Topology of the germ plasm and development of primordial germ cells in inverted amphibian eggs

Abstract. Inverted Xenopus eggs have reduced numbers of primordial germ cells (PGCs). The extent of the reduction varies from spawning to spawning. Histologic examination revealed that PGC counts were lowest in inverted eggs which displayed the greatest amount of shift in the vegetal mass of large yolk platelets, although the germ plasm itself always remained localized in the egg's original vegetal hemi-sphere. Even at blastulation the germ plasm continued to be localized in the egg's original vegetal hemisphere. In many cases, however, it was confined to the periphery of the embryo, which probably accounts for the reduced PGC number in some tadpoles. In other cases it may have been dispersed and therefore not detectable in histologic analyses.
Although the altered site of involution in inverted embryos did not influence PGC development, subsequent cell movement patterns apparently did. Those embryos which displayed the largest degree of pattern reversal at the tail-bud stage also exhibited the most extreme reduction in PGC numbers. A brief cold shock (4° C, 10 min) prior to first cleavage leads to a further reduction in PGC numbers in inverted embryos, probably as a result of the displace-ment of the germ plasm away from its original vegetal pole location.     

Topology of the germ plasm and development of primordial germ cells in inverted amphibian eggs

Inverted Xenopus eggs have reduced numbers of primordial germ cells (PGCs). The extent of the reduction varies from spawning to spawning. Histologic examination revealed that PGC counts were lowest in inverted eggs which displayed the greatest amount of shift in the vegetal mass of large yolk platelets, although the germ plasm itself always remained localized in the egg's original vegetal hemisphere. Even at blastulation the germ plasm continued to be localized in the egg's original vegetal hemisphere. In many cases, however, it was confined to the periphery of the embryo, which probably accounts for the reduced PGC number in some tadpoles. In other cases it may have been dispersed and therefore not detectable in histologic analyses. Although the altered site of involution in inverted embryos did not influence PGC development, subsequent cell movement patterns apparently did. Those embryos which displayed the largest degree of pattern reversal at the tail-bud stage also exhibited the most extreme reduction in PGC numbers. A brief cold shock (4 degrees C, 10 min) prior to first cleavage leads to a further reduction in PGC numbers in inverted embryos, probably as a result of the displacement of the germ plasm away from its original vegetal pole location.     

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.     

The cause of the decreased number of primordial germ cells in albino Xenopus resides not in the micro-environment but in the presumptive PGC

The number of primordial germ cells (PGC) in albino tadpoles of Xenopus is significantly decreased as compared with that of the wild-type. Whether the decreased number of PGC is caused by the presumptive PGC (pPGC) themselves or the micro-environment surrounding those cells in the albino, or both was investigated in the present study. [³H]thymidine-labeled pPGC of wild-type and albino were implanted into unlabeled, host neurulae of wild-type br albino and wild-type, respectively. Labeled PGC in the genital ridges of experimental tadpoles were examined by autoradiography. There were no significant differences in the proportion of tadpoles with labeled PGC and in the average number of those PGC between the albino and wild-type tadpoles, into which wild-type pPGC had been implanted. The proportion in wild-type tadpoles with albino pPGC was much lower than that in wild-type tadpoles with wild-type pPGC. These results suggest that the pPGC of the albino and not the micro-environment are responsible for the decreased number of PGC.     

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.     

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.     

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.     

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.     

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.     

Genetic and developmental evidence for a repressed genital primordium in Drosophila melanogaster

Agametic, a maternal-effect mutation, causes the absence of germ cells in approximately 40% of the gonads of flies derived from homozygous females. The nature of the deficiency in the eggs produced by these flies was examined. Ultrastructural abnormalities were seen in the polar granules of some eggs shortly after fertilization. Although a normal number of pole cells form, some are abnormal with degenerating polar granules and nuclear bodies and they contain myeloid bodies. The pole cells reach the gonads and at 14 hr of development all the gonads contain germ cells. However, in 40% of the gonads the germ cells become necrotic and disappear. Thus, the source of agametic gonads in the adult is embryonic death of pole cells in some gonads. To test whether this gonadal death is an autonomous deficiency of the mutant pole cells, mosaic pole cell populations were produced by reciprocal pole cell transplantation. In both types of transplants, the mutant pole cells died autonomously. In eight instances gonads containing only donor pole cells were obtained. Since mutant pole cells die when wild-type pole cells normally begin dividing, we suggest that the lesion affects the ability of these mutant pole cells to reenter the cell cycle.     

Drosophila pericentrin‐like protein promotes the formation of primordial germ cells

Primordial germ cells (PGCs) are the precursors to the adult germline stem cells that are set aside early during embryogenesis and specified through the inheritance of the germ plasm, which contains the mRNAs and proteins that function as the germline fate determinants. In Drosophila melanogaster, formation of the PGCs requires the microtubule and actin cytoskeletal networks to actively segregate the germ plasm from the soma and physically construct the pole buds (PBs) that protrude from the posterior cortex. Of emerging importance is the central role of centrosomes in the coordination of microtubule dynamics and actin organization to promote PGC development. We previously identified a requirement for the centrosome protein Centrosomin (Cnn) in PGC formation. Cnn interacts directly with Pericentrin‐like protein (PLP) to form a centrosome scaffold structure required for pericentriolar material recruitment and organization. In this study, we identify a role for PLP at several discrete steps during PGC development. We find PLP functions in segregating the germ plasm from the soma by regulating microtubule organization and centrosome separation. These activities further contribute to promoting PB protrusion and facilitating the distribution of germ plasm in proliferating PGCs.     

