This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Aflatoonian, B. Articles by Moore, H. Search for Related Content PubMed PubMed Citation Articles by Aflatoonian, B. Articles by Moore, H.

Focus on Stem Cells

Germ cells from mouse and human embryonic stem cells

Centre for Stem Cell Biology, University of Sheffield, Sheffield S10 2UH, UK

Correspondence should be addressed to H Moore; Email: h.d.moore{at}shef.ac.uk

	Abstract

Top Abstract Introduction Development of primordial germ... Embryonic stem cells to... Gamete determination The germ cell niche... Conclusion Acknowledgements References

 
Mammalian gametes are derived from a founder population of primordial germ cells (PGCs) that are determined early in embryogenesis and set aside for unique development. Understanding the mechanisms of PGC determination and differentiation is important for elucidating causes of infertility and how endocrine disrupting chemicals may potentially increase susceptibility to congenital reproductive abnormalities and conditions such as testicular cancer in adulthood (testicular dysgenesis syndrome). Primordial germ cells are closely related to embryonic stem cells (ESCs) and embryonic germ (EG) cells and comparisons between these cell types are providing new information about pluripotency and epigenetic processes. Murine ESCs can differentiate to PGCs, gametes and even blastocysts – recently live mouse pups were born from sperm generated from mESCs. Although investigations are still preliminary, human embryonic stem cells (hESCs) apparently display a similar developmental capacity to generate PGCs and immature gametes. Exactly how such gamete-like cells are generated during stem cell culture remains unclear especially as in vitro conditions are ill-defined. The findings are discussed in relation to the mechanisms of human PGC and gamete development and the biotechnology of hESCs and hEG cells.

	Introduction

Top Abstract Introduction Development of primordial germ... Embryonic stem cells to... Gamete determination The germ cell niche... Conclusion Acknowledgements References

 
Detailed investigations of the earliest stages of germ cell development in the human are curbed by the practical and ethical difficulties associated with obtaining human tissue samples. An in vitro model system that can recapitulate the development of human germ cells and gametes would be an extremely valuable research tool, which might lead in due course to novel reproductive toxicological assays and eventually new clinical applications to overcome infertility. Embryonic stem cells (ESCs) are defined as pluripotent stem-cell lines derived from early embryos before the formation of the tissue germ layers (Smith 2006). ESCs are derived usually from the pre-implantation blastocyst and exhibit indefinite proliferative capacity under appropriate conditions in vitro (Evans & Kaufman 1981, Martin 1981, Thomson et al. 1998, Draper et al. 2004). In the last few years, landmark investigations have demonstrated that murine embryonic stem cells (mESCs) can differentiate to primordial germ cell (PGCs) and subsequently early gametes and blastocysts (Hubner et al. 2003, Geijsen et al. 2004). Recently, immature sperm cells derived from mESCs in culture have generated live offspring (Nayernia et al. 2006a, 2006b). Preliminary data indicate that human embryonic stem cells (hESCs) most likely display a similar developmental capacity (Clark et al. 2004, Aflatoonian et al. 2005). This ESC technology offers great potential for new types of reproductive investigations including a readily accessible system to investigate early stages of human gametogenesis including epigenetic modifications of the germline (Allegrucci et al. 2005). Indeed, a range of pluripotent stem cell lines have now been derived from different stages of germ cell development in the mouse and human (Fig. 1). Here, we review the derivation of germ cells and their formation from embryonic stem cells in vitro and discuss how these investigations may provide fresh insight into some of the mechanisms of human germ cell development.

View larger version (33K): [in this window] [in a new window]   Figure 1 Thegermcellcycle. Primordialgermcells (PGCs) are first specified inthe proximal epiblast (1) and migrate intothe genital ridge ofthe developing gonad (2). Embryonic germ cell lines have been derived by transformation of PGCs in vitro. In the female, germ cells enter meiosis and remain in an arrested state until follicular development occurs after puberty (3). In the male, germ cells retain mitotic capacity for proliferation (3) and pluripotent murine spermatogonial and multipotent adult germ stem cell lines have been reported. From the inner cell mass of the pre-implantation blastocyst, pluripotent embryonic stem cells have been derived (4).

