Production of donor-derived offspring by transfer of primordial germ cells in Japanese quail.

We transfused concentrated primordial germ cells (PGCs) of the black strain (D: homozygous for the autosomal incomplete dominant gene, D) of quail into the embryos of the wild-type plumage strain (WP: d+/d+) of quail. The recipient quail were raised until sexual maturity and a progeny test of the putative germline chimeras was performed to examine the donor gamete-derived offspring (D/d+). Thirty-one percent (36/115) of the transfused quail hatched and 21 (13 females and 8 males) of them reached maturity. Five females and 2 males were germline chimeras producing donor gamete-derived offspring. Transmission rates of the donor derived gametes in the chimeric females and males were 1.8-8.3% and 2.6-63.0%, respectively. Germline chimeric and the other putative chimeric males were also test-mated with females from the sex-linked imperfect albino strain (AL: d+/d+, al/W, where al indicates the sex-linked imperfect albino gene on the Z chromosome, and W indicates the W chromosome) for autosexing of W-bearing spermatozoa: No albino offspring were born.     

Histochemical identification and behavior of quail primordial germ cells injected into chick embryos by the intravascular route.

The behavior of quail primordial germ cells (PGC) after injection into chick embryos by the intravascular route was examined. The quail (donor) PGC, taken from the bloodstream of quail embryos (recipient) at stage 13-14, were injected into the vitelline vessels of chick embryos (recipient) at stage 15. In the recipient embryos, the PGC of the quail and the chick were histochemically distinguished by a double-staining technique involving a lectin, from Wistaria floribunda (WFA) and the PAS reaction. One day after injection, quail PGC appeared in the prospective gonadal region of recipient chick embryos, being localized among the recipient chick PGC. This result indicates that a staining technique specific for WFA lectin is useful for identification of quail PGC and that quail PGC can be transferred by a vascular route for the production of germline chimeras.     

Immunomagnetic purification of viable primordial germ cells of Japanese quail (Coturnix japonica).

Immunomagnetic cell sorting (MACS) with the monoclonal antibody (mAb) QCR1 was compared with the Ficoll density-gradient centrifugation system (FICS) in terms of the efficiency of enrichment of quail (Coturnix japonica) primordial germ cells (PGCs) from blood. The purified PGCs were tested for their ability to settle in the chick (Gallus domesticus) embryonic gonad. Blood containing 60-100 PGCs microliter-1 was taken from the dorsal aorta of quail embryos at Hamburger and Hamilton's stages 14-16. The amount and concentration of PGCs in the PGC-rich fraction purified by MACS were greater than in the fraction purified by FICS. Purified quail PGCs were transfused into chick embryos at stages 14-16 and immunohistochemically stained with mAb QCRI on day 8 of chick development. Transfused PGCs purified by either MACS or FICS were positively stained in the chick embryonic gonads.     

Production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos

The possibility of producing quail germline chimeras by the transfer of gonadal primordial germ cells (gPGCs) into recipient embryos was investigated. Japanese quail of the black (D: homozygous for the autosomal incomplete dominant gene D) and wild-type plumage (WP: d+/d+) strains were used as donors and recipients, respectively. Gonadal cells were retrieved from the gonads of 5-day-old D embryos, and gPGCs were enriched by magnetism-activated cell sorting. Fresh (noncultured) gPGCs or those isolated after culture for 3 days with gonadal stromal cells present in the mixed cell population were introduced into the dorsal aorta of 2-day-old recipient WP embryos. Hatchability of the recipient embryos was 23.7% (31/131) and 34.4% (31/90) for those transfused with cultured or noncultured gPGCs, respectively. Of the hatched quail, 28 acquired sexual maturity; among these animals, 7.1% (1/14) and 21.4% (3/14) of those that received cultured or noncultured gPGCs, respectively, were proved to be germline chimeras. The percentage of germline transmission to the donor-derived gametes in the chimeras that received cultured and noncultured gPGCs were 1.9 and 2.2-4.7%, respectively. In conclusion, quail gPGCs retrieved from 5-day-old embryos were thus transmitted in the germline after their transfer to quail embryos of a different strain. This property of the gPGCs was not adversely affected by culture for up to 3 days.     

