PCNA Regulates Primordial Follicles Assembly Figure 5. PCNA RNAi effciency and specificity is validated in cultured fetal mouse ovaries, and down-regulation of PCNA in fetal mouse ovaries increases the number of primordial follicles. (A) PCNA and Oas1 mRNA levels were determined by real-time PCR in 18.5 dpc ovaries transfected with nontargeting siRNAs (Control) or siRNAs against PCNA for 96 hours. (B) PCNA protein levels were determined by Western blot in 18.5 dpc ovaries transfected for 96 hours. A representative image of four independent experiments is shown. (C) Follicles/oocytes were detected immunohistochemically in sections of PCNA RNAi or control ovaries. MVH is a protein that specifically expresses in the cytoplasm of germ cells. Bar: 25 mm. (D) Quantification of primordial and primary follicles in PCNA RNAi and control ovaries from five independent experiments in different animals. Each bar represents a mean6s.d. of 16 (A) and 20 (D) ovaries. #: P,0.05, unpaired t test, *: P,0.05, **: P,0.01, student’s t-test. doi:10.1371/journal.pone.0016046.g005 ovaries [12], have been proposed for oocyte loss, with the definitive elucidation of oocyte extrusion being impossible because of technologies available at present [12]. We thus analysed the expression of apoptosis- and autophagy-related genes after PCNA siRNAs transfection, to understand which mechanism(s) is/are responsible for the increased oocytes in mouse ovaries around primordial follicle formation after down-regulation of PCNA expression. All the autophagy markers tested, LC3I, LC3II, Atg5, and Becn1 [59] did not show any changes after PCNA RNAi (Fig. 6C). However, apoptotic oocytes, detected using the TUNEL assay, were observed in both PCNA RNAi and control ovaries at 48 and 96 hours after siRNA transfection (Fig. 6D, arrows). Significantly fewer apoptotic oocytes were noted in PCNA RNAi ovaries compared to controls (Fig. 6E). These results indicate that the increase in oocytes in PCNA RNAi ovaries results from decreased oocyte apoptosis rather than autophagy. PCNA has been reported to participate in apoptosis of somatic cells [51,57,60]. Several genes from the Bcl-2 family [14,61,62,63], TNF pathway [61,64], caspase family [32], and PAR family [65] have been shown to be involved in the regulation of oocyte PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e16046