Cell Division In Embryos Research Articles

Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation.

In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AX (γH2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of γH2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis.

Dynamics of abscisic acid and indole-3-acetic acid during the early-middle stage of seed development in Rosa hybrida

Concentrations of endogenous abscisic acid (ABA) and indole-3-acetic acid (IAA) in Rosa hybrida seed coat and embryos were determined at 28, 35, 42, and 49 days after pollination (DAP), a period encompassing the early-middle stages of seed development. No studies on rose have ever documented simultaneous change in ABA and IAA during these developmental phases in both seed coat and embryo. Plant growth regulators were extracted and then quantified by using high performance liquid chromatography based on solid phase extraction purification. In both the seed coat and embryo, ABA content decreased from 28 DAP (4.39 and 1.36 pmol mg−1, respectively) and onward. Endogenous IAA in seed coat followed the same trend. In contrast, IAA in embryo began to increase at 28 DAP (2.06 pmol mg−1), peaked at 42 DAP (5.06 pmol mg−1), and then declined dramatically at 49 DAP (1.17 pmol mg−1). In embryo, the IAA/ABA ratio was always >1.0 and showed a tendency to increase from 28 DAP to the maximum significant rate at 42 DAP (9.20). The ABA decrease associated with increased IAA levels in embryo could be a result of crosstalk between these two phytohormones. Such a change in the IAA/ABA ratio may signal the end of endodormancy caused by ABA at the pre-cotyledonary stage and the start of increased embryo cell division during the cotyledonary stage, which also results in increased hip weight.

A New Model System to Explore the Mechanisms and Functions of Global Cortical Contraction Waves in Oocyte and Embryo Cell Divisions

Cortical contractions are observable in cell divisions in many species, from meiotic divisions in Ascidian and starfish eggs to the early mitotic divisions of the Xenopus embryo. There have been many hypotheses concerning the function of these contractions, from generating pressure gradients to localizing mRNAs. Importantly, the physical and molecular mechanisms underlying these global contractions remain unknown, but would be essential in providing insights into their functions.As a system well-suited for live imaging and physical manipulations, we are studying the prominent contraction that occurs at the highly asymmetric meiotic division in starfish oocytes. In this system, the contraction occurs immediately before the division but its importance for the division remains controversial. Therefore, we set out to understand the molecular details and mechanics of the cortical contraction.Detailed analysis of the oocytes' shape and cytoplasmic flows revealed that the contraction wave is a flattening of the cell cortex which progresses across the whole oocyte towards the site of division with increasing intensity. This results in a strong and varied cytoplasmic flow. Visualizing the localization of components of the cytoskeleton indicates that F-actin and non-muscle myosin II are recruited to the cortical area currently undergoing contraction.To test the involvement of myosin II in the contraction, we applied the myosin II inhibitor blebbistatin that strongly hindered the contraction wave. Our analysis of the inhibition phenotype will reveal important details of the mechanisms of the contraction wave and allow us to determine the contraction waves' function. We will search for additional factors controlling the contraction wave for which an important clue is that changing the nuclear position prior to contraction repositions the axis of contraction, suggesting the involvement of nuclear factors.

In vitro auxin treatment promotes cell division and delays endoreduplication in developing seeds of the model legume species Medicago truncatula

The role of auxins in the morphogenesis of immature seeds of Medicago truncatula was studied, focusing on the transition from the embryo cell division phase to seed maturation. We analyzed seed development in vitro, by flow cytometry, and through the determination of the kinetics of seed fresh weight and size. Thus, seeds were harvested at 8, 10 and 12 days after pollination and cultured in vitro on a medium either without auxin or supplemented with indole‐3‐butyric acid (IBA) or naphthalene acetic acid (NAA) at 1 mg l−1. All parameters studied were determined every 2 days from the start of in vitro culture. The results showed that both auxins increased the weight and size of seeds with NAA having a stronger effect than IBA. We further demonstrated that the auxin treatments modulate the transition between mitotic cycles and endocycles in M. truncatula developing seed by favoring sustained cell divisions while simultaneously prolonging endoreduplication, which is known to be the cytogenetical imprint of the transition from the cell division phase to the storage protein accumulation phase during seed development.

