Human Reproduction Update, Vol.10, No.3 pp.197-206, 2004
© European Society of Human Reproduction and Embryology 2004; all rights reserved
Experimental approaches to the study of primordial germ cell lineage and proliferation
Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Rome 1 To whom correspondence should be addressed at: Dipartimento di Sanità Pubblica e Biologia Cellulare, Università di Roma Tor Vergata, Via Montpellier 1, 00173 Roma, Italy. e-mail: defelici{at}uniroma2.it
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Abstract |
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 TOP Abstract IntroductionSegregation of germ cell...PGC’s proliferationConclusionsAcknowledgementsReferences |
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New information regarding primordial germ cell (PGC’s) segregation and proliferation over the last decade is reviewed. Advances have been obtained in the mouse but current knowledge of human PGC’s remains scant. Questions still fully or partially unresolved about the emergence of the germline in mammals are addressed. (i) When and where is the germ line set aside in the embryo? (ii) How is the germ line segregated from the somatic lineages? (iii) Which factors guide PGC’s to the gonadal ridges? (iv) Which factors regulate PGC’s proliferation? The main purpose of this review is to outline the information obtained using mainly in vitro culture systems about two aspects of these processes namely the segregation of PGC’s and their proliferation.
Key words: differentiation/meiosis/mitosis/primordial germ cell/proliferation
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Introduction |
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TOPAbstractIntroductionSegregation of germ cell...PGC’s proliferationConclusionsAcknowledgementsReferences |
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The establishment of the germ cell line in the embryo involves the segregation of the primordial germ cells (PGC’s) from the somatic lineages, migration of PGC’s to the gonad ridges, proliferation of PGC’s and finally their differentiation within the gonads.
In the mouse embryo, this period covers 
7 days from 5.5 days post coitus (dpc) when the first inductive events of the germ cell lineage are likely to occur in the epiblast, to 12.5 dpc when PGC’s, upon ending gonad colonization, differentiate according to the embryonic sex (Figure 1).
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In humans, at
22 days, PGC’s (also known as gonocytes) have been demonstrated in the endoderm of allantois and in the mesenchyme of the stalk (Falin, 1969
). At 4.5 weeks of gestation, the colonization of the gonadal ridges has already taken place and at 7 weeks the testis and ovary appear differentiated (Francavilla et al., 1990; Rabinovici and Jaffe, 1990). Germ cells are now called oogonia and pro-spermatogonia and are characterized by high proliferation. Around 11–12 weeks gestational age, oogonia begin entering into meiosis (oocyte I) while prospermatogonia cease mitotic division at 18–20 weeks gestation. This brief outline describing the embryonic history of the germ line in the mouse and human embryo highlights some of the questions still in part or fully unresolved about the emergence of the germ line in mammals, for example: (i) When and where is the germ line set aside in the embryo? (ii) How is the germ line segregated from the somatic lineages? (iii) Which factors guide PGC’s to the gonadal ridges? (iv) Which factors regulate PGC’s proliferation? (v) Which factors control PGC’s differentiation in the gonads and what signals the beginning of meiosis in the female PGC’s and the mitotic arrest in G1 in the male PGC’s?
The main purpose of this review is to outline the information obtained using mainly in vitro culture systems with particular attention to results obtained in the last decade about two aspects of these processes in the mouse embryo (with some reference to the scant information we have about human PGC’s), namely the segregation of PGC’s and their proliferation. Other recent reviews can be consulted to find more general and comprehensive information and discussion about PGC’s development (see for example, Wylie, 1999; De Felici, 2000, 2001; Starz-Gaiano and Lehmann, 2001; McLaren, 2003).
