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Sample records for affect embryo development

  1. Factors affecting the development of somatic cell nuclear transfer embryos in Cattle.

    PubMed

    Akagi, Satoshi; Matsukawa, Kazutsugu; Takahashi, Seiya

    2014-01-01

    Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.

  2. Early development of Xenopus embryos is affected by simulated gravity

    NASA Technical Reports Server (NTRS)

    Yokota, Hiroki; Neff, Anton W.; Malacinski, George M.

    1994-01-01

    Early amphibian (Xenopus laevis) development under clinostat-simulated weightlessness and centrifuge-simulated hypergravity was studied. The results revealed significant effects on (i) 'morphological patterning' such as the cleavage furrow pattern in the vegetal hemisphere at the eight-cell stage and the shape of the dorsal lip in early gastrulae and (ii) 'the timing of embryonic events' such as the third cleavage furrow completion and the dorsal lip appearance. Substantial variations in sensitivity to simulated force fields were observed, which should be considered in interpreting spaceflight data.

  3. Paternal benzo[a]pyrene exposure affects gene expression in the early developing mouse embryo.

    PubMed

    Brevik, Asgeir; Lindeman, Birgitte; Rusnakova, Vendula; Olsen, Ann-Karin; Brunborg, Gunnar; Duale, Nur

    2012-09-01

    The health of the offspring depends on the genetic constitution of the parental germ cells. The paternal genome appears to be important; e.g., de novo mutations in some genes seem to arise mostly from the father, whereas epigenetic modifications of DNA and histones are frequent in the paternal gonads. Environmental contaminants which may affect the integrity of the germ cells comprise the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). B[a]P has received much attention due to its ubiquitous distribution, its carcinogenic and mutagenic potential, and also effects on reproduction. We conducted an in vitro fertilization (IVF) experiment using sperm cells from B[a]P-exposed male mice to study effects of paternal B[a]P exposure on early gene expression in the developing mouse embryo. Male mice were exposed to a single acute dose of B[a]P (150 mg/kg, ip) 4 days prior to isolation of cauda sperm, followed by IVF of oocytes from unexposed superovulated mice. Gene expression in fertilized zygotes/embryos was determined using reverse transcription-qPCR at the 1-, 2-, 4-, 8-, and blastocyst cell stages of embryo development. We found that paternal B[a]P exposure altered the expression of numerous genes in the developing embryo especially at the blastocyst stage. Some genes were also affected at earlier developmental stages. Embryonic gene expression studies seem useful to identify perturbations of signaling pathways resulting from exposure to contaminants, and can be used to address mechanisms of paternal effects on embryo development.

  4. [Some factors affecting in vitro development of porcine embryo reconstitution from somatic cells nuclear transfer].

    PubMed

    Zhang, De Fu; Wang, Ying; Chen, Yin; Wang, Kai; Schellander, Karl; Lin, Cai Lu

    2003-02-01

    In this paper, a study on reconstitution of porcine oocytes by using nuclear transfer with cumulus cells(CC) and fibroblast cells(FC) was carried out. Reconstituted oocytes which were the fusion with CC and showed a cleavage rate of 56.7%, developed to morula(11.7%) and blastocysts(6.7%) phases which were higher than those derived from the fusion with FC(p < 0.05). The results of this study also involved the effects of oocyte collection method and maturational age of recipient oocytes during the in vitro development of nuclear-transfer embryos which were reconstructed with cultured cumulus cells. The cumulus cells synchronized in G0/G1 phases through serum-starvation culture, were transferred into enucleated oocytes which were collected by aspiration or dissection method and cultured for 33 or 44 h. Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulsation and 6-DMAP, and cultured for 6 days. As for the oocytes collection methods, activation treatment in the presence of cytochalasin B did not affect the developmental rate of embryos reconstituted with 44-h-mature recipients. However, the development rate of reconstituted embryos with 33-h-mature recipients was significantly higher(p < 0.05) by activation with the combination of electric pulsation and 6-DMAP. These results suggest that reconstituted porcine embryos derived from cultured cumulus cells can develop to the blastocyst stage and that the development of the former could be improved by reconstruction with young oocyte cytoplast after the activation with the combination of electric pulsation and 6-DMAP.

  5. Nickel affects gill and muscle development in oriental fire-bellied toad (Bombina orientalis) embryos.

    PubMed

    Park, Chan Jin; Song, Sang Ha; Kim, Dae Han; Gye, Myung Chan

    2017-01-01

    The developmental toxicity of nickel was examined in the embryos of Bombina orientalis, a common amphibian in Korea. Based on a standard frog embryo teratogenesis assay, the LC50 and EC50 for malformation of nickel after 168h of treatment were 33.8μM and 5.4μM, respectively. At a lethal concentration (100μM), nickel treatment decreased the space between gill filaments and caused epithelial swelling and abnormal fusion of gill filaments. These findings suggest that nickel affects the functional development of gills, leading to embryonic death. At sublethal concentrations (1-10μM), nickel produced multiple embryonic abnormalities, including bent tail and tail dysplasia. At 10μM, nickel significantly decreased tail length and tail muscle fiber density in tadpoles, indicating inhibition of myogenic differentiation. Before hatching, the pre-muscular response to muscular response stages (stages 26-31) were the most sensitive period to nickel with respect to tail muscle development. During these stages, MyoD mRNA was upregulated, whereas myogenic regulatory factor 4 mRNA was downregulated by 0.1μM nickel. Calcium-dependent kinase activities in muscular response stage embryos were significantly decreased by nickel, whereas these activities were restored by exogenous calcium. In tadpoles, 10μM nickel significantly decreased the expression of the myosin heavy chain and the 12/101 muscle marker protein in the tail. Expression was restored by exogenous calcium. Our results indicate that nickel affects muscle development by disrupting calcium-dependent myogenesis in developing B. orientalis embryos.

  6. Crumbs affects protein dynamics in anterior regions of the developing Drosophila embryo.

    PubMed

    Firmino, João; Tinevez, Jean-Yves; Knust, Elisabeth

    2013-01-01

    Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.

  7. Bisphenol A affects early bovine embryo development and metabolism that is negated by an oestrogen receptor inhibitor

    PubMed Central

    Choi, Bom-Ie; Harvey, Alexandra J.; Green, Mark P.

    2016-01-01

    Increasing evidence supports an association between exposure to endocrine disruptors, such as the xenoestrogen bisphenol A (BPA), a commonly used plasticiser, and the developmental programming of offspring health. To date however animal studies to investigate a direct causal have mainly focussed on supra-environmental BPA concentrations, without investigating the effect on the early embryo. In this study we investigated the effect of acute BPA exposure (days 3.5 to 7.5 post-fertilisation) at environmentally relevant concentrations (1 and 10 ng/mL) on in vitro bovine embryo development, quality and metabolism. We then examined whether culturing embryos in the presence of the oestrogen receptor inhibitor fulvestrant could negate effects of BPA and 17β-oestradiol (E2). Exposure to BPA or E2 (10 ng/mL) decreased blastocyst rate and the percentage of transferrable quality embryos, without affecting cell number, lineage allocation or metabolic gene expression compared to untreated embryos. Notably, blastocysts exposed to BPA and E2 (10 ng/mL) displayed an increase in glucose consumption. The presence of fulvestrant however negated the adverse developmental and metabolic effects, suggesting BPA elicits its effects via oestrogen-mediated pathways. This study demonstrates that even acute exposure to an environmentally relevant BPA concentration can affect early embryo development and metabolism. These may have long-term health consequences on an individual. PMID:27384909

  8. Dose of recombinant FSH and oestradiol concentration on day of HCG affect embryo development kinetics.

    PubMed

    Muñoz, Manuel; Cruz, María; Humaidan, Peter; Garrido, Nicolás; Pérez-Cano, Inmaculada; Meseguer, Marcos

    2012-10-01

    During follicular growth, the follicle is exposed to an almost ever-changing composition of isoforms of FSH and LH, which causes a number of different and divergent biological effects. Through a time-lapse system, embryo kinetics were examined following the use of FSH only (recombinant FSH, rFSH) and gonadotrophins containing LH activity (human menopausal gonadotrophin, HMG, and FSH+HMG) in oocyte donors. No significant differences were seen between the three groups (for rFSH, HMG and rFSH+HMG, t2 was 27.8h, 27.9h and 27.5h respectively). Moreover, although embryos obtained with rFSH showed an increase in the proportions of optimal timings of development, the differences observed were not significant, as shown by the percentages of embryos inside/outside these kinetic variables. In contrast, for gonadotrophin dosage and oestradiol concentration, this study observed differences in embryo development kinetics for some of the variables evaluated, which allowed the description of an optimal range of gonadotrophin dosage and oestradiol concentration. However, these kinetic differences did not translate into important distinctions in the proportion of optimal embryos with a higher implantation potential.

  9. Inhibition of Rac1 GTPase activity affects porcine oocyte maturation and early embryo development

    PubMed Central

    Song, Si-Jing; Wang, Qiao-Chu; Jia, Ru-Xia; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2016-01-01

    Mammalian oocyte asymmetric division relies on the eccentric positioning of the spindle, resulting in the polar body formation. Small signaling G protein Rac1 is a member of GTPases, which regulates a diverse array of cellular events, including the control of cell growth, cytoskeletal reorganization, and the activation of protein kinases. However, effects of Rac1 on the porcine oocyte maturation and early embryo development are not fully understood. In present study we investigated the role of Rac1 in oocyte maturation and embryo cleavage. We first found that Rac1 localized at the cortex of the porcine oocytes, and disrupting the Rac1 activities by treating with NSC 23766 led to the failure of polar body emission. In addition, a majority of treated oocytes exhibited abnormal spindle morphology, indicating that Rac1 may involve into porcine oocyte spindle formation. This might be due to the regulation of Rac1 on MAPK, since p-MAPK expression decreased after NSC 23766 treatments. Moreover, we found that the position of most meiotic spindles in treated oocytes were away from the cortex, indicating the roles of Rac1 on meiotic spindle positioning. Our results also showed that inhibition of Rac1 activity caused the failure of early embryo development. Therefore, our study showed the critical roles of Rac1 GTPase on porcine oocyte maturation and early embryo cleavage. PMID:27694954

  10. Estradiol and endocrine disrupting compounds adversely affect development of sea urchin embryos at environmentally relevant concentrations.

    PubMed

    Roepke, Troy A; Snyder, Mark J; Cherr, Gary N

    2005-01-26

    Environmental endocrine disrupting compounds (EDCs) are a wide variety of chemicals that typically exert effects, either directly or indirectly, through receptor-mediated processes, thus mimicking endogenous hormones and/or inhibiting normal hormone activities and metabolism. Little is known about the effects of EDCs on echinoderm physiology, reproduction and development. We exposed developing sea urchin embryos (Strongylocentrotus purpuratus and Lytechinus anamesus) to two known EDCs (4-octylphenol (OCT), bisphenol A (BisA)) and to natural and synthetic reproductive hormones (17beta-estradiol (E2), estrone (E1), estriol (E3), progesterone (P4) and 17alpha-ethynylestradiol (EE2)). In addition, we studied two non-estrogenic EDCs, tributyltin (TBT) and o,p-DDD. Successful development to the pluteus larval stage (96 h post-fertilization) was used to define EDC concentration-response relationships. The order of compound potency based on EC50 values for a reduction in normal development was as follows: TBT(L. anamesus)>OCT>TBT(S. purpuratus)>E2>EE2>DDD>BisA>P4>E1>E3. The effect of TBT was pronounced even at concentrations substantially lower than those commonly reported in heavily contaminated areas, but the response was significantly different in the two model species. Sea urchin embryos were generally more sensitive to estrogenic EDCs and TBT than most other invertebrate larvae. Stage-specific exposure experiments were conducted to determine the most sensitive developmental periods using blastula, gastrula and post-gastrula (pluteus) stages. The stage most sensitive to E2, OCT and TBT was the blastula stage with less overall sensitivity in the gastrula stage, regardless of concentration. Selective estrogen receptor modulators (SERMs) were added to the experiments individually and in combination with estrogenic EDCs to interfere with potential receptor-mediated actions. Tamoxifen, a partial ER agonist, alone inhibited development at concentrations as low as 0.02 ng

  11. Undernutrition affects embryo quality of superovulated ewes.

    PubMed

    Abecia, J A; Forcada, F; Palacín, I; Sánchez-Prieto, L; Sosa, C; Fernández-Foren, A; Meikle, A

    2015-02-01

    To determine the effect of undernutrition on embryo production and quality in superovulated sheep, 45 ewes were allocated into two groups to be fed diets that provided 1.5 (control, C; n = 20) or 0.5 (low nutrition, L; n = 25) times daily requirements for maintenance, from oestrous synchronization with intravaginal sponges to embryo collection. Embryos were collected 7 days after the onset of oestrus (day 0). Low nutrition resulted in lower live weight and body condition at embryo collection (P < 0.05). Diet (P < 0.01) and day of sampling (P < 0.001) significantly affected plasma non-esterified fatty acid (NEFA) and insulin concentrations. Plasma leptin concentrations decreased on day 7 only in L ewes. A significant effect of dietary treatment (P < 0.05) and day (P < 0.0001) was observed on plasma insulin-like growth factor (IGF)-I concentrations. The number of recovered oocytes and embryos did not differ between the groups (L: 15.4 ± 0.4; C: 12.4 ± 0.4). Recovery rate was lower (P < 0.05) in the L (60%) than in the C group (73%). The total number of embryos and number of viable-transferable embryos (5.0 ± 0.3 and 3.4 ± 0.3 embryos, respectively) of the L group were lower (P < 0.1) when compared with controls (8.4 ± 0.4 and 6.2 ± 0.4 embryos, respectively). Undernutrition during the period of superovulation and early embryonic development reduced total and viable number of embryos. These effects might be mediated by disruption of endocrine homeostasis, oviduct environment and/or oocyte quality.

  12. Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression.

    PubMed

    Mohammed, Rabeea H; Sweetman, Dylan

    2016-01-17

    Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining. In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18. This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

  13. Dimethylsulfoxide and conjugated linoleic acids affect bovine embryo development in vitro.

    PubMed

    Stinshoff, Hanna; Wilkening, Sandra; Hanstedt, Ana; Bollwein, Heinrich; Wrenzycki, Christine

    2014-01-01

    Conjugated linoleic acids (CLA) are employed to overcome the bovine periparturitional negative energy balance. Especially of interest are trans10,cis12 -linoleic acid (t10c12-CLA) and cis9,trans11-linoleic acid (c9t11-CLA). Their impact on embryonic development, though, is not clear. Here, effects of both above-mentioned CLA on bovine in vitro-produced embryos were assessed. Zygotes (n=2098) were allocated to one of seven groups: cultured with 50 or 100µM of either c9t11-CLA or t10c12-CLA, with 14 or 28mM DMSO or without supplement (control). Messenger RNA analysis of target gene transcripts (IGF1R, IGFBP2, IGFBP4, CPT2, ACAA1, ACAA2, FASN, SCD) via RT-qPCR was performed in single blastocysts. Cleavage rates did not differ, whereas development rates were decreased in both t10c12-supplemented groups in comparison to the unsupplemented group (31.7% ±2.2 control vs 20.2% ±2.0 50µM t10c12 vs 21.0% ±2.8 100µM t10c12). Compared with the unsupplemented group, SCD was expressed at a lower level in embryos cultured with 50µM c9t11-CLA. The relative amount of several transcripts was increased in embryos cultured with 14mM DMSO in comparison to those that developed in the presence of 50µM t10c12-CLA (IGFBP2, ACAA1, CPT2, FASN, SCD) or 50µM c9t11-CLA (IGF1R, IGFBP2, ACAA1, CPT2, FASN, SCD). The molecular analyses show that CLA influence embryonic fat metabolism.

  14. Biopsy of embryos produced by in vitro fertilization affects development in C57BL/6 mouse strain

    PubMed Central

    Sugawara, Atsushi; Ward, Monika A.

    2012-01-01

    Preimplantation genetic diagnosis (PGD) is considered highly successful in respect to its accuracy in detecting genetic anomalies but the effects of embryo biopsy on embryonic/fetal growth and development are less known, particularly in conjunction with in vitro fertilization (IVF). Here, we compared biopsied (B) and non-biopsied (NB) mouse embryos for their developmental competence. Embryos C57BL/6 (B6) and B6D2F2 (F2) generated by IVF were subjected to single blastomere biopsy at the 4-cell stage, and were either cultured for 120 h and subjected to differential inner cell mass (ICM) and trophoblast (T) staining, or were transferred into the uterine tubes of surrogate mothers after 72 h of culture, to examine their pre- and post-implantation development, respectively. Non-biopsied embryos from the same IVF cohorts served as controls. Embryo biopsy negatively affected preimplantation development to blastocyst in C57BL/6 (69 vs 79%, P<0.01) but not in B6D2F1 mice (89 vs 91%, P=NS). Although B6 embryos had lower total cell number than F2 (B6: 47 and 61 vs. F1: 53 and 70; B and NB, respectively, P<0.05) there were no differences between B and NB blastocysts in %ICM (B6: 19.8 vs 19.8; F2: 20.9 vs 20.4, P=NS) and ICM:T ratio (B6: 4.7 vs 4.7; F2: 4.4 vs. 4.7) in both mouse strains. Post-implantation development to live fetuses of B embryos as compared to NB counterparts was impaired in C57BL/6 (6 vs 18%, P<0.001) but not in B6D2F1 mice (26 vs 35%, P=NS). We conclude that blastomere biopsy impairs embryonic/fetal development in mice known to be sensitive to in vitro culture and manipulations. Such mice model infertile couples with poor quality gametes seeking help in assisted reproduction technologies (ART) clinics. PMID:23174776

  15. Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana.

    PubMed

    Tsukamoto, Tatsuya; Palanivelu, Ravishankar

    2010-11-01

    Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the pollen tube to the female gametophyte, followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte not only regulates these steps but also controls the subsequent initiation of seed development. Previously, we reported that loss of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the female reproductive tissues causes a delay in initiation of seed development. From these studies, however, it was unclear if embryos derived from fertilization of lre-5 gametophytes continued to lag behind wild type during seed development. Additionally, it was not determined if the delay in initiation of seed development had any lingering effects during seed germination. Finally, it was not known if loss of LORELEI function affects seedling development given that LORELEI is expressed in eight-day-old seedlings. Here, we showed that despite a delay in initiation, lre-5/lre-5 embryos recover, becoming equivalent to the developing wild-type embryos beginning at 72 hours after pollination. Additionally, lre-5/lre-5 seed germination, and seedling and root development are indistinguishable from wild type indicating that loss of LORELEI is tolerated, at least under standard growth conditions, in vegetative tissues.

  16. Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana

    PubMed Central

    Tsukamoto, Tatsuya

    2010-01-01

    Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the pollen tube to the female gametophyte, followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte not only regulates these steps but also controls the subsequent initiation of seed development. Previously, we reported that loss of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the female reproductive tissues causes a delay in initiation of seed development. From these studies, however, it was unclear if embryos derived from fertilization of lre-5 gametophytes continued to lag behind wild-type during seed development. Additionally, it was not determined if the delay in initiation of seed development had any lingering effects during seed germination. Finally, it was not known if loss of LORELEI function affects seedling development given that LORELEI is expressed in eight-day-old seedlings. Here, we showed that despite a delay in initiation, lre-5/lre-5 embryos recover, becoming equivalent to the developing wild-type embryos beginning at 72 hours after pollination. Additionally, lre-5/lre-5 seed germination, and seedling and root development are indistinguishable from wild-type indicating that loss of LORELEI is tolerated, at least under standard growth conditions, in vegetative tissues. PMID:21051955

  17. Transporters involved in pH and K+ homeostasis affect pollen wall formation, male fertility, and embryo development

    DOE PAGES

    Padmanaban, Senthilkumar; Czerny, Daniel D.; Levin, Kara A.; ...

    2017-02-23

    Flowering plant genomes encode multiple cation/H+ exchangers (CHXs) whose functions are largely unknown. AtCHX17, AtCHX18, and AtCHX19 are membrane transporters that modulate K+ and pH homeostasis and are localized in the dynamic endomembrane system. Loss of function reduced seed set, but the particular phase(s) of reproduction affected was not determined. Pollen tube growth and ovule targeting of chx17chx18chx19 mutant pollen appeared normal, but reciprocal cross experiments indicate a largely male defect. Although triple mutant pollen tubes reach ovules of a wild-type pistil and a synergid cell degenerated, half of those ovules were unfertilized or showed fertilization of the egg ormore » central cell, but not both female gametes. Fertility could be partially compromised by impaired pollen tube and/or sperm function as CHX19 and CHX18 are expressed in the pollen tube and sperm cell, respectively. When fertilization was successful in self-pollinated mutants, early embryo formation was retarded compared with embryos from wild-type ovules receiving mutant pollen. Thus CHX17 and CHX18 proteins may promote embryo development possibly through the endosperm where these genes are expressed. The reticulate pattern of the pollen wall was disorganized in triple mutants, indicating perturbation of wall formation during male gametophyte development. Lastly, as pH and cation homeostasis mediated by AtCHX17 affect membrane trafficking and cargo delivery, these results suggest that male fertility, sperm function, and embryo development are dependent on proper cargo sorting and secretion that remodel cell walls, plasma membranes, and extracellular factors.« less

  18. Arsenic exposure affects embryo development of sea urchin, Paracentrotus lividus (Lamarck, 1816).

    PubMed

    Gaion, Andrea; Scuderi, Alice; Pellegrini, David; Sartori, Davide

    2013-11-01

    Toxicity tests were performed with embryos of Paracentrotus lividus to investigate the toxicological effect of two arsenic species: arsenate (As(V)), expected to be more toxic, and dimethyl-arsinate (DMA) expected to be less toxic. Exposures to toxicants were performed at different developmental stages in order to identify the most sensitive phase of embryological development. Statistical analysis revealed a high significance of each factor (Molecule, Concentration and Time of exposure) and their interaction for the dependent variable "Percentage of normal-shaped plutei". In particular, the 8 cell stage was the most sensitive to arsenic; at a concentration of 50 μg L(-1) DMA proved to be more toxic than As(V), resulting in nearly 50 % of normal-shaped plutei against the 74 % recorded for As(V). Starting the administration of arsenic at the morula stage, arsenate proved to be significantly more toxic when compared to DMA.

