Mouse Primordial Germ Cells Research Articles

Gene Transfection of Mouse Primordial Germ Cellsin Vitroand Analysis of Their Survival and Growth Control

We evaluated electroporation, liposome-mediated transfection, and the calcium phosphate (CaPO4) coprecipitation method for gene transfection of mouse primordial germ cells (PGCs) in culture as a prelude to the investigation of molecular mechanisms of the germ cell development. We found that electroporation severely damaged PGCs, and the efficiency of liposome-mediated transfection was very low. In contrast, using the CaPO4coprecipitation method, 18% of PGCs transfected with plasmid pSV-LT expressed simian virus 40 large tumor antigen (SV 40 T-Ag) transiently. However, we did not detect any effects on the proliferation and survival of PGCs obtained from the embryonic gonads at 11.5 days postcoitum (d.p.c.) during 2 days of culture after the transfection. PGCs isolated from the 11.5-d.p.c. gonads change from spread- to round-shape and exhibit growth arrest during a few days of culture, and these rounded PGCs quickly disappear from the culture. We found that the transfection and expression of Bcl-xlor adenovirus type 2 E1B 19,000-molecular-weight protein (E1B 19K) significantly promoted the survival of PGCs and retarded the disappearance of rounded PGCs from the culture system. These results suggest that the Bcl-xlor E1B 19K can prevent the apoptosis of PGCs and inhibit the cell death of the rounded PGCs in culture.

A rapid and quantitative assay of adhesiveness of mouse primordial germ cells that can be applied to many types of early embryonic cell

Cell migration is an essential component of embryogenesis. In order to move from one location to another, cells must quantitatively alter their adhesiveness to the different substrata they encounter. Little is known about these changes in adhesive strengths, nor how they are controlled. Here we describe a combination of simple, quantitative, and reproducible methods to partially purify primordial germ cells from different stages of early mouse embryos, and compare their strengths of adhesion to different extracellular matrix glycoproteins. Primordial germ cells arise very early in the mouse embryo, and migrate through the tissues of the embryo to reach the site of gonad formation. Here they assemble into sex cords and start to differentiate into male or female gametes. Although the focus here is germ line cells, in principle this method can be used on any population of cells in the early embryo.

Purification of Primordial Germ Cells from TNAPβ-geoMouse Embryos Using FACS-gal

Tissue nonspecific alkaline phosphatase (TNAP), the product of theAkp2locus, is expressed in mouse primordial germ cells (PGC) for an extensive period during embryogenesis. Mice with theAkp2tm1Sormutant allele of TNAP expresslacZ(β-galactosidase; β-gal) under control of theAkp2locus. PGCs were purified fromAkp2tm1Sorembryos using fluorescence activated cell sorting of β-gal expressing cells (FACS-gal). Analysis of the purified cells by alkaline phosphatase staining and immunocytochemistry with anti-c-kitantibody demonstrated that highly (98%) purified PGCs can be isolated using this method. This technique will facilitate experiments that require highly purified preparations of PGCs including cell culture and gene expression analyses.

A novel serine/threonine kinase gene, Gek1, is expressed in meiotic testicular germ cells and primordial germ cells.

We have isolated a novel serine/ threonine kinase gene designated Gek1 from mouse primordial germ cell-derived embryonic germ cell. Gek1 is preferentially expressed in meiotic testicular germ cells and primordial germ cells. Gek1 mRNA is also detected in several other tissues, including hematopoietic organs in adult mice and central nervous system in embryos. The Gek1 cDNA encodes a protein with the consensus sequence of the catalytic domain of protein kinases in its N-terminal region. The deduced amino acid sequence of Gek1 in the kinase domain is related to those encoded by the Saccharomyces cerevisiae STE20, CDC15, and Drosophila melanogaster ninaC. The patterns of expression and the structural features of Gek1 suggest that the gene product is involved in signal transduction or nuclear division of germ cells and other proliferating cells. We also show that Gek1 locates on chromosome 11, near the wr locus, showing neuronal and reproductive defects.

