Fig. 3

Epigenetic marks in platypus spermatocytes. Platypus total testis suspensions were PFA fixed for surface spreading and immunostained using antibodies against repressive histone marks H2AFY (panels a-d and e), H3K9me3 f, H3K27me3 g, followed by sex chromosome specific BAC e or chromosome 6 paint f DNA FISH. Late prophase I nuclei show variable H2AFY staining patterns positioned adjacent the NLB and restricted to SYCP1 positive nuclei b. H2AFY configurations include dispersed fibrillar arms a, more restricted foci e, and larger para-nucleolar blocks c and d. Y5 c and X5 d were frequently observed to co-localise with H2AFY while other elements were not observed to colocalise with H2AFY e. To merge DNA FISH signals with H2AFY panels the background image (non FISH signal) was subtracted and colour changed as both immunostaining and FISH probes were Alexa-488 tagged. FISH signal image adjustment was performed using standard PowerPoint tools. f Chromosome 6 maintains attachment to the NLB with regions of the p arm where the rDNA resides colocalising with H3K9me3. g H3K27me3 staining occurs in SYCP1 positive nuclei close to the NLB. Scale bars = 10 μm. PFA paraformaldehyde, FISH fluorescence in situ hybridisation