Arrest Of Embryo Development Research Articles

Reciprocal compatibility within the genus Pisum L. as studied in F1 hybrids: 1. Crosses involving P. sativum L. subsp. sativum

Seven diverse accessions of peas including Pisum fulvum Boiss. et Noe (WL2140), Pisum abyssinicum A. Br. (VIR2759) and Pisum sativum L. subsp. elatius (Bieb.) Schmahl. were crossed reciprocally with a testerline WL1238 of the cultivated pea, P. sativum L. subsp. sativum. Efficiency of crosses (the average number of hybrid seeds per cross) in reciprocal directions and general characteristics of reciprocal F1 hybrids, such as F1 seed mass, pollen and seed fertility, height, yield and biomass, were compared. P. fulvum and P. abyssinicum showed strong reproductive barriers with P. sativum subsp. sativum, both prezygotic, as estimated by crossing efficiency, and postzygotic, manifested as hybrid sterility and weakness. Among crosses of five accessions of wild P. sativum subsp. elatius with the testerline, only hybrids with CE1 (Crimea) showed no difference in reciprocal combinations. Reciprocal hybrids with CE1 and JI1794 (Holan Heights) showed fully fertile pollen. In other cases reciprocal differences were registered as to pollen fertility and general vigour. Two special cases with wild peas as seed parents were observed: abnormal hybrids with VIR320 (Palestine) and arrest of embryo development in crosses with L100 (Be’er Sheva). In all cases of reciprocal differences except for those involving accession 721 (Mt. Carmel), vigour and fertility of hybrids were higher when WL1238 (P. sativum subsp. sativum) was used as seed parent. Paternal plastid DNA was registered in hybrids with WL2140 and VIR320 as seed parents. Reproductive barriers within P. sativum L. showed a complicated pattern scarcely corresponding to any intraspecies taxonomy.

Treatment with Ca2+ ionophore improves embryo development and outcome in cases with previous developmental problems: a prospective multicenter study

Does calcium ionophore treatment (A23187, calcimycin) improve embryo development and outcome in patients with a history of developmental problems/arrest? Application of A23187 leads to increased rates of cleavage to 2-cell stage, blastocyst formation and clinical pregnancy/live birth. Studies on lower animals indicate that changes in intracellular free calcium trigger and regulate the events of cell division. In humans, calcium fluctuations were detected with a peak shortly before cell division. Interestingly, these calcium oscillations disappeared in arrested embryos. Mitotic division blocked with a Ca(2+) chelator could be restored by means of ionophores in an animal model. This prospective, multicenter (five Austrian centers), uncontrolled intervention study (duration 1 year) includes 57 patients who provided informed consent. Inclusion criteria were complete embryo developmental arrest in a previous cycle (no transfer), complete developmental delay (no morula/blastocyst on Day 5), or reduced blastocyst formation on Day 5 (≤15%). Severe male factor patients and patients with <30% fertilization rate after ICSI were excluded because these would be routine indications for ionophore usage. The total of the 57 immediately preceding cycles in the same patients constituted the control cycles/control group. In the treatment cycles, all metaphase II-oocytes were exposed to a commercially available ready-to-use ionophore for 15 min immediately after ICSI. After a three-step washing procedure, in vitro culture was performed as in the control cycles, up to blastocyst stage when achievable. Fertilization rate did not differ (75.4 versus 73.2%); however, further cleavage to 2-cell stage was significantly higher (P < 0.001) in the ionophore group (98.5%) when compared with the control cycles (91.9%). In addition, significantly more (P < 0.05) blastocysts formed on Day 5 in the study compared with the control group (47.6 versus 5.5%, respectively) and this was associated with a significant increase (P < 0.01) in the rates of implantation (44.4 versus 12.5%), clinical pregnancy (45.1 versus 12.8%) and live birth (45.1 versus 12.8%). All babies born at the time of writing (22/28) were healthy. The frequency of patients showing embryo developmental problems was expected to be low; therefore, a multicenter approach was chosen in order to increase sample size. In one-third of the cycles, the clinician or patient requested a change of stimulation protocol; however, this did not influence the developmental rate of embryos. This is the first evidence that developmental incompetence of embryos is an additional indication for ionophore treatment. The present approach is exclusively for overcoming cleavage arrest. No funding received. T.E. reports fees from Gynemed, outside the submitted work. All co-authors have no interest to declare.