	Development of primordial germ cells

Top Abstract Introduction Development of primordial germ... Embryonic stem cells to... Gamete determination The germ cell niche... Conclusion Acknowledgements References

Specification of PGCs, their proliferation, maintenance, then differentiation to primary oocytes and prospermatogonia (precursor spermatogonia stem cells) all have a profound bearing on the number and function of germ cells that are available subsequently for gametogenesis. Over many years, the activity of tissue non-specific alkaline phosphatase (TNAP) has been used to mark PGCs and monitor their transition from the base of the allantois, through the hindgut to the dorsal body wall where they enter into the genital ridges of the gonadal anlagen (McLaren 2003). The high expression of this enzyme in PGCs is a feature also shared with ESCs, embryonic germ (EG) cells and embryonal carcinoma (EC) cells, all of which have pluripotent capabilities. PGC migration (whether active or passive) occurs at about 7–10 days post-conception (dpc) in the mouse and between weeks 5 and 8 of human gestation (Freeman 2003, Molyneaux & Wylie 2005). Because of the difficulties of undertaking detailed investigations of early human fetal development, relatively little is known of the specification of human PGCs, although it is probable that common signalling pathways occur in mammals and possibly all vertebrates (Donovan & de Miguel 2003). In the mouse, PGCs arise from the proximal epiblast, a region of the early mammalian embryo that also contributes to the first blood lineages of the embryonic yolk sac (Ginsburg et al. 1990). Recent studies indicate that as early as 6.25 d.p.c, germline competence can be identified in a founder population of perhaps as few as six epiblast cells that express the protein Blimp1 (B-lymphocyte-induced maturation protein 1, McLaren & Lawson 2005, Ohinata et al. 2005, Vincent et al. 2005). Blimp1 was first identified as a transcriptional repressor that enables the further differentiation of immunoglobulin-secreting plasma cells by inhibiting the expression of genes involved in alternative B-cell development. Mutant null-allele mice lacking Blimp1 generate very few PGCs and those that develop lack normal migratory behaviour, unlike the cells of wild-type individuals where PGCs will multiply rapidly as they migrate to the genital ridges to eventually become non-migratory gonocytes (Vincent et al. 2005). In females, the gonocyte surrounded by a cortical interstitial layer initiates meiosis and becomes a primary oocyte and follicle, thereby ending precursor proliferative potential. In males, the gonocyte surrounded by the fetal sex cord of the gonadal ridge (pre-seminiferous tubules) arrests in G0/G1 of mitosis as a prospermatogonium, but retains a proliferative precursor potential. Following birth, prospematogonia migrate to the basement membrane of the seminiferous tubule and differentiate into spermatogonial stem cells (SSCs). Like adult stem cells, SSCs can both self-renew and provide daughter cells, which differentiate into one or more terminal cell types (Brinster 2002).

Germ cell competence is induced in the murine proximal epiblast in response to signals emanating from the extra embryonic ectoderm including the synergistic action of the growth factors, particularly bone morphogenic proteins (BMP) 4 and 8b; both members of the transforming growth factor-ß (TGF-ß) superfamily of secreted proteins (Shimasaki et al. 2004). Mature BMP4 is a dimer that binds to and signals through heteromeric receptor complexes and downstream SMAD proteins. However, BMP4 or BMP8b alone are unable to induce PGCs from cultured epiblast, while they can when combined, which suggests that signalling for various BMPs may occur through separate receptor complexes. BMP2, a close relative of BMP4, is expressed in visceral endoderm at the time of PGC specification, and inactivation of BMP2 results in a reduction in PGC number, revealing a function of visceral endoderm in PGC generation in the mouse at least (Lawson et al. 1999, Ying et al. 2001, de Sousa Lopes et al. 2004, Shimasaki et al. 2004).

The genes fragilis and stella also have key roles in germ cell competency and development. Fragilis is a transmembrane protein and part of a larger interferon-inducible family of genes that is evolutionarily conserved and has human homologues. Interferon-inducible proteins such as fragilis have an anti-proliferative function and may serve to increase the length of the cell cycle in PGCs. As germ cell fate is induced, there is only transient expression of fragilis, but this gene is also expressed in ESCs and embryonic germ (EG) cells, suggesting a potential role in pluripotency status (Saitou et al. 2002). Similarly, stella may have a function during the development of pluripotency and is associated with chromatin remodelling or RNA processing. It is expressed in the oocyte, through pre-implantation embryo development and in germ cell tumours (Payer et al. 2003). Stella-positive nascent germ cells exhibit repression of homeobox genes, which may explain their escape from a somatic cell fate and the retention of pluripotency (Saitou et al. 2002). Transgenic mice have been generated that express a green fluorescent protein (GFP) – stella reporter transgene, which appears to accurately mark PGC development (Payer et al. 2006).

A number of other factors have been implicated in PGC derivation and maintenance. Immunohistochemical analyses demonstrate that mouse vasa homologue (mvh) protein was exclusively expressed in PGCs just after their colonisation of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females (Toyooka et al. 2000). The tyrosine-kinase receptor c-kit and its ligand, stem cell factor (SCF), are also essential for the maintenance of PGCs in both sexes. In the adult testis, the c-kit receptor is re-expressed in differentiating spermatogonia, but not in spermatogonial stem cells, whereas SCF is expressed by Sertoli cells under follicle-stimulating hormone stimulation (Rossi et al. 2000).

