Spatiotemporal localization of germ plasm RNAs during zebrafish oogenesis

In zebrafish, primordial germ cells (PGCs) are determined by a specialized maternal cytoplasm, the germ plasm, which forms at the distal ends of the cleavage furrows in 4-cell embryos. The germ plasm includes maternal mRNAs from the germline-specific genes such as vasa and nanos1, and vegetally localized dazl RNA is also incorporated into the germ plasm. However, little is known about the distributions and assembly mechanisms of germ plasm components, especially during oogenesis. Here we report that the germ plasm RNAs vasa, nanos1, and dazl co-localize with the mitochondrial cloud (MC) and are transported to the vegetal cortex during early oogenesis. We found that a mitochondrial cloud localization element (MCLE) previously identified in the 3' untranslated region (3'UTR) of Xenopus Xcat2 gene can direct RNA localization to the vegetal cortex via the MC in zebrafish oocytes. In addition, the RNA-binding protein Hermes is a component of the MC in zebrafish oocytes, as is the case in Xenopus. Moreover, we provide evidence that the dazl 3'UTR possesses at least three types of cis-acting elements that direct multiple steps in the localization process: MC localization, anchorage at the vegetal cortex, and localization at the cleavage furrows. Taken together, the data show that the MC functions as a conserved feature that participates in transport of the germ plasm RNAs in Xenopus and zebrafish oocytes. Furthermore, we propose that the germ plasm components are assembled in a stepwise and spatiotemporally-regulated manner during oogenesis and early embryogenesis in zebrafish.     

Ultrastructural features of gametogenesis during the life cycle in Branchiura sowerbyi Beddard (Oligochaeta,Tubificidae)

An ultrastructural analysis of the gametogenetic phases in Branchiura sowerbyi, a tubificid oligochaete, has been accomplished. These phases mostly conform to the usual pattern for the family, however, some interesting peculiarities are pointed out. The regression of sexual apparatus after reproductive period and its regeneration up to a new period of sexual maturity, has been followed throughout the year.     

Intermolecular interactions of homologs of germ plasm components in mammalian germ cells

In some species such as flies, worms, frogs and fish, the key to forming and maintaining early germ cell populations is the assembly of germ plasm, microscopically distinct egg cytoplasm that is rich in RNAs, RNA-binding proteins and ribosomes. Cells which inherit germ plasm are destined for the germ cell lineage. In contrast, in mammals, germ cells are formed and maintained later in development as a result of inductive signaling from one embryonic cell type to another. Research advances, using complementary approaches, including identification of key signaling factors that act during the initial stages of germ cell development, differentiation of germ cells in vitro from mouse and human embryonic stem cells and the demonstration that homologs of germ plasm components are conserved in mammals, have shed light on key elements in the early development of mammalian germ cells. Here, we use FRET (Fluorescence Resonance Energy Transfer) to demonstrate that living mammalian germ cells possess specific RNA/protein complexes that contain germ plasm homologs, beginning in the earliest stages of development examined. Moreover, we demonstrate that, although both human and mouse germ cells and embryonic stem cells express the same proteins, germ cell-specific protein/protein interactions distinguish germ cells from precursor embryonic stem cells in vitro; interactions also determine sub-cellular localization of complex components. Finally, we suggest that assembly of similar protein complexes may be central to differentiation of diverse cell lineages and provide useful diagnostic tools for isolation of specific cell types from the assorted types differentiated from embryonic stem cells.     

The mRNA coding for Xenopus glutamate receptor interacting protein 2 (XGRIP2) is maternally transcribed, transported through the late pathway and localized to the germ plasm

Using a large-scale in situ hybridization screening, we found that the mRNA coding for Xenopus glutamate receptor interacting protein 2 (XGRIP2) was localized to the germ plasm of Xenopus laevis. The mRNA is maternally transcribed in oocytes and, during maturation, transported to the vegetal germ plasm through the late pathway where VegT and Vg1 mRNAs are transported. In the 3'-untranslated region (UTR) of the mRNA, there are clusters of E2 and VM1 localization motifs that were reported to exist in the mRNAs classified as the late pathway group. With in situ hybridization to the sections of embryos, the signal could be detected in the cytoplasm of migrating presumptive primordial germ cells (pPGCs) until stage 35. At stage 40, when the cells cease to migrate and reach the dorsal mesentery, the signal disappeared. A possible role of XGRIP2 in pPGCs of Xenopus will be discussed.     