  19. Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis

    PubMed Central

    Kapoor, Sanjay

    2013-01-01

    Rice MADS29 has recently been reported to cause programmed cell death of maternal tissues, the nucellus, and the nucellar projection during early stages of seed development. However, analyses involving OsMADS29 protein expression domains and characterization of OsMADS29 gain-of-function and knockdown phenotypes revealed novel aspects of its function in maintaining hormone homeostasis, which may have a role in the development of embryo and plastid differentiation and starch filling in endosperm cells. The MADS29 transcripts accumulated to high levels soon after fertilization; however, protein accumulation was found to be delayed by at least 4 days. Immunolocalization studies revealed that the protein accumulated initially in the dorsal-vascular trace and the outer layers of endosperm, and subsequently in the embryo and aleurone and subaleurone layers of the endosperm. Ectopic expression of MADS29 resulted in a severely dwarfed phenotype, exhibiting elevated levels of cytokinin, thereby suggesting that cytokinin biosynthesis pathway could be one of the major targets of OsMADS29. Overexpression of OsMADS29 in heterologous BY2 cells was found to mimic the effects of exogenous application of cytokinins that causes differentiation of proplastids to starch-containing amyloplasts and activation of genes involved in the starch biosynthesis pathway. Suppression of MADS29 expression by RNAi severely affected seed set. The surviving seeds were smaller in size, with developmental abnormalities in the embryo and reduced size of endosperm cells, which also contained loosely packed starch granules. Microarray analysis of overexpression and knockdown lines exhibited altered expression of genes involved in plastid biogenesis, starch biosynthesis, cytokinin signalling and biosynthesis. PMID:23929654

  20. Does fine sediment source as well as quantity affect salmonid embryo mortality and development?

    PubMed

    Sear, D A; Jones, J I; Collins, A L; Hulin, A; Burke, N; Bateman, S; Pattison, I; Naden, P S

    2016-01-15

    Fine sediments are known to be an important cause of increased mortality in benthic spawning fish. To date, most of the research has focussed on the relationship between embryo mortality and the quantity of fine sediment accumulated in the egg pocket. However, recent evidence suggests a) that the source of fine sediment might also be important, and b) that fitness of surviving embryos post-hatch might also be impacted by the accumulation of fine sediments. In this paper, we report an experiment designed to simulate the incubation environment of brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). During the experiment, the incubating embryos were exposed to different quantities of fine (<63 μm) sediment derived from four different sources; agricultural topsoils, damaged road verges, eroding river channel banks and tertiary level treated sewage. Results showed that mass and source are independently important for determining the mortality and fitness of alevin. Differences between species were observed, such that brown trout are less sensitive to mass and source of accumulated sediment. We demonstrate for the first time that sediment source is an additional control on the impact of fine sediment, and that this is primarily controlled by the organic matter content and oxygen consumption of the catchment source material.

  1. A mutation in Arabidopsis seedling plastid development1 affects plastid differentiation in embryo-derived tissues during seedling growth.

    PubMed

    Ruppel, Nicholas J; Logsdon, Charles A; Whippo, Craig W; Inoue, Kentaro; Hangarter, Roger P

    2011-01-01

    Oilseed plants like Arabidopsis (Arabidopsis thaliana) develop green photosynthetically active embryos. Upon seed maturation, the embryonic chloroplasts degenerate into a highly reduced plastid type called the eoplast. Upon germination, eoplasts redifferentiate into chloroplasts and other plastid types. Here, we describe seedling plastid development1 (spd1), an Arabidopsis seedling albino mutant capable of producing normal green vegetative tissues. Mutant seedlings also display defects in etioplast and amyloplast development. Precocious germination of spd1 embryos showed that the albino seedling phenotype of spd1 was dependent on the passage of developing embryos through the degreening and dehydration stages of seed maturation, suggesting that SPD1 is critical during eoplast development or early stages of eoplast redifferentiation. The SPD1 gene was found to encode a protein containing a putative chloroplast-targeting sequence in its amino terminus and also domains common to P-loop ATPases. Chloroplast localization of the SPD1 protein was confirmed by targeting assays in vivo and in vitro. Although the exact function of SPD1 remains to be defined, our findings reveal aspects of plastid development unique to embryo-derived cells.

  2. Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

    PubMed Central

    Bernal-Ulloa, Sandra Milena; Heinzmann, Julia; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Winkler, Sylke; Pache, Dorit; Baulain, Ulrich; Lucas-Hahn, Andrea; Niemann, Heiner

    2016-01-01

    High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency. PMID:26926596

  3. Reduced heart rate and cardiac output differentially affect angiogenesis, growth, and development in early chicken embryos (Gallus domesticus).

    PubMed

    Branum, Sylvia R; Yamada-Fisher, Miho; Burggren, Warren

    2013-01-01

    An increase in both vascular circumferential tension and shear stress in the developing vasculature of the chicken embryo has been hypothesized to stimulate angiogenesis in the developing peripheral circulation chorioallantoic membrane (CAM). To test this hypothesis, angiogenesis in the CAM, development, and growth were measured in the early chicken embryo, following acute and chronic topical application of the purely bradycardic drug ZD7288. At hour 56, ZD7288 reduced heart rate (f(H)) by ~30% but had no significant effect on stroke volume (~0.19 ± 0.2 μL), collectively resulting in a significant fall in cardiac output (CO) from ~27 ± 3 to 18 ± 2 μL min(-1). Mean f(H) at 72 h of development was similarly significantly lowered by acute ZD7288 treatment (250 μM) to 128 ± 0.3 beats min(-1), compared with 174.5 ± 0.3 and 174.7 ± 0.8 beats min(-1) in control and Pannett-Compton (P-C) saline-treated embryos, respectively. Chronic dosing with ZD7288-and the attendant decreases in f(H) and CO-did not change eye diameter or cervical flexion (key indicators of development rate) at 120 h but significantly reduced overall growth (wet and dry body mass decreased by 20%). CAM vessel density index (reflecting angiogenesis) measured 200-400 μm from the umbilical stalk was not altered, but ZD7288 reduced vessel numbers-and therefore vessel density-by 13%-16% more distally (500-600 μm from umbilical stalk) in the CAM. In the ZD7288-treated embryos, a decrease in vessel length was found within the second branch order (~300-400 μm from the umbilical stock), while a decrease in vessel diameter was found closer to the umbilical stock, beginning in the first branch order (~200-300 μm). Paradoxically, chronic application of P-C saline also reduced peripheral CAM vessel density index at 500 and 600 μm by 13% and 7%, respectively, likely from washout of local angiogenic factors. In summary, decreased f(H) with reduced CO did not slow development rate but reduced embryonic

  4. MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro.

    PubMed

    Feng, Ruizhi; Sang, Qing; Zhu, Yan; Fu, Wei; Liu, Miao; Xu, Yan; Shi, Huijuan; Xu, Yao; Qu, Ronggui; Chai, Renjie; Shao, Ruijin; Jin, Li; He, Lin; Sun, Xiaoxi; Wang, Lei

    2015-03-03

    Previous work from our laboratory demonstrated the existence of miRNAs in human follicular fluid. In the current study, we have sought to identify miRNAs that might affect oocyte/embryo quality in patients undergoing intracytoplasmic sperm injection and to investigate their roles in in vitro fertilization outcomes in mouse oocytes. 53 samples were classified as Group 1 (high quality) if the day-3 embryos had seven and more cells or as Group 2 (low quality) if the embryos had six and fewer cells. TaqMan Human microRNAs cards and qRT-PCR were performed to verify differently expressed miRNAs. The function of the corresponding miRNA was investigated in mouse oocytes by injecting them with miRNA-inhibitor oligonucleotides. We found that hsa-miR-320a and hsa-miR-197 had significantly higher expression levels in the Group 1 follicular fluids than in Group 2 (p = 0.0073 and p = 0.008, respectively). Knockdown of mmu-miR-320 in mouse oocytes strongly decreased the proportions of MII oocytes that developed into two-cell and blastocyst stage embryos (p = 0.0048 and p = 0.0069, respectively). Wnt signaling pathway components had abnormal expression level in miR-320 inhibitor-injected oocytes. This study provides the first evidence that miRNAs in human follicular fluid are indicative of and can influence embryo quality.

  5. 141 BOVINE EMBRYO DEVELOPMENT RATES ARE AFFECTED WHEN OOCYTES ARE MATURED IN DIFFERENT VIALS CONTAINING HEPES/BICARBONATE BUFFERED MEDIUM.

    PubMed

    Hashem, N; Secher, J O; Pryor, J H; Long, C R; Looney, C R; Avery, B; Hyttel, P; Stroebech, L

    2016-01-01

    Laboratory ware for the in vitro-produced embryos is generally made from embryo-tested plastic instead of glass. The quality of the plastic is crucial for the outcome because plastic is often toxic to gametes (Nijs et al. 2009 Fertil. Steril. 92, 527-535). In addition, gas molecules permeate through the plastic at a rate that depends on a variety of factors, such as diffusion coefficient and thickness of the plastic. In an incubator with appropriate concentration of CO2 and vented culture vessels, the gas permeability of the plastic is not important. When oocytes are transported outside a controlled atmosphere, gas permeability, toxicity, and oocyte cumulus cell CO2 metabolism could perturb the outcome. Medium containing bicarbonate buffer increases pH outside of a controlled atmosphere within minutes, whereas medium buffered with HEPES maintains suitable pH for hours. Previously, we tested that gas permeability differs among plastic vials and glass vials with no cumulus-oocyte complexes (COC) by measuring pH after 2, 5, and 24h at the same temperature. The objective of this study was to compare pH post-maturation, blastocyst development rates on Day 8 post-IVF (Day 0=IVF) between 2 different 1.2-mL polypropylene cryovials (A: VWR DK, 479-1219; B: Sigma-Aldrich, St. Louis, MO, USA, CLS430289), glass vial (VWR DK, NSCAC4015-96), and 4-well plate (4WP) as control (Thermo Fisher Scientific, Waltham, MA, USA, 144444). A total of 1135 abattoir-derived COC in Exp. 1 and 133 in Exp. 2 were divided equally between the treatments (20-25 COC per vessel). Vials/4WP contained 0.8/0.5mL of BO-IVM HEPES, a HEPES/bicarbonate medium (IVF Bioscience; BO-HEPES-IVM, UK). Maturation lasted 22 to 24h at 38.8°C in an incubator with either a humidified atmosphere of 5.5% CO2 in air (Exp. 1) or with no CO2 contact (Exp. 2). In Exp. 1, oocyte vials were matured without a vial lid while in Exp. 2 vial lids were closed. Statistical analysis was performed with chi-square and mean±SD. In Exp

  6. Effects of Fluoxetine on Human Embryo Development

    PubMed Central

    Kaihola, Helena; Yaldir, Fatma G.; Hreinsson, Julius; Hörnaeus, Katarina; Bergquist, Jonas; Olivier, Jocelien D. A.; Åkerud, Helena; Sundström-Poromaa, Inger

    2016-01-01

    The use of antidepressant treatment during pregnancy is increasing, and selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed antidepressants in pregnant women. Serotonin plays a role in embryogenesis, and serotonin transporters are expressed in two-cell mouse embryos. Thus, the aim of the present study was to evaluate whether fluoxetine, one of the most prescribed SSRI antidepressant world-wide, exposure influences the timing of different embryo developmental stages, and furthermore, to analyze what protein, and protein networks, are affected by fluoxetine in the early embryo development. Human embryos (n = 48) were randomly assigned to treatment with 0.25 or 0.5 μM fluoxetine in culture medium. Embryo development was evaluated by time-lapse monitoring. The fluoxetine-induced human embryo proteome was analyzed by shotgun mass spectrometry. Protein secretion from fluoxetine-exposed human embryos was analyzed by use of high-multiplex immunoassay. The lower dose of fluoxetine had no influence on embryo development. A trend toward reduced time between thawing and start of cavitation was noted in embryos treated with 0.5 μM fluoxetine (p = 0.065). Protein analysis by shotgun mass spectrometry detected 45 proteins that were uniquely expressed in fluoxetine-treated embryos. These proteins are involved in cell growth, survival, proliferation, and inflammatory response. Culturing with 0.5 μM, but not 0.25 μM fluoxetine, caused a significant increase in urokinase-type plasminogen activator (uPA) in the culture medium. In conclusion, fluoxetine has marginal effects on the timing of developmental stages in embryos, but induces expression and secretion of several proteins in a manner that depends on dose. For these reasons, and in line with current guidelines, the lowest possible dose of SSRI should be used in pregnant women who need to continue treatment. PMID:27378857

  7. Ion currents in embryo development.

    PubMed

    Tosti, Elisabetta; Boni, Raffaele; Gallo, Alessandra

    2016-03-01

    Ion channels are proteins expressed in the plasma membrane of electrogenic cells. In the zygote and blastomeres of the developing embryo, electrical modifications result from ion currents that flow through these channels. This phenomenon implies that ion current activity exerts a specific developmental function, and plays a crucial role in signal transduction and the control of embryogenesis, from the early cleavage stages and during growth and development of the embryo. This review describes the involvement of ion currents in early embryo development, from marine invertebrates to human, focusing on the occurrence, modulation, and dynamic role of ion fluxes taking place on the zygote and blastomere plasma membrane, and at the intercellular communication between embryo cell stages.

  8. Physical influences on embryo development.

    PubMed

    Deeming, D C; Rowlett, K; Simkiss, K

    1987-01-01

    There is a critical period between 3 and 7 days of incubation when the absence of turning in eggs of the domestic fowl leads to increased mortality and decreased embryo growth. This critical period coincides with the time of subembryonic fluid formation, and it is suggested that the absence of turning leads to the presence of unstirred layer effects in fluid secretion. This fluid deficiency persists throughout the subsequent development of the embryo. Experiments on shell-less culture systems support this interpretation in preference to other explanations of embryo death in unturned eggs, which usually refer to chorion adhesion to shell membranes.

  9. Mutation in the Arabidopisis thaliana DEK1 calpain gene perturbs endosperm and embryo development while over-expression affects organ development globally.

    PubMed

    Lid, Stein Erik; Olsen, Lene; Nestestog, Ragnhild; Aukerman, Milo; Brown, Roy C; Lemmon, Betty; Mucha, Mark; Opsahl-Sorteberg, Hilde-Gunn; Olsen, Odd-Arne

    2005-06-01

    A T-DNA insertion in the Arabidopsis thaliana DEK1 gene, encoding a calpain-like cysteine proteinase with a predicted membrane anchor, causes unorganized embryo development displaying irregular mitotic divisions in the embryo proper and suspensor. Embryo development is arrested at the globular stage, and the embryo proper lacks a defined protoderm. In the endosperm, the aleurone-like peripheral cell layer is partly or completely lacking. The Arabidopsis DEK1 wild-type transcript is expressed evenly throughout the endosperm and the embryo in developing seed as determined using in situ hybridization. The conclusion that the observed phenotype is caused by a T-DNA insertion in the Arabidopsis DEK1 gene is confirmed by complementation with the Arabidopisis DEK1 genomic sequence, as well as analysis of a second T-DNA insertion allele. Over-expression of the Arabidopsis DEK1 gene coding sequence under the control of the 35S promoter causes a number of developmental phenotypes, including a global lack of trichomes, leaves exhibiting improper dorsiventral symmetry and aberrant cell organization in flowers. We interpret the data to suggest a role for DEK1 in providing cells with positional clues for an appropriate developmental context within plant tissues.

  10. The ART of studying early embryo development: progress and challenges in ruminant embryo culture.

    PubMed

    Lonergan, Pat; Fair, Trudee

    2014-01-01

    The study of preimplantation mammalian embryo development is challenging due to difficulties in accessing in vivo-derived embryos in large numbers at the early stages and the inability to culture embryos in vitro much beyond the blastocyst stage. Nonetheless, embryos exhibit an amazing plasticity and tolerance when it comes to adapting to the environment in which they are cultured. They are capable of developing in media ranging in composition from simple balanced salt solutions to complex systems involving serum and somatic cells. At least a proportion of the blastocysts that develop in culture are developmentally competent as evidenced by the fact that live offspring have resulted following transfer. However, several studies using animal models have shown that such embryos are sensitive to environmental conditions that can affect future pre- and post-natal growth and developmental potential. This review summarises some key aspects of early embryo development and the approaches taken to study this important window in early life.

  11. Biosensors for detecting stress in developing embryos

    NASA Astrophysics Data System (ADS)

    Purdey, Malcolm S.; Saini, Avishkar; McLennan, Hanna J.; Pullen, Benjamin J.; Schartner, Erik P.; Sutton-McDowall, Melanie L.; Thompson, Jeremy G.; Monro, Tanya M.; Nicholls, Stephen J.; Abell, Andrew D.

    2016-12-01

    Reactive Oxygen Species (ROS) cause DNA damage and defective function in sperm and also affects the developmental competence of embryos. It is therefore critical to monitor ROS in sperm, oocytes and developing embryos. In particular, hydrogen peroxide (H2O2) is a ROS important to normal cell function and signalling as well as its role in oxidative stress. Here we report the development of a fluorescent sensor for H2O2 using carboxyperoxyfluor-1 (CPF1) in solution and attached to a glass slide or multi-mode optical fibre. CPF1 increases in fluorescence upon reaction with H2O2 to non-invasively detect H2O2 near developing embryos. These probes are constructed by immobilising CPF1 to the optical fibre tip a polyacrylamide layer. Also reported is a new dual optical fibre sensor for detecting both H2O2 and pH that is functional at biologically concentrations of H2O2 and can sense pH to 0.1 units. This research shows promise for the use of optical fibre sensors for monitoring the health of developing embryos. Furthermore, these sensors are applicable for use beyond embryos such as detecting stress in endothelial cells involved in cardiovascular dysfunction.

  12. Factors affecting the cryosurvival of mouse two-cell embryos.

    PubMed

    Critser, J K; Arneson, B W; Aaker, D V; Huse-Benda, A R; Ball, G D

    1988-01-01

    A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Embryo development in dairy cattle.

    PubMed

    Lonergan, Pat; Fair, Trudee; Forde, Niamh; Rizos, Dimitrios

    2016-07-01

    During the past 50 years, the fertility of high-producing lactating dairy cows has decreased, associated with intensive selection for increased milk production. The physiological and metabolic changes associated with high milk production, including decreased (glucose, insulin, IGF-I) or increased (nonesterified fatty acids, ketone bodies) concentrations of circulating metabolites during nutrient partitioning associated with negative energy balance as well as uterine and nonuterine diseases have been linked with poor reproductive efficiency. Fertilization is typically above 80% and does not seem to be the principal factor responsible for the low fertility in dairy cows. However, early embryonic development is compromised in high-producing dairy cows, as observed by most embryonic losses occurring during the first 2 weeks after fertilization and may be linked to compromised oocyte quality due to a poor follicular microenvironment, suboptimal reproductive tract environment for the embryo, and/or inadequate maternal-embryonic communication. These and other factors related to embryo development will be discussed.

  14. 182 MATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES WITH FOLLICLE FLUID VARYING IN ESTRADIOL CONTENT AFFECTS CUMULUS CELL EXPANSION WITHOUT AFFECTING SUBSEQUENT EMBRYO DEVELOPMENT IN VITRO.

    PubMed

    Harl, A W; Larimore, E L; Al Naib, A; Wooldridge, L K; Ealy, A D; Perry, G A; Rhoads, M L

    2016-01-01

    The objective of this work was to determine how characteristics of bovine follicle fluid (FF; especially oestradiol content) affect cumulus cell expansion and oocyte competence. In the first study, FF was collected from abattoir-derived ovaries and pooled separately for large follicles (≥10mm) and small follicles (≤3mm). A portion of the FF from each category was charcoal stripped. These 4 types of FF were then used as the primary ingredient (75% vol/vol) in oocyte maturation media. A separate control group lacking FF but containing BSA was included to monitor potential impacts of protein on outcomes (control; without FF). Some of the cumulus-oocyte complexes (COC; n=250) were matured in individual drops for analysis of cumulus expansion (photographed and measured at 0 and 21h of maturation). Other COC (n=770) were matured in groups of 12 to 25 in the previously described media, and then subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 8 post-fertilization. Cumulus cell expansion was greatest when COC were matured in medium containing FF from large follicles, wherein it even exceeded the controls (P<0.02). Maturation in FF from small follicles resulted in cumulus expansion that was intermediate between large and control. Maturation in charcoal-stripped FF severely restricted cumulus cell expansion (P<0.05) compared with those matured in untreated FF. Despite the observed improvement in cumulus cell expansion, COC that had been matured in media containing FF were less likely to cleave (P<0.05) and also less likely to develop to the blastocyst stage (P<0.01) than those matured in control medium. Cleavage and blastocyst rates did not differ among any of the maturation media containing FF. In the second study, oestrous cycles of 9 crossbred cows were synchronized and FF samples were collected 36 to 42h after prostaglandin F2α injection. Samples from individual cows were categorized as having high

  15. Decreased maternal and fetal cholesterol following maternal bococizumab (anti-PCSK9 monoclonal antibody) administration does not affect rat embryo-fetal development.

    PubMed

    Campion, Sarah N; Han, Bora; Cappon, Gregg D; Lewis, Elise M; Kraynov, Eugenia; Liang, Hong; Bowman, Christopher J

    2015-11-01

    Bococizumab is a humanized monoclonal IgG2Δa antibody against proprotein convertase subtilisin/kexin type 9 (PCSK9) for the treatment of hyperlipidemia. The evaluation of potential effects on embryo-fetal development was conducted in the rat. In a pharmacokinetic/pharmacodynamic study bococizumab was administered intravenously to pregnant Sprague-Dawley (SD) rats (n = 8/group) at 0, 10, 30, and 100 mg/kg during organogenesis. Maternal and fetal bococizumab, total cholesterol and HDL concentrations were determined. Bococizumab was well tolerated and there were no effects on ovarian or uterine parameters. Maternal and fetal bococizumab exposure increased with increasing dose, with a corresponding dose-dependent decrease in fetal cholesterol levels. Maternal cholesterol levels were decreased significantly, with reductions that were of a similar magnitude regardless of dose. In the definitive embryo-fetal development study bococizumab was administered to pregnant SD rats (n = 20/group) at 0, 10, 30, and 100 mg/kg and no adverse maternal or developmental effects were observed up to 100 mg/kg. These studies have provided an appropriate and relevant safety assessment of bococizumab in pregnant rats to inform human risk assessment, demonstrating no adverse effects on embryo-fetal development at magnitudes greater than anticipated clinical exposure and in the presence of maximal reductions in maternal cholesterol and dose-dependent reductions in fetal cholesterol.

  16. Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos.

    PubMed

    Ding, Li-Jun; Tian, Hai-Bin; Wang, Jing-Jun; Chen, Juan; Sha, Hong-Ying; Chen, Jian-Quan; Cheng, Guo-Xiang

    2008-12-01

    The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used

  17. Cryosurvival of in vitro produced embryos as affected by health status effect of oocyte donor cow.

    PubMed

    Hosseini, S M; Hajian, M; Asgari, V; Forouzanfar, M; Ostadhosseini, S; Moulavi, F; Abedi, P; Kiani, M; Vash N, N T; Safahani-Langroodi, M; Nasr-Esfahani, M H

    2013-01-01

    In vitro embryo production and embryo vitrification of genetically superior cows that culled inevitably due to health problems can accelerate genetic progress. This study was carried out to investigate whether maternal age and health status effects of high genetic merit cows affect cryosurvival and developmental competence of IVP embryos. In this sense, the effects of ageing and four common culling causes of dairy cows [repeat breeding (RPB), udder problems (UPM), chronic endometritis (CRE), and lameness (LAM)] on in vitro embryo development, and in vivo developmental competence after embryo vitrification were evaluated. The mean number of oocytes obtained per cow did not vary significantly between donors indifferent groups. Cleavage rates in RPB (86.0+/-4.2%), SEN (81.3+/-2.5%) and CRE (77.6+/-6.3%) cows which were comparable to control (95.9+/-1.5%) but were significantly higher than the related rate of UPM donors (50.6+/-2.6%). Importantly, there was no significant difference between the blastocyst rates of different groups. Mean overall survival rate was not different between the groups and was not affected by the blastocyst production rate. There was no significant difference between pregnancy rates of different groups. The results of the present study indicated that in cattle, neither ageing, nor these four diseases affect ovarian potential in terms of the yield and quality of in vitro embryo development.