Role of stem cell factor in somatic-germ cell interactions during prenatal oogenesis.

During embryogenesis germ cells originate from primordial germ cells (PGCs). The development of mammalian PGCs involves a number of complex events (formation and segregation of PGC precursors, PGC migration and proliferation) which lead to the differentiation of oocytes or prospermatogonia (for a review see De Feliciet al., 1992). During recent years developments in methods for isolation, purification and culture of mouse PGCs have led to significant progress in the understanding of molecular mechanisms of migration, proliferation and differentiation of these cells (for reviews see De Felici, 1994; and De Felici & Pesce, 1994a). In this paper we describe the key role played by stem cell factor (SCF) in PGC development and early folliculogenesis.

Dose-dependent induction of recessive mutations with N-ethyl- N-nitrosourea in primordial germ cells of male mice

Using a specific locus test, we previously found that N-ethyl- N-nitrosourea (ENU) induces recessive mutations at a relatively high rate in male mouse primordial germ cells (PGC) at 8.5, 10.5 and 13.5 days of development (G8.5, G10.5 and G13.5). A large difference was observed on the induced mutation rate between 30 and 50 mg/kg ENU in 10.5-day PGC. We therefore carried out specific locus tests to ascertain whether ENU induces recessive mutations in a dose-dependent manner in G8.5 and G10.5 PGC. We also gave multiple doses of 25 mg/kg ENU using an 18-h interval, the approximate doubling time of PGC at these developmental stages, to test for an additive effect on the induced mutations rate. A dose-dependent induction of recessive mutations by ENU was observed in both G8.5 and G10.5 PGC, and multiple dosing of 25 mg/kg ENU showed an additive effect. Comparing these results to data on spermatogonial stem cells, we conclude the capacity to repair ENU-induced premutagenic damages is less effective in male mouse PGC at these developmental stages than in spermatogonial stem cells.

MesP1: a novel basic helix-loop-helix protein expressed in the nascent mesodermal cells during mouse gastrulation.

A subtractive hybridization strategy was used to isolate putative genes involved in the development of mouse primordial germ cells (PGC). Complimentary DNA was amplified on RNA isolated from the base of the allantois where PGC are located in the 7.5 days post coitum (dpc) mouse embryo. It was then subtracted by hybridization with cDNA amplified on RNA of the anterior region where PGC are absent. A novel gene thus isolated is designated as Mesp1 and encodes a possible transcription factor MesP1 containing a basic helix-loop-helix motif. Its earliest expression was observed at the onset of gastrulation, as early as 6.5 dpc, in the nascent mesodermal cells that first ingressed at the end of the primitive streak. These expressing cells in the lateral and extraembryonic mesoderm showed a wing-shaped distribution. Its initial expression was soon down-regulated at 7.5 dpc before the completion of gastrulation, except at the proximal end of the primitive streak which included the extraembryonic mesoderm and the base of allantois. At 8 dpc, the expression at the base of the allantois moved laterally. This distribution between 7.0 and 8.0 dpc was similar to that of PGC detected by the alkaline phosphatase activity. However, the expression of Mesp1 was down-regulated thereafter, when PGC entered in the migration stage. After birth, Mesp1 expression was detected only in mature testes, but in a different isoform from that expressed in the embryo. Mesp1 was mapped to the mid region of chromosome 7, near the mesodermal deficiency gene (mesd). However, a Southern hybridization study clearly showed that Mesp1 was distinctly different from mesd. The amino acid sequence and its expression pattern suggest that MesP1 plays an important role in the development of the nascent mesoderm including PGC.

