The function of female germline stem cells (FGSCs, also called oogonial stem cells) in the adult mammalian ovary is currently debated in the scientific community. As the evidence to support or discard the possible crucial role of this new class of germ cells in mammals has been extensively discussed, in this review, we wonder which could be their origin. We will assume that FGSCs are present in the post-natal ovaries and speculate as to what origin and characteristics such cells could have. We believe that the definition of these features might shed light on future experimental approaches that could clarify the ongoing debate.
From their first description in the human embryo, just 100 years ago (Felix 1911, Fuss 1911, 1912), the studies on primordial germ cells (PGCs), the embryonic precursors of the adult gametes, have remained limited to the reproductive field. In the last decade, interest in the natural development of PGCs has increased, as PGCs possess many of the secrets of genome reprogramming and stemness plasticity. Until now, in mammals, no ‘magic’ germ plasma has been identified in the egg to determine the germ cell fate like that occurring in other animal species (i.e. Drosophila, Caenorhabditis elegans and Xenopus laevis). At least in mice, PGCs are derived from epiblast cells that are undergoing somatic lineage differentiation. To establish the germline, the somatic program must be inhibited and pluripotency reacquired. Before and after reaching their final residence in the gonadal ridges, PGCs undergo unique genome-wide modifications, including imprinting erasure, that enable them to embark into gametogenesis (Hackett et al. 2012, Magnusdottir et al. 2012). In particular, in female mammals, PGCs, called in some species oogonia after their settling into the gonadal ridges, proliferate to give rise to primary meiotic oocytes, which are subsequently enclosed in primordial follicles. As in mammals proliferating PGCs/oogonia seem to disappear from the ovary after the fetal or early post-natal period, the possibility of forming new oocytes and follicles should be precluded. Conversely, in male mammals, but also in females of other species (i.e. Drosophila, C. elegans and sea urchin), PGCs give rise to germ stem cells (GSCs) able to self-renew and to give rise to meiotic germ cells. The presence of GSCs or female germline stem cells (FGSCs), also termed oogonial stem cells, in the mammalian ovary and consequently the possibility of forming new oocytes and follicles also after birth, termed ‘neo-oogenesis’, have been hotly debated at the beginning of the last century and after the recent papers from J Tilly's group (Johnson et al. 2004, 2005a). In this review, we will not discuss the old and new results in favour or against ‘neo-oogenesis’ in mammals because these have recently been thoroughly addressed (Tilly et al. 2009, De Felici 2010, Oatley & Hunt 2012, Woods et al. 2012). We will instead assume that FGSCs are present in the post-natal ovaries, and we will speculate about what origin and characteristics such cells could have. We believe that the definition of these features might shed light on future experimental approaches that could clarify the ongoing debate.
PGC formation outside the gonads is the first stage of gametogenesis in all animal species
In all animal species, the early precursors of the oogonia/oocytes are the PGCs. PGCs are set aside from the somatic cell lineages early in the embryo before they are enclosed within the developing gonads. With the exception of some species of echinoderms in which there are no follicular cells around the oocyte (Bottger et al. 2004), the gonadal cells will give rise to follicular/granulosa cells of the follicle, the structure inside which single oocytes develop and mature.
In the embryo, the formation of cells far from their final residence is a common feature of some pluripotent stem cells such as, the neural crest cells and the haematopoietic stem cells. PGCs are potentially totipotent cells due to their unique ability of generating the mature gametes that give rise to the zygote upon fertilization. It is likely that the formation of cells with multiple differentiations requires special microenvironments or ‘niches’ suitable for maintaining the undifferentiated state of stem cells. As a matter of fact, in all species where GSCs are present in the ovary (such as Drosophila and C. elegans), they derive from PGCs and become enclosed into a ‘niche’ in which they can undergo self-renewal and generate differentiating germ cells (Matova & Cooley 2001, Yamashita 2010).
These considerations favour the view that the putative mammalian FGSCs should originate outside the ovary and that a ‘niche’ should exist inside the ovary where the stemness of such cells is maintained. Otherwise, we should think that in the post-natal ovary, the conditions for PGC formation from other type of cells should be periodically or continuously recreated. But what could be such conditions?
