Dynamic expression patterns of <Emphasis Type="Italic">vasa</Emphasis> during embryogenesis in the cricket <Emphasis Type="Italic">Gryllus bimaculatus</Emphasis>

The specification of germ cells during embryogenesis is an important issue in the development of metazoans. In insects, the mode of germ cell specification appears to be highly variable among species and molecular data are not sufficient to provide an evolutionary perspective to this issue. Expression of vasa can be used as a germ line marker. Here, we report the isolation of a vasa-like gene in a hemimetabolous insect, the cricket Gryllus bimaculatus (Gb'vas), and its expression patterns during oogenesis and embryogenesis. Gb'vas is preferentially expressed in the germarium and the expression of Gb'vas is detectable throughout vitellogenesis including mature eggs subjected to oviposition, suggesting that Gb'vas is maternally contributed to the cricket eggs. The zygotic expression of Gb'vas appears to start at the mid blastoderm stage in the posterior region of the egg, expanding in a developing germ anlage. In early germbands, an intense expression of Gb'vas is restricted to the posterior end. In later embryos, Gb'vas expression extends over the whole body and then distinctly localized to the embryonic gonad at the stage immediately before hatching. These results suggest that, in the cricket, germ cells are specified early in development at the posterior end of an early germband, as proposed by Heymons (1895) based on cytological criteria.     

Cloning and characterization of naringenin 8-prenyltransferase, a flavonoid-specific prenyltransferase of Sophora flavescens

Prenylated flavonoids are natural compounds that often represent the active components in various medicinal plants and exhibit beneficial effects on human health. Prenylated flavonoids are hybrid products composed of a flavonoid core mainly attached to either 5-carbon (dimethylallyl) or 10-carbon (geranyl) prenyl groups derived from isoprenoid (terpenoid) metabolism, and the prenyl groups are crucial for their biological activity. Prenylation reactions in vivo are crucial coupling processes of two major metabolic pathways, the shikimate-acetate and isoprenoid pathways, in which these reactions are also known as a rate-limiting step. However, none of the genes responsible for the prenylation of flavonoids has been identified despite more than 30 years of research in this field. We have isolated a prenyltransferase gene from Sophora flavescens, SfN8DT-1, responsible for the prenylation of the flavonoid naringenin at the 8-position, which is specific for flavanones and dimethylallyl diphosphate as substrates. Phylogenetic analysis shows that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. The gene expression of SfN8DT-1 is strictly limited to the root bark where prenylated flavonoids are solely accumulated in planta. The ectopic expression of SfN8DT-1 in Arabidopsis thaliana resulted in the formation of prenylated apigenin, quercetin, and kaempferol, as well as 8-prenylnaringenin. SfN8DT-1 represents the first flavonoid-specific prenyltransferase identified in plants and paves the way for the identification and characterization of further genes responsible for the production of this large and important class of secondary metabolites.     

Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis

Primordial germ cells (PGCs) are derived from a population of pluripotent epiblast cells in mice. However, little is known about when and how PGCs acquire the capacity to differentiate into functional germ cells, while keeping the potential to derive pluripotent embryonic germ cells and teratocarcinomas. In this investigation, we show that epiblast cells and PGCs can establish colonies of spermatogenesis after transfer into postnatal seminiferous tubules of surrogate infertile mice. Furthermore, we obtained normal fertile offspring by microinsemination using spermatozoa or spermatids derived from PGCs harvested from fetuses as early as 8.5 days post coitum. Thus, fetal male germ cell development is remarkably flexible, and the maturation process, from epiblast cells through PGCs to postnatal spermatogonia, can occur in the postnatal testicular environment. Primordial germ cell transplantation techniques will also provide a novel tool to assess the developmental potential of PGCs, such as those manipulated in vitro or recovered from embryos harboring lethal mutations.     

Genetic and epigenetic properties of mouse male germline stem cells during long-term culture

Although stem cells are believed to divide infinitely by self-renewal division, there is little evidence that demonstrates their infinite replicative potential. Spermatogonial stem cells are the founder cell population for spermatogenesis. Recently, in vitro culture of spermatogonial stem cells was described. Spermatogonial stem cells can be expanded in vitro in the presence of glial cell line-derived neurotrophic factor (GDNF), maintaining the capacity to produce spermatogenesis after transplantation into testis. Here, we examined the stability and proliferative capacity of spermatogonial stem cells using cultured cells. Spermatogonial stem cells were cultured over 2 years and achieved approximately 10(85)-fold expansion. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, spermatogonial stem cells retained the euploid karyotype and androgenetic imprint during the 2-year experimental period, and produced normal spermatogenesis and fertile offspring. However, the telomeres in spermatogonial stem cells gradually shortened during culture, suggesting that they are not immortal. Nevertheless, the remarkable stability and proliferative potential of spermatogonial stem cells suggest that they have a unique machinery to prevent transmission of genetic and epigenetic damages to the offspring, and these characteristics make them an attractive target for germline modification.     