PRIMORDIAL GERM CELL-DERIVED MOUSE EMBRYONIC GERM (EG) CELLS IN VITRORESEMBLE UNDIFFERENTIATED STEM CELLS WITH RESPECT TO DIFFERENTIATION CAPACITY AND CELL CYCLE DISTRIBUTION

Embryonic germ (EG) cells of line EG-1 derived from mouse primordial germ cells were investigated for their in vitrodifferentiation capacity. By cultivation as embryo-like aggregates EG-1 cells differentiated into cardiac, skeletal muscle and neuronal cells accompanied by the expression of tissue-specific genes and proteins as shown by RT-PCR analysis and indirect immunofluorescence. In comparison to embryonic stem (ES) cells of line D3 the efficiency of differentiation into cardiac and muscle cells was comparatively low, whereas spontaneous neuronal differentiation was more efficient than in D3 cells. Furthermore, the distribution of cell cycle phases as a parameter for the differentiation state was analysed in undifferentiated EG cells and ES cells and compared to data obtained for embryonic carcinoma (EC) cells of line P19 and differentiated, epithelioid EPI-7 cells. Flow cytometric analysis revealed similar cell cycle phase distributions in EG, EC and ES cells. In contrast, the somatic differentiated EPI-7 cells showed a longer G 1-phase and shorter S- and G 2/M-phases. Together, our results demonstrate that the differentiation state and capacity of EG cells in vitroresemble that of totipotent ES cells.

A combination of Buffalo rat liver cell-conditioned medium, forskolin and membrane-bound stem cell factor stimulates rapid proliferation of mouse primordial germ cells in vitro similar to that in vivo.

Recent studies have shown that stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and the enhancement of cAMP levels increase proliferation and survival of mouse primordial germ cells (PGC) in vitro. Even after the addition of these factors, however, it is still not possible to obtain proliferation of PGC at a rapid rate similar to that in vivo, suggesting the presenge of other growth factor(s) in vivo. We previously reported that tumor necrosis factor-α stimulates proliferation of PGC at earlier migration stages. We now show that the use of SI/SI4-m220 feeder cells and the addition of a medium conditioned with Buffalo rat liver cells and forskolin to the culture medium stimulate PGC obtained from 8.5 days post coitum embryos to proliferate in culture at a rate comparable to that in vivo. Under such conditions, proliferation of PGC continued several days past the timing of growth arrest in vivo; however, it did stop afterwards. Such proliferating PGC continue to express c-kit and Oct-3 proteins. The characteristics of the culture medium and the requirement of feeder cells were different from those for embryonic stem (ES) cells, suggesting that these rapidly proliferated PGC are not transformed into ES-like EG cells.

Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells.

Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse primordial germ cells (PGC) in culture. The receptor complex for LIF comprises LIF-binding subunit and non-binding signal transducer, gp130. The gp130 was originally identified as a signal-transducing subunit of interleukin (IL)-6 and later also found to be a functional component of receptor complexes for other LIF-related cytokines (oncostatin M [OSM], ciliary neurotrophic factor [CNTF] and IL-11). In this study, we have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. OSM was able to fully substitute for LIF; both cytokines promoted the proliferation of migratory PGC (mPGC) and enhanced the viability of postmigratory (colonizing) PGC (cPGC) when cultured on SI/SI4-m220 cells. Interestingly, IL-11 stimulated mPGC growth comparable to LIF and OSM, but did not affect cPGC survival. IL-6 and CNTF did not affect PGC. In addition, a combination of IL-6 and soluble IL-6 binding subunit (sIL-6R), which is known to activate intracellular signaling via gp130, fully reproduced the LIF action of PGC. Both in the presence and absence of LIF, addition of neutralizing antibody against gp130 in culture remarkably blocked cPGC survival. These results suggest a pivotal role of gp130 in PGC development, especially that it is indispensable for cPGC survival as comparable to the c-KIT-mediated action. We have further demonstrated that a combination of LIF with forskolin or retinoic acid, a potent mitogen for PGC, supported the proliferation of PGC, leading to propagation of the embryonic stem cell-like cells, termed embryonic germ (EG) cells. Since EG cells were also obtained by using OSM or the IL-6/sIL-6R complex in place of LIF, a significant contribution of gp130-mediated signaling in EG cell formation was further suggested.