Selective decrease of chick embryonic primordial germ cells in vivo and in vitro by soft X-ray irradiation

The feasibility of soft (low-energy) X-ray irradiation as a means of depleting the endogenous primordial germ cell(s) (PGC) of chicken embryos, to improve the efficiency of germ cell-mediated transgenesis, was investigated. Eggs were subjected to a non-irradiated control treatment and embryos were exposed for 40s to soft X-ray at 15, 16.5, or 18 kV ( approximately 1.5, 1.65, and 1.8 Gy, respectively). Exposure of stage X embryos to each dose of X-ray resulted in a reduction of approximately 50% in the number of PGC apparent at stage 28, whereas the total number of gonadal cells was unaffected. Irradiation (16.5 kV) of embryos at stage 9 or 14 also resulted in similar decreases in the number of PGC with no effect on the total number of gonadal cells. Irradiation did not affect embryo hatchability, compared with the non-irradiated control treatment, although the hatch rate increased with the age of embryos at the time of irradiation. Exposure of gonadal cells isolated from stage 28 embryos to X-ray (16.5 kV, approximately 0.8 Gy) prevented the increase in PGC number during subsequent culture for 10 days; the increase in the total number of gonadal cells was not affected. In conclusion, exposure of chicken embryos to a low dose of soft X-rays is effective for depleting the endogenous PGC population without affecting embryo hatchability or somatic cell viability.     

Ultraviolet irradiation of Rana pipiens embryos delays the migration of primordial germ cells into the genital ridges

Ultraviolet (UV) irradiation of the vegetal pole of anuran embryos at the two-cell stage has been reported to cause aberrant cleavage as well as a subsequent reduction in germ cell numbers. In this study, we find no correlation between UV-induced cleavage abnormalities and the absence of primordial germ cells in Rana pipiens tadpoles examined at stage 25. On the other hand, some tadpoles from a population which was lacking primordial germ cells at stage 25 subsequently contained germ cells. These late-appearing germs cells exhibited damaged mitochondria, autophagosomes, and secondary lysosomes, while surrounding somatic cells were morphologically normal. We suggest that these cytoplasmic abnormalities resulted from an effect of the initial UV irradiation of germ plasm. We conclude that one effect of UV irradiation of germ plasm is to delay or inhibit the migration of primordial germ cells into the genital ridges.     

Simple method for isolation of primordial germ cells from chick embryos.

A simple one step centrifugation method was developed for purification of primordial germ cells (PGCs) of chick embryos. PGCs, constituting less than 0.1% of the total blood cells of stage 13-14 embryos that contained a microliter amount of blood, were concentrated at the interface of a 6.3% (w/v) and 14.4% (w/v) Ficoll bilayer by centrifugation at 800 x g for 30 min. the purity of these PGCs was 86%, which was 22 times that obtained previously.     

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.     

Lectin histochemistry shows fucosylated glycoconjugates in the primordial germ cells of Xenopus embryos.

Previous works have shown that glycoconjugates with terminal fucose (Fuc) are located in the primordial germ cells (PGCs) of some mammals and might play a role in the migration and adhesion processes during development. The aim of this work was to identify the terminal Fuc moieties of Xenopus PGCs by means of three Fuc-binding lectins: from asparagus pea (LTA), gorse seed (UEA-I), and orange peel fungus (AAA). The histochemical procedures were also carried out after deglycosylation pretreatments: beta-elimination with NaOH to remove O-linked oligosaccharides; incubation with PNGase F to remove N-linked carbohydrate chains; and incubation with alpha(1,2)- and alpha(1,6)-fucosidase. The PGCs were always negative for LTA and UEA-I, two lectins that have the highest affinity for Fuc alpha(1,2)-linked. However, the PGCs were strongly labeled with AAA, which preferentially binds to Fuc with alpha(1,3) or alpha(1,4) linkages and to Fuc alpha(1,6)-linked to the proximal N-acetylglucosamine. There was fainter labeling with AAA when the sections were preincubated with alpha(1,6)-fucosidase, but the labeling remained strong when the sections were pretreated with alpha(1,2)fucosidase. When the beta-elimination procedure was carried out, the PGC labeling with AAA was slight. If the PNGase F incubation was performed, the PGCs remained moderately positive for AAA. These data suggest that the Xenopus PGCs have Fuc moieties in O- and N-linked oligosaccharides, including Fuc alpha(1,6) linked to the innermost GlcNAc, and that the Fuc was not in alpha(1,2)-linkage.     

