Spatio-temporal distribution of the protein of Xenopus vasa homologue (Xenopus vasa-like gene 1, XVLG1) in embryos

In order to know when the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) first appears in germ line cells and whether the protein is also present in somatic cells as is vasa protein in Drosophila , the spatio-temporal distribution of the protein in Xenopus embryos was carefully investigated by fluorescent microscopy. Part of the observation was performed by whole-mount immunocytochemistry and immunoblotting. A distinct fluorescence of XVLG1 protein was first recognized in a juxta-nuclear location of germ line cells or presumptive primordial germ cells (pPGC) at stage 12 (late gastrula) and remained associated with the pPGC or primordial germ cells (PGC) throughout the following stages until stage 46 (feeding tadpole). In contrast, weak fluorescence was seen in the animal hemisphere rather than in the vegetal hemisphere of cleaving embryos and in the perinuclear region of somatic cells at stages 10–42 (early gastrula to young tadpole), respectively. Nearly the same pattern as revealed by fluorescence was seen by whole-mount immunocytochemistry, except that a small amount of XVLG1 protein seemed to be present in the germ plasm and pPGC of embryos earlier than stage 12. The presence of the protein in the somatic cells and the PGC was also shown by immunoblotting.     

A trial for induction of supernumerary primordial germ cells in Xenopus tadpoles by injecting RNA of Xenopus vasa homologue into germline cells of 32-cell embryos

Whether overexpression of Xenopus vasa homologue or Xenopus vasa-like gene 1 (XVLG1) in germline cells of Xenopus embryos can induce supernumerary primordial germ cells (PGC) at tadpole stage was investigated. XVLG1 RNA (0.1-2.0 ng) and beta-gal RNA (0.5 ng) were injected into one of, usually, four germ plasm-bearing cells (GPBC) of 32-cell embryos, with the beta-gal RNA (2.0 ng) serving as both lineage tracer and control for XVLG1 RNA. The total number of PGC, including X-gal-stained and unstained PGC of injected and uninjected GPBC origins respectively, was examined in the experimental tadpoles developed from the injected embryos. The injected RNA, XVLG1 and beta-gal RNA, were translated, resulting in a large amount of corresponding proteins in presumptive PGC (pPGC) as well as in somatic cells derived from the injected GPBC. Nevertheless, the average number of total PGC per tadpole found in the experimental tadpoles from the XVLG1 RNA-injected embryos was not significantly different from that of beta-gal RNA-injected ones, irrespective of the injected dose of XVLG1 RNA. This indicates that the extra XVLG1 protein in pPGC is not sufficient to increase the number of PGC in the tadpoles.     

Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells

In order to understand the role of the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) in germ line cells, an attempt was made to perturb the function of the protein with the anti-vasa antibody 2L-13. The 2L-13 or the control antibody was microinjected with a lineage tracer (FITC-dextran-lysine, FDL) into single vegetal blastomeres containing the germ plasm of Xenopus 32-cell embryos, the descendants of which were destined to differentiate into a small number of primordial germ cells (PGC) and a large number of somatic cells, mostly of endodermal tissues at the tadpole stage. No significant effect of the injection of the antibodies on FDL-labeled, presumptive PGC (pPGC) was observed in embryos until stage 37/38. However, FDL-labeled PGC were not observed in almost all the 2L-13 antibody-injected tadpoles, although a similar number of labeled somatic cells were always present. As 2L-13 antibody specifically reacts with XVLG1 protein in the embryos by immunoblotting, the present results suggest that the antibody perturbed the function of XVLG1 protein in the pPGC, resulting in failure of PGC differentiation at the tadpole stage.     