Autonomous Regulation of Proliferation and Growth Arrest in Mouse Primordial Germ Cells Studied by Mixed and Clonal Cultures

In culture, mouse primordial germ cells (PGCs) proliferate and undergo growth arrest with a time course similar to thatin vivo.It is unclear whether this behavior is regulated autonomously or by coexisting somatic cells. We performed mixed culture experiments using PGCs from 8.5- and 11.5-d.p.c. embryos and found no interaction between the PGCs and somatic cells at the two stages. Next, we carried out clonal culture of PGCs and examined the proliferation of and morphological change in individual clones. Such clonal culture did not reveal any subpopulation of PGCs with an increased growth rate or less differentiated characteristics, which might have been suggested by formation of the embryonic germ cell lines. Our results suggest that there is an autonomous regulation of growth and cell shape change in PGCs which occur as stochastical events but are not strictly timed by the number of cell divisions.

The Role of Interleukin-4 in the Regulation of Mouse Primordial Germ Cell Numbers

Interleukin-4 (IL-4), a pleiotropic cytokine, stimulates a dose-dependent increase in the number of mouse primordial germ cells in culture. Results from bromodeoxyuridine incorporation assays suggest that IL-4 acts as a survival factor rather than as a mitogen for primordial germ cells in this system. Studies on the embryonic expression patterns of IL-4 and its receptors, using RT–PCR and ELISA, show that IL-4 and its receptors are present at the correct time and place to influence PGC numbersin vivo.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates adenylate cyclase and promotes proliferation of mouse primordial germ cells

During migration and for about 2 days after their arrival in the gonadal ridges, primordial germ cells (the embryonic precursors of gametes of the adult animal) proliferate actively. Certain growth factors, such as stem cell factor and leukemia inhibitory factor, seem to be essential for survival, proliferation and possibly differentiation of mouse primordial germ cell in vivo and/or in vitro. Similarly, increase in intracellular cAMP is followed by a marked enhancement of primordial germ cell proliferation, at least in culture. In the present study, we show that pituitary adenylate cyclase-activating polypeptides (PACAP-27 and PACAP-38), two neuropeptides of the secretin-glucagon-vasoactive intestinal polypeptide-GH-releasing hormone family, stimulate in vitro proliferation of mouse primordial germ cells, bind to primordial germ cells and gonadal somatic cells (possibly to type I PACAP receptor) and activate adenylate cyclase in the same cells. Moreover, PACAP-like immunoreactivity was found in gonadal ridges, mostly on germ cell surface. In conclusion, evidence is provided that PGC proliferation can be stimulated by certain bioactive polypeptides, thus suggesting a novel regulatory role for such compounds in early gonad development.

Purification of Mouse Primordial Germ Cells by MiniMACS Magnetic Separation System

During migration toward gonadal ridges, primordial germ cells (PGCs; the earliest identifiable germ cells in the embryo) are very few in number, move along different tissues, and are not identifiable by morphological criteria alone. Here we report the use of the magnetic cell sorter MiniMACS as a tool for the isolation of such rare cells from 10.5- to 13.5-days post coitum mouse embryos. Cells stained sequentially by TG-1 (a monoclonal IgM antibody known to bind to the surface of PGCs) and superparamagnetic microbeads coated with secondary anti-mouse IgM antibody were separated on a magnetic column. Unlabeled cells (somatic cells) pass through the column, while labeled cells (germ cells) are retained. The retained cells can be eventually easily eluted and immediately used for biochemical studies or grown in suitable in vitro culture systems.

