In vivo detection of a novel endogenous etheno–DNA adduct derived from arachidonic acid and the effects of antioxidants on its formation

Previous studies showed that 7-(1′,2′-dihydroxyheptyl)-substituted etheno DNA adducts are products of reactions with the epoxide of (E)-4-hydroxy-2-nonenal, an oxidation product of ω-6 polyunsaturated fatty acids (PUFAs). In this work, we report the detection of 7-(1′,2′-dihydroxyheptyl)-1,N⁶-ethenodeoxyadenosine (DHHedA) in rodent and human tissues by two independent methods: a ³²P-postlabeling/HPLC method and an isotope dilution liquid chromatography–electrospray ionization–tandem mass spectrometry method, demonstrating for the first time that DHHedA is a background DNA lesion in vivo. We showed that DHHedA can be formed upon incubation of arachidonic acid with deoxyadenosine, supporting the notion that ω-6 PUFAs are the endogenous source of DHHedA formation. Because cyclic adducts are derived from the oxidation of PUFAs, we subsequently examined the effects of antioxidants, α-lipoic acid, Polyphenon E, and vitamin E, on the formation of DHHedA and γ-hydroxy-1,N²-propanodeoxyguanosine (γ-OHPdG), a widely studied acrolein-derived adduct arising from oxidized PUFAs, in the livers of Long Evans Cinnamon (LEC) rats. LEC rats are afflicted with elevated lipid peroxidation and prone to the development of hepatocellular carcinomas. The results showed that although the survival of LEC rats was increased significantly by α-lipoic acid, none of the antioxidants inhibited the formation of DHHedA, and only Polyphenon E decreased the formation of γ-OHPdG. In contrast, vitamin E caused a significant increase in the formation of both γ-OHPdG and DHHedA in the livers of LEC rats.     

Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ cells,bind Tudor and protect from transposable elements

Germ cells give rise to all cell lineages in the next‐generation and are responsible for the continuity of life. In a variety of organisms, germ cells and stem cells contain large ribonucleoprotein granules. Although these particles were discovered more than 100 years ago, their assembly and functions are not well understood. Here we report that glycolytic enzymes are components of these granules in Drosophila germ cells and both their mRNAs and the enzymes themselves are enriched in germ cells. We show that these enzymes are specifically required for germ cell development and that they protect their genomes from transposable elements, providing the first link between metabolism and transposon silencing. We further demonstrate that in the granules, glycolytic enzymes associate with the evolutionarily conserved Tudor protein. Our biochemical and single‐particle EM structural analyses of purified Tudor show a flexible molecule and suggest a mechanism for the recruitment of glycolytic enzymes to the granules. Our data indicate that germ cells, similarly to stem cells and tumor cells, might prefer to produce energy through the glycolytic pathway, thus linking a particular metabolism to pluripotency.     

Primordial germ cell development in zebrafish

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. These cells follow a characteristic developmental path that is manifested in specialized regulation of basic cell functions and behavior making them an attractive system for studying cell fate specification, differentiation and migration. This review summarizes studies aimed at understanding the development of this cell population in zebrafish and compares these results with those obtained in other model organisms.     

Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patterning

Pole cells and posterior segmentation in Drosophila are specified by maternally encoded genes whose products accumulate at the posterior pole of the oocyte. Among these genes is tudor (tud). Progeny of hypomorphic tud mothers lack pole cells and have variable posterior patterning defects. We have isolated a null allele to further investigate tud function. While no pole cells are ever observed in embryos from tud-null mothers, 15% of these embryos have normal posterior patterning. OSKAR (OSK) and VASA (VAS) proteins, and nanos (nos) RNA, all initially localize to the pole plasm of tud-null oocytes and embryos from tud-null mothers, while localization of germ cell-less (gcl) and polar granule component (pgc), is undetectable or severely reduced. In embryos from tud-null mothers, polar granules are greatly reduced in number, size, and electron density. Thus, tud is dispensable for somatic patterning, but essential for pole cell specification and polar granule formation.     

