Figure 4. Validation of RNAi system in cultured fetal mouse ovaries. (A–D) Efficiency of delivery siRNAs into ovarian cells was monitored by transfecting cultured mouse ovaries with Cy3-labeled nontargeting siRNAs. Ovaries from 18.5 dpc fetal mice were cultured for 96 hours in the presence (A, C) or absence (B, D) of transfection reagent metafectene (2 ul/ml) and Cy3-labeled siRNAs against luciferase GL2 at 100 nM. The Cy3 fluorescence was detected either in cryostat sections (A and B) or single cells of ovaries (C and D) after 96 hours of transfection. Single ovarian cells were obtained by mechanically dispersing the ovaries and depositing on microscope slides by centrifugation for fluorescence detection. Bar: (B) 100 mm and (D) 10 mm. (E–H)The toxicity of RNAi reagents was determined by the inspection of morphology of ovaries receiving various treatments, and counting apoptotic cells labeled by TUNEL in treated ovaries. Sections from 18.5 dpc ovaries cultured for 96 hours in vitro (E) in medium alone, (F) medium with metafectene, (G) medium with nontargeting siRNAs, and (H) medium with nontargeting siRNAs and metafectene. (Arrow: apoptotic oocytes). Bar: 100 mm. (I) mRNA expression of Oas1, a classical interferon target gene, was measured to determine interferon response PCNA Regulates Primordial Follicles Assembly by real-time PCR in ovaries receiving various treatments. Each bar represents a mean6s.d. of 16 ovaries from three independent experiments in different animals. doi:10.1371/journal.pone.0016046.g004 response after transfection, the mRNA level of 29-59 oligoadeny- late syhthase1 (Oas1), a classical interferon target gene [43,44], was measured by real-time PCR. A similar Oas1 mRNA level was observed in ovaries treated or untreated with metafectene, non- targeting siRNAs, or both (Fig. 4I). More importantly, after a 96- hour transfection of 18.5 dpc ovaries with siRNAs targeting PCNA, PCNA mRNAs were significantly reduced to 38% of the control level (P,0.05), while the Oas1 mRNA level was not significantly different from that in the concurrent control ovaries (P.0.05, Fig. 5A) and PCNA proteins were obviously reduced (Fig. 5B). These observations demonstrate that under our experimental conditions, PCNA expression can be down-regulated efficiently and specifically by RNAi technology without invoking interferon response and cellular toxicity in fetal mouse ovaries. Down-regulation of PCNA by RNAi increases oocyte survival and thus primordial follicle assembly, but retards the development of primordial follicles into primary follicles To understand the consequences of PCNA RNAi on the development of ovarian follicles, primordial and primary follicles were counted separately in sections from 18.5 dpc ovaries transfected with PCNA siRNAs or non-targeting siRNAs (control) for 96 hours (Figs. 5C, D). The ovaries transfected with PCNA siRNAs exhibited significantly more total (P,0.05) and primordial follicles (P,0.05) than the control ovaries. However, significantly fewer primary follicles were observed in PCNA RNAi ovaries than in the controls (P,0.01, Figs. 5C, D). Thus, these data indicate that knockdown of PCNA in mouse ovaries around follicle formation increases primordial follicle assembly, but delays the transition of primordial to primary follicles. An increase in the number of primordial follicles in neonatal mouse ovaries has been attributed to the increased number of oocytes during follicle formation [52]. We thus determined whether or not PCNA RNAi could promote oocyte survival. We counted oocytes in sections of cultured ovaries harvested every 24 hours after PCNA RNAi initiation compared with those in non-targeting siRNAs transfected control ovaries (Figs. 6A, B). A steady decrease in the number of oocytes was observed in both PCNA RNAi and control ovaries with the extension of culture (Fig. 6B). Strikingly, this decrease was much less severe in PCNA RNAi ovaries, with significantly more oocytes being observed at 72 and 96 hours after PCNA RNAi initiation when compared with the concurrent controls, respectively (P,0.05, Fig. 6B). Consistently, down-regulation by RNAi in 18.5 dpc mouse ovaries of Ing1b, a PCNA-binding protein in UV-induced apoptosis regulation [57], significantly increased the number of oocytes and primordial follicles than in control ovaries (Fig. S1B–E). Altogether, these results suggest that down-regulation of PCNA by RNAi in mouse ovaries around follicle assembly is able to alleviate oocyte loss, which could promote the formation of primordial follicles and delay the transition of primordial to primary follicles. The increase of oocytes in PCNA RNAi ovaries is due to decreased apoptosis of oocytes during follicle formation Several possible mechanisms, including apoptosis [7,8,9,10,11], autophagic cell death [10,12,58], and oocyte extrusion from PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16046