Autonomy in specification of primordial germ cells and their passive translocation in the sea urchin.

The process of germ line determination involves many conserved genes, yet is highly variable. Echinoderms are positioned at the base of Deuterostomia and are crucial to understanding these evolutionary transitions, yet the mechanism of germ line specification is not known in any member of the phyla. Here we demonstrate that small micromeres (SMics), which are formed at the fifth cell division of the sea urchin embryo, illustrate many typical features of primordial germ cell (PGC) specification. SMics autonomously express germ line genes in isolated culture, including selective Vasa protein accumulation and transcriptional activation of nanos; their descendants are passively displaced towards the animal pole by secondary mesenchyme cells and the elongating archenteron during gastrulation; Cadherin (G form) has an important role in their development and clustering phenotype; and a left/right integration into the future adult anlagen appears to be controlled by a late developmental mechanism. These results suggest that sea urchin SMics share many more characteristics typical of PGCs than previously thought, and imply a more widely conserved system of germ line development among metazoans.

Glucocorticoid receptor activation following elevated oocyte cortisol content is associated with zygote activation, early embryo cell division, and IGF system gene responses in rainbow trout

Increased in ovo cortisol content of rainbow trout oocytes from ~3·5 to ~5·0 ng.oocyte(-1) before fertilization enhances the growth of embryos and juveniles and changes the long-term expression pattern of IGF-related genes. This study used embryos reared from oocytes enriched with cortisol and the glucocorticoid receptor (GR) antagonist, RU486, to determine whether the growth-promoting actions of cortisol involve GR protein activation and modulation of gr expression. Whole-mount in situ immunohistofluorescence studies of zygotes showed that enhanced oocyte cortisol increased the immunofluorescent GR signal and activated the relocation of GR from a general distribution throughout the cytoplasm to an accumulation in the peri-nuclear cytoplasm. In ovo cortisol treatment increased the number of embryonic cells within 48-h post-fertilization, and RU486 partially suppressed this cortisol stimulation of cell duplication. In addition, there was complex interplay between the expression of gr and igf system-related genes spatiotemporally in the different treatment groups, suggesting a role for GR in the regulation of the expression of development. Taken together, these findings indicate an essential role for GR in the regulation of epigenomic events in very early embryos that promoted the long-term growth effects of the embryos and juvenile fish. Moreover, the pretreatment of the oocyte with RU486 had a significant suppressive effect on the maternal mRNA transcript number of gr and igf system-related genes in oocytes and very early stage embryos, suggesting an action of antagonist on the stability of the maternal transcriptome.

Timing of cell division in human cleavage-stage embryos is linked with blastocyst formation and quality

