volume of 10 ml, and each reaction contained 5 ml SYBR PREmis EX TaqTM (2x), 0.4 ml forward and reverse PCR primers (10 mM), 0.2 ml ROX reference Dye II, 1 ml cDNA and 3 ml ddH2O. The PCR program consisted of an initial step of 10 seconds at 95uC, followed by 40 cycles of denaturing at 95uC for 5 seconds and extension at 60uC for 31 seconds. All PCR primers used were listed in Table S1. Morphometric evaluation of oogenesis and folliculogenesis To identify oocytes and follicles, ovarian sections were immunohistochemically labeled with an antibody recognized MVH, a protein specifically expressed in germ cells [51], and Hoechst 33342 for nuclei of ovarian cells. The sections were analyzed using an Olympus fluorescence microscope. All cells labeled by MVH antibodies (oocytes) were counted, no matter whether they were enclosed by granulosa cells or not. Based on their localization, oocytes were designed as naked oocytes, or in large cysts, small cysts, primordial or primary follicles according to previous reports [7,52]. Germline cysts are clusters of intercon- nected germline cells. Based on the number of oocytes in each cluster, cysts were classed as small cysts if they had 2–4 oocytes, and large cysts if they contained more than 4 oocytes. Naked oocytes were those that did not locate in cysts and had 0–1 granulosa cells at the surroundings. The oocytes enclosed by at least two squamous granulosa cells were termed primordial follicles. Based on the number of enclosing granulosa cells, primordial follicles were further divided into three categories: type1, the oocytes were enclosed by 2–3 granulosa cells, type 2, by 4–5, and type 3, by more than 5. Oocytes surrounded primarily by a single layer of cuboidal granulosa cells were designated in primary follicles. Statistical analysis In each experiment group, 4–6 ovaries were used, and all experiments were repeated 2–5 times. For each ovary, oocytes, somatic cells or follicles were counted in at least 3 randomly chosen ovarian sections. To eliminate errors in oocyte identifica- tion and counting, all analyses were performed in a double-blind PCNA Regulates Primordial Follicles Assembly manner by two or three individuals and the data were pooled. For real-time PCR and Western blot analysis, two or three cultured ovaries per group were pooled to form one sample, and repeated at least three times for statistical analysis. Chi-square Goodness-of- Fit test was used for correlation analysis. All comparisons between two groups were performed using the paired student’s t-test except quantitative data of real-time PCR were analyzed using unpaired t test or one-way ANOVA with unpaired student’s t-test. P,0.05 was considered significant. Results PCNA expression varies during the development of fetal and neonatal mouse ovaries In fetal mouse ovaries, meiosis initiates at 13.5 dpc, progresses to pachytene of meiosis I at 16.5 dpc in most oocytes, and reaches and arrests at diplotene at 1 dpp in almost all oocytes [5,7]. Primordial follicle assembly starts at 17.5 dpc and continues during the following 4–5 days, with approximately 30% of oocytes enclosed in primordial follicles at 1 dpp (data not shown). To understand which proteins regulate the progression of meiotic prophase I and primordial follicle formation, 2D-PAGE and MALDI-TOF/TOF technology were used to identify proteins differentially expressed in 13.5 dpc, 16.5 dpc, and 1 dpp fetal or neonatal mouse ovaries. PCNA is one of the differentially expressed proteins identified. PCNA is highly expressed in 13.5 dpc ovaries, and significantly decreased with the development of fetal ovaries (Fig. 1A). This finding was confirmed by Western blot analysis using an anti-PCNA monoclonal antibody (Fig. 1B). To more thoroughly understand the pattern of expression and to determine the cell localization of PCNA protein during the development of fetal and neonatal ovaries, an immunohistochem- istry technique was applied to sections of 13.5 dpc, 16.5 dpc, 18.5 dpc, 1 dpp, 3 dpp, and 5 dpp ovaries. PCNA protein was detected in both oocytes (round) and somatic cells (fusiform or cube) in all the ovaries examined (Fig. 2A). However, the number of oocytes and somatic cells expressing PCNA varied distinctly in ovaries of various developmental stages (Fig. 2B). In 13.5 dpc ovaries, almost all of the oocytes (97%) were labeled by PCNA antibody. This Figure 1. Differential expression of PCNA protein in fetal mouse ovaries. (A) Expression of PCNA (arrow) in fetal and neonatal mouse ovaries decreased from 13.5 dpc to 1 dpp detected by 2D-PAGE. (B) The decreasing expression of PCNA in mouse fetal ovaries from 13.5 dpc to 1 dpp was confirmed by Western blot analysis using a monoclonal antibody against mouse PCNA. doi:10.1371/journal.pone.0016046.g001 PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16046