Ectopic formation of primordial germ cells by transplantation of the germ plasm: Direct evidence for germ cell determinant in Xenopus

In many animals, the germ line is specified by a distinct cytoplasmic structure called germ plasm (GP). GP is necessary for primordial germ cell (PGC) formation in anuran amphibians including Xenopus. However, it is unclear whether GP is a direct germ cell determinant in vertebrates. Here we demonstrate that GP acts autonomously for germ cell formation in Xenopus.EGFP-labeled GP from the vegetal pole was transplanted into animal hemisphere of recipient embryos. Cells carrying transplanted GP (T-GP) at the ectopic position showed characteristics similar to the endogenous normal PGCs in subcellular distribution of GP and presence of germ plasm specific molecules. However, T-GP-carrying-cells in the ectopic tissue did not migrate towards the genital ridge. T-GP-carrying cells from gastrula or tailbud embryos were transferred into the endoderm of wild-type hosts. From there, they migrated into the developing gonad. To clarify whether ectopic T-GP-carrying cells can produce functional germ cells, they were identified by changing the recipients, from the wild-type Xenopus to transgenic Xenopus expressing DsRed2. After transferring T-GP carrying cells labeled genetically with DsRed2 into wild-type hosts, we could find chimeric gonads in mature hosts. Furthermore, the spermatozoa and eggs derived from T-GP-carrying cells were fertile. Thus, we have demonstrated that Xenopus germ plasm is sufficient for germ cell determination.     

Genetic diversity in European and Mediterranean faba bean germ plasm revealed by RAPD markers

Broadening of the genetic base and systematic exploitation of heterosis in faba bean requires reliable information on the genetic diversity in the germ plasm. Three groups of faba bean inbred lines were examined by means of RAPDs (random amplified polymorphic DNAs) assays: 13 European small-seeded lines, 6 European large-seeded lines, and 9 Mediterranean lines. Out of 59 primers, 35 were informative and yielded 365 bands, 289 of which were polymorphic with a mean of 8.3 bands per primer. Monomorphic bands were omitted from the analyses and genetic distances (GD) were estimated via the coefficient of Jaccard. The mean GD among the European small-seeded lines was significantly greater than those among the lines of the other two groups. Repeatability of GD estimates was high. Cluster (UPGMA) and principal coordinate analyses identified European small-seeded lines and Mediterranean lines as distinct groups with European large-seeded lines located in between. The results are in harmony with published archaeobotanical findings. We conclude that RAPDs are useful for classification of germ plasm and identification of divergent heterotic groups in faba bean.     

Bazooka regulates microtubule organization and spatial restriction of germ plasm assembly in the Drosophila oocyte

Localization of the germ plasm to the posterior of the Drosophila oocyte is required for anteroposterior patterning and germ cell development during embryogenesis. While mechanisms governing the localization of individual germ plasm components have been elucidated, the process by which germ plasm assembly is restricted to the posterior pole is poorly understood. In this study, we identify a novel allele of bazooka (baz), the Drosophila homolog of Par-3, which has allowed the analysis of baz function throughout oogenesis. We demonstrate that baz is required for spatial restriction of the germ plasm and axis patterning, and we uncover multiple requirements for baz in regulating the organization of the oocyte microtubule cytoskeleton. Our results suggest that distinct cortical domains established by Par proteins polarize the oocyte through differential effects on microtubule organization. We further show that microtubule plus-end enrichment is sufficient to drive germ plasm assembly even at a distance from the oocyte cortex, suggesting that control of microtubule organization is critical not only for the localization of germ plasm components to the posterior of the oocyte but also for the restriction of germ plasm assembly to the posterior pole.     

Transfer of germ plasm from the secondary to the primary gene pool in pennisetum

Summary Germ plasm from the A-genome of Pennisetum purpureum Schum. (AABB) of the secondary gene pool was transferred to cultivated pearl millet (AA) [P. glaucum (L.) R. Br.] by pollinating cytoplasmicnuclear male-sterile (cms) pearl millet with fertile allohexaploid pearl millet x P. purpureum hybrids (AAAABB). Certain allohexaploids used as pollinators on cms pearl millet resulted in 14-chromosome diploid pearl millet progenies. Three types of diploid pearl millet plants were produced in addition to the expected 28-chromosome AAAB-genome plants: (1) cms plants with only the A-genome, (2) cms plants with the A- and A-genomes, and (3) fertile plants with the A- and A-genomes. The latter group has allowed the utilization of genes for fertility restoration, stiff stalk, maturity, height, and morphological characteristics from the A-genome of P. purpureum in the pearl millet breeding program. Production of monoploid gametes by the allohexaploids appeared to be genetically controlled.     