  18. Prolonged oral treatment with an essential amino acid L-leucine does not affect female reproductive function and embryo-fetal development in rats.

    PubMed

    Mawatari, Kazunori; Katsumata, Toyohisa; Uematsu, Masanobu; Katsumata, Tomoyoshi; Yoshida, Junichi; Smriga, Miro; Kimura, Takeshi

    2004-09-01

    L-leucine, an essential amino acid, is one of the most popular ingredients in dietary supplements. To investigate a possibility of its embryo-fetal toxicity in rats, 11- to 12-week old dams were orally administered an aqueous solution of L-leucine at doses of 300 or 1000 mg/kg body weight on gestational days 7-17. Body weight and feed intake was evaluated throughout the whole course of pregnancy (days 0-20). L-Leucine did not influence body weight, but at a dose of 1000 mg/kg, slightly enhanced feed intake on days 14 and 18 of pregnancy. Caesarean section (day 20) revealed no influences on the litter size and weight of live-born fetuses, the number of corpora lutea, implantation index or the quality of placenta, and the minor increase in feed intake was considered irrelevant to the pregnancy outcomes. Fetuses were evaluated in a battery of external, visceral and skeletal examinations. No effects of L-leucine on gender ratio and external abnormalities, and no significant treatment-related variations in visceral and skeletal pathologies were observed. These results suggested that L-leucine, administered orally during organogenesis at doses up to 1000 mg/kg body weight, did not affect the outcome of pregnancy and did not cause fetotoxicity in rats.

  19. ROCK inhibition prevents early mouse embryo development.

    PubMed

    Duan, Xing; Chen, Kun-Lin; Zhang, Yu; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2014-08-01

    ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development.

  20. Equine cloning: in vitro and in vivo development of aggregated embryos.

    PubMed

    Gambini, Andrés; Jarazo, Javier; Olivera, Ramiro; Salamone, Daniel F

    2012-07-01

    The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.

  1. The Roles of Glutathione Peroxidases during Embryo Development.

    PubMed

    Ufer, Christoph; Wang, Chi Chiu

    2011-01-01

    Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency - in contrast to all other GPx family members - leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis on

  2. Lipidome signatures in early bovine embryo development.

    PubMed

    Sudano, Mateus J; Rascado, Tatiana D S; Tata, Alessandra; Belaz, Katia R A; Santos, Vanessa G; Valente, Roniele S; Mesquita, Fernando S; Ferreira, Christina R; Araújo, João P; Eberlin, Marcos N; Landim-Alvarenga, Fernanda D C

    2016-07-15

    Mammalian preimplantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding membrane lipid profile fluctuation during this crucial period is fundamental to address mechanisms governing embryogenesis. Therefore, the aim of the present work was to perform a comprehensive assessment of stage-specific lipid profiles during early bovine embryonic development and associate with the mRNA abundance of lipid metabolism-related genes (ACSL3, ELOVL5, and ELOVL6) and with the amount of cytoplasmic lipid droplets. Immature oocytes were recovered from slaughterhouse-derived ovaries, two-cell embryos, and eight- to 16-cell embryos, morula, and blastocysts that were in vitro produced under different environmental conditions. Lipid droplets content and mRNA transcript levels for ACSL3, ELOVL5, and ELOVL6, monitored by lipid staining and quantitative polymerase chain reaction, respectively, increased at morula followed by a decrease at blastocyst stage. Relative mRNA abundance changes of ACSL3 were closely related to cytoplasmic lipid droplet accumulation. Characteristic dynamic changes of phospholipid profiles were observed during early embryo development and related to unsaturation level, acyl chain length, and class composition. ELOVL5 and ELOVL6 mRNA levels were suggestive of overexpression of membrane phospholipids containing elongated fatty acids with 16, 18, and 20 carbons. In addition, putative biomarkers of key events of embryogenesis, embryo lipid accumulation, and elongation were identified. This study provides a comprehensive description of stage-specific lipidome signatures and proposes a mechanism to explain its potential relationship with the fluctuation of both cytoplasmic lipid droplets content and mRNA levels of lipid metabolism-related genes during early bovine embryo development.

  3. Retarded Embryo Development 1 (RED1) regulates embryo development, seed maturation and plant growth in Arabidopsis.

    PubMed

    Du, Qian; Wang, Huanzhong

    2016-07-20

    Plant seeds accumulate large amounts of protein and carbohydrate as storage reserves during maturation. Thus, understanding the genetic control of embryo and seed development may provide bioengineering tools for yield improvement. In this study, we report the identification of Retarded Embryo Development 1 (RED1) gene in Arabidopsis, whose two independent T-DNA insertion mutant lines, SALK_085642 (red1-1) and SALK_022583 (red1-2), show a retarded embryo development phenotype. The embryogenesis process ceases at the late heart stage in red1-1 and at the bent-cotyledon stage in red1-2, respectively, resulting in seed abortion in both lines. The retarded embryo development and seed abortion phenotypes reverted to normal when RED1 complementation constructs were introduced into mutant plants. Small red1-2 homozygous plants can be successfully rescued by culturing immature seeds, indicating that seed abortion likely results from compromised tolerance to the desiccation process associated with seed maturation. Consistent with this observation, red1-2 seeds accumulate less protein, and the expression of two late embryo development reporter transgenes, LEA::GUS and β-conglycinin::GUS, was significantly weak and started relatively late in the red1-2 mutant lines compared to the wild type. The RED1 gene encodes a plant specific novel protein that is localized in the nucleus. These results indicate that RED1 plays important roles in embryo development, seed maturation and plant growth.

  4. Photobiomodulation of early mouse embryo development

    NASA Astrophysics Data System (ADS)

    Sviridova-Chailakhyan, T. A.; Fakhranurova, L. I.; Simonova, N. B.; Khramov, R. N.; Manokhin, A. A.; Paskevich, S. I.; Chailakhyan, L. M.

    2008-04-01

    The effect of artificial sunlight (AS) from a xenon source and of converted AS with an additional orange-red luminescent (λ MAX=626 nm) component (AS+L) on the development of mouse zygotes was investigated. A plastic screen with a photoluminophore layer was used for production of this orange-red luminescent (L) component. A single short-term (15 min) exposure produced a long-term stable positive effect on early embryo development of mice, which persisted during several days. After exposure to AS+L, a stimulating influence on preimplantation development was observed, in comparison with the control group without AS exposure. The positive effects were as follows: increase in percent of embryos (P <= 0.05) developed to the blastocyst stage (96.2 %) with hatching from the zona pellucida (80.8 %) within 82-96 hours in vitro compared to the control (67.1 % and 28.8 %, respectively).

  5. Trichostatin A affects histone acetylation and gene expression in porcine somatic cell nucleus transfer embryos.

    PubMed

    Cervera, R P; Martí-Gutiérrez, N; Escorihuela, E; Moreno, R; Stojkovic, M

    2009-11-01

    Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming after somatic cell nucleus transfer (SCNT) and may underlie the observed reduced viability of cloned embryos. In the current study, we tested the effects of the histone deacetylase-inhibitor trichostatin A (TSA) on preimplantation development and on histone acetylation and the gene expression of nucleus transfer (NT) porcine (Sus scrofa) embryos. Our results showed that 5 nM TSA for 26 h after reconstitution resulted in embryos (NTTSA) that reached the blastocyst stage at a higher level (48.1% vs. 20.2%) and increased number of cells (105.0 vs. 75.3) than that of the control (NTC) embryos. In addition, and unlike the NTC embryos, the treated embryos displayed a global acetylated histone H4 at lysine 8 profile similar to the in vitro-fertilized (IVF) and cultured embryos during the preimplantation development. Finally, we determined that several transcription factors exert a dramatic amount of genetic control over pluripotency, including Oct4, Nanog, Cdx2, and Rex01, the imprinting genes Igf2 and Igf2r, and the histone deacetyltransferase gene Hdac2. The NT blastocysts showed similar levels of Oct4, Cdx2, and Hdac2 but lower levels of Nanog than those of the IVF blastocyst. However, the NTTSA blastocysts showed similar levels of Rex01, Igf2, and Igf2r as those of IVF blastocysts, whereas the NTC blastocysts showed significantly lower levels for those genes. Our results suggest that TSA improves porcine SCNT preimplantation development and affects the acetylated status of the H4K8, rendering acetylation levels similar to those of the IVF counterparts.

  6. Effect of Embryo Density on In Vitro Development and Gene Expression in Bovine In Vitro-fertilized Embryos Cultured in a Microwell System

    PubMed Central

    SUGIMURA, Satoshi; AKAI, Tomonori; HASHIYADA, Yutaka; AIKAWA, Yoshio; OHTAKE, Masaki; MATSUDA, Hideo; KOBAYASHI, Shuji; KOBAYASHI, Eiji; KONISHI, Kazuyuki; IMAI, Kei

    2012-01-01

    Abstract To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 µl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density. PMID:23154384

  7. Effect of embryo density on in vitro development and gene expression in bovine in vitro-fertilized embryos cultured in a microwell system.

    PubMed

    Sugimura, Satoshi; Akai, Tomonori; Hashiyada, Yutaka; Aikawa, Yoshio; Ohtake, Masaki; Matsuda, Hideo; Kobayashi, Shuji; Kobayashi, Eiji; Konishi, Kazuyuki; Imai, Kei

    2013-01-01

    To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 μl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density.

  8. Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development.

    PubMed

    Lee, W-J; Lee, J-H; Jeon, R-H; Jang, S-J; Lee, S-C; Park, J-S; Lee, S-L; King, W-A; Rho, G-J

    2017-02-13

    Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm-transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro-fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.

  9. Role of melatonin in embryo fetal development.

    PubMed

    Voiculescu, S E; Zygouropoulos, N; Zahiu, C D; Zagrean, A M

    2014-01-01

    Melatonin is an indoleamine produced by the pineal gland and secreted in a circadian manner. In the past few decades, research over this topic has been enhanced. Melatonin has many important roles in the human physiology: regulator of the circadian rhythms, sleep inducer, antioxidant, anticarcinogenic. This paper reviews the involvement of melatonin in embryo fetal development. The pineal gland develops completely postpartum, so both the embryo and the fetus are dependent on the maternal melatonin provided transplacentally. Melatonin appears to be involved in the normal outcome of pregnancy beginning with the oocyte quality and finishing with the parturition. Its pregnancy night-time concentrations increase after 24 weeks of gestation, with significantly high levels after 32 weeks. Melatonin receptors are widespread in the embryo and fetus since early stages. There is solid evidence that melatonin is neuroprotective and has a positive effect on the outcome of the compromised pregnancies. In addition, chronodisruption leads to a reproductive dysfunction. Thus, the influence of melatonin on the developing human fetus may not be limited to the entertaining of circadian rhythmicity, but further studies are needed.

  10. Cells, embryos and development in space

    NASA Technical Reports Server (NTRS)

    Krikorian, A. D.

    1984-01-01

    Work continues to focus on the demonstrable totipotency of cultured somatic cells of various higher plants and has examined the conditions which regulate this propensity to be controllably released. This was done with special reference to cells obtained from cultured explants of daylily and carrot. For purposes of identifying the variables in question, work was carried out almost exclusively in liquid media. The events that intervene between the aseptic isolation of tissue explants, the culture of small derived units and free cells and the propagation in large numbers of adventive or somatic embryos to plantlets were traced and certain definitive stages at which control is exercised were identified. In daylily, morphologically competent units are now propagated with a high degree of precision in rotated liquid cultures in bulk, and under the conditions of continuous neutralized gravity, the development progresses so that embryo-plantlets are obtained.

  11. Pollination and embryo development in Brassica rapa L. in microgravity

    NASA Technical Reports Server (NTRS)

    Kuang, A.; Popova, A.; Xiao, Y.; Musgrave, M. E.

    2000-01-01

    Plant reproduction under spaceflight conditions has been problematic in the past. In order to determine what aspect of reproductive development is affected by microgravity, we studied pollination and embryo development in Brassica rapa L. during 16 d in microgravity on the space shuttle (STS-87). Brassica is self-incompatible and requires mechanical transfer of pollen. Short-duration access to microgravity during parabolic flights on the KC-135A aircraft was used initially to confirm that equal numbers of pollen grains could be collected and transferred in the absence of gravity. Brassica was grown in the Plant Growth Facility flight hardware as follows. Three chambers each contained six plants that were 13 d old at launch. As these plants flowered, thin colored tape was used to indicate the date of hand pollination, resulting in silique populations aged 8-15 d postpollination at the end of the 16-d mission. The remaining three chambers contained dry seeds that germinated on orbit to produce 14-d-old plants just beginning to flower at the time of landing. Pollen produced by these plants had comparable viability (93%) with that produced in the 2-d-delayed ground control. Matched-age siliques yielded embryos of equivalent developmental stage in the spaceflight and ground control treatments. Carbohydrate and protein storage reserves in the embryos, assessed by cytochemical localization, were also comparable. In the spaceflight material, growth and development by embryos rescued from siliques 15 d after pollination lagged behind the ground controls by 12 d; however, in the subsequent generation, no differences between the two treatments were found. The results demonstrate that while no stage of reproductive development in Brassica is absolutely dependent upon gravity, lower embryo quality may result following development in microgravity.

  12. Pollination and embryo development in Brassica rapa L. in microgravity.

    PubMed

    Kuang, A; Popova, A; Xiao, Y; Musgrave, M E

    2000-03-01

    Plant reproduction under spaceflight conditions has been problematic in the past. In order to determine what aspect of reproductive development is affected by microgravity, we studied pollination and embryo development in Brassica rapa L. during 16 d in microgravity on the space shuttle (STS-87). Brassica is self-incompatible and requires mechanical transfer of pollen. Short-duration access to microgravity during parabolic flights on the KC-135A aircraft was used initially to confirm that equal numbers of pollen grains could be collected and transferred in the absence of gravity. Brassica was grown in the Plant Growth Facility flight hardware as follows. Three chambers each contained six plants that were 13 d old at launch. As these plants flowered, thin colored tape was used to indicate the date of hand pollination, resulting in silique populations aged 8-15 d postpollination at the end of the 16-d mission. The remaining three chambers contained dry seeds that germinated on orbit to produce 14-d-old plants just beginning to flower at the time of landing. Pollen produced by these plants had comparable viability (93%) with that produced in the 2-d-delayed ground control. Matched-age siliques yielded embryos of equivalent developmental stage in the spaceflight and ground control treatments. Carbohydrate and protein storage reserves in the embryos, assessed by cytochemical localization, were also comparable. In the spaceflight material, growth and development by embryos rescued from siliques 15 d after pollination lagged behind the ground controls by 12 d; however, in the subsequent generation, no differences between the two treatments were found. The results demonstrate that while no stage of reproductive development in Brassica is absolutely dependent upon gravity, lower embryo quality may result following development in microgravity.

  13. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    PubMed

    Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

    2012-01-01

    Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

  14. Decoupling morphological development from growth in periodically cooled zebra finch embryos.

    PubMed

    Olson, Christopher R; Vleck, Carol M; Adams, Dean C

    2008-07-01

    Temperature affects growth and development, and morphometry can provide a quantitative description of how temperature changes affect the resulting phenotype. We performed a morphometric analysis on zebra finch (Taeniopygia guttata) embryos that were either exposed to periodic cooling to 20 or 30 degrees C throughout incubation over a background temperature of 37.5 degrees C, or were incubated at a constant temperature of 37.5 degrees C. Using a principle components analysis, we found that the relationship between the multivariate size (first principle component) and dry embryo mass depended upon the thermal treatment to which the developing embryos were exposed. Periodic cooling resulted in a smaller embryo mass, but had no effect on the multivariate size of the embryo. This suggests that the growth of phenotypic traits such as the length of long bones and the skull are less affected by temperature than is growth of other soft tissues such as muscle and organs that contribute to body mass.

  15. Development of interspecies cloned embryos in yak and dog.

    PubMed

    Murakami, Masao; Otoi, Takeshige; Wongsrikeao, Pimprapar; Agung, Budiyanto; Sambuu, Rentsenkhand; Suzuki, Tatsuyuki

    2005-01-01

    Interspecies nuclear transfer (NT) could be an alternative to replicate animals when supply of recipient oocytes is limited or in vitro embryo production systems are incomplete. In the present study, embryonic development was assessed following interspecies NT of donor cumulus cells derived from yak and dog into the recipient ooplasm of domestic cow. The percentages of fusion and subsequent embryo development to the eight-cell stage of interspecies NT embryos were comparable to those of intraspecies NT embryos (cow-cow NT embryos). The percentage of development to blastocysts was significantly lower (p < 0.05) in yak-cow NT embryos than that in cow-cow NT embryos (10.9% vs. 39.8%). In dog-cow NT embryos, only one embryo (0.4%) developed to the blastocyst stage. These results indicate that interspecies NT embryos possess equally developmental competence to the eight-cell stage as intraspecies NT embryos, but the development to blastocysts is very low when dog somatic cells are used as the donor nuclei.

  16. Epigenetics in fertilization and preimplantation embryo development.

    PubMed

    Rivera, Rocio Melissa; Ross, Jason Wayne

    2013-12-01

    Epigenetic reprogramming of the parental genomes upon fertilization is required for proper embryonic development. It has long been appreciated that asymmetric distribution of histone modifications as well as differences in the level of DNA methylation exist between the parental pronuclei in mammalian zygotes and during preimplantation development. The speed at which the paternal genome is demethylated after entering the oocyte and the fact that rapid demethylation occurs in the absence of DNA replication have led many to hypothesize that a DNA demethylase must exist. However, such an enzyme has not been found. That the genome of mammalian preimplantation embryos undergo a wave of global demethylation was first reported 25 years ago but only in the past three years has data surfaced that can partially explain the elusive nature of this phenomenon. In addition to the global reorganization of the methylation and histone modification patterns, oocyte development prior to germinal vesicle breakdown involves the production of numerous small RNA, including miRNA. Despite their presence, miRNA functional activity is thought to be limited in the mature mouse oocyte. Additionally, molecular signatures in the 3' untranslated region of maternally expressed transcripts may impact mRNA stability during the transcriptionally quiescent period following germinal vesicle breakdown and prior to the maternal to zygote transition. In this review, we reference some of the recent works which attempt to shed light into the importance of the dynamic epigenetic landscape observed during oocyte maturation and preimplantation embryo development in mammals.

  17. In vitro bovine embryo production in a synthetic medium: embryo development, cryosurvival, and establishment of pregnancy.

    PubMed

    Moreno, D; Neira, A; Dubreil, L; Liegeois, L; Destrumelle, S; Michaud, S; Thorin, C; Briand-Amirat, L; Bencharif, D; Tainturier, D

    2015-10-15

    The aim of this study was to develop an in vitro embryo culture medium without either fetal calf serum or BSA, using various growth factors and cytokines (GFs-CYKs; IGF-I, IGF-II, bFGF, LIF, GM-CSF, TGF-β1, and PDGF-BB), and other molecules with surfactant and embryotrophic properties, such as recombinant albumin (RA) and hyaluronan (HA). The first part of the study was dedicated to define the best combination of GFs-CYKs + RA + HA for optimal embryonic development. Next, we compared development rates and embryo quality (inner cell mass [ICM]-to-total cell number [TCN] ratio), and postthaw survival and hatching rates using this synthetic medium (T1) and a control medium: synthetic oviduct fluid + BSA + ITS (insulin, transferrin, and selenium). The blastocyst rates were significantly higher with T1 than those with the control at 7 and 8 days after fertilization. There was no significant difference in TCN or the ICM/TCN ratio between the two treatments. Survival and hatching rates 48 hours after thawing were similar for both treatments. Finally, nine embryo transfers were conducted using fresh and previously frozen Day-7 blastocysts to evaluate the in vivo viability of embryos produced in this synthetic medium; four gestations were obtained from six fresh embryos and one gestation from three frozen embryos. In conclusion, the fetal calf serum and BSA-free medium, supplemented with GFs-CYKs + RA + HA, improved embryo development and gave comparable ICM/TCN ratios and postthaw survival rates to the control with BSA. Fresh and frozen embryos produced in this medium are viable for embryo transfer. This fully synthetic method of embryo culture is a useful means of reducing the risk of disease transmission via embryo transfer.

  18. Effect of supplementation of different growth factors in embryo culture medium with a small number of bovine embryos on in vitro embryo development and quality.

    PubMed

    Ahumada, C J; Salvador, I; Cebrian-Serrano, A; Lopera, R; Silvestre, M A

    2013-03-01

    When embryos are cultured individually or in small groups, blastocyst yield efficiency and quality are usually reduced. The aim of this work was to investigate the effect of supplementation of the embryo culture medium (CM) with several growth factors (GFs) on embryo development and apoptosis rate when a reduced number of embryos were in vitro cultured. Two experimental studies (ES) were carried out. In ES 1, five treatments were tested to study the effect of GF on embryo development: Control (∼30 to 50 embryos cultured in 500 μl of CM); Control 5 (Five embryos cultured in 50 μl microdrops of CM), without addition of GF in either of the two control groups; epidermal GF (EGF); IGF-I; and transforming GF-α (TGF-α) (Five embryos were cultured in 50 μl microdrops of CM with 10 ng/ml EGF, 10 ng/ml IGF-I or 10 ng/ml TGF-α, respectively). In ES 2, following the results obtained in ES 1, four different treatments were tested to study their effect on embryo development and quality (number of cells per blastocyst and apoptotic rate): Control; Control 5; EGF, all three similar to ES 1; EGF + IGF-I group (five embryos cultured in 50 μl microdrops of CM with 10 ng/ml EGF and 10 ng/ml IGF-I). In both ESs, it was observed that a higher proportion of embryos cultured in larger groups achieved blastocyst stage than embryos cultured in reduced groups (22.6% v. 14.0%, 12.6% and 5.3% for Control v. Control 5, IGF-I, TGF-α groups in ES 1, and 24.9% v. 17.1% and 19.0% for Control v. Control 5 and EGF in ES 2, respectively; P < 0.05), with the exception of embryos cultured in medium supplemented with EGF (18.5%) or with EGF + IGF-I (23.5%), in ES 1 and ES 2, respectively. With regard to blastocyst quality, embryos cultured in reduced groups and supplemented with EGF, alone or combined with IGF-I, presented lower apoptosis rates than embryos cultured in reduced groups without GF supplementation (11.6% and 10.5% v. 21.9% for EGF, EGF + IGF-I and Control 5 groups, respectively; P

  19. Genetic analysis of traits affecting the success of embryo transfer in dairy cattle.

    PubMed

    König, S; Bosselmann, F; von Borstel, U U; Simianer, H

    2007-08-01

    The primary aim of this study was to estimate variance components for traits related to embryo transfer (ET) by applying generalized linear mixed models (GLMM) for different distributions of traits (normal, binomial, and Poisson) in a synergistic context. Synergistic models were originally developed for traits affected by several genotypes, denoted as maternal, paternal, and direct effects. In the case of ET, the number of flushed ova (FO) only depends on a donor's maternal genetic effect, whereas paternal fertility must be considered for other embryo survival traits, such as the number of transferable embryos (TE), the number of degenerated embryos (DE), the number of unfertilized oocytes (UO), and the percentage of transferable embryos (PTE). Data for these traits were obtained from 4,196 flushes of 2,489 Holstein cows within 4 regions of northwest Germany from January 1998 through October 2004. Estimates of maternal heritability were 0.231 for FO, 0.096 for TE, 0.021 for DE, 0.135 for UO, and 0.099 for PTE, whereas the relative genetic impact of the paternal component was near zero. Estimates of the genetic correlations between the maternal and the paternal component were slightly negative, indicating a genetic antagonism. For the analysis of pregnancy after ET, 8,239 transfers to 6,819 different Holstein-Friesian recipients were considered by applying threshold methodology. The direct heritability for pregnancy in the recipient after ET was 0.056. The relative genetic impact of maternal and paternal components on pregnancy of recipients describing a donor's and a sire's ability to produce viable embryos was below 1%. The genetic correlations of the direct effect of the recipient with the sire of embryos (paternal effect) and the donor cow (maternal effect) for pregnancy after ET were -0.32 and -0.14, respectively. With the exception of FO and PTE (-0.17), estimates of genetic correlations among traits for the maternal site were distinctly positive, especially

  20. Extracellular pancuronium affects sodium current in chick embryo sensory neurones.

    PubMed Central

    Maestrone, E.; Magnelli, V.; Nobile, M.; Usai, C.