Location of micropyles and early embryonic development of the two-spotted cricket Gryllus bimaculatus (Insecta, Orthoptera)

Early embryogenesis of the two-spotted cricket Gryllus bimaculatus was examined by scanning electron microscopy and several fluorescence staining methods, with special reference to these four issues: (i) the location of micropyles; (ii) the transfer of the female pronucleus following meiosis; (iii) the timing of cellularization; and (iv) the process of the germ primordium formation. Between two and four micropyles lie in the mid-ventral region of the egg. The egg nucleus is at the mid-dorsal periphery of the new laid egg, and meiosis resumes and is completed there. The female pronucleus moves to the mid-ventral side, and fertilization occurs there. Energid starts to proliferate and migrates to the periphery of the egg, initiating blastoderm formation. Actin caps surround each superficial nucleus. Cellularization occurs during the blastoderm stage. At a late blastoderm stage, nuclei aggregate in both the posterolateral patch-like regions of the egg to form a germ primordium. The germ primordium looks like a pair of dumbbells. Both the patches shift towards the ventral side and fuse into a germ primordium. The germ primordium contracts to produce a clearly delineated germ band. Observations on distribution patterns of F-actin indicate that, all through the process, the germ primordium retains that unity, and is not separated into two parts.     

CD9 is a surface marker on mouse and rat male germline stem cells

Spermatogenesis is dependent on a small population of stem cells. Despite the biological significance of spermatogonial stem cells, their analysis has been hampered by their scarcity. However, spermatogonial stem cells can be enriched by selection with an antibody against cell-surface molecules. In this investigation, we searched for new antigens expressed on spermatogonial stem cells. Using the spermatogonial transplantation technique, we examined expression of the CD9 molecule, which is commonly expressed on stem cells of other tissues. Selection of both mouse and rat testis cells with anti-CD9 antibody resulted in 5- to 7-fold enrichment of spermatogonial stem cells from intact testis cells, indicating that CD9 is commonly expressed on spermatogonial stem cells of both species. Therefore, CD9 may be involved in the common machinery in stem cells of many self-renewing tissues, and the identification of a common surface antigen on spermatogonial stem cells of different species has important implications for the development of a technique to enrich stem cells from other mammalian species.     

Regulation of mouse spermatogonial stem cell self-renewing division by the pituitary gland

Spermatogenesis originates in spermatogonial stem cells, which have the unique mode of replication. It is considered that a single stem cell can produce two stem cells (self-renewing division), one stem and one differentiating (asymmetric division), or two differentiating cells (differentiating division). However, little is known regarding how each type of division is regulated. In this investigation, we focused on the analysis of self- renewing division and examined the effect of the pituitary gland using two models of stem cell self-renewing division. In the first experiment using newborn mice, the administration of GnRH- analogue, which represses the release of gonadotropin, reduced the number of stem cells during postnatal testicular development, suggesting that the pituitary gland enhances stem cell self- renewing division. In the second experiment, however, the number of stem cells increased dramatically in hypophysectomized adult recipients after spermatogonial transplantation. Thus, the pituitary gland affects the self-renewing division of stem cells, but these contradictory results suggest that its role may be different depending on the stage of the testicular development.     

Mass spectrometric analysis of posttranslational modifications of a carrot extracellular glycoprotein

Expression of extracellular dermal glycoprotein (EDGP) is induced by biotic or abiotic stress. The amino acid sequence alignment showed that EDGP shared significant homology with proteins from legumes, tomato, Arabidopsis, wheat, and cotton. These proteins are involved in signal transduction or stress response systems. Most of the Cys residues in these proteins are conserved, suggesting that they share similar tertiary structures. Surface plasmon resonance (SPR) analysis shows that EDGP binds a soybean 4-kDa hormone-like peptide (4-kDa peptide) in vitro and reduction of EDGP decreased significantly the binding activity, implying that posttranslational modifications are important for its function. Therefore, we investigated the posttranslational modifications in EDGP using mass spectrometry. As the result, six disulfide bonds in EDGP were identified: Cys(70)-Cys(158), Cys(84)-Cys(89), Cys(97)-Cys(113), Cys(100)-Cys(108), Cys(201)-Cys(426), and Cys(332)-Cys(378). In addition, the N-terminal glutamine was cyclized into pyroglutamic acid. All four putative glycosylation sites were occupied by N-linked glycans, which have similar masses of m/z 1171. Finally, measuring the mass of the native protein showed that the posttranslational modifications of EDGP (pI 9.5) involved only disulfide bonds, N-terminal modification, and glycosylation.     

Evolutionary and environmental effects on the geographical adaptation of herbivory resistance in native and introduced <Emphasis Type="Italic">Solidago altissima</Emphasis> populations

To understand rapid evolution in plant resistance to herbivory, it is critical to determine how the genetic correlation among resistances varies genetically and/or environmentally. We conducted a reciprocal transplant experiment of tall goldenrod, Solidago altissima with multiple replicates within the native range (USA) and the introduced range (Japan) to explore the differences in phenotypic traits of resistance to multiple herbivorous insects and their relationships between and within the countries. The Japanese plants were more resistant to the lace bug, Corythucha marmorata, which had recently invaded Japan, but were more susceptible to other herbivorous insects compared to the USA plants. An antagonistic relationship was found between plant resistances to lace bugs and other herbivorous insects in both USA and Japanese plants. In addition, this relationship was more obvious in gardens with a high level of foliage damage than in gardens with a low level of foliage damage by other herbivorous insects. An antagonistic relationship between resistances to aphids and lace bugs was also observed in USA gardens, but not in Japanese garden. These results suggest that the strength of constraints on the evolution of plant resistance due to genetic trade-offs may differ among biotic environments, including community structure of herbivorous insects. Therefore, differences in herbivorous insect communities between the native and introduced ranges can result in the rapid evolution of greater resistance in plants in the introduced range than in the native range.