Evidence for conserved DNA and histone H3 methylation reprogramming in mouse, bovine and rabbit zygotes

Epigenetics & Chromatin Evidence for conserved DNA and histone H3 methylation reprogramming in mouse, bovine and rabbit zygotes Konstantin Lepikhov 2 Valeri Zakhartchenko 1 Ru Hao 1 Feikun Yang 1 Christine Wrenzycki 0 Heiner Niemann 3 Eckhard Wolf - 1 Joern Walter 2 0 University of Veterinary Medicine, Clinic for Cattle, Reproductive Medicine Unit , 30173 Hannover , Germany 1 Department of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University , 81377 Munich , Germany 2 University of Saarland, Natural Sciences - Technical Faculty III , Biological Sciences, Genetics/Epigenetics, 66123 Saarbrucken , Germany 3 Institute of Animal Breeding (FAL), Department of Biotechnology , Holtystrasse 10, Mariensee, 31535 Neustadt , Germany Background: In mammals the parental genomes are epigenetically reprogrammed after fertilization. This reprogramming includes a rapid demethylation of the paternal (sperm-derived) chromosomes prior to DNA replication in zygotes. Such active DNA demethylation in the zygote has been documented for several mammalian species, including mouse, rat, pig, human and cow, but questioned to occur in rabbit. Results: When comparing immunohistochemical patterns of antibodies against 5-methyl-cytosine, H3K4me3 and H3K9me2 modifications we observe similar pronuclear distribution and dynamics in mouse, bovine and rabbit zygotes. In rabbit DNA demethylation of the paternal chromosomes occurs at slightly advanced pronuclear stages. We also show that the rabbit oocyte rapidly demethylates DNA of donor fibroblast after nuclear transfer. Conclusion: Our data reveal that major events of epigenetic reprogramming during pronuclear maturation, including mechanisms of active DNA demethylation, are apparently conserved among mammalian species. - Background DNA methylation in CpG dinucleotides is an important epigenetic signal controlling heterochromatin formation, genomic imprinting, X-inactivation and gene expression [1]. DNA methylation patterns are subject to genomewide epigenetic reprogramming at certain developmental stages, particularly during certain phases of germ cell and early embryonic development [2]. After fertilization, DNA methylation of sperm and oocyte-derived chromosomes is largely erased. While Southern blot studies of DNA methylation in repetitive elements in mouse gametes suggested that sperm chromosomes are more hypermethylated than those of oocytes [3], a recent analysis using methylated DNA immunoprecipitation revealed equally low DNA methylation levels in both sperm and oocytes, at least in the promoter regions [4]. Hence the observed decrease of DNA methylation during early embryonic development apparently largely reflects demethylation of (some) repetitive elements. Upon further development DNA methylation again increases in cells of the inner cell mass, while cells of the trophectoderm remain rather hypomethylated [5,6]. The dynamics of DNA demethylation during early preimplantation development have been thoroughly investigated by a number of research groups in different mammalian species. Immunohistochemical studies on mouse zygotes using antibodies against 5-methyl-cytosine (-5meC) showed a rapid loss of DNA methylation exclusively in the paternal pronucleus. The reactivity of the -5meC antibody starts to diminish around the early pronuclear stage 2 (PN2) when the protamine-histone exchange is completed (approximately three hours post fertilization). At early PN4 (approximately 8 to 10 hours post fertilization) the 5meC signal is completely absent from the paternal pronucleus [6-8]. Bisulfite sequencing of zygotic DNA confirmed these rapid demethylation events for some single copy sequences and repetitive elements but revealed that imprinting control regions of imprinted genes and certain classes of repeat sequences remain refractory to such general demethylation [9-11]. Pronounced active demethylation of paternal DNA was not only found in mouse but also reported for rat, pig, human and, to a lesser extent, for bovine zygotes [12-15]. It is therefore considered as a general early epigenetic reprogramming event in mammalian development. However, the biological function of this process remains unclear. It has been proposed as being important for early transcriptional control, or as serving as a mechanism to reduce accumulation of transgenerational epigenetic effects propagated through the male germ line [6,7,1618]. The concept of paternal pronuclear demethylation as a general hallmark of early mammalian development was challenged by reports stating that this process is lacking in rabbit, ovine and pig zygotes [15,19-21]. On the contrary, other experiments demonstrated the capability of mature ovine oocytes to demethylate mouse sperm DNA introduced by intracytoplasmic sperm injection (ICSI) [22]. Moreover, Zhang et al showed a partial loss of DNA methylation at centromeric satellite repeats in rabbit zygotes following ICSI [23]. While these data suggest the existence of DNA demethylation activity in rabbit and ovine oocytes, it remained unclear whether the paternal pronucleus is subject to such demethylation in naturally derived zygotes. Concomitant with pronuclear DNA methylation reprogramming specific alterations in histone modifications have been observed in early mouse embryos. On the paternal chromosomes protamines are rapidly exchanged by acetylated histones which subsequently become monomethylated at position H3K4 [6,24]. This process coincides with paternal DNA demethylation in the mouse zygote. In addition, particular histone modifications such as di/trimethylation at H3K9, H4K20 and H3K27 are only present on the maternal chromosomes [24-27]. This asymmetry between parental genomes, particular of DNA methylation and H3K9me2, persists until at least the twocell stage of mouse embryo development [6,24,28,29]. Direct comparative epigenetic studies are still scarce for mammalian species. In our studies we therefore analyzed the dynamics of H3K9me2 and H3K4me3 methylation along with DNA methylation alterations in mouse, bovine and rabbit zygotes. Results and discussion Dynamics of DNA methylation in mouse, bovine and rabbit zygotes To compare the developmental dynamics of DNA methylation in pronuclei of mouse, rabbit and bovine zygotes we performed indirect immunofluorescence using well-characterized -5meC-specific monoclonal antibody [30]. Mouse and bovine zygotes were obtained by in vitro fertilization. Rabbit zygotes were derived from superovulated females, naturally mated with males. In all three species we found a clear asymmetry of -5meC staining between the parental pronuclei at advanced stages of zygotic development (more than 6 hours after fertilization). Whereas the DNA of the maternal pronuclei and polar bodies retained a strong reactivity with the antibody, this reactivity was greatly reduced in all paternal pronuclei of the three species (Figure 1A). While the observed loss of DNA methylation sign (...truncated)