recombination patterns between male and female meiosis; second, more permissive checkpoints in females than males; third, the age- related loss of chromosome cohesion in female gametes; and fourth, the difference in the ability to detect the consequences of meiotic division errors [5,11–15,34]. We found here that Separase phosphosite mutation leads to genome instability and partial depletion in female PGCs, whereas Separase phosphosite mutation caused genome instability and complete abolishment in male PGCs [28]. The differential depletion of PGCs between males and females indicates that there are obvious sexually dimorphic effects of Separase de- regulation. Combined with the fact that Cohesin, the substrate of Separase, is one of the reasons for sex-specific differences of gamete aneuploidy [12–15], we think that sexually dimorphic effects of Separase de-regulation may serve as additional possibilities of sex-specific differences in aneuploidy incidences during gametogenesis. Sexually dimorphic effects of Separase de-regulation on PGCs may be correlated with gender-specific Securin expression After confirming the identical expression levels of mitotic checkpoint regulators and their downstream effectors in male and female genital ridges, we demonstrated that some established factors are not plausible for the gender discrepancy seen in PGC genome instability and depletion. Separase is redundantly controlled by Securin and phosphorylation [30]. Phosphosite mutations caused Separase deregulation exclusively in PGCs [28] and early embryonic cells [29] because they contain insufficient amount of Securin to regulate Separase. Here we provided evidence that the presence or absence of Securin affected the phenotype of Separase mutation in both ES cells and PGCs. Collectively, we believe that Securin expression is correlated to Separase phosphosite mutation-mediated defects in PGCs. The facts that Securin expression affects Separase mutation- mediated defects, and the existence of gender-specific Securin expression discrepancy, suggested that, the sexually dimorphic effects of Separase de-regulation on PGCs may be correlated with the gender-specific Securin expression. Females had considerably more Securin than males, which may alleviate the phosphosite mutation-mediated Separase malfunction, resulting in fewer chromosome segregation error occurrences and thus less PGC death in females than in males. How is the Securin gender-specific discrepancy formed? As we know, sex determination changes the gene expression of PGCs [7,48]. Further investigations are required to decide whether sex determination affects Securin expression or not. Materials and Methods Ethics Statement Animal studies were carried out in an SPF animal facility accredited by the Association for the Assessment and Accredita- tion of Laboratory Animal Care, and all animal protocols were approved by the Animal Care and Use Committee of the Model Animal Research Center, the host for the National Resource Center for Mutant Mice in China, Nanjing University. Generation of mice with Separase inhibitory phosphosite mutation Mice with conditional Separase inhibitory phosphosite mutation were generated, genotyped and sex determined as described [30]. For genotyping, 1–2 mm sections of tail tip were dissolved in Separase and Oogenesis 0.1 ml of 50 mM Tris (pH 8.0), 100 mM EDTA, 0.5% SDS, and 0.5 mg/ml proteinase K (Roche) solution at 55uC over night with vigorous shaking. The DNA was purified by phenol/chloroform extraction followed by ethanol precipitation and then dissolved in 0.2 ml of TE buffer. Genotyping PCR was carried out using rTaq (Takara). Primers used for the analysis were as follows: for Separase, pz228a: 59-cct tct cta acc cag gta gg-39, pz228b: 59-aag agc tct acc tac ctc agg-39, and pz228c: 59-atc gca tcg agc gag cac gta ctc-39. Pz228a/b amplifies S1121A allele, and pz228b/c S1121A-flox-Puro. Sexing of the embryos was performed with a PCR assay on genomic DNA for the presence or absence of Sry, a gene only present on the Y chromosome [49]. Histological analysis Standard histological procedures were followed to prepare fetal gonads and ovaries. In brief, fetal gonads were fixed in 4% paraformaldehyde solution (Sigma), and ovaries were fixed in 10% neutral buffered formalin (Sigma). The specimens were dehydrat- ed through a graded series of ethanol washes, cleared in Histo- Clear, embedded in wax, and sectioned by standard techniques. Sections (4 mm thick) were dewaxed and stained with PAS- hematoxylin or hematoxylin and eosin (H&E). Antibodies Primary antibodies used for immunofluorescence and Western blotting were mouse anti-a-Tubulin (Sigma), mouse anti-b- Tubulin (clone E7; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), mouse anti-P53 and rabbit anti-Bax (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti- active Caspase-3, mouse anti-Aurora B, and rabbit anti-Mad2 (BD Biosciences PharMingen, San Diego, CA), rabbit anti-phospho- Histone H3 (S10) (Cell Signaling Technology, Danvers, MA), mouse anti-c-Tubulin (Sigma-aldrich, St. Louis, MO), mouse anti- Securin (Novocastra, Newcastle, UK), and mouse anti-Mvh (a kind gift from Dr. Toshiaki Noce, Mitsubishi-Kasei Institute of Life Science, Tokyo, Japan). The following secondary antibodies were used: Cy3- and FITC- conjugated anti-rabbit IgG and Cy3- and FITC-conjugated anti- mouse antibodies (Jackson ImmunoResearch Laboratories, Inc.). Western blot analysis and immunostaining The genital ridges or urogenital ridges were collected and lysed on ice in lysis buffer (1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M sodium chloride, 2 mM EDTA, 0.01 M sodium phosphate, pH 7.2) supplemented with protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride. Protein con- centration was determined with Bio-Rad DC protein assay (Bio- Rad). Equal amounts of total protein were separated on denaturing polyacrylamide gel and transferred to polyvinylidene difluoride transfer membrane (Bio-Rad). Blots were probed with the indicated primary and appropriate secondary antibodies and detected using ECL chemiluminescence (GE Healthcare). For immunoassaying, the dewaxed histological sections of the fatal gonads were antigen-retrieved by immersion of grids bearing sections in 0.1 M sodium citrate buffer, pH 6.0, at 95uC for 10 min, permeabilized with 0.4% Triton X-100 in phosphate- buffered saline (PBS) for 10 min at room temperature, covered with blocking solution (5% goat serum in PBS) for 1 h at 37uC, and incubated with the indicated primary antibodies in blocking solution for overnight at 4uC. Primary antibodies were stained with appropriate secondary antibodies for 60 min at 37uC, followed by 4, 6-diamidino-2-phenylindole for 5 min at room temperature. Fluorescence analysis was carried out with a BX51 Olympus fluorescence microscope connected to a DP 20 digital PLoS ONE | www.plosone.org 8 April 2011 | Volume 6 | Issue 4 | e18763