Another set of genes involved in germ cell development is DAZ (deleted in azoospermia) genes. Men with deletions encompassing the Y-chromosome DAZ genes have few or no germ cells, indicating they are defective in the formation or maintenance of germ cells. A DAZ homolog, DAZL (DAZ-Like), found in diverse organisms, including humans is required for germ cell development in males and females. Significantly, PUM2, a human homologue of pumilio, a protein required to maintain germ line stem cells in Drosophila and Caenorhabditis elegans, forms a stable complex with DAZ through the same functional domain required for RNA binding, protein–protein interactions (Moore et al. 2003) suggesting mechanisms of germ cell development are highly conserved.

The POU domain transcription factor Oct4 has been shown to have a role in PGC survival. It has been known for some time that this transcription factor is crucial for maintaining pluripotency in the inner cell mass of the blastocyst and in ESCs. By use of conditional gene targeting with the Cre/LoxP system, Kehler et al.(2004) showed that loss of Oct4 leads to apoptosis (Kehler et al. 2004). In human fetal tissue, Oct4 is strongly expressed in migrating PGCs as well as in human germ cell tumours and EG cells (Looijenga et al. 2003, Rajpert-De Meyts et al. 2004). Oct4 expression is down-regulated rapidly in the human female gonad and silenced as oocytes enter the first meiotic prophase. In contrast, the same process occurs much more gradually in the male with Oct4 expression often persisting in some gonocytes and infantile spermatogonia. This observation has prompted the suggestion that differential germ cell expression of Oct4 between the sexes might contribute to the fact that germ-cell derived cancer is much higher in men than women (Rajpert-De Meyts et al. 2003).

Thus, some of the genes that play a crucial role in germ cell differentiation are Blimp1, Stella, fragilis, c-Kit, vasa and DAZL. Their expression is stage-specific, therefore, allowing solid identification of germ cells at different developmental phases. In addition to the expression of these genes, other markers associated with germ cell development are non-specific alkaline phosphatase activity, the stage-specific embryonic antigen 1, the transcription factor Oct4 and beta1- and alpha6-integrins (Lacham-Kaplan 2004; Fig. 2).

View larger version (22K): [in this window] [in a new window]   Figure 2 Schematic diagram of the developmental stages of mammalian germ cell development and some of the factors and genes (italics) involved. The genes have been used as markers of PGC and gamete development from ESCs in culture.

	Embryonic stem cells to germ cells

Top Abstract Introduction Development of primordial germ... Embryonic stem cells to... Gamete determination The germ cell niche... Conclusion Acknowledgements References

While investigations with hESCs are more preliminary, they also show that spontaneous or induced differentiation in culture can generate cells displaying mRNA expression profiles and cell surface markers consistent with PGCs (Clark et al. 2004, Clark & Reijo Pera 2006) and of subsequent meiosis. In our lab, PGCs (and on rare occasions, early spermatid cells but so far not oocytes) have been identified after the appropriate culture conditions (Aflatoonian et al. 2005).

	Gamete determination

Top Abstract Introduction Development of primordial germ... Embryonic stem cells to... Gamete determination The germ cell niche... Conclusion Acknowledgements References

 
During murine and human ESC culture, markers of female germ cells are expressed in both XX and XY cell lines (Toyooka et al. 2003, Geijsen et al. 2004). The prevailing hypothesis to date has been that whatever their complement of sex chromosomes germ cells are cell – autonomous and intrinsically programmed to undergo meiosis, enter prophase and develop as oocytes, unless otherwise prevented from doing so by a meiosis inhibiting factor(s) (McLaren 2003). In this case, it would be unsurprising for both male and female ESC lines to display female germ cell markers, since culture conditions may be sub-optimal and lack meiosis inhibition. However, recent studies (Bowles et al. 2006, Koubova et al. 2006) indicate that instead of an intrinsic programme to enter meiosis, germ cells respond to the external signal of RA and its metabolism. Thus, in the embryonic mouse ovary, RA induces germ cells to express the pre-meiotic marker Stra-8 (stimulated by retinoic acid 8) and initiate meiosis. By contrast, in the embryonic mouse testis, RA is metabolised and inactivated by the P450 enzyme CYP26 (B1) thereby preventing early germ cell entry into meiosis with down-regulation of genes such as SCP3 (synaptonemal complex protein; associated with meiotic events) and the induction into the alternative pathway of mitotic arrest as G0/G1 prospermatogonia. Therefore, the induction of presumptive PGCs into meiosis in culture medium containing RA might be expected although local concentrations within cell aggregates may differ significantly and affect the timing. When RA has not been added as a supplement to basal medium, the spontaneous induction of PGCs from ESCs might potentially still arise if RA is endogenously generated by ESC derivatives. Concentrations of RA in plasma or serum (or serum replacement) that supplement ESC culture medium are normally in the 2–8 nM range (Eckhoff & Nau 1990), much lower than the 1–5 µM concentrations often used with induction protocols. However, the sensitivity of cells to RA can vary considerably depending on composition of medium.

