ULTRASTRUCTURE OF THE 'GERMINAL PLASM' IN XENOPUS EMBRYOS AFTER CLEAVAGE

The endodermal location of 'germinal plasm'-bearing cells (GPBCs) and the ultrastructure of the 'germinal plasm' were studied in Xenopus laevis embryos at gastrula, neurula, tailbud and younger tadpole stages. Primordial germ cells (PGCs) of feeding tadpoles were also observed ultrastructurally.
GPBCs were found in the inner endoderm and in the yolk plug region at the late gastrula stage, in the middle and in the dorsal part of the endoderm cell mass at the late neurula and late tailbud stages, respectively. At the younger tadpole stage they were observed in the uppermost dorsal part of the endoderm. Germinal granules were always present in GPBCs at all stages examined but were not found in PGCs of feeding tadpoles. Irregularly shaped-stringlike bodies (ISBs) which seemed to have changed from germinal granules were first noticed in GPBCs at the late neurula stage, and were still present in PGCs of tadpoles, while 'granular materials' were not seen in GPBCs until the feeding tadpole stages. These facts and ultrastructural similarities shared by these organelles lead us to conclude that the change of the germinal granule through ISB, to the 'granular material' takes place during the differentiation of GPBCs into PGCs.     

Ectopic germline cells in embryos of Xenopus laevis

Whether all descendants of germline founder cells inheriting the germ plasm can migrate correctly to the genital ridges and differentiate into primordial germ cells (PGCs) at tadpole stage has not been elucidated in Xenopus. We investigated precisely the location of descendant cells, presumptive primordial germ cells (pPGCs) and PGCs, in embryos at stages 23-48 by whole-mount in situ hybridization with the antisense probe for Xpat RNA specific to pPGCs and whole-mount immunostaining with the 2L-13 antibody specific to Xenopus Vasa protein in PGCs. Small numbers of pPGCs and PGCs, which were positively stained with the probe and the antibody, respectively, were observed in ectopic locations in a significant number of embryos at those stages. A few of the ectopic PGCs in tadpoles at stages 44-47 were positive in TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) staining. By contrast, pPGCs in the embryos until stage 40, irrespective of their location and PGCs in the genital ridges of the tadpoles at stages 43-48 were negative in TUNEL staining. Therefore, it is evident that a portion of the descendants of germline founder cells cannot migrate correctly to the genital ridges, and that a few ectopic PGCs are eliminated by apoptosis or necrosis at tadpole stages.     

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".     

Presumptive Primordial Germ Cells (pPGCs) and PGCs in Tadpoles from UV-irradiated embryos of Xenopus

In order to determine whether or not tadpoles that once lacked primordial germ cells (PGCs) in the genital ridges and dorsal mesentery as a result of ultraviolet (UV) irradiation subsequently contained germ cells at more advanced stages of larval development, the numbers of presumptive PGCs or PGCs were carefully examined in Xenopus tadpoles at Nieuwkoop and Faber's stage 35/36–52 that developed normally from UV-irradiated eggs.
No late-appearing germ cells were observed in almost all the UV-irradiated tadpoles examined at stages 49–52. This same population had completely lacked PGCs at about stage 46. Moreover, presumptive PGCs (pPGCs) or cells with granular cytoplasm that reacted with a monoclonal antibody specific for the germ plasm of cleaving Xenopus eggs stayed in the central part of the endoderm cell mass in the irradiated tadpoles at stage 35/36, when the majority of those cells were located in the dorsal part of the endoderm in unirradiated controls. Furthermore, in the irradiated embryos pPGCs were demonstrated to decrease in number with development and eventually to disappear in tadpoles at about stage 40. The results strongly suggest that UV irradiation under the conditions used here totally eliminated germline cells from the irradiated animals.     