Noninvasive markers of embryo quality are being sought to improve IVF success. The present study aimed to discover possible associations between embryo division kinetics in the cleavage stage, the subsequent ability of human embryos to reach the blastocyst stage and the resulting blastocyst morphology. A retrospective cohort study analysed 834 embryos from 165 oocyte donation couples using a time-lapse monitoring system that allowed the recording of the exact timings for key events related to embryo development. Timing parameters were categorized into four quartiles. The probability of an embryo developing to a blastocyst was linked to a strict chronology of development. To further evaluate the relationships between these morphokinetic parameters and subsequent blastocyst formation, the ensuing blastocyst morphology was compared with a viability score based on a hierarchical classification of the cleavage-stage morphokinetic parameters. It is concluded that the kinetics of early embryo development and the potential for human embryos to develop to the blastocyst stage on day 5 are closely related and that time-lapse-based evaluation of the exact timing of early events in embryo development is a promising tool for the prediction of blastocyst formation and quality according to the proposed model.Assisted reproduction treatment has helped several thousand infertile couples to start a family. Even though treatment is presently very successful, there is still scope for improvement in the process of selecting the embryos for transfer that have the highest implantation potential. An improved selection procedure may be able to increase the pregnancy rates, or alternatively, to reduce the number of embryos transferred while maintaining high pregnancy rates.We report the results after using an incubator with time-lapse video recording and image analysis to determine the timing of key events during embryo development. The events were assessed as potential markers of embryo morphology and development. One of the major factors that has already helped to increase success rates is extended embryo culture and blastocyst transfer on day 5 of development, because at this stage the embryo has stronger developmental competence. Also embryo/uterine synchrony is better achieved by a blastocyst-stage than a cleavage-stage embryo. However, extended culture in vitro is time consuming and extended exposure to artificial culture conditions in vitro may stress the embryo and could have negative side effects. For that reason, we sought evidence in the data obtained from the time-lapse monitoring system for a link between timing of early embryo cell divisions and the probability of subsequent development of embryos into blastocysts. The purpose of this was to avoid extended culture if blastocyst development could instead be reliably predicted from time-lapse images of the early cleavage states.

Regulation of Arabidopsis embryo and endosperm development by the polypeptide signaling molecule CLE8.

The plant seed is a major nutritional source for humans as well as an essential embryo development and dispersal unit. To ensure proper seed formation, fine spatial and temporal coordination between the embryo, endosperm, and maternal seed components must be achieved. However, the intercellular signaling pathways that direct the synchronous development of these tissues are poorly understood. Here we show that the Arabidopsis thaliana peptide ligand CLAVATA3/embryo surrounding region-related8 (CLE8) is exclusively expressed in young embryos and endosperm, and that it acts cell and noncell autonomously to regulate basal embryo cell division patterns, endosperm proliferation, and the timing of endosperm differentiation. CLE8 positively regulates expression of the transcription factor gene Wuschel-like homeobox8 (WOX8), and together CLE8 and WOX8 form a signaling module that promotes seed growth and overall seed size. These results demonstrate that seed development is coordinated by a secreted peptide ligand that plays a key early role in orchestrating cell patterning and proliferation in the embryo and endosperm.

The Arabidopsis APC4 subunit of the anaphase‐promoting complex/cyclosome (APC/C) is critical for both female gametogenesis and embryogenesis

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that is involved in regulating cell-cycle progression. It has been widely studied in yeast and animal cells, but the function and regulation of the APC/C in plant cells are largely unknown. The Arabidopsis APC/C comprises at least 11 subunits, only a few of which have been studied in detail. APC4 is proposed to be a connector in the APC/C in yeast and animals. Here, we report the functional characterization of the Arabidopsis APC4 protein. We examined three heterozygous plant lines carrying apc4 alleles. These plants showed pleiotropic developmental defects in reproductive processes, including abnormal nuclear behavior in the developing embryo sac and aberrant cell division in embryos; these phenotypes differ from those reported for mutants of other subunits. Some ovules and embryos of apc4/+ plants also accumulated cyclin B protein, a known substrate of APC/C, suggesting a compromised function of APC/C. Arabidopsis APC4 was expressed in meristematic cells of seedlings, ovules in pistils and embryos in siliques, and was mainly localized in the nucleus. Additionally, the distribution of auxin was distorted in some embryos of apc4/+ plants. Our results indicate that Arabidopsis APC4 plays critical roles in female gametogenesis and embryogenesis, possibly as a connector in APC/C, and that regulation of auxin distribution may be involved in these processes.