When hESCs are cultured the process of sex determination seems even more dysregulated as markers of both male and female germ cell development have been detected regardless of the sex of the cell line (Clark et al. 2004, Aflatoonian et al. 2005). The implication from these findings is that RA or possibly other factors affecting meiosis and gamete determination are present in the culture conditions possibly generated by male and female hESCs. For example, for male germ cell development in vivo, seminiferous epithelium with Sertoli cells is required. This is normally dependent on the expression of the transcription factor SRY, the gene of which is normally located on the Y chromosome. Therefore, for XX hESCs to generate presumptive male germ cells either an artefactual differentiation of Sertoli cells occurs, for example, by female supporting cells differentiating to a Sertoli-like phenotype (Adams & McLaren 2002) or meiosis inhibitor factors are produced (or metabolised in the case of RA) from a different origin. Interestingly, in vitamin A-deficient adult mice, where only spermatogonia and Sertoli cells populate the seminiferous epithelium, the addition of retinoids such as RA and retinol may induce the resumption of spermatogenesis (Baleato et al. 2005).

	The germ cell niche and ESCs

Top Abstract Introduction Development of primordial germ... Embryonic stem cells to... Gamete determination The germ cell niche... Conclusion Acknowledgements References

 
An intriguing aspect of the generation of ESC-derived germ cells in vitro is how the normal checkpoints of the developmental process, which would naturally span a timeline from early fetal development to puberty and lead to arrest of cells at different stages, seem to be overcome and then apparently compressed into a relatively much short culture period. It has been suggested that in the absence of environmental cues, germ cells may develop according to an intrinsic clock. Using stella–GFP murine ESCs, Payer et al.(2006) showed somewhat surprisingly that ESC colonies displayed a well-defined subpopulation of Stella-positive cells and unlike the uniform distribution of cells expressing Oct4 or TNAP. Significantly, there was overlap of expression of Oct4 and stella in some cells and, since this combination of markers identifies germ cells it may indicate a population of germ cell-like ESCs (Payer et al. 2006). Whether stella-positive ESCs are destined to form PGCs in the mouse and whether their specification requires subsequent exposure to a set of environmental cues, awaits further investigation but these results suggest that there is perhaps an inherent propensity of ESC culture systems to form PGCs. Thereafter, these cells may create a stem cell niche continuum for induction of germ cell development.

Exactly how ESC cultures may mimic the somatic environment that encapsulates either developing oogonia (the follicle) or the sperm stem cell (seminiferous epithelium) is unclear. The appropriate growth factor and hormonal microenvironment required to support and sustain these complex niches probably depends to some extent on the type of culture system adopted for the differentiation process. ESCs are cultured usually in two basic ways. Monolayer adherent cultures of ESCs can be allowed to differentiate directly to form an appropriate niche or ESCs can be induced to aggregate to form embryoid bodies that form a more three-dimensional microenvironment. In conjunction with these two culture methods, ESCs can be initially co-cultured with feeder layers (i.e. mouse embryonic fibroblasts and neonatal gonadal cell culture) or conditioned-medium from feeder cells and various growth factor and serum supplementation. Generally, these culture conditions attempt to create an environment conducive for germ-cell proliferation and differentiation but these conditions are far from being specific. Apart from the absence of a defined culture medium, the various procedures for passaging ESCs (i.e. enzymatically with trypsin to single cells or as clumps with collagenase or manually), the influence of feeder cells and extracellular matrix (e.g. Matrigel) and the cell line itself can all contribute to whether germ cells are specified. If a progenitor cell population is not selected (i.e. using fluorescent reporter systems), then only a small proportion of cells (usually less than 5%) will normally display PGC or gonocyte/spermatogonia markers, but the fact that some cells progress further to a post-meiotic stage seems remarkable given the complex hormonal requirements of gamete development. Interestingly, oestradiol production (50–100 pg/ml) was detected after 12 days in mESC cultures that generated follicle-like structures (Hubner et al. 2003), while appreciable levels (60 pg/ml) of dihydrotestosterone can be detected in hESCs cultures that generate PGCs and spermatids (Aflatoonian & Moore, unpublished). Presently, the source of steroido-genesis is unclear. It may originate from additives to the culture medium (i.e. serum or serum replacement supplement) or gonadal cell types (granulosa, Leydig cells) that co-differentiate with germ cells. This appeared to be the case for oocytes generated from mESCs where increased oestradiol levels were correlated with increased proportion of apparent follicular cells (Hubner et al. 2003). Alternatively, steroids may be produced from non-reproductive cell phenotypes (e.g. liver or adrenal cells) that could conceivably differentiate from ESCs in culture at the same time as germ cells.

