Structural characterization and distribution of zebrafish germ plasm during interphase and mitosis

In zebrafish, germ cells are responsible for transmitting the genetic information from one generation to the next. During the first cleavages of zebrafish embryonic development, a specialized part of the cytoplasm known as germ plasm, is responsible of committing four blastomeres to become the progenitors of all germ cells in the forming embryo. Much is known about how the germ plasm is spatially distributed in early stages of primordial germ cell development, a process described to be dependant on microtubules and actin. However, little is known about how the material is inherited after it reorganizes into a perinuclear location, or how is the symmetrical distribution regulated in order to ensure proper inheritance of the material by both daughter cells. It is also not clear whether there is a controlled mechanism that regulates the number of granules inherited by the daughter cells, or whether it is a random process. We describe the distribution of germ plasm material from 4hpf to 24hpf in zebrafish primordial germ cells using Vasa protein as marker. Vasa positive material appears to be conglomerate into 3 to 4 big spherical structures at 4hpf. While development progresses, these big structures become smaller perinuclear granules that reach a total number of approximately 30 at 24hpf. We investigated how this transformation occurs and how the minus-end microtubule dependent motor protein Dynein plays a role in this process. Additionally, we describe specific colocalization of microtubules and perinuclear granules during interphase and more interestingly, during all different stages of cell division. We show that distribution of granules follow what seems to be a regulated distribution: during cells division, daughter cells inherit an equal number of granules. We propose that due to the permanent colocalization of microtubular structures with germinal granules during interphase and cell division, a coordinated mechanism between these structures may ensure proper distribution of the material among daughter cells. Furthermore, we show that exposure to the microtubule-depolymerizing drug nocodazole leads to disassembly of the germ cell nuclear lamin matrix, chromatin condensation, and fusion of granules to a big conglomerate, revealing dependence of granular distribution on microtubules and proper nuclear structure.

Germ Cell Transplantation in Felids: A Potential Approach to Preserving Endangered Species

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mitochondrial inheritance and germ line determination in a bivalve species with Doubly Uniparental Inheritance (DUI)

Mitochondria are inherited maternally in most metazoans. However, in some bivalves, two mitochondrial lineages are present: one transmitted through eggs (F), the other through sperm (M). This is called Doubly Uniparental Inheritance (DUI). During male embryo development, spermatozoon mitochondria aggregate and end up in the primordial germ cells, while they are dispersed in female embryos. The molecular mechanisms of segregation patterns are still unknown. In the DUI species Ruditapes philippinarum, I examined sperm mitochondria distribution by MitoTracker, microtubule staining and TEM, and I localized germ line determinants with immunocytochemical analysis. I also analyzed the gonad transcriptome, searching for genes involved in reproduction and sex determination. Moreover, I analyzed an M-type specific open reading frame that could be responsible for maintenance/degradation of M mitochondria during embryo development. These transcripts were also localized in tissues using in situ hybridization. As in Mytilus, two distribution patterns of M mitochondria were detected in R. philippinarum, supporting that they are related to DUI. Moreover, the first division midbody concurs in positioning aggregated M mitochondria on the animal-vegetal axis of the male embryo: in organisms with spiral segmentation this zone is not involved in further cleavages, so aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area where germ plasm is transferred, suggesting their contribution in male germ line formation. The finding of reproduction and ubiquitination transcripts led to formulate a model in which ubiquitination genes stored in female oocytes during gametogenesis would activate sex-gene expression in the early embryonic developmental stages (preformation). Only gametogenetic cells were labeled by in situ hybridization, proving their specific transcription in developing gametes. Other than having a role in sex determination, some ubiquination factors could also be involved in mitochondrial inheritance, and their differential expression could be responsible for the different fate of sperm mitochondria in the two sexes.

