Materials and Methods (Fig. 7B). In PCNA RNAi ovaries, significantly more type 1 (P,0.01) and type 2 (P,0.05) primordial follicles were observed than in the controls, while the number of type 3 primordial follicles was similar to that in control ovaries (P.0.05, Figs. 7B, C). To detect whether or not the observed phenomenon that primordial follicles are enclosed by fewer granulosa cells after PCNA RNAi caused by the disruption of interactions between oocyte-granulosa cells or defects in oocytes, the expression of genes that play key roles in primordial follicle formation, e.g. Figa, Nobox, Lhx8, and NGFb [49,66,67,68], were analyzed by real-time PCR at 60 and 96 hours after PCNA RNAi. With the increase in oocyte number and primordial follicle assembly, these genes increased consistently after PCNA RNAi (Fig. 8C). These results indicate that primordial follicles with fewer granulosa cells after PCNA RNAi did not have defects in the ability of oocytes to recruit granulosa cells. Significantly fewer primary follicles were observed in PCNA RNAi ovaries than in the controls (Fig. 5D). We thus investigated the consequences of PCNA RNAi on the development of primordial follicles by detecting the expression of several genes (Gdf9, Kit, Kitl, ZP3, and Nobox) [66,69], which were reported to be activated during development of primordial follicles after PCNA RNAi. Gdf9, Kit, Kitl, and Nobox were all decreased after PCNA RNAi for 96 hours, which is consistent with fewer primary follicles (Fig. 8C). To understand whether down-regulation of PCNA delays the transition of primordial to primary follicles, we PCNA Regulates Primordial Follicles Assembly analyzed the number of primordial and primary follicles in PCNA RNAi ovaries transfected for 6 and 8 days. Following RNAi for 6 and 8 days, primary follicles were still fewer in PCNA RNAi ovaries than in controls (Fig. S2A), however, the difference between PCNA RNAi and the control ovaries became small with the extension of culture time (Fig. S2B). Altogether, these results demonstrate that down-regulation of PCNA in mouse ovaries around primordial follicle formation delays the transition of primordial to primary follicles. Discussion The expression of PCNA proteins during the development of fetal and neonatal mouse ovaries was carefully analyzed (Figs. 1, 2, 3). A typical V-shape expression pattern, i.e., decreasing expression in ovaries from 13.5–18.5 dpc and increasing expression in ovaries from 18.5 dpc to 5 dpp was observed by Western blot analysis (Fig. 2C). This finding is consistent with the expression of PCNA mRNA detected by real-time PCR analysis (Fig. 2D), and corroborated by the observation on the number of PCNA expressed oocytes and somatic cells in ovary sections (Figs. 2A, B). The decreasing PCNA expression in mouse ovaries aged from 13.5–18.5 dpc is further supported by a function analysis, showing that the expression of non-degradable PCNA arrested meiosis at the early pachytene stage in mouse testes [37] and oocytes in the pachytene stage were found mainly in 16.5 dpc Figure 8. Down-regulation of PCNA increases the number of primordial follicles with fewer granulosa cells, and delays the transition of primordial follicles to primary follicles. (A) Based on the number of granulosa cells enclosing oocytes, the primordial follicles were grouped into three types, with type 1 having 2–3, type 2 4–5, and type 3 more than 5 granulosa cells. (B) Quantification of each type of primordial follicles in 18.5 dpc fetal mouse ovaries transfected with siRNAs targeting PCNA mRNA (RNAi) or nontargeting siRNAs (Control) for 96 hours. (C) The mRNA level of genes which regulated primordial follicle formation and transition of primordial to primary follicles were determined by real-time PCR in 18.5 dpc ovaries transfected with siRNAs against PCNA (RNAi) or nontargeting siRNAs (Control) for 60 and 96 hours. Each bar represents a mean6s.d. of 16 ovaries from three (B) or five (C) independent experiments in different animals. #: P,0.05, unpaired t-test, *: P,0.05, **: P,0.01, student’s t-test, compared with concurrent controls. doi:10.1371/journal.pone.0016046.g008 PLoS ONE | www.plosone.org 11 January 2011 | Volume 6 | Issue 1 | e16046