In vitro culture of mouse primordial germ cells

Germ cells were isolated from mouse fetal gonads 11 1/2-16 1/2 days post coitum (dpc), and exposed to various methods of in vitro culture. From 13 1/2 dpc onwards, both male and female germ cells survived well at 37 degrees C for several days. During the culture period the proportion of female germ cells in meiosis increased and later stages of meiotic prophase were seen. The gonadal environment is therefore not essential for the progress of meiosis. Male germ cells in vitro did not enter meiosis. Germ cells isolated from gonads 11 1/2 or 12 1/2 dpc did not survive at 37 degrees C in any of the three culture systems used (Petri dishes, microtest plate wells, drops under oil); cell density, substrate and culture medium were varied, and several additives tested, but no improvement in viability was detected. Below 30 degrees C, on the other hand, 11 1/2 and 12 1/2 day germ cells survived in vitro for at least a week. They did not enter meiosis in culture, but continued to undergo mitotic proliferation.     

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.     

Isolation of mouse primordial germ cells

Primordial germ cells (PGCs) were obtained from fetal mouse gonads of both sexes days post coitum (dpc), either by collagenase treatment, or by mechanical procedures with or without prior EDTA treatment. With mechanical procedures alone, yield was relatively low and many of the cells released were dead. After EDTA treatment, both yield and viability were significantly improved. Collagenase treatment gave the best yield of cells, since the entire gonad was disaggregated, but contamination with somatic cells was substantial, and the adhesive properties of the germ cells were altered by the treatment. When cells released following EDTA treatment were fractionated on a simple Percoll gradient, several thousand viable PGCs per gonad could be obtained in 2–3 h, with not more than 10–20% somatic cell contamination.     

小鼠原生殖细胞体外培养及其应用研究

原生殖细胞（ｐｒｉｍｏｒｄｉａｌｇｅｒｍｃｅｌｌ,ＰＧＣ）是胚胎生殖谱系最原始形式的细胞,在体胚胎迁移期ＰＧＣ增殖极为旺盛。体外培养的小鼠迁移期ＰＧＣ在饲养层细胞和三种生长因子（干细胞生长因子、碱性成纤维细胞生长因子及白血病抑制因子）的共同作用下,可发展为长期增殖并维持不分化状态的胚胎性干细胞,即胚胎生殖细胞（ｅｍｂｒｙｏｎｉｃｇｅｒｍｃｅｌｌ,ＥＧ）,具全能性发育潜能。ＥＧ建系成功对于研究生殖细胞发育以及寻找新的转基因动物操作的有效载体具有重要价值。     

Epigenetic reprogramming in mouse primordial germ cells

Genome-wide epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. We show here that mouse primordial germ cells (PGCs) exhibit dynamic changes in epigenetic modifications between days 10.5 and 12.5 post coitum (dpc). First, contrary to previous suggestions, we show that PGCs do indeed acquire genome-wide de novo methylation during early development and migration into the genital ridge. However, following their entry into the genital ridge, there is rapid erasure of DNA methylation of regions within imprinted and non-imprinted loci. For most genes, the erasure commences simultaneously in PGCs in both male and female embryos, which is completed within 1 day of development. Based on the kinetics of this process, we suggest that this is an active demethylation process initiated upon the entry of PGCs into the gonadal anlagen. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which new parental imprints are established subsequently. Some repetitive elements, however, show incomplete erasure, which may be essential for chromosome stability and for preventing activation of transposons to reduce the risk of germline mutations. Aberrant epigenetic reprogramming in the germ line would cause the inheritance of epimutations that may have consequences for human diseases as suggested by studies on mouse models.     

Myogenic potential of mouse primordial germ cells

Primordial germ cells are the only stem cells that retain true developmental totipotency after gastrulation, express markers typical of totipotent/pluripotent status and are able both in vivo and in vitro to give rise to pluripotent stem cells as EC and EG cells. We have therefore explored the possibility of the trans-differentiation of mouse PGCs to a myogenic lineage by transplanting them directly or after in vitro culture into a regenerating muscle and by culturing them on monolayers of differentianting muscle cells. The results obtained suggest that mouse PGCs may trans-differentiate into myogenic cells, provided that their somatic environment is preserved. This occurs at an estimated frequency of 0.01%, which is no higher than that reported for stem cells of adult tissues.     

Synthesis of glycoconjugates in mouse primordial germ cells

The synthesis of protein-bound carbohydrates has been studied in primordial germ cells (PGCs) and in somatic cells of 12.5 to 13.5-days-postcoitum (dpc) fetal mouse gonads. Both cell types were shown to synthesize asparagine-linked glycopeptides and glycosaminoglycans (GAGs). In addition, PGCs also synthesize lactosaminoglycans (LAGs) although in different proportions in female and male germ cells. Female PGCs, which at 13.5 dpc are entering meiosis, synthesize mainly LAGs, and minor amounts of hyaluronic acid (HA) and chondroitin sulfate (CS). Male germ cells, on the other hand, synthesize mainly CS. Furthermore, somatic cells of fetal gonads synthesize HA as the major class of GAGs. It is suggested that the activation of LAG synthesis in developing germ cells might be related to the beginning of meiosis. Moreover, we propose that HA synthesis might be developmentally regulated in somatic cells of the gonad, in order to regulate the establishment of specific interactions with germ cells.     