Combined third-harmonic generation and four-wave mixing microscopy of tissues and embryos

Nonlinear microscopy can be used to probe the intrinsic optical properties of biological tissues. Using femtosecond pulses, third-harmonic generation (THG) and four-wave mixing (FWM) signals can be efficiently produced and detected simultaneously. Both signals probe a similar parameter, i.e. the real part of the third-order nonlinear susceptibility χ(3). However THG and FWM images result from different phase matching conditions and provide complementary information. We analyze this complementarity using calculations, z-scan measurements on water and oils, and THG-FWM imaging of cell divisions in live zebrafish embryos. The two signals exhibit different sensitivity to sample size and clustering in the half-wavelength regime. Far from resonance, THG images reveal spatial variations |Δχ(3)(−3ω;ω,ω,ω)| with remarkable sensitivity while FWM directly reflects the distribution of χ(3)(−2ω1 + ω2;ω1, –ω2, ω1). We show that FWM images provide χ(3) maps useful for proper interpretation of cellular THG signals, and that combined imaging carries additional structural information. Finally we present simultaneous imaging of intrinsic THG, FWM, second-harmonic (SHG) and two-photon-excited fluorescence (2PEF) signals in live Caenorhabditis elegans worms illustrating the information provided by multimodal nonlinear imaging of unstained tissue.

Growth-Arrest-Specific Protein 2 Inhibits Cell Division in Xenopus Embryos

BackgroundGrowth-arrest-specific 2 gene was originally identified in murine fibroblasts under growth arrest conditions. Furthermore, serum stimulation of quiescent, non-dividing cells leads to the down-regulation of gas2 and results in re-entry into the cell cycle. Cytoskeleton rearrangements are critical for cell cycle progression and cell division and the Gas2 protein has been shown to co-localize with actin and microtubules in interphase mammalian cells. Despite these findings, direct evidence supporting a role for Gas2 in the mechanism of cell division has not been reported.Methodology and Principal FindingsTo determine whether the Gas2 protein plays a role in cell division, we over-expressed the full-length Gas2 protein and Gas2 truncations containing either the actin-binding CH domain or the tubulin-binding Gas2 domain in Xenopus laevis embryos. We found that both the full-length Gas2 protein and the Gas2 domain, but not the CH domain, inhibited cell division and resulted in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that Gas2 function is mediated by microtubule binding. Gas2 co-localized with microtubules at the cell cortex of Gas2-injected Xenopus embryos using cryo-confocal microscopy and co-sedimented with microtubules in cytoskeleton co-sedimentation assays. To investigate the mechanism of Gas2-induced cell division arrest, we showed, using a wound-induced contractile array assay, that Gas2 stabilized microtubules. Finally, electron microscopy studies demonstrated that Gas2 bundled microtubules into higher-order structures.Conclusion and SignificanceOur experiments show that Gas2 inhibits cell division in Xenopus embryos. We propose that Gas2 function is mediated by binding and bundling microtubules, leading to cell division arrest.

TopBP1 Deficiency Causes an Early Embryonic Lethality and Induces Cellular Senescence in Primary Cells

TopBP1 plays important roles in chromosome replication, DNA damage response, and other cellular regulatory functions in vertebrates. Although the roles of TopBP1 have been studied mostly in cancer cell lines, its physiological function remains unclear in mice and untransformed cells. We generated conditional knock-out mice in which exons 5 and 6 of the TopBP1 gene are flanked by loxP sequences. Although TopBP1-deficient embryos developed to the blastocyst stage, no homozygous mutant embryos were recovered at E8.5 or beyond, and completely resorbed embryos were frequent at E7.5, indicating that mutant embryos tend to die at the peri-implantation stage. This finding indicated that TopBP1 is essential for cell proliferation during early embryogenesis. Ablation of TopBP1 in TopBP1(flox/flox) mouse embryonic fibroblasts and 3T3 cells using Cre recombinase-expressing retrovirus arrests cell cycle progression at the G(1), S, and G(2)/M phases. The TopBP1-ablated mouse cells exhibit phosphorylation of H2AX and Chk2, indicating that the cells contain DNA breaks. The TopBP1-ablated mouse cells enter cellular senescence. Although RNA interference-mediated knockdown of TopBP1 induced cellular senescence in human primary cells, it induced apoptosis in cancer cells. Therefore, TopBP1 deficiency in untransformed mouse and human primary cells induces cellular senescence rather than apoptosis. These results indicate that TopBP1 is essential for cell proliferation and maintenance of chromosomal integrity.