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Segregation of germ cell lineage: an in vitro point of view |
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TOPAbstractIntroductionSegregation of germ cell...PGC’s proliferationConclusionsAcknowledgementsReferences |
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Taking the embryo to pieces to solve a puzzle
In the mouse embryo, PGC’s are initially detectable at 7.25 dpc in the base of the allantois as a cluster of 40–50 cells expressing tissue-non-specific alkaline phosphatase (TNAP) (Ginsburg et al., 1990; MacGregor et al., 1995). The lack of markers of PGC’s precursors before this time made it difficult to define the genetic basis of germ cell lineage segregation in the mouse. In fact, TNAP is also present in somatic cells that surround PGC’s as well as other surface markers such as the oligosaccharide SSEA-1 (very similar or identical to the antigen recognized by the antibodies TG-1, EMA-1 and 4C9) and the c-Kit receptor. Even oct4, a gene that is primarily expressed in pluripotent lineages and thought to have a role in germ cell development [in this case driven by a germ cell-specific distal enhancer (
E); Yeom et al., 1996] shows widespread expression in early embryos. So far, all studies performed in vitro which aimed to investigate the segregation of the germ cell lineage suffered such limitations. Several studies suggested that in contrast to most of the non-mammalian species, the determination of PGC’s fate in mammals is independent of germ line-specific determinants; it occurs instead through an inductive process (for reviews see Extavour and Akam, 2003; McLaren, 2003). But how it happens has been a mystery until now. Results obtained from in vitro culture experiments gave important insights into some of the mechanisms of such processes. The formation of putative PGC’s in isolated pieces of pre-and early gastrulation embryos in culture has been an experimental in vitro approach used to investigate this topic. Early studies showed that at 7 dpc most of PGC’s precursors are located in the mid-region of the primitive streak, as indicated by the formation of alkaline phosphatase (APase)-positive cells in explants of this region after 24 h of culture (Snow, 1981). Interestingly, 7.5 dpc embryos were unable to replace the missing PGC’s precursors, indicating that by this time the process of PGC’s segregation has been completed. After 20 years, more refined analyses of PGC’s formation in culture performed on explanted fragments of 5.5 dpc epiblasts revealed that interactions between extraembryonic ectoderm and epiblast induced the conditions required for determination of bona fide PGC’s (identified by high APase activity, 4C9 and Oct4 positivity; Yoshimizu et al., 2001). These results confirmed studies of fate-mapping of epiblast cells in vivo showing that the precursors of PGC’s are located in the epiblast right next to the extraembryonic ectoderm (proximal epiblast) (Lawson and Hage, 1994; Tam and Zhou, 1996). Moreover, they suggested that factors acting on the proximal epiblast of pre- and early gastrulating embryos (5.5 dpc) play critical roles in germ cell determination. Three such factors, Bmp4, Bmp8b (produced by the extraembryonic ectoderm; Ying and Zhao, 2001) and Bmp2 (produced by the visceral or primitive endoderm, Coucouvanis and Martin, 1999), were rapidly identified on the basis of targeted mutagenesis (Lawson et al., 1999; Ying et al., 2000) and in vitro culture studies. Ying et al. (2001) showed that a significant number of APase and E-Oct4-GFP (a truncated Oct4 promoter expressed specifically in germ cells; Yeom et al, 1996; Anderson et al., 1999)-positive putative PGC’s (34 per epiblast) were induced in vitro from fragments of proximal epiblast of 6.0–6.25 dpc embryos after co-culture for 3 days on COS cells producing Bmp4 and Bmp8b. Using targeted gene mutation, Lawson et al. (1999) and Fujiwara et al. (2001) found that Bmp4 is also essential for allantois development and the localization and survival of PGC’s. At the same time, we found that in vitro, Bmp4 treatment enables recruitment of epiblast cells to a PGC’s phenotype by a multi-step process involving an initial Bmp4-dependent pre-commitment and followed by stages of Bmp4-independent PGC’s phenotypic determination and expansion (Pesce et al., 2002). The importance of a community effect in the initial PGC’s commitment by Bmp4 was indicated by the fact that putative PGC’s (30–40 per epiblast) formed only if epiblast fragments were maintained intact. We provided further evidence that Bmp4 may act on PGC’s precursors and on PGC’s through Smad1/4 signalling pathway; a notion later confirmed by Tremblay et al. (2001) and Hayashi et al. (2002), who reported that Smad1 signalling pathway is critical for initial commitment of the germ cell lineage from mouse epiblast.