The proliferative capacity of germ cells in culture will depend to some extent on their fate. Since, at least in vivo germ cells are programmed by default to enter meiosis, the efficiency at which ESC cultures will generate germ cells will depend on the initial rate of differentiation to PGCs and subsequently whether factors are present to divert development down spermatogenesis when mitotic proliferation of prospermatogonia can potentially occur. Recent studies (Hamra et al. 2005, Ryu et al. 2005) indicate that the self-renewal of these spermatogonial stem cells (SSCs) in rodents (and possibly all mammals) is dependent on glial cell line-derived neurotrophin factor (GDNF), the GDNF-family receptor -1 and basic fibroblast growth factor, providing candidate factors to be tested in ESC systems.

In the mouse, pluripotency of ESCs is mirrored also by EG cells (Matsui et al. 1992, Resnick et al. 1992) but additionally extends to a population of SSCs in the neonatal mouse testis (Kanatsu-Shinohara et al. 2004) and perhaps more remarkably in adult mouse testis (Guan et al. 2006). These adult SSCs can be transformed in culture to ES-like cell lines displaying pluripotency both in vitro and significantly in vivo. When injected into mouse pre-implantation blastocysts they contribute to the development of all the major organs and display germline transmission (Guan et al. 2006) and therefore are termed multipotent adult germline stem cells. The obvious conservation in many of the mechanisms for germ cell development in mammals suggests that it might be possible to develop similar cell lines from human adult spermatogonial populations although the translation from the murine to the human system may not be straightforward. While human EG cell lines have been reported (Shamblott et al. 1998), their proliferative and pluripotent capacity presently as true EG cells has been questioned (Turnpenny et al. 2003, Aflatoonian & Moore 2005).

Since the evidence is becoming more compelling that in culture ESCs can generate PGCs and germ cells, an important question (and the subject of considerable debate) is whether germ cells can be derived (or transdifferentiated) from adult stem cells residing outside the gonad? In the mouse, it has been claimed that germ cells can be derived from bone marrow (BM) and peripheral blood cells (Johnson et al. 2004, 2005, Nayernia et al. 2006b) and BM-derived germ cells can repopulate the ovarian follicular reserve (Johnson et al. 2004, 2005). However, the latter investigations were criticised (Telfer et al. 2005) and subsequently have not been substantiated (Eggan et al. 2006). Germ-cell derivation from pig fetal skin cells in vitro has also been reported (Dyce et al. 2006) with the formation of morphological structures resembling follicles, that secrete oestradiol and progesterone; and oocyte-like cells displaying markers of germ cells (oct4, vasa, GDF9b) or exhibiting the meiosis marker, SCP3 and displaying apparent parthenogenetic embryo structures. Clearly, if any these recent reports can be fully corroborated, they may indicate that PGC and germ cell formation in vitro and possibly in vivo is much more plastic than previously believed (Fig. 3). At present, it is very difficult to reconcile some of these observations with known reproductive physiology in mammals, and an alternative explanation is that detection of many germ cell markers is due to aberrant expression or detection in culture and not due to true germ cell phenotype. The use of a wider battery of specific protein and functional phenotypic assays are required to resolve this issue.

View larger version (28K): [in this window] [in a new window]   Figure 3 Schematic diagram showing the relationship between in vivo development of germ cells and the in vitro pluripotent stem cell lines that have been reported to be derived from mouse and human cells (ESC and EG) and their developmental potential. The relationship between ESC, EG ad AGS cells still remains unclear although they express similar markers of pluripotency.

	Conclusion

Top Abstract Introduction Development of primordial germ... Embryonic stem cells to... Gamete determination The germ cell niche... Conclusion Acknowledgements References

	Acknowledgements

Top Abstract Introduction Development of primordial germ... Embryonic stem cells to... Gamete determination The germ cell niche... Conclusion Acknowledgements References

 
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 5 May 2006
First decision 7 August 2006
Accepted 1 September 2006

	References

Top Abstract Introduction Development of primordial germ... Embryonic stem cells to... Gamete determination The germ cell niche... Conclusion Acknowledgements References

 

Adams IR & McLaren A 2002 Sexually dimorphic development of mouse primordial germ cells: switching from oogenesis to spermatogenesis. Development 129 1155–1164.[Abstract/Free Full Text]

Aflatoonian B & Moore H 2005 Human primordial germ cells and embryonic germ cells, and their use in cell therapy. Current Opinion in Biotechnology 16 530–535.[CrossRef][Web of Science][Medline]

Aflatoonian B, Fazeli A, Ruban L, Andrews P & Moore H 2005 Human embryonic stem cells differentiate to primordial germ cells as determined by gene expression profiles and antibody markers. Proceedings of 21st Annual Meeting of the European Society for Human Reproduction and Embryology, Copenhagen. Human Reproduction 20 (Supplement 1) i6.