    1994-01-01

    1. The action of pancuronium on transmembrane sodium conductance was investigated in dorsal root ganglion neurones of chick embryos. The Na+ current was measured by use of the patch-clamp technique in whole-cell configuration. 2. Externally perfused pancuronium (50 microM to 1 mM) reversibly inhibited the current by a fast mechanism of action. Inhibition was concentration-dependent (with a half-effective dose of 170 microM) but not voltage-dependent. 3. The activation and inactivation kinetics of the Na+ current were estimated in pancuronium and in control solution by fitting experimental data with a Hodgkin-Huxley theoretical model. 4. The activation time constant tau m, at negative membrane voltages, was larger in the presence of pancuronium than in the control. In contrast, the inactivation time constant tau h was smaller during drug perfusion at membrane voltages < -10 mV. The steady-state inactivation h infinity was not affected by pancuronium. 5. These results suggest that pancuronium may reduce the sodium current by interacting with the sodium channels in both the resting and open states. PMID:8012707

  1. Embryos generated from oocytes lacking complex N- and O-glycans have compromised development and implantation

    PubMed Central

    Grasa, Patricia; Kaune, Heidy; Williams, Suzannah A

    2012-01-01

    Female mice generating oocytes lacking complex N- and O-glycans (double mutants (DM)) produce only one small litter before undergoing premature ovarian failure (POF) by 3 months. Here we investigate the basis of the small litter by evaluating ovulation rate and embryo development in DM (Mgat1F/FC1galt1F/F:ZP3Cre) and Control (Mgat1F/FC1galt1F/F) females. Surprisingly, DM ovulation rate was normal at 6 weeks, but declined dramatically by 9 weeks. In vitro development of zygotes to blastocysts was equivalent to Controls although all embryos from DM females lacked a normal zona pellucida (ZP) and ∼30% lacked a ZP entirely. In contrast, in vivo preimplantation development resulted in less embryos recovered from DM females compared with Controls at 3.5 days post coitum (dpc) (3.2±1.3 vs 7.0±0.6). Furthermore, only 45% of mated DM females contained embryos at 3.5 dpc. Of the preimplantation embryos collected from DM females, approximately half were morulae unlike Controls where the majority were blastocysts, indicating delayed embryo development in DM females. Post-implantation development in DM females was analysed to determine whether delayed preimplantation development affected subsequent development. In DM females at 5.5 dpc, only ∼40% of embryos found at 3.5 dpc had implanted. However, at 6.5 dpc, implantation sites in DM females corresponded to embryo numbers at 3.5 dpc indicating delayed implantation. At 9.5 dpc, the number of decidua corresponded to embryo numbers 6 days earlier indicating that all implanted embryos progress to midgestation. Therefore, a lack of complex N- and O-glycans in oocytes during development impairs early embryo development and viability in vivo leading to delayed implantation and a small litter. PMID:22919046

  2. Cadmium affects retinogenesis during zebrafish embryonic development

    SciTech Connect

    Hen Chow, Elly Suk; Yu Hui, Michelle Nga; Cheng, Chi Wa; Cheng, Shuk Han

    2009-02-15

    Ocular malformations are commonly observed in embryos of aquatic species after exposure to toxicants. Using zebrafish embryos as the model organism, we showed that cadmium exposure from sphere stage (4 hpf) to end of segmentation stage (24 hpf) induced microphthalmia in cadmium-treated embryos. Embryos with eye defects were then assessed for visual abilities. Cadmium-exposed embryos were behaviorally blind, showing hyperpigmentation and loss of camouflage response to light. We investigated the cellular basis of the formation of the small eyes phenotype and the induction of blindness by studying retina development and retinotectal projections. Retinal progenitors were found in cadmium-treated embryos albeit in smaller numbers. The number of retinal ganglion cells (RGC), the first class of retinal cells to differentiate during retinogenesis, was reduced, while photoreceptor cells, the last batch of retinal neurons to differentiate, were absent. Cadmium also affected the propagation of neurons in neurogenic waves. The neurons remained in the ventronasal area and failed to spread across the retina. Drastically reduced RGC axons and disrupted optic stalk showed that the optic nerves did not extend from the retina beyond the chiasm into the tectum. Our data suggested that impairment in neuronal differentiation of the retina, disruption in RGC axon formation and absence of cone photoreceptors were the causes of microphthalmia and visual impairment in cadmium-treated embryos.

  3. Embryo transfer day does not affect the initial maternal serum β-hCG levels: A retrospective cohort study.

    PubMed

    Dahiya, Mona; Rupani, Karishma; Yu, Su Ling; Fook-Chong, Stephanie M C; Siew Fui, Diana Chia; Rajesh, Hemashree

    2017-03-18

    The aim of this study is to compare the serum β-hCG values post transfer of a cleavage stage embryo versus a blastocyst stage embryo at equal time intervals post oocyte retrieval (OR) in clinically pregnant patients, and to ascertain a β-hCG value to predict pregnancy outcomes. This is a retrospective cohort study of 560 women with clinical pregnancy who underwent an embryo transfer performed at either the cleavage stage or the blastocyst stage of embryo development between January 2003 and June 2014 at the Center for Assisted Reproduction (CARE), Singapore General Hospital. The serum β-hCG level was measured on day 17 post OR. The β-hCG values were not significantly different in the cleavage stage versus the blastocyst stage embryos (mean±SD: 387±486IU/L D3 vs. 352±268IU/L D5, p=0.96, median value 297 in both groups). Our study suggests that the initial maternal serum β-hCG values were not affected by the day of transfer of the embryos since assessing the β-hCG at equivalent points after transfer should not lead to a significant difference assuming the progress and development of the embryos occurred as expected.

  4. Fatty acid breakdown in developing embryos of Brassica napus L.

    PubMed

    Chia, T; Rawsthorne, S

    2000-12-01

    Developing Brassica napus embryos are primarily concerned with the accumulation of storage products, namely oil, starch and protein. The presence of fatty acid catabolic pathways in the background of this biosynthetic activity was investigated. Enzymes involved in the process of lipid mobilization, such as malate synthase and isocitrate lyase, are detectable towards the late stages of embryo development. [(14)C]Acetate feeding experiments also reveal that fatty acid catabolism becomes increasingly functional as the embryo matures.

  5. Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

    NASA Astrophysics Data System (ADS)

    Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

    2015-11-01

    Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in

  6. Oxamflatin Treatment Enhances Cloned Porcine Embryo Development and Nuclear Reprogramming*

    PubMed Central

    Mao, Jiude; Zhao, Ming-Tao; Whitworth, Kristin M.; Spate, Lee D.; Walters, Eric M.; O'Gorman, Chad; Lee, Kiho; Samuel, Melissa S.; Murphy, Clifton N.; Wells, Kevin; Rivera, Rocio M.

    2015-01-01

    Abstract Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low cloning efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro–fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the cloned embryos, indicating these genes were successfully reprogrammed. However, compared to the lack of methylation of XIST in day-7 IVF embryos, a higher methylation level in day-7 cloned embryos was observed, implying that X chromosomes were activated in day-7 IVF blastocysts, but were not fully activated in cloned embryos, i.e., reprogramming of XIST was delayed. A time-course analysis of XIST DNA methylation on day-13, -15, -17, and -19 in vivo embryos revealed that XIST methylation initiated at about day 13 and was not completed by day 19. The methylation of the XIST gene in day-19 control cloned embryos was delayed again when compared to in vivo embryos. However, methylation of XIST in Oxamflatin-treated embryos was comparable with in vivo embryos, which further demonstrated that Oxamflatin could accelerate the delayed reprogramming of XIST gene and thus might improve cloning efficiency. PMID:25548976

  7. Genes affecting novel seed constituents in Limnanthes alba Benth: transcriptome analysis of developing embryos and a new genetic map of meadowfoam

    PubMed Central

    Cooper, Laurel D.; Kishore, Venkata K.; Knapp, Steven J.; Kling, Jennifer G.

    2015-01-01

    The seed oil of meadowfoam, a new crop in the Limnanthaceae family, is highly enriched in very long chain fatty acids that are desaturated at the Δ5 position. The unusual oil is desirable for cosmetics and innovative industrial applications and the seed meal remaining after oil extraction contains glucolimnanthin, a methoxylated benzylglucosinolate whose degradation products are herbicidal and anti-microbial. Here we describe EST analysis of the developing seed transcriptome that identified major genes involved in biosynthesis and assembly of the seed oil and in glucosinolate metabolic pathways. mRNAs encoding acyl-CoA Δ5 desaturase were notably abundant. The library was searched for simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). Fifty-four new SSR markers and eight candidate gene markers were developed and combined with previously developed SSRs to construct a new genetic map for Limnanthes alba. Mapped genes in the lipid biosynthetic pathway encode 3-ketoacyl-CoA synthase (KCS), Δ5 desaturase (Δ5DS), lysophosphatidylacyl-acyl transferase (LPAT), and acyl-CoA diacylglycerol acyl transferase (DGAT). Mapped genes in glucosinolate biosynthetic and degradation pathways encode CYP79A, myrosinase (TGG), and epithiospecifier modifier protein (ESM). The resources developed in this study will further the domestication and improvement of meadowfoam as an oilseed crop. PMID:26038713

  8. In vitro fertilization and embryo development in pigs.

    PubMed

    Abeydeera, L R

    2001-01-01

    Considerable progress has been made in the in vitro production of pig embryos using improved methods for in vitro maturation (IVM) and fertilization (IVF). Despite the progress, polyspermic penetration remains a problem for in vitro-matured oocytes. Variation among boars, ejaculates and IVF protocols used in different laboratories appears to influence the incidence of polyspermy. Recent studies indicate that oviduct cells and their secretions play a role in reducing polyspermy. Very early attempts to culture in vivo-derived pig embryos met with little success and most were arrested at the four-cell stage. At present, many culture media are available that can overcome the four-cell block and support development to the blastocyst stage. In contrast, blastocyst development of in vitro-produced (IVP) embryos in these culture media varies significantly. Significant differences in morphology and numbers of cells have been observed in in vitro-produced blastocysts compared with in vivo-derived blastocysts. Surgical transfer of in vitro-produced embryos to recipient animals has resulted in acceptable pregnancy rates with moderate litter sizes. Although several systems are available for the generation of in vitro-produced embryos, the problems of polyspermy and poor embryo survival prevent large-scale production of embryos. Further research should be directed to improve oocyte and embryo quality, and to develop methods to minimize polyspermy through development of better IVM, IVF and embryo culture techniques.

  9. Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

    PubMed

    Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

    2016-09-01

    The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes

  10. Metabolite profiling reveals clear metabolic changes during somatic embryo development of Norway spruce (Picea abies).

    PubMed

    Businge, Edward; Brackmann, Klaus; Moritz, Thomas; Egertsdotter, Ulrika

    2012-02-01

    Progress on industrial-scale propagation of conifers by somatic embryogenesis has been hampered by the differences in developmental capabilities between cell lines, which are limiting the capture of genetic gains from breeding programs. In this study, we investigated the metabolic events occurring during somatic embryo development in Norway spruce to establish a better understanding of the fundamental metabolic events required for somatic embryo development. Three embryogenic cell lines of Norway spruce (Picea abies (L.) Karst) with different developmental capabilities were studied during somatic embryo development from proliferation of proembryogenic masses to mature somatic embryos. The three different cell lines displayed normal, aberrant and blocked somatic embryo development. Metabolite profiles from four development stages in each of the cell lines were obtained using combined gas chromatography-mass spectrometry. Multivariate discriminant analyses of the metabolic data revealed significant metabolites (P  ≤  0.05) for each development stage and transition. The results suggest that endogenous auxin and sugar signaling affects initial stages of somatic embryo development. Furthermore, the results highlight the importance of a timed stress response and the presence of stimulatory metabolites during late stages of embryo development.

  11. Effects of brief hypoxia and hyperoxia on tissue element levels in the development chick embryo

    SciTech Connect

    Richards, M.P.; Stock, M.K.; Metcalfe, J. Oregon Health Sciences Univ., Portland )

    1991-03-15

    Brief hypoxia or hyperoxia has been shown to affect growth and metabolism of chick embryos during the later stages of development. The objective of this experiment was to alter the availability of oxygen to chick embryos developing in ovo and to determine the effects on tissue levels of Zn, Cu, Fe and Mn. Hypoxia reduced embryo, heart, brain and liver wts (wet wt), whereas, hyperoxia increased embryo, heart, lung and liver wts compared to normoxic controls. Chorioallantoic membrane (CAM) wt was increased by hypoxia and reduced by hyperoxia. Livers from hyperoxic embryos contained more Zn, Fe and Mn and less Cu than livers from hypoxic or normoxic embryos. Tissue levels of Zn, Cu, Fe and Mn were reduced in brains from hypoxic compared to hyperoxic or normoxic embryos. Hyperoxia increased the concentrations of Zn and Cu in CAM; whereas, hypoxia reduced the levels of Zn and Fe. The amounts of Zn and Cu were increased in hyperoxic compared to normoxic lungs. Hearts from hyperoxic embryos had more Zn, Cu and Mn than hypoxic or normoxic hearts. Hypoxic yolk sac contained more Zn, Cu and Mn than hyperoxic or normoxic yolk sac. Except for yolk sac, the amounts of Zn, Cu, Fe and Mn in tissues from normoxic embryos increased from day 15 to day 18 of incubation in concert with tissue growth. The authors conclude that the availability of O{sub 2} to the developing chick embryo affects tissue trace element levels either through its effects on tissue growth or via effects on the regulation of trace element uptake and assimilation by the tissues.

  12. Storage oil breakdown during embryo development of Brassica napus (L.).

    PubMed

    Chia, Tansy Y P; Pike, Marilyn J; Rawsthorne, Stephen

    2005-05-01

    In this study it is shown that at least 10% of the major storage product of developing embryos of Brassica napus (L.), triacylglycerol, is lost during the desiccation phase of seed development. The metabolism of this lipid was studied by measurements of the fate of label from [1-(14)C]decanoate supplied to isolated embryos, and by measurements of the activities of enzymes of fatty acid catabolism. Measurements on desiccating embryos have been compared with those made on embryos during lipid accumulation and on germinating seedlings. Enzymes of beta-oxidation and the glyoxylate cycle, and phosphoenolpyruvate carboxykinase were present in embryos during oil accumulation, and increased in activity and abundance as the seeds matured and became desiccated. Although the activities were less than those measured during germination, they were at least comparable to the in vivo rate of fatty acid synthesis in the embryo during development. The pattern of labelling, following metabolism of decanoate by isolated embryos, indicated a much greater involvement of the glyoxylate cycle during desiccation than earlier in oil accumulation, and showed that much of the (14)C-label from decanoate was released as CO(2) at both stages. Sucrose was not a product of decanoate metabolism during embryo development, and therefore lipid degradation was not associated with net gluconeogenic activity. These observations are discussed in the context of seed development, oil yield, and the synthesis of novel fatty acids in plants.

  13. Retinoic Acid Signaling Is Essential for Valvulogenesis by Affecting Endocardial Cushions Formation in Zebrafish Embryos.

    PubMed

    Li, Junbo; Yue, Yunyun; Zhao, Qingshun

    2016-02-01

    Retinoic acid (RA) plays important roles in many stages of heart morphogenesis. Zebrafish embryos treated with exogenous RA display defective atrio-ventricular canal (AVC) specification. However, whether endogenous RA signaling takes part in cardiac valve formation remains unknown. Herein, we investigated the role of RA signaling in cardiac valve development by knocking down aldh1a2, the gene encoding an enzyme that is mainly responsible for RA synthesis during early development, in zebrafish embryos. The results showed that partially knocking down aldh1a2 caused defective formation of primitive cardiac valve leaflets at 108 hpf (hour post-fertilization). Inhibiting endogenous RA signaling by 4-diethylaminobenzal-dehyde revealed that 16-26 hpf was a key time window when RA signaling affects the valvulogenesis. The aldh1a2 morphants had defective formation of endocardial cushion (EC) at 76 hpf though they had almost normal hemodynamics and cardiac chamber specification at early development. Examining the expression patterns of AVC marker genes including bmp4, bmp2b, nppa, notch1b, and has2, we found the morphants displayed abnormal development of endocardial AVC but almost normal development of myocardial AVC at 50 hpf. Being consistent with the reduced expression of notch1b in endocardial AVC, the VE-cadherin gene cdh5, the downstream gene of Notch signaling, was ectopically expressed in AVC of aldh1a2 morphants at 50 hpf, and overexpression of cdh5 greatly affected the formation of EC in the embryos at 76 hpf. Taken together, our results suggest that RA signaling plays essential roles in zebrafish cardiac valvulogenesis.

  14. Early embryo development in Fucus distichus is auxin sensitive

    NASA Technical Reports Server (NTRS)

    Basu, Swati; Sun, Haiguo; Brian, Leigh; Quatrano, Ralph L.; Muday, Gloria K.

    2002-01-01

    Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [(3)H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development.

  15. Sex determination of duck embryos: observations on syrinx development

    USGS Publications Warehouse

    Wilson, Robert E.; Sonsthagen, Sarah A.; Franson, J. Christian

    2013-01-01

    Ducks exhibit sexual dimorphism in vocal anatomy. Asymmetrical ossification of the syrinx (bulla syringealis) is discernable at about 10 days of age in male Pekin duck (Anas platyrhynchos domestica) embryos, but information is lacking on the early development of the bulla in wild ducks. To evaluate the reliability of this characteristic for sexing developing embryos, we examined the syrinx of dead embryos and compared results with molecular sexing techniques in high arctic nesting Common Eiders (Somateria mollissima). Embryos 8 days or older were accurately (100%) sexed based on the presence/absence of a bulla, 2 days earlier than Pekin duck. The use of the tracheal bulla can be a valuable technique when sex identification of embryos or young ducklings is required.

  16. Effects of embryo-derived exosomes on the development of bovine cloned embryos

    PubMed Central

    Qu, Pengxiang; Qing, Suzhu; Liu, Ruiqi; Qin, Hongyu; Wang, Weiwei; Qiao, Fang; Ge, Hui; Liu, Jun; Zhang, Yong; Wang, Yongsheng

    2017-01-01

    The developmental competence of in vitro cultured (IVC) embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT) embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE), as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development), but also following growth to term (in vivo development). Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation. PMID:28350875

  17. Factors affecting oocyte quality and quantity in commercial application of embryo technologies in the cattle breeding industry.

    PubMed

    Merton, J S; de Roos, A P W; Mullaart, E; de Ruigh, L; Kaal, L; Vos, P L A M; Dieleman, S J

    2003-01-15

    With the introduction of multiple ovulation, embryo recovery and transfer techniques (MOET) plus embryo freeze-thaw methods in the early 1980s, the breeding industry has the tools in hand to increase the number of calves from donors of high genetic merit. In the early 1990s, the introduction of ovum pick-up followed by in vitro embryo production (OPU-IVP) opened up even greater possibilities. Using these technologies, we challenge biological mechanisms in reproduction. Where normally one oocyte per estrous cycle will develop to ovulation, now numerous other oocytes that otherwise would have degenerated are expected to develop into an embryo. Completion of oocyte growth and pre-maturation in vivo before final maturation both appear to be essential phases in order to obtain competence to develop into an embryo and finally a healthy offspring. In order to increase oocyte quality and quantity in embryo production technologies, current procedures focus primarily on improving the homogeneity of the population of oocytes with regard to growth and state of pre-maturation at the start of a treatment. In the case of MOET, dominant follicle removal (DFR) before superovulation treatment improves the number of viable embryos per session from 3.9 to 5.4 in cows but not in heifers and a prolonged period of follicle development obtained by preventing release of the endogenous LH surge increases the number of ova but not the number of viable embryos per session. In the case of OPU-IVP, the frequency of OPU clearly affects quantity and quality of the collected oocytes and FSH stimulation prior to OPU every 2 weeks resulted in 3.3 embryos per session. Analysis of 7,800 OPU sessions demonstrated that the oocyte yield is dependent on the team, in particular, the technician manipulating the ovaries. It is concluded that an increased understanding of the processes of oocyte growth, pre- and final maturation will help to improve the efficiency of embryo technologies. However, somewhere we

  18. The efficacy of the well of the well (WOW) culture system on development of bovine embryos in a small group and the effect of number of adjacent embryos on their development.

    PubMed

    Kang, Sung-Sik; Ofuji, Sosuke; Imai, Kei; Huang, Weiping; Koyama, Keisuke; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

    2015-06-01

    The aim of the present study was to clarify the efficacy of the well of the well (WOW) culture system for a small number of embryos and the effect of number of adjacent embryos in a WOW dish on blastocyst development. In conventional droplet culture, embryos in the small-number group (5-6 embryos/droplet) showed low blastocyst development compared with a control group (25-26 embryos/droplet). However, small and large numbers of embryos (5-6 and 25 embryos, respectively) in a WOW dish showed no significant differences in cleavage, blastocyst rates, and mean cell number in blastocysts compared with the control group (25-30 embryos/droplet). In addition, the number of adjacent embryos in a WOW dish did not affect the development to blastocysts and cell number in blastocysts. In conclusion, a WOW dish can provide high and stable blastocyst development in small group culture wherever embryos are placed in microwells of the WOW dish.