The mode and molecular mechanisms of the migration of presumptive PGC in the endoderm cell mass of Xenopus embryos

We investigated the mode of migration of presumptive primordial germ cells (pPGC) in the endoderm cell mass of Xenopus embryos at stages 7-40. The molecules underlying the migration were also studied cytochemically and immunocytologically. By examining the relative positions of pPGC and somatic cells derived from the single, fluorescein-dextran lysine (FDL)-injected, germ plasm-bearing cells of stage 6 embryos, pPGC in embryos at stages 7-23 and those at stages later than 24 were assumed to passively and actively migrate in the endoderm cell mass, respectively. This assumption was supported by the observation that F-actin, essential for active cell migration, was recognized on pPGC of the latter stages, but never on those of the former ones. In addition, the molecule like CXC chemokine receptor 4 (CXCR4) found on directionally migrating PGC in mouse and zebrafish, probably Xenopus CXCR4 (xCXCR4), was detected on pPGC only at latter stages. Accordingly, F-actin and xCXCR4, and probably beta1-integrin and collagen type IV, which are indispensable for the formation of F-actin, are thought to be involved in the active migration of pPGC in the endoderm cell mass.     

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.     

Cause of the decreased number of PGC in albino Xenopus: analysis of the number and position of pPGC in albino embryos during and after cleavage

In order to understand the cause for the decreased number of primordial germ cells (PGC) in Xenopus albino (a(p)/a(p)) tadpoles, the number of presumptive PGC (pPGC) in the albino and wild-type embryos at Nieuwkoop and Faber's stages 6-37/38 were examined using the antibody specific to germ plasm. The positions of pPGC in the endodermal cell mass in embryos of both types at stages 28 and 33/34 were also observed to learn the migratory behavior of pPGC. The number of pPGC in the albino increased up to stage 28 with development, but decreased thereafter. In contrast, the number in the wild-type increased to stage 33/34 as development proceeded, and the number of pPGC in stage 33/34 embryos reached nearly that of PGC of the feeding tadpoles in the same batches. Judging from the positions of pPGC, the migration of pPGC from the median part through the lateral to the dorsal part of the endodermal cell mass in the albino was suspected to be somewhat later than that in the wild-type. These results, together with the results in previous studies, suggest that the decreased number of PGC in the albino would be closely related to the sudden decrease in number of pPGC at stage 33/34, as well as to the ectopic position of pPGC in endodermal cell mass, the latter of which had already been demonstrated to be responsible for the differentiation into PGC.     

Gonadal morphogenesis and sex differentiation in the viviparous fish Chapalichthys encaustus (Teleostei, Cyprinodontiformes, Goodeidae)

This study describes the structural and ultrastructural characteristics of gonadal sex differentiation and expression of Vasa, a germline marker, in different developmental stages of embryos and newborn fry of the barred splitfin Chapalichthys encaustus, a viviparous freshwater teleost endemic to Mexico. In stage 2 embryos, the gonadal crest was established; gonadal primordia were located on the coelomic epithelium, formed by scarce germ and somatic cells. At stage 3, the undifferentiated gonad appeared suspended from the mesentery of the developing swimbladder and contained a larger number of germ and somatic cells. At stages 4 and 5, the gonads had groups of meiotic and non-meiotic germ cells surrounded by somatic cells; meiosis was evident from the presence of synaptonemal complexes. These stages constituted a transition towards differentiation. At stage 6 and at birth, the gonad was morphologically differentiated into an ovary or a testis. Ovarian differentiation was revealed by the presence of follicles containing meiotic oocytes, and testicular differentiation by the development of testicular lobules containing spermatogonia in mitotic arrest, surrounded by Sertoli cells. Nuage, electron-dense material associated with mitochondria, was observed in germ cells at all gonadal stages. The Vasa protein was detected in all of the previously described stages within the germ-cell cytoplasm. This is the first report on morphological characteristics and expression of the Vasa gene during sexual differentiation in viviparous species of the Goodeidae family. Chapalichthys encaustus may serve as a model to study processes of sexual differentiation in viviparous fishes and teleosts.     