Transcriptional and translational regulators of gene expression in germ cell development

Germ cell development is a highly coordinated process driven, in part, by regulatory mechanisms that control gene expression. Not only transcription, but also translation, is under regulatory control to direct proper germ cell development. In this dissertation, I have focused on two regulators of germ cell development. One is the homeobox protein RHOX10, which has the potential to be both a transcriptional and translational regulator in mouse male germ cell development. The other is the RNA-binding protein, Hermes, which functions as a translational regulator in Xenopus laevis female germ cell development. ^ Rhox10 is a member of reproductive homeobox gene X-(linked (Rhox) gene cluster, of which expression is developmentally regulated in developing mouse testes. To identify the cell types and developmental stages in which Rhox10 might function, I characterized its temporal and spatial expression pattern in mouse embryonic, neonatal, and adult tissues. Among other things, this analysis revealed that both the level and the subcellular localization of RHOX10 are regulated during germ cell development. To understand the role of Rhox10 in germ cell development, I generated transgenic mice expressing an artificial microRNA (miRNA) targeting Rhox10. While this artificial miRNA robustly downregulated RHOX10 protein expression in vitro, it did not significantly reduce RHOX10 expression in vivo. So I next elected to knockdown RHOX10 levels in spermatogonial stem cells (SSCs), which I found highly express both Rhox10 mRNA and RHOX10 protein. Using a recently developed in vitro culture system for SSCs combined with a short-hairpin RNA (shRNA) approach, I strongly depleted RHOX10 expression in SSCs. These RHOX10-depleted cells exhibited a defect in the ability to form stem cell clusters in vitro. Expression profiling analysis revealed many genes regulated by Rhox10, including many meiotic genes, which could be downstream of Rhox10 in a molecular pathway that controls SSC differentiation. ^ RNA recognition motif (RRM) containing protein, Hermes is localized in germ plasm, where dormant mRNAs are also located, of Xenopus oocytes, which implicates its role in translational regulator. To understand the function of Hermes in oocyte meiosis, I used a morpholino oligonucleotide (MO) based knockdown approach. Microinjection of Hermes MO into fully grown oocytes, which are arrested in meiotic prophase, caused acceleration of oocytes reentry into meiosis (i.e., maturation) upon progesterone induction. Using a candidate approach, I identified at least three targets of Hermes: Ringo/Spy, Xcat2, and Mos. Ringo/Spy and Mos are known to have functions in oocyte maturation, while Ringo/Spy, Xcat2 mRNA are localized in the germ plasm of oocytes, which drives germ cell specification after fertilization. This led me to propose that Hermes functions in both oocyte maturation and germ cell development through its ability to regulate 3 crucial target mRNAs. ^

Germ Cell Transplantation in Felids: A Potential Approach to Preserving Endangered Species

With the exception of the domestic cat, all members of the family Felidae are considered either endangered or threatened. Although not yet used for this purpose, spermatogonial stem cell (SSC) transplantation has a high potential to preserve the genetic stock of endangered species. However, this technique has not previously been established in felids. Therefore, we developed the necessary procedures to perform syngeneic and xenogeneic SSC transplants (eg, germ cell [GC] depletion in the recipient domestic cats, enrichment and labeling of donor cell suspension, and the transplantation method) in order to investigate the feasibility of the domestic cat as a recipient for the preservation and propagation of male germ plasm from wild felids. In comparison with busulfan treatment, local x-ray fractionated radiation was a more effective approach to depleting endogenous spermatogenesis. The results of both syngeneic and xenogeneic transplants revealed that SSCs were able to successfully colonize and differentiate in the recipient testis, generating elongated spermatids several weeks posttransplantation. Specifically, ocelot spermatozoa were observed in the cat epididymis 13 weeks following transplantation. As donor GCs from domestic cats and ocelots were able to develop and form mature GCs in the recipient environment seminiferous tubules, these findings indicate that the domestic cat is a suitable recipient for SSC transplantation. Moreover, as modern cats descended from a medium-size cat that existed approximately 10 to 11 million years ago, these results strongly suggest that the domestic cat could be potentially used as a recipient for generating and propagating the genome of wild felids.

Differentiation of peanut seedborne potyviruses and curcumoviruses by RT-PCR

Seedborne peanut viruses pose important constraints to peanut production and safe movement of germ plasm. They also pose a risk of accidental introduction into previously disease-free regions. We have developed reverse transcription-polymerase chain reaction (RT-PCR) assays based on identical cycling parameters which identified peanut stripe, Peanut mottle, Peanut stunt, and Cucumber mosaic viruses through production of specific DNA fragments of 234 bp, 327 bp, 390 bp, and 133 bp, respectively. Assay sensitivity in the picogram range was achieved. The two potyviruses and two cucumoviruses could be differentiated using duplex RT-PCR assays. These assays should be useful for testing peanut leaves or seeds for virus identification in epidemiological studies, seed testing or in post-entry quarantine.