Taken together these results have brought us closer to understanding how the germ cell lineage is likely to be segregated in mammals. As reported above, however, it was not possible to establish with absolute certainty whether the putative PGC’s formed in culture received a complete germ cell specification. Very recently, Saitou et al. (2002) published an elegant paper in which they compared gene expression between single PGC’s founders and their somatic neighbours of the extraembryonic mesoderm and identified fragilis, a gene that encodes an interferon-inducible protein likely involved in homotypic cell adhesion. Although fragilis is expressed at low levels by all epiblast cells, it is transiently up-regulated only in a subpopulation of epiblast clustered cells. These cells represent presumptive PGC’s which are presumably made responsive to interferons (IFN’s) by exposure to high doses of Bmp4 and which begin to express another gene called stella (also identified by Sato et al., 2002 es Pgc7 and by Bortvin et al., 2003 es Dpp3). A putative DNA binding protein, stella, is thereafter expressed by migrating PGC’s and apparently represents the first gene to mark the onset of germ cell competence. Using in vitro experiments, Saitou et al. (2002) found that extraembryonic ectoderm was also able to induce a high level of fragilis, TNAP and oct4 in some cells of distal epiblast fragments (which do not normally give rise to PGC’s), but none of these cells showed expression of stella. This suggests that additional signal(s) may be required for the final stages of PGC’s specification in vivo (Lawson et al., 1999; McLaren, 1999) which some in vitro conditions are perhaps unable to supply.
On this basis, the following model of PGC’s segregation in the mouse embryo can be drawn (Figure 2). First, in response to signals (Bmp4 and Bmp8b) from the extraembryonic ectoderm and from the primitive endoderm (Bmp2), cells in the proximal epiblast are programmed to become common precursors of the extraembryonic mesoderm and PGC’s (5.5–6.5 dpc). A second signal (possibly IFN’s) then imposes high levels of fragilis expression on a few of these cells located in a ‘niche’ inside the extraembryonic mesoderm cells and primed by exposure to high Bmp level; eventually a third signal switches on stella in such cells and determines their final specification as PGC’s (7–7.5 dpc). Very recently two papers (Payer et al., 2003 and Bortvin et al., 2004) using stella-deficient mice showed, however, that stella is not required for germ cell formation re-opening the search for genes specifically involved in such processes.
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Germ cells from ES cells: a default or an inducible process?
New insights as to precisely how these inductive signals work might come from recent in vitro experiments aimed at obtaining germ cells from embryonic stem (ES) cells in vitro. Three papers by Hübner et al. (2003), Toyooka et al. (2003) and Geijesen et al. (2004) showed that it is possible to induce mouse ES cells to differentiate into mature germ cells. Since in vitro the formation of oocytes and sperm from ES cells should also presumably follow PGC’s formation, it is interesting to analyse how this process seems to occur in such culture systems. Hübner et al. (2003), using gcOct4-GFP ES cells (XY or XX) in which GFP expression is driven by a germ cell-specific Oct4 enhancer (E), reported that ES cells plated on culture dishes and maintained in ES cell medium without any feeder cells and growth factors, besides the factors present in the serum, begin to progressively differentiate into colonies containing germ cells at different stages of development. At 7 days of culture, 25% of ES cells expressed the reporter gene. An undetermined fraction of these cells on the basis of their oct4+ c-kit+ expression and elongated morphology and absence of vasa and scp3 expression (two genes expressed by post-migratory and meiotic germ cells) was considered to be migratory PGC’s, while other cells (oct4+ c-kit– and vasa+ scp3–) were believed to be post-migratory PGC’s. In a few days these cells progressively disappear from the culture. The remaining cells showed the characteristics of primary oocytes. This extraordinary process, never observed before in >20 years of studies on ES cells, seems to occur without any influence of external compounds and organization of a ‘niche’ for PGC’s specification. Unfortunately, the