camera (Olympus Corporation, Japan) and using 206, 406, or 636 1.4 numerical aperture oil immersion objectives at room temperature. The images were collected with software DP controller (Olympus Corporation, Japan), and processed with Adobe Photoshop 7.0 (Adobe Systems Incorporated, San Jose, CA). For the quantitative evaluation of Mvh-positive cells, serial sections of female genital ridges were stained for DNA, Mvh, and phospho-Histone 3. Mvh-positive cells from at least three sections selected by every two sections in at least three embryos (six genital ridges) were scored for each time point. For the quantitative evaluation of mitotic index, serial sections of female genital ridges were stained for DNA, Mvh, and phospho-Histone 3, Mvh single positive cell, as well as Mvh and phospho-Histone 3 double positive cells were scored from at least three sections selected by every two sections in at least three embryos (six genital ridges) for each time point. The mitotic indices of the female PGCs were the percentage of the number of Mvh and phospho-Histone 3 double positive cells divided by the total number of Mvh-positive cells. Whole-mount staining of gonads for PGCs Dissected genital ridges were fixed with 4% paraformaldehyde, washed with deionized water and then stained for alkaline- phosphatase activity with a-naphtyl phosphate (Sigma)/fast red TR (Sigma) solution [28,50]. Collection and culture of germ cells and chromosome spreads Genital ridges at 11.5 dpc were minced and treated with 0.25% Trypsin/EDTA for 30 min at 37uC to dissociate the cells. The resulting cell suspensions were plated on a feeder layer formed by irradiated LIF-expressing SNL fibroblasts in 15% FBS/DMEM for 2 d. The cells were harvested via trypsin digestion after 6 h nocodazole (65 ng/ml) treatment and subjected to AP staining. AP-positive cells were collected through a mouth-pipette. PGC chromosome spread was performed as described [30]. In brief, the collected AP-positive cells were washed once with 16PBS, treated with hypotonic solution (0.8% sodium citrate) at 37uC for 30 min, and fixed with Carnoy’s Fixative (75% methanol, 25% acetic acid) for 3610 min. The fixative was dropped onto the slides. The dried slides were stained with Giemsa stain (Sigma) for 30 min. Culture of mouse ES cells and chromosome spreads Mouse ES cells were cultured on a feeder layer formed by irradiated LIF-expressing SNL fibroblasts in 15% FBS/DMEM (Gibco) [30]. Chromosome spread of mouse ES cells was performed as for PGC. For ES cell aneuploidy assay, we treated the cells with 65 ng/ml of nocodazole for 6 h, removed the nocodazole for 12 h, and then treated the cells again with nocodazole for 12 h to prepare chromosome drop. The chromo- some numbers of the metaphase spreads for each type of the cells were counted. Fertilization assay Female mice were superovulated by intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (PMSG; Calbiochem) followed 46 h later by intraperitoneal injection of 5 IU of human chorinonic gonadotropin (hCG; Calbiochem), they were then References Separase and Oogenesis mated immediately with known fertile male mice. Two-cell stage embryos were collected by flushing the oviducts 43–47 h post hCG. The embryos were stained for DNA. The fertilization rates were the percentage of the number of the 2-cell stage embryos divided by the total number of the collected embryos. Statistical analysis Statistical analysis was performed with Excel 2002 (Microsoft, Redmond, Washington, USA) software. The mean value is shown with standard error. Student’s t-test was used for the comparison of two independent groups (*p,0.05, **p,0.001). Supporting Information Figure S1 Photographs of ovaries. Representative photos of the ovaries from four-week and one-year-old wild-type and SeparaseS1121A littermates. Arrows indicate the degenerated ovaries of the one-year-old mutant. Bar = 20.0 mm. (TIF) Figure S2 Abnormal Aurora B expression in mutant female PGCs. A. Aurora B expression in female PGCs at 13.0 dpc observed by immuno-staining with Aurora B (red), Mvh (green) and DNA (blue). Arrows indicate the Aurora B foci. Bar = 0.5 mm. B. Quantitative analysis of Aurora B-positive PGCs with abnormal nuclei from control and mutant female gonads of 13 dpc. Serial sections of female genital ridges were stained for DNA, Mvh, and Aurora B. All Mvh-positive cells with abnormal nuclei and the Mvh and Aurora B double positive cells with abnormal nuclei 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). (TIF) Figure S3 Mutant female PGCs proceeded to meiotic oocytes normally. A. Meiosis of female PGCs at 13.5 dpc detected by immuno-staining with phospho-H2AX-c (green), Mvh (red) and DNA (blue). Bar = 2.0 mm. B. Quantitative analysis of phospho-H2AX-c-positive female PGCs from wild-type and SeparaseS1121A female gonads of 13.5 dpc. Serial sections of female genital ridges were stained for DNA, Mvh, and phospho- H2AX-c. All Mvh-positive cells and the Mvh and phospho- H2AX-c double positive cells were scored from at least three sections in at least three embryos (six genital ridges). The mean value is shown with standard error. (TIF) Acknowledgments We thank Drs. Yu Liu, Jiankang Jin, Yulong He, and Mr. J. Philippe York for critical reading of the manuscript and discussion. We also thank Jinmei Chen for technical assistance. We are especially grateful to Dr. Toshiaki Noce of Mitsubishi-Kasei Institute of Life Science, Japan, for the ant-Mvh antibody. Author Contributions Conceived and designed the experiments: XH XT. Performed the experiments: JX MW XG BH YD JZ XT XH. Analyzed the data: XH XT. Wrote the paper: XH XT. 1. Uhlmann F, Lottspeich F, Nasmyth K (1999) Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 400(6739): 37–42. 2. Uhlmann F, Wernic D, Poupart MA, Koonin EV, Nasmyth K (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103(3): 375–386. PLoS ONE | www.plosone.org 9 April 2011 | Volume 6 | Issue 4 | e18763