Germ plasm in Caenorhabditis elegans, Drosophila and Xenopus

Special cytoplasm, called germ plasm, that is essential for the differentiation of germ cells is localized in a particular region of Caenorhabditis elegans, Drosophila and Xenopus eggs. The mode of founder cell formation of germline, the origin and behavior of the germline granules, and the molecules localized in germline cells are compared in these organisms. The common characteristics of the organisms are mainly as follows. First, the founder cells of germline are established before the intiation of gastrulation. Second, the germline granules or their derivatives are always present in germline cells or germ cells throughout the life cycle in embryos, larvae, and adults. Lastly, among the proteins localized in the germ plasm, only Vasa protein or its homolog is detected in the germline cells or germ cells throughout the life cycle. As the protein of vasa homolog has been reported to be also localized in the germline-specific structure or nuage in some of the organisms without the germ plasm, the possibility that the mechanism for differentiation of primordial germ cells is basically common in all organisms with or without the germ plasm is discussed.     

Differential subcellular sequestration of proapoptotic and antiapoptotic proteins and colocalization of Bcl-x(L) with the germ plasm, in Xenopus laevis oocytes

Apoptosis is an important element of normal embryonic development and gametogenesis in invertebrate and vertebrate species. Although the components of apoptotic machinery are present in Xenopus laevis fully grown stage VI oocytes and eggs, apoptosis in the developing Xenopus ovary is limited to the somatic cells with no indication of apoptosis in the germ cells. Considering the possibility that Xenopus previtellogenic oocytes might lack the components of the apoptotic pathway, we analyzed Xenopus Stage I oocytes for the presence of the proapoptotic factors Bax and tumor suppressor p53, and antiapoptotic factors Bcl-x(L) and mitochondrial heat shock protein 60 (Hsp60). We found that pro- and antiapoptotic proteins are present in Xenopus oocytes but, surprisingly, they are located in distinct subcellular compartments with proapoptotic proteins Bax and p53 being sequestered in the oocyte nucleus and antiapoptotic protein Bcl-x(L) sequestered in the cytoplasm and highly enriched in the METRO region of the mitochondrial cloud, where it colocalized with the germ plasm, and Hsp60 colocalizing with all mitochondria. The absence of apoptosis in Xenopus early oogenesis is maybe due to differential sequestration of pro- and antiapoptotic molecules.     

Morphohistochemical changes in hepatocytes during the life cycle of the European eel

The comparative analysis of morphological, histochemical and cytochemical patterns of eel (Anguilla anguilla L.) hepatocytes reveals clear differences between two stages of its life cycle, i.e. the trophic stage (yellow eel) and reproductive stage (silver eel). The storage of glycogen prevails in the yellow eel, whilst lipids appear to be remarkably increased in the silver eel, in which some hepatocytes also show glycogen-rich areas. Generally, in the silver eel dehydrogenase and acid phosphatase activities seem greater and different distribution of the reaction products is present; on the contrary, a lower G6PDH activity is observed. The electron microscopy characteristics and distribution of both cellular organelles and reserve materials reflect the modifications found at light microscopy. The ultrastructural patterns provide further evidence for the heterogeneity of liver parenchyma in silver eel. In particular, the coexistence of nuclei showing a different degree of chromatin compactness is also accounted for by the quantitative cytochemical data on the nuclear DNA after Feulgen reaction and intercalation with propidium iodide at low and high concentrations. With regard to the DNA content, the hepatocytes in the silver eel as well as in the yellow eel are mainly 2c. However, some 4c values are also found, which according to the literature can be ascribed to cells in G2 phase. The present data may express the onset of different functional requirements during the reproductive stage in comparison with the trophic one. Moreover, our results are consistent with modifications found by other authors as a consequence of interruption of nourishment and during gonad maturation, i.e. two phenomena characterizing the transition from yellow to silver eel.     