expression of fragilis and stella in such cells was not detected so that it remains to be determined if all stages of germ cell specification occur in such a system. Toyooka et al. (2003) used a similar strategy but a quite different culture system. They used ES (XY) cell lines in which a fragment of mvh, a gene encoding the protein VASA specifically expressed by differentiating germ cells, was replaced by the reporter genes laZ-neo or GFP-neo. The expression of the reporter genes (the signal for germ cell formation) during the formation of ES cell aggregates named embryoid bodies (EB’s) was highly increased when ES cells were co-aggregated with cells producing Bmp4 or Bmp8b. After 1 day of culture, all EB’s showed mvh+ cells; these cells express increasing levels of mRNA for fragilis, stella and oct4 and also express GCNA1 and SCP3 (two proteins characteristic of pre- and meiotic germ cells). When placed in a suitable environment, they gave rise to sperm. It is not clear why mvh+ ES cells express markers of pre-migratory (fragilis, stella and oct4), migratory (stella and oct4) and post-migratory (GCNA1 and SCP3 and VASA protein itself) PGC’s so rapidly (1 day) and apparently at the same time. Timing deregulation of PGC’s development seems evident. In any case, this ES cell system should ideally permit the examination of a large number of PGC’s-competent cells in vitro without the need to isolate them from the embryo. It should be interesting to study whether these cells behave in culture as ex vivo PGC’s and to investigate if they can give rise to oocytes when left in culture (as in the study by Hübner et al., 2003) or placed into a female environment. It has to be pointed out that, unlike Hübner et al. (2003), in the culture system described by Toyooka et al. (2003) the production of germ cells from ES cells depends on the constant stimulation of Bmp4, together with a unique three-dimensional configuration of the EB’s. The latter could produce a community effect mimicking close cell association in epiblast tissues in the embryo. In fact, the authors report that this condition is essential for formation of germ cells from ES and that this does not occur if ES are cultured as cell monolayers (the condition used by Hübner and co-workers. Similarly to Toyooka et al. (2003), Geijesen et al. (2004), isolated bona fide PGC’s from EB’s using retinoic acid treatment, and derived continuously growing lines of embryonic germ (EG) cells. Moreover, they claimed that it was possible to isolate from EB’s cultured for 20 days haploid male germ cells, which, microinjected into oocytes, gave rise to blastocysts.
Although these results could represent a real breakthrough for the biology of reproduction and the stem cell biology, they must be still considered with caution. The efficiency and the reproducibility of these processes are not well-defined, the gene expression and behaviour of PGC’s were not entirely what may be expected and the temporal sequence of events also appears disrupted.
Human PGC’s
In human embryos, the region in which PGC’s are first recognizable at 21–22 days of gestation is the same as that in the mouse, the wall of the yolk sac near the developing allantois. Their number has been estimated at 50–90, fairly similar to the mouse. According to the mouse model, at this time, it is likely that human PGC’s have been already segregated from the epiblast as extraembryonic mesoderm. Like mouse PGC’s, human PGC’s are characterized by high activity of the enzyme APase (Figure 3) and the presence of the SSEA-1 antigen. Glycogen particles and lipid droplets are commonly detectable in human PGC’s, but not in the mouse. These inclusions confer PAS (periodic acid staining) positivity to human PGC’s and it is likely that they may be used as energy reserves during their migration to the gonadal ridges.
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Interestingly, genes demonstrated to be crucial for PGC’s segregation in the mouse or other species, such as Oct4 and Vasa, are expressed by human PGC’s (Goto et al., 1999
and 2001; Castrillion et al., 2000).
Lines of ES have recently been obtained from human blastocysts (Thomson et al., 1998), and it is therefore possible that derivation of germ cells from such cell lines will be attempted soon. While we were writing this review, an online paper by Clark et al. (2004) claimed the spontaneous differentiation of germ cells from human ES cells.
