A study on cell lineage,especially the germ cell line,in embryos of the teleost fish,Barbus conchonius

Summary Lucifer Yellow-Dextran labelling of lower layer cells (LLC), sometimes together with upper layer cells (ULC), of the 64-cellBarbus conchonius embryo resulted in labelled primordial germ cells (PGCs) at 12 h after fertilization (a.f.) in about 25% of cases. The presence of labelled PGCs was independent of the location of the injected blastomere with respect to the later orientation of the embryonic axis. After injection of an ULC alone, however, labelled PGCs were never found. Also, the distribution of labelled somatic cells differed between the ULC- and LLC-injected embryos. When we found fluorescent PGCs, only a few of them were labelled, suggesting that either a single predecessor exists earlier than the 64-cell stage or that the formation of germ cells is a polyclonal process. Tracing the fluorescent cells at successive stages of development shows an extensive mixing with unlabelled cells during the epiboly stage, which might well be the cause of partly unpredictable cell lineages. The chance of being committed to a specific fate is different for the ULC and LLC descendants. This might be due to relatively limited cell mixing between these two cell populations.     

Origin of Germ Cells and Early Differentiation of Gonads in the Starfish, Asterina pectinifera

The origin of the germ cells and the development of the genital system in the annually spawning starfish, Asterina pectinifera , were studied by light and electron microscopy. Characteristic germ cells were first characterized in gonads after spawning: the gonia are larger than somatic cells, have large nuclei (with electron-lucent nucleoplasm), and show mitochondrial aggregation associated with nuage (electron-dense bodies). In young starfish without gonads similar cells were detected in the haemal sinus, where they were termed primordial germ cells (PGCs). Brachiolariae and metamorphosed juveniles had a cellular cluster in the coelomic epithelium, near the hydroporic canal. The cluster was comprised of cells endowed with the above-mentioned characteristics of the germ cells. The germ cell counts indicated that PGCs migrate from the aboral haemal sinus near the hydroporic canal, through the haemal sinus to the gonads, where they settle, proliferate, and differentiate into gonia.     

Segregation of primordial germ cells: Their numbers and fate during early development of Barbus conchonius (Cyprinidae,Teleostei) as indicated by 3H-thymidine incorporation

³H-Thymidine incorporation experiments in Barbus conchonius showed that presumptive primordial germ cells (PGCs) terminated their mitotic activity between midepibolys, and late epiboly. At the ten-somite stage, shortly after labeling of PGCs by uptake of ³H-thymidine became arrested, they could be recognized by their relatively large size and large nucleus. They were located in two longitudinal rows of cells between mesoderm and periblast, always at the same distance to the left and right of the notochord. Contact with the endoderm was not observed before the 16- to 23-somite stage. The numbers of PGCs were small (mean number, 18–19) and remained small for nearly 3 weeks. Mitotic activity was not observed in PGCs during that period; thereafter, rapid proliferation began. There is no evidence for active migration of PGCs; it is assumed that they are merely translocated passively together with their surrounding tissues. No specific constituents were detected with histochemical methods for glycogen, alkaline phosphatase, and RNA. Electron microscopy revealed the presence of “nuage” around the nucleus of PGCs. This material corresponded with perinuclear dense bodies as seen with light microscopy from the 19-somite stage onward. It is concluded that presumptive PGCs segregate from the somatic cells between midepiboly and late epiboly, before the three germ layers have been formed, and that locations of PGCs in the endodermal or mesodermal layer may be merely transitory stages during their translocation toward the gonadal primordia.     

Differentiation of primordial germ cells during embryogenesis of Allacma fusca (L.) (Collembola: Symphypleona)

The youngest primordial germ cells (PGCs) of Allacma fusca (L.) (Collembola: Sminthuridae) can be identified in embryos at the blastoderm stage as scattered in the yolk mass. They are arranged in pairs connected via intercellular bridges and dispersed among the yolk granules over a relatively small area but they never form multicellular clusters. With progressing development, the mesoderm of the germ band differentiates, the PGCs migrate to the abdominal part of the germ band and enter among mesoderm cells making two clusters of cells in the left and right parts of the abdomen. The mesoderm cells neighbouring the PGC cluster differentiate into a one-layered gonad envelope and produce a thin basal lamina separating the gonad from the rest of the mesoderm. The PGCs are still connected in pairs. At the end of the embryonic development, the gonads have regular spherical shapes and are enclosed within the envelope built up by a layer of flat somatic cells. Now, the PGCs do not occur only in pairs, but chains of cells connected with a sequence of intercellular bridges can also be seen.     

