Fig 2: Injection of the YWHA-inhibitory peptide and Ywha mRNA antisense morpholinos against mRNA for each Ywha isoform. For each injection condition and the uninjected controls in this series of experiments, the number of cells with GVBD is represented as a percent of the total number of cells studied (indicated at the top the corresponding bar). See the Methods section for experimental methods. a Percent germinal vesicle breakdown (GVBD) in mouse oocytes injected with the YWHA-inhibitory peptide R18, water or uninjected. The data is combined from two repeated experiments. Fisher’s exact test and pairwise comparison of the percentage of GVBD in the control uninjected cells and control water-injected cells revealed no significant difference between those groups, while a significantly greater percentage of GVBD was noted for oocytes injected with R18 compared to control water-injected cells (* in A). b Percent GVBD in oocytes injected with antisense morpholinos against each of the seven mammalian Ywha mRNA isoforms as well as the standard nonsense moprholino that was designed to have no effect. The data is combined from two repeated experiments. A significantly greater percentage of GVBD was noted for oocytes injected with morpholino targeting Ywhah mRNA compared to control non-sense morpholino (* in B)

Fig 3: Breeding of Ywhah and Ywhae oocyte-specific and global gene knockout mice with wild-type males. The number of pups for each female of a given geneotype is indicated for the first two litters. In vivo breding illustrates the range of offspring number in mice of different females mated with different wild-type males. Here, the number of pups produced by females with oocyte-specific inactivation of Ywhah or Ywhae and global inactivation of Ywhah (a) falls within the normal range of litter sizes of mice without gene inactivation (here mice containing ZP3 or ACTB Cre, and mice with floxed Ywhah or Ywhae gene (LoxP/LoxP) and no Cre (b). However, females with global inactivation of Ywhae did not produce pups (last group in (a), see Results and Discussion)

Fig 4: Expression of fluorescently-tagged YWHAH and YWHAE in mouse oocytes. Immature oocytes from wild type ICR(CD-1) mice were microinjected with mCherry-YWHAH (a) or EGFP-YWHAE mRNA (b). The corresponding brightfield images are shown at the right and the oocyte nucleus is outlined with a dashed black line. The images are representative of results from 16 different oocytes expressing mCherry-YWHAH from five different experiments and 12 oocytes expressing EGFP-YWHAE from three different experiments. c Representative images of three oocytes simultaneously injected with mCherry-YWHAH and EGFP-YWHAE mRNA. Both isoforms are expressed in the same cell and the merged emission channels suggests a very similar distribution for both isoforms. The white squares shown in Cell 2 are the regions of interest used to determine the average Pearson’s correlation coefficient for this cell (see Results). The nucleus was imaged at higher magnification in Cell 3. There is a clear distinction between the cytoplasm and the nucleus, with the nucleus displaying less fluorescence than the cytoplasm and the nucleolus displaying little fluorescence. YWHAH and YWHAE are primarily cytoplasmic in an immature oocyte. All of seven other injected cells were similar. The scale bars represent 10 μm

Fig 5: Diagrammatic summary of the production of Ywhah oocyte-specific conditional knockout. Exons 1 and 2 of the Ywhah gene are represented by orange boxes. The targeting vector and homology arms are indicated. The vector was electroporated into C57BL/6 ES cells, chimeras were produced, and germ line transmission in C57BL/6 J was confirmed. Exon 2 is removed following Cre recombination (see Methods)