Characterization for plant height and flowering date in the biological species oat

The use of wild oat races in artificial hybridization with cultivated oat (Avena sativa L.) has been used as a way of increasing the variability. This work aimed to identify the variability for plant height and flowering date of groups of cultivated oat genotypes, wild introductions of A. fatua L. and segregating populations of natural crosses between A. sativa and A. fatua. Wide genetic variability was observed for both traits in the groups and between them. The wild group of A. fatua L. showed high plants with early maturity, but in the segregating group there was reduced plant height and early maturity. The wild introductions of A. fatua L. studied in this work can be used in oat breeding programs to increase genetic variability by transferring specific characters into the cultivated germ plasm.

Occurrence and ultrastructural characterization of nuage during oogenesis and early spermatogenesis of Piaractus mesopotamicus Holmberg, 1887 (Teleostei)

Foi investigada a ocorrência e caracterizada ultra-estruturalmente a eliminação de material nuclear eletrondenso (nuage) para o citoplasma durante a ovogênese e durante os estágios iniciais da espermatogênese de Piaractus mesopotamicus, um peixe do Pantanal Matogrossense de ciclo reprodutivo sazonal. Constataram-se nas células germinativas femininas dois momentos de eliminação desse material, na ovogônia e no ovócito em fase perinucleolar. Nas células masculinas, material com morfologia e comportamento muito semelhante foi encontrado na espermatogônia. em todos os casos, o material associou-se a mitocôndrias. A possível função desse material foi discutida.

Pathogenic variation in Colletotrichum gloeosporioides infecting Stylosanthes spp. in a center of diversity in Brazil

Pathogenic variation in Colletotrichum gloeosporioides infecting species of the tropical pasture legume Stylosanthes at its center of diversity was determined from 296 isolates collected from wild host population and selected germ plasm of S. capitata, S. guianensis, S. scabra, and S. macrocephala in Brazil. A putative host differential set comprising 11 accessions was selected from a bioassay of 18 isolates on 19 host accessions using principal component analysis. A similar analysis of anthracnose severity data for a subset of 195 isolates on the 11 differentials indicated that an adequate summary of pathogenic variation could be obtained using only five of these differentials. of the five differentials, S. seabrana 'Primar' was resistant and S. scabra 'Fitzroy' was susceptible to most isolates. A cluster analysis was used to determine eight natural race clusters using the 195 isolates. Linear discriminant functions were developed for eight race clusters using the 195 isolates as the training data set, and these were applied to classify a test data set of the remaining 101 isolates. All except 11 isolates of the test data set were classified into one of the eight race clusters. Over 10% of the 296 isolates were weakly pathogenic to all five differentials and another 40% were virulent on just one differential. The unclassified isolates represent six new races with unique virulence combinations, of which one isolate is virulent on all five differentials. The majority of isolates came from six field sites, and Shannon's index of diversity indicated considerable variation between sites. Pathogenic diversity was extensive at three sites where selected germ plasm were under evaluation, and complex race clusters and unclassified isolates representing new races were more prevalent at these sites compared with sites containing wild Stylosanthes populations.

Variance Effective Population Size for Dioecious Species

The concept of effective population size (N(e)) is an important measure of representativeness in many areas. In this research, we consider the statistical properties of the number of contributed gametes under practical situations by adapting Crow and Denninston's (1988) N(e) formulas for dioecious species. Three sampling procedures were considered. In all circumstances, results show that as the offspring sex ratio (r) deviates from 0.5, N(e) values become smaller, and the efficiency of gametic control for increasing N(e) is reduced. For finite populations, where all individuals are potentially functional parents, the reduction in N(e) due to an unequal sex ratio can be compensated for through female gametic control when 0.28 <= r <= 0.72. This outcome is important when r is unknown. When only a fraction of the individuals in a population is taken for reproduction, N(e) is meaningful only if the size of the reference population is clearly defined. Gametic control is a compensating factor in accession regeneration when the viability of the accession is around 70 or 75%. For germ-plasm collection, when parents are a very small fraction of the population, maximum N(e) will be approximately 47 and 57% of the total number of offspring sampled, with female gametic control, r varying between 0.3 and 0.5, and being constant over generations.