Formation of a large Vasa-positive germ granule and its inheritance by germ cells in the enigmatic Chaetognaths

Chaetognaths (arrow worms) are abundant hermaphrodite marine organisms whose phylogenetic position amongst protostomes and deuterostomes is still debated. Ancient histological observations dating from a century ago described the presence in eggs of a large granule, presumed to be a germ plasm, and its probable inheritance in four primary germ cells (PGCs). Using videomicroscopy, electron microscopy and immunocytochemistry (labelling with anti-Vasa antibodies) we have followed the cycle of aggregation and dispersion of germ plasm and nuage material in eggs, embryos, PGCs and oocytes in several species of benthic (Spadella) and planctonic (Sagitta) chaetognaths. In these animals, germ cells and gametes can be observed in vivo throughout the 1-2 month life cycle. After describing internal fertilization in live animals we show that the single large (15 microm diameter) germ granule forms by a spiralling aggregation movement of small germ islands situated in the vegetal cortex at the time of first mitosis. We also demonstrate that the granule forms autonomously in unfertilized activated eggs or fertilized egg fragments. Once formed, the germ granule first associates with the cleavage furrow and is segregated into one of the first two blastomeres. The germ granule is then translocated from the cortex to the mitotic spindle during 3(rd) cleavage and remains in the single most-vegetal blastomere until the 32-cell stage. At the 64-cell stage the germ granule is partitioned as nuage material into two founder PGCs and further partitioned into four PGCs situated at the tip of the archenteron during gastrulation. These four PGCs migrate without dividing to reach the transverse septum, then proliferate and differentiate into oocytes and spermatocytes of two ovaries and two testes. We noted that germ plasm and nuage material were associated with mitochondria, the nucleus, the spindle and the centrosome during some stages of development and differentiation of the germ line. Finally, we demonstrate that a Vasa-like protein is present in the germ granule, in PGCs and in the electron-dense material associated with the germinal vesicle of oocytes. These features stress the conservation of cellular and molecular mechanisms involved in germ cell determination.     

A light- and electron-microscopic study on the migration of primordial germ cells in the teleost,Oryzias latipes

Summary The primordial germ cells (PGCs) of Oryzias latipes in migration to the gonadal anlage have been investigated by light and electron microscopy. The ultrastructure of the PGCs, which occur in the subendodermal space on the syncytial periblast, differ conspicuously from that of the surrounding endodermal cells. After the PGCs move to the cavity between lateral plate and ectoderm, they are taken into the somatomesodermal layer and transferred to the dorsal mesentery where they form gonadal anlage with mesodermal cells. During their translocation to the dorsal mesentery through the somatic mesoderm, apparently without formation of pseudopods, the PGCs are completely surrounded by mesodermal cells. Since these conditions seem unfavorable to the active translocation of the PGCs to the dorsal mesentery, it is more likely that the PGCs are transferred passively by the morphogenic activity of the lateral-plate mesoderm.Counts of the number of the PGCs revealed that they are mitotically dormant during the migratory period. After the completion of the migration, they regain their proliferative activity. The PGCs in the female proliferate more actively than those in the male, which provides the first morphological indication of sex differentiation in this species of fish.     