Effects of feeder cells and growth factors on the proliferation of mouse primordial germ cells

There are various kinds of stem cell which have the ability to self-renew under appropriate microenvironments and growth factors. In vitro culture of stem cells may provide a tool for elucidating the mechanism of self-renewal of these cells. Hematopoietic B cell progenitor cell proliferation is supported on the born marrow-derived cell line, ST2. Embryonic stem (ES) cells derived from mouse blastocysts acquire their ability for self-renewal and pluripotency on mouse embryonic fibroblast cells (MEF). In the present study, we examined effects of these 2 different kinds of feeder cells, in the presence of growth factors, on the ability of mouse primordial germ cells (PGCs) collected at 8.5 d post coitum (dpc) to proliferate and acquire of pluripotency. The addition of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF), ST2, but not MEF, supported the proliferation of the PGCs for at least 5 d in culture. The presence of either LIF or bFGF renders a lower ability to support the proliferation of PGCs. Reverse transcription-PCR analysis shows that both feeder cells transcribed the soluble form of stem cell factor (SCF) but not membrane-bound SCF, which is an established factor related to the proliferation and pluripotency of PGCs. These results suggest that both hematopoietic stem cells and PGCs make use of the common growth factors for their growth in culture. In addition, the inability of MEF to affect proliferation of PGCs may suggest that the initial culture conditions for the proliferation could be different between ES cells and PGCs in the mouse.

Retinoic Acid Is a Potent Growth Activator of Mouse Primordial Germ Cells in Vitro

Effects of retinoic acid (RA) on the growth of mouse primordial germ cells (PGC) were studied using an in vitro coculture system. Addition of RA to the culture medium markedly increased the number of PGC of the migratory phase and also significantly retarded the depletion of gonadal PGC. We observed stimulation of mitotic activity by RA treatment at all stages of PGC examined (8.5, 11.5, and 13.5 days postcoitum), even in the absence of feeder cells. From these results, we conclude that RA affects PGC directly to promote their survival and proliferation and that the RA-induced intracellular signal may have a crucial role in the development of PGC.

Immunoaffinity Purification of Migratory Mouse Primordial Germ Cells

Primordial germ cells (PGCs) are the earliest identifiable germ cells in the embryo. It has not previously been possible to purify PGCs from mammalian embryos at early stages of development (before they arrive in the gonads), when such cells are very low in number, are moving along different tissues toward the gonadal ridges, and are not identifiable by morphological criteria alone. We report here that the use of PGC-specific TG-1 antibody in combination with immunoaffinity adhesion to plastic plates coated with anti-mouse IgM secondary antibody allows the isolation of reasonably pure yields of migratory PGCs from 9.5- to 11.5-dpc mouse embryos.

The germline: familiar and newly uncovered properties.

Transplantations of both Drosophila pole cells and mouse primordial germ cells suggest that the pregonadal germline of these two organisms is not pluripotent. In mouse PGCs, however, the potential for activating pluripotency is clearly present, as seen in the relative case of deriving EG cells from migratory-stage PGCs. EG cells are derived from altering the in vitro growth conditions of PGCs by the addition of one factor. Conceivably, straying of PGCs in vivo could also lead to novel fates if they migrated into a suitable environment. Aberrant PGC migration underlies models for the origin of several tumor types. How are migratory-stage PGCs prevented from adopting alternative fates in wild-type development? One solution is to link accurate migration into the gonads with germ cell survival. One mechanism of accomplishing this is through the action of pleiotrophic factors such as Steel. Steel is both a proliferation and migration factor; sterility in Steel mutants is due to poor germ cell survival and improper migration. Networking of migratory PGCs is another mechanism to reduce the chances of individual germ cells straying from the migration path. Postmigratory germ cells apparently undergo another restriction on potency--novel imprinting. Assuming that EG methylation profiles accurately reflect modifications made in their founder PGCs, then "erased" pluripotent EG lines suggest that PGCs can be diverted from the germline lineage. Direct assays on PGCs show that the unmethylated phenotype at Igf2r region 2 is characteristic of late germ cells. The maintenance of germline-like methylation with a switch in PGC fate is somewhat analogous to the situation in lag mutants from Volvox, which retain large gonidial-sized cells that can nevertheless initiate somatic differentiation. Just as it is not possible to readjust cell size with a subsequent change in gonidial differentiation, it may be difficult, without gametogenesis, to reimpose methylation modifications once they have been erased. In addition, the methylation status of some sites/loci may be inconsequential to developmental gene activity in germ cells or EG cells. Recent studies indicate that the methylation imprint at Igf2r is not interpreted at functional level until embryogenesis (51). In summary, we have reviewed some distinctive properties of the development of germ cells, from their early segregation, mitotic and meiotic profile, sex determination, to their migration and sequestration into gonads. Some constraints on development of the germline likely serve to maintain its integrity until maturation, when the gametic genomes must be capable of initiating embryonic development. To accomplish this ultimate goal, germ cells in some organisms may retain a bias towards pluripotency throughout development.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of leukemia inhibitory factor and its receptor in mouse primordial germ cell growth

