Separase phosphosite mutation leads to genome instability and primordial germ cell depletion during oogenesis

Abstract

To ensure equal chromosome segregation and the stability of the genome during cell division, Separase is strictly regulated primarily by Securin binding and inhibitory phosphorylation. By generating a mouse model that contained a mutation to the inhibitory phosphosite of Separase, we demonstrated that mice of both sexes are infertile. We showed that Separase deregulation leads to chromosome mis-segregation, genome instability, and eventually apoptosis of primordial germ cells (PGCs) during embryonic oogenesis. Although the PGCs of mutant male mice were completely depleted, a population of PGCs from mutant females survived Separase deregulation. The surviving PGCs completed oogenesis but produced deficient initial follicles. These results indicate a sexual dimorphism effect on PGCs from Separase deregulation, which may be correlated with a gender-specific discrepancy of Securin. Our results reveal that Separase phospho-regulation is critical for genome stability in oogenesis. Furthermore, we provided the first evidence of a pre-zygotic mitotic chromosome segregation error resulting from Separase deregulation, whose sex-specific differences may be a reason for the sexual dimorphism of aneuploidy in gametogenesis.

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 µm. 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 Separase^S1121A mice. Two bands are visible. The lower bands in both lanes are the wild-type Separase allele; the upper band is the Separase^S1121A allele. E. Quantitative analysis of the fertilization rate of four-week-old mice (n = 4) (** p<0.001, T test).

Figure 2. Partial depletion of PGCs in female Meox-2-cre/Separase S1121A mutant mice.

A. Whole-mount TNAP staining of female gonads of 13.0 dpc. TNAP-positive cells are red. Bar = 20.0 µm. B. Western blot detection of Mvh of gonads at 13.0 dpc. C. Mvh immuno-staining of gonads at 13.0 dpc. Mvh-positive cells are red. Bar = 4.0 µm. D. Quantitative analysis of Mvh-positive cells in female gonads of different stages. Serial sections of female genital ridge were stained for DNA and Mvh. The Mvh-positive cells were scored from at least three sections in at least three embryos (at least six genital ridges) for each time point. The mean value is shown with standard error (** p<0.001, T test).

Figure 3. Abnormal mitotic chromosome segregation in PGCs of female Meox-2-cre/Separase S1121A mutant mice.

A. The graph shows the calculated mitotic indices of the female PGCs from wild-type and Separase^S1121A gonads of various ages as indicated (n = 3) (** p<0.001, T test). B. Aberrant mitotic configurations of female PGCs at 13.0 dpc detected by immuno-staining with Mvh (green/red) and DNA (blue). Inset: higher magnification of DAPI-stained misaligned chromosome and lagging chromosome. Bar = 0.5 µm. C. Chromosome spreads of cultured female PGCs at 13.0 dpc after 6 h nocodazole treatment. Arrows indicated the prematurely separated sister chromatids. Bar = 1.0 µm. D. Quantitative analysis of premature chromosome segregation from chromosome spreads of cultured female PGCs at 13.0 dpc after 6 h nocodazole treatment (n = 4) (** p<0.001, T test).

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 µm. B. Quantitative analysis of micronuclei of female PGCs from wild-type and Separase^S1121A 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 µm. D. Centrosome number in female PGCs visualized by immuno-staining with γ-Tubulin (red), Mvh (green) and DNA (blue). Red dots (arrows) of γ-Tubulin staining indicate centrosomes. Bar = 0.5 µm. 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 γ-Tubulin. The Mvh-positive cells with three or more γ-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).

Figure 5. More Securin expression in female PGCs.

A. Securin levels of PGCs at 13.0 dpc observed by immuno-staining with Securin (red), Mvh (green) and DNA (blue). Bar = 5.0 µm. B. Quantitative analysis of Securin-positive PGCs in male and female gonads at 13.0 dpc. Serial sections of female genital ridges were stained for DNA, Mvh, and Securin. All Mvh-positive cells and the Mvh and Securin double positive cells 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. Western blot detection of Mad2, Aurora B, P53, and Bax of gonads at 13.0 dpc. D. Western blot detection of Mvh and Securin of gonads at 13.0 dpc.

Figure 6. Securin expression is correlated to Separase-mediated defects.

A. Quantitative analysis of karyotyping of the four types of ES cells (n = 3) (* p<0.05, T test). B. Western blot detection of Mvh and Securin of urogenital ridges at 10.5 dpc.