On the fate of primordial germ cells injected into early mouse embryos

Primordial germ cells (PGCs) are the founder cells of the germline. Via gametogenesis and fertilisation this lineage generates a new embryo in the next generation. PGCs are also the cell of origin of multilineage teratocarcinomas. In vitro, mouse PGCs can give rise to embryonic germ (EG) cells – pluripotent stem cells that can contribute to primary chimaeras when introduced into pre-implantation embryos. Thus, PGCs can give rise to pluripotent cells in the course of the developmental cycle, during teratocarcinogenesis and by in vitro culture. However, there is no evidence that PGCs can differentiate directly into somatic cell types. Furthermore, it is generally assumed that PGCs do not contribute to chimaeras following injection into the early mouse embryo. However, these data have never been formally published. Here, we present the primary data from the original PGC-injection experiments performed 40 years ago, alongside results from more recent studies in three separate laboratories. These results have informed and influenced current models of the relationship between pluripotency and the germline cycle. Current technologies allow further experiments to confirm and expand upon these findings and allow definitive conclusions as to the developmental potency of 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.     

GalNAc moieties in O-linked oligosaccharides of the primordial germ cells of Xenopus embryos

Glycoconjugates could play a role in cell adhesion and migration mechanisms, including the locomotive movements of the primordial germ cells (PGCs) during the development of the embryo. In the present work, we have studied by lectin histochemistry the presence of N-acetylgalactosamine (GalNAc) in the glycans of the Xenopus PGCs, as a first approach to identifying their glycoconjugates which could be involved in the migration mechanism. The PGCs were negative for three of the GalNAc-binding lectins employed (from soybean, SBA; from lima bean, LBA; and from snail, HPA). However, when sialic acid (NeuAc) was previously removed by acid hydrolysis, SBA and HPA, but not LBA, labeled the PGCs, except if the staining was combined with the beta-elimination procedure. This suggests the presence of GalNAc alpha(1,3)-linked to galactose (Gal) in O-linked oligosaccharides, in a subterminal position to NeuAc. As the PGCs were always negative for LBA, the absence of fucose alpha(1,2)-linked to subterminal Gal is suggested. With the lectin from horse gram (DBA), the PGCs were stained, although beta-elimination turned the cells negative and acid hydrolysis increased the labeling, suggesting that GalNAc(alpha)(1,3)GalNAc was in O-linked glycans in terminal and subterminal to NeuAc position.     

Interleukin-2 induces the proliferation of mouse primordial germ cells in vitro

Primordial germ cells (PGCs) are the stem cell precursors of the germ line. Several growth factors contribute to enlarging the PGC population by acting as mitogens, survival factors or both. Interleukin-2 (IL-2) has a growth-promoting activity for T and B-lymphocytes, but its role in PGCs had not yet been studied. Here, we show that PGCs isolated from 10.5, 11.5 and 12.5 day postcoitum (dpc) mouse embryos constitutively express the three subunits (alpha, beta and gamma) of the IL-2 receptor (IL-2R). In contrast, IL-2 mRNA was not detected in these cells. However, the addition of recombinant IL-2 to the culture medium increased the number of PGCs in vitro via a mitogenic effect, as indicated by bromodeoxyuridine incorporation assays. Neutralization of the IL-2 receptor using anti-IL-2R subunit antibodies inhibited this IL-2-mediated proliferative effect on PGCs from 11.5 dpc embryos. Together, these data are indicative of a paracrine effect of IL-2 on PGC proliferation. In this regard, we also compared the effect of IL-2 with other compounds such as basic fibroblast growth factor (bFGF), steel factor, leukemia inhibitory factor and forskolin, and found that the degree of proliferation induced by IL-2 was similar to that induced by bFGF and forskolin. These observations support the notion that similar patterns of molecular signaling may underlie the developmental pathways of hematopoietic and germ stem cell precursors.     