LOCATION AND ULTRASTRUCTURE OF PRIMORDIAL GERM CELLS (PGCs) IN AMBYSTOMA MEXICANUM

The location and ultrastructure of the primordial germ cells (PGCs) were studied in Ambystoma mexicanum larvae of stages 23 to 47.
PGCs were found in the spaces between the endodermal cell mass and the lateral plate mesoderm at stages 23 to 35. Some of the PGCs at stage 35, and most of them at stages 40 and 42, were located near the Wolffian duct. At stages 46 and 47 all the PGCs were situated in the genital ridges. Cilia, which have hitherto never been reported in PGCs, were occasionally seen in PGCs of Ambystoma from stage 23 till stage 46.
No "germinal plasm" was found in the PGCs prior to stage 40. Specific structures or "nuage material", corresponding to the germinal granules or their derivatives in Xenopus , were first recognized in the vicinity of the nucleus at stage 40. Between stages 40 and 46, the amount of "nuage material" markedly increased. It was finally localized mainly in "intermitochondrial spaces". A possible transfer of material from the nucleus to the cytoplasm or vice versa through nuclear pores was first noticed at stage 40, the material concerned being quite similar in ultrastructure to the "nuage material".     

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.     

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.     

Isolation and identification of germ cells from fetal bovine ovaries

Gonadal cell suspensions were made from bovine fetuses of 35–55-, 56–80-, and 80–130-day age groups corresponding to the periods predominated by primordial germ cells (PGCs), oogonia, and meiotic cells, respectively. Germ cells identified on morphological criteria prior to their isolation from suspensions were compared histochemically and morphologically with cells in cryosections, impression smears, and semithin sections of similar gonads. Oocytes were distinguished by their chromosomal configurations in cell spreads. In suspensions from 35–55-day fetuses, cells considered to be PGCs stood out by their size, large nucleus, intracytoplasmic vesicles, and occasional blebbing. The somatic cells were smaller and contained little cytoplasm and few vesicles. In bovine gonads, in contrast to murine gonads, alkaline phosphatase (AP) activity was not specific enough to identify germ cells once they had entered the gonad. In ovaries from the 56–80-day age group, cells similar to PGCs, but slightly larger and with more cytoplasmic vesicles, were identified as oogonia. The cytoplasmic vesicles stained positively for lipid. In ovaries of 80–130-day fetuses, oogonia, oocytes, degenerating germ cells, and multinucleate germ cells were recognized. Degenerating germ cells exhibited a variety of morphological characteristics and were consistently positive for acid-phosphatase activity. Binucleate germ cells appeared around day 85 of gestation, while multinucleate germ cells were seen from day 95. It was concluded that bovine mitotic germ cells can be isolated from gonadal cell suspensions and that the best time to recover them is between 50 and 70 days of gestation. © 1994 Wiley-Liss, Inc.     

The formation of primordial germ cells from germline cells in spherical embryos derived from the blastodisc of 2-cell embryos in goldfish, Carassius auratus

The property of primordial germ cells (PGCs) in fragmented goldfish embryos was investigated. When 1- and 2- cell embryos were cut at several perpendicular levels at the animal-vegetal axis, cells expressing vas mRNA were observed in the resultant embryos derived from all kinds of animal fragments. Blastodisc fragments from the 1- to 2-cell stage developed to spherical embryos containing yolk body with a yolk syncytial layer (YSL). Germ ring and no tail expression were not observed in the spherical embryo. When the spherical embryo labeled with tracer dye or GFP-nos1 3'UTR mRNA was transplanted onto the animal part of the blastoderm in a host embryo at the blastula stage, PGCs of spherical embryo origin were detected around the gonadal ridges in the resultant embryos which developed normally. These results suggest that small animal fragments should contain factors sufficient for PGC differentiation and that PGCs differentiate without mesoderm induction, since mesoderm is not induced in a spherical embryo.     

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.     

Expression of axolotl DAZL RNA, a marker of germ plasm: widespread maternal RNA and onset of expression in germ cells approaching the gonad

How germ cell specification occurs remains a fundamental question in embryogenesis. The embryos of several model organisms contain germ cell determinants (germ plasm) that segregate to germ cell precursors. In other animals, including mice, germ cells form in response to regulative mechanisms during development. To investigate germ cell determination in urodeles, where germ plasm has never been conclusively identified, we cloned a DAZ-like sequence from axolotls, Axdazl. Axdazl is homologous to Xdazl, a component of Xenopus germ plasm found in the vegetal pole of oocytes and eggs. Axdazl RNA is not localized in axolotl oocytes, and, furthermore, these oocytes do not contain the mitochondrial cloud that localizes Xdazl and other germ plasm components in Xenopus. Maternal Axdazl RNA is inherited in the animal cap and equatorial region of early embryos. At gastrula, neurula, and tailbud stages, Axdazl RNA is widely distributed. Axdazl first shows cell-specific expression in primordial germ cells (PGCs) approaching the gonad at stage 40, when nuage (germ plasm) appears in PGCs. These results suggest that, in axolotls, germ plasm components are insufficient to specify germ cells.     

