colleagues demonstrated that Shugoshin can prevent Separase activation independently of Securin [26]. Sun and coworkers found that Cohesin cleavage by Separase was bridged by DNA in a sequence-nonspecific manner [27]. By generating a knock-in mouse model expressing non- phosphorylatable Separase with a S1121A point mutation, we have explored the Separase phospho-regulation at organismal level. We observed that both male and female mutant mice are infertile. We first determined that the sterility of males arose from the failure of spermatogenesis caused by exclusively depletion of PGCs in which Securin was expressed at low levels. This meant that the inhibition of Separase in PGCs was dependent mainly on inhibitory phosphorylation [28]. Upon further investigation of the female infertility, we uncovered that the Separase phosphosite mutation leads to reduced folliculogenesis to give birth to deficient initial follicles, which results from partial depletion of female PGC. Furthermore, we defined a gender-specific discrepancy of Securin, which may explain the sexual dimorphism of the Separase mutation-mediated PGC defects. Results Separase S1121A point mutation resulted in deficient initial follicles, decreased ovulation and less fertilization We recently reported our initial observations of mice carrying a non- phosphorylable Separase allele [28,29]. The crosses between Separ- ase+/S1121A-Puro mice and Meox2+/Cre mice yielded mutants carrying a point mutation in Separase, Meox2+/Cre/Separase+/S1121A. Mating analysis revealed that, in contrast to their normal fertile littermates, including Meox2+/+Separase+/+ (wild type), Meox2+/+/ Separase+/S1121A-flox-Puro, or Meox2+/Cre/Separase+/+, both male and female Meox2+/Cre/Separase+/S1121A mice (hereafter named mutant) were sterile. Previously, we determined that the infertility of mutant males was due to spermatogenesis failure characterized by smaller testes and lack of gametes [28]. To our surprise, the ovaries of mutant female mice developed without apparent abnormalities (Figure S1). Also, the mutant oocytes developed into different kinds of follicles, including primordial, primary, preantral, and antral follicles, and they produced atretic follicles and corpora lutea as shown in Figure 1A. These results suggested that female mutant mice had normal folliculogenesis. Nevertheless, quantitative assay indicated that the three-week- old mutant mice were deficient in initial follicle numbers (Figure 1B). We therefore assessed the ovulation of the Separase point mutation mice. We induced superovulation with exogenous gonadotropins in immature Separase control and mutant mice at 3 weeks of age to evaluate the oocyte production of each female. The results showed that mutant mice produced fewer oocytes (39.3361.53) compared to those of control mice (58.3362.52) (Figure 1C), indicating that the mutant mice ovulated fewer eggs. Genotyping PCR demonstrated that eggs from mutant mice contained the Separase mutation (Figure 1D). We then performed a fertilization assay. Consistently, Separase mutant mice had a lower fertilization rate (71.9463.59) than control mice (93.7762.67) (Figure 1E). Subsequently, the mutant ovaries degenerated (Figure S1), and mutant follicles exhausted faster from aging than those of control mice (data not shown). It is noteworthy that the fertilization success rate of the mutants was lower than that of the controls, suggesting there may be some defects in the mutant oocytes that affected their fertilization. In addition, although the mutant mice displayed normal folliculogen- esis, and the mutant oocytes were capable of being fertilized, mutant mice were unable to bear young because of the critical role Separase and Oogenesis the phosphorylation of Separase plays in early embryogenesis, when Securin expression is very low [29]. The Separase S1121A point mutation led to the partial depletion of female PGCs Folliculogenesis is a process succeeding oogenesis; like sper- matogenesis, it starts from ,45 PGCs around 7.0 days post- conception (dpc) during embryogenesis [7]. We followed the female development over time. Using PGC-specific, tissue non- specific alkaline phosphatase (TNAP) staining, we found that both control and mutant E11.5 genital ridges of the female were stained identically to that of the male (data not shown). As development progressed, fewer red TNAP-positive cells remained in mutant female gonads compared to those of controls. However, compared to mutant male samples, more TNAP-positive cells were present in mutant female genital ridges (Figure 2A). We further examined the situation by Western blot and immunostaining with mouse vasa homologue (Mvh), another PGC-specific marker. As shown in Figure 2B, both male and female mutant genital ridges expressed less Mvh than their control, but the decrease of Mvh level in the female mutant was significantly less than that in the male mutant. In situ immunostaining showed that many Mvh-positive cells remained in female gonads (Figure 2C & D). These results indicated that, in contrast to the complete depletion of PGCs seen in male mutant mice [28], the Separase mutation only partially impaired the development of female PGC, resulting in deficient initial follicles but not in complete PGC depletion. Separase S1121A point mutation induced aberrant mitosis and mitotic arrest of female PGCs We found that Separase point mutation led to developmental defects in male PGCs, which exhibited mitotic arrest resulting from premature chromosome segregation and an aberrant mitosis- activated spindle assembly checkpoint [28]. This suggests that developmental defects induced by Separase point mutation in female mutant PGCs may also be the consequences of mitotic errors and mitotic arrest. Therefore, mitosis in the PGCs of female mutant mice was carefully observed by immunostaining for DNA, Mvh, and phospho-Histone 3, one of the mitosis markers. As expected, normal mitotic cell configurations in the different mitosis stages were visualized (data not shown). The female PGC mitotic arrest was evidenced by quantitative evaluation showing that the mitotic indices of mutant PGCs increased slightly at 11.5 dpc and became significantly higher than that of control PGCs from 12.5 dpc to 13.5 dpc (Figure 3A). Similarly, the mitotic arrest was accompanied by aberrant mitosis characterized by mis-aligned and lagging chromosomes (Figure 3B), which are the typical mitotic errors resulting from premature chromosome segregation. Furthermore, sister chromatids remained intact in the control PGCs but prematurely separated in the mutant PGCs (Figure 3C & D). Like the mutant male PGCs, the mutant female PGCs with abnormal nuclei adapted to sustained mitotic checkpoint activa- tion featured with abnormal Aurora B staining [28] (Figures S2A & B). Separase S1121A point mutation caused genome instability of mutant female PGCs It has been previously described that Securin2/2/Separase+/S1121A double mutant ES cells [30], Hela cells over-expressing human S1126A mutant Separase [31], and male Separase S1121A mutant PGCs undergo aberrant mitosis and mitotic arrest, resulting in aneuploidy [28]. These facts led us to test whether surviving PLoS ONE | www.plosone.org 2 April 2011 | Volume 6 | Issue 4 | e18763