Isolation of a factor inducing pole cell formation from Drosophila embryos

Summary

An attempt to isolate an ooplasmic factor active in inducing pole cells in Drosophila embryos is described. With the help of a bioassay system, we demonstrated that RNA extracted from embryos was active in inducing pole cells. These RNA-induced pole cells were morphologically identical to the normal ones. In addition, a local application of cycloheximide suggests that translation in the posterior pole cytoplasm is a precondition for pole cell formation.     

Analysis of localization and reorganization of germ plasm in Xenopus transgenic line with fluorescence‐labeled mitochondria

Germ plasm is found in germ‐line cells of Xenopus and thought to include the determinant of primordial germ cells (PGCs). As mitochondria is abundant in germ plasm, vital staining of mitochondria was used to analyze the movement and function of germ plasm; however, its application was limited in early cleavage embryos. We made transgenic Xenopus, harboring enhanced green fluorescent protein (EGFP) fused to the mitochondria transport signal (Dria‐line). Germ plasm with EGFP‐labeled mitochondria was clearly distinguishable from the other cytoplasm, and retained mostly during one generation of germ‐line cells in Dria‐line females. Using the Dria‐line, we show that germ plasm is reorganized from near the cell membrane to the perinuclear space at St. 9, dependent on the microtubule system.     

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.     

The culture and establishment of embryonic germ （EG） cell lines from Chinese mini swine

As a part of a basic research project on Xeno-transplantion, we have been engaged in the derivation of embryonic stem cell lines from Chinese mini swine. Here, we reported for the first time the establishment of two porcine EG cell lines (BPEG1 and BPEG2) from primordial germ cells of genital ridges of a 28 anda 27 d embryos respectively. Their pluripotent nature has been identified by colony morphology, marker characterization as well as by in vitro and in vivo differentiation. These porcine EG cells are potentially useful for further basic studies.     

Mitochondrial trafficking through Rhot1 is involved in the aggregation of germinal granule components during primordial germ cell formation in Xenopus embryos

In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline‐Mt). However, the role of germline‐Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline‐Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C‐terminal mitochondrial transmembrane domain, inhibited the aggregation of germline‐Mt during early development. In Rhot1‐inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail‐bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.     

Generation of stella-GFP transgenic mice: a novel tool to study germ cell development

The relationship between germ cells and pluripotent embryonic stem (ES) cells is of particular interest, together with approaches to generate primordial germ cell (PGCs) from ES cells. A critical requirement in these experiments is the ability to unambiguously detect PGCs with the use of, for example, reporter genes. The currently available transgenic reporters do not show exclusive expression in PGCs at their earliest developmental stages. Here we describe the use of germline-restricted expression of stella, which is currently the best marker gene for PGCs. We generated two stella-GFP reporters and show that both transgenes surpass other reporters in terms of timing and specificity of expression in PGCs. Additionally, we demonstrate the usefulness of stella-GFP during the derivation of PGCs from ES cells.     

Genetic changes from introgression of highland Mexican germ plasm into a Corn Belt Dent population of maize

Summary A backcross population (NZS1) of maize (Zea mays L.) was produced by crossing a highland Mexican population with the elite Corn Belt Dent synthetic AS3, and then by backcrossing to AS3. S₁ lines, S₂ lines, and S₂ testcrosses with an elite tester were used to compare the means, correlations, genetic variances, and predicted gains from selection of NZS1 and AS3 for grain yield, grain moisture at harvest, root and stalk lodging in a cool, temperate environment in New Zealand. The S₁ and S₂ lines from NZS1 had lower mean grain yields, higher levels of root lodging and higher mean grain moistures than the S₁ and S₂ lines from AS3. Mean grain yields of testcrosses of NZS1 and AS3 were similar, but NZS1 testcrosses had higher levels of root lodging. Genotypic variances estimated from S₁ and S₂ lines were larger for grain yield and root lodging for NZS1, smaller for grain moisture, and similar for stalk lodging. Predicted gains from selection for grain yield using intrapopulation methods based on the additive-genetic variance were larger for NZS1, but predicted gains for testcross selection were similar for the two populations. Lines with high combining ability for grain yield and acceptable grain moisture in combination with the tester occurred in NZS1. Because of the higher additive-genetic variance and the occurrence of lines with high combining ability for grain yield, we concluded that populations including highland Mexican germ plasm should be valuable for recurrent selection programs in New Zealand and in other cool, temperate regions.