  19. The effect of molybdenum on the in vitro development of mouse preimplantation embryos.

    PubMed

    Bi, Cong-Ming; Zhang, Yu-Ling; Liu, Feng-Jun; Zhou, Tie-Zhong; Yang, Zi-Jun; Gao, Shen-Yang; Wang, Shu-De; Chen, Xiao-Li; Zhai, Xiao-Wei; Ma, Xue-Gang; Jin, Li-Jun; Wang, Shen

    2013-04-01

    The object of this study was to investigate the effect of molybdenum on the development of mouse preimplantation embryos cultured in vitro. Zygotes were flushed from one outbred mouse strain (Kunming), and then were cultured in potassium simplex optimized medium (KSOM) containing 0, 5, 10, 20, 40, 80, 120, and 160 µg/ml of molybdenum for 5 days until the mid-blastocyst stage. The addition of ≤ 20 µg/ml molybdenum did not affect the blastocyst and birth rates. Molybdenum at doses of 40 µg/ml and higher significantly decreased the cleavage, blastocyst and birth rates, the average cell number, and significantly increased the proportion of degenerative blastocysts. At 120 µg/ml molybdenum inhibited the blastocysts development to birth. At 160 µg/ml molybdenum caused overall developmental arrest (up to 16-cells) of embryos and their massive degeneration. In conclusion, molybdenum negatively affected the development of embryos in a dose-dependent manner. With lower doses (≤ 20 µg/ml), mouse embryos were not apparently damaged. With very high doses (≥ 40 µg/ml), embryo quality significantly decreased. This assessment of the effect of molybdenum on the preimplantation embryo is an initial survey of toxicological risk.

  20. Superovulatory response and embryo development in ewes treated with two doses of bovine somatotropin.

    PubMed

    Carrera-Chávez, J M; Hernández-Cerón, J; López-Carlos, M A; Lozano-Domínguez, R R; Molinar, F; Echavarría-Cháirez, F G; Bañuelos-Valenzuela, R; Aréchiga-Flores, C F

    2014-12-30

    This study evaluated whether the administration of 50 and 100mg bovine somatotropin (bST) at the start of synchronization and at the time of natural mating in ewes improves the ovulation rate, embryonic development and pregnancy rate of transferred embryos. Forty-eight donors were assigned to three treatments: the bST-100 treatment (n=15) received 100mg bST at the start of synchronization and at natural mating, the bST-50 treatment (n=15) received 50mg bST on the same schedule as the previous group, and the control (n=18) did not receive any bST. Two embryos were transferred to each recipient (n=121): 35 received embryos from bST-100; 50 received embryos from bST-50, and 36 received embryos from the control. The superovulatory rate, percentage of recovered structures, cleavage rate, percentage of transferable embryos, embryo quality and development and pregnancy rate were analyzed using the GENMOD procedure of SAS. The number of corpora lutea and the cell number were analyzed using the GLM procedure of SAS. The insulin and IGF-1 concentrations were analyzed with ANOVA for repeated measures. The bST application did not affect the superovulatory rate, number of corpora lutea and recovered structures (P>0.05). The numbers of transferable embryos and embryos reaching the blastocyst were higher (P≤0.01) in the bST-50 (96.4±3.6% and 69.0±7.8%) than the bST-100 (93.0±4.5% and 27.2±38.9%) and control (87.7±5.4% and 50.4±6.4%) groups. The insulin and IGF-1 concentrations were higher (P<0.05) in the bST-treated groups, but the insulin concentration was higher (P<0.05) in the bST-100 group than in the bST-50 group. The pregnancy rate was similar (P=0.21) in ewes receiving embryos from the two treatments [bST-50, (70.0%); bST-100, (62.5%), and control, (56.6%)]. The administration of 50mg bST at the start of synchronization and at natural mating in superovulated ewes was concluded to enhance the proportion and development of transferable embryos. However, bST did not

  1. Chronic toxicity of copper on embryo development in Chinese toad, Bufo gargarizans.

    PubMed

    Xia, Kun; Zhao, Hongfeng; Wu, Minyao; Wang, Hongyuan

    2012-06-01

    This study examined the effects of copper exposure on embryonic development of Chinese toad, Bufo gargarizans. Firstly, the LC(50) values from 24 to 96 h of exposure were 3.61×10(-6) M, by means of a 4 d toxicity test with B. gargarizans embryos. Secondly, Chinese toad embryos were exposed to 10(-9)-10(-6) M copper from mid gastrula stage to operculum completion stage. Measurements included mortality, tadpole weight, tadpole total length, growth retardation, duration of different embryo stages and malformation. Embryonic survival was not affected by copper. Relative to control tadpoles, significantly decreased weight and total length were found at 10(-9)-10(-6) M reduced percentage of the embryos in right operculum stage after 10 d exposure to copper and reduced percentage of embryos in operculum completion stage after 12 d exposure to copper were also observed. Moreover, the duration of embryonic development increased at neural, circulation and operculum development stage in copper-treated groups. For the scanning microscope and histological observation, the abnormalities were malformation of wavy dorsal fin, flexural tail, curvature body axis, yolk sac oedema and reduced pigmentation in the yolk sac. Histopathological changes in olfactory, retinal epithelium and skin were also observed. DNA strand breaks exposed to the copper were analyzed by DNA ladder. In conclusion, copper induced toxic effects on B. gargarizans embryos. The present study indicated chronic toxicity tests may provide more accurate way in formulating the "safe levels" of heavy metals to amphibian.

  2. Selection for rapid embryo development correlates with embryo exposure to maternal androgens among passerine birds

    USGS Publications Warehouse

    Schwabl, H.; Palacios, M.G.; Martin, T.E.

    2007-01-01

    Greater offspring predation favors evolution of faster development among species. We hypothesized that greater offspring predation exerts selection on mothers to increase levels of anabolic androgens in egg yolks to achieve faster development. Here, we tested whether (1) concentrations of yolk androgens in passerine species were associated with offspring predation and (2) embryo and nestling development rates were associated with yolk androgen concentrations. We examined three androgens that increase in potency along the synthesis pathway: androstenedione (A4) to testosterone (T) to 5??- dihydrotestosterone (5??-DHT). Concentrations of none of these steroids were related to clutch size; only A4 was allometrically related to egg volume. Species that experience greater predation showed higher yolk concentrations of T and 5??-DHT. Higher concentrations of T and particularly 5??-DHT were strongly correlated with faster development during the embryo period and less so during the nestling period. Development rates were most strongly correlated with 5??-DHT, suggesting that potency increases along the androgen synthesis pathway and that effects are mediated by the androgen receptor pathway. These results are consistent with the hypothesis that selection for faster development by time-dependent offspring mortality may be achieved epigenetically by varying embryo exposure to maternal anabolic steroids. ?? 2007 by The University of Chicago. All rights reserved.

  3. From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula.

    PubMed

    Wang, Xin-Ding; Song, Youhong; Sheahan, Michael B; Garg, Manohar L; Rose, Ray J

    2012-01-01

    • The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. • We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. • Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. • Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.

  4. The effects of strontium on skeletal development in zebrafish embryo.

    PubMed

    Pasqualetti, Sara; Banfi, Giuseppe; Mariotti, Massimo

    2013-10-01

    The strontium is an alkaline earth metal found in nature as trace element. Chemically similar to calcium, it is known to be involved in the human bone mineral metabolism. The strontium ranelate has been approved in therapy as drug with both anti-resorption and anabolic effects on bone tissues. Since few data in vivo are available, we used Danio rerio as animal model to evaluate the effects of strontium on skeletal development. First, toxicity assay performed on zebrafish embryos estimated the LC50 around 6mM. Since several zebrafish bones are formed from cartilage mineralization, we evaluated whether strontium affects cartilage development during embryogenesis. Strontium does not perturb the development of the cartilage tissues before the endochondral osteogenesis takes place. About the mineralization process, we evidentiated an increase of vertebral mineralization respect to controls at lower strontium concentrations whereas higher concentration inhibited mineral deposition in dose dependent fashion. Our results evidentiated, in addition, that the calcium/strontium rate but not the absolute level of strontium modulates the mineralization process during embryonic osteogenesis. Zebrafish represents an excellent animal model to study the role of micronutrients in the development of the tissues/organs because the ions are not absorbed by intestine but assumed by skin diffusion.

  5. Effect of MEM vitamins and forskolin on embryo development and vitrification tolerance of in vitro-produced pig embryos.

    PubMed

    Cuello, C; Gomis, J; Almiñana, C; Maside, C; Sanchez-Osorio, J; Gil, M A; Sánchez, A; Parrilla, I; Vazquez, J M; Roca, J; Martinez, E A

    2013-01-30

    The aims of this study were (1) to determine the effect of in vitro maturation (IVM) medium supplementation with MEM vitamins on in vitro embryo development and sensitivity to vitrification of Day 6 blastocysts and (2) to evaluate whether the addition of forskolin to in vitro culture (IVC) medium enhances blastocyst survival following Super Open Pulled Straw (SOPS) vitrification. Cumulus-oocyte complexes (COCs; n=4000) were matured with 0.0% or 0.05% (v/v) MEM vitamins. After 44h of IVM, the oocytes were in vitro fertilized, and presumptive zygotes were cultured. At Day 5 of IVC, embryos from both experimental groups were cultured for 24h with 0 or 10μM forskolin, achieving a 2×2 factorial design. The blastocyst formation rate was assessed on Day 6, and subsets of samples from the four experimental groups were vitrified (n=469) or kept fresh (n=546). Fresh and vitrified-warmed blastocysts were cultured for 24h prior to embryo survival and total blastocyst cell number assessment. The MEM vitamins increased (P<0.001) the blastocyst formation rate at Day 6, but they did not affect embryo survival after vitrification. In contrast, the addition of forskolin to the culture medium enhanced (P<0.05) the blastocyst vitrification tolerance. The total blastocyst cell number was similar among the groups. In conclusion, supplementation with 0.05% MEM vitamins improved the blastocyst formation rate, and the addition of 10μM forskolin to the culture medium increased survival in Day 6 in vitro-produced blastocysts after SOPS vitrification.

  6. Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos.

    PubMed

    Cisterna, B; Flach, F; Vecchio, L; Barabino, S M L; Battistelli, S; Martin, T E; Malatesta, M; Biggiogera, M

    2008-01-01

    In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM) soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in pre-mRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

  7. Fusion of blastomeres in mouse embryos under the action of femtosecond laser radiation. Efficiency of blastocyst formation and embryo development

    NASA Astrophysics Data System (ADS)

    Osychenko, A. A.; Zalesskii, A. D.; Krivokharchenko, A. S.; Zhakhbazyan, A. K.; Ryabova, A. V.; Nadtochenko, V. A.

    2015-05-01

    Using the method of femtosecond laser surgery we study the fusion of two-cell mouse embryos under the action of tightly focused femtosecond laser radiation with the fusion efficiency reaching 60%. The detailed statistical analysis of the efficiency of blastomere fusion and development of the embryo up to the blastocyst stage after exposure of the embryos from different mice to a femtosecond pulse is presented. It is shown that the efficiency of blastocyst formation essentially depends on the biological characteristics of the embryo, namely, the strain and age of the donor mouse. The possibility of obtaining hexaploid embryonal cells using the methods of femtosecond laser surgery is demonstrated.

  8. Danio rerio embryos on Prozac - Effects on the detoxification mechanism and embryo development.

    PubMed

    Cunha, V; Rodrigues, P; Santos, M M; Moradas-Ferreira, P; Ferreira, M

    2016-09-01

    In the past decade the presence of psychopharmaceuticals, including fluoxetine (FLU), in the aquatic environment has been associated with the increasing trend in human consumption of these substances. Aquatic organisms are usually exposed to chronic low doses and, therefore, risk assessments should evaluate the effects of these compounds in non-target organisms. Teleost fish possess an array of active defence mechanisms to cope with the deleterious effects of xenobiotics. These include ABC transporters, phase I and II of cellular detoxification and oxidative stress enzymes. Hence, the present study aimed at characterising the effect of FLU on embryo development of the model teleost zebrafish (Danio rerio) concomitantly with changes in the detoxification mechanisms during early developmental phases. Embryos were exposed to different concentrations of FLU (0.0015, 0.05, 0.1, 0.5 and 0.8μM) for 80hours post fertilization. Development was screened and the impact in the transcription of key genes, i.e., abcb4, abcc1, abcc2, abcg2, cyp1a, cyp3a65, gst, sod, cat, ahr, pxr, pparα, pparβ, pparγ, rxraa, rxrab, rxrbb, rxrga, rxrgb, raraa, rarab, rarga evaluated. In addition, accumulation assays were performed to measure the activity of ABC proteins and antioxidant enzymes (CAT and Cu/ZnSOD) after exposure to FLU. Embryo development was disrupted at the lowest FLU concentration tested (0.0015μM), which is in the range of concentrations found in WWTP effluents. Embryos exposed to higher concentrations of FLU decreased Cu/Zn SOD, and increased CAT (0.0015 and 0.5μM) enzymatic activity. Exposure to higher concentrations of FLU decreased the expression of most genes belonging to the detoxification system and upregulated cat at 0.0015μM of FLU. Most of the tested concentrations downregulated pparα, pparβ, pparγ, and raraa, rxraa, rxrab, rxrbb rxrgb and ahr gene expression while pxr was significantly up regulated at all tested concentrations. In conclusion, this study

  9. RAPID COMMUNICATION: Nerve growth factor influences cleavage rate and embryo development in sheep.

    PubMed

    Crispo, M; Dos Santos-Neto, P C; Vilariño, M; Mulet, A P; de León, A; Barbeito, L; Menchaca, A

    2016-10-01

    Recent information about Nerve growth factor (NGF), a protein traditionally associated to the nervous system that regulates survival and maturation of developing neurons, suggests that it may exert action also on different levels in the reproductive system. The aim of this study was to evaluate the effect of NGF added during in vitro oocyte maturation, fertilization or in vitro embryo development in sheep. Nerve growth factor was supplemented to the culture medium at 0, 100, or 1,000 ng/mL, during either in vitro maturation (Exp. 1), in vitro fertilization (Exp. 2), or in vitro culture (Exp. 3). In addition, NGF mRNA expression was determined in cumulus cells and oocytes. Nerve growth factor induced early cleavage when added during oocyte maturation or fertilization, improved embryo development when added during fertilization, and had no significant effect when added during embryo culture. In general, the effect was more evident with 100 rather than 1,000 ng/mL (P < 0.05). Expression of endogenous NGF was not detected in oocytes, and increased in cumulus cells when 1,000 ng/mL of NGF was added during fertilization, but not during maturation and embryo culture. In conclusion, the addition of NGF during oocyte maturation and fertilization affects in vitro cleavage and embryo development in sheep. We suggest a possible effect of this growth factor on oocyte maturation and mainly on the fertilization process.

  10. MicroRNA processing machinery in the developing chick embryo.

    PubMed

    Carraco, Gil; Gonçalves, Ana N; Serra, Carlos; Andrade, Raquel P

    2014-11-01

    Gene expression regulation during embryo development is under strict regulation to ensure proper gene expression in both time and space. The involvement of microRNAs (miRNA) in early vertebrate development is documented and inactivation of different proteins involved in miRNA synthesis results in severe malformations or even arrests vertebrate embryo development. However, there is very limited information on when and in what tissues the genes encoding these proteins are expressed. Herein, we report a detailed characterization of the expression patterns of DROSHA, DGCR8, XPO5 and DICER1 in the developing chick embryo, from HH1 (when the egg is laid) to HH25 (5-days incubation), using whole mount in situ hybridization and cross-section analysis. We found that these genes are co-expressed in multiple tissues, mostly after stage HH4. Before early gastrulation DICER1 expression was never detected, suggesting the operation of a Dicer-independent pathway for miRNA synthesis. Our results support an important role for miRNAs in vertebrate embryo development and provide the necessary framework to unveil additional roles for these RNA processing proteins in development.

  11. Mouse embryo motion and embryonic development from the 2-cell to blastocyst stage using mechanical vibration systems.

    PubMed

    Asano, Yuka; Matsuura, Koji

    2014-06-01

    We investigated the effect of mechanical stimuli on mouse embryonic development from the 2-cell to blastocyst stage to evaluate physical factors affecting embryonic development. Shear stress (SS) applied to embryos using two mechanical vibration systems (MVSs) was calculated by observing microscopic images of moving embryos during mechanical vibration (MV). The MVSs did not induce any motion of the medium and the diffusion rate using MVSs was the same as that under static conditions. Three days of culture using MVS did not improve embryonic development. MVS transmitted MV power more efficiently to embryos than other systems and resulted in a significant decrease in development to the morula or blastocyst stage after 2 days. Comparison of the results of embryo culture using dynamic culture systems demonstrated that macroscopic diffusion of secreted materials contributes to improved development of mouse embryos to the blastocyst stage. These results also suggest that the threshold of SS and MV to induce negative effects for mouse embryos at stages earlier than the blastocyst may be lower than that for the blastocyst, and that mouse embryos are more sensitive to physical and chemical stimuli than human or pig embryos because of their thinner zona pellucida.

  12. Determinant molecular markers for peri-gastrulating bovine embryo development.

    PubMed

    Hue, Isabelle

    2016-01-01

    Peri-gastrulation defines the time frame between blastocyst formation and implantation that also corresponds in cattle to elongation, pregnancy recognition and uterine secretion. Optimally, this developmental window prepares the conceptus for implantation, placenta formation and fetal development. However, this is a highly sensitive period, as evidenced by the incidence of embryo loss or early post-implantation mortality after AI, embryo transfer or somatic cell nuclear transfer. Elongation markers have often been used within this time frame to assess developmental defects or delays, originating either from the embryo, the uterus or the dam. Comparatively, gastrulation markers have not received great attention, although elongation and gastrulation are linked by reciprocal interactions at the molecular and cellular levels. To make this clearer, this peri-gastrulating period is described herein with a focus on its main developmental landmarks, and the resilience of the landmarks in the face of biotechnologies is questioned.

  13. Effects of sediment cover on survival and development of white sturgeon embryos

    USGS Publications Warehouse

    Kock, T.J.; Congleton, J.L.; Anders, P.J.

    2006-01-01

    A simple, inexpensive apparatus (embryo incubation unit [EIU]) was developed and used to assess the relationship between sediment cover (Kootenai River sediments, 97% by weight in the 0.83-mm- to 1.0-mm-diameter range) and survival of white sturgeon Acipenser transmontanus embryos in the laboratory. An apparatus-testing trial assessed the effects of two sediment depths (5 and 20 mm), three EIU ventilation hole sizes (4.8, 6.8, and 9.5 mm) providing three levels of intrasediment flow, and EIU location (upstream or downstream in laboratory troughs) on embryo survival at two above-substrate flow velocities (0.05 and 0.15 m/s). A second trial assessed the effects of sediment cover duration (5-mm sediment cover for 4, 7, 9, 11, or 14 d, with a ventilation hole size of 9.5 mm and a flow velocity of 0.17 m/s) on mean embryo survival and larval length and weight. In the apparatus-testing trial, embryo survival was reduced (P < 0.0001) to 0-5% under sediment covers of either 5 or 20 mm in both the higher-flow and lower-flow troughs; survival in control EIUs without sediments exceeded 80%. Survival was not significantly affected by ventilation hole size but was weakly affected by EIU location. In the second trial, embryo survival was negatively correlated (P = 0.001) with increasing duration of sediment cover and was significantly higher for embryos covered for 4 d (50% survival) or 7 d (30% survival) than for those covered for 9, 11, or 14 d (15-20% survival). Sediment cover also delayed hatch timing (P < 0.0001) and decreased mean larval length (P < 0.0001). Our results suggest that sediment cover may be an important early life stage mortality factor in rivers where white sturgeon spawn over fine-sediment substrates. ?? Copyright by the American Fisheries Society 2006.

  14. FOXO1, FOXO3, AND FOXO4 are differently expressed during mouse oocyte maturation and preimplantation embryo development.

    PubMed

    Kuscu, Nilay; Celik-Ozenci, Ciler

    2015-01-01

    Preimplantation embryo development is affected by its environment. FoxO transcription factors are regulated by PI3K/Akt signaling pathway that essentially supports growth and development. FoxO transcription factors are at the interface of crucial cellular processes, orchestrating programs of gene expression that regulate apoptosis, cell-cycle arrest, oxidative stress resistance, DNA repair, glucose metabolism, and differentiation. In the presence of growth factors, FoxO transcription factors are localized in the cytoplasm, whereas under stress conditions they move to the nucleus and trigger transcriptional activities of their target genes. The aim of the present study is to investigate whether FoxO transcription factors are present during in vivo oocyte maturation and preimplantation embryo development. Presence and localizations of FoxO1, FoxO3 and FoxO4 proteins have been determined with immunofluorescence staining. Our results have confirmed that FoxO1, FoxO3 and FoxO4 proteins are differentially expressed in prophase I, metaphase I, metaphase II oocytes, as well as in fertilized oocyte, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst. FoxOs translocate to nucleus in embryos with developmental delay. Our findings indicate that FoxO transcription factors are present during both oocyte and embryo in vivo maturation and provide fundamental knowledge that FoxOs may regulate in vitro embryo development under stress conditions.

  15. Development of bovine embryos derived from reproductive techniques.

    PubMed

    Alberto, Míryan L V; Meirelles, Flavio V; Perecin, Felipe; Ambrósio, Carlos E; Favaron, Phelipe O; Franciolli, André L R; Mess, Andrea M; Dos Santos, José M; Rici, Rose E G; Bertolini, Marcelo; Miglino, Maria A

    2013-01-01

    Assisted reproduction techniques have improved agricultural breeding in the bovine. However, important development steps may differ from the situation in vivo and there is a high mortality rate during the first trimester of gestation. To better understand these events, we investigated the development of embryos and fetal membranes following fixed-time AI (FTAI), IVF and nuclear transfer (NT). The onset of yolk-sac development was not normal in cloned embryos. Later steps differed from conditions in vivo in all three groups; the yolk-sac was yellowish and juxtaposed with the amniotic membrane. Vascularisation of the chorioallantoic membrane was relatively late and low in NT gestations, but normal in the others. The overall development of the embryos was normal, as indicated by morphology and regression analysis of growth rate. However, NT conceptuses were significantly smaller, with the livers in some embryos occupying the abdominal cavity and others exhibiting heart abnormalities. In conclusion, the yolk-sac and the cardiovascular system seem to be vulnerable to morphogenetic alterations. Future studies will focus on gene expression and early vascularisation processes to investigate whether these changes may be responsible for the high incidence of intrauterine mortality, especially in clones.

  16. Set up of a serum-free culture system for bovine embryos: embryo development and quality before and after transient transfer.

