Separase and Oogenesis Figure 1. Reduced follicles, decreased ovulation and decreased fertilization of female Meox-2-cre/Separase S1121A mutant mice. A. Hematoxylin-eosin staining of ovary sections obtained from three-week-old mice. Bar = 5.0 mm. B. Quantitative analysis of different kinds of follicles of three-week-old mice (n = 3) (** p,0.001, T test). C. Quantitative analysis of super-ovulation of four-week-old mice (n = 5) (** p,0.001, T test). D. Genotyping of superovulated oocytes from four-week-old wild-type and heterozygous SeparaseS1121A mice. Two bands are visible. The lower bands in both lanes are the wild-type Separase allele; the upper band is the SeparaseS1121A allele. E. Quantitative analysis of the fertilization rate of four- week-old mice (n = 4) (** p,0.001, T test). doi:10.1371/journal.pone.0018763.g001 female PGCs with the Separase point mutation manifested genome instability. PGC nuclei were carefully checked by immunostaining for DNA and Mvh. Micronuclei appeared in female mutant PGCs (Figure 4A). Quantitative evaluation of E11.5 to E14.5 samples showed that the proportion of mutant PGCs with micronuclei increased slightly at 11.5 dpc, then dramatically from 12.5 to 14.5 dpc. Meanwhile, all control samples had few PGCs with abnormal nuclei (Figure 4B), underscoring that it was the Separase point mutation that led to the genome instability of female PGCs, as in male PGCs. Examination of these PGCs by immunostaining for c-Tubulin revealed that many of them contained multiple centrosomes (Figure 4D & E), confirming the genome instability of the female PGCs. Interestingly, Separase is also involved with the regulation of centrosome duplication [32]. Therefore, multiple centrosomes and abnormal nuclei may be the results of chromosome mis- segregation, or abnormal centrosome duplication, potentially mediated by the deregulation of Separase. The mutant female PGCs underwent apoptosis in the same way as male mutant PGCs, as shown by caspase-3 activation (Figure 4C). Upon finishing mitotic proliferation, PGCs entered a premeiotic stage. In the male genital ridge, meiosis proceeded no further, and the germ cells entered mitotic arrest as G0/G1 prospermatogonia. In the female genital ridge, however, the germ cells committed to meiosis and passed through leptotene, zygotene, and pachytene stages before arresting in diplotene around the time of birth [7]. We were very interested in the meiotic situation of the mutant female PGCs that bear the Separase mutation. Examination by immunostaining with the meiotic-specific marker phospho-H2AX- c showed that some of the Mvh-positive cells were co-stained by phospho-H2AX-c in both female control and mutant PGCs of E13.5 (Figure S3A). There was no significant difference in the percentage of phospho-H2AX-c-positive cells over Mvh-positive cells (Figure S3B), indicating that the PGCs suffering from Separase mutation have developed into normal meiotic oocytes. Sex-specific differences in Securin levels Although the Separase mutation induced similar mitotic defect in male and female PGCs, the above results indicated that there was an apparent sex-specific difference in the efficiency of abnormal PGC depletion. Similar to the meiotic aneuploidy, this discrepancy may be due to the fact that the checkpoint functions more effectively in spermatogenesis than in oogenesis [33,34]. Thus, we postulated that the gender effects of Separase mutation- PLoS ONE | www.plosone.org 3 April 2011 | Volume 6 | Issue 4 | e18763