Separase and Oogenesis Figure 2. Partial depletion of PGCs in female Meox-2-cre/Separase S1121A mutant mice. A. Whole-mount TNAP staining of female gonads of 13.0 dpc. TNAP-positive cells are red. Bar = 20.0 mm. B. Western blot detection of Mvh of gonads at 13.0 dpc. C. Mvh immuno-staining of gonads at 13.0 dpc. Mvh-positive cells are red. Bar = 4.0 mm. D. Quantitative analysis of Mvh-positive cells in female gonads of different stages. Serial sections of female genital ridge were stained for DNA and Mvh. The Mvh-positive cells were scored from at least three sections in at least three embryos (at least six genital ridges) for each time point. The mean value is shown with standard error (** p,0.001, T test). doi:10.1371/journal.pone.0018763.g002 mediated mitotic defects could be due to a relaxed mitotic checkpoint. To test this possibility, we carefully checked the mitotic checkpoint regulators, Aurora B and Mad2, and the downstream effectors, P53 and Bax, which played crucial roles in Separase mutation-mediated male PGC depletion [28]. The results failed to prove our hypothesis, as both male and female gonads had equal level of the mitotic regulators and effectors (Figure 5C), suggesting there may be other mechanisms involved in the gender discrepancy of Separase mutation-mediated defects. Because Securin and inhibitory phosphorylation act redundant- ly to control Separase activity [30], we asked whether there was gender discrepancy in Securin levels. To this end, we checked the levels of Securin in PGCs by in situ immunostaining with Securin, Mvh and DNA. Interestingly, we observed that considerably fewer Securin-positive PGCs with low level of Securin were present in male genital ridges than in female (Figure 5A & B). To further confirm these intriguing results, the overall level of Securin in genital ridges was normalized against Mvh. Examination by Western blot showed that there was considerably more Securin in female genital ridges than in male genital ridges (Figure 5D), indicating that there was a sex-specific difference in Securin production of PGCs. Securin levels were correlated to Separase S1121A point mutation-mediated genome instability We suspected that the sex-specific difference in Securin is correlated with the sexual dimorphism effects of Separase S1121A point mutation on PGCs. We first tried to provide convincing evidence by carefully comparing the phenotype of wild-type, Securin+/+/Separase+/S1121A, Securin2/2/Separase+/+, and Se- curin2/2/Separase+/S1121A ES cells. As we observed before, all the cells grew well without aneuploidy [30]. Because double mutant ES cells undergo premature chromosome segregation and grow slower once challenged with one of the spindle poisons, nocodazole [30], we doubted that we would be able to catch the anouploidy of the double mutant cells because of selection against aneuploidy. Therefore, we double-challenged the cells with nocodazole to induce chromosome mis-segregation. Interestingly, the double mutant cells displayed a significantly higher percentage of aneuploidy (30.7064.06) than wild-type (19.1864.10), Se- curin+/+/Separase+/S1121A (20.2963.74), or Securin2/2/Separ- ase+/+ (20.3664.25) cells (Figure 6A). These results indicated that the Separase mutation did not cause defects in the presence of Securin in ES cells, but it did lead to chromosome segregation errors in the absence of Securin. Therefore, Securin affected the Separase mutation-mediated defects in ES cells. By TNAP staining, we demonstrated that the Securin2/2/ Separase+/+, Securin+/2/Separase+/+, and Securin+/2/Separ- ase+/S1121A mice have normal PGCs at 10.5 dpc, while the Securin2/2/Separase+/S1121A mice do not, suggesting that Securin expression affected Separase mutation-induced defects on PGCs [28]. To further confirm these results, the overall levels of Mvh and Securin in urogenital ridges of 10.5 dpc were examined by Western blot. Consistently, Separase mutation led to PLoS ONE | www.plosone.org 4 April 2011 | Volume 6 | Issue 4 | e18763