    PubMed

    George, F; Daniaux, C; Genicot, G; Verhaeghe, B; Lambert, P; Donnay, I

    2008-03-15

    It is well known that serum in culture medium negatively affects blastocyst quality. The objective of this work was to develop and test a serum-free culture medium which could improve embryo quality, measured by the resistance to freezing, lipid and glutathione content of the resulting blastocysts, as well as the ability of the blastocysts to elongate after transient transfer to recipient cows. In a first experiment we showed that adding a mixture of insulin, transferrin and selenium to serum-free Synthetic Oviduct Fluid medium (SOF-ITS) improved embryo development and quality. In the second experiment, the addition of BSA to SOF-ITS further improved blastocyst development. Moreover, a reduction in lipid content of morulae was observed in SOF-ITS-BSA by comparison with morulae cultured with serum (SOF-FCS). The resistance to freezing measured by hatching rates 24h post-thawing was also improved for blastocysts with a diameter between 160 and 180 microm cultured in SOF-ITS-BSA by comparison to those produced with serum. In order to evaluate the redox potential of the embryos, reduced glutathione content (GSH) was evaluated both before and after cryopreservation. A significant decrease in glutathione was observed after freezing, whatever the culture medium, but no difference was observed between culture conditions. Transient transfers were performed and elongated D-13 embryos were recovered. Elongation was more pronounced and the embryonic disk more often visible in embryos cultured in SOF-ITS-BSA than in embryos cultured with FCS. In conclusion, the serum-free system we developed to produce in vitro bovine embryos meets the developmental and qualitative requirements for a large-scale use.

  17. Hybrid embryos produced by transferring panda or cat somatic nuclei into rabbit MII oocytes can develop to blastocyst in vitro.

    PubMed

    Wen, Duan-Cheng; Bi, Chun-Ming; Xu, Ying; Yang, Cai-Xia; Zhu, Zi-Yu; Sun, Qing-Yuan; Chen, Da-Yuan

    2005-08-01

    The developmental potential of hybrid embryos produced by transferring panda or cat fibroblasts into nucleated rabbit oocytes was assessed. Both the panda-rabbit and the cat-rabbit hybrid embryos were able to form blastocysts in vitro. However, the rates of attaining the two-cell, four-cell, eight-cell, morula, or blastocyst stages for panda-rabbit hybrids were significantly greater than those of cat-rabbit hybrids (P<0.05). Transferring the rabbit fibroblasts into nucleated rabbit oocytes, 31.0% of the blastocyst rate was obtained, which was significantly higher than that of both the panda-rabbit and the cat-rabbit hybrid embryos (P<0.05). Whether or not the second polar body (PB2) was extruded from the one-cell hybrid embryos (both panda-rabbit and cat-rabbit hybrids) significantly affected their developmental capacity. Embryos without an extruded PB2 showed a higher capacity to develop into blastocysts (panda-rabbit: 19.2%; cat-rabbit: 4.3%), while embryos with extruded PB2 could only develop to the morula stage. The hybrid embryos formed pronucleus-like structures (PN) in 2-4 hr after activation, and the number of PN in one-cell embryos varied from one to five. Tracking of the nucleus in the egg after fusion revealed that the somatic nucleus could approach and aggregate with the oocyte nucleus spontaneously. Chromosome analysis of the panda-rabbit blastocysts showed that the karyotype of the hybrid embryos (2n=86) consisted of chromosomes from both the panda (2n=42) and the rabbit (2n=44). The results demonstrate that (1) it is possible to produce genetic hybrid embryos by interspecies nuclear transfer; (2) the developmental potential of the hybrid embryos is highly correlated to the donor nucleus species; and (3) the hybrid genome is able to support the complete preimplantation embryonic development of the hybrids.

  18. Use of infrared imaging for investigation of chicken embryo development

    NASA Astrophysics Data System (ADS)

    Frye, Ryan A.; Hsieh, Sheng-Jen; Girón Palomares, José Benjamín D.

    2011-05-01

    The focus of this study is two-fold: first, to investigate the feasibility of thermal imaging for characterizing the development of chicken embryos; and second, to compare the effects of photo periods of 11 hours of light followed by 11 hours of darkness (11-11) versus 24 hours of darkness (24 dark) during the incubation cycle on embryo development. Previous reported work has used invasive methods, such as ultrasound, tomography, and MRI to study chicken embryos with some success. However, very little work has been reported on use of thermography, which is a non-invasive method. Results suggest that use of a cooling-heating-cooling cycle can reveal the anatomy of chicken embryos. A statistical comparison of image data from the two photo periods found no difference in the average cooling rates. However, the 11-11 group of eggs did hatch earlier overall than 24-dark group. Of the hatched eggs, all the chickens from the 24-dark group appeared to be in normal physical condition. However, two of the chickens from the 11-11 group appeared to have leg weakness shortly after hatching. Of these, one fully recovered the next day and the second remains the same after two days of observation. In addition, the second chicken took about 48 hours to fully emerge from its shell.

  19. Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation.

    PubMed

    Li, Xiang-Yun; Jia, Qing; Di, Ke-Qian; Gao, Shu-Min; Wen, Xiao-Hui; Zhou, Rong-Yan; Wei, Wei; Wang, Li-Ze

    2007-03-01

    The aim of this study was to determine whether the number of passages affected the developmental pluripotency of embryonic stem (ES) cells as measured by the attainment of adult fertile mice derived from embryonic stem (ES) cell/tetraploid embryo complementation. Thirty-six newborns were produced by the aggregation of tetraploid embryos and hybrid ES cells after various numbers of passages. These newborns were entirely derived from ES cells as judged by microsatellite DNA, coat-color phenotype, and germline transmission. Although 15 survived to adulthood, 17 died of respiratory failure, and four were eaten by their foster mother. From the 15 mice that reached adulthood and that could reproduce, none arose from ES cells at passage level 15 or more. All 15 arose from cells at passages 3-11. Our results demonstrate that the number of passages affects the developmental pluripotency of ES cells.

  20. Chromosome remodeling and differentiation of tetraploid embryos during preimplantation development.

    PubMed

    Park, Mi-Ryung; Lee, Ah-Reum; Bui, Hong-Thuy; Park, Chankyu; Park, Keun-Kyu; Cho, Ssang-Goo; Song, Hyuk; Kim, Jae-Hwan; Nguyen, Van Thuan; Kim, Jin-Hoi

    2011-07-01

    Although it is known that the tetraploid embryo contributes only to the placenta, the question of why tetraploid embryos differentiate into placenta remains unclear. To study the effect of electrofusion on the development of mouse tetraploid oocytes, mouse two-cell embryos were fused and cultured in vitro in Chatot-Ziomek-Bavister medium. After electrofusion, two chromosome sets from the tetraploid blastomere were individually duplicated before nuclear fusion. At 8-10 hr after electrofusion, each chromosome set was condensing and the nuclear membrane was breaking down. Around 12-14 hr after electrofusion, the two chromosome sets had combined together and had reached the second mitotic metaphase, at this point with 8n sets of chromosomes. Interestingly, we discovered that expression of OCT4, an inner cell mass cells biomarker, is lost by the tetraploid expanded blastocysts, but that CDX2, a trophectoderm cells biomarker, is strongly expressed at this stage. This observation provides evidence clarifying why tetraploid embryos contribute only to trophectoderm.

  1. Lipid transport to avian oocytes and to the developing embryo.

    PubMed

    Schneider, Wolfgang J

    2016-05-01

    Studies of receptor-mediated lipoprotein metabolic pathways in avian species have revealed that physiological intricacies of specific cell types are highly analogous to those in mammals. A prime example for the power of comparative studies across different animal kingdoms, elucidated in the chicken, is that the expression of different lipoprotein receptors in somatic cells and oocytes are the key to oocyte growth. In avian species, yolk precursor transport from the hen's liver to rapidly growing oocytes and the subsequent transfer of yolk nutrients via the yolk sac to the developing embryo are highly efficient processes. Oocytes grow from a diameter of 5 mm to 2.5-3 cm in only 7 days, and the yolk sac transfers nutrients from the yolk stored in the mature oocyte to the embryo within just 2 weeks. The underlying key transport mechanism is receptor-mediated endocytosis of macromolecules, i.e., of hepatically synthesized yolk precursors for oocyte growth, and of mature yolk components for embryo nutrition, respectively. Recently, the receptors involved, as well as the role of lipoprotein synthesis in the yolk sac have been identified. As outlined here, lipoprotein degradation/resynthesis cycles and the expression of lipoprotein receptors are not only coordinated with the establishment of the follicular architecture embedding the oocyte, but also with the generation of the yolk sac vasculature essential for nutrient transfer to the embryo.

  2. AGL15, a MADS domain protein expressed in developing embryos.

    PubMed Central

    Heck, G R; Perry, S E; Nichols, K W; Fernandez, D E

    1995-01-01

    To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo. PMID:7549483

  3. Lipid transport to avian oocytes and to the developing embryo

    PubMed Central

    Schneider, Wolfgang J.

    2016-01-01

    Abstract Studies of receptor-mediated lipoprotein metabolic pathways in avian species have revealed that physiological intricacies of specific cell types are highly analogous to those in mammals. A prime example for the power of comparative studies across different animal kingdoms, elucidated in the chicken, is that the expression of different lipoprotein receptors in somatic cells and oocytes are the key to oocyte growth. In avian species, yolk precursor transport from the hen's liver to rapidly growing oocytes and the subsequent transfer of yolk nutrients via the yolk sac to the developing embryo are highly efficient processes. Oocytes grow from a diameter of 5 mm to 2.5-3 cm in only 7 days, and the yolk sac transfers nutrients from the yolk stored in the mature oocyte to the embryo within just 2 weeks. The underlying key transport mechanism is receptor-mediated endocytosis of macromolecules, i.e., of hepatically synthesized yolk precursors for oocyte growth, and of mature yolk components for embryo nutrition, respectively. Recently, the receptors involved, as well as the role of lipoprotein synthesis in the yolk sac have been identified. As outlined here, lipoprotein degradation/resynthesis cycles and the expression of lipoprotein receptors are not only coordinated with the establishment of the follicular architecture embedding the oocyte, but also with the generation of the yolk sac vasculature essential for nutrient transfer to the embryo. PMID:26585559

  4. Erythroid development in the mammalian embryo.

    PubMed

    Baron, Margaret H; Vacaru, Andrei; Nieves, Johnathan

    2013-12-01

    Erythropoiesis is the process by which progenitors for red blood cells are produced and terminally differentiate. In all vertebrates, two morphologically distinct erythroid lineages (primitive, embryonic, and definitive, fetal/adult) form successively within the yolk sac, fetal liver, and marrow and are essential for normal development. Red blood cells have evolved highly specialized functions in oxygen transport, defense against oxidation, and vascular remodeling. Here we review key features of the ontogeny of red blood cell development in mammals, highlight similarities and differences revealed by genetic and gene expression profiling studies, and discuss methods for identifying erythroid cells at different stages of development and differentiation.

  5. Tongue Growth during Prenatal Development in Korean Fetuses and Embryos

    PubMed Central

    Hong, Soo Jeong; Cha, Bong Geun; Kim, Yeon Sook; Lee, Suk Keun; Chi, Je Geun

    2015-01-01

    Background: Prenatal tongue development may affect oral-craniofacial structures, but this muscular organ has rarely been investigated. Methods: In order to document the physiology of prenatal tongue growth, we histologically examined the facial and cranial base structures of 56 embryos and 106 fetuses. Results: In Streeter’s stages 13–14 (fertilization age [FA], 28 to 32 days), the tongue protruded into the stomodeal cavity from the retrohyoid space to the cartilaginous mesenchyme of the primitive cranial base, and in Streeter’s stage 15 (FA, 33 to 36 days), the tongue rapidly swelled and compressed the cranial base to initiate spheno-occipital synchondrosis and continued to swell laterally to occupy most of the stomodeal cavity in Streeter’s stage 16–17 (FA, 37 to 43 days). In Streeter’s stage 18–20 (FA, 44 to 51 days), the tongue was vertically positioned and filled the posterior nasopharyngeal space. As the growth of the mandible and maxilla advanced, the tongue was pulled down and protruded anteriorly to form the linguomandibular complex. Angulation between the anterior cranial base (ACB) and the posterior cranial base (PCB) was formed by the emerging tongue at FA 4 weeks and became constant at approximately 124°–126° from FA 6 weeks until birth, which was consistent with angulations measured on adult cephalograms. Conclusions: The early clockwise growth of the ACB to the maxillary plane became harmonious with the counter-clockwise growth of the PCB to the tongue axis during the early prenatal period. These observations suggest that human embryonic tongue growth affects ACB and PCB angulation, stimulates maxillary growth, and induces mandibular movement to achieve the essential functions of oral and maxillofacial structures. PMID:26471340

  6. HUWE1 plays important role in mouse preimplantation embryo development and the dysregulation is associated with poor embryo development in humans

    PubMed Central

    Chen, L. J.; Xu, W. M.; Yang, M.; Wang, K.; Chen, Y.; Huang, X. J.; Ma, Q. H.

    2016-01-01

    HUWE1 is a HECT domain containing ubiquitin ligase implicated in neurogenesis, spermatogenesis and cancer development. The purpose of the current study is to investigate the role of HUWE1 in early embryo development. Here we demonstrate that Huwe1 is expressed in both nucleus and cytoplasm of preimplantation mouse embryos as well as gametes. Hypoxia (5% O2) treatment could significantly increase Huwe1 expression during mouse embryo development process. HUWE1 knockdown inhibited normal embryonic development and reduced blastocyst formation, and increased apoptotic cell numbers were observed in the embryos of HUWE1 knockdown group. Human embryo staining result showed that reduced HUWE1 staining was observed in the poor-quality embryos. Furthermore, Western blot result showed that significantly reduced expression of HUWE1 was observed in the villi of miscarriage embryos compared with the normal control, indicating that reduced expression of HUWE1 is related to poor embryo development. Oxidative reagent, H2O2 inhibited HUWE1 expression in human sperm, indicating that HUWE1 expression in sperm is regulated by oxidative stress. In conclusion, these results suggest that HUWE1 protein could contribute to preimplantation embryo development and dysregulated expression of HUWE1 could be related to poor embryo development and miscarriage in IVF clinic. PMID:27901130

  7. Effect of eggshell temperature during incubation on embryo development, hatchability, and posthatch development.

    PubMed

    Lourens, A; van den Brand, H; Meijerhof, R; Kemp, B

    2005-06-01

    An experiment was conducted to study the effects of different eggshell temperature (EST) profiles during incubation on embryo mortality, hatchability, and embryo development. Furthermore, chicks from different EST profiles were reared under low and high housing temperatures to investigate subsequent posthatch growth and rectal temperature. Two batches of eggs were used in this experiment. Hatching eggs were subjected to 36.7 or 37.8 degrees C EST during the first week, to 37.8 degrees C EST during the second week, and to 37.8 or 38.9 degrees C EST during the third week of incubation. Posthatch housing temperature decreased from 35 degrees C at d 1 to 30 degrees C at d 7 (high) or decreased from 30 degrees C at d 1 to 25 degrees C at d 7 (low). The difference between machine temperature and EST (DT) was used to illustrate the effect of EST on heat production during incubation. DT differed per batch, and was smallest when eggs were incubated at 36.7 degrees C instead of 37.8 degrees C during wk 1. High EST during wk 3 of incubation (38.9 degrees C instead of 37.8 degrees C) reduced DT only in batch 2. Embryo development was most retarded in eggs incubated at 36.7 degrees C EST compared with at 37.8 degrees C during the first week of incubation. However, highest hatchability and embryo development were always found when EST was maintained at 37.8 degrees C constantly throughout incubation. Chicks that hatched from eggs incubated at low EST during wk 1 of incubation had lower rectal temperature after hatching, especially under low housing temperatures, and this effect lasted until 7 d posthatch in batch 1. The highest rectal temperatures were always found in chicks incubated at 37.8 degrees C EST constantly throughout incubation. Eggs and chicks from different batches require different environmental conditions for optimal embryo development, hatchability, and posthatch growth. Rearing temperature and incubation conditions affect the ability of young chicks to maintain

  8. Nanomaterial Toxicity Screening in Developing Zebrafish Embryos

    EPA Science Inventory

    To assess nanomaterial vertebrate toxicity, a high-content screening assay was created using developing zebrafish, Danio rerio. This included a diverse group of nanomaterials (n=42 total) ranging from metallic (Ag, Au) and metal oxide (CeO2, CuO, TiO2, ZnO) nanoparticles, to non...

  9. Altered development of Xenopus embryos in a hypogeomagnetic field.

    PubMed

    Mo, Wei-Chuan; Liu, Ying; Cooper, Helen M; He, Rong-Qiao

    2012-04-01

    The hypogeomagnetic field (HGMF; magnetic fields <200 nT) is one of the fundamental environmental factors of space. However, the effect of HGMF exposure on living systems remains unclear. In this article, we examine the biological effects of HGMF on the embryonic development of Xenopus laevis (African clawed frog). A decrease in horizontal third cleavage furrows and abnormal morphogenesis were observed in Xenopus embryos growing in the HGMF. HGMF exposure at the two-cell stage, but no later than the four-cell stage, is enough to alter the third cleavage geometry pattern. Immunofluorescent staining for α-tubulin showed reorientation of the spindle of four-cell stage blastomeres. These results indicate that a brief (2-h) exposure to HGMF is sufficient to interfere with the development of Xenopus embryos at cleavage stages. Also, the mitotic spindle could be an early sensor to the deprivation of the geomagnetic field, which provides a clue to the molecular mechanism underlying the morphological and other changes observed in the developing and/or developed embryos.

  10. 75 FR 69717 - In the Matter of: Edentify, Inc., Embryo Development Corp., Enclaves Group, Inc., Energytec, Inc...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-15

    ..., Inc., Embryo Development Corp., Enclaves Group, Inc., Energytec, Inc., Enesco Group, Inc... securities of Embryo Development Corp. because it has not filed any periodic reports since the period...

  11. Myoinositol Improves Embryo Development in PCOS Patients Undergoing ICSI

    PubMed Central

    2016-01-01

    The aim of this study was to investigate the activity of myoinositol, in a court of 217 PCOS women undergoing intracytoplasmic sperm injection (ICSI), on pregnancy rate, embryo development, estradiol, and progesterone concentration in blood serum, superoxide dismutase (SOD), and catalase (CAT) in follicular fluid. Concerning the court of patient, 112 (groups I and II) out of 217 were PCOS women, whereas group III consisted of healthy subjects (not PCOS). Group I patients were treated with 400 μg of folic acid per day for 3 months before ICSI, whereas group II patients received 4000 mg of myoinositol and 400 μg of folic acid per day for 3 months before ICSI. Group II revealed a shorter embryo/blastocyst development period between microinjection and 5-cell stage compared to group I. The difference in SOD concentration between groups I and II and between groups II and III was statistically significant. In group II, 34.62% of pregnancies were obtained, whereas in group I this number reached 20% (NS). Myoinositol increased embryo development dynamics and accelerated blastocyst stage reaching time; however, no effect was shown on clinical pregnancy. Furthermore, it restored SOD concentration, lowered in PCOS women, but did not exert any effect on CAT concentration. PMID:27777587

  12. Chick embryogenesis: a unique platform to study the effects of environmental factors on embryo development.

    PubMed

    Yahav, S; Brake, J

    2014-01-01

    Bird embryogenesis takes place in a relatively protected environment that can be manipulated especially well in domestic fowl (chickens) where incubation has long been a commercial process. The embryonic developmental process has been shown to begin in the oviduct such that the embryo has attained either the blastodermal and/or gastrulation stage of development at oviposition. Bird embryos can be affected by "maternal effects," and by environmental conditions during the pre-incubation and incubation periods. "Maternal effects" has been described as an evolutionary mechanism that has provided the mother, by hormonal deposition into the yolk, with the potential to proactively influence the development of her progeny by exposing them to her particular hormonal pattern in such a manner as to influence their ability to cope with the expected wide range of environmental conditions that may occur post-hatching. Another important aspect of "maternal effects" is the effect of the maternal nutrient intake on progeny traits. From a commercial broiler chicken production perspective, it has been established that greater cumulative nutrient intake by the hen during her pullet rearing phase prior to photostimulation resulted in faster growing broiler progeny. Generally, maternal effects on progeny, which have both a genetic and an environmental component represented by yolk hormones deposition and embryo nutrient utilization, have an important effect on the development of a wide range of progeny traits. Furthermore, commercial embryo development during pre-incubation storage and incubation, as well as during incubation per se has been shown to largely depend upon temperature, while other environmental factors that include egg position during storage, and the amount of H2O and CO2 lost by the egg and the subsequent effect on albumen pH and height during storage have become important environmental factors to be considered for successful embryogenesis under commercial conditions

  13. Nerve growth factor regulates axial rotation during early stages of chick embryo development.

    PubMed

    Manca, Annalisa; Capsoni, Simona; Di Luzio, Anna; Vignone, Domenico; Malerba, Francesca; Paoletti, Francesca; Brandi, Rossella; Arisi, Ivan; Cattaneo, Antonino; Levi-Montalcini, Rita

    2012-02-07

    Nerve growth factor (NGF) was discovered because of its neurotrophic actions on sympathetic and sensory neurons in the developing chicken embryo. NGF was subsequently found to influence and regulate the function of many neuronal and non neuronal cells in adult organisms. Little is known, however, about the possible actions of NGF during early embryonic stages. However, mRNAs encoding for NGF and its receptors TrkA and p75(NTR) are expressed at very early stages of avian embryo development, before the nervous system is formed. The question, therefore, arises as to what might be the functions of NGF in early chicken embryo development, before its well-established actions on the developing sympathetic and sensory neurons. To investigate possible roles of NGF in the earliest stages of development, stage HH 11-12 chicken embryos were injected with an anti-NGF antibody (mAb αD11) that binds mature NGF with high affinity. Treatment with anti-NGF, but not with a control antibody, led to a dose-dependent inversion of the direction of axial rotation. This effect of altered rotation after anti NGF injection was associated with an increased cell death in somites. Concurrently, a microarray mRNA expression analysis revealed that NGF neutralization affects the expression of genes linked to the regulation of development or cell proliferation. These results reveal a role for NGF in early chicken embryo development and, in particular, in the regulation of somite survival and axial rotation, a crucial developmental process linked to left-right asymmetry specification.

  14. beta-Catenin in early development of the lancelet embryo indicates specific determination of embryonic polarity.

    PubMed

    Yasui, Kinya; Li, Guorong; Wang, Yong; Saiga, Hidetoshi; Zhang, Peijun; Aizawa, Shinichi

    2002-12-01

    The lancelet (amphioxus) embryo develops from a miolecithal egg and starts gastrulation when it is approximately 400 cells in size, in a fashion similar to that of some non-chordate deuterostomes. Throughout this type of gastrulation, the embryo develops characteristics such as the notochord and hollow nerve cord that commonly appear in chordates. beta-Catenin is an important factor in initiating body patterning. The behavior and developmental pattern of this protein in early lancelet development was examined in this study. Cytoplasmic beta-catenin was localized to the animal pole after fertilization and then was incorporated asymmetrically into the blastomeres during the first cleavage. Asymmetric distribution was observed at least until the 32-cell stage. The first nuclear localization was at the 64-cell stage, and involved all of the cells. At the initial gastrula stage, however, concentrated beta-catenin was found on the dorsal side. LiCl treatment affected the asymmetric pattern of beta-catenin during the first cleavage. LiCl also changed distribution of nuclear beta-catenin at the initial gastrula stage: distribution extended to cells on the animal side. Apparently associated with this change, expression domains of goosecoid, lhx3 and otx also changed to a radially symmetric pattern centered at the animal pole. However, LiCl-treated embryos were able to establish embryonic polarity. The present study suggests that in the lancelet embryo, polarity determination is independent of dorsal morphogenesis.