A Possibility of an in Vitro Differentiation of Primordial Germ Cells (PGCs) from Blastomeres Containing "Germinal Plasm" of Early Cleavage Stage in Xenopus laevis

The blastomeres containing the "germinal plasm" were isolated from 32-cell stage Xenopus embryos and cultured in vitro for various periods of time till the control embryos developed to stage 28, 33/34, 40 and 45, respectively. The cells containing the plasm in the 'stage-28', '33/34' and '40' explants were similar in external shape, and in distribution in the spherical endodermal cell mass to the presumptive primordial germ cells (pPGCs) in normal embryos of the corresponding stages. In addition, the cells in explants as well as the pPGCs were separated by a large intercellular space from the surrounding endodermal cells. The change in proportion of the compact or the loosely structured germinal granules and the irregularly shaped-stringlike bodies (ISBs) occurred in the cells of the explants with the prolongation of the culture period. In the cells of the 'stage-45' explant as well as in the PGCs of normal stage-45 tadpoles the ISBs and "granular materials" replace those germinal granules. These facts lead to the conclusion that the change of the germinal granules through the ISBs, to the "granular materials", noticed in the normal course of differentiation of pPGCs into PGCs (see (1)), also takes place in the cells of the explants during the culture. Therefore, it is likely that the cells in the explants are genuine pPGCs or PGCs. This is the first demonstration of a possibility of the in vitro differentiation of PGCs from the blastomeres containing the "germinal plasm" of early cleavage stage.     

Developmental regulation of locomotive activity in Xenopus primordial germ cells

Primordial germ cells (PGCs) arise in the early embryo and migrate toward the future gonad through species‐specific pathways. They are assumed to change their migration properties dependent on their own genetic program and/or environmental cues, though information concerning the developmental change in PGC motility is limited. First, we re‐examined the distribution of PGCs in the endodermal region of Xenopus embryos at various stages by using an antibody against Xenopus Daz‐like protein, and found four stages of migration, namely clustering, dispersing, directionally migrating and re‐aggregating. Next, we isolated living PGCs at each stage and directly examined their morphology and locomotive activity in cell cultures. PGCs at the clustering stage were round in shape with small blebs and showed little motility. PGCs in both the dispersing and the directionally migrating stages alternated between the locomotive phase with an elongated morphology and the pausing phase with a rugged morphology. The locomotive activity of the elongated PGCs was accompanied by the persistent formation of a large bleb at the leading front. The duration of the locomotive phase was shortened gradually with the transition from the dispersing stage to the directionally migrating stage. At the re‐aggregating stage, PGCs became round in shape and showed no motility. Thus, we directly showed that the locomotive activity of PGCs changes dynamically depending upon the migrating stage. We also showed that the locomotion and blebbing of the PGCs required F‐actin, myosin II activity and RhoA/Rho‐associated protein kinase (ROCK) signaling.     

Changes in the morphology of the germinal dense bodies in primordial germ cells of the teleost,Oryzias latipes

Summary In many organisms, the germinal dense bodies (GDBs) are known to be organelles unique to the cells of germ-line. In the present study, GDBs in primordial germ cells (PGCs) of the teleost, Oryzias latipes, were examined by electron microscopy. An obvious change was noticed in the morphology of GDBs. In PGCs situated in the endoderm, GDBs consisted of a loosely woven strand-like structure, whereas, GDBs in PGCs in the gonadal anlage, which were amorphous bodies of various sizes and shapes, were composed of electron-dense fine fibrils. The changes in the morphology of GDBs proceeded gradually according to the progress of the stages in migration of the PGCs. GDBs of intermediate morphology were found. The change in the morphology of the GDBs began at the stage of movement of the PGCs from endoderm to mesoderm. It is suggested that the differentiation of PGCs proceeds during their migratory stages under the influence of surrounding somatic cells.     

OBSERVATIONS OF PRIMORDIAL GERM CELLS IN THE TURTLE EMBRYO (CARETTA CARETTA): LIGHT AND ELECTRON MICROSCOPIC STUDIES

Primordial germ cells (PGCs) in the turtle embryo ( Caretta caretta ) were observed with light and transmission electron microscopes. Identification of the PGCs for light microscopy was made by the periodic acid-Schiff (PAS) technique. PGCs were first found in the yolk-sac endoderm through the 5th to 6th day of development. PGCs freed from the endoderm then migrated to the root area of the dorsal mesentery and the coelomic angle between the 7th and the 11th day of development, and finally settled down in the gonadal anlage by the 14th day. Turtle PGCs were characterized by a large size (16 μm in diameter) and large nuclei with distinct nucleoli, and by the presence of large numbers of lipid droplets, yolk platelets and glycogen particles in the cytoplasm. Cell organelles were well-developed in PGCs at later stages. Amoeboid features of the PGCs were observed in the mesenchyme, indicating active locomotion. PGCs were usually surrounded or encircled by neighboring somatic cells. No intravascular PGCs were detected at any stage of development examined.     

