KDa protein which is well-conserved in all eukaryotic species from yeast to human. The expression of PCNA has been reported in fetal and adult ovaries in several arthropods and mammals, but with considerable variation [15,16,17,18,19,20,21,22]. In Marsu- penaeus japonicus, the expression of PCNA mRNA changes significantly with the development of the ovaries [15]. PCNA expression is found in oogonia and oocytes in meiosis I prophase, and increases during oocyte growth in zebrafish [19]. In rabbit ovaries, only part of the oocytes in cysts and primordial follicles are PCNA-positive, and most oocytes stained positive for PCNA in developmental follicles [21]. In adult pig, bovine, and baboon ovaries, PCNA staining was absent from granulosa cells and oocytes of the quiescent primordial follicles, but was intensive in most granulosa cells and oocytes in primary-to-large antral follicles [23,24,25]. Oktay et al. reported that in rat ovaries, the expression of PCNA was not detected in granulosa cells or oocytes in primordial follicles, but increased with the initiation of follicle growth [17]. In two recent papers, however, oocytes in all stages of follicles, including primordial follicles, were labeled by PCNA antibody in adult rat ovaries, and PCNA staining was suggested as a marker for ovarian follicle counts [16,18]. Recently, the distinct expression of PCNA was reported with the development of fetal and newborn rat ovaries, with PCNA-positive oocytes observed at high percentages from 14.5 dpc to 1 day post-partum (dpp), decreasing after birth, and increasing during primordial follicle formation [22]. In mouse ovaries, a complicated PCNA expression pattern was reported in a recent study, in which PCNA staining was intensive in oocytes at 1 dpp when primordial follicles actively assembly, absent in oocytes in all follicles from 14–25 dpp, and absent in primordial, but not primary or secondary follicles from 7–12 dpp and 50–200 dpp [20]. Thus, in mammals the expression of PCNA in oocytes of various meiotic I prophase before primordial follicle formation and in primordial follicles is incom- pletely understood. Distinct expression usually suggests an important role of interested proteins during biological processes, and it has been shown that PCNA serves as a key factor in many essential cellular processes, such as DNA replication, DNA repair, sister-chromatid cohesion, DNA damage avoidance, cell cycle control, and cell survival [26,27,28,29]. PCNA has also been suggested to be a key regulator during the development of ovarian follicles [30], but the exact function of PCNA in meiosis, particularly in primordial follicle formation, remains unknown. In mammals, functions of a protein/gene in the development of germ cells and ovaries are usually studied by gene manipulation [31,32], antibody neutralization [33,34], or chemical treatment [35,36]. However, targeted deletion of PCNA in mice resulted in embryonic death before the initiation of meiosis [37]; antibodies supplied in culture medium may not neutralize PCNA activity in ovaries because of the localization to the nucleus of this protein, and chemicals specifically inhibiting PCNA are not available. Fortunately, Wang and Roy successfully suppressed gene expres- sion in cultured newborn hamster ovaries using RNA interference (RNAi), which provides a convenient and powerful strategy to explore functions of an interested gene during female meiosis and primordial follicle formation [38]. In the present study, we detected expression of PCNA in fetal and neonatal mouse ovaries during meiotic prophase progression and primordial follicle formation, and explored the possible roles played by this protein in primordial follicle formation using a modified RNAi approach [38]. A V-shaped expression of PCNA in both oocytes and somatic cells was observed in ovaries from 13.5–18.5 dpc and from 18.5 dpc to 5 dpp, which is closely correlated with the meiotic prophase I progression from pre- PCNA Regulates Primordial Follicles Assembly leptotene to pachytene and from pachytene to diplotene. Inhibition of the increased expression of PCNA in ovaries around primordial follicle formation alleviates apoptosis of oocytes and increases primordial follicle assembly. Thus, this report provides strong evidence that PCNA plays a crucial role in oocyte loss during ovarian follicle formation. Materials and Methods Animals Male and female ICR mice, which have been used widely for reproduction research [39,40], were purchased from the National Rodent Laboratory Animal Center (Shanghai Branch, China). All mice were maintained according to the Institutional Animal Care and Use Committee of University of Science and Technology of China. Fetal or neonatal mouse ovaries were obtained from the pregnant mice between 13.5–19.5 dpc (0 dpp) and 1–5 dpp. Noon of the day when the copulatory plug was found was designated as 0.5 dpc or 0 dpp. 2D-PAGE, MALDI-TOF/TOF and identification of PCNA To identify proteins differentially expressed in 13.5 dpc, 16.5 dpc, and 1 dpp mouse ovaries, 2D-PAGE, MALDI-TOF/TOF, and Statistical analysis was carried out as described [41]. Differentially expressed proteins were identified by the mass chromatographic/mass chromatographic spectra, and results of mass were cross-referenced with the International Protein Index mouse database (http://www.ebi.ac.uk/IPI/IPIhelp.html). Only the database entry exhibiting the highest number of match in peptides was included. Immunohistochemistry Ovaries or testes were fixed in 10% formaldehyde for 12– 24 hours before paraffin embedding, and 5 mm paraffin sections were attached to microscope slides. Paraffin sections were heated at 60uC for 2 hours. Following de-paraffinization, sections were re-hydrated in a series of graded ethanol/water solutions, then boiled in 10 mM citric acid (pH 6.0) at 95–100uC for 10 minutes followed by incubation in 3% hydrogen peroxide (H2O2) for 10 minutes. The tissues were blocked by BDT (3% BSA, 10% normal donkey serum in TBS) and incubated with a mouse anti-PCNA monoclonal antibody (Zhong Shan Goldenbridge Biotechnology, Beijing, China; 1:100), or rabbit anti-MVH antibody (A kindly gift from Professor Toshiaki Noce, Mitsubishi Kagaku of life Sciences, Japan; 1:1000) or a mouse anti-Ki-67 monoclonal antibody (Zhong Shan Goldenbridge Biotechnology, Beijing, China; 1:100) overnight at 4uC. After rinsing thoroughly with TBS, the sections were incubated with a biotinylated and streptomycin- labeled goat anti-mouse antibody (Maixin Bio, KIT-5010, Fujian, China) for 15 minutes at room temperature or an Alexa 488- conjugated donkey anti-rabbit gamma globulin antibody (Molec- ular Probes, A21206, CA, USA; 1:250) for 1 hour at 37uC. PCNA expression in sections was detected by the reaction of peroxidase with 3,39-diaminobenzidine tetrahydrochloride (DAB) and ana- lyzed using a Olympus BX61 fluorescence microscope. Western blot analysis Lysates from ovaries in vivo or cultured in vitro were separated on 15% SDS polyacrylamide gels and the proteins were then transferred to nitrocellulose membranes (Amersham Biosciences, RPR303D). The membranes were blocked in TBST (0.5% Tween-20 in TBS) containing 5% nonfat milk powder for 1 hour, incubated overnight with a mouse anti-PCNA monoclonal antibody (1:100), a mouse anti-beta actin monoclonal antibody PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16046