  15. Effects of acoustic levitation on the development of zebrafish, Danio rerio, embryos.

    PubMed

    Sundvik, Maria; Nieminen, Heikki J; Salmi, Ari; Panula, Pertti; Hæggström, Edward

    2015-09-04

    Acoustic levitation provides potential to characterize and manipulate material such as solid particles and fluid in a wall-less environment. While attempts to levitate small animals have been made, the biological effects of such levitation have been scarcely documented. Here, our goal was to explore if zebrafish embryos can be levitated (peak pressures at the pressure node and anti-node: 135 dB and 144 dB, respectively) with no effects on early development. We levitated the embryos (n = 94) at 2-14 hours post fertilization (hpf) for 1000 (n = 47) or 2000 seconds (n = 47). We compared the size and number of trunk neuromasts and otoliths in sonicated samples to controls (n = 94), and found no statistically significant differences (p > 0.05). While mortality rate was lower in the control group (22.3%) compared to that in the 1000 s (34.0%) and 2000 s (42.6%) levitation groups, the differences were statistically insignificant (p > 0.05). The results suggest that acoustic levitation for less than 2000 sec does not interfere with the development of zebrafish embryos, but may affect mortality rate. Acoustic levitation could potentially be used as a non-contacting wall-less platform for characterizing and manipulating vertebrae embryos without causing major adverse effects to their development.

  16. Effects of acoustic levitation on the development of zebrafish, Danio rerio, embryos

    PubMed Central

    Sundvik, Maria; Nieminen, Heikki J.; Salmi, Ari; Panula, Pertti; Hæggström, Edward

    2015-01-01

    Acoustic levitation provides potential to characterize and manipulate material such as solid particles and fluid in a wall-less environment. While attempts to levitate small animals have been made, the biological effects of such levitation have been scarcely documented. Here, our goal was to explore if zebrafish embryos can be levitated (peak pressures at the pressure node and anti-node: 135 dB and 144 dB, respectively) with no effects on early development. We levitated the embryos (n = 94) at 2–14 hours post fertilization (hpf) for 1000 (n = 47) or 2000 seconds (n = 47). We compared the size and number of trunk neuromasts and otoliths in sonicated samples to controls (n = 94), and found no statistically significant differences (p > 0.05). While mortality rate was lower in the control group (22.3%) compared to that in the 1000 s (34.0%) and 2000 s (42.6%) levitation groups, the differences were statistically insignificant (p > 0.05). The results suggest that acoustic levitation for less than 2000 sec does not interfere with the development of zebrafish embryos, but may affect mortality rate. Acoustic levitation could potentially be used as a non-contacting wall-less platform for characterizing and manipulating vertebrae embryos without causing major adverse effects to their development. PMID:26337364

  17. Inclusion of bovine lipoproteins and the vitamin E analogue, Trolox, during in vitro culture of bovine embryos changes both embryo and fetal development.

    PubMed

    Rooke, J A; Watt, R G; Ashworth, C J; McEvoy, T G

    2012-01-01

    This experiment investigated effects of lipoproteins and Trolox (vitamin E analogue) on bovine embryo and fetal development. The treatments were: in vitro culture (IVC) in synthetic oviducal fluid alone (SOF); with bovine lipoproteins (2% v/v; SOFLP); with Trolox (100μM; SOFT); and with lipoproteins and Trolox (SOFLPT). In vitro culture with lipoproteins increased fatty acid content of blastocysts (P<0.001) whereas inclusion of Trolox had no effect (P>0.05). Whereas lipoproteins reduced zygote development to blastocysts (P=0.03), Trolox facilitated increased development (P<0.001) and counteracted the reduction observed with lipoproteins (interaction, P=0.009). Lipoproteins also compromised (P<0.001) but presence of Trolox (P>0.05) had no effect on blastocyst morphological grade. Pregnancy rates resulting from synchronous transfer of IVP embryos were not affected by IVC treatment. At Day 70 of pregnancy, compared with SOF, fetal weight was lower in SOFLP but not SOFLPT (interaction, P<0.001). Liver weight (g kg(-1) fetal weight) was greater (P=0.03) in treatments containing Trolox. Placentome numbers were greater in SOF and SOFLPT compared with SOFLP and SOFT (interaction, P=0.002); superior embryo grades were also associated with increased numbers of placentomes (P=0.024). In conclusion, the interactive effects of lipoprotein and Trolox inclusion on in vitro embryo development were also evident in fetal development at Day 70.

  18. Development of rat tetraploid and chimeric embryos aggregated with diploid cells.

    PubMed

    Shinozawa, T; Sugawara, A; Matsumoto, A; Han, Y-J; Tomioka, I; Inai, K; Sasada, H; Kobayashi, E; Matsumoto, H; Sato, E

    2006-11-01

    In the present study, we examined the preimplantation and postimplantation development of rat tetraploid embryos produced by electrofusion of 2-cell-stage embryos. Developmental rate of tetraploid embryos to morula or blastocyst stage was 93% (56/60) and similar to that found in diploid embryos (95%, 55/58). After embryo transfer, rat tetraploid embryos showed implantation and survived until day 8 of pregnancy, however the conceptuses were aberrant on day 9. In mouse, tetraploid embryos have the ability to support the development of blastomeres that cannot develop independently. As shown in the present study, a pair of diploid blastomeres from the rat 8-cell-stage embryo degenerated immediately after implantation. Therefore, we examined whether rat tetraploid embryos have the ability to support the development of 2/8 blastomeres. We produced chimeric rat embryos in which a pair of diploid blastomeres from an 8-cell-stage green fluorescent protein negative (GFP-) embryo was aggregated with three tetraploid blastomeres from 4-cell GFP-positive (GFP+) embryos. The developmental rate of rat 2n(GFP-) <--> 4n(GFP+) embryos to the morula or blastocyst stages was 93% (109/117) and was similar to that found for 2n(GFP-) <--> 2n(GFP+) embryos (100%, 51/51). After embryo transfer, 2n(GFP-) <--> 4n(GFP+) conceptuses were examined on day 14 of pregnancy, the developmental rate to fetus was quite low (4%, 4/109) and they were all aberrant and smaller than 2n(GFP-) <--> 2n(GFP+) conceptuses, whereas immunohistochemical analysis showed no staining for GFP in fetuses. Our results suggest that rat tetraploid embryos are able to prolong the development of diploid blastomeres that cannot develop independently, although postimplantation development was incomplete.

  19. Dose-dependent effect of melatonin on postwarming development of vitrified ovine embryos.

    PubMed

    Succu, Sara; Pasciu, Valeria; Manca, Maria E; Chelucci, Sara; Torres-Rovira, Laura; Leoni, Giovanni G; Zinellu, Angelo; Carru, Ciriaco; Naitana, Salvatore; Berlinguer, Fiammetta

    2014-05-01

    After cryopreservation, embryos become sensitive to the oxidative stress, resulting in lipid peroxidation, membrane injury, and structural destruction. The present study aimed to assess the effect of increasing concentration of melatonin during postwarming culture on embryo's ability to restore its functions after cryopreservation. In vitro-produced blastocysts were vitrified, warmed, and cultured in vitro in TCM 199 with 5 different supplementations: control (CTR): 10% fetal calf serum; bovine serum albumin (BSA): 0.04% (wt/vol) BSA; and MEL(-3), MEL(-6), MEL(-9): BSA plus melatonin 10(-3), 10(-6), and 10(-9) M. The medium with the highest melatonin concentration had the highest trolox equivalent antioxidant capacity, whose values were comparable with those determined in plasma sampled from adult ewes (8.7 ± 2.4 mM). The other media had lower trolox equivalent antioxidant capacity values (P < 0.01), below the range of the plasma. At the same time, embryos cultured with the highest melatonin concentration reported a lower in vitro viability, as evaluated by lower re-expansion and hatching rates, and lower total cell number compared with the other groups (P < 0.05). Their metabolic status was also affected, as evidenced by higher oxidative and apoptotic index and lower ATP concentration. The beneficial effects of melatonin on embryo development during postwarming culture were observed only at low concentration (10(-9) M). These results suggest that melatonin at high concentration may exert some degree of toxic activity on pre-implantation embryos. Thus, the dose at which the embryos are exposed is pivotal to obtain the desiderate effect.

  20. Genetic selection of embryos that later develop the metabolic syndrome.

    PubMed

    Edwards, M J

    2012-05-01

    THE BARKER HYPOTHESIS: Is an excellent explanation of the process where human and animal foetuses exposed to malnutrition, either by maternal malnutrition or placental insufficiency, are metabolically programmed, with selective stunting of cell differentiation and organ growth. With the postnatal excess of nutrition observed in developed countries, this irreversible programming causes metabolic syndrome, including obesity, type 2 diabetes, and hypertension. Metabolic programming involves epigenetic changes including imprinting which might be transmitted through more than one generation rather than being completely re-set or erased during reproduction. The Barker hypothesis was supported by epidemiological data that recognised no excess fetal or postnatal mortality when pregnant women were starved during the Dutch famine in World War II. This argued against the "thrifty genotype" theory introduced in 1962, which proposed that starvation selected against members of the population with less "thrifty" genes, but the survivors who had "thrifty" genes developed metabolic syndrome if they were subsequently over-nourished. EMBRYONIC/FETAL SELECTION: Embryos or early foetuses could be selected very early in pregnancy on the basis of their genotype, by maternal malnutrition, hypertension, obesity or other causes of placental insufficiency. The genotype that allows embryos, or cells within them, to survive a less hospitable environment in the decidua after implantation might contribute to the later development of metabolic syndrome. This article hypothesises that an adverse intrauterine environment, caused by maternal malnutrition or placental insufficiency, kills a proportion of embryos and selects a surviving population of early embryos whose growth in utero is retarded by their genotype, their environment or a combination of both. The metabolic syndrome follows if the offspring is over-nourished later in life. The embryonic selection hypothesis presented here could be

  1. IVF and embryo transfer: historical origin and development.

    PubMed

    Biggers, John D

    2012-08-01

    IVF and embryo transfer for the treatment of human infertility has now resulted in the birth of over 4 million babies. The technique did not arise as a quantum event but was built on the efforts of many earlier workers in the fields of reproductive endocrinology and development. One should remember the famous saying of Isaac Newton: 'If I have seen further than most, it is because I have stood on the shoulder's of giants'. Ethical and moral issues have always arisen when investigators study early mammalian development, particularly human development. This paper documents these earlier studies and also draws attention to the ethical and moral arguments that inevitably arose.

  2. [Interaction between calcium and lead affects the toxicity to embryo of zebrafish (Danio rerio)].

    PubMed

    Chen, Zhong-Zhi; Zhu, Lin; Yao, Kun; Wang, Xiu-Juan; Ding, Jun-Nan

    2009-04-15

    This study tested the hypothesis that increased Ca2+ content increases the sensitivity of the developing embryos and larvae of zebrafish (Danio rerio) to Pb. And the aim of the study was to investigate the extent to which calcium can individually mitigate lead ion toxicity based on the concept of biotic ligand model (BLM). Embryos of the zebrafish were exposed to various Pb concentrations. Chemical characteristics of water and representative toxicological endpoints of zebrafish embryo were recorded. And general growth retardation as a major toxicological endpoint was used for analysis at 72 h due to its sensitivity and facility. The BLM software of Visual MINTEQ (Version 2.5.2) was employed to calculate the chemical speciation in the solution. The results showed that when Ca2+ concentration increased from 0.25 mmol/L to 2.00 mmol/L, the toxicity of lead on embryos of zebrafish (Danio rerio) decreased markedly after 72 h. And a large part of these decrease can be explained by the positive linear relations between EC50{Pb2+}/EC50[Pb]T (expressed as lead ion activity/dissolved total concentration) and activity/total concentration of Ca2+, through which the influence of Ca2+ on toxicity could be predicted. The results support the assumptions of the BLM and associated with competition between lead and calcium for binding on transport and toxic action sites on biological surfaces. However, when Ca2+ concentration increased from 2.00 mmol/L to 4.00 mmol/L, the toxicity of lead on embryos of zebrafish (Danio rerio) seemed to be constant at 72 h.

  3. Fusion of blastomeres in mouse embryos under the action of femtosecond laser radiation. Efficiency of blastocyst formation and embryo development

    SciTech Connect

    Osychenko, A A; Zalesskii, A D; Krivokharchenko, A S; Zhakhbazyan, A K; Nadtochenko, V A; Ryabova, A V

    2015-05-31

    Using the method of femtosecond laser surgery we study the fusion of two-cell mouse embryos under the action of tightly focused femtosecond laser radiation with the fusion efficiency reaching 60%. The detailed statistical analysis of the efficiency of blastomere fusion and development of the embryo up to the blastocyst stage after exposure of the embryos from different mice to a femtosecond pulse is presented. It is shown that the efficiency of blastocyst formation essentially depends on the biological characteristics of the embryo, namely, the strain and age of the donor mouse. The possibility of obtaining hexaploid embryonal cells using the methods of femtosecond laser surgery is demonstrated. (extreme light fields and their applications)

  4. Culture of bovine embryos in polyester mesh sections: the effect of pore size and oxygen tension on in vitro development.

    PubMed

    Somfai, T; Inaba, Y; Aikawa, Y; Ohtake, M; Kobayashi, S; Akai, T; Hattori, H; Konishi, K; Imai, K

    2010-12-01

    The purpose of this study was to assess the feasibility of polyester mesh culture for the in vitro production of bovine embryos, as polyester mesh is an alternative way for tracking individual embryos throughout culture using time-lapse cinematography (TLC). Bovine embryos were isolated during in vitro culture using sections of three different polyethylene terephthalate (PET) mesh products. In vitro matured and fertilized bovine oocytes were cultured in the 217 × 217, 230 × 230 or 238 × 238-μm openings of PET mesh sections or in simple micro-drops (control) for 7 days under either 20% or 5% O(2) tensions. No difference in embryo developmental rates was found between the culture groups in terms of cleavage, blastocyst formation and blastocyst expansion irrespective of O(2) tension. In contrast, under 20% O(2) tension, blastocysts that developed in PET mesh with 217 × 217-μm opening had significantly higher numbers of total and trophectoderm (TE) cells than control embryos; however, the numbers and proportions of inner cell mass (ICM) cells did not differ. Under 5% O(2) tension, no difference was found among the culture groups in the numbers of total, ICM and TE cells in embryos. All three PET mesh products investigated in this study were proven to be effective to prevent embryo movement. The results demonstrate that bovine embryos can be cultured in PET mesh sections without negative side-effects and suggest that embryo distance determined by the mesh affects embryo quality at atmospheric oxygen tension. Polyethylene terephthalate mesh with 217 × 217-μm openings was found to be the most suitable for further application in TLC.

  5. Ecdysone Mediates the Development of Immunity in the Drosophila Embryo

    PubMed Central

    Tan, Kiri Louise; Vlisidou, Isabella; Wood, Will

    2014-01-01

    Summary Beyond their role in cell metabolism, development, and reproduction, hormones are also important modulators of the immune system. In the context of inflammatory disorders, systemic administration of pharmacological doses of synthetic glucocorticoids (GCs) is widely used as an anti-inflammatory treatment [1, 2]. However, not all actions of GCs are immunosuppressive, and many studies have suggested that physiological concentrations of GCs can have immunoenhancing effects [3–7]. For a more comprehensive understanding of how steroid hormones regulate immunity and inflammation, a simple in vivo system is required. The Drosophila embryo has recently emerged as a powerful model system to study the recruitment of immune cells to sterile wounds [8] and host-pathogen dynamics [9]. Here we investigate the immune response of the fly embryo to bacterial infections and find that the steroid hormone 20-hydroxyecdysone (20-HE) can regulate the quality of the immune response and influence the resolution of infection in Drosophila embryos. PMID:24794300

  6. Amino-modified polystyrene nanoparticles affect signalling pathways of the sea urchin (Paracentrotus lividus) embryos.

    PubMed

    Pinsino, Annalisa; Bergami, Elisa; Della Torre, Camilla; Vannuccini, Maria Luisa; Addis, Piero; Secci, Marco; Dawson, Kenneth A; Matranga, Valeria; Corsi, Ilaria

    2017-03-01

    Polystyrene nanoparticles have been shown to pose serious risk to marine organisms including sea urchin embryos based on their surface properties and consequently behaviour in natural sea water. The aim of this study is to investigate the toxicity pathways of amino polystyrene nanoparticles (PS-NH2, 50 nm) in Paracentrotus lividus embryos in terms of development and signalling at both protein and gene levels. Two sub-lethal concentrations of 3 and 4 μg/mL of PS-NH2 were used to expose sea urchin embryos in natural sea water (PS-NH2 as aggregates of 143 ± 5 nm). At 24 and 48 h post-fertilisation (hpf) embryonic development was monitored and variations in the levels of key proteins involved in stress response and development (Hsp70, Hsp60, MnSOD, Phospho-p38 Mapk) as well as the modulation of target genes (Pl-Hsp70, Pl-Hsp60, Pl-Cytochrome b, Pl-p38 Mapk, Pl-Caspase 8, Pl-Univin) were measured. At 48 hpf various striking teratogenic effects were observed such as the occurrence of cells/masses randomly distributed, severe skeletal defects and delayed development. At 24 hpf a significant up-regulation of Pl-Hsp70, Pl-p38 Mapk, Pl-Univin and Pl-Cas8 genes was found, while at 48 hpf only for Pl-Univin was observed. Protein profile showed different patterns as a significant increase of Hsp70 and Hsp60 only after 48 hpf compared to controls. Conversely, P-p38 Mapk protein significantly increased at 24 hpf and decreased at 48 hpf. Our findings highlight that PS-NH2 are able to disrupt sea urchin embryos development by modulating protein and gene profile providing new understandings into the signalling pathways involved.

  7. Aging and the environment affect gamete and embryo potential: can we intervene?

    PubMed

    Meldrum, David R; Casper, Robert F; Diez-Juan, Antonio; Simon, Carlos; Domar, Alice D; Frydman, Rene

    2016-03-01

    Optimal maturation of the oocyte depends on its environment and determines embryo competence, because the embryonic genome is not active until the cleavage stage and new mitochondria are not produced until blastulation. Adverse environmental factors include aging, andropause, oxidative stress, obesity, smoking, alcohol, and psychologic stress, whereas androgen supplementation, a prudent diet, exercise, nutritional supplements, and psychologic interventions have beneficial effects. Mitochondrial function and energy production deteriorate with age, adversely affecting ovarian reserve, chromosome segregation, and embryo competence. In aging mice, the mitochondrial cofactor coenzyme Q10 reverses most of these changes. Early human experience has been encouraging, although only a small study using a shorter duration of intervention compared with the murine model has been carried out. Mitochondrial metabolic stress can result in an abnormal compensatory increase in mitochondrial DNA, which can be assessed in biopsied blastomeres of trophectoderm as a predictive biomarker of implantation failure. Psychologic stress may reduce oocyte competence by shifting blood flow away from the ovary as part of the classic "fight or flight" physiologic response, and methods to reduce stress or the body's reaction to stress improve pregnancy success. Enhancing oocyte competence is a key intervention that promises to reduce the number of euploid embryos failing to produce viable deliveries.

  8. Nutritional effects on oocyte and embryo development in mammals: implications for reproductive efficiency and environmental sustainability

    PubMed Central

    Ashworth, Cheryl J.; Toma, Luiza M.; Hunter, Morag G.

    2009-01-01

    The environment in which a breeding female lives prior to conception and during the early stages of her pregnancy has striking effects on oocytes developing in the ovarian follicle and on early embryos in the reproductive tract. Of the various environmental factors known to affect oocyte and embryo development, altered nutrition during this critical period has been particularly well studied. Alterations in the quantity of food consumed or the composition of the diet imposed solely during the pre-mating period affect oocyte maturity, blastocyst yield, prenatal survival and the number of offspring born alive. Importantly, nutrition at this time also affects the quality of embryos and resultant offspring, with increasing evidence from a variety of species showing that peri-conception nutrition can alter behaviour, cardiovascular function and reproductive function throughout post-natal life. In livestock species, it is important to devise nutritional strategies that improve reproductive efficiency and the quality of offspring but that do not add to the environmental footprint of the production system and which recognize likely changes in feedstuff availability arising from predicted changes in climate. PMID:19833647

  9. In vitro embryo development and blastocyst hatching rates following vitrification of river buffalo embryos produced from oocytes recovered from slaughterhouse ovaries or live animals by ovum pick-up.

    PubMed

    Manjunatha, B M; Gupta, P S P; Ravindra, J P; Devaraj, M; Nandi, S

    2008-03-03

    The present study was undertaken to determine whether the source of oocytes (ovum pick up versus slaughterhouse ovaries) affected in vitro embryo production and embryo survival (as measured by blastocyst hatching rates) following vitrification in buffaloes (Bubalus bubalis). Oocytes recovered from live buffaloes (n=6) by ovum pick up (OPU) and by manual aspiration from slaughterhouse ovaries were in vitro matured, fertilized and cultured to blastocyst stage under same culture conditions. Vitrification of blastocysts was carried out in two steps at 24 degrees C. Embryos were equilibrated in 10% EG+10% DMSO+0.3 M sucrose in base medium for 4 min. Subsequently, the embryos were transferred into 25% EG+25% DMSO+0.3 M sucrose in base medium for 45 s and then the embryos were loaded into straws and immersed in liquid nitrogen. Following warming, blastocysts were cultured in vitro for 48 h to assess hatching. Oocytes derived from live animals by OPU resulted in a significantly higher blastocyst yield then those derived from slaughterhouse ovaries (30.6+/-4.3 versus 18.5+/-1.8). Blastocyst hatching rates following vitrification of buffalo embryos produced from the oocytes collected from live animals by OPU was significantly higher than the oocytes collected from slaughterhouse ovaries (52.8+/-4.2 versus 40.2+/-4.4). In conclusion, the present study showed that source of oocytes (OPU versus slaughterhouse ovaries) affects the in vitro embryo development and blastocyst hatching rates following vitrification of embryos in buffaloes.