Direct Evidence for the Presence of Germ Cell Determinant in Vegetal Pole Cytoplasm of Xenopus laevis and in a Subcellular Fraction of It

To test for the presence of germ cell determinant in Xenopus embryos, vegetal pole cytoplasm containing the "germ plasm", or a subcellular fraction of it, was microinjected into single somatic blastomeres isolated from 32-cell embryos. Injected or non-injected (control) blastomeres were cultured in ³H-thymidine until normal control embryos reached the neurula stage. The labeled explants were then implanted into unlabeled host neurulae, which were allowed to develop to the tadpole stage. Labeled PGCs of explant origin in the genital ridges of the experimental tadpoles were examined by autoradiography.
Isolated blastomeres were injected with vegetal pole cytoplasm of 32-cell embryos or with a 20,000 g pellet made from vegetal pole cytoplasm of 2-cell embryos. Labeled PGCs were found in 7.6% and 2.3% of the experimental tadpoles, respectively. No labeled PGCs were found in the control tadpoles, except for one tadpole in the first experiment. These results strongly suggest that the vegetal pole cytoplasm and its subcellular fractions act as germ cell determinant.     

Mass isolation of primordial germ cells from transgenic rainbow trout carrying the green fluorescent protein gene driven by the vasa gene promoter

Mass isolation of live primordial germ cells (PGCs) was demonstrated for the first time in ectothermal vertebrates. To establish a stem cell-mediated gene transfer system in fish, a stem cell line that retains the ability to develop into gametes is necessary. PGCs are well suited for use as the initial material for such a stem cell line. We established transgenic rainbow trout (Oncorhynchus mykiss) strains carrying the green fluorescent protein (GFP) gene driven by a rainbow trout vasa-like gene (RtVLG) promoter/enhancer. Because GFP expression was specific to the PGCs, PGCs were successfully visualized in all developmental stages examined. Isolated genital ridges containing GFP-labeled PGCs were enzymatically dissociated. To isolate PGCs from the complex pools of dissociated genital ridges, GFP-labeled cells were sorted by flow cytometry. The sorted GFP-positive cells were large and round with a large nucleus, typical characters of PGC morphology. The expression of RtVLG was detected only in the GFP-positive cell population, confirming that these cells were PGCs. This simple and efficient technique to purify a large number of viable PGCs opens the way for establishing a stem cell line, which can differentiate into the germline. The purified PGCs would also be a novel tool for cellular and molecular study of vertebrate germline stem cells.     

不同时期鸡胚原始生殖细胞分离的研究

采用Ficoll密度梯度离心,酶解离两种方法在鸡胚孵化的第14期、19期、28期,分离、培养鸡胚中的原始生殖细胞(PGCs)。探索PGCs分离、培养的适宜时期及方法,以期获得较多数量,较高活力的PGCs作介导生产转基因鸡。结果表明：1．提取、分离PGCs的最佳时期依次为19期、28期。2．两种分离方法均能分离到一定数量的PGCs细胞。但在19期和28期,酶解离法分离到的PGCs的相对数量较多,存活时间较长,是一种较适宜的分离方法。     

The unique responses of the primordial germ cells in the fish Oryzias latipes to gamma-rays

The effects of gamma-radiation on the development of the primordial germ cells (PGCs) of medaka embryos (Oryzias latipes) during the early stages of development were quantitatively examined and compared to the effects on the intestinal cells. The PGCs develop in three stages: an extra-gonadal proliferative stage (1-2.5 days after fertilization), a mitotically inactive stage after the termination of the migration into the gonad (2.5-4.5 days), and an extensive proliferative stage (between 4.5 days and hatching). A dose-rate effect was absent in the PGCs, regardless of their mitotic activity, when dose rates were 2.5 and 0.14 Gy/min. The radiation effect on the PGCs was not reduced by hypoxia and was not enhanced by heat treatment during the proliferating stages. Conversely, radiation resistance was induced in the PCGs during the mitotocally inactive stage by hypoxia and, unexpectedly, by heat treatment. From the present data, we conclude that the PGCs have a small repair ability, and we discuss the radiation resistance induced in the PGCs by hypoxia and heat treatment.     

The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.     

