI. 2n, gametic diploid cells (panel a-b); 4n, meiotic spermatocytes (panel c-d); and n spermatid cells (panel e-f). D) Colocalisation of BrUTP labelled transcript with UBF in round spermatids in the presence of α-amanitin. Transcripts were detected by monoclonal anti-BrdU and UBF with anti-UBF a) DAPI stained nuclei; b) BrUTP labelling; c) UBF localisation; and d) merge of b and c.