A host beetle pheromone regulates development and behavior in the nematode Pristionchus pacificus.

Nematodes and insects are the two most speciose animal phyla and nematode-insect associations encompass widespread biological interactions. To dissect the chemical signals and the genes mediating this association, we investigated the effect of an oriental beetle sex pheromone on the development and behavior of the nematode Pristionchus pacificus. We found that while the beetle pheromone is attractive to P. pacificus adults, the pheromone arrests embryo development, paralyzes J2 larva, and inhibits exit of dauer larvae. To uncover the mechanism that regulates insect pheromone sensitivity, a newly identified mutant, Ppa-obi-1, is used to reveal the molecular links between altered attraction towards the beetle pheromone, as well as hypersensitivity to its paralyzing effects. Ppa-obi-1 encodes lipid-binding domains and reaches its highest expression in various cell types, including the amphid neuron sheath and excretory cells. Our data suggest that the beetle host pheromone may be a species-specific volatile synomone that co-evolved with necromeny.

Identification of enzymes for adenosine-to-inosine editing and discovery of cytidine-to-uridine editing in nucleus-encoded transfer RNAs of Arabidopsis.

In all organisms, transfer RNAs (tRNAs) contain numerous modified nucleotides. For many base modifications in tRNAs, the functional significance is not well understood, and the enzymes performing the modification reactions are unknown. Here, we have studied members of a family of putative nucleotide deaminases in the model plant Arabidopsis (Arabidopsis thaliana). We show that two Arabidopsis genes encoding homologs of yeast (Saccharomyces cerevisiae) tRNA adenosine deaminases catalyze adenosine-to-inosine editing in position 34 of several cytosolic tRNA species. The encoded proteins (AtTAD2 and AtTAD3, for tRNA-specific adenosine deaminase) localize to the nucleus and interact with each other in planta in bimolecular fluorescence complementation and coimmunoprecipitation assays. Both AtTAD2 and AtTAD3 are encoded by essential genes whose knockout is lethal and leads to arrested embryo development at the globular stage. Knockdown mutants for AtTAD2 and AtTAD3 display reduced growth and inefficient editing from adenosine to inosine in six nucleus-encoded tRNA species. Moreover, upon comparison of DNA and complementary DNA sequences, we discovered cytidine-to-uridine RNA editing in position 32 of two nucleus-encoded serine tRNAs, tRNA-serine(AGA) and tRNA-serine(GCT). This adds a unique type of RNA editing to the modifications occurring in nuclear genome-encoded RNAs in plants.

Injection of ligand-free gold and silver nanoparticles into murine embryos does not impact pre-implantation development.

Intended exposure to gold and silver nanoparticles has increased exponentially over the last decade and will continue to rise due to their use in biomedical applications. In particular, reprotoxicological aspects of these particles still need to be addressed so that the potential impacts of this development on human health can be reliably estimated. Therefore, in this study the toxicity of gold and silver nanoparticles on mammalian preimplantation development was assessed by injecting nanoparticles into one blastomere of murine 2 cell-embryos, while the sister blastomere served as an internal control. After treatment, embryos were cultured and embryo development up to the blastocyst stage was assessed. Development rates did not differ between microinjected and control groups (gold nanoparticles: 67.3%, silver nanoparticles: 61.5%, sham: 66.2%, handling control: 79.4%). Real-time PCR analysis of six developmentally important genes (BAX, BCL2L2, TP53, OCT4, NANOG, DNMT3A) did not reveal an influence on gene expression in blastocysts. Contrary to silver nanoparticles, exposure to comparable Ag+-ion concentrations resulted in an immediate arrest of embryo development. In conclusion, the results do not indicate any detrimental effect of colloidal gold or silver nanoparticles on the development of murine embryos.

TFIIB co-localizes and interacts with α-tubulin during oocyte meiosis in the mouse and depletion of TFIIB causes arrest of subsequent embryo development.