The ter mutation first causes primordial germ cell deficiency in ter/ter mouse embryos at 8 days of gestation

The ter (teratoma) mutation causes primordial germ cell (PGC) deficiency in ter/ter embryos at 9.5–12.5 days of post-coitum (dpc) in mouse strains 129/Sv- ter and LTXBJ- ter . To study the effects of the ter mutation on the PGC development more precisely, we examined the PGC number and distribution in 7.5–12.5 dpc embryo of ter congenic C57BL/6J- ter strain using their complete serial sections. The ter genotypes of embryos were identified by the polymerase chain reaction (PCR) polymorphisms of the microsatellite DNA of the Grl -1 locus mapped near the ter locus. Results showed that: (i) the PGC number in ter/ter embryos was similar to those of + / ter and + / + embryos at 7.5 dpc, and did not increase at 8.0–12.5 dpc, although those of normal littermates did usually; (ii) the PGC migration to genital ridges was never affected in all embryos; and (iii) the ter genotype difference in the PGC numbers was not recognized between + / ter and + / + embryos. We concluded that the ter mutation does not affect the PGC appearance around 7.5 dpc, but first causes PGC deficiency around 8.0 dpc at the beginning of their migration and proliferation, suggesting that the normal function of the ter gene may be essential for the proliferation or survival mechanisms of PGC.     

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.     

The stabilization of beta-catenin leads to impaired primordial germ cell development via aberrant cell cycle progression

Primordial germ cells (PGCs) are germ cell precursors that are committed to sperm or oocytes. Dramatic proliferation during PGC development determines the number of founder spermatogonia and oocytes. Although specified to a germ lineage, PGCs produce pluripotent embryonic germ (EG) cells in vitro and testicular teratomas in vivo. Wnt/beta-catenin signaling regulates pluripotency and differentiation in various stem cell systems, and dysregulation of this signaling causes various human cancers. Here, we examined the role of Wnt/beta-catenin signaling in PGC development. In normal PGC development, Wnt/beta-catenin signaling is suppressed by the GSK3beta-mediated active degradation of beta-catenin and the low expression of canonical Wnt molecules. The effects of aberrant activation of Wnt/beta-catenin signaling in PGCs were analyzed using mice carrying a deletion of the exon that encodes the GSK3beta phosphorylation sites in the beta-catenin locus. Despite the potential activity of Wnt/beta-catenin signaling in stem cell maintenance and carcinogenesis in various cell lineages, teratomas were not induced in the mice expressing the nuclear-localized beta-catenin in PGCs. Instead, the mutant mice showed germ cell deficiency caused by the delayed cell cycle progression of the proliferative phase PGCs. Our results show that the suppression of Wnt/beta-catenin signaling is a prerequisite for the normal development of PGCs.     

Chromatin in diapause of the silkworm Bombyx mori L.: thermal parthenogenesis and normal development

Having used hematoxylin as a stain, some features of silkworm embryo chromatin in diapause have been studied in normal and parthenogenetic development. With found direct correlation between the number of interphase chromatin grains and the number of chromosomes in the nucleus, we examined cell polyploidization in the embryo at diapause stage. Polyploidization by parthenogenesis is not reducible to endomitotic doubling of the chromosome set because it comprises 6n-nuclei. Explanation of more diverse range of polyploid cells in parthenogenesis needs to consider the fusion of cleavage nuclei that is carried out by the cytoplasmic karyogamic mechanism in the absence of fertilization. For the first time on squash preparations, in diapausing embryo, we have identified primary germ cells (PGC) that are characterized by less compact chromatin, especially in the zygotic form of development, a larger size of the nucleus and cytoplasm, and irregular number and size of nucleoli. Evaluation of PGC ploidy in parthenogenesis by calculation of "loose" chromatin grains in diapause is possible and testifies polyploidization in embryo germ-line. This explains the inevitable admixture of tetraploid eggs in diploid parthenoclone grain and its absence in normal development. Cytological method used has revealed a spiral arrangement of chromatin grains on the inner surface of the nucleus at different levels of ploidy.     

SCN— Blocks Hardening of the Fertilization Envelope of Sea Urchin Eggs and Adhesion of Blastomeres

Sea urchin eggs kept in artificial sea water (ASW) containing 0.01–0.3 M NaSCN in place of NaCI from within 2 min after insemination formed thin, enlarged fertilization envelopes, which were broken on mild agitation of egg suspensions more easily than those formed in Ca²⁺-free ASW. The blastomeres of almost all embryos derived from eggs treated with 0.2M SCN^— for 1 hr dissociated spontaneously, and did not reassociate with other blastomeres appreciably. Thus SCN^— probably denaturated some compound(s) participating in blastomere binding and hardening of the fertilization envelope. Abnormal arrangements of blastomeres, probably due to incomplete blastomere dissociation, were observed in embryos derived from eggs treated with 0.1 M SCN^— for 1 hr. Treatment of fertilized or unfertilized eggs with 0.05–0.1 M SCN^— for a short period caused concentration-dependent block of morphogenic processes such as formation of the archenteron and pluteus arms in the post-hatching period. The effects of SCN^— on morphogenesis were not inhibited by furosemide or 4,4'-diisothiocyano 2,2'-disulfonic stilbene. Presumably, the denaturation of several compounds in the egg surface by SCN^— causes abnormal morphogenesis of embryos. The inhibitory effects of SCN^— on hardening of the fertilization envelope, blastomere binding and morphogenesis were greater in the absence of Ca²⁺.