PCNA Regulates Primordial Follicles Assembly usually had a small number of BrdU-positive granulosa cells (Fig. 7A). As expected, a majority of BrdU-positive somatic cells were cubic granulosa cells in primary follicles (Fig. 7A, black arrow). These observations were consistent with the expression of Ki-67 protein (Fig. S2C). To further understand whether PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes, without affecting somatic cells in mouse ovaries, oocytes, and somatic cells from cultured ovaries were isolated and used for detection of the expression of apoptosis-related genes. The purity of isolated oocytes and somatic cells were assayed by real-time PCR of Figa and Cyp19 [49,50]. As expected, Cyp19 and Figa were expressed at very low levels in oocytes and somatic cells, respectively, demonstrating the high degree of purity (Fig. 7B). At 60 and 96 hours after PCNA RNAi, the pattern of expression of almost all apoptosis-related genes in isolated oocytes (Fig. 6F) was almost identical to that in PCNA RNAi ovaries, whereas only a slight and inconsistent expression of these genes was detected in somatic cells (Figs. 7C, D). These results indicate that PCNA RNAi mainly affected oocyte, but not somatic cell survival, in cultured ovaries and further supports the notion that proliferating somatic cells may not participate in the process of primordial follicle formation. Down-regulation of PCNA leads to more primordial follicles enclosed by fewer granulosa cells and delays the transition of primordial to primary follicles In mouse ovaries, primordial follicle formation starts with two or three granulosa cells surrounding one oocyte, followed by more pre-granulosa cells migrating towards and enclosing the oocyte continuously [5]. To study the function of PCNA during primordial follicle formation more precisely, we classified primor- dial follicles arbitrarily into three categories, as described in the Figure 7. Down-regulation of PCNA increases oocyte survival, but decreases proliferation of somatic cells without effects on survival of somatic cells. (A) Proliferating somatic cells were labeled by an anti-BrdU monoclonal antibody in sections of control or PCNA RNAi ovaries, sampled at 24, 48, 72, and 96 hours after siRNA transfection, Respectively. BrdU was added into the culture medium 24 hours before ovary harvest. White arrows: BrdU-positive somatic cells; black arrows; BrdU positive granulosa cells; white circle: the areas showing that primordial follicles formed inactively; black box: a primary follicle showing that most granulosa cells are BrdU-positive; red box: a primary follicle showing a few BrdU- positive granulosa cells. Bar: 50 mm. (B) mRNA levels of Cyp19, a maker of somatic cells, and Figa, a marker of oocytes, were determined by real-time PCR in purified oocytes and somatic cells (S cells) in 18.5 dpc ovaries transfected with nontargeting siRNAs (Control) or siRNAs against PCNA for 60 and 96 hours. (C and D) mRNA expression of apoptosis-related genes was quantified by real-time PCR in purified oocytes and somatic cells at 60 (C) and 96 (D) hours after transfection. Each bar represents a mean6s.d. of 16 (B, C and D) ovaries from three or four independent experiments in different animals. #: P,0.05, ##: P,0.01, unpaired t test, compared with concurrent controls. doi:10.1371/journal.pone.0016046.g007 PLoS ONE | www.plosone.org 10 January 2011 | Volume 6 | Issue 1 | e16046