PCNA Regulates Primordial Follicles Assembly Figure 2. PCNA protein and mRNA level varies with the development of mouse ovaries. (A) The expression and localization of PCNA protein in fetal and neonatal mouse ovaries was detected immunohistochemically using a monoclonal antibody against mouse PCNA in ovary sections (Arrow: PCNA positive oocytes; Arrowhead: PCNA positive somatic cells). Bar: 25 mm. (B) Quantification of oocytes or somatic cells labeled by PCNA antibodies in mouse ovaries. The expression of PCNA protein (C) and mRNA (D) varied in fetal and neonatal mouse ovaries detected by Western blot and real-time PCR. Each bar represents a mean6s.d. of 24 (B) and 12 (D) ovaries from two or three independent experiments in different animals. doi:10.1371/journal.pone.0016046.g002 number decreased with the development of ovaries and reached the lowest in 18.5 dpc ovaries, in which 18% of oocytes stained positive for PCNA. Then, the PCNA-positive oocytes increased rapidly to 54% in 1 dpp ovaries and reached a plateau in 3 dpp ovaries (.92%), at which time primordial follicles start growing. For somatic cells expressing PCNA, a similar pattern, but much fewer cells, were observed in ovaries of different developmental stages examined when compared to PCNA-positive oocytes (Fig. 2B). These observations were consistent with the expression of PCNA protein detected by Western blot analysis (Fig. 2C), and corroborated the findings on PCNA mRNA levels during ovary development (Fig. 2D). To investigate the correlation between PCNA expression and development of germ cells in meiotic prophase I, double-staining was performed using SCP3 and PCNA antibodies. Based on the localization of SCP3 in microspreading preparations of oocytes, the different stages of meiotic prophase I could be easily distinguished [53], unfortunately PCNA could not be detected by immunostaining in these preparations (probably due to the extraction of PCNA protein from the nuclei). To preserve nuclear PCNA, oocytes were cytospun onto microscope slides for SCP3 and PCNA staining. The oocytes in pre-leptotene, leptotene, zygotene, and diplotene, but not in pachytene, were observed to be labeled by PCNA antibody (Figs. 3A, B). Similarly, we noted PCNA staining of spermatogonia and leptotene spermatocytes, but not pachytene spermatocytes in adult mouse testis sections (Fig. S1A), which is consistent with a previous report [54]. Thus, our results indicated rigorous dynamic expression of PCNA in oocytes during meiotic prophase I progression and primordial follicle formation. Liposome-mediated delivery of siRNAs into cultured fetal mouse ovarian cells down-regulates PCNA expression efficiently and specifically The RNAi strategy has been widely and intensively used in gene/protein function analysis in various systems, in vivo and in vitro [55], and recently in cultured hamster ovaries [38], but it has rarely been applied to the studies of gene/protein function in PLoS ONE | www.plosone.org 5 January 2011 | Volume 6 | Issue 1 | e16046