Allegrucci C, Thurston A, Lucas E & Young L 2005 Epigenetics and the germline. Reproduction 129 137–149.[Abstract/Free Full Text]

Anway MD, Cupp AS, Uzumcu M & Skinner MK 2005 Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308 1466–1469.[Abstract/Free Full Text]

Baillie HS, Pacey AA & Moore HDM 2003 Environmental chemicals and the threat to male fertility in mammals: evidence and perspective. In Conservaion Biology 8. Reproductive Science and Integrated Conservation, pp 57–66. Eds WV Holt, AR Pickard, JC Rodgers & DE Wilat. Cambridge, UK: Cambridge University Press.

Baleato RM, Aitken RJ & Roman SD 2005 Vitamin A regulation of BMP4 expression in the male germ line. Developmental Biology 286 78–90.[CrossRef][Web of Science][Medline]

Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S, Yashiro K, Chawengsaksophak K, Wilson MJ, Rossant J, et al. 2006 Retinoid signaling determines germ cell fate in mice. Science 312 516–600.[Abstract/Free Full Text]

Brinster RL 2002 Germline stem cell transplantation and transgenesis. Science 296 2174–2176.[Abstract/Free Full Text]

Clark AT & Reijo Pera RA 2006 Modeling human germ cell development with embryonic stem cells. Regenerative Medicine 1 85–93.[CrossRef][Web of Science][Medline]

Clark AT, Bodnar MS, Fox M, Rodriquez RT, Abeyta MJ, Firpo MT & Pera RA 2004 Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Human Molecular Genetics 13 727–739.[Abstract/Free Full Text]

de Sousa Lopes SM, Roelen BA, Monteiro RM, Emmens R, Lin HY, Li E, Lawson KA & Mummery CL 2004 BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo. Genes & Development 18 1838–1849.[Abstract/Free Full Text]

Donovan PJ & de Miguel MP 2003 Turning germ cells into stem cells. Current Opinion in Genetics & Development 13 463–471.[CrossRef][Web of Science][Medline]

Draper JS, Moore HD, Ruban LN, Gokhale PJ & Andrews PW 2004 Culture and characterization of human embryonic stem cells. Stem Cells and Development 13 325–336.[CrossRef][Web of Science][Medline]

Dyce PW, Wen L & Li J 2006 In vitro germline potential of stem cells derived from fetal porcine skin. Nature Cell Biology 8 384–390.[CrossRef][Web of Science][Medline]

Eckhoff C & Nau H 1990 Identification and quantitation of all-trans-and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma. Journal of Lipid Research 31 1445–1454.[Abstract]

Eggan K, Jurga S, Gosden R, Min IM & Wagers AJ 2006 Ovulated oocytes in adult mice derive from non-circulating germ cells. Nature 441 1109–1114.[CrossRef][Medline]

Evans MJ & Kaufman MH 1981 Establishment in culture of pluripotential cells from mouse embryos. Nature 292 154–156.[CrossRef][Medline]

Freeman B 2003 The active migration of germ cells in the embryos of mice and men is a myth. Reproduction 125 635–643.[Abstract]

Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K & Daley GQ 2004 Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427 106–107.[CrossRef][Medline]

Ginsburg M, Snow MH & McLaren A 1990 Primordial germ cells in the mouse embryo during gastrulation. Development 110 521–528.[Abstract/Free Full Text]

Guan K, Nayernia K, Maier MS, Wagner S, Dressel R, Lee JH, Nolte J, Wolf F, Li M, Engel W & Hasenfuss G 2006 Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 440 1199–1203.[CrossRef][Medline]

Hamra FK, Chapman KM, Nguyen DM, Williams-Stephens AA, Hammer RE & Garbers DL 2005 Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture. PNAS 102 17430–17435.[Abstract/Free Full Text]

Harun R, Ruban L, Matin M, Draper J, Jenkins NM, Liew GC, Andrews PW, Li TC, Laird SM & Moore HD 2006 Cytotrophoblast stem cell lines derived from human embryonic stem cells and their capacity to mimic invasive implantation events. Human Reproduction 21 1349–1358.[Abstract/Free Full Text]

Hay DC, Sutherland L, Clark J & Burdon T 2004 Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells 2 225–235.

