PCNA Regulates Primordial Follicles Assembly Figure 6. Down-regulation of PCNA increases the number of oocytes by alleviating the apoptosis of oocytes in fetal mouse ovaries. (A) Oocytes (arrow) were detected in sections of 18.5 dpc ovaries transfected with siRNAs targeting PCNA mRNA (RNAi) or nontargeting siRNAs (control) for 48 or 96 hours and immunohistochemically labeled with MVH antibodies (green). Bar: 100 mm. (B) The number of oocytes decreased gradually with time of transfection in PCNA RNAi or control ovaries, while the decrease in control ovaries was more dramatic than in PCNA RNAi ovaries. (C) Autophagy-related proteins were determined by Western blot in 18.5 dpc ovaries transfected with siRNAs against PCNA (RNAi) or nontargeting siRNAs (Control) for 96 hours. A representative image of three independent experiments with similar results was shown. (D) Apoptotic oocytes (arrow) were identified by the detection of DNA fragmentation with TUNEL agents in 18.5 dpc fetal mouse ovaries transfected with siRNAs targeting PCNA or nontargeting siRNAs for 48 and 96 hours. Bar: 50 mm. (E) Quantification of TUNEL positive oocytes in PCNA RNAi or control ovaries. (F) mRNA expression of apoptosis-related genes was quantified by real-time PCR in PCNA RNAi and control ovaries 96 hours after transfection. Each bar represents a mean6s.d. of 20 (B), 12 (D) and 16 (E) ovaries from three or five independent experiments in different animals. #: P,0.05, ##: P,0.01, unpaired t test, *: P,0.05, **: P,0.01, student’s t-test, compared with concurrent controls. doi:10.1371/journal.pone.0016046.g006 survival. We therefore investigated the expression of apoptosis- related genes after PCNA RNAi. As shown in Fig. 6F, the mRNA levels of known pro-apoptotic genes (Bax [Bcl-2 family], caspase-3 [caspase family], and TNFa and TNFR2 [TNF pathway], except caspase-7 [caspase family] and Par6 [PAR family]), decreased significantly in PCNA RNAi ovaries (P,0.05). The expression of Bcl-2 (Bcl-2 family), an anti-apoptotic gene, increased following PCNA RNAi (P,0.01). These results indicate that PCNA could regulate oocyte survival mediated by modulating the expression of apoptosis-associated genes. PCNA RNAi slightly decreases the proliferation of somatic cells that are not involved in primordial follicle formation During primordial follicle formation, most pre-granulosa cells migrate and differentiate into squamous granulosa cells without proliferation [5,7]. To determine whether or not altered proliferation of somatic cells could affect primordial follicle assembly and development, we measured cell proliferation in PCNA RNAi ovaries using BrdU incorporation as a marker of DNA replication. BrdU-positive somatic cells were observed in sections of cultured ovaries harvested every 24 hours after transfection initiation in both control and PCNA RNAi ovaries. In the first 72 hours after siRNA transfection, PCNA RNAi ovaries showed a slight decrease in the number of BrdU-positive somatic cells, when compared with the controls (Fig. 7A). However, because these BrdU-positive cells in both PCNA RNAi and control ovaries were cubic and mainly localized in the areas where the primordial follicle assembly was not active (Fig. 7A, white circle, white arrow), it is reasonable to suggest that these somatic cells were not participating in primordial follicle assembly. At 96 hours, significantly more primary follicles were observed in control ovaries and most granulosa cells in these follicles were BrdU-positive (Fig. 7A, red box). However, only a few primary follicles were observed in PCNA RNAi ovaries and these follicles PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e16046