PGCs are specified from pluripotent stem cells by inductive external signals
In mouse embryos, the germ cell lineage emerges from the pluripotent epiblast prior to gastrulation. In response to BMPs and WNTs from adjacent tissues, few cells in the posterior region of the proximal epiblast become the founders of the germline. Establishment of the germ cell fate relies on a distinct transcriptional program and a set of epigenetic regulators (Hackett et al. 2012, Magnusdottir et al. 2012, Saitou et al. 2012). In particular, Prdm1 (also known as Blimp1) is necessary to repress the ongoing transcriptional program of somatic cells and Prdm14 activates the re-expression of pluripotency genes (Kurimoto et al. 2008, Yamaji et al. 2008). Several attempts have been made to reproduce PGC specification and meiotic entry in vitro from different sources of pluripotent cells. However, only very recently healthy offspring were born from PGCs specified in vitro, under chemically defined conditions, from embryonic stem cells and induced pluripotent stem cells (Hayashi et al. 2011, 2012). A critical requirement to recapitulate PGC specification in vitro is to induce the pluripotent cells to adopt a transient epiblast-like state (Hayashi et al. 2011, 2012). In addition, a further requirement for obtaining functional oocytes from such in vitro specified PGCs is their aggregation with freshly isolated pre-follicular cells (Hayashi et al. 2011, 2012). When imagining a potential origin for the putative FGSCs of mammals, we find it rather unlikely that epiblast-like cells are present within the adult ovary. It is difficult to imagine how somatic cells within the adult ovary could generate the appropriate environment to induce other somatic cells to become competent to adopt a germ cell fate. In other words, while the existence of FGSCs in mammals may find parallelisms with germline development in other organisms, the transdifferentiation and reprogramming of ovarian somatic cells into PGCs currently lack any logical precedent and await experimental proof.
FGSCs could originate from undifferentiated PGCs or a subpopulation of PGC precursors
On the basis of the evidence reported in the previous sections, we would favour the possibility that if FGSCs exist in the post-natal mammalian ovaries, they should originate from pre-meiotic PGCs probably already undergoing imprint erasure. FGSCs, however, should not be simple undifferentiated PGCs but rather distinct ones. Within the developing ovary, a ‘niche’ that allows very few PGCs to acquire some characteristics not normally present in female fetal germ cells such as cell cycle quiescence and self-renewal capability should be established (Fig. 1, Upper panel). From this perspective, it will be critical to elucidate which are the differential molecular mechanisms that allow a PGC to escape the signalling that induces meiotic commitment and instead of oogonia characteristics induces female PGCs to adopt a gonocyte-like phenotype. It might be that the quiescent state of putative mouse FGSCs is maintained through modulation of chromatin structure, particularly by histone deacetylation, and through inhibition of CDK2 (Johnson et al. 2005b, Lee et al. 2007). In the male germ line, spermatogonial stem cells (SSCs) originating from PGCs are intrinsically pluripotent and posses both cell cycle quiescence and self-renewal capability; in SSCs, however, imprinting is already partly re-established (Davis et al. 2000, Kerjean et al. 2000). Two other types of pluripotent stem cells that originate from PGCs either in vitro or in vivo are the embryonic germ (EG) cells and the embryonic carcinoma (EC) cells respectively. EG and EC cells posses self-renewal capability and variable imprinting status but are actively proliferating cells (Donovan & de Miguel 2003). At least in mice, the core pluripotency network relies on three transcription factors Pou5f1, Sox2 and Nanog, while components of the LIF-STAT3 and BMP4-ID interact with the core members to maintain the self-renewal potential of the pluripotent stem cells. Self-renewal of rodent SSCs is greatly influenced by the niche factor glial cell line-derived neurotrophic factor (GDNF; Oatley et al. 2007). While Pou5f1, Sox2 and Nanog genes are highly expressed in mouse PGCs, they do not seem to possess active LIF-STAT3 and BMP4-ID nor GDNF-dependent pathways (Durcova-Hills et al. 2008). Two BMPs termed Dpp and Gbb function synergistically to maintain GSCs in Drosophila while a NOTCH signalling cascade is essential in maintaining GSCs in C. elegans (for a review, see Wong et al. (2005)). Activation of similar pathways could be essential for acquisition of FGSC self-renewal capability from PGCs. Interestingly, evidence is arising for a role of Notch signalling in the regulation of mouse folliculogenesis also (Johnson et al. 2001, Trombly et al. 2009).