Expression of Dazl and Vasa in turtle embryos and ovaries: evidence for inductive specification of germ cells

SUMMARY In bilaterian animals, germ cells are specified by the inductive/regulative mode or the predetermined (germ plasm) mode. Among tetrapods, mammals and urodeles use the inductive mode, whereas birds and anurans use the predetermined mode. From histological data it has been predicted that some reptiles including turtles use the inductive mode. Examining turtle oocytes, we find that Dazl RNA, Vasa RNA, and Vasa protein are not localized, suggesting that germ plasm is not present. In turtle embryos at somite stages, primordial germ cells (PGCs) expressing Dazl lie on a path from the lateral posterior extraembryonic endoderm through the gut to the gonad as previously described. In gastrulating embryos, cells expressing Dazl are found in the blastoporal plate and subsequently below the blastoporal plate, indicating that PGCs are generated at the equivalent of the early posterior primitive streak of mammals. Vasa RNA is expressed in somatic cells of gastrula to early somite stages, and Vasa RNA and protein are expressed in PGCs of later embryos. Taken together the evidence strongly suggests that turtles, and other reptiles (lacertoid lizards) with the same location of PGCs in embryos, use the inductive mode of germ cell specification. Phylogenetic analysis of the available evidence supports the following hypotheses: (1) the inductive mode is basal among reptiles, indicating that this mode was maintained as basal tetrapods evolved to amniotes, (2) the predetermined mode arose twice within reptiles, and (3) the induced mode may be used in several lepidosaurs whose PGCs are located in an unusual pattern distributed around the embryo.     

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.     

Refinement of gene expression patterns in the early Xenopus embryo

During blastula and gastrula stages of Xenopus development, cells become progressively and asynchronously committed to a particular germ layer. We have analysed the expression of genes normally expressed in ectoderm, mesoderm or endoderm in individual cells from early and late gastrula embryos, by both in situ hybridization and single-cell RT-PCR. We show that at early gastrula stages, individual cells in the same region may express markers of two or more germ layers, and 'rogue' cells that express a marker outside its canonical domain are also observed at these stages. However, by the late gastrula stage, individual cells express markers that are more characteristic of their position in the embryo, and 'rogue' cells are seen less frequently. These observations exemplify at the gene expression level the observation that cells of the early gastrula are less committed to one germ layer than are cells of the late gastrula embryo. Ectodermal cells induced to form mesendoderm by the addition of Activin respond by activating expression of different mesodermal and endodermal markers in the same cell, recapitulating the response of marginal zone cells in the embryo.     

银鲫种系细胞标记分子Vasa: cDNA克隆及其抗体制备

种系细胞始自胚胎发育早期,是动物生殖及生殖工程的基础。为研究鱼类的种系细胞提供标记分子,我们克隆并鉴定了银鲫的vasacDNA即Cagvasa。CagvasacDNA全长2771碱基(nt),编码的蛋白为银鲫Vasa即CagVasa,全长701个氨基酸(aa)。CagVasa蛋白与已知Vasa蛋白的结构特征一致:在N端有14个RGG重复序列,在C端Vasa所特有的8个功能域俱全。银鲫Vasa与鲤鱼、斑马鱼、陆生脊椎动物和果蝇的Vasa蛋白分别有95%,89%,61%-66%和50%的同源性。卵巢切片的RNA原位杂交揭示,Cagvasa限于种系细胞,且表达水平呈现出低-高-低的动态变化:即两头低(卵原细胞跟Ⅳ期成熟卵子),中间高(Ⅱ-Ⅲ期卵子)。为分析鱼类种系细胞提供手段,我们用310aa的N端序列产生细菌的重组蛋白来免疫大白兔,获得了抗Vasa的多克隆抗体αVasa。Western免疫印迹表明,αVasa特异性地识别一个鱼类性腺的蛋白,该蛋白的分子量为75kD,仅见于银鲫的性腺和卵子。卵巢切片的组织免疫荧光共聚焦显微分析表明,抗体αVasa只对种系细胞染色:卵原细胞着色最深,卵母细胞和早期的卵子都浓染,成熟卵则浅染。类似情况亦见之于精子发生早期阶段的雄性种系细胞。卵巢和精巢的体细胞则不着色。因此,Cagvasa编码的当是Vasa同源蛋白,为银鲫种系细胞的第一个标记分子。我们的研究表明,抗体αVasa染色灵敏度高,特异性好,当是鉴别银鲫及其它鲤科鱼类的种系细胞的有效手段     

