Figure 5

Cell death induced by the microinjection and electroporation technique. A-D. The Live-Dead assay in intact seminiferous tubules assessed by confocal microscopy projected from 14 superimposed sections, using calcein fluorescence of all living cells (green) compared to the ethidium homodimer fluorescence (red) of the dead cell nuclei. A. Basal conditions after 24 h incubation. B. Basal conditions after 48 h incubation. C. After 24 h incubation following DNA microinjection. D. After 24 h incubation following DNA microinjection and electroporation. Note only under these conditions is the evidence of significant cell death. E-G. TUNEL analysis of cryosections from incubated seminiferous tubules. All nuclei are labelled blue with DAPI. The TUNEL assay used tetramethyl rhodamine to label nuclei with nicked DNA (red). E. Tubule after 48 h incubation under basal conditions – no obvious DNA damage. F. Tubule after 48 h incubation following microinjection of 10 μM annexin V to block phagocytosis – some limited DNA damage in mural cells. G. Tubule after 48 h incubation following DNA injection – negligible DNA damage evident.