PCNA Regulates Primordial Follicles Assembly Figure 3. The expression of PCNA is associated with the development of oocytes in fetal mouse ovaries. (A) Examples of oocytes at every sub-stages of meiotic prophase prepared by cytospinning from fetal mouse ovaries and immunostained for SCP3 (Green) and PCNA (Red). Bar: 10 mm. (B) The percentage of PCNA positive oocytes was negatively correlated with the frequency of pachytene oocytes during the development of fetal and neonatal mouse ovaries. Each bar represents a mean6s.d. of data from 24 or 16 ovaries from two independent experiments in different animals. R = 20.95, Chi-Square Goodness-of-Fit test. doi:10.1371/journal.pone.0016046.g003 cultured mouse ovaries. It is thus necessary to validate the RNAi technology in cultured mouse ovaries. We first examined the efficiency of delivery of siRNAs to cultured mouse ovaries by using Cy3-labeled non-targeting siRNAs as an indicator. Ninety-six hours following metafectene-mediated transfection of cultured 18.5 dpc mouse ovaries with Cy3-labeled non-targeting siRNAs, bright Cy3 fluorescence was observed in almost all of oocytes and somatic cells in cryostat sections (Figs. 4A, B). This is confirmed by the observation of single cells from ovaries treated with Cy3- labeled siRNAs, in which 92% of oocytes and somatic cells showed intense Cy3 fluorescence (Figs. 4C, D). These data showed that siRNAs could be delivered efficiently into ovarian cells and sustained at a considerable concentration in the cells throughout the culture period under our experimental conditions. Another major concern of RNAi technology is toxicity and interferon response invoked by RNAi reagents [56]. We tested the toxicity of the RNAi reagents on cultured fetal mouse ovaries by inspection of ovarian morphology and the fraction of apoptotic cells indicated by TUNEL labeling. After a 96-hour treatment on 18.5 dpc ovaries with either transfection reagent (metafectene) alone, non-targeting siRNAs alone, or both together following our transfection protocols, the morphology and size of ovaries and the number of apoptotic cells in sections were indistinguishable from the untreated controls (Figs. 4E–H). To evaluate the interferon PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16046