PRC1 function during oogenesis is required for embryonic development. (A) Microinjection of myc-tagged Rnf2 mRNA into Ring1^m−z+/Rnf2^m−z+ zygotes leads to de novo PRC1 complex formation. Immunofluorescence analyses of control and Ring1^m−z+/Rnf2^m−z+ zygotes microinjected with water or with myc-tagged Rnf2 mRNA. Embryos were stained with anti-myc antibody to detect injected myc-tagged Rnf2, and with anti-Cbx2 and anti-Bmi1 antibodies to visualize reconstitution of chromatin-bound PRC1. For some embryos, focal planes of two parental pronuclei were merged into one image. Bar, 20 μm. (B) Microinjection of 25 ng of myc-tagged Rnf2 mRNA into early zygotes does not alleviate the developmental arrest at the two-cell stage. Diagram shows developmental potential of control and Ring1^m−z+/Rnf2^m−z+ embryos at embryonic day 3.5 that had been microinjected at the early zygote stage with water or Rnf2 mRNA.

Aberrant gene expression in Ring1/Rnf2 dm GV oocytes and impaired ZGA in Ring1^m−z+/Rnf2^m−z+ two-cell embryos. (A) Analyses of ZGA by transcriptome profiling of untreated, α-amanitin-treated control, and Ring1^m−z+/Rnf2^m−z+ late two-cell embryos. Venn diagram shows overlap among α-amanitin-sensitive probe sets (≥1.5-fold in untreated vs. α-amanitin-treated; P-value < 0.05) in control (dark-green) and Ring1^m−z+/Rnf2^m−z+ (light green) embryos. (B) Analyses of gene expression in wild-type, Ring1 mutant, Rnf2 mutant, and Ring1/Rnf2 dm GV oocytes. Venn diagrams show overlap among probe sets either up-regulated or down-regulated in Rnf2 mutant, Ring1 mutant, and Ring1/Rnf2 dm GV oocytes compared with wild type (≥1.5-fold; P-value < 0.05). (C) Origin of transcripts misregulated in Ring1^m−z+/Rnf2^m−z+ late two-cell embryos. (Left diagram) For probe sets up-regulated in Ring1^m−z+/Rnf2^m−z+ versus control two-cell embryos, the majority was expressed in control and Ring1/Rnf2 dm GV oocytes (blue), with some up-regulated in Ring1/Rnf2 dm GV oocytes (dark red). Only a minority is de novo transcribed (α-amanitin-sensitive) in Ring1^m−z+/Rnf2^m−z+ two-cell embryos (light green). (Right diagram) For probe sets down-regulated in Ring1^m−z+/Rnf2^m−z+ versus control two-cell embryos, the majority is not de novo transcribed (α-amanitin-sensitive) to a level observed in control embryos (light and dark green). Only a few probe sets were down-regulated in Ring1/Rnf2 dm GV oocytes (light red). For all comparisons, a ≥1.5-fold difference with a P-value of <0.05 was used.

Transcripts of the developmental regulator Eomes, derepressed in Ring1/Rnf2 dm oocytes, are only translated after fertilization. (A) Quantitative real-time PCR analysis of Eomes transcripts in growing oocytes (from primary and secondary follicles), GV oocytes, zygotes, and two-cell embryos that are wild type or single or double deficient for maternal Ring1 and/or Rnf2. Transcript levels were normalized to LnmB1 control. Error bars indicate standard deviation based on two to three biological replicates. (B) Immunofluorescence staining for Eomes in control and Ring1/Rnf2 dm GV and M-II oocytes, zygotes, and two-cell embryos. Numbers of oocytes or embryos analyzed at each stage are indicated in brackets. Bar, 20 μm.

Loss of maternal Ring1 and Rnf2 impairs early embryonic development beyond the two-cell stage. (A) Differential interference contrast images of Ring1^m−z+/Rnf2^m+z+ (further used as control; C) and Ring1^m−z+/Rnf2^m−z+ (DM) embryos at embryonic day 3.5. (B) Average number of Ring1^−/− (C) and Ring1/Rnf2 dm (DM) oocytes and Ring1^−/− (C) and Ring1^m−z+/Rnf2^m−z+ (DM) embryos isolated per female mouse at the indicated developmental stages. The number of female mice analyzed is shown in brackets. (C) Cartoons illustrating developmental potential of embryos with different maternal and/or zygotic deficiencies for Ring1 and Rnf2. (^m−) Maternal deficiency; (^z−) zygotic deficiency; (^z+) zygotic proficiency from either paternal or both parental origins. Coloring of (pro)nuclei indicates wild-type alleles of Ring1 and Rnf2 ([red] maternal; [blue] paternal; [green] maternal and paternal). The right panel shows microinjection of Rnf2 mRNA. Yellow and white cytoplasms indicate the presence or absence of Ring1/Rnf2 proteins, respectively.

