Fig 2: 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 3: 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)

Fig 4: FRET analysis of interaction of YWHAH with CDC25B and YWHAH with YWHAE. a ICR(CD1) oocytes were co-injected with mCherry-YWHAH and EGFP-CDC25mRNA. Expression of EGFP-CDC25B protein provides the donor fluorescence (green) in the FRET analyses. The mCherry-YWHAH serves as the acceptor fluorescent probe (red). Images show the donor emission with donor excitation (488 nm), the acceptor emission with donor excitation, and the acceptor emission with acceptor excitation (543 nm), and the corrected FRET image. Data from three regions of interest (shown by white boxes) were used to calculate the FRET efficiency, which, in this cell averaged 72%, and is visually represented in the heat map at the right. The figure is representative of 12 oocytes collected from three different females in three experiments. b ICR(CD1) oocytes were co-injected with mCherry-YWHAH and EGFP-YWHAE. EGFP-YWHAE protein provides the donor fluorescence (green) in the FRET analyses. The mCherry-YWHAH serves as the acceptor fluorescent probe (red). Images show the donor emission with donor excitation (488 nm), the acceptor emission with donor excitation, and the acceptor emission with acceptor excitation (543 nm), and the corrected FRET image. Data from three regions of interest (show by white boxes) was used to calculate the FRET efficiency, which in this cell averaged 27%, and is visually represented in the heat map at the right. The figure is representative of five oocytes examined in two experiments

Fig 5: 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)