Developmental potential of mouse primordial germ cells

There are distinctive and characteristic genomic modifications in primordial germ cells that distinguish the germ cell lineage from somatic cells. These modifications include, genome-wide demethylation, erasure of allele-specific methylation associated with imprinted genes, and the re-activation of the X chromosome. The allele-specific differential methylation is involved in regulating the monoallelic expression, and thus the gene dosage, of imprinted genes, which underlies functional differences between parental genomes. However, when the imprints are erased in the germ line, the parental genomes acquire an equivalent epigenetic and functional state. Therefore, one of the reasons why primordial germ cells are unique is because this is the only time in mammals when the distinction between parental genomes ceases to exist. To test how the potentially imprint-free primordial germ cell nuclei affect embryonic development, we transplanted them into enucleated oocytes. Here we show that the reconstituted oocyte developed to day 9.5 of gestation, consistently as a small embryo and a characteristic abnormal placenta. The embryo proper also did not progress much further even when the inner cell mass was 'rescued' from the abnormal placenta by transfer into a tetraploid host blastocyst. We found that development of the experimental conceptus was affected, at least in part, by a lack of gametic imprints, as judged by DNA methylation and expression analysis of several imprinted genes. The evidence suggests that gametic imprints are essential for normal development, and that they can neither be initiated nor erased in mature oocytes; these properties are unique to the developing germ line.     

In vitro differentiation of germ cells from stem cells: a comparison between primordial germ cells and in vitro derived primordial germ cell-like cells

Stem cells are unique cell types capable to proliferate, some of them indefinitely, while maintaining the ability to differentiate into a few or any cell lineages. In 2003, a group headed by Hans R. Schöler reported that oocyte-like cells could be produced from mouse embryonic stem (ES) cells in vitro. After more than 10 years, where have these researches reached? Which are the major successes achieved and the problems still remaining to be solved? Although during the last years, many reviews have been published about these topics, in the present work, we will focus on an aspect that has been little considered so far, namely a strict comparison between the in vitro and in vivo developmental capabilities of the primordial germ cells (PGCs) isolated from the embryo and the PGC-like cells (PGC-LCs) produced in vitro from different types of stem cells in the mouse, the species in which most investigation has been carried out. Actually, the formation and differentiation of PGCs are crucial for both male and female gametogenesis, and the faithful production of PGCs in vitro represents the basis for obtaining functional germ cells.     

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.     

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.     

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,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.     

X chromosome activity in mouse XX primordial germ cells

In the early epiblast of female mice, one of the two X chromosomes is randomly inactivated by a Xist-dependent mechanism, involving the recruitment of Ezh2-Eed and the subsequent trimethylation of histone 3 on lysine 27 (H3K27me3). We demonstrate that this random inactivation process applies also to the primordial germ cell (PGC) precursors, located in the proximal region of the epiblast. PGC specification occurs at about embryonic day (E)7.5, in the extraembryonic mesoderm, after which the germ cells enter the endoderm of the invaginating hindgut. As they migrate towards the site of the future gonads, the XX PGCs gradually lose the H3K27me3 accumulation on the silent X chromosome. However, using a GFP transgene inserted into the X chromosome, we observed that the XX gonadal environment (independently of the gender) is important for the substantial reactivation of the inactive X chromosome between E11.5 and E13.5, but is not required for X-chromosome reactivation during the derivation of pluripotent embryonic germ cells. We describe in detail one of the key events during female PGC development, the epigenetic reprogramming of the X chromosome, and demonstrate the role of the XX somatic genital ridge in this process.     

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.     

Hematopoietic development of primordial germ cell-derived mouse embryonic germ cells in culture.

Induction of hematopoiesis in an embryonic germ (EG) cell line derived from mouse primordial germ cells (PGCs) was examined. When single undifferentiated EG-1 cells were inoculated directly into the methylcellulose medium, both primitive and definitive erythropoiesis were seen in embryoid bodies derived from the EG cells as observed in ES cells, and production of myeloid cell lineages was stimulated by IL-3. These results indicate that EG cells acquired in vitro potency to differentiate toward hematopoietic cells, although they were derived from PGC and are distinct from inner cell mass-derived ES cells with regard to gene expression and patterns of DNA methylation corresponding to genomic imprinting. It turns out that they are useful for study of cell differentiation in the animals whose ES cells are not available.     

Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells

Epigenetic modifications are crucial for the identity and stability of cells, and, when aberrant, can lead to disease. During mouse development, the genome-wide epigenetic states of pre-implantation embryos and primordial germ cells (PGCs) undergo extensive reprogramming. An improved understanding of the epigenetic reprogramming mechanisms that occur in these cells should provide important new information about the regulation of the epigenetic state of a cell and the mechanisms of induced pluripotency. Here, we discuss recent findings about the potential mechanisms of epigenetic reprogramming, particularly genome-wide DNA demethylation, in pre-implantation mouse embryos and PGCs.