Hubner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J, Strauss JF III, Boiani M & Scholer HR 2003 Derivation of oocytes from mouse embryonic stem cells. Science 300 1251–1256.[Abstract/Free Full Text]

Johnson J, Canning J, Kaneko T, Pru JK & Tilly JL 2004 Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 428 145–150.[CrossRef][Medline]

Johnson J, Bagley J, Skaznik-Wikiel M, Lee HJ, Adams GB, Niikura Y, Tschudy KS, Tilly JC, Cortes ML, Forkert R et al. 2005 Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. Cell 122 303–315.[CrossRef][Web of Science][Medline]

Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y, Toyokuni S et al. 2004 Generation of pluripotent stem cells from neonatal mouse testis. Cell 119 1001–1012.[CrossRef][Web of Science][Medline]

Kehler J, Tolkunova E, Koschorz B, Pesce M, Gentile L, Boiani M, Lomeli H, Nagy A, McLaughlin KJ, Scholer HR & Tomilin A 2004 Oct4 is required for primordial germ cell survival. EMBO Report 5 1078–1083.[CrossRef][Web of Science][Medline]

Koubova J, Menke DB, Zhou Q, Capel B, Griswold MD & Page DC 2006 Retinoic acid regulates sex-specific timing of meiotic initiation in mice. PNAS 103 2474–2479.[Abstract/Free Full Text]

Lacham-Kaplan O 2004 In vivo and in vitro differentiation of male germ cells in the mouse. Reproduction 128 147–152.[Abstract/Free Full Text]

Lacham-Kaplan O, Chy H & Trounson A 2006 Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes. Stem Cells 24 266–273 (Epub 2005 Aug 18).[CrossRef][Web of Science][Medline]

Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, Korving JP & Hogan BL 1999 Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes & Development 13 424–436.[Abstract/Free Full Text]

Looijenga LH, Stoop H, de Leeuw HP, de Gouveia Brazao CA, Gillis AJ, van Roozendaal KE, van Zoelen EJ, Weber RF, Wolffenbuttel KP, van Dekken H, et al. 2003 POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors. Cancer Research 63 2244–2250.[Abstract/Free Full Text]

Martin GR 1981 Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. PNAS 78 7634–7638.[Abstract/Free Full Text]

Matsui Y, Zsebo K & Hogan BL 1992 Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70 841–847.[CrossRef][Web of Science][Medline]

McLaren A 2003 Primordial germ cells in the mouse. Developmental Biology 262 1–15.[CrossRef][Web of Science][Medline]

McLaren A & Lawson KA 2005 How is the mouse germ-cell lineage established? Differentiation 73 435–437.[CrossRef][Web of Science][Medline]

Molyneaux K & Wylie C 2005 Primordial germ cell migration. International Journal of Developmental Biology 48 537–543.[CrossRef][Web of Science]

Moore FL, Jaruzelska J, Fox MS, Urano J, Firpo MT, Turek PJ, Dorfman DM & Pera RA 2003 Human Pumilio-2 is expressed in embryonic stem cells and germ cells and interacts with DAZ (Deleted in AZoospermia) and DAZ-like proteins. PNAS 100 538–543.[Abstract/Free Full Text]

Nayernia K, Nolte J, Michelmann HW, Lee JH, Rathsack K, Drusenheimer N, Dev A, Wulf G, Ehrmann IE, Elliott DJ, et al. 2006a In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Developmental Cell 11 125–132.[CrossRef][Web of Science][Medline]

Nayernia K, Lee JH, Drusenheimer N, Nolte J, Wulf G, Dressel R, Gromoll J & Engel W 2006b Derivation of male germ cells from bone marrow stem cells. Laboratory Investigation 86 654–663.[CrossRef][Web of Science][Medline]

Ohinata Y, Payer B, O’Carroll D, Ancelin K, Ono Y, Sano M, Barton SC, Obukhanych T, Nussenzweig M, Tarakhovsky A, et al. 2005 Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 436 207–213.[CrossRef][Medline]

Payer B, Saitou M, Barton SC, Thresher R, Dixon JP, Zahn D, Colledge WH, Carlton MB, Nakano T & Surani MA 2003 Stella is a maternal effect gene required for normal early development in mice. Current Biology 13 2110–2117.[CrossRef][Web of Science][Medline]

Payer B, Chuva de Sousa Lopes SM, Barton SC, Lee C, Saitou M & Surani MA 2006 Generation of stella-GFP transgenic mice: a novel tool to study germ cell development. Genesis 44 75–83.[CrossRef][Web of Science][Medline]

Rajpert-De Meyts E, Bartkova J, Samson M, Hoei-Hansen CE, Frydelund-Larsen L, Bartek J & Skakkebaek NE 2003 The emerging phenotype of the testicular carcinoma in situ germ cell. Acta Pathologica, Microbiologica, et Immunologica Scandinavica 111 267–278.

