A maternal store of macroH2A is removed from pronuclei prior to onset of somatic macroH2A expression in preimplantation embryos

Abstract

MacroH2A histones are variants of canonical histone H2A that are conserved among vertebrates. Previous studies have implicated macroH2As in epigenetic gene-silencing events including X chromosome inactivation. Here we show that macroH2A is present in developing and mature mouse oocytes. MacroH2A is localized to chromatin of germinal vesicles (GV) in both late growth stage (lg-GV) and fully grown (fg-GV) stage oocytes. In addition, macroH2A is associated with the chromosomes of mature oocytes, and abundant macroH2A is present in the first polar body. However, maternal macroH2A is lost from zygotes generated by normal fertilization by the late 2 pronuclei (2PN) stage. Normal embryos at 2-, 4-, and 8-cell stages lack macroH2A except in residual polar bodies. MacroH2A protein expression reappears in embryos after the 8-cell stage and persists in morulae and blastocysts, where nuclear macroH2A is present in both the trophectodermal and inner cell mass cells. We followed the loss of macroH2A from pronuclei in parthenogenetic embryos generated by oocyte activation. Abundant macroH2A is present upon the metaphase II plate and persists through parthenogenetic anaphase, but macroH2A is progressively lost during pronuclear decondensation prior to synkaryogamy. Examination of embryos generated by intracytoplasmic sperm injection (ICSI) revealed that macroH2A is associated exclusively with female pronuclei prior to loss in late pronucleus stage embryos. These results outline a surprising finding that a maternal store of macroH2A is removed from the maternal genome prior to synkaryogamy, resulting in embryos that execute three to four mitotic divisions in the absence of macroH2A prior to the onset of embryonic macroH2A expression.

Genome organization and epigenetic marks in mouse germinal vesicle oocytes

“…Regarding heterochromatin localization, our observations (summarized in Fig. 8) confirm previous data obtained mostly through immunostainings (Chang et al, 2005;De La Fuente et al, 2004;Garagna et al, 2004; Longo et respectively) during this transition. This is consistent with the number of centromeric spots previously reported by Garagna and coworkers (2004) in fully matured GV oocytes (about 15 detected with CREST antibodies).…”

“…In interphasic somatic cells, these regions are usually forming chromocenters that represent clusters of the pericentric regions of different chromosomes, surrounded by the centromeres of the corresponding chromosomes (Bannister et al, 2001;Lachner et al, 2001). In GV oocytes, it has already been demonstrated that HP1 (a or b) proteins -known to be associated with pericentromeric heterochromatin (Bannister et al, 2001;Lachner et al, 2001)-co-localize with highly condensed areas of chromatin in the nucleoplasm of NSN and SN oocytes as well as around the NLB of SN oocytes (Chang et al, 2005;Liu et al, 2012;Meglicki et al, 2008;Wang et al, 2008). It was also shown that centromeres cluster around the NLB in SN oocytes only (De La Fuente et al, 2004;Garagna et al, 2004;Liu et al, 2012;Longo et al, 2003;Zuccotti et al, 2005), suggesting that pericentromeric heterochromatin distribution is reorganized during the NSN to SN transition.…”

Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development

“…Embryos were prepared for IF as previously described (Chang et al, 2005;Lin et al, 2010). The following antibodies were used: H3.3 (Abnova H00003021-M01; Abcam ab62642), H3 (Abcam ab1971), H4K16ac (Millipore 07-329), H3K36me2 (Abcam ab9049), H1 (Abcam ab1938; ab62884), H3K4me3 (Millipore 04-745), H3K9me2 (Abcam ab1220), H3K9me3 (Abcam ab8898), H3K27me3 (Millipore 07-449), HP1β (Santa Cruz sc-10212), H4K5/8/12ac (Upstate 04-557), Pan-H4ac (Millipore 06-866), Mof (Santa Cruz Biotechnology sc-13677), Cdx2 (Biogenex Laboratories MU392) and Oct4 (Pou5F1; Santa Cruz Biotechnology sc-9081).…”

Immunocytochemistry in Early Mammalian Embryos

“…Embryos made by somatic nuclear transfer utilize the same chromatin remodeling mechanisms. [62,63] HDAC1 is expressed in preimplantation embryos , where its expression inversely correlates with changes in the acetylation state of histone H4K5 during preimplantation development HDAC1 is involved in the formation of a chromatin-mediated transcriptionally repressive state that initiates in the late two-cell embryo [31] ICC of late zygotes shows that constitutive heterochromatin is only maternally labeled by H3K9me3 and HP1…”