  10. Avian egg odour encodes information on embryo sex, fertility and development.

    PubMed

    Webster, Ben; Hayes, William; Pike, Thomas W

    2015-01-01

    Avian chemical communication is a rapidly emerging field, but has been hampered by a critical lack of information on volatile chemicals that communicate ecologically relevant information (semiochemicals). A possible, but as yet unexplored, function of olfaction and chemical communication in birds is in parent-embryo and embryo-embryo communication. Communication between parents and developing embryos may act to mediate parental behaviour, while communication between embryos can control the synchronicity of hatching. Embryonic vocalisations and vibrations have been implicated as a means of communication during the later stages of development but in the early stages, before embryos are capable of independent movement and vocalisation, this is not possible. Here we show that volatiles emitted from developing eggs of Japanese quail (Coturnix japonica) convey information on egg fertility, along with the sex and developmental status of the embryo. Specifically, egg volatiles changed over the course of incubation, differed between fertile and infertile eggs, and were predictive of embryo sex as early as day 1 of incubation. Egg odours therefore have the potential to facilitate parent-embryo and embryo-embryo interactions by allowing the assessment of key measures of embryonic development long before this is possible through other modalities. It also opens up the intriguing possibility that parents may be able to glean further relevant information from egg volatiles, such as the health, viability and heritage of embryos. By determining information conveyed by egg-derived volatiles, we hope to stimulate further investigation into the ecological role of egg odours.

  11. Embryonic genotype and inbreeding affect preimplantation development in cattle.

    PubMed

    Lazzari, G; Colleoni, S; Duchi, R; Galli, A; Houghton, F D; Galli, C

    2011-05-01

    Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7-8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle.

  12. Enzymatic Metabolism of Vitamin A in Developing Vertebrate Embryos

    PubMed Central

    Metzler, Melissa A.; Sandell, Lisa L.

    2016-01-01

    Embryonic development is orchestrated by a small number of signaling pathways, one of which is the retinoic acid (RA) signaling pathway. Vitamin A is essential for vertebrate embryonic development because it is the molecular precursor of the essential signaling molecule RA. The level and distribution of RA signaling within a developing embryo must be tightly regulated; too much, or too little, or abnormal distribution, all disrupt embryonic development. Precise regulation of RA signaling during embryogenesis is achieved by proteins involved in vitamin A metabolism, retinoid transport, nuclear signaling, and RA catabolism. The reversible first step in conversion of the precursor vitamin A to the active retinoid RA is mediated by retinol dehydrogenase 10 (RDH10) and dehydrogenase/reductase (SDR family) member 3 (DHRS3), two related membrane-bound proteins that functionally activate each other to mediate the interconversion of retinol and retinal. Alcohol dehydrogenase (ADH) enzymes do not contribute to RA production under normal conditions during embryogenesis. Genes involved in vitamin A metabolism and RA catabolism are expressed in tissue-specific patterns and are subject to feedback regulation. Mutations in genes encoding these proteins disrupt morphogenesis of many systems in a developing embryo. Together these observations demonstrate the importance of vitamin A metabolism in regulating RA signaling during embryonic development in vertebrates. PMID:27983671

  13. Development of porcine tetraploid somatic cell nuclear transfer embryos is influenced by oocyte nuclei.

    PubMed

    Fu, Bo; Liu, Di; Ma, Hong; Guo, Zhen-Hua; Wang, Liang; Li, Zhong-Qiu; Peng, Fu-Gang; Bai, Jing

    2016-02-01

    Cloning efficiency in mammalian systems remains low because reprogramming of donor cells is frequently incomplete. Nuclear factors in the oocyte are removed by enucleation, and this removal may adversely affect reprogramming efficiency. Here, we investigated the role of porcine oocyte nuclear factors during reprogramming. We introduced somatic cell nuclei into intact MII oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. We then examined the influence of the oocyte nucleus on tetraploid SCNT embryo development by assessing characteristics including pronucleus formation, cleavage rate, and blastocyst formation. Overall, tetraploid SCNT embryos have a higher developmental competence than do standard diploid SCNT embryos. Therefore, we have established an embryonic model in which a fetal fibroblast nucleus and an oocyte metaphase II plate coexist. Tetraploid SCNT represents a new research platform that is potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming.

  14. Imidacloprid Exposure Suppresses Neural Crest Cells Generation during Early Chick Embryo Development.

    PubMed

    Wang, Chao-Jie; Wang, Guang; Wang, Xiao-Yu; Liu, Meng; Chuai, Manli; Lee, Kenneth Ka Ho; He, Xiao-Song; Lu, Da-Xiang; Yang, Xuesong

    2016-06-15

    Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests found on crops and fleas on pets. However, it is still unclear whether imidacloprid exposure could affect early embryo development-despite some studies having been conducted on the gametes. In this study, we demonstrated that imidacloprid exposure could lead to abnormal craniofacial osteogenesis in the developing chick embryo. Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull. We found that the imidacloprid exposure retards the development of gastrulating chick embryos. HNK-1, PAX7, and Ap-2α immunohistological stainings indicated that cranial NCCs generation was inhibited after imidacloprid exposure. Double immunofluorescent staining (Ap-2α and PHIS3 or PAX7 and c-Caspase3) revealed that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in the developing neural tube and by altering expression of EMT-related adhesion molecules (Cad6B, E-Cadherin, and N-cadherin) in the developing neural crests. We also determined that imidacloprid exposure suppressed cranial NCCs migration and their ability to differentiate. In sum, we have provided experimental evidence that imidacloprid exposure during embryogenesis disrupts NCCs development, which in turn causes defective cranial bone development.

  15. Influence of nitric oxide during maturation on bovine oocyte meiosis and embryo development in vitro.

    PubMed

    Schwarz, Kátia R L; Pires, Pedro R L; Adona, Paulo R; Câmara de Bem, Tiago H; Leal, Cláudia L V

    2008-01-01

    The effect of s-nitroso-n-acetyl-l,l-penicillamine (SNAP, a nitric oxide donor) during in vitro maturation (IVM) on nuclear maturation and embryo development was investigated. The effect of increasing nitric oxide (NO) during prematuration or maturation, or both, on embryo development was also assessed. 10(-3) m SNAP nearly blocked oocytes reaching metaphase II (MII) (7%, P < 0.05) while 10(-5) m SNAP showed intermediate proportions (55%). For 10(-7) m SNAP and controls (without SNAP), MII percentages were similar (72% for both, P > 0.05), but superior to the other treatment groups (P < 0.05). Blastocyst development, however, was not affected (38% for all treatments, P < 0.05). TUNEL-positive cells in hatched blastocysts (Day 9) increased when IVM included 10(-5) m SNAP (8 v. 3 to 4 cells in the other treatments, P > 0.05), without affecting total cell numbers (240 to 291 cells, P > 0.05). When oocytes were prematured followed by IVM with or without 10(-7) m SNAP, during either culture period or both, blastocyst development was similar (26 to 40%, P > 0.05). When SNAP was included during both prematuration and IVM, the proportion of Day 9 hatched embryos increased (28% v. 14 to 19% in the other treatments, P < 0.05). Apoptotic cells, however, increased when SNAP was included (6 to 10 cells) in comparison to prematuration and maturation without SNAP (3 cells, P < 0.05). NO may be involved in meiotic progression and apoptosis during embryo development.

  16. Serotonin metabolism in directly developing frog embryos during paternal care.

    PubMed

    Ten Eyck, Gary R; Ronan, Patrick J; Renner, Kenneth J; Summers, Cliff H

    2005-11-11

    Central serotonin (5-HT) metabolism during embryogenesis and a 3-day post-hatching period was analyzed using high performance liquid chromatography in the directly developing frog, Eleutherodactylus coqui. This anuran bypasses the free-swimming larval stage and embryos hatch as miniature frogs in the adult phenotype. During embryogenesis and for a short time immediately after hatching, male E. coqui provide paternal care by brooding and guarding eggs/embryos to prevent desiccation and predation. Serotonin and its catabolite, 5-HIAA, were measured from whole brain during embryogenesis and at 3 days post-hatch to identify critical periods in 5-HT development and to determine the relationship between 5-HT and life history events such as hatching and frog dispersal from the nest site. Serotonergic activity was highest during the early-mid embryonic stages as indicated by the ratio of 5-HIAA/5-HT, a general indicator of turnover and metabolism. There were significant increases in tissue concentrations of 5-HT during the latest or terminal embryonic stage, just prior to hatching, and also at 3 days post-hatch, shortly before neonates disperse into the rainforest. These two increases probably represent different functional requirements during development. The first may occur as a result of the surge of development in the 5-HT system during late embryogenesis that occurs in E. coqui and the second may be from the increase demand in sensory and motor neural development required before dispersal from the nest site.

  17. Temporal patterns of tyrosine nitration in embryo heart development

    PubMed Central

    Viera, Liliana; Radmilovich, Milka; Vargas, Marcelo R.; Dennys, Cassandra N.; Wilson, Landon; Barnes, Stephen; Franco, Maria Clara; Beckman, Joseph S.; Estévez, Alvaro G.

    2012-01-01

    Tyrosine nitration is a biomarker for the production of peroxynitrite and other reactive nitrogen species. Nitrotyrosine immunoreactivity is present in many pathological conditions including several cardiac diseases. Because the events observed during heart failure may recapitulate some aspects of development, we tested whether nitrotyrosine is present during normal development of the rat embryo heart and its potential relationship in cardiac remodeling through apoptosis. Nitric oxide (NO) production is highly dynamic during development, but whether peroxynitrite and nitrotyrosine are formed during normal embryonic development has received little attention. Rat embryo hearts exhibited strong nitrotyrosine immunoreactivity in endocardial and myocardial cells of the atria and ventricles from E12 to E18. After E18, nitrotyrosine staining faded and disappeared entirely by birth. Tyrosine nitration in the myocardial tissue coincided with elevated protein expression of nitric oxide synthases (eNOS and iNOS). The immunoreactivity for these NOS isoforms remained elevated even after nitrotyrosine had disappeared. Tyrosine nitration did not correlate with cell death or proliferation of cardiac cells. Analysis of tryptic peptides by MALDI-TOF shows that nitration occurs in actin, myosin, and the mitochondrial ATP synthase alpha chain. These results suggest that reactive nitrogen species are not restricted to pathological conditions but may play a role during normal embryonic development. PMID:23195686

  18. A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro.

    PubMed

    Zhao, Shuan; Liu, Zhen-Xing; Gao, Hui; Wu, Yi; Fang, Yuan; Wu, Shuai-Shuai; Li, Ming-Jie; Bai, Jia-Hua; Liu, Yan; Evans, Alexander; Zeng, Shen-Ming

    2015-07-15

    No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation.

  19. Supplementation of insulin-transferrin-selenium to embryo culture medium improves the in vitro development of pig embryos.

    PubMed

    Das, Ziban Chandra; Gupta, Mukesh Kumar; Uhm, Sang Jun; Lee, Hoon Taek

    2014-08-01

    Insulin, transferrin and selenium (ITS) supplementation to oocyte maturation medium improves the post-fertilization embryonic development in pigs. ITS is also commonly used as a supplement for the in vitro culture (IVC) of embryos and stem cells in several mammalian species. However, its use during IVC of pig embryos has not been explored. This study investigated the effect of ITS supplementation to IVC medium on the in vitro development ability of pig embryos produced by parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). We observed that ITS had no significant effect on the rate of first cleavage (P > 0.05). However, the rate of blastocyst formation in ITS-treated PA (45.3 ± 1.9 versus 27.1 ± 2.3%), IVF (31.6 ± 0.6 versus 23.5 ± 0.6%) and SCNT (17.6 ± 2.3 versus 10.7 ± 1.4%) embryos was significantly higher (P < 0.05) than those of non-treated controls. Culture of PA embryos in the presence of ITS also enhanced the expansion and hatching ability (29.1 ± 3.0 versus 18.2 ± 3.8%; P < 0.05) of blastocysts and increased the total number of cells per blastocyst (53 ± 2.5 versus 40.9 ± 2.6; P < 0.05). Furthermore, the beneficial effect of ITS on PA embryos was associated with significantly reduced level of intracellular reactive oxygen species (ROS) (20.0 ± 2.6 versus 46.9 ± 3.0). However, in contrast to PA embryos, ITS had no significant effect on the blastocyst quality of IVF and SCNT embryos (P > 0.05). Taken together, these data suggest that supplementation of ITS to the IVC medium exerts a beneficial but differential effect on pig embryos that varies with the method of embryo production in vitro.

  20. Emergence and development of gut motility in the chicken embryo

    PubMed Central

    Chevalier, N. R.; Fleury, V.; Dufour, S.

    2017-01-01

    The gastrointestinal tract transports the food bolus by peristalsis. Gut motility starts at an early age in the developing embryo, well before it is required for nutrition of the organism. We present a comprehensive kinematic study of the emergence and physiological development of gut motility in all regions of the lower digestive tract of the chicken embryo from embryonic days E5 through E9. We characterized motility emergence time, propagation patterns, speed, frequency and amplitude of peristalsis waves. We found that the emergence of an uninterrupted circular ring of smooth muscle correlated with the appearance of propagative contractile waves, at E6 in the hindgut and midgut, and at E9 in the caecal appendix. We show that peristalsis at these stages is critically dependent on calcium and is not mediated by neurons as gut motility is insensitive to tetrodotoxin and takes place in the hindgut in the absence of neurons. We further demonstrate that motility also matures in ex-vivo organ culture. We compare our results to existing literature on zebrafish, mouse and human motility development, and discuss their chronological relationship with other major developmental events occurring in the chicken embryonic gut at these stages. Our work sets a baseline for further investigations of motility development in this important animal model. PMID:28222167

  1. Emergence and development of gut motility in the chicken embryo.

    PubMed

    Chevalier, N R; Fleury, V; Dufour, S; Proux-Gillardeaux, V; Asnacios, A

    2017-01-01

    The gastrointestinal tract transports the food bolus by peristalsis. Gut motility starts at an early age in the developing embryo, well before it is required for nutrition of the organism. We present a comprehensive kinematic study of the emergence and physiological development of gut motility in all regions of the lower digestive tract of the chicken embryo from embryonic days E5 through E9. We characterized motility emergence time, propagation patterns, speed, frequency and amplitude of peristalsis waves. We found that the emergence of an uninterrupted circular ring of smooth muscle correlated with the appearance of propagative contractile waves, at E6 in the hindgut and midgut, and at E9 in the caecal appendix. We show that peristalsis at these stages is critically dependent on calcium and is not mediated by neurons as gut motility is insensitive to tetrodotoxin and takes place in the hindgut in the absence of neurons. We further demonstrate that motility also matures in ex-vivo organ culture. We compare our results to existing literature on zebrafish, mouse and human motility development, and discuss their chronological relationship with other major developmental events occurring in the chicken embryonic gut at these stages. Our work sets a baseline for further investigations of motility development in this important animal model.

  2. The effect of sevoflurane on developing A/J strain mouse embryos using a whole-embryo culture system--the incidence of cleft lip in culture embryos.

    PubMed

    Yamada, Morimasa; Yamamoto, Naoki; Ohgami, Saori; Kanazawa, Mayuko; Harada, Jun; Ohno, Norikazu; Natsume, Nagato

    2014-03-01

    A/J strain mice have a high spontaneous incidence of cleft lip (ICL) and/or palate. The primary palate-related effects of sevoflurane on developing A/J strain mouse embryos (embryos) were studied using a whole-embryo culture (WEC) system. This system could separate the direct effects of sevoflurane from those that are maternally mediated. A total of 205 10.5-d embryos were cultured for 24 h in either a control group (control gas: 95% O2 and 5% CO2) or sevoflurane-administered groups (1/4, 1/2, and 1 minimum alveolar concentration (MAC) with control gas) for 8 h. After 16 h, 11.5-d culture embryos were examined in terms of crown-rump length, number of somites, and protein content. Crown-rump length in the 1 MAC was significantly shorter than in the control group (p < 0.05). Protein content in the 1/2 MAC (p < 0.05) and 1 MAC (p < 0.001) was significantly lower than in the control group. The ICL showed no significant differences between each group. (The ICL rose with an increase in the sevoflurane concentration, but this was not significant). The positive findings in this study indicate that a WEC system is useful for studying the mechanisms of ICL (teratogenicity) associated with sevoflurane.

  3. Polycyclic aromatic hydrocarbons disrupt axial development in sea urchin embryos through a beta-catenin dependent pathway.

    PubMed

    Pillai, Murali C; Vines, Carol A; Wikramanayake, Athula H; Cherr, Gary N

    2003-04-15

    Sea urchin (Lytechinus anemesis) embryos were used as an experimental system to investigate the mechanisms of the developmental toxicity of creosote, one of the most widely used wood preserving chemicals, as well as some of its polycyclic aromatic hydrocarbon (PAH) constituents (phenanthrene, fluoranthene, fluorene, pyrene and quinoline). Data suggest that creosote and PAHs affect axial development and patterning in sea urchin embryos by disrupting the regulation of beta-catenin, a crucial transcriptional co-activator of specific target genes in the Wnt/wg signaling pathway. When ciliated blastula stage embryos were exposed to these compounds, they developed into exogastrulae with completely evaginated archentera, demonstrating that these chemicals disrupt axial development and patterning. This response occurred in a dose-dependent fashion, with the EC(50) of creosote for complete exogastrulation being 1.57 ppm, while the EC(50)s of the PAHs ranged from 0.41 ppm (2.0 microM) to 4.33 ppm (33.5 microM). Morphologically, the exogastrulae that developed from embryos exposed to creosote and PAHs appeared to be identical to those that resulted from exposure to lithium chloride, a classical agent known to induce vegetalization and exogastrulation in sea urchin embryos. Immunological studies using antibodies against beta-catenin, a multi-functional protein known to be involved in cell-cell adhesion and cell fate specification during embryonic development, revealed high levels of nuclear accumulation of beta-catenin by cells of creosote- and PAH-exposed embryos, irrespective of their positions in the developing embryo. Dissociated embryonic cells cultured in the presence of these agents rapidly responded in a similar fashion. Since beta-catenin accumulation occurs in nuclei of several types of cancer cells, it is possible this may be a general mechanism by which PAHs affect a variety of different cell types.

  4. Development of interspecies nuclear transfer embryos reconstructed with argali (Ovis ammon) somatic cells and sheep ooplasm.

    PubMed

    Pan, Xiaoyan; Zhang, Yanli; Guo, Zhiqin; Wang, Feng

    2014-02-01

    Interspecies nuclear transfer has already achieved success in several species, which shows great potential in recovery and conservation of endangered animals. The study was conducted to establish an efficient system for in vitro argali (Ovis ammon)-sheep embryo reconstruction via interspecies somatic cell nuclear transfer (iSCNT). The competence of domestic sheep cytoplasts to reprogram the adult argali fibroblast nuclei was evaluated, and the effects of enucleation methods and donor cell passage and cell state on the in vitro development of argali-sheep cloned embryos were also examined. Sheep oocytes could support argali and sheep fibroblast cell nuclei transfer and develop to blastocysts in vitro. Oocytes matured for 21–23 h and enucleated by chemically assisted enucleation (CAE) had a higher enucleation rate than blind enucleation (BE), but the development rate of iSCNTembryos was the same (P>0.05). Moreover, passage numbers of fibroblast cells <10, as well as the cell cycle stages did not affect the development rate of iSCNT reconstructed embryos. Thus sheep cytoplasm successfully supports argali nucleus development to blastocyst stage after optimising the nuclear transfer procedure, which indicates that iSCNT can be used to conserve endangered argali in the near future.

  5. PRDM16 expression in the developing mouse embryo.

    PubMed

    Horn, Kristin H; Warner, Dennis R; Pisano, Michele; Greene, Robert M

    2011-02-01

    PRDM16 is a member of the PR domain-containing protein family and is associated with various disease states including myelodysplastic syndrome and adult T-cell leukemia, as well as developmental abnormalities such as cleft palate. It is also known to act as a regulator of cell differentiation. Expression analysis of PRDM16 is limited, especially within the developing embryo. The current study evaluated the temporal and spatial localization of PRDM16 during early mouse development (embryonic days 8.5-14.5). PRDM16 was first detected on E9.5 in a limited number of tissues and by E14.5, was expressed in a broad range of developing tissues including those of the brain, lung, kidney, and gastrointestinal tract. The expression pattern is consistent with a role for PRDM16 in the development of multiple tissues. Collectively, these studies are the first to characterize the expression of the PRDM16 gene during early murine development.

  6. Failure to turn eggs during incubation: effects on embryo weight, development of the chorioallantois and absorption of albumen.

    PubMed

    Tullett, S G; Deeming, D C

    1987-06-01

    Turning eggs during incubation is essential for good hatchability. In the present paper additional effects on the development of the chorioallantois, absorption of albumen and growth of the embryo are recorded. The ability of an unturned egg to hatch was not affected by egg weight, egg shell porosity or water loss during incubation. The ability of the chorioallantois to spread around the inner surface of the inner shell membrane and the degree of absorption of the residual albumen affected the growth of the embryo and its ability to hatch. Unturned eggs hatched later than eggs which were turned throughout incubation.

  7. The influence of growth factors on the development of preimplantation mammalian embryos.

    PubMed

    Díaz-Cueto, L; Gerton, G L

    2001-01-01

    The development of the preimplantation mammalian embryo from a fertilized egg to a blastocyst capable of implanting in the uterus is a complex process. Cell division must be carefully programmed. The embryonic genome must be activated at the appropriate stage of development, and the pattern of gene expression must be carefully coordinated for the initiation of the correct program of differentiation. Cell fates must be chosen to establish specific cell types such as the inner cell mass and the trophectoderm, which give rise to the embryo proper and the placenta, respectively. This review summarizes recent findings concerning the influence of growth factors on the development of preimplantation mammalian embryos. Maternal factors secreted into the lumen of the female reproductive tract as well as substances synthesized by the developing embryo itself help to regulate this process. Studies of embryos in culture and investigations using homologous recombination to create embryos and animals null for specific genes have enabled the identification of several growth factors that appear essential for preimplantation mammalian embryo development. Some of the factors are required maternal factors; others are embryo-derived autocrine and paracrine factors. Studies using molecular biology are beginning to identify differences in the patterns of genes expressed by naturally derived embryos and those developing in culture. The knowledge gained from studies on growth factors, media, embryonic development, and gene expression should help improve culture conditions for embryos and will provide for safer outcomes from assisted reproductive procedures in human and animal clinics.

  8. Tissue densities in developing avian embryos. [under acceleration stresses

    NASA Technical Reports Server (NTRS)

    Smith, A. H.; Abbott, U. K.; Morzenti, A.

    1984-01-01

    The density changes in the components of the incubated egg, the embryo, and the embryo's body parts were measured in the course of 21 days of incubation. In the first two-thirds of the incubation period there is a sequence of increasing density among egg contents: amniotic fluid, embryo, yolk, and albumin. As a result, the embryo is located at the bottom of the amniotic fluid, but at the top of the albumin. This position provides the embryo with mechanical protection and a proximity to the egg's air cell. The observed density changes and the asymmetry of these changes among various body parts of the embryo suggest a functional relationship. The density distributions among the body parts are particularly important in gravitational investigations of embryogenesis since they will produce forces tending to dislocate parts of the embryo.

  9. Passive cellular microrheology in developing fruit fly embryos

    NASA Astrophysics Data System (ADS)

    Crews, Sarah; Ma, Xiaoyan; Lawrence, Stacey; Hutson, M. Shane

    2012-02-01

    The development of fruit fly (Drosophila) embryos involves spatial and temporal regulation of cellular mechanical properties. These properties can be probed in vivo using laser hