Pin1 regulates the timing of mammalian primordial germ cell proliferation

Primordial germ cells (PGCs) give rise to male and female germ cells to transmit the genome from generation to generation. Defects in PGC development often result in infertility. In the mouse embryo, PGCs undergo proliferation and expansion during and after their migration to the gonads from 8.5 to 13.5 days post coitum (dpc). We show that a peptidyl-prolyl isomerase, Pin1, is involved in the regulation of mammalian PGC proliferation. We discovered that both the male and female Pin1(-/-) mice had profound fertility defects. Investigation of the reproductive organs revealed significantly fewer germ cells in the adult Pin1(-/-) testes and ovaries than in wild type or heterozygotes, which resulted from Pin1(-/-) males and females being born with severely reduced number of gonocytes and oocytes. Further studies in 8.5 to 13.5 dpc Pin1(-/-) embryos showed that PGCs were allocated properly at the base of the allantois, but their cell expansion was progressively impaired, resulting in a markedly reduced number of PGCs at 13.5 dpc. Analyses using markers of cell cycle parameters and apoptosis revealed that Pin1(-/-) PGCs did not undergo cell cycle arrest or apoptosis. Instead, Pin1(-/-) PGCs had a lower BrdU labeling index compared with wild-type PGCs. We conclude that PGCs have a prolonged cell cycle in the absence of Pin1, which translates into fewer cell divisions and strikingly fewer Pin1(-/-) PGCs by the end of the proliferative phase. These results indicate that Pin1 regulates the timing of PGC proliferation during mouse embryonic development.     

Formation and cultivation of medaka primordial germ cells

Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka (Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model.     

Activin and TGFbeta limit murine primordial germ cell proliferation

Mammalian primordial germ cells (PGCs) proliferate as they migrate from their initial location in the extraembryonic mesoderm to the genital ridge, the gonadal anlage. Once in the genital ridge, PGCs cease dividing and differentiate according to their gender. To identify ligands that might limit PGC proliferation, we analyzed growth factor receptors encoded in RNA obtained from purified germ cells shortly after their arrival in the genital ridge. Receptors for two members of the TGFbeta superfamily were found, TGFbeta1 and activin. As the signal-transducing domains of both receptor systems are highly conserved, the effects of both TGFbeta1 and activin on PGCs would be expected to be similar. We found that both ligands limited the accumulation of germ cells in primary PGC cultures. BrdU incorporation assays demonstrated that either ligand inhibits PGC proliferation. These results suggest that these signal transduction pathways are important elements of the mechanism that determines germ cell endowment.     

Autocrine and paracrine mechanisms regulating primordial germ cell proliferation

Although several mitogens and survival factors have been previously shown to act on primordial germ cells (PGCs) in culture, it is not clear whether they are responsible for controlling proliferation of PGCs in the embryo. We show here that during their migratory phase, PGCs do not express FGF-4, FGF-8, or FGF-17, but these FGFs are expressed by neighboring cells. Thus, any FGF action on migrating PGCs would appear to be through a paracrine mechanism. We found that after entering into the gonads, PGCs start to express FGF-4 and FGF-8. On this basis, we hypothesize that FGF signaling is involved in both a paracrine manner in initiating PGC proliferation during their migration and an autocrine manner in sustaining PGC proliferation after their arrival in the gonads. We then studied the role of soluble stem cell factor (SCF), which acts as a survival factor or a mitogen in culture, to determine whether it interacts with FGFs. We found that SCF has a complex effect on PGC proliferation. On one hand, soluble SCF promoted PGC proliferation synergistically with FGF in the absence of membrane-bound SCF. Conversely, soluble SCF inhibited FGF-stimulated proliferation of PGCs in the presence of membrane-bound SCF. We account for these findings in a model involving regulation of PGC proliferation, in which SCF modulates the response to FGFs.     

Guidance of primordial germ cell migration by the chemokine SDF-1

The signals directing primordial germ cell (PGC) migration in vertebrates are largely unknown. We demonstrate that sdf-1 mRNA is expressed in locations where PGCs are found and toward which they migrate in wild-type as well as in mutant embryos in which PGC migration is abnormal. Knocking down SDF-1 or its receptor CXCR4 results in severe defects in PGC migration. Specifically, PGCs that do not receive the SDF-1 signal exhibit lack of directional movement toward their target and arrive at ectopic positions within the embryo. Finally, we show that the PGCs can be attracted toward an ectopic source of the chemokine, strongly suggesting that this molecule provides a key directional cue for the PGCs.     

Time-lapse analysis reveals different modes of primordial germ cell migration in the medaka Oryzias latipes

Previous studies have shown that medaka primordial germ cells (PGC) are first distinguishable by olvas expression during late gastrulation, and that they migrate to the gonadal region through the lateral plate mesoderm. Here, we demonstrate that medaka nanos expression marks the germ line at early gastrulation stage. By marking the germ line with green fluorescent protein (GFP) fused to the nanos 3' untranslated region, we were able to visualize the behavior of PGC using time-lapse imaging. We show that there are three distinct modes of PGC migration that function at different stages of development. At early gastrulation stage, PGC actively migrate towards the marginal zone, a process that requires the function of a chemokine receptor, CXCR4. However, at late gastrulation stage, PGC change the mode and direction of their movement, as they are carried towards the midline along with somatic cells undergoing convergent movements. After aligning bilaterally, PGC actively migrate to the posterior end of the lateral plate mesoderm. This posterior movement depends on the activity of both HMGCoAR and a ligand of CXCR4, SDF-1a. These results demonstrate that PGC undergo different modes of migration to reach the prospective gonadal region of the embryo.     

Growth factors in mouse primordial germ cell migration and proliferation

Information obtained mainly from in vitro culture studies and genetic analysis of mouse mutants White spotting and Steel indicate a pivotal role of growth factors in the development of mouse primordial germ cells (PGCs). While stem cell factor (SCF) and TGFβ1 seem to have a role in PGC migration (as an adhesion factor and a chemoattractant, respectively), the former is certainly required for PGC survival in vitro and probably in vivo as well. Recent findings suggest that the mechanism by which SCF supports PGC survival is by preventing PGC apoptosis. A similar action appears to be exerted by leukemia inhibitory factor (LIF), a further growth factor influencing PGC growth in culture.PGC proliferation seems to be mainly induced by cAMP dependent mechanisms, but futther investigations are needed to clarify the interrelationships among the different molecular pathways activated by SCF, LIF, cAMP and other putative PGC growth factors (i.e. bFGF). Stimulation of long-term proliferation of PGCs, leading to derivation of ES-like cells (embryonal germ cells) obtained by using a combination of growth factors (bFGF, SCF and LIF), opens new intriguing perspectives for such studies and transgenic technology.     

Specification of primordial germ cells in medaka (Oryzias latipes)

Background  

Primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. Two modes of germ line specification have been described: the inheritance of asymmetrically-localized maternally provided cytoplasmic determinants and the induction of the PGC fate by other cell types.     

MCM9 deficiency delays primordial germ cell proliferation independent of the ATM pathway

Maintenance of genome integrity is crucial for the germline, and this is reflected by lower mutation rates in gametes than somatic cells. Germ cells at different stages employ different DNA damage response (DDR) mechanisms. In response to certain DNA repair defects, primordial germ cells (PGCs) either undergo apoptosis or delayed proliferation, although little is known about the underlying mechanisms that govern these outcomes. Here, we report genetic studies of DDR pathways that underlie germ cell depletion in mice mutant for minichromosome maintenance 9 (Mcm9), a gene that plays a role in homologous recombination repair (HRR). Germ cell depletion in these mice is a result of reduced PGC numbers both before and after they arrive in the primitive gonads. This reduction was attributable to reduced proliferation, not apoptosis, and this response was independent of ATM‐CHK2‐TRP53‐P21 signaling. This mechanism of PGC depletion differs from that in Fancm mutants, which also display reduced PGC depletion that is partially orchestrated by the ATM‐TRP53‐P21 pathway. Germ cell depletion in mice doubly deficient for FANCM and MCM9 was additive, indicating that the damage caused by each mutation triggers different DDR pathways to slow the cell cycle as a means to preserve genomic integrity. genesis 53:678–684, 2015. © 2015 Wiley Periodicals, Inc.     

Guidance of primordial germ cell migration

Primordial germ cells (PGCs), the progenitors of the gametes, migrate from the position where they are specified towards the region where the gonad develops. To reach their target, the PGCs obtain directional cues from cells positioned along their migration path. One such cue, the chemokine SDF-1, has recently been found to be critical for proper PGC migration in zebrafish and in mice. In Drosophila, too, a molecule that is structurally related to chemokine receptors and is important for PGC migration has been identified. The ability to visualize chemokine-guided migration at a high resolution in vivo in these model organisms provides a unique opportunity to study this process, which is relevant for many events in normal development and disease.     

DMRT1/Dmrt1, the sex determining or sex differentiating gene in Vertebrata

Although the phenomenon of sexual dimorphism is widespread in vertebrates, the molecular mechanism of sex-determination is not the same across animal phyla, in contrast to other areas of developmental biology. Recent extensive studies, however, have given proof of evolutionarily conserved function in genes which share a novel DNA binding DM domain, primarily identified in two invertebrate sex regulatory genes: doublesex of Drosophila melanogaster and mab-3 of Caenorhabditis elegans. Their mammalian autosomal homologue, DMRT1, first isolated in humans, was further discovered in genomes of various vertebrate species and appears to be involved in similar aspects of sexual development. Its precise role is still speculated, thus identification of sex reversal mutations, functional studies as well as determination of the sex-specific expression profile during embryogenesis are still being undertaken. Is this a sex determining rather than a sex differentiating gene? Is it involved in a dosage-sensitive mechanism? On what level does it function in the hierarchy of the sexual regulatory gene cascade? Recent results are discussed in this paper.     

Nuclear transplantation of male primordial germ cells in the mouse

We examined the developmental ability of enucleated eggs receiving embryonic nuclei and male primordial germ cells (PGCs) in the mouse. Reconstituted eggs developed into the blastocyst stage only when an earlier 2-cell nucleus was transplanted (36%) but very rarely if the donor nucleus was derived from a later 2-cell, 8-cell, or inner cell mass of a blastocyst (0-3%). 54-100%, 11-67%, 6-43% and 6-20% of enucleated eggs receiving male PGCs developed to 2-cell, 4-cell, 8-cell and blastocyst stage, respectively, in culture. The overall success rate when taking into account the total number of attempts at introducing germ cells was actually 0-6%. Live fetuses were not obtained after transfer of reconstituted eggs to recipients, although implantation sites were observed. The developmental ability of reconstituted eggs in relation to embryonic genome activation and genomic imprinting is discussed.     

Migration and proliferation of primordial germ cells in the rat

Information about early primordial germ cell (PGC) formation and migration in rats is lacking. In utero developed and in vitro cultivated whole rat embryos were studied on days 10-13 postcoitum (p.c.). The development of the PGCs was investigated in serial sections stained for alkaline phosphatase activity. On postcoital day 10, PGCs were found in the invaginating visceral yolk sac endoderm and at the base of the allantois. At day 11 p.c. PGCs were mostly found in the ventral and lateral gut wall or in the mesenchyme between the gut and the future genital ridges. At day 12 p.c. most of the PGCs (94%) could be localised in the mesenchyme or in the future genital ridges. On postcoital day 13 almost all PGCs had reached the now-well-developed genital ridges. Quantitative measurements showed an increase in the number of PGCs from 84 at day 10 p.c. up to 2,768 at day 13 p.c. Only slight differences were found between in vivo and in vitro embryos with respect to the number of PGCs and their developmental pattern. The in vitro culture of whole rat embryos enables the discrimination between the effects of indirect (maternal) and direct action of PGC-toxic agents.     

Migration,proliferation and potency of primordial germ cells

In most species, the cells allocated to the germ line, the primordial germ cells (PGCs) arise very early in embryo-genesis, and migrate to join the somatic cells at the site where the gonad will form. In three widely studied animals; the mouse, the frog and Drosophila, the PGCs associate with the developing gut, from which they migrate during the period of organogenesis to the gonads. During this migration, the germ cell population increases by an amount which is more or less constant for a particular species. Genes important in the control of PGC migration and population are being identified in two ways. In invertebrates, and to a lesser extent in mice, genetic approaches have identified important loci or gene products. Culturing PGCs in a variety of conditions has been an alternative approach in mouse embryos. From these latter studies, it is now known that a number of growth factors, released from surrounding tissues, control many aspects of PGC behaviour, including their proliferation, migration, potency, and survival. Attention is also focusing on changes in PGC adhesiveness during migration.     

Repression of primordial germ cell differentiation parallels germ line stem cell maintenance

In Drosophila, primordial germ cells (PGCs) are set aside from somatic cells and subsequently migrate through the embryo and associate with somatic gonadal cells to form the embryonic gonad. During larval stages, PGCs proliferate in the female gonad, and a subset of PGCs are selected at late larval stages to become germ line stem cells (GSCs), the source of continuous egg production throughout adulthood. However, the degree of similarity between PGCs and the self-renewing GSCs is unclear. Here we show that many of the genes that are required for GSC maintenance in adults are also required to prevent precocious differentiation of PGCs within the larval ovary. We show that following overexpression of the GSC-differentiation gene bag of marbles (bam), PGCs differentiate to form cysts without becoming GSCs. Furthermore, PGCs that are mutant for nanos (nos), pumilio (pum) or for signaling components of the decapentaplegic (dpp) pathway also differentiate. The similarity in the genes necessary for GSC maintenance and the repression of PGC differentiation suggest that PGCs and GSCs may be functionally equivalent and that the larval gonad functions as a "PGC niche".     