Mariusz Ratajezak and his group proposed the intriguing possibility that cells with self-renewal capability may already be present as a fraction of the founder PGC population (Kucia et al. 2006, Trombly et al. 2009; Fig. 1, Lower panel). These cells would be ‘epiblast-like PGC precursors’ originating at the base of the allantois, the anatomical location shared by specified PGCs and haematopoietic precursors, that would remain in an undifferentiated state and potentially migrate along with the ‘normal’ PGCs. Within the bone marrow, the existence of such cells characterized by very small size (about 2–4 μm) and so termed very small embryonic-like (VSEL) stem cells is accepted, but a demonstration of their ability to commit to the germ cell fate is, to the best of our knowledge, totally lacking. Lineage tracing experiments could shed light into the origin and differentiation potential of the VSEL stem cells, although this could be difficult to implement if these cells have no unique gene to distinguish their identity. It would also be extremely informative to know which fate VSEL stem cells would acquire if cultured under conditions that induce PGCs' specification or transformation into EG or ES.
The isolation of fresh FGSCs in mouse and in human ovaries relies on the binding to live cells of an antibody raised against the C-terminus of Ddx4, or Vasa homologue (Zou et al. 2009, Pacchiarotti et al. 2010, Woods et al. 2012). Although the presence of plasma membrane Ddx4 remains questionable until a clear image of the plasmalemma distribution of the protein is obtained and an attempt to isolate FGSCs from Ddx4 null mice is performed, immunopurification of FGSCs has been reproduced independently in many laboratories worldwide. This finding enables the implementation of several experimental approaches to investigate whether PGCs are able to produce FGSCs or contain a subpopulation of FGSC-producing cells. One is to systematically investigate to what extent the cell surface variant of Ddx4 may be expressed within the bulk population of PGCs. Eventually, it would be interesting to determine whether Vasa exposure on the membrane is associated with any particular germ cell state. Another possibility would be to culture PGCs under the same conditions used to culture the putative FGSCs and observe whether colonies of highly proliferating oogenic cells appear. Finally, a purified population of PGCs could be injected into ovaries to investigate whether de novo folliculogenesis occurs. This last experimental approach has been recently used by Zhang et al. (2012) and by us and will be discussed in the next section.
Might pre-meiotic PGCs remain and form follicle-enclosed oocytes in the post-natal ovary?
Most probably, if FGSCs originate from PGCs, pre-meiotic PGCs or a subpopulation of PGC precursors mentioned earlier would remain in the post-natal ovaries. The environment of the adult ovary should then be suitable to recapitulate all the stages of ovogenesis from PGCs to fully grown oocytes, including the crucial process of follicle formation. Information about the mechanisms that control this process is still scarce, but if ‘neo-oogenesis‘ occurs in the adult ovary, precursors of follicular cells should also be present and their differentiation should be coordinated with oocyte formation. Lei & Spradling (2013) have recently reported that PGCs divide mitotically and form nests of associated cells that partially fragment prior to meiosis. Thus, retention in small cysts might represent the way to preserve undifferentiated PGCs within the post-natal ovary. Some evidence exists that in the mouse and in some primate species, including humans pre-meiotic proliferating PGCs/oogonia remain in the post-natal ovary during the first period after birth perhaps until puberty (Motta & Makabe 1982, McClellan et al. 2003, Johnson et al. 2004, Telfer 2004, Niikura et al. 2009, Byskov et al. 2011, Zhang et al. 2012). The question of whether any of these proliferating germ cells become follicle-enclosed oocytes remains open.