Repression of primordial germ cell differentiation parallels germ line stem cell maintenance

In Drosophila, primordial germ cells (PGCs) are set aside from somatic cells and subsequently migrate through the embryo and associate with somatic gonadal cells to form the embryonic gonad. During larval stages, PGCs proliferate in the female gonad, and a subset of PGCs are selected at late larval stages to become germ line stem cells (GSCs), the source of continuous egg production throughout adulthood. However, the degree of similarity between PGCs and the self-renewing GSCs is unclear. Here we show that many of the genes that are required for GSC maintenance in adults are also required to prevent precocious differentiation of PGCs within the larval ovary. We show that following overexpression of the GSC-differentiation gene bag of marbles (bam), PGCs differentiate to form cysts without becoming GSCs. Furthermore, PGCs that are mutant for nanos (nos), pumilio (pum) or for signaling components of the decapentaplegic (dpp) pathway also differentiate. The similarity in the genes necessary for GSC maintenance and the repression of PGC differentiation suggest that PGCs and GSCs may be functionally equivalent and that the larval gonad functions as a "PGC niche".     

鸟类原生殖细胞的研究 Ⅰ.鸡胚生殖新月区原生殖细胞超微结构的观察

作者观察了鸡胚生殖新月区的原生殖细胞(PGC)的超微结构。PGC为圆形或椭圆形,13—16μm,有丰富的伪足和微绒毛,尚可见到相邻PGC存在桥粒样结构。细胞核为圆形、椭圆形及分叶状,并呈多处凹陷。与同期胚的其它细胞相比,胞质内细胞器相当丰富且较成熟。观察到有大量微丝。上述PGC的形态,除了细胞桥粒样结构及微丝很少见到报道外,其它特征与鸟类PGC的超微记载相一致。 作者首次观察到PGC中有一种特殊颗粒(即电子致密小体),它自核内产生,进入核周池,并借核膜破裂的方式进入胞质。这种颗粒可能就是生殖颗粒,而由该颗粒在胞质中聚集所构成的特殊高电子致密区可能就是生殖质。从而从形态学上提供了鸟类具有生殖质的证据。     

Distribution and space-time relationship of proteoglycans in the extracellular matrix of the migratory pathway of primordial germ cells in mouse embryos.

In this paper we present an in situ ultrastructural cytochemical study on the distribution and spatial-temporal expression of proteoglycans (PGs) in the extracellular matrix of the migratory pathway of mouse primordial germ cells (PGCs) during the different phases of migration, by the use of the cationic dye ruthenium hexammine trichloride (RHT). Embryos of 9, 10, 11 and 12 days of development were used. The treatment with RHT revealed PGs as electron dense layers, granules, and filaments. Whereas granules prevailed in the extracellular spaces of the migratory route during the whole migratory process, the amount of filamentous structures increased during the migration phase of PGCs. At the end of the migratory process the surface of the PGCs lost its reaction by RHT. There were differences in the size of the granules of PGs at the initial migratory period (9-day-old embryos) as compared with the other days of gestation. There was a strong reaction for PGs in the extracellular spaces, expressed as a meshwork of granules interconnected by filaments, as well as reaction on the basement membranes during the peak of the PGCs migration in 10-day-old embryos. These results support the hypothesis that these molecules may have an important role in the migration of PGCs, although the precise mechanism involved in this process is not yet clear.     

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.     

Ultrastructural Evidence that Chick Primordial Germ Cells Leave the Blood-Vascular System Prior to Migrating to the Gonadal Anlagen

It is known that chick primordial germ cells (PGCs), after separation from the endoderm in early embryonic development, temporarily circulate via the blood-vascular system and eventually migrate to the gonadal anlagen. However, direct evidence that circulating PGCs leave the blood vessels is lacking. The purpose of present study is to describe the ultrastructural features of PGCs as they emerge from the blood vessels. PGCs leaving the blood vessels were first examined with semi-thin sections stained with toluidine blue. Then, some of the sections were re-embedded in Epon 812, and sectioned for electron microscopy. PGCs were observed emerging from the capillaries in the region posterior to the omphalomesenteric arteries of the embryo, between the splanchnic mesoderm and open-gut endoderm, at stages 15–18 (about 2.5 days of incubation). Ultrastructurally, PGCs exhibited the protruding, bulge-like cytoplasmic processes through the endothelial gaps in the capillary walls. Prior to emerging, intravascular PGCs seemed to stick to the endothelium of the blood vessels. Thus, our results offer ultrastructural evidence that the circulating PGCs exit the blood vessels prior to migrating to the gonadal anlagen.     

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.