Rajpert-De Meyts E, Hanstein R, Jorgensen N, Graem N, Vogt PH & Skakkebaek NE 2004 Developmental expression of POU5F1 (OCT-3/4) in normal and dysgenetic human gonads. Human Reproduction 19 1338–1344.[Abstract/Free Full Text]

Resnick JL, Bixler LS, Cheng L & Donovan PJ 1992 Long-term proliferation of mouse primordial germ cells in culture. Nature 359 550–551.[CrossRef][Medline]

Rossi P, Sette C, Dolci S & Geremia R 2000 Role of c-kit in mammalian spermatogenesis. Journal of Endocrinological Investigation 23 609–615.[Web of Science][Medline]

Ryu BY, Kubota H, Avarbock MR & Brinster RL 2005 Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. PNAS 102 14302–14307 (Epub 2005 Sep 23).[Abstract/Free Full Text]

Saitou M, Barton SC & Surani MA 2002 A molecular programme for the specification of germ cell fate in mice. Nature 418 293–300.[CrossRef][Medline]

Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan -PJ, Blumenthal PD, Huggins GR & Gearhart JD 1998 Derivation of pluripotent stem cells from cultured human primordial germ cells. PNAS 95 13726–13731.[Abstract/Free Full Text]

Shimasaki S, Moore RK, Otsuka F & Erickson GF 2004 The bone morphogenetic protein system in mammalian reproduction. Endocrine Reviews 25 72–101.[Abstract/Free Full Text]

Skakkebaek NE, Rajpert-De Meyts E & Main KM 2001 Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Human Reproduction 16 972–978.[Abstract/Free Full Text]

Smith A 2006 A glossary for stem-cell biology. Nature 441 1060.[CrossRef][Web of Science]

Telfer EE, Gosden RG, Byskov AG, Spears N, Albertini D, Andersen CY, Anderson R, Braw-Tal R, Clarke H, Gougeon A, et al. 2005 On regenerating the ovary and generating controversy. Cell 122 821–822.[CrossRef][Web of Science][Medline]

Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS & Jones JM 1998 Embryonic stem cell lines derived from human blastocysts. Science 282 1145–1147.[Abstract/Free Full Text]

Tiido T, Rignell-Hydbom A, Jonsson B, Giwercman YL, Rylander L, Hagmar L & Giwercman A 2005 Exposure to persistent organochlorine pollutants associates with human sperm Y:X chromosome ratio. Human Reproduction 20 1903–1909.[Abstract/Free Full Text]

Toyooka Y, Tsunekawa N, Takahashi Y, Matsui Y, Satoh M & Noce T 2000 Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development. Mechanisms of Development 93 139–149.[CrossRef][Web of Science][Medline]

Toyooka Y, Tsunekawa N, Akasu R & Noce T 2003 Embryonic stem cells can form germ cells in vitro. PNAS 100 11457–11462.[Abstract/Free Full Text]

Turnpenny L, Brickwood S, Spalluto CM, Piper K, Cameron IT, Wilson DI & Hanley NA 2003 Derivation of human embryonic germ cells: an alternative source of pluripotent stem cells. Stem Cells 21 598–609.[CrossRef][Web of Science][Medline]

Vincent SD, Dunn NR, Sciammas R, Shapiro-Shalef M, Davis MM, Calame K, Bikoff EK & Robertson EJ 2005 The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse. Development 132 1315–1325.[Abstract/Free Full Text]

Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, Zwaka TP & Thomson JA 2002 BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nature Biotechnology 20 1261–1264.[CrossRef][Web of Science][Medline]

Ying Y, Qi X & Zhao GQ 2001 Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8b signaling pathways. PNAS 98 7858–7862.[Abstract/Free Full Text]

This article has been cited by other articles:

				X. Bai, D. Silvius, E. D. Chan, D. Escalier, and S. X. Xu Identification and characterization of a novel testis-specific gene CKT2, which encodes a substrate for protein kinase CK2 Nucleic Acids Res., May 1, 2009; 37(8): 2699 - 2711. [Abstract] [Full Text] [PDF]

				M. F. Gjerstorff, L. Harkness, M. Kassem, U. Frandsen, O. Nielsen, M. Lutterodt, K. Mollgard, and H. J. Ditzel Distinct GAGE and MAGE-A expression during early human development indicate specific roles in lineage differentiation Hum. Reprod., October 1, 2008; 23(10): 2194 - 2201. [Abstract] [Full Text] [PDF]

				H. Fulka, I. Barnetova, T. Mosko, and J. Fulka Epigenetic analysis of human spermatozoa after their injection into ovulated mouse oocytes Hum. Reprod., March 1, 2008; 23(3): 627 - 634. [Abstract] [Full Text] [PDF]

				C. G. M. Extavour Evolution of the bilaterian germ line: lineage origin and modulation of specification mechanisms Integr. Comp. Biol., November 1, 2007; 47(5): 770 - 785. [Abstract] [Full Text] [PDF]

				L. E Young Focus on stem cells in reproduction. Reproduction, November 1, 2006; 132(5): 671 - 672. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Aflatoonian, B. Articles by Moore, H. Search for Related Content PubMed PubMed Citation Articles by Aflatoonian, B. Articles by Moore, H.