Fig. 1. Asymmetric localisation of Ezh2/EED and the effect of Ezh2 depletion. (A) Schematic representation of oocyte maturation and preimplantation development (see left panels). The second meiotic division commences at fertilisation and the two parental genomes remain separate as pronuclei until the first cleavage division. Development can also be initiated by activation of oocytes without fertilisation, followed by the suppression of second polar body extrusion by cytochalasin B to generate diploid parthenogenetic embryos. To deplete the oocytes of maternal Ezh2, transgenic mice with the Ezh2 conditional alleles, Ezh2^F/F, were crossed with ZP3 Cre recombinase transgenic animals to delete the Ezh2^F/F alleles specifically in the growing oocyte (see far right panel). Zp3 is expressed prior to the completion of the first meiotic division. Embryos depleted of maternal Ezh2 (right panels) were compared with those lacking both the maternal and embryonic Ezh2 (shown in the panel adjacent to the far-right panel). (B) Schematic depiction of development of zygotes at 0-3, 3-6 and 6-10 hours post fertilisation (hpf) (top line), with the corresponding immunostaining shown immediately below them. The haploid pronuclei inherited from the sperm and the oocyte can be distinguished morphologically (Hogan et al., 1994). Male and female pronuclei, and the second polar body (PB) are marked. All images in green show antibody staining, red shows DNA staining and yellow shows merged images. Ezh2 is first associated preferentially with the female pronucleus and the PB at 0-3 hpf. At 3-6 hpf, Ezh2 can also be detected in the paternal pronucleus, and by 6-10 hpf, both male and female pronuclei show Ezh2 (white arrow heads). (C) Depicts a zygote depleted of maternally inherited Ezh2. Oocytes depleted of maternally inherited Ezh2 and fertilised by wild-type sperm show Ezh2 by immunostaining at the four-cell stage, indicating initiation of embryonic transcription of Ezh2. Note that the pronuclei in Ezh2 depleted zygotes appear to be slightly larger and less compact than in controls shown in 1B. (D) Eed is also asymmetrically localised to the female pronucleus (right panels). However, in Ezh2-depleted zygotes, asymmetric Eed localisation to the female pronucleus is highly reduced to virtually absent. Thus, asymmetrical localisation of Eed is apparently dependent on the maternal inheritance of Ezh2.

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