Identification of a new isoform of eEF2 whose phosphorylation is required for completion of cell division in sea urchin embryos

Elongation factor 2 (eEF2) is the main regulator of peptide chain elongation in eukaryotic cells. Using sea urchin eggs and early embryos, two isoforms of eEF2 of respectively 80 and 83kDa apparent molecular weight have been discovered. Both isoforms were identified by immunological analysis as well as mass spectrometry, and appeared to originate from a unique post-translationally modified protein. Accompanying the net increase in protein synthesis that occurs in early development, both eEF2 isoforms underwent dephosphorylation in the 15min period following fertilization, in accordance with the active role of dephosphorylated eEF2 in regulation of protein synthesis. After initial dephosphorylation, the major 83kDa isoform remained dephosphorylated while the 80kDa isoform was progressively re-phosphorylated in a cell-cycle dependent fashion. In vivo inhibition of phosphorylation of the 80kDa isoform impaired the completion of the first cell cycle of early development implicating the involvement of eEF2 phosphorylation in the exit from mitosis.

Sea Urchin Embryotoxicity Test for Environmental Contaminants—Potential Role of the MRP Proteins

The main goals of this study were (1) to standardize a simple and reliable embryotoxicity test for environmental contaminants and (2) to evaluate the presence and possible protective role of the multidrug resistance-associated (MRP) protein-mediated multixenobiotic defense in two Mediterranean sea urchin species, Paracentrotus lividus and Arbacia lixula. Toxic end-point used was the success of the first cell division in sea urchin embryos. Embryotoxicities of three environmentally relevant contaminants: mercuric chloride (HgCl2, 0.05–6 μM), trybutiltin (TBT, 2.5–500 nM), and oxybenzone (OXI, 0.1–100 μM); as well as seawater samples collected from the polluted and unpolluted locations, were determined and compared. A. lixula embryos were more sensitive to all three toxic compounds, and both P. lividus and A. lixula embryos were highly sensitive to TBT at nanomolar concentrations (EC50 49 ± 5 and 36.8 ± 3 nM, respectively). Inhibition of MRP protein by specific inhibitor MK571 caused significant increase in embryotoxic potency of HgCl2 (EC50 0.697 ± 0.03 and 0.245 ± 0.04 μM, respectively), TBT (EC50 24 ± 3 and 7.4 ± 1 nM, respectively) and polluted seawater sample, but not of OXI or unpolluted, natural seawater. Therefore, our results demonstrated for the first time the protective relevance of MRP proteins in early development of these two Mediterranean sea urchin species. Finally, the embryotoxicity protocol described in this study represents a simple and rapid bioassay for determination of environmentally relevant seawater contamination.

A new type of specialized morphophysiological dormancy and seed storage behaviour in Hydatellaceae, an early-divergent angiosperm family.

Recent phylogenetic analysis has placed the aquatic family Hydatellaceae as an early-divergent angiosperm. Understanding seed dormancy, germination and desiccation tolerance of Hydatellaceae will facilitate ex situ conservation and advance hypotheses regarding angiosperm evolution. Seed germination experiments were completed on three species of south-west Australian Hydatellaceae, Trithuria austinensis, T. bibracteata and T. submersa, to test the effects of temperature, light, germination stimulant and storage. Seeds were sectioned to examine embryo growth during germination in T. austinensis and T. submersa. Some embryo growth and cell division in T. austinensis and T. submersa occurred prior to the emergence of an undifferentiated embryo from the seed coat ('germination'). Embryo differentiation occurred later, following further growth and a 3- to 4-fold increase in the number of cells. The time taken to achieve 50 % of maximum germination for seeds on water agar was 50, 35 and 37 d for T. austinensis, T bibracteata and T. submersa, respectively. Seeds of Hydatellaceae have a new kind of specialized morphophysiological dormancy in which neither root nor shoot differentiates until after the embryo emerges from the seed coat. Seed biology is discussed in relation to early angiosperm evolution, together with ex situ conservation of this phylogenetically significant group.

