Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis

Primordial germ cells (PGCs) are derived from a population of pluripotent epiblast cells in mice. However, little is known about when and how PGCs acquire the capacity to differentiate into functional germ cells, while keeping the potential to derive pluripotent embryonic germ cells and teratocarcinomas. In this investigation, we show that epiblast cells and PGCs can establish colonies of spermatogenesis after transfer into postnatal seminiferous tubules of surrogate infertile mice. Furthermore, we obtained normal fertile offspring by microinsemination using spermatozoa or spermatids derived from PGCs harvested from fetuses as early as 8.5 days post coitum. Thus, fetal male germ cell development is remarkably flexible, and the maturation process, from epiblast cells through PGCs to postnatal spermatogonia, can occur in the postnatal testicular environment. Primordial germ cell transplantation techniques will also provide a novel tool to assess the developmental potential of PGCs, such as those manipulated in vitro or recovered from embryos harboring lethal mutations.     

Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture.

Steel factor (SF) and LIF (leukemia inhibitory factor) synergistically promote the proliferation and survival of mouse primordial germ cells (PGCs), but only for a limited time period in culture. We show here that addition of bFGF to cultures in the presence of membrane-associated SF and LIF enhances the growth of PGCs and allows their continued proliferation beyond the time when they normally stop dividing in vivo. They form colonies of densely packed, alkaline phosphatase-positive, SSEA-1-positive cells resembling undifferentiated embryonic stem (ES) cells in morphology. These cultures can be maintained on feeder layers for at least 20 passages, and under appropriate conditions give rise to embryoid bodies and to multiple differentiated cell phenotypes in monolayer culture and in tumors in nude mice. PGC-derived ES cells can also contribute to chimeras when injected into host blastocysts. The long-term culture of PGCs and their reprogramming to pluripotential ES cells has important implications for germ cell biology and the induction of teratocarcinomas.     

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.     

Requirement of Bmp8b for the generation of primordial germ cells in the mouse

In the mouse embryo, the generation of primordial germ cells (PGCs) from the epiblast requires a bone morphogenetic protein-4 (BMP4) signal from the adjacent extraembryonic ectoderm. In this study, we report that Bmp8b, a member of the Gbb-60A class of the BMP superfamily, is expressed in the extraembryonic ectoderm in pregastrula and gastrula stage mouse embryos and is required for PGC generation. A mutation in Bmp8b on a mixed genetic background results in the absence of PGCs in 43% null mutant embryos and severe reduction in PGC number in the remainder. The heterozygotes are unaffected. On a largely C57BL/6 background, Bmp8b null mutants completely lack PGCs, and Bmp8b heterozygotes have a reduced number of PGCs. In addition, Bmp8b homozygous null embryos on both genetic backgrounds have a short allantois, and this organ is missing in some more severe mutants. Since Bmp4 heterozygote embryos have reduced numbers of PGCs, we used a genetic approach to generate double-mutant embryos to study interactions of Bmp8b and Bmp4. Embryos that are double heterozygotes for the Bmp8b and Bmp4 mutations have similar defects in PGC number as Bmp4 heterozygotes, indicating that the effects of the two BMPs are not additive. These findings suggest that BMP4 and BMP8B function as heterodimers and homodimers in PGC specification in the mouse.     

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.     

Migratory and postmigratory mouse primordial germ cells behave differently in culture

In all vertebrate groups, the progenitors of the germ line, the primordial germ cells (PGCs) arise extragonadally and move to the developing gonad early in embryonic development. We have examined the behavior of isolated pregonadal and gonadal PGCs in vitro on feeder layers of an embryo-derived cell line. Histochemically and serologically identified pregonadal germ cells are found to be actively motile in vitro and, furthermore, show behavior characteristic of invasive cells. PGCs isolated from the developing gonad, however, show little locomotory activity and are not invasive on the same cellular substrate. These observations suggest that PGCs undergo a major change in phenotype at the time of their entry into the gonad anlagen.     

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。     

Serum-free culture of murine primordial germ cells and embryonic germ cells

Fetal calf serum (FCS) has usually been used for culture of embryonic stem (ES) cell as a component of the culture medium. However, FCS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. Recently, a chemically-defined serum replacement, Knockout Serum Replacement (KSR), was developed to maintain ES cells in an undifferentiated state. In this experiment, we examined the effects of KSR on the growth and differentiation of primordial germ cells (PGCs) and embryonic germ (EG) cells. PGCs were collected 8.5 days postcoitum (dpc) from B6D2F1 (C57BL/6JxDBA/2J) female mice mated with B6D2F1 males. Most of the PGCs that were cultured in FCS-supplemented medium (FCS medium) had alkaline phosphatase (AP) activity and acquired a fibroblast cell shape. In contrast, PGCs in KSR-supplemented medium (KSR medium) proliferated, maintaining round and stem cell-like morphology. In addition, EG cells were established more easily from PGCs cultured in KSR medium than from PGCs cultured in FCS medium. The percentage of undifferentiated colonies of EG cells was significantly higher in KSR medium than in FCS medium. The germ line chimera was also produced from EG cells established in KSR medium. These results suggest that KSR can be used for sustaining an undifferentiated state of PGCs and EG cells in vitro.     

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.     

