Proliferating Cell Nuclear Antigen (PCNA) Regulates Primordial Follicle Assembly by Promoting Apoptosis of Oocytes in Fetal and Neonatal Mouse Ovaries Bo Xu1., Juan Hua1., Yuanwei Zhang 1 , Xiaohua Jiang 1 , Huan Zhang1, Tieliang Ma 1 , Wei Zheng 1 , Rui Sun 1 , Wei Shen2, Jiahao Sha3, Howard J. Cooke4, Qinghua Shi1* 1 Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, China, 2 Department of Animal and Poultry Science, University of Guelph, Ontario, Canada, 3 Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China, 4 MRC Human Genetics Unit and Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, United Kingdom Abstract Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFa and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer guanulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV- induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries. Citation: Xu B, Hua J, Zhang Y, Jiang X, Zhang H, et al. (2011) Proliferating Cell Nuclear Antigen (PCNA) Regulates Primordial Follicle Assembly by Promoting Apoptosis of Oocytes in Fetal and Neonatal Mouse Ovaries. PLoS ONE 6(1): e16046. doi:10.1371/journal.pone.0016046 Editor: Hongmei Wang, Institute of Zoology, Chinese Academy of Sciences, China Received October 9, 2010; Accepted December 3, 2010; Published January 6, 2011 Copyright: 2011 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Basic Research Program (2006CB504003, 2007CB947401) of China (973), and the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX1-YW-R-51, KSCX2-EW-R-07). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: qshi@ustc.edu.cn . These authors contributed equally to this work. Introduction Development of germ cells in female rodents initiates with the migration and colonization of primordial germ cells (PGCs) from the yolk sac to the urogenital ridges [1]. Once the PGCs reach the gonadal anlagen at about 8.5 days post-coital (dpc), the PGCs proliferate continuously until they enter meiosis, after which the cells are referred to as oocytes [2,3]. Oocytes undergo first meiotic prophase and arrest at diplotene at approximately 17.5 dpc in the mouse until ovulation [4,5]. Primordial follicles are crucial for fertility of mammalian females throughout their entire reproductive life [5,6,7] and are formed in neonatal mouse ovaries. During primordial follicle formation, large cysts (a special cluster of oocytes) have been proposed to break into smaller cysts, and this process is repeated until a few individual oocytes remain [7]. Some of the individual oocytes are finally packaged into primordial follicles, and two- thirds of oocytes die in this process [5,7]. Several possible mechanisms, including apoptosis [7,8,9,10,11], autophagic cell death [10,11,12], and oocyte extrusion from ovaries [12], have been proposed for oocyte loss, with apoptosis being the major mechanism revealed by almost all in vivo and in vitro studies by different groups [7,8,9,13,14]. This has been corroborated by observations in mouse models after deletion of apoptosis- regulating genes [13,14]. Autophagic oocyte death was observed by two groups under specific culture conditions, where individual isolated oocytes but not intact ovaries were cultured in vitro [10,11], or newborn mouse ovaries were cultured in the absence of serum [12]. Recently, based on analysis of static images of mouse ovaries, Rodrigues et al. [12] suggested that oocyte extrusion from cultured ovaries could result in loss of a significant fraction of oocytes during primordial follicle formation, however, current technolo- gies can not definitively elucidate of this process. To understand which proteins regulate promordial follicle formation, we used 2D-PAGE and MALDI-TOF/TOF to identify proteins differentially expressed around the time of primordial follicle formation. Proliferating cell nuclear antigen (PCNA) is one of the differentially expressed proteins identified. PCNA is a 36 PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16046