Effects of Bleomycin on microspore embryogenesis in Brassica napus and detection of somaclonal variation using AFLP molecular markers

Bleomycin, a glycopeptide antibiotic produced by the bacterium Streptomyces verticillus, has been demonstrated to be an effective mutagen in Arabidopsis thaliana. The objective of the present study was to determine the effect of bleomycin on embryo production and to assess the genetic variation of the doubled haploid (DH) populations by amplified fragment length polymorphism (AFLP). The effects of bleomycin on microspore embryogenesis and cell division were investigated using three concentrations of bleomycin in five semi-winter genotypes of Brassica napus viz. T8, T10, B409, P30, and DH1142. Inclusion of bleomycin in the culture medium at a concentration of 0.1 μg ml−1 for 30 min significantly improved embryo production and cell division in all five genotypes. Embryo production was induced at rates two- and four-fold higher than controls after bleomycin treatment. Fifty plants regenerated by microspore embryogenesis treated with bleomycin in addition to non-treated controls of T8, T10, and B409 were selected for AFLP analysis. The results suggest that microspore culture is capable of producing 0.095–0.114% genetic variation, and there was no effect of bleomycin treatment on genetic stabilisation of doubled haploid populations versus the non-treated control.

278 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON THE NORMALITY OF BOVINE EMBRYOS PRODUCED IN VITRO

We previously reported that follicular wave synchronization by dominant follicle removal on Day 5 and the start of a superstimulatory treatment on Day 7 after ovum pick-up (OPU) was effective to increase oocyte quality (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The present study was designed to examine the effect of superstimulatory treatment-induced follicular wave synchronization on quality of embryos obtained by OPU and in vitro production. Japanese Black cows were reared under the same feeding and environmental conditions and 2 OPU sessions were conducted in each cow. The first OPU session was performed in 7 cows at arbitrary days of estrous cycle using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner. Then, follicles larger than 8 mm in diameter were aspirated and CIDR was inserted on Day 5 (the day of first OPU session = Day 0). The cows then received 30 mg of FSH twice a day from Days 7 to 10 in decreasing doses (4, 4, 3, 3, 2, 2, 1, 1 mg per shot) by i.m. injections. Cloprostenol (PGF; 0.75 mg) was administered in the morning of Day 9. The second OPU session was performed 48 h after PGF administration (Day 11) and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Grade 1 and 2 cumulus oocyte complexes were in vitro matured, fertilized (IVF), and cultured as described by Imai et al. (2006 J. Reprod. Dev. 52, Suppl. S19-29). Some zygotes were fixed and stained to check their sperm penetration. Embryo development was monitored by time-lapse cinematography for 168 h after IVF. Cleavage pattern of embryos was classified morphologically into normal and abnormal (including those with multiple fragments, protrusions, 3 to 4 blastomeres, and uneven cell division) groups at their first cleavage. Normal penetration rate of second OPU session was significantly (P < 0.05) higher than that of the first OPU session. There were no differences in the mean percentage of total blastocyst and grade 1 blastocyst rates between the first (45.2 and 46.9%, respectively) and second (47.5 and 41.8%, respectively) OPU sessions. However, the rates of blastocysts developing from embryos that were beyond the 4-cell stage at 48 h after IVF was significantly (P < 0.05) higher after the second OPU session (81.2%) than after the first OPU session (67.4%). Furthermore, a significant difference (P < 0.05) was found in the rates of normal cleavage at the first cell division in embryos that developed to the blastocyst stage between the first and second OPU sessions (53.3% and 73.9%, respectively). These results indicate that superstimulatory treatment-induced follicular wave synchronization improved the normality of fertilization and development of cattle oocytes obtained by OPU. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