Long-term culture and characterization of goat primordial germ cells

While the culture and identification of primordial germ cells (PGCs) in mice is established, only limited investigations on PGCs in livestock have been reported. This study was performed to characterize goat PGCs after culture and cryopreservation. Goat PGCs were isolated from Day 32 fetuses and cultured on a continuous cell line of murine embryonal fibroblasts (STO) as feeder-cells in the presence of leukemia inhibitory factor (LIF). The PGCs proliferated slowly and showed colony formation in early passages. Frozen-thawed PGCs continued to proliferate when stem cell factor (SCF) was added to the culture medium. However, differentiation into epithelial-like polygonal cells or neuronal cells was observed after 1 or 2 passages. The PGCs of 1 female and 1 male cell line were characterized by immunocytochemistry. The PGCs showed positive staining for anti stage-specific embryonic antigen-1 (SSEA-1) and FMA-1 (monoclonal antibody produced against a glycoprotein cell surface antigen of the embryonal carcinoma Nulli SCC1), whereas the reactivity to alkaline phosphatase (AP), an established marker for PGCs in mice, was inconsistent. After differentiation, PGCs lost their positive reaction to SSEA-1, EMA-1 and AP. In conclusion, SSEA-1 and EMA-1 can be used as reliable markers for identifying goat PGCs in addition to morphological criteria. The results indicate that goat PGCs can be kept in long-term culture without losing their morphological characteristics and their positive reaction to SSEA-1 and EMA-1, thus providing a promising source of donor-karyoplasts for nuclear transfer procedures.     

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.     

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.     

Derivation of embryonic germ cells from post migratory primordial germ cells, and methylation analysis of their imprinted genes by bisulfite genomic sequencing

The embryonic germ cell (EGCs) of mice is a kind of pluripotent stem cell that can be generated from pre- and post-migratory primordial germ cells (PGCs). Most previous studies on DNA methylation of EGCs were restricted to 12.5 days post coitum (dpc). This study was designed to establish and characterize murine EGC lines from migrated PGCs as late as 13.5 dpc and to estimate the degrees of methylation of their imprinted genes as well as of the non-imprinted locus, Oct4, using an accurate and quantitative method of measurement. We established five independent EGC lines from post migratory PGCs of 11.5-13.5 dpc from C57BL/6xDBA/2 F1 hybrid mouse fetuses. All the EGCs exhibited the typical features of pluripotent cells including hypomethylation of the Oct4 regulatory region. We examined the methylation status of three imprinted genes; Igf2, Igf2r and H19 in the five EGC lines using bisulfite genomic sequencing analysis. Igf2r was almost unmethylated in all the EGC lines irrespective of the their sex and stage of isolation; Igf2 and H19 were more methylated than Igf2r, especially in male EGCs. Moreover, EGCs derived at 13.5 dpc exhibited higher levels of DNA methylation than those from earlier stages. These results suggest that in vitro derived EGCs acquire different epigenotypes from their parental in vivo migratory PGCs, and that sex-specific de novo methylation occurs in the Igf2 and H19 genes of EGCs.     

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.     

Isolation of viable mouse primordial germ cells by antibody-directed flow sorting

The germ line represents a cell type of unique interest in mammals because it retains complete genotypic totipotency while undergoing significant phenotypic differentiation. Analysis of the mechanism that underlies the maintenance of this totipotency requires the ability to isolate and study all stages of the germ cell lineage. The primordial germ cells (PGC) are the earliest identifiable germ cells in the embryo. It has not previously been possible to isolate PGC in sufficient numbers and purity to facilitate biochemical and/or molecular analysis. We report here that the use of a monoclonal antibody in combination with flow cytometry does permit the isolation of reasonably large and pure yields of viable mouse PGC.     

Differentiation of murine premigratory primordial germ cells in culture.

In the mouse embryo, primordial germ cells first appear in the extraembryonic mesoderm and divide rapidly while migrating to the fetal gonad. Shortly after their arrival in the gonad, germ cells sexually differentiate as proliferation ceases. Previous studies have established that primordial germ cells proliferate and migrate in feeder layer culture. To explore cellular regulation of fetal germ cell development, we have used germ cell nuclear antigen 1 (GCNA1), a marker normally expressed only in postmigratory germ cells, to investigate the developmental potency of both pre- and postmigratory cells in this culture system. We found that explanted premigratory germ cells will initiate expression of this marker and are, therefore, capable of undertaking some aspects of gonocyte differentiation without intimate exposure to the fetal gonad. We have also tested whether postmigratory gonocytes are stable in culture. As detected by either alkaline phosphatase or GCNA1, we did not detect long-term survival of either prospermatogonia or oogonia under conditions that support the survival, proliferation, and differentiation of earlier premigratory cells. These observations are consistent with an autonomous cellular mechanism governing the initial stages of gonocyte differentiation, and suggest that differentiation towards gonocytes is accompanied by a change in requirements for cell survival.     

Impaired meiotic competence in putative primordial germ cells produced from mouse embryonic stem cells

There are still several unanswered questions and problems about the recently claimed possibility of producing functional germ cells in vitro from pluripotent embryonic stem cells (ESCs). In the present paper, we compared by single-cell analysis the capability of putative primordial germ cells (PGCs), produced in vitro from ESCs, and that of endogenous PGCs isolated from embryos, to enter and progress through meiotic prophase I. Using a protocol previously reported to be suitable to produce female germ cells from mouse ESC monolayers, we first identified putative PGCs by analysing the expression pattern of several markers such as SSEA1, APase, OCT4, NANOG, MVH and SCP3 of pre- and post migratory PGCs. Next, after isolation of such cells from culture, we tested their meiotic capability. The evaluation at 2-5 days of culture of the number of cells showing meiotic nuclear SCP3 staining in cytospreads showed that it remained nearly constant in the putative PGCs, whereas it increased markedly in endogenous PGCs. Moreover, we observed that in putative PGCs, the nuclear distribution or expression of SCP3 and other meiotic markers such as DMC1, gH2AX and SCP1 were always highly abnormal in comparison to that observed in endogenous cultured PGCs. We conclude that although the formation of cells showing characteristics of PGCs can occur efficiently from ESCs in vitro, these cells possess impaired capability to enter and progress through meiotic prophase I.