The histochemical identification of primordial germ cells in diploid parthenogenetic mouse embryos

An attempt has been made to improve the early post-implantation development potential of diploid parthenogenetic mouse embryos by transferring parthenogenetic blastocysts to one uterine horn of a pseudopregnant recipient and a similar number of fertilized embryos to the contralateral horn. In control studies, diploid parthenogenetic embryos were transferred to both uterine horns of appropriate recipients. Unfortunately no obvious advantage appeared to be gained by carrying out the former manoeuvre. A significant improvement in the development potential of the parthenogenones could have indicated that their poor post-implantation survival might have been associated with a deficiency, possibly of hormonal origin, in the functioning of their decidual reaction. However, sufficient somite-containing parthenogenetic embryos were obtained in this study to allow a comparison to be made between them and fertilized embryos that were morphologically at a comparable stage of development. The parthenogenones were found to have a markedly smaller crown-rump length than their fertilized counterparts. A high proportion of both the parthenogenetic and fertilized embryos were subsequently fixed and appropriately stained in order to localize alkaline phosphatase activity. The analysis of this material clearly demonstrated that parthenogenetic mouse embryos are in fact capable of producing primordial germ cells. The latter were recognized by their morphology, histochemical staining appearance, and characteristic location, being found in the early 'turned' embryos within the dorsal mesentery in close proximity to the developing gut tube, and in the more advanced limb-bud stage embryos within the gonadal ridges.     

Galactosides and sialylgalactosides in O-linked oligosaccharides of the primordial germ cells in Xenopus embryos

The primordial germ cells (PGCs) are covered by surface glycoconjugates; some of them, like galactose residues recognized by peanut agglutinin (PNA), have been reported to be implicated in the PGC migration process. The aim of this work was the characterization of galactosides and sialylgalactosides in N- and O-linked oligosaccharides of Xenopus PGCs. Galactose(Gal)- and sialic acid(Neu5Ac)-binding lectin cytochemistry, in combination with chemical and enzymatic deglycosylation methods, were used. PGCs were slightly labeled with PNA, RCA-I and BSI-B₄, which suggests the presence of the sequences Gal(1,4)GlcNAc and Gal(1,3)Gal. Moreover, there was no labeling when -elimination pre-treatment was performed, suggesting that galactosides were in O-linked oligosaccharides. The strong staining with DSA was probably due to GlcNAc. Furthermore, sialylgalactosides with the sequence Neu5Ac(2,3)Gal(1,4)GlcNAc in O-linked oligosaccharides have been shown by means of MAA, PNA and RCA-I.     

Ontogenesis of primordial germ cells

In sexually reproducing animals all gametes of either sex arise from primordial germ cells (PGC). PGC represent a small cell population, appearing early during embryo development. They represent a key cell population responsible for the survival and the evolution of a species. Indeed, the production of gametes will assure fertilisation and therefore the establishment of the next generation. Until recently only few laboratories were working on PGC biology. A new interest emerged since these cells have the ability to function as pluripotent stem cells when established as cell lines. Indeed, like embryonic stem cells (ESC), embryonic germ cells (EGC) are able to differentiate in a wide variety of tissues. In vivo, EGC are able, after injection into a host blastocyst cavity to colonise the inner cell mass and to participate in embryonic development. In vitro studies in human and mouse have also shown their capacity to differentiate into a large variety of cell types allowing the study of processes involved in cardiomyocyte, haematopoietic, neuronal and myogenic differentiation pathways. We present here the last updates of PGC ontogeny focusing mainly on the murine model.     

Histochemical identification of primordial germ cells in diandric and digynic triploid mouse embryos

Diandric and digynic triploid mouse embryos were isolated in the morning on day 10 of gestation. The embryos were separated from their extraembryonic membranes, and the latter were analysed cytogenetically by G-banding to establish the ploidy and sex chromosome constitution of these embryos. The diandric triploid embryos were produced by the technique of nuclear micromanipulation. Females were mated with male mice with a morphologically distinguishable "marker" chromosome to confirm the diandric status of these embryos. Digynic triploid and normal diploid embryos were isolated from LT/Sv strain females. These females spontaneously ovulate both primary and secondary oocytes, which are fertilisable and give rise to digynic triploid and normal diploid embryos, respectively. All the embryos were serially sectioned and processed in order to demonstrate the presence of alkaline phosphatase enzyme activity. This histochemical technique allowed primordial germ cells to be readily recognised, due to their characteristic location, cellular morphology, and staining appearance. Primordial germ cells were found in all the embryos studied, being located within the visceral yolk sac, at the base of the allantois, and/or in association with the wall or mesentery of the hindgut. The total number of germ cells present was established in nine diandric triploids and in five digynic triploids. The findings presented here represent the first demonstration that primordial germ cells can differentiate in either diandric or digynic triploid mammalian embryos.