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PGC’s proliferation |
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TOPAbstractIntroductionSegregation of germ cell...PGC’s proliferationConclusionsAcknowledgementsReferences |
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Increasing in number: external signals and intrinsic clock
During the migratory period, and for 2–3 days after their arrival in the gonadal ridges, mouse PGC’s proliferate actively. In about eight replication cycles (doubling time of 16 h; Tam and Snow, 1981), their number increases from 50–100 at the beginning of migration to 20 000 at 13.5 dpc (Tam and Snow, 1981). Interestingly, the doubling time of PGC’s precursors is much shorter <7 h (Lawson and Hage, 1994), and the putative function of IFN’s during PGC’s formation (see previous section) may be also to slow down the cell cycle in the nascent PGC’s since such compounds are able to induce anti-proliferation signals. Isolation of mouse PGC’s from 8.5 to 13.5 dpc and culturing them in various culture systems (De Felici, 1998a,b) allowed us to test the effects of a variety of compounds on their survival and proliferation. Early experiments with culture medium conditioned by different embryonic tissues revealed that the gonadal ridges release soluble factors that increase PGC’s numbers (Godin et al., 1990). This has led to a search for purified growth factors that mimic these effects and that are therefore candidates for these roles in vivo. Several growth factors are now known to affect PGC’s growth in vitro (Table I). While some factors are primarily stimulatory or inhibitory, most are likely to have more complex functions in PGC’s growth. For example, the observation that PGC’s isolated from their somatic environment undergo apoptotic degeneration in culture (Pesce et al., 1993; Pesce and De Felici, 1994) led to the finding that some of these factors (i.e. KL and LIF) act by preventing PGC’s apoptosis (Pesce et al. 1993), thus suggesting that the survival and proliferation of PGC’s in vivo are strictly dependent on a variety of growth factor activities. But how relevant is the action of each of these growth factors in vivo? Is the in vitro response of PGC’s to some of these factors misleading? In fact, in most cases it is not known whether these factors play a physiological role in vivo. One exception is the KL–Kit interaction. Steel and W (dominant white spotting) were originally identified as genetic loci, in which the mutation caused sterility, anaemia and lack of melanocytes. The products of these two loci were later identified as the tyrosine kinase receptor Kit, the product of the W locus, and its ligand, Steel factor (SF), also known as Kit ligand (KL) or Stem cell factor (SCF), the product of the Steel locus. Studies on cultured PGC’s extensively reviewed elsewhere (De Felici, 2000, 2001), showed that the KL–Kit interaction is required for PGC’s survival/proliferation. Knockout mice lacking other growth factors which were able to influence PGC’s growth in vitro (bFGF knockout mice, Ortega et al., 1998; LIF knockout mice, Stewart et al., 1992; TGFß knockout mice, for a review, see Dünker and Krieglstein, 2000; activin knockout mice, Matzuk et al., 1995) or their receptors (LIFR knockout mice, Ware et al., 1995; gp130 knockout mice, Molyneaux et al., 2003b) did not show apparent alteration in PGC’s numbers or have defects in the initial PGC’s formation (BMP knockout mice, see above) that precludes analysis at later stages. In addition, in vitro culture experiments showed that PGC’s are able to autonomously regulate their growth timing. In fact, cultured PGC’s stop proliferating at the time corresponding to 12–13 dpc in vivo independently of the influence of the somatic cell monolayers (Ohkubo et al., 1996). Moreover, the increased proliferation of PGC’s in the presence of single growth factors never exceeds their normal in vivo proliferation rate and does not significantly alter the timing of their growth arrest (for a review, see De Felici, 2001). It seems therefore that in normal conditions PGC’s are able to integrate external signals maintaining their characteristic proliferation rate and to measure the time of proliferation by an intrinsic clock. A stochastic model in which causal oscillations of cell cycle controlling factors gradually increase the probability of growth arrest has been proposed to explain this PGC’s behaviour (Ohkubo et al., 1996). In any case, to understand how such complex processes may occur requires dissection of intracellular signalling pathways of PGC’s proliferation.