Differentiation of mouse primordial germ cells into female or male germ cells.

Mouse primordial germ cells (PGCs) migrate from the base of the allantois to the genital ridge. They proliferate both during migration and after their arrival, until initiation of the sex-differentiation of fetal gonads. Then, PGCs enter into the prophase of the first meiotic division in the ovary to become oocytes, while those in the testis become mitotically arrested to become prospermatogonia. Growth regulation of mouse PGCs has been studied by culturing them on feeder cells. They show a limited period of proliferation in vitro and go into growth arrest, which is in good correlation with their developmental changes in vivo. However, in the presence of multiple growth signals, PGCs can restart rapid proliferation and transform into pluripotent embryonic germ (EG) cells. Observation of ectopic germ cells and studies of reaggregate cultures suggested that both male and female PGCs show cell-autonomous entry into meiosis and differentiation into oocytes if they were set apart from the male gonadal environments. Recently, we developed a two-dimensional dispersed culture system in which we can examine transition from the mitotic PGCs into the leptotene stage of the first meiotic division. Such entry into meiosis seems to be programmed in PGCs before reaching the genital ridges and unless it is inhibited by putative signals from the testicular somatic cells.     

Regulation of primordial germ cell development in the mouse

Primordial germ cells (PGCs) are the founders of the gametes. They arise at the earliest stages of embryonic development and migrate to the gonadal ridges, where they differentiate into oogonia/oocytes in the ovary, and prospermatogonia in the testis. The present article is a review of the main studies undertaken by the author with the aim of clarifying the mechanisms underlying the development of primordial germ cells. Methods for the isolation and purification of migratory and post-migratory mouse PGCs devised in the author's laboratory are first briefly reviewed. Such methods, together with the primary culture of PGCs onto suitable cell feeder layers, have allowed the analysis of important aspects of the control of their development, concerning in particular survival, proliferation and migration of mouse PGCs. Compounds and growth factors affecting PGC numbers in culture have been identified. These include survival anti-apoptotic factors (SCF, LIF) and positive regulators of proliferation (cAMP, PACAPs, RA). Evidence has been provided that the motility of migrating PGCs relies on integrated signals from extracellular matrix molecules and the surrounding somatic cells. Moreover, homotypic PGC-PGC interaction has been evidenced that might play a role in PGC migration and in regulating their development. Several molecules (i.e. integrins, specific types of oligosaccharides, E-cadherin, the tyrosine kinase receptor c-kit) have been found to be expressed on the surface of PGCs and to mediate adhesive interactions of PGCs with the extracellular matrix, somatic cells and neighbouring PGCs.     

In vitro survival and proliferation of porcine primordial germ cells

Primordial germ cells (PGC) collected from the genital ridge of Day 25 porcine embryos were cultured on STO feeder cells in medium with or without supplemented growth factors. The effects on porcine PGC proliferation of leukemia inhibitory factor (LIF), LIF + stem cell factor (SCF) or LIF + SCF + basic fibroblast growth factor (bFGF), growth factors shown to be essential for in vitro survival and proliferation of murine PGC, were tested. After histochemical staining, both freshly collected and cultured PGC expressed alkaline phosphatase activity. With or without supplemented growth factors, porcine PGC survived and proliferated in culture for at least 5 d. None of the growth factors tested markedly enhanced in vitro growth of porcine PGC. These results suggest that growth factors provided by either the STO feeder layer or the cultured PGC themselves are sufficient to support in vitro survival and proliferation of porcine PGC. With the support of STO cells, addition of growth factors shown to be essential for the in vitro growth of murine PGC is not required for survival and proliferation of cultured porcine PGC.     

Cellular and molecular aspects of mouse primordial germ cell migration and proliferation in culture.

The development of mouse primordial germ cells is followed from their first appearance in the extraembryonic mesoderm of the posterior amniotic fold (7 dpc embryo) to their settlement in the genital ridges (12.5 dpc embryo). The role of fibronectin as adhesive substrate and/or in stimulating cell motility during PGC migration is discussed. Recent papers showing how PGCs migrate when cultured in vitro on cellular monolayers are reviewed. The process of PGC homing is proposed to be controlled by chemotaxis as well by developmentally regulated cell-to-cell interactions. Finally, evidence that survival and proliferation of PGCs is strictly dependent on growth factors such as LIF and MGF, and possibly on a cAMP-dependent mechanism is reported.