Essential role of brc-2 in chromosome integrity of germ cells in C. elegans

brc-2, an ortholog of BRCA2 in Caenorhabditis elegans, is essential in the maintenance of genetic integrity. In C. elegans, cellular location correlates with meiotic progression, and transgene-induced cosuppression is observed in the germ line but not in somatic cells. We used these unique features to dissect the role of brc-2 in the germ line from that in somatic cells. In situ hybridization of wild type animals revealed that brc-2 gene expression was higher in oocytes than in other germline cells, and was barely detectable in mitotic cells. In contrast, germ cells containing multicopies of the brc-2 transgene showed no significant in situ hybridization signal at any oogenesis stage, confirming that brc-2 expression was functionally cosuppressed in the transgenic germ line. RAD-51 foci formation in response to DNA damage was abrogated in brc-2-cosuppressed germ cells, whereas wild-type germ cells showed strong RAD-51 foci formation. These germ cells exhibited massive chromosome fragmentation and decompaction instead of six bivalent chromosomes in diakinesis. Accordingly, lethality was observed after the early stage of germline development. These results suggest that brc-2 plays essential roles in chromosome integrity in early prophase, and therefore is crucial in meiotic progression and embryonic survival.     

Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells

To obtain a reliable molecular probe to trace the origin of germ cell lineages in birds, we isolated a chicken homolog (Cvh) to vasa gene (vas), which plays an essential role in germline formation in Drosophila. We demonstrate the germline-specific expression of CVH protein throughout all stages of development. Immunohistochemical analyses using specific antibody raised against CVH protein indicated that CVH protein was localized in cytoplasm of germ cells ranging from presumptive primordial germ cells (PGCs) in uterine-stage embryos to spermatids and oocytes in adult gonads. During the early cleavages, CVH protein was restrictively localized in the basal portion of the cleavage furrow. About 30 CVH-expressing cells were scattered in the central zone of the area pellucida at stage X, later 45-60 cells were found in the hypoblast layer and subsequently 200-250 positive cells were found anteriorly in the germinal crescent due to morphogenetic movement. Furthermore, in the oocytes, CVH protein was predominantly localized in granulofibrillar structures surrounding the mitochondrial cloud and spectrin protein-enriched structure, indicating that the CVH-containing cytoplasmic structure is the precursory germ plasm in the chicken. These results strongly suggest that the chicken germline is determined by maternally inherited factors in the germ plasm.     

Growth of mouse oocytes to maturity from premeiotic germ cells in vitro

In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12-14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6-7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16-18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.     

DNA hypomethylation circuit of the mouse oocyte-specific histone H1foo gene in female germ cell lineage

The oocyte-specific subtype of the linker histone H1 is H1FOO, which constitutes a major part of oocyte chromatin. H1foo is expressed in growing oocytes, through fertilization, up until the two-cell embryo stage, when it is subsequently replaced by somatic H1 subtypes. To elucidate whether an epigenetic mechanism is involved in the limited expression of H1foo, we analyzed the dynamics of the DNA methylation status of the H1foo locus in germ and somatic cells. We identified a tissue-dependent and differentially methylated region (T-DMR) upstream of the H1foo gene, which was hypermethylated in sperm, somatic cells, and stem cell lines. This region was specifically unmethylated in the ovulated oocyte, where H1foo is expressed. 5-Aza-2'-deoxycytidine treatments and luciferase assays provided in vitro evidence that DNA methylation plays a role in repressing H1foo in nonexpressing cells. DNA methylation analyses of fetal germ cells revealed the T-DMR to be hypomethylated in female and male germ cells at Embryonic Day 9.5 (E9.5), whereas it was highly methylated in somatic cells at this stage. Intriguingly, the unmethylated status was continuously observed throughout oogenesis at E9.5, E12.5, E15.5, E18.5, in mature oocytes, and after fertilization, in E3.5 blastocysts. In comparison, male germ cells acquired methylation beyond E18.5. These data demonstrate a continuously unmethylated circuit at the H1foo locus in the female germline.