Fig 1.
Impaired fertility of mice with conditional Rb1 inactivation in oocytes.
(A) Representative images of immunostaining with antibodies to Rb1 and phospho-Rb1 in ovarian cross-sections from wild-type adult mice. Normal IgG was used as a negative control. Arrows indicate different stages of developing follicles (i.e. primordial, primary, and secondary/preantral) and the scale bar = 50μm. (B) Representative images of co-immunofluorescence staining for Rb1 and the germ cell marker Ddx4 in ovarian cross-sections from adult control and Rb1-cKO mice. DNA was labeled with DAPI. Scale bar = 50μm. (C) Number of pups born from mating of control and Rb1-cKO females with wild-type males at 2–4 months and 4–6 months after pairing, n = 7 mice of each genotype and * denotes significantly different at P<0.05.
Fig 2.
Ovarian tumor formation in mice with conditional Rb1 inactivation in oocytes.
(A) Representative image of the reproductive tract from an Rb1-cKO mouse. Scale bar = 1cm. (B) Average weight of normal and abnormal ovaries from Rb1-cKO mice. Data are mean±SEM and n = 4 mice of each genotype. *denotes significantly different at P<0.05. (C) Incidence of ovarian tumor formation at 3 months of age in control (Rb1fl/fl, Rb1fl/+), heterozygous Rb1-cKO (Ddx4-Cre;Rb1fl/+ and Ddx4-Cre;Rb1+/∆) and homozygous Rb1-cKO (Ddx4-Cre;Rb1fl/∆ or Rb1-cKO) female mice.
Fig 3.
Formation of ovarian teratomas in mice with conditional Rb1 inactivation in oocytes.
(A-C) Representative images of hematoxylin and eosin (H&E) stained cross-sections from ovaries of control mice (A), Rb1-cKO mice with classical teratoma (B), and Rb1-cKO mice with cystic teratoma (C). Scale bars = 100μm (A) and 500μm (B and C). (D) Representative images of H&E stained cross-sections from an ovary with classical teratoma displaying endoderm, ectoderm and mesoderm-derived tissues. Scale bar = 50μm. (E) Representative images of H&E stained cross-sections from an ovary with cystic teratoma. Scale bar = 50μm.
Fig 4.
Aberrant follicle development in mice with conditional Rb1 inactivation in oocytes.
(A) Representative images of hematoxylin and eosin (H&E) stained cross-sections of ovaries from PD 7 control and Rb1-cKO females. (B) Quantitative comparison of the number of primary and secondary follicles at PD 7. (C) Representative images for H&E stained cross-sections from PD 14 control and Rb1-cKO ovaries. (D) Quantitative comparison of the number of secondary/preantral and antral follicles at PD 14. (E) Representative images of H&E stained cross-sections from PD 21 control and Rb1-cKO ovaries. (F) Quantitative comparison of the number of preantral and antral follicles at PD 21. (G) Representative images of H&E stained cross-sections from PD 35 control and Rb1-cKO ovaries. (H and I) Quantitative comparison of the number of antral follicles (H) and corpora lutea (CL; panel I). Data are mean±SEM of for at least 3 mice per group. Scale bars = 20μm for A, and 50μm for B, C, and D.
Fig 5.
Propensity for parthenogenetic activation is not increased in Rb1 deficient oocytes.
(A) Representative bright field images of MII arrested oocytes from control and Rb1-cKO mice after 48 h of culture. Oocytes from control mice treated with 7% ethanol (EtOH) served as a positive control. (B) Quantitative comparison of the percent parthenogenetic activation (cleavage to 2-cell stage) after 48 h in culture for eggs from control and Rb1-cKO females. (C) Representative images of MII oocytes from control and Rb1-cKO mice immunostained for α-Tubulin and possessing a normal MII spindle (green) and chromosome alignment (blue). (D) Quantitative comparison of germinal vesicle (GV) stage oocytes recovered from control and Rb1-cKO mice 44–45 h after PMSG stimulation. (E and F) Quantitative comparison of the number of oocytes undergoing GV breakdown (GVBD) (E) and first polar body (PB) extrusion after 2–17 h in culture (F). Data represent mean±SEM for 3 mice of each genotype; *denotes significant difference at P<0.05.
Fig 6.
Premature resumption of meiosis in preantral follicles of mice with conditional Rb1 inactivation in oocytes.
(A) Representative images of agarose gels from genotypic analysis of teratomas and host somatic tissues for the Ddx4-Cre transgene. (B-C) Representative images of hematoxylin and eosin (H&E) stained cross-sections of normal follicles in ovaries from control and Rb1-cKO mice at PD 21 and 35 (B) and abnormal follicles from Rb1-cKO mice (C). Arrows indicate abnormal oocytes with condensed chromatin. Arrowhead indicates polar body in an apparent 2-cell embryo. (D) Percentage of Rb1-cKO mice at advancing postnatal ages (n = 6–7 mice at each age point and 33 mice total) that contained abnormal oocytes in ovarian cross-sections (blue line and left vertical axis) and the average number of abnormal oocytes observed per cross-section (red line and right vertical axis). (E) Age distribution of Rb1-cKO mice (n = 10) that developed ovarian teratomas (OT).