Anti-5meC immunostaining on rabbit zygotes at various pronuclear stages. Lane (A): Example of a very early zygote (n = 4) (approximately PN0) just after the penetration of sperm into the oocyte. The decondensing sperm chromatin is strongly stained with the α-5meC antibody (in contrast to sperm heads). Two haploid sets of maternal chromosomes are still present in the ooplasm and the second polar body is not yet formed. Lane (B): Example of a zygote (n = 3) with newly formed pronuclei (approximately PN2). Both paternal and maternal pronuclei at this stage (approximately 3 to 4 hours after fertilization) show similar α-5meC signal intensities. Lane (C) shows an example of a zygote with expanded pronuclei (approximately PN3-4, 8 to 10 hours post-fertilization, n > 20). The α-5meC signal is strongly different between the two parental pronuclei, the paternal chromatin almost devoid of antibody staining. Panels: DNA – images of propidium iodide staining; 5meC – images of α-5meC monoclonal antibody staining; DNA/5meC – merged images. The arrows in the DNA staining panels indicate sperm heads still attached to the zygote. The highly compacted DNA of these sperm heads is not accessible to α-5meC staining in contrast to the just-penetrated sperm shown in (A). Scale bar 20 μm.