Aberrant cell cycle progression in Ring1^m−z+/Rnf2^m−z+ embryos. (A) Ring1^m−z+/Rnf2^m−z+ zygotes stained with DAPI show a delay in pronuclear formation at 5 h post-insemination. For some embryos, focal planes of two parental pronuclei were merged into one image. Bars, 20 μm. (B) Timing of cell cycle phases of two-cell embryos and definitions for early, mid, late, and very late two-cell stages. (C) Quantification of DNA synthesis in control and Ring1^m−z+/Rnf2^m−z+ two-cell and four-cell embryos cultured in the presence of EdU for the indicated intervals. EdU was quantified by measuring total fluorescent signal in both nuclei of two-cell embryos or in two randomly chosen nuclei of a four-cell embryo (indicated by two adjacent bars).

Transcriptional misregulation of canonical PRC1 target genes in Ring1/Rnf2 dm oocytes. (A) GO analysis of genes up-regulated (red) or down-regulated (green) in Ring1/Rnf2 dm GV oocytes. Fold overrepresentation indicates the observed percentage of up-regulated genes in a particular GO category over the percentage expected on the basis of all GO-annotated genes on the array. P-values indicate significance of enrichment. (B) Box plot showing log2 fold change of expression in Ring1/Rnf2 dm over control GV oocytes for all probe sets (black) and for probe sets classified as promoter H3K27me3-positive (dark gray) or H3K27me3-negative (light gray) in chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) studies in human sperm (1:1 orthologs) (Hammoud et al. 2009), mouse ESCs, and MEFs (Mikkelsen et al. 2007). (C) Percent of probe sets detected (ON) or not detected (OFF) during normal oogenesis (Pan et al. 2005) and early embryonic development (Zeng et al. 2004; Zeng and Schultz 2005) among not changed, up-regulated, or down-regulated probe sets in Ring1/Rnf2 dm GV oocytes compared with control (≥1.5-fold; P-value < 0.05).

Ring1/Rnf2 expression during oogenesis defines maternal cytoplasmic and chromatin contributions required for embryonic development. (A) Cartoon illustrating generation of hybrid embryos by exchanging matPN between early zygotes with different genotypes. Wild-type paternal pronuclei (patPN) are not exchanged. (B) Diagram showing developmental potential (scored at embryonic day 4.5) of reconstructed zygotes. Control groups include (1) control (Ring1^m−z+) zygote containing a matPN from another control zygote (cyto-C/matPN-C) and (2) Ring1^m−z+/Rnf2^m−z+ zygote containing a matPN from another Ring1^m−z+/Rnf2^m−z+ zygote (cyto-DM/matPN-DM). Experimental groups include (1) matPN of control zygote transferred into Ring1^m−z+/Rnf2^m−z+ zygote (cyto-DM/matPN-C) and (2) matPN of Ring1^m−z+/Rnf2^m−z+ zygote transferred into control zygote (cyto-C/matPN-DM). Numbers of reconstructed embryos analyzed are shown in brackets. (C) Cartoon illustrating generation of hybrid embryos by exchanging M-II chromosomes between M-II oocytes with different genotypes. M-II oocytes were activated to produce parthenogenetic embryos. (D) Diagram showing developmental potential (scored at embryonic day 4.5) of parthenogenetically activated reconstructed M-II oocytes. Control group includes (enucleated) control (Ring1 mutant) M-II oocyte containing chromosomes from another control M-II oocyte (cyto-C/M-II-C). Experimental groups include (1) chromosomes of control M-II oocyte transferred into (enucleated) Ring1/Rnf2 dm M-II oocyte (cyto-DM/M-II-C) and (2) chromosomes of Ring1/Rnf2 dm M-II oocyte transferred into (enucleated) control M-II oocyte (cyto-C/M-II-DM). Numbers of reconstructed embryos analyzed are shown in brackets. Note that parthenogenic activation causes a developmental arrest more frequently at the zygotic than at the two-cell stage compared with embryonic activation by sperm fusion. (E,F) Immunofluorescence staining for H2AK119ub1 in wild-type GV and MII oocytes and in Ring1/Rnf2 dm GV oocytes. Numbers of oocytes analyzed are indicated in brackets.