Figure 2

CCND2 and P27 are maintained in GCs of primordial follicles but decrease as follicles initiate growth. (A) Immunofluorescent localisation of CCND2 (red) in the prepubertal mouse ovary. High power images show strong (persistent) nuclear CCND2 staining in GCs of primordial follicles (PF; arrows). Transitional follicles (T) show nuclei with variable CCND2 (arrow heads) whereas larger primary plus staged follicles (P+) show weak CCND2 expression. (B) Nuclear CCND2 protein expression in GCs by follicle stage. Each point represents the % positive pixels for all GC nuclei in an individual follicle. (C) Relative expression of Ccnd2 mRNA by qPCR in d4, d8 and d16 ovaries. (D) Immunofluorescent localisation of P27 (green) in the prepubertal mouse ovary. High power images (lower panels) show strong nuclear P27 staining in GCs of primordial follicles (PF; arrows). Transitional follicles (T) show nuclei with variable P27 whereas larger secondary staged follicles (S) show weak P27 expression. (E) Nuclear P27 protein expression in GCs by follicle stage. Each point represents the % positive pixels for all GC nuclei in an individual follicle. (F) Relative expression of p27 mRNA by qPCR in d4, d8 and d16 ovaries. (G) Co-localisation of CCND2 and P27 in the prepubertal mouse ovary. After image processing (d4 and d8; lower panels), only positive pixels in the green and red channels are shown, highlighting co-localisation in white. A negative IgG control (d16; lower right) is shown. (H) P27/CCND2 ratio in individual GCs by follicle stage. Each point represents the % positive pixels for P27 relative to % positive pixels for CCND2 an individual nucleus. Numbers in parentheses refer to the GC number range for each follicle stage. (I) Western blot of P27 and CCND2 following co-immunoprecipitation from d4 and d16 ovaries. Protein samples (12.5 µg) were immunoprecipitated using anti- P27 (IP) or non-specific rabbit IgG (IgG; control). Non-immunoprecipitated protein (5 µg) was used as positive control (IN). Gel images have been cropped from originals provided in Fig. S2. PF, primordial; T, transitional; P, primary; P+, primary plus, S, secondary follicle. Nuclei in A, D, G are labelled with DAPI (blue). Data in B, E, H are medians ± interquartile ranges and differences are relative to the follicle stage in parentheses. Points in red and blue represent measurements from d4 and d8 ovaries, respectively. Data in C, F are means ± SEM (n = 5 ovaries/age) and differences are relative to d4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.