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Dissection of signalling pathways: old and new strategies
In other species, natural mutations have been of major help in elucidating the molecular pathways underlying germ cell survival and proliferation. However, few natural mutations have been described which affect PGC’s number in the mouse gonadal ridges (Table II). Among these, the already mentioned white and steel have been demonstrated to result in a defective KL/Kit system, leading to a marked reduction of PGC’s survival/proliferation and perhaps migration (Mintz and Russell, 1957; McCoshen and McCallion, 1975; Buher et al., 1993); gcd (germ cell deficient, Pellas et al., 1991) affects PGC’s proliferation (Agoulnik et al., 2002), ter results in increased PGC’s apoptosis (Takabayashi et al., 2001). Whereas gcd causes a complete deletion of a gene called pog encoding for a protein believed to be important for gene regulation (Agoulnik et al., 2002), ter acts through an unknown anti-apoptotic factor produced by the gonadal somatic cells (Takabayashi et al., 2001).
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Although, as we discussed above, in vitro conditions may exaggerate or be misleading as to the importance of certain factors, quantitative modelling of in vitro systems allows exploration of the complex signal integration necessary to control cell survival and proliferation. For PGC’s, however, this has so far been difficult. Until recently most of the information on such processes has been restricted to the effect on PGC’s growth in culture of compounds and inhibitors known to affect more or less specifically components of signalling pathways. For example, studies carried out in our laboratory showed that dbcAMP or cAMP agonists such as forskolin and cholera toxin markedly stimulate PGC’s proliferation (De Felici et al., 1993
), whereas activators of protein kinase C such as 12-O-tetrodecanoylphorbul-13-acetates (TPA) or 1-oleolyl-2-acetyl-glycerol (OAG) did not influence PGC’s numbers (De Felici and Pesce, 1994). More recently, a wide spectrum of inhibitors for key signalling pathways of cell growth such as PI3K (wortmannin and LY294002), Src (PP2 and SU6656), MEK/MAPK (PD98059 and U0126) and mTOR/FRAP (rapamycin) has been studied on 8.5 dpc PGC’s (De Miguel et al., 2002). While PI3K inhibitors had no effect on PGC’s growth after 7 days of culture, all other inhibitors reduced significantly the PGC’s number. Though these studies may give useful information on some of the pathways involved in the control of PGC’s survival and proliferation, they have several drawbacks—the variability of the possible protocol of exposure, the inhibitors, the specificity and toxicity of the inhibitors—and doubts may be aroused about the physiological significance of certain effects. In any case, the results obtained from such experiments need to be integrated and supported by other analyses. For example, it is now possible to study the expression of genes and proteins involved in cell cycle control or apoptosis in a purified population of PGC’s before and after culture using RT–PCR and mini-blotting. In this regard, the immuno-magnetic cell purification method employed to isolate PGC’s from freshly collected tissues (Pesce and De Felici, 1995) has been recently adapted to recover and purify PGC’s from co-culture on embryonic fibroblasts, the most common method for PGC’s culture (our unpublished results). This should be useful to analyse at molecular levels a long-term response of PGC’s to various stimuli.