The structure of the germinal dense bodies (nuages) during differentiation of the male germ line of the teleost,Oryzias latipes

Summary The germinal dense body (GDB) in the teleost, Oryzias latipes, an organelle unique to the cells of germ line, is regarded as a counterpart of nuage material in amphibians and mammals. In the study described herein, GDBs in male germ line cells were examined by electron microscopy. GDBs existed continuously in the cytoplasm of primordial germ cells (PGCs), prespermatogonia, type-A spermatogonia and early type-B spermatogonia. But they became rudimentary in late type-B spermatogonia and early spermatocytes, and no longer occurred in spermatids. Differences in the morphology of GDBs of PGCs and male germ cells were also noted. In PGCs of indifferent gonads, about 50% of GDBs were amorphous bodies of fine electron-dense fibrils, whereas in spermatogonia amorphous bodies decreased in number and GDBs of strand-like structure were more frequent. The change in the morphology of GDBs began when the sex differentiation of gonads became evident, and proceeded gradually in prespermatogonia. No obvious differences in morphology of GDBs were noted between prespermatogonia in the fry at later stages of development and spermatogonia in adult fish.     

Origin of the gonad in the juvenile of a solitary ascidian, Ciona intestinalis

In the just-metamorphosed juveniles of Ciona intestinalis, a round mass of tissue debris derived from the resorbed tadpole tail is situated in the broad space enclosed by the peritoneal membrane and the epidermis around the ventral side of the esophagus. In living juveni es, the origin of the gonad rudiment was traced back to the mass of tissue debris. Electron microscopically, the round mass was a clump of irregular-shaped phagocytotic cells engulfing degenerated cell fragments. On the surface of the cell clump, a small number of singly occurring round cells were found and identified as primordial germ cells on the basis of morphological continuity to obvious germ cells in later stages. Presence of nuage around the nucleus characterized the germ cells. In a few days the germ cells assembled to form a solid slender body (gonad rudiment) together with smaller somatic cells. The gonad rudiment left the space around the esophagus, moving into the narrow mesenteric space connecting the stomach and intestine on the fourth day after metamorphosis. It gradually increased in size by proliferation of the germ cells and somatic cells. The solid gonad rudiment changed into an oval vesicle with an eccentrically located cavity on about the seventh day after metamorphosis. The vesicle comprised a thinner wall made of a simple epithelium without germ cells and a thicker wall containing germ cells and somatic cells.     

Fine structural observations on the origin and associations of primordial germ cells of the mouse.

This study explores the origin of primordial germ cells (PGCs) of the mouse and examines their morphology and associations with other cells during early development. PGCs have been selectively stained by the alkaline phosphatase histochemical reaction and viewed by light and electron microscopy from the time they are first detectable in the yolk sac endoderm until they enter the gonadal ridges. There are conflicting reports as to whether the PGCs originate from endodermal cells or whether they originate elsewhere and subsequently enter the endoderm. The observations in the present study favor the premise that PGCs of the mouse do not originate in the endoderm. Furthermore, it was observed that PGCs undergo specific changes in morphology during the developmental period studied and this was interpreted to mean that, although PGCs are set aside early in development as a distinct cell line, they also continue to become more specialized within time. The germ cell line is rather unusual in that it does not exist as a discrete tissue but, instead, resides within various other tissues during its life history. This apparent dependence upon somatic cells is maintained even in adult animals and may be important in serving to maintain or modify the environment of the germ cells.     

