to 1 dpp mouse ovaries (Fig. 3B). In contrast, the finding that all oocytes in adult mouse ovaries were labeled by PCNA antibody affirms the increasing PCNA expression from 18.5 dpc to 5 dpp [16,18]. Interestingly, the differential expression of PCNA during the development of fetal and neonatal mouse ovaries was tightly correlated with the progression of meiotic prophase I and the development of follicles (Fig. 3). The frequency of PCNA-positive oocytes decreased in parallel with the decrease of oocytes in pre- leptotene, leptotene, and zygotene stages. This can be perfectly explained by the findings that the oocytes in these stages are undertaking DNA synthesis and repair [70], and PCNA plays a central role in DNA replication and repair as the processing factor of DNA polymerase [71]. This finding is also supported by the observation that oocytes at pachytene were not labeled by PCNA antibodies (Fig. 3A), and this variation in expression of PCNA was also found in fetal rat ovaries [22]. With the progression of meiotic prophase I, PCNA proteins were again detected in oocytes at diplotene, and the frequency of PCNA-positive oocytes increased rapidly with the accumulation of diplotene oocytes. The time of re- expression of PCNA in oocytes is coincident with the initiation of primordial follicle formation, followed by a rapid increase and maintenance of a high frequency of PCNA-positive oocytes during active primordial follicle formation. An increase in PCNA expression during primordial follicle formation has also been detected in mouse and rat ovaries by others [18,20,22]. The increased expression of PCNA in oocytes around the initiation of primordial follicle formation, and the observation that two-thirds of oocytes die during primordial follicle assembly [5,7] suggest a role of PCNA in the regulation of oocyte fate, i.e., death or survival to form primordial follicles. After inhibition of PCNA expression by RNAi in ovaries during primordial follicle formation, surviving oocytes were significantly increased and apoptotic oocytes were correspondingly reduced (Figs. 6A–E). Overall, with the development of oocytes in meiotic prophase I, PCNA might play a central role in DNA replication and repair before pachytene stage, and then participate in regulation of apoptosis at diplotene stage during primordial follicle assembly. This was consistent with the increased expression of anti-apoptosis genes and suppression of pro-apoptotic genes (Figs. 6F, 8C). PCNA has been reported to participate actively in the regulation of apoptosis, either by promoting pro-apoptotic proteins, such as Ing1b or suppressing anti-apoptotic proteins, including Gadd45, MyD118, and CR6, in somatic cells [51,57,60]. Moreover, binding of Ing1b to PCNA has been shown to regulate the induction of apoptosis by UV irradiation [57], and Ing1b RNAi ovaries, like PCNA reduced ovaries, exhibited more oocytes and primordial follicles (Figs. 5D, 6B, Fig. S1B–E). These results not only indicate that Ing1b participates in apoptosis of oocytes, but also corroborate the observation that PCNA can regulate apoptosis of oocytes in fetal and neonatal mouse ovaries. In mammals studied so far, germ cell loss is a striking feature of primordial follicle formation [5,7]. In fetal and neonatal mouse ovaries, it was observed that approximately two-thirds of oocytes undergo cell death, whereas surviving oocytes develop into primordial follicles [5.7]. Several possible mechanisms, including apoptosis [7,8,9,10,11], autophagic cell death [10,11,12] and cell extrusion from ovaries [12], have been proposed for oocyte loss, with apoptosis of oocytes 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]. Recently, autophagic oocyte death was observed by two groups when individual isolated oocytes but not intact ovaries were cultured in vitro [10,11], even though, the apoptotic cell death was PCNA Regulates Primordial Follicles Assembly also found to be the major mechanism of oocyte death, and when newborn mouse ovaries were cultured in the absence of serum [12]. Recently, based on analysis of static images of mouse ovaries at various developmental points, Rodrigues et al. (2009) suggested that oocyte extrusion could result in loss of a significant fraction of oocytes during primordial follicle formation [12]. However, current technologies cannot definitively elucidate this process, as pointed out by the authors. Although multiple mechanisms might function simultaneously, our results, in agreement with many other studies, support the notion that apoptosis rather than autophagic cell death is a major mechanism for oocyte loss during primordial follicle assembly (Figs. 6C–F, 7B–D), and PCNA may participate actively in oocyte loss by regulating oocyte apoptosis. The exact mechanism by which PCNA regulates apoptosis of oocytes during primordial follicle formation remains unknown. One possible explanation is that a surveillance mechanism may exist in ovaries around the time of primordial follicle formation and oocytes that do not satisfy the mechanism are eliminated through apoptosis. PCNA, perhaps in a manner analogous to P63, may serve as a component of this mechanism. Deletion of P63 in mouse ovaries protects oocytes from irradiation-induced apoptosis [72,73]. Whether or not and how PCNA can protect oocytes with genetic damage from apoptosis remains to be tested. Nevertheless, our results provide strong evidence that PCNA plays an important role in apoptosis-mediated oocyte loss during primordial follicle formation in neonatal mouse ovaries. Accompanying a decreased rate rate of apoptotic oocytes, more primordial follicles were observed in PCNA RNAi ovaries (Figs. 5C, D). This is consistent with earlier reports that deregulation in the expression of apoptosis-related genes from the caspase family, Bcl-2 family, and TNF pathway, which are responsible for oocyte survival, could lead to alteration of follicle assembly. For example, deletion of caspase-2 and Smpd-1 resulted in more surviving oocytes and promoted follicle formation in neonatal mouse ovaries [31,32]. Knockout of Bcl-2 and Bcl-XL in mice decreased the viability of oocytes around the time of primordial follicle formation [13,62]. Bax has also been shown to participate in the regulation of oocyte apoptosis during follicle assembly by several studies [61,63], even though Greenfeld et al. reported that the increased oocytes and follicles in Bax knockout mice possibly resulted from the decreased apoptosis of PGCs prior to PGC colonization in the gonadal ridge [61]. TNFa and TNFR2 have also shown to regulate primordial follicle assembly through promoting oocyte apoptosis [64,74]. Considering the decreasing rate of oocyte apoptosis and the increasing number of primordial follicles in PCNA RNAi mouse ovaries, we propose that PCNA promotes primordial follicles assembly by down-regulation of oocyte apoptosis during the perinatal stage. Primordial follicles are formed by an individual oocyte enclosed within a single layer of squamous granulosa cells in neonatal mouse ovaries [7]. An oocyte arrested in the diplotene stage interacts with surrounding somatic cells (also called pre-granulosa cells) which progressively enclose the oocyte and ultimately transform into squamous granulosa cells around it to form a primordial follicle [7]. Pre-granulosa and squamous granulosa cells arrest in the G0 stage and undergo a growth phase until the transition from a primordial to a primary follicle, during which proliferation is exceedingly slow [75]. Thus, proliferating somatic cells are not likely to participate in primordial follicle assembly. Consistently, although somatic cell proliferation decreased slightly following PCNA RNAi, more primordial follicles were still formed (Fig. 7A, Fig. S2C). These results indicate that altered proliferation of somatic cells following PCNA RNAi in neonatal mouse ovaries did not affect primordial follicle assembly. PLoS ONE | www.plosone.org 12 January 2011 | Volume 6 | Issue 1 | e16046