TFIIB (transcription factor IIB) is a transcription factor that provides a bridge between promoter-bound TFIID and RNA polymerase II, and it is a target of various transcriptional activator proteins that stimulate the pre-initiation complex assembly. The localization and/or attachment matrix of TFIIB in the cytoplast is not well understood. This study focuses on the function of TFIIB and its interrelationship with α-tubulins in a mouse model. During oocyte maturation TFIIB distributes throughout the entire nucleus of the germinal vesicle (GV). After progression to GV breakdown (GVBD), TFIIB and α-tubulin co-localize and accumulate in the vicinity of the condensed chromosomes. During the MII stage, the TFIIB signals are more concentrated at the equatorial plate and the kinetochores. Colcemid treatment of oocytes disrupts the microtubule (MT) system, although the TFIIB signals are still present with the altered MT state. Injection of oocytes with TFIIB antibodies and siRNAs causes abnormal spindle formation and irregular chromosome alignment. These findings suggest that TFIIB dissociates from the condensed chromatids and then tightly binds to microtubules from GVBD to the MII phase. The assembly and disassembly of TFIIB may very well be associated with and driven by microtubules. TFIIB maintains its contact with the α-tubulins and its co-localization forms a unique distribution pattern. Depletion of Tf2b in oocytes results in a significant decrease in TFIIB expression, although polar body extrusion does not appear to be affected. Knockdown of Tf2b dramatically affects subsequent embryo development with more than 85% of the embryos arrested at the 2-cell stage. These arrested embryos still maintain apparently normal morphology for at least 96h without any obvious degeneration. Analysis of the effects of TFIIB in somatic cells by co-transfection of BiFC plasmids pHA-Tf2b and pFlag-Tuba1α further confirms a direct interaction between TFIIB and α-tubulins.

Intraspecific and interspecific crossability in three Ziziphus species (Rhamnaceae)

Three cultivated species of Ziziphus Mill. [Z. jujuba Mill., Z. mauritiana Lam. and Z. spina-christi (L.) Willd.] comprising a total of seventeen cultivars/genotypes were selected for a mating system study. Phenological stage determination showed an overlap in blooming between the three species during June. Intraspecific cross compatibility was evaluated for Z. jujuba and Z. mauritiana, and interspecific crosses between the three species were carried out during two flowering seasons. Following intraspecific hand cross-pollination, pollen tube formation and growth to the ovule 24 h after pollination (HAP) were observed in all three species. Although very low seed set was obtained following intraspecific Z. jujuba crosses, histological studies showed normal embryo formation and endosperm development, suggesting that double fertilization had occurred and that embryo development arrest occurred later. Following interspecific hand pollination, in vivo pollen grain germination and the formation of pollen tubes and their growth to the ovule 24 HAP were observed in compatible crosses, which also set fruit and seed. Viable embryos were obtained from Z. mauritiana × Z. jujuba, Z. mauritiana × Z. spina-christi and Z. spina-christi × Z. mauritiana crosses. Putative hybrids from these crosses were germinated in vitro, and the resultant plantlets were studied using flow cytometric analysis. Indications of true hybrid origin were obtained based on total 2C-DNA content of plantlets, but they died during the hardening-off process. This study showed that gene flow among these species is possible, thus increasing the potential that interspecific crosses can be used for genetic crop improvement.

A galactosyltransferase acting on arabinogalactan protein glycans is essential for embryo development in Arabidopsis

Arabinogalactan proteins (AGPs) are a complex family of cell-wall proteoglycans that are thought to play major roles in plant growth and development. Genetic approaches to studying AGP function have met limited success so far, presumably due to redundancy within the large gene families encoding AGP backbones. Here we used an alternative approach for genetic dissection of the role of AGPs in development by modifying their glycan side chains. We have identified an Arabidopsis glycosyltransferase of CAZY family GT31 (AtGALT31A) that galactosylates AGP side chains. A mutation in the AtGALT31A gene caused the arrest of embryo development at the globular stage. The presence of the transcript in the suspensor of globular-stage embryos is consistent with a role for AtGALT31A in progression of embryo development beyond the globular stage. The first observable defect in the mutant is perturbation of the formative asymmetric division of the hypophysis, indicating an essential role for AGP proteoglycans in either specification of the hypophysis or orientation of the asymmetric division plane.