A major obstacle to study signalling pathways in PGC’s is the difficulty in manipulating these cells using the tools of modern molecular biology. In some cases, introduction or deletion of genes into mouse PGC’s in vivo has been achieved by targeted mutagenesis and transgenesis. A summary of targeted mutations resulting in altered survival or proliferation of PGC’s is reported in Table III. Two such mutations, the targeted mutation of Pin1 (Atchison et al., 2003) and the conditional loss of Pten in PGC’s (Kimura et al., 2003), will be discussed at length not only since the mechanism implicated in the genetic defects has been supported by in vitro culture experiments, but also since they have allowed the identification of two possible key molecules of PGC’s proliferation. Pin1 is a proline isomerase, implicated in the control of the cell cycle through its specific interaction with proteins (i.e. Cdc25c, Tau and the transcriptional factor CF2) that are phosphorylated at Ser/Thr-Pro motifs to promote their degradation (Luoh et al., 1996; Yaffe et al., 1997). Pin–/– mice have significantly reduced numbers of PGC’s which has been explained by the prolonged duration of the G1 phase (Atchison et al., 2003). Preliminary results from our laboratory show that a specific inhibitor of Pin1, called PinB, decreases significantly the growth of PGC’s in culture (V.Nazzicone and M.De Felici, unpublished data). Pten is a tumour suppressor gene encoding a lipid phosphatase which plays a crucial role in the regulation of cellular proliferation, differentiation, apoptosis and migration (for a review, see Di Cristoforo and Pandolfi, 2000). A Cre-LoxP conditional gene-targeting system in which Cre was knocked into the PGC’s-specific TNAP gene (Lomeli et al., 2000) was adopted to analyse the function of PTEN in PGC’s. Both in vivo and in vitro, Pten-null PGG showed increased proliferation and enhanced ability to give rise to tumours in vivo or tumorigenic cells (EG cells) in vitro (Kimura et al., 2003). One of the important roles of PTEN is to induce G1 arrest through the suppression of the PI3K–Akt pathway (Li and Sun, 1998; Ramaswamy et al., 1999; Sun et al., 1999). Therefore, it seems reasonable that increased numbers of Pten-null PGC’s show high proliferation activity. How this led to a high level of tumour formation from PGC’s remains to be clarified and will be discussed below. Two other recent studies of targeted mutation showed that PGC’s numbers in vivo were unaffected by mutation of the PI3K binding site on Kit (Tyr719), one of the main receptors mentioned above present on the PGC’s membrane able to activate PI3K (Blume-Jensen et al., 2000; Kissel et al., 2000), thus suggesting that in PGC’s Kit may act through multiple pathways, e.g. that of MAPK’s (De Miguel et al., 2002).
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Although studies of targeted mutation may unravel some of the molecular pathways controlling PGC’s proliferation, they are still relatively time-consuming and laborious. An alternative approach to such analyses is represented by transfection of genes or by interfering with specific mRNA transduction directly in PGC’s in culture. Early transfection of genes into PGC’s in vitro resulted in low efficiency and the transfection procedures drastically reduced PGC’s viability (Watanabe et al., 1997
). However, recent data by De Miguel et al. (2002) demonstrate that some types of retroviruses (i.e. MLV eco and ALV) can now be used to manipulate gene expression in mouse PGC’s and to analyse many aspects of their behaviour that can be studied in vitro. For example, De Miguel et al. used such technology to over-express AKT in PGC’s, a key kinase downstream of the activation of several tyrosine kinase receptors including c-Kit. The results showed that the infection vector improved PGC’s survival, demonstrating that AKT is required for PGC’s survival. In our laboratory, we introduced oligo-antisense for PTEN mRNA in proliferating PGC’s using liposomes (Behrens et al, 2003). The results obtained matched very well those reported by Kimura et al. in Pten–/– mice discussed above; the proliferation of PGC’s and their ability to give rise to tumorigenic cells (EG cells) in culture was significantly increased. It is unlikely that the augmented proliferation is the only cause of the increased frequency of tumour formation. In fact, the loss of PTEN function itself is not sufficient for enhanced EG colony formation; in fact no EG cell formation was observed without the addition of LIF (Behrens et al., 2003; Kimura et al., 2003). Conditions favouring the maintenance of an undifferentiated status of PGC’s are likely to play a critical role in such abnormality. Taken together, the results obtained by the combination of these various technologies permits a first sketch of possible molecular pathways controlling PGC’s cell cycle (Figure 4).
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Human PGC’s
Human PGC’s proliferate actively during migration but mainly after their arrival in the gonadal ridges. In the human embryo, however, after entering into the gonads, PGC’s are called oogonia or pre-spermatogonia. The mitotic proliferation of oogonia and prospermatogonia lasts 3–4 weeks. In the ovary, the estimation of the maximal oogonia number is 3x106 per ovary at 16–20 weeks. It is likely that this number resulting from the work of Baker et al. (1963), is an overestimation that should be reduced by 
20–30% (Baker et al., 1963; Rabinovici and Jaffe, 1990). A characteristic of human oogenesis over this period is that the developmental stages of the germ cells are not synchronized. In fact, until 3 months of fetal life, oogonia and primary oocytes in different stages of meiosis co-exist (Kurilo, 1981).