Functional Analyses of LONELY GUY Cytokinin-Activating Enzymes Reveal the Importance of the Direct Activation Pathway in Arabidopsis

Cytokinins play crucial roles in diverse aspects of plant growth and development. Spatiotemporal distribution of bioactive cytokinins is finely regulated by metabolic enzymes. LONELY GUY (LOG) was previously identified as a cytokinin-activating enzyme that works in the direct activation pathway in rice (Oryza sativa) shoot meristems. In this work, nine Arabidopsis thaliana LOG genes (At LOG1 to LOG9) were predicted as homologs of rice LOG. Seven At LOGs, which are localized in the cytosol and nuclei, had enzymatic activities equivalent to that of rice LOG. Conditional overexpression of At LOGs in transgenic Arabidopsis reduced the content of N(6)-(Delta(2)-isopentenyl)adenine (iP) riboside 5'-phosphates and increased the levels of iP and the glucosides. Multiple mutants of At LOGs showed a lower sensitivity to iP riboside in terms of lateral root formation and altered root and shoot morphology. Analyses of At LOG promoter:beta-glucuronidase fusion genes revealed differential expression of LOGs in various tissues during plant development. Ectopic overexpression showed pleiotropic phenotypes, such as promotion of cell division in embryos and leaf vascular tissues, reduced apical dominance, and a delay of leaf senescence. Our results strongly suggest that the direct activation pathway via LOGs plays a pivotal role in regulating cytokinin activity during normal growth and development in Arabidopsis.

Somatic Embryogenesis in Juniperus Procera using Juniperus Communis as a Model

The study of somatic embryogenesis in Juniperus communis has been conducted as a preliminary study for the further development of somatic embryogenesis, micropropagation and long-term conservation/cryopreservation in Juniperus procera, which is economically and ecologically important and endangered forest species in Ethiopia. The aim for this particular research was initially an adaptation of optimum half strength lithium chloride-sodium propionate (LP) medium protocol for growth and proliferation of embryogenic culture. But as other conifer species it was difficult to stop proliferation of somatic embryo cell division and bring them to maturation stage. However using optimal concentration of ABA (32 mg/l), it was possible to get maturation response in most of the cultures. Additional study on the effect of seed extraction to the growing embryogenic culture showed no effect on mature somatic embryos. Rather they were showing faster growth which increases embryogenic mass of each culture. It was also observed that green premature seeds of Juniperus are suitable for the initiation and proliferation of somatic embryo. Key words: Cryopreservation, Juniperus communis, Juniperus procera, Somatic embryogenesis.

Phylogenetic conservation of cytostatic factor related genes in the ascidian Ciona intestinalis

In all vertebrates, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase-I, others at prophase-I. Fertilization induces completion of meiosis and entry into the first mitotic division. Several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (CSF) and the maturation promoting factor (MPF) in metaphase I or II. Recently, we proposed the oocytes of ascidian Ciona intestinalis as a new model to study the meiotic division. Here, taking advantage of the recent publication of the C. intestinalis genome, we presented a phylogenetic analysis of key molecular components of the CSF-related machinery. We showed that the Mos/MAP kinase pathway is perfectly conserved in ascidians. We demonstrated the presence of a CSF-like activity in metaphase-I arrested C. intestinalis oocytes able to block cell division in two-cell embryos. We further investigated the regulation of CSF by demonstrating that both CSF and MPF inactivation, at the exit of metaphase-I, are independent from protein synthesis, indicating the absence of short-lived factors that regulate metaphase stability, as in other invertebrate species. The results obtained suggest that meiotic regulation in C. intestinalis resembles that of vertebrates, such as Xenopus accordingly to the position of this organism in the evolutionary tree.