Embryonic Diapause: Advances in Understanding the Enigma of Seasonal Delayed Implantation

Embryonic diapause is an evolutionary strategy by which a reversible arrest in embryo development occurs. In its two forms, facultative and obligate, it assures that offspring are born when optimal maternal and environmental conditions are present to ensure maximal survival. We have explored obligate delayed implantation in the mink (Neovison vison) over four decades: first by evaluation of the environmental regulation, then by determination of the pituitary factors that maintain diapause and provoke implantation followed by exploration of the ovarian contribution to the process. As the uterine environment is the proximal regulator of diapause, we employed a strategy of global gene analysis to discover differentially expressed pathways during embryo arrest and reactivation. These trials revealed that the synthesis of polyamines was increased in the uterus with reactivation of the embryo in vivo. Subsequent experiments demonstrated that the polyamine, putrescine, was capable of inducing escape of the embryo from obligate diapause, providing strong evidence that the paucity of polyamines induces developmental arrest, and reactivation is coupled to renewed uterine and/or embryonic synthesis of these polycations.

Pno1 Tissue-Specific Expression and Its Functions Related to the Immune Responses and Proteasome Activities

Pno1 is a protein that plays a role in proteasome and ribosome neogenesis in yeast. So far, its functions in mammalian cells have not been investigated. To understand its function in mammals, we performed in situ hybridization analysis of Pno1 expression in different development stages and generated Pno1 gene knockout (KO) and transgenic (Tg) mice lineages. The results showed early lethality of homozygous Pno1 KO lineage caused, as demonstrated in parallel by ex vivo experiments, by arrest of embryo development before compaction stage. Though, heterozygous (HET) mice with 50% of normal Pno1 mRNA concentration were fertile and showed no obvious anomalies. The lymphoid organs of HET mice were normal in size, weight and cellularity, with normal T and B cell subpopulations. TCR-triggered activation and proliferation of HET T cells were normal. Proteasome activities in HET organs were uncompromised. Tg mice with actin promoter-driven Pno1 expression were also fertile, with no apparent anomalies, although they expressed 2–5-fold higher Pno1 mRNA levels. The lymphoid organs of Tg mice were of normal size, weight and cellularity with normal T and B cell sub-populations. TCR-triggered activation and proliferation of Tg T cells were normal. Tg organs and tissues presented normal proteasome activity as did their wild type counterparts. Tagged Pno1 over-expression in L cells and density gradient fractionation established that Pno1 existed in large complexes with sedimentation rates between 20S and 26S, bigger than mature 26S proteasomes. Pno1 in fractions did not coincide with 40S or 60S ribosome subunits. Our study indicates that Pno1 is essential for cellular functions, but only a small percentage of its normal level is sufficient, and excessive amounts are neither harmful nor useful. The nature of the large complexes it associates with remains to be identified, but it is certain that they are not mature proteasomes or ribosomes.

LONO1 encoding a nucleoporin is required for embryogenesis and seed viability in Arabidopsis.

Early embryogenesis in Arabidopsis (Arabidopsis thaliana) is distinguished by a predictable pattern of cell divisions and is a good system for investigating mechanisms of developmental pattern formation. Here, we identified a gene called LONO1 (LNO1) in Arabidopsis in which mutations can abolish the first asymmetrical cell division of the zygote, alter planes and number of cell divisions in early embryogenesis, and eventually arrest embryo development. LNO1 is highly expressed in anthers of flower buds, stigma papilla of open flowers, and embryo and endosperm during early embryogenesis, which is correlated with its functions in reproductive development. The homozygous lno1-1 seed is not viable. LNO1, a homolog of the nucleoporin NUP214 in human (Homo sapiens) and Nup159 in yeast (Saccharomyces cerevisiae), encodes a nucleoporin protein containing phenylalanine-glycine repeats in Arabidopsis. We demonstrate that LNO1 can functionally complement the defect in the yeast temperature-sensitive nucleoporin mutant nup159. We show that LNO1 specifically interacts with the Arabidopsis DEAD-box helicase/ATPase LOS4 in the yeast two-hybrid assay. Furthermore, mutations in AtGLE1, an Arabidopsis homolog of the yeast Gle1 involved in the same poly(A) mRNA export pathway as Nup159, also result in seed abortion. Our results suggest that LNO1 is a component of the nuclear pore complex required for mature mRNA export from the nucleus to the cytoplasm, which makes LNO1 essential for embryogenesis and seed viability in Arabidopsis.

Maternal insulin resistance causes oxidative stress and mitochondrial dysfunction in mouse oocytes

Insulin resistance (IR) and hyperinsulinemia compromise fertility in females and are well-recognized characteristics of anovulatory women with polycystic ovary syndrome. Patients with IR and hyperinsulinemia undergoing ovarian stimulation for IVF are at increased risks of impaired oocyte developmental competence, implantation failure and pregnancy loss. However, the precise underlying mechanism remains unknown. We investigated how IR impairs oocyte quality and early embryonic development by an insulin-resistant mouse model. Oocyte quality, fertilization and embryonic development were analyzed. Furthermore, oxidant stress products and mitochondrial function were evaluated by quantitative real-time PCR and immunofluorescence. An imbalance between oxidants and antioxidants revealed by increased concentrations of reactive oxygen species, and a decreased concentration of glutathione (GSH) and a decreased GSH/GSSG ratio resulted in oxidative stress (OS) and impaired mitochondrial function in germinal vesicle (GV) and metaphase II (MII) oocytes of insulin-resistant mice. MII oocytes displayed a decrease in the ATP content and the mitochondrial DNA (mtDNA) copy number. In contrast, GV oocytes were characterized by a high ATP content concomitant with increased clustering of mitochondria and a high inner mitochondrial membrane potential. GV oocytes from insulin-resistant mice showed early stage apoptosis, and fewer MII oocytes could be retrieved from these mice and were of poor quality associated with decreased fertilization and an arrest of embryo development with increased fragmentation. Abnormal spindles and misaligned chromosomes of MII oocyte were significantly increased in IR and hyperinsulinemia mice compared with the control mice. IR contributes to OS and disrupts mitochondrial function in mouse oocytes. This may impair the accurate transmission of mtDNA from one generation to the next. Therefore, our results suggest that OS and mitochondrial dysfunction are responsible for poor oocyte quality of insulin-resistant mice, and may provide novel targets to improve low fertility in females with IR.

Endosperm cellularization defines an important developmental transition for embryo development

The endosperm is a terminal seed tissue that is destined to support embryo development. In most angiosperms, the endosperm develops initially as a syncytium to facilitate rapid seed growth. The transition from the syncytial to the cellularized state occurs at a defined time point during seed development. Manipulating the timing of endosperm cellularization through interploidy crosses negatively impacts on embryo growth, suggesting that endosperm cellularization is a critical step during seed development. In this study, we show that failure of endosperm cellularization in fertilization independent seed 2 (fis2) and endosperm defective 1 (ede1) Arabidopsis mutants correlates with impaired embryo development. Restoration of endosperm cellularization in fis2 seeds by reducing expression of the MADS-box gene AGAMOUS-LIKE 62 (AGL62) promotes embryo development, strongly supporting an essential role of endosperm cellularization for viable seed formation. Endosperm cellularization failure in fis2 seeds correlates with increased hexose levels, suggesting that arrest of embryo development is a consequence of failed nutrient translocation to the developing embryo. Finally, we demonstrate that AGL62 is a direct target gene of FIS Polycomb group repressive complex 2 (PRC2), establishing the molecular basis for FIS PRC2-mediated endosperm cellularization.