Delocalization of polar plasm components caused by grandchildless mutations, gs(1)N26 and gs(1)N441, in Drosophila melanogaster

Two maternal-effect grandchildless (gs) mutations of Drosophila melanogaster, gs(1)N26 and gs(1)N441, cause delay in nuclear arrival at the polar plasm. In mutant embryos, polar plasm loses its ability to induce pole cells during retarded nuclear migration to the posterior pole of embryos. In the present study, it was shown that in N26 and N441 embryos, mitochondrial large rRNA (mtlrRNA), an essential factor for pole cell formation, is delocalized during the delay in nuclear arrival. This suggests that the loss of mtlrRNA causes failure of the mutants to form pole cells. Furthermore, it was shown that all of the other polar plasm components examined, namely Vasa protein, Germ cell-less protein, nanos mRNA and Polar granule component RNA start to be delocalized during the delay in nuclear arrival. This suggests that polar plasm integrity is not maintained in mutant embryos. It was finally shown that Vas is also delocalized in embryos that are inhibited to form pole cells by reducing the amount of mtlrRNA. This indicates that the segregation of polar plasm into pole cells is required to maintain polar plasm integrity. The mechanism regulating polar plasm integrity in embryos is discussed.     

Ultrastructural Study of Relationships between Germinal Bodies and Mitochondria in Apostichopus japonicus (Echinodermata: Holothuroidea) and Pleuronectes asper (Teleostei: Pleuronectidae)

The relationships between germinal bodies and mitochondria were studied in the holothurian Apostichopus japonicus and the flounder Pleuronectes asper using TEM. In the gonial cells of both species the mitochondria are arranged around germinal bodies and are in contact with the latter. A gradual disappearance of the outer membrane is found in the mitochondria that interact with the germinal substance. Later on, dispersion of the globules of the mitochondrial matrix containing mitochondrial cristae occurs. It is supposed that the substance of the mitochondrial matrix takes part in the development and functioning of the germinal plasm in both invertebrates and vertebrates.     

Ultrastructural changes in the abdominal midgut of the mosquito, Culiseta melanura, during the gonotrophic cycle

Abdominal midguts of the mosquito, Culiseta melanura, were examined by light and electron microscopy 1 hr-14 days days after blood feeding. Epithelial cells were drastically altered from columnar to squamous in form after engorgement, and returned to columnar by day 4 after feeding. Accumulation of mitochondria along brush borders of digestive cells, followed by the appearance of large secondary lysosomes, accompanied blood digestion. Evidence was obtained that myelin-like material in the lysosomes, probably the result of mitochondrial autolysis, is extruded into the lumen. Digestive cells resumed their pre-blood meal appearance by 10-14 days post-engorgement. Regenerative cells were scattered throughout the basal portion of the epithelium, along with endocrine cells. Other midgut cells containing large, microvilli-lined apical cavities were identified in most specimens. No evidence of division or differentiation was obtained for any cell types.     

Pix1 and Pix2 are novel WD40 microtubule-associated proteins that colocalize with mitochondria in Xenopus germ plasm and centrosomes in human cells

In many animals, the germ line develops from a distinct mitochondria-rich region of embryonic cytoplasm called the germ plasm. However, the protein composition of germ plasm and its formation remain poorly understood, except in Drosophila. Here, we show that Xpat, a recently identified protein component of Xenopus germ plasm, interacts via its C-terminal domain with a novel protein, xPix1. Xpat and xPix1 are co-expressed in ovaries, eggs and early embryos and colocalize to the mitochondrial cloud and germ plasm in stage I and stage VI oocytes, respectively. Although Xpat appears unique to Xenopus, Pix proteins, which contain an N-terminal WD40 domain and C-terminal coiled-coil, are widely conserved. In humans, two proteins, Pix1 and Pix2, are expressed at varying levels in different cancer cell lines. Importantly, as well as localizing to mitochondria, human Pix proteins localize to centrosomes and associate with microtubules in vitro and in vivo. Although, Pix proteins are stably expressed through the cell cycle, Pix2 concentrates on microtubule structures in mitosis and microinjection of Pix antibodies interferes with cell division. Based on these data, we propose that Pix1 and Pix2 are microtubule-associated adaptor proteins that likely contribute to a range of developmental and cell division processes.