In an effort to ascertain the existence of FGSCs in post-natal mouse ovaries, it has recently been shown that adult mouse ovaries are able to support the formation of new follicles when provided with pre-meiotic female PGCs and companion pre-follicular cells; transplanted PGCs were, however, able to form follicles only with their own pre-follicular cells and these latter only with the transplanted PGCs (Zhang et al. 2012). Although from these and other results, the authors concluded that ‘neo-oogenesis’ does not occur in adult mouse ovaries; nevertheless, these results give a positive answer to the important question as to whether the adult ovary is able to support ‘neo-oogenesis’ from PGCs. In our view, the fact that transplanted embryonic ovarian cells did not give rise to ‘chimeric follicles’ (Zhang et al. 2012) may be attributed to a higher likelihood of association of the transplanted germ cells with their accompanying follicular cells than with host follicular cells, as extensive evidence exists in the literature that somatic cells from fetal or early post-natal ovaries do support exogenous oocyte development (Eppig & Wigglesworth 2000, Hayashi et al. 2012). In line with this, in experiments carried out in collaboration with J Tilly's group, we observed that 3 days after injecting disaggregated E11.5 gonadal cells from transgenic mice with germline-specific GFP expression into adult ovaries (Yeom et al. 1996), GFP+ germ cells were surrounded by morphologically undifferentiated somatic cells probably from the transplanted tissues (Fig. 2). Similarly, when the putative FGSCs were immunopurified with an antibody raised against the C-terminal extreme of Ddx4, expanded in vitro, and transplanted into the ovaries of mouse adult females, they formed chimeric follicles with the host somatic cells and gave rise to fertilizable oocytes for some oestrus cycles (Zou et al. 2009, White et al. 2012). On the other hand, as reported earlier, when PGC-like cells obtained in vitro from epiblast-like cells, or natural E12.5 PGCs, were re-aggregated with somatic cells of the embryonic ovaries and transplanted under the ovarian bursa or the kidney capsule of nude mice, they underwent ovogenesis in a single wave (Matoba & Ogura 2011, Hayashi et al. 2012). Altogether, these results suggest that PGCs may survive and orchestrate folliculogenesis within post-natal ovaries and that the type of somatic cells in which they are enclosed (their own or that of the host) may determine whether they undergo single or multiple waves of maturation.
Cumulative evidence suggests the existence of a novel germ cell population with the potential to self-renew and perhaps to give rise to new oocytes within the adult ovary of mice and humans. Morphological criteria have not yet matched their biochemical characterization. The lack of a distinct gene expression pattern has so far precluded lineage tracing studies to establish their origin and fate. We favour the view that such cells originate from pre-meiotic PGCs or a subpopulation of PGC precursors remained in the post-natal ovaries. The characterization of such germ cell population in the human ovary potentially increases the possibilities of preserving fertility of women undergoing chemotherapy before or during reproductive age and also restoring their fertility under conditions of premature ovarian failure. This perspective should be kept in mind when controversy is raised around the longstanding dogma of oogenesis in mammals.
Materials and Methods of the experiments cited in the text
C57BL/6 WT females were mated with TgOG2 (Charles River, Wilmington, MA, USA) (Yeom et al. 1996) males and killed on E11.5; gonadal ridges were collected and disaggregated as described previously (Pesce & De Felici 1995). Total cells from four gonads were pelleted and injected into adult ovaries of WT C57BL/6 females as described in Zou et al. (2009). Three recipients were killed 3 days after the injection, and their ovaries were fixed overnight in 4% paraformaldehyde at 4 °C. After embedding in paraffin, serial sections were cut as described in Johnson et al. (2005a, 2005b). Sectioned ovaries were stained for anti-GFP with WT and TgOG2 sections in parallel in every slide. Lack of primary antibody did not produce positive staining. Every section from the transplanted ovaries was analysed. Counterstaining was performed with haematoxylin.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the review.
This review did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
F Barrios holds a Newton International Fellowship from the Royal Society (UK) and thanks Prof. M Azim Surani for laboratory space. The authors thank Dr Yuichi Niikura for technical assistance and Dr Jonathan Tilly for discussion.
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