Lack of cytochrome c in Arabidopsis decreases stability of Complex IV and modifies redox metabolism without affecting Complexes I and III

We studied the role of cytochrome c (CYTc), which mediates electron transfer between Complexes III and IV, in cellular events related with mitochondrial respiration, plant development and redox homeostasis. We analyzed single and double homozygous mutants in both CYTc-encoding genes from Arabidopsis: CYTC-1 and CYTC-2. While individual mutants were similar to wild-type, knock-out of both genes produced an arrest of embryo development, showing that CYTc function is essential at early stages of plant development. Mutants in which CYTc levels were extremely reduced respective to wild-type had smaller rosettes with a pronounced decrease in parenchymatic cell size and an overall delay in development. Mitochondria from these mutants had lower respiration rates and a relative increase in alternative respiration. Furthermore, the decrease in CYTc severely affected the activity and the amount of Complex IV, without affecting Complexes I and III. Reactive oxygen species levels were reduced in these mutants, which showed induction of genes encoding antioxidant enzymes. Ascorbic acid levels were not affected, suggesting that a small amount of CYTc is enough to support its normal synthesis. We postulate that, in addition to its role as an electron carrier between Complexes III and IV, CYTc influences Complex IV levels in plants, probably reflecting a role of this protein in Complex IV stability. This double function of CYTc most likely explains why it is essential for plant survival.

Embryonic Diapause Is Conserved across Mammals

Embryonic diapause (ED) is a temporary arrest of embryo development and is characterized by delayed implantation in the uterus. ED occurs in blastocysts of less than 2% of mammalian species, including the mouse (Mus musculus). If ED were an evolutionarily conserved phenomenon, then it should be inducible in blastocysts of normally non-diapausing mammals, such as domestic species. To prove this hypothesis, we examined whether blastocysts from domestic sheep (Ovis aries) could enter into diapause following their transfer into mouse uteri in which diapause conditions were induced. Sheep blastocysts entered into diapause, as demonstrated by growth arrest, viability maintenance and their ED-specific pattern of gene expression. Seven days after transfer, diapausing ovine blastocysts were able to resume growth in vitro and, after transfer to surrogate ewe recipients, to develop into normal lambs. The finding that non-diapausing ovine embryos can enter into diapause implies that this phenomenon is phylogenetically conserved and not secondarily acquired by embryos of diapausing species. Our study questions the current model of independent evolution of ED in different mammalian orders.

Stimulation or Inhibition of AMPK Causes Arrest of Mouse Embryo Development.

Stimulation or Inhibition of AMPK Causes Arrest of Mouse Embryo Development.

Abstract 1141: p19Arf represses Pdgfrβ-driven pericyte proliferation in vivo

Abstract p19Arf, encoded by the mouse Arf gene, is a critical tumor suppressor with roles regulating cell cycle arrest in embryo development and suppressing oncogenesis. Previous studies established that p19Arf controls pericyte-like cells in the hyaloid vascular system (HVS) in the primary vitreous of the developing eye. Arf-deficient mice have hyperplasic expansion of pericytes in the vitreous, a developmental defect driven by excessive Pdgfrβ signaling. We have used complementary studies of cultured cells and mouse embryos to further explore the functional interactions between p19Arf and Pdgfrβ. To address whether Pdgfrβ acts as a mitogenic or a survival factor in the Arf-deficient mouse eye, we quantified cell proliferation and apoptosis at 13.5 days post coitum (dpc) in ArfGFP/GFP embryos that had or lacked Pdgfrβ. Cell proliferation, measured by BrdU incorporation, was increased by two-fold in the vitreous in the presence of Pdgfrβ. The presence or absence of Pdgfrβ did not change the small number of apoptotic cells in the vitreous of the eye. To explore mechanisms by which Arf expression impeded Pdgfrβ signaling, we used the 10T1/2 pericyte-like cell culture model to show that retroviral expression of p19Arf significantly repressed Pdgfrβ protein. Conversely, shRNA-mediated knockdown of p19Arf enhanced Pdgfrβ in serial-passaged mouse embryo fibroblasts (MEFs). In vivo measurements of Arf mRNA extracted from vitreous samples isolated by laser capture microdissection (LCM) from mouse embryos at 13.5 dpc demonstrated increased Pdgfrβ mRNA in Arf-deficient embryos as compared to wild-type. To confirm that p19Arf repressed Pdgfrβ transcription, we demonstrated that both mature and primary Pdgfrβ RNA transcripts were repressed in 10T1/2 cells. Co-transfection of p19Arf with a luciferase reporter plasmid driven by the Pdgfrβ promoter demonstrated that a 1.6kb segment of DNA conferred Arf-dependent regulation. Chromatin immunoprecipitation (ChIP) showed that p19Arf reduced RNA Pol II binding to the proximal Pdgfrβ promoter. Overall our data show that, in the absence of Arf, Pdgfrβ drives excess proliferation in cells that would normally express Arf in the developing eye. p19Arf acts to repress Pdgfrβ transcription, in part by blocking RNA Pol II binding to the proximal Pdgfrβ promoter. We are currently using additional ChIP assays to identify how the presence or absence of Arf influences the binding of several transcription factors known or suspected of regulating Pdgfrβ expression, and we are exploring the importance of the Arf-Pdgfrβ pathway deregulation in cancer models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1141. doi:10.1158/1538-7445.AM2011-1141