The pleiotropic cytokine leukemia inhibitory factor (LIF) is able to promote the growth of mouse primordial germ cells (PGCs) in culture. It is unclear whether LIF acts directly on PGCs or indirectly via feeder cells or embryonic somatic cells. To understand the role of LIF in PGC growth, we have carried out molecular and cell culture analyses to investigate the role of both the LIF ligand and its receptor in PGC development. LIF is able to stimulate PGC growth independently of the presence of feeder cells supporting the hypothesis that LIF acts directly on PGCs to promote their growth. We show here that transcripts for the low-affinity LIF receptor (LIFR), an integral component of the functional LIF receptor complex, are expressed in the developing gonad. Fluorescence-activated cell sorter (FACS) analysis, using an anti-LIFR antiserum, demonstrates that LIFR is present on the surface of PGCs, suggesting that PGCs are likely to be a direct target of LIF action in culture. Signalling via LIFR is essential for PGC growth in culture since the anti-LIFR antiserum, which blocks LIF binding to its receptor, abolishes PGC survival in culture. Two LIF-related cytokines, namely oncostatin M and ciliary neurotrophic factor, can also promote PGC growth in culture in addition to LIF. Thus one or more of these LIFR-dependent cytokines may play an important role in PGC development in mice.

X-chromosome activity of the mouse primordial germ cells revealed by the expression of an X-linked lacZ transgene

We have determined the timing of the inactivation and reactivation of the X chromosome in the mouse primordial germ cells (PGCs) by monitoring the expression of an X-linked HMG-lacZ reporter gene. PGCs were identified by their distinct alkaline phosphatase activity and they were first localised in the primitive streak and allantoic bud of the 7.5-day gastrulating embryo. Although inactivation of the transgene was found in some PGCs at these sites, at least 85% of the population were still expressing the lacZ gene. This suggests that, although X-inactivation has commenced during gastrulation, the majority of PGCs still possess two active X chromosomes. Transgene activity remained unchanged during the relocation of PGCs to the hindgut endoderm, but decreased abruptly when PGCs left the hindgut and migrated through the mesentery. X-inactivation was completed during the migration of PGCs, but not simultaneously for the whole population. The first wave of PGCs entering the genital ridge at 9.5 days did not immediately re-activate the silent transgene until about 24 hours later. Re-activation of the transgene took place in over 80% of PGCs entering the genital ridge at 10.5-13.5 days p.c., preceding the entry into meiosis. About 90% of the meiotic germ cells in the 14.5-15.5 day fetal ovary expressed the transgene. Similar profiles of transgene activity were observed in PGCs of embryos that have inherited the lacZ transgene from different parents, showing unequivocally that X-inactivation in the germ cell lineage is not related to parental legacy.(ABSTRACT TRUNCATED AT 250 WORDS)