Following PCNA RNAi, primordial follicles with fewer granulosa cells (types 1 and 2 primordial follicles) significantly increased and those with more than five granulosa cells (type 3 primordial follicles) did not change (Figs. 8A, B). These observations may be explained partly by the following. (1) The number of surviving oocytes increased markedly, whereas the number of surviving pre-granulosa cells remained unchanged during primordial follicle assembly after PCNA RNAi (Figs. 6A–E, 7A, Fig. S2C). (2) In PCNA RNAi ovaries, some oocytes may maintain a normal capacity to recruit somatic cells and thus form type 3 primordial follicles, whereas others have a reduced capacity to recruit somatic cells and thus form types 1 and 2 primordial follicles. This suggestion needs to be tested experimentally. Although more primordial follicles were observed after PCNA RNAi, the number of primary follicles decreased (Fig. 5D, Fig. S2A), accompanied by decreased expression of several key regulators of the transition from primordial to primary follicles (Fig. 8C). This inhibition of primordial follicle development could be explained by the decreased proliferation of granulosa cells after downregulation of PCNA (Fig. 7A, box). As the culture time increased, the difference in primary oocyte number between PCNA RNAi and control ovaries decreased, which can be attributed to the decreasing effect of PCNA siRNAs on proliferation of granulosa cells. In mice, developing follicles regulate the development of primordial follicles through secretion of TGF-b superfamily members, e.g. anti-Mullerian hormone, but these factors are not likely to regulate primordial follicle assembly in fetal and neonatal mouse ovaries according to the temporal development of follicles [76]. Therefore, in PCNA RNAi ovaries, although the number of primordial follicles increased, the development of these follicles was impaired. In conclusion, the present study has demonstrated a dynamic expression of PCNA in germ cells and somatic cells during the development of fetal and neonatal mouse ovaries, which is tightly correlated with the progression of meiotic prophase I and assembly of primordial follicles. PCNA expression gradually decreased with meiosis progression from the pre-leptotene to pachytene stage, and steadily increased in diplotene oocytes around the time of primordial follicle formation when massive oocyte loss occurred. Suppressing the increase of PCNA by RNAi reduces apoptosis-mediated oocyte loss, and increases the number of primordial follicles. Although the exact mechanism by which PCNA RNAi promotes oocyte survival in ovaries around primordial follicle formation is unknown at the present time, our results indicate that PCNA plays a functional role during the well-known physiological process of germ cell loss around primordial follicle formation in mouse ovaries. Supporting Information Figure S1 The expression of PCNA is associated with the development of spermatocytes in adult testes, and down-regulation of Ing1b, a binding protein of PCNA in the regulation of the apoptosis induction, increases the References 1. De Felici M, Scaldaferri ML, Lobascio M, Iona S, Nazzicone V, et al. (2004) Experimental approaches to the study of primordial germ cell lineage and proliferation. Hum Reprod Update 10: 197–206. 2. McLaren A (2000) Germ and somatic cell lineages in the developing gonad. Mol Cell Endocrinol 163: 3–9. 3. Monk M, McLaren A (1981) X-chromosome activity in foetal germ cells of the mouse. J Embryol Exp Morphol 63: 75–84. 4. Borum K (1967) Oogenesis in the mouse. A study of the origin of the mature ova. Exp Cell Res 45: 39–47. 5. Pepling ME (2006) From primordial germ cell to primordial follicle: mammalian female germ cell development. Genesis 44: 622–632. 6. Kezele P, Nilsson E, Skinner MK (2002) Cell-cell interactions in primordial follicle assembly and development. Front Biosci 7: d1990–1996. PCNA Regulates Primordial Follicles Assembly number of oocytes and primordial follicles. (A) An example of sections of paraffin-embedded testes from adult mice displayed differential staining of PCNA protein in spermatogonia (S), leptotene (L), and pachytene spermatocytes (P). Bar: 50 mm and 5 mm. (B) Follicles/oocytes were detected immunohistochem- ically in sections of 18.5 dpc ovaries transfected with nontargeting siRNAs (Control) or siRNAs (RNAi) against Ing1b for 96 hours. Bar: 25 mm. Quantification of oocytes (C) and follicles (D) in Ing1b RNAi and control ovaries. (E) Ing1b mRNA level was detected by real-time PCR in the Ing1b RNAi and control ovaries. Each bar represents a mean6s.d. of 20 (B), 12 (C) and 16 (D) ovaries from three or five independent experiments in different animals. #: P,0.05, unpaired t-test, *: P,0.05, student’s t-test. (TIF) Figure S2 Down-regulation of PCNA delays the transi- tion of primordial to primary follicles and decreases proliferation of somatic cells. The index of primary follicles (A) and the number of follicles (B) were determined in PCNA RNAi and control ovaries transfected for 6 and 8 days. (C) Proliferating somatic cells were labeled with an anti-Ki-67 monoclonal antibody in sections of control or PCNA RNAi ovaries, sampled at 24, 48, 72, and 96 hours after siRNA transfection of 18.5 dpc ovaries, respectively. Ki-67-positive cells were cubic and mainly localized in the areas where the primordial follicle assembly was not active during the first 72 hours (black arrow), and in primray follicles at 96 hours (white arrow) after transfection. Bar: 50 mm. Each bar represents a mean6s.d. of 12 ovaries from three independent experiments in different animals. *: P,0.05, student’s t-test, compared with concurrent controls. (TIF) Table S1 Primers used for quantifying the levels of various transcripts present in mouse ovary. (PDF) Acknowledgments We thank Dr. Peter Moens (Department of Biology, York University, Toronto, Canada) for his generous gift of SCP1 and SCP3 antibodies, Dr. Toshiaki Noce (Mitsubishi Kagaku of life Sciences, Tokyo, Japan) for kindly providing us with antibodies against MVH, and Dr. Hans-Uwe Simon (Institute of Pharmacology, University of Bern, Bern, Switzerland) for generous gift of antibodies against Atg5. We also thank Dr. Cheng Wang (University of Nebraska Medical Center, Nebraska, USA) for his suggestions on ovary culture and RNAi experiments. Author Contributions Conceived and designed the experiments: BX JH QHS. Performed the experiments: BX JH YWZ XHJ HZ TM WZ RS. Analyzed the data: BX JH. Contributed reagents/materials/analysis tools: JHS. Wrote the paper: BX JH QHS. Modification of the manuscript: WS HJC. 7. Pepling ME, Spradling AC (2001) Mouse ovarian germ cell cysts undergo programmed breakdown to form primordial follicles. Dev Biol 234: 339–351. 8. De Pol A, Vaccina F, Forabosco A, Cavazzuti E, Marzona L (1997) Apoptosis of germ cells during human prenatal oogenesis. Hum Reprod 12: 2235–2241. 9. Coucouvanis EC, Sherwood SW, Carswell-Crumpton C, Spack EG, Jones PP (1993) Evidence that the mechanism of prenatal germ cell death in the mouse is apoptosis. Exp Cell Res 209: 238–247. 10. Lobascio AM, Klinger FG, Scaldaferri ML, Farini D, De Felici M (2007) Analysis of programmed cell death in mouse fetal oocytes. Reproduction 134: 241–252. 11. De Felici M, Lobascio AM, Klinger FG (2008) Cell death in fetal oocytes: many players for multiple pathways. Autophagy 4: 240–242. PLoS ONE | www.plosone.org 13 January 2011 | Volume 6 | Issue 1 | e16046