Regulation of plant growth and metabolism by the TOR kinase

The TOR (target of rapamycin) kinase is present in nearly all eukaryotic organisms and regulates a wealth of biological processes collectively contributing to cell growth. The genome of the model plant Arabidopsis contains a single TOR gene and two RAPTOR (regulatory associated protein of TOR)/KOG1 (Kontroller of growth 1) and GβL/LST8 (G-protein β-subunit-like/lethal with Sec thirteen 8) genes but, in contrast with other organisms, plants appear to be resistant to rapamycin. Disruption of the RAPTOR1 and TOR genes in Arabidopsis results in an early arrest of embryo development. Plants that overexpress the TOR mRNA accumulate more leaf and root biomass, produce more seeds and are more resistant to stress. Conversely, the down-regulation of TOR by constitutive or inducible RNAi (RNA interference) leads to a reduced organ growth, to an early senescence and to severe transcriptomic and metabolic perturbations, including accumulation of sugars and amino acids. It thus seems that plant growth is correlated to the level of TOR expression. We have also investigated the effect of reduced TOR expression on tissue organization and cell division. We suggest that, like in other eukaryotes, the plant TOR kinase could be one of the main contributors to the link between environmental cues and growth processes.

Polyamines Are Implicated in the Emergence of the Embryo from Obligate Diapause

Embryonic diapause is a poorly understood phenomenon of reversible arrest of embryo development prior to implantation. In many carnivores, such as the mink (Neovison vison), obligate diapause characterizes each gestation. Embryo reactivation is controlled by the uterus by mechanisms that remain elusive. Because polyamines are essential regulators of cell proliferation and growth, it was hypothesized that they trigger embryo reactivation. To test this, mated mink females were treated with α-difluoromethylornithine, an inhibitor of ornithine decarboxylase 1, the rate-limiting enzyme in polyamine biosynthesis, or saline as a control during the first 5 d of reactivation. This treatment induced polyamine deprivation with the consequence of rearrest in embryo cell proliferation. A mink trophoblast cell line in vitro subjected to α-difluoromethylornithine treatment likewise displayed an arrest in cell proliferation, morphological changes, and intracellular translocation of ornithine decarboxylase 1 protein. The arrest in embryo development deferred implantation for a period consistent with the length of treatment. Successful implantation and parturition ensued. We conclude that polyamine deprivation brought about a reversible rearrest of embryo development, which returned the mink embryo to diapause and induced a second delay in embryo implantation. The results are the first demonstration of a factor essential to reactivation of embryos in obligate diapause.

Day 2 embryo transfer (ET) and day 3 ET afford similar reproductive outcomes in the poor responder

A retrospective review of 237 initial, fresh nondonor IVF cycles in which all embryos generated during the cycle were transferred on either day 2 (n = 109) or day 3 (n = 128) were evaluated with regards to reproductive outcomes. Patients who underwent a day 2 ET had similar conception (18% vs. 16%; relative risk [RR], 1.1; 95% confidence interval [CI], 0.64-1.95), clinical pregnancy (13% vs. 16%; RR, 0.8; 95% CI, 0.44-1.55), implantation (6% vs. 7%; RR, 0.9; 95% CI, 0.50-1.68), and live-birth (10% vs. 16%; RR, 0.7; 95% CI, 0.32-1.29) rates as those who underwent a day 3 ET.