An important finding from the few in vitro culture experiments carried out with human PGC’s is that they respond to the same compounds (forskolin, retinoic acid) and growth factors (KL, bFGF, LIF) reported to stimulate the survival and proliferation of mouse PGC’s (Shamblott et al., 1998; our unpublished observations). Most importantly, like mouse PGC’s, human PGC’s give rise to pluripotent embryonal germ (EG) cells when cultured in vitro in the presence of the correct cocktail of such compounds and growth factors (Shamblott et al., 1998), suggesting that the mechanisms controlling PGC’s growth in mammals are largely conserved and that mouse PGC’s represent a suitable experimental model to study the PGC’s biology.
Analysis of gene expression in human PGC’s and the study of mutations resulting in reduction or absence of fertility may help to confirm or disprove such similarities. For example, c-Kit is expressed by both male and female germ cells at 13–21 weeks of gestation. Mutations in the c-kit gene affect both haematopoietic and melanocyte lineages in humans, but to date no association with infertility has been documented as it has been in mice. On the other hand, c-kit is strongly up-regulated in some types of germ cell testicular tumour which are believed to originate from PGC’s (Bokemeyer et al., 1996; reviewed by de Kretser and Damjanov, 1998), suggesting that it plays a crucial role in the control of human PGC’s survival/proliferation as in mice. In this regard, Oct4 was found to be expressed in all human germ cell tumours containing undifferentiated cells (Looijenga et al., 2003). In Fanconi’s anaemia (FA), individuals are characterized by several congenital abnormalities including decreased fertility (for a review, see D’Andrea and Grompe, 2003). As reported above, targeted mutation of Francc in the mice results in significantly slower proliferation of PGC’s (Nadler and Braun, 2000), suggesting again shared control mechanisms of PGC’s proliferation in these species. Finally, in trisomy 16 mouse, an animal model of Down’s syndrome leading frequently to sub- or infertility, a delay in migration and reduction of PGC’s number was observed (Leffler et al., 1999).
Gene expression studies on single human PGC’s obtained from 10 week embryos have been reported (Goto et al., 1999, 2001). The preparation of cDNA libraries and microarrays from human PGC’s should be valuable resources for researchers in this field.
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Conclusions |
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TOPAbstractIntroductionSegregation of germ cell...PGC’s proliferationConclusionsAcknowledgementsReferences |
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In vitro studies have provided important insights into the biology of PGC’s and will continue to supply information which can be obtained only by the study of isolated cells in culture. Of particular importance have been the experiments that have clarified the mechanisms of PGC’s segregation and those showing that germ cells can be obtained from ES cells in culture. However, other tools are now available with which to investigate the factors governing PGC’s development in the embryo. Among the most promising of these are the new technologies allowing PGC’s transfection in culture and the production of mice carrying targeted mutations in which specific genes have been deleted or mutated. In this respect, conditional gene-targeting systems in which genes are knocked out specifically in PGC’s will be particularly useful. By removing or altering the function of a specific molecule and describing the influence of that mutation in vivo, a more direct examination of factors affecting PGC’s segregation, proliferation, migration and differentiation will be possible. However, the possibility of alternative signalling pathways operating in the embryo may make analysis of some of these mutants difficult, and ultimately a combination of both in vivo and in vitro studies will possibly be necessary before a clear picture of PGC’s biology comes to light.
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Acknowledgements |
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TOPAbstractIntroductionSegregation of germ cell...PGC’s proliferationConclusionsAcknowledgementsReferences |
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We are grateful to Mr Graziano Bonelli for his expert assistance with the preparation of figures and Ms Maddalena Vecchione for aid in the preparation of the manuscript. This work was supported by MURST National Project 2000 and 2002 and EU grant No. QIK4-CT-02403.
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TOPAbstractIntroductionSegregation of germ cell...PGC’s proliferationConclusionsAcknowledgementsReferences |
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