Separase and Oogenesis Figure 4. Genome instability in PGCs of female Meox-2-cre/Separase S1121A mutant mice. A. Micronucleus configuration of female PGCs at 13.0 dpc observed by immuno-staining with Mvh (green) and DNA (blue). Arrows indicate the micronuclei. Bar = 0.5 mm. B. Quantitative analysis of micronuclei of female PGCs from wild-type and SeparaseS1121A gonads of various ages as indicated. Serial sections of female genital ridges were stained for DNA and Mvh. All the Mvh-positive cells and the Mvh-positive cells with micronuclei were scored from at least three sections in at least three embryos (six genital ridges) for each time point. The mean value is shown with standard error (** p,0.001, T test). C. Representative of apoptotic PGCs detected by active Caspase-3 (red)-positive PGC with abnormal nucleus (blue) configuration. Bar = 2.0 mm. D. Centrosome number in female PGCs visualized by immuno-staining with c-Tubulin (red), Mvh (green) and DNA (blue). Red dots (arrows) of c-Tubulin staining indicate centrosomes. Bar = 0.5 mm. E. Quantitative analysis of abnormal centrosomes of female PGCs at 13.0 dpc. Serial sections of female genital ridges were stained for DNA, Mvh, and c-Tubulin. The Mvh-positive cells with three or more c-Tubulin foci were scored from at least three sections in at least three embryos (six genital ridges). The mean value is shown with standard error (** p,0.001, T test). doi:10.1371/journal.pone.0018763.g004 somatic cells with dually controlled Separase, PGCs have insufficient Securin to fully inhibit Separase and, therefore, their inhibition of Separase is dependent mainly on inhibitory phosphorylation. Without Securin regulation, Separase is well regulated in all kinds of cells in vivo [30,42–45], whereas Separase functions poorly in PGCs without phospho-regulation, resulting in the premature segregation of sister chromotids and other chromosome segregation errors, including chromosome misalign- ment and mis-segregation. These errors lead to genome instability and gametogenesis failure. Our results indicated that Separase phospho-regulation is crucial for gametogenesis and only game- togenesis. Separase deregulation in mitosis of gametogenesis is a potential origin of aneuploidy The vast majority of aneuploidy during gametogenesis comes from meiotic division [5,10,34,46,47], which produces chromo- some segregation errors including non-disjunction in MI and premature chromosome segregation in MII. Our present study provides the first evidence that PGCs undergo mitotic chromo- some segregation errors at the beginning of gametogenesis. Therefore, during gametogenesis, chromosome segregation errors come not only from meiosis but also from mitosis. The meiotic chromosome segregation errors result from abnormalities in the connection between homologous chromo- somes and sister chromatids as well as related events such as pairing, synapsis, and recombination [11–15]. In contrast, the mitotic chromosome errors we described here occurred due to de- regulation of Separase, the upstream protease of cohesion. We have therefore uncovered a potential origin for aneuploidy during gametogenesis. Sexually dimorphic effect of Separase de-regulation on PGCs may be an additional possibility of sex-specific differences in aneuploidy incidences during oogenesis It is well known that there are sex-specific differences in aneuploidy incidence during gametogenesis. Different possibilities account for the sex-specific differences: first, the differences in the PLoS ONE | www.plosone.org 6 April 2011 | Volume 6 | Issue 4 | e18763