Phylogeny of rice genomes with emphasis on origins of allotetraploid species

The rice genus, Oryza, which comprises 23 species and 9 recognized genome types, represents an enormous gene pool for genetic improvement of rice cultivars. Clarification of phylogenetic relationships of rice genomes is critical for effective utilization of the wild rice germ plasm. By generating and comparing two nuclear gene (Adh1 and Adh2) trees and a chloroplast gene (matK) tree of all rice species, phylogenetic relationships among the rice genomes were inferred. Origins of the allotetraploid species, which constitute more than one-third of rice species diversity, were reconstructed based on the Adh gene phylogenies. Genome types of the maternal parents of allotetraploid species were determined based on the matK gene tree. The phylogenetic reconstruction largely supports the previous recognition of rice genomes. It further revealed that the EE genome species is most closely related to the DD genome progenitor that gave rise to the CCDD genome. Three species of the CCDD genome may have originated through a single hybridization event, and their maternal parent had the CC genome. The BBCC genome species had different origins, and their maternal parents had either a BB or CC genome. An additional genome type, HHKK, was recognized for Oryza schlechteri and Porteresia coarctata, suggesting that P. coarctata is an Oryza species. The AA genome lineage, which contains cultivated rice, is a recently diverged and rapidly radiated lineage within the rice genus.

A critique of the theory of evolution,

Half-title: Princeton university. The Louis Clark Vanuxem foundation lectures for 1915-1916.

Wolbachia infections are distributed throughout insect somatic and germ line tissues

Wolbachia are intracellular microorganisms that form maternally-inherited infections within numerous arthropod species. These bacteria have drawn much attention, due in part to the reproductive alterations that they induce in their hosts including cytoplasmic incompatibility (CI), feminization and parthenogenesis. Although Wolbachia's presence within insect reproductive tissues has been well described, relatively few studies have examined the extent to which Wolbachia infects other tissues. We have examined Wolbachia tissue tropism in a number of representative insect hosts by western blot, dot blot hybridization and diagnostic PCR. Results from these studies indicate that Wolbachia are much more widely distributed in host tissues than previously appreciated. Furthermore, the distribution of Wolbachia in somatic tissues varied between different Wolbachia/host associations. Some associations showed Wolbachia disseminated throughout most tissues while others appeared to be much more restricted, being predominantly limited to the reproductive tissues. We discuss the relevance of these infection patterns to the evolution of Wolbachia/host symbioses and to potential applied uses of Wolbachia.

INTENSITY-MODULATED AND 3D-CONFORMAL RADIOTHERAPY FOR WHOLE-VENTRICULAR IRRADIATION AS COMPARED WITH CONVENTIONAL WHOLE-BRAIN IRRADIATION IN THE MANAGEMENT OF LOCALIZED CENTRAL NERVOUS SYSTEM GERM CELL TUMORS

Purpose: To compare the sparing potential of cerebral hemispheres with intensity-modulated radiotherapy (IMRT) and three-dimensional conformal radiotherapy (3D-CRT) for whole-ventricular irradiation (WVI) and conventional whole-brain irradiation (WBI) in the management of localized central nervous system germ cell tumors (CNSGCTs). Methods and Materials: Ten cases of patients with localized CNSGCTs and submitted to WVI by use of IMRT with or without a ""boost"" to the primary lesion were selected. For comparison purposes, similar treatment plans were produced by use of 3D-CRT (WVI with or without boost) and WBI (opposed lateral fields with or without boost), and cerebral hemisphere sparing was evaluated at dose levels ranging from 2 Gy to 40 Gy. Results: The median prescription dose for WVI was 30.6 Gy (range, 25.2-37.5 Gy), and that for the boost was 16.5 Gy (range, 0-23.4 Gy). Mean irradiated cerebral hemisphere volumes were lower for WVI with IMRT than for 3D-CRT and were lower for WVI with 3D-CRT than for WBI. Intensity-modulated radiotherapy was associated with the lowest irradiated volumes, with reductions of 7.5%, 12.2%, and 9.0% at dose levels., compared with 3D-CRT. Intensity-modulated radiotherapy provided of 20, 30, and 40 Gy, respectively statistically significant reductions of median irradiated volumes at all dose levels (p = 0.002 or less). However, estimated radiation doses to peripheral areas of the body were 1.9 times higher with IMRT than with 3D-CRT. Conclusions: Although IMRT is associated with increased radiation doses to peripheral areas of the body, its use can spare a significant amount of normal central nervous system tissue compared with 3D-CRT or WBI in the setting of CNSGCT treatment. (C) 2010 Elsevier Inc.