Analysis of the molecular dynamics of medaka nuage proteins by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching

The nuage is a unique organelle in animal germ cells that is known as an electron-dense amorphous structure in the perinuclear region. Although the nuage is essential for primordial germ cell (PGC) determination and development, its roles and functions are poorly understood. Herein, we report an analysis of the diffusion properties of the olvas gene product of the medaka fish (Oryzias lapites) in PGCs prepared from embryos, using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Olvas-green fluorescent protein (GFP) localized in granules thought to be nuages, and exhibited a constraint movement with two-component diffusion constants of 0.15 and 0.01 microm(2).s(-1). On the other hand, cytosolic Olvas-GFP was also observed to have a diffusion movement of 7.0 microm(2).s(-1). Interestingly, Olvas-GFP could be expressed in HeLa cells, and formed granules that were similar to nuages in medaka PGCs. Olvas-GFP also exhibited a constraint movement in the granules and diffused in the cytosol of HeLa cells, just as in the medaka embryo. The other two gene products, Nanos and Tudor of the medaka, which are known as constituents of the nuage, could also be expressed in HeLa cells and formed granules that colocalized with Olvas-GFP. Nanos-GFP and Tudor-GFP exhibited constraint movement in the granules and diffused in the cytosol of HeLa cells. These results suggest that these granules in the HeLa cell are not simple aggregations or rigid complexes, but dynamic structures consisting of several proteins that shuttle back and forth between the cytosol and the granules.     

Growth factors sustain primordial germ cell survival, proliferation and entering into meiosis in the absence of somatic cells

It is known that mammalian primordial germ cells (PGCs), the precursors of oocytes and prospermatogonia, depend for survival and proliferation on specific growth factors and other undetermined compounds. Adhesion to neighboring somatic cells is also believed to be crucial for preventing PGC apoptosis occurring when they lose appropriate cell to cell contacts. This explains the current impossibility to maintain isolated mouse PGCs in culture for periods longer than a few hours in the absence of suitable cell feeder layers producing soluble factors and expressing surface molecules necessary for preventing PGTC apoptosis and stimulating their proliferation. In the present paper, we identified a cocktail of soluble growth factors, namely KL, LIF, BMP-4, SDF-1, bFGF and compounds (N-acetyl-L-cysteine, forskolin, retinoic acid) able to sustain the survival and self-renewal of mouse PGCs in the absence of somatic cell support. We show that under culture conditions allowing PGC adhesion to an acellular substrate, such growth factors and compounds were able to prevent the occurrence of significant levels of apoptosis in PGCs for two days, stimulate their proliferation and, when LIF was omitted from the cocktail, allow most of them to enter into and progress through meiotic prophase I. These results consent for the first time to establish culture conditions for purified mammalian PGCs in the absence of somatic cell support and should make easier the molecular dissection of the processes governing the development of such cells crucial for early gametogenesis.     

Dye-coupling and the formation and fate of the hypoblast in the teleost fish embryo, Barbus conchonius.

The present report describes Lucifer Yellow (LY) transfer between the syncytial layer of the yolk cell (YSL) and blastodermal cells during epiboly in the teleost fish Barbus conchonius. The fate of a group of labeled cells is described until germ layer formation. At the onset of epiboly, LY seems to be transferred from the YSL to all blastodermal cells. Between 10% and 40% epiboly, dye-coupling appears to be restricted to the marginal region. Within 60 min individually labeled cells are distributed among unlabeled cells within the blastoderm. Between 40% and 60% epiboly, we observed a ring-shaped group of labeled cells, which probably have involuted during early gastrulation. Consequently, this cell group may correlate with the leading edge of the hypoblast layer within the germ ring. At 60% epiboly and later, the blastodermal cells are dye-uncoupled from the YSL. A gradual translocation of the ring-shaped hypoblast towards a dorsally located bar-like structure is observed between 50% and 100% epiboly. At 100% epiboly, fluorescent cells were located in contact with the YSL within the embryo proper, with the brightest fluorescence in the future head region. The translocation is due to dorsalwards convergent cell movements during the gastrulation process. The appearance of the hypoblast as a dye-coupled cell layer may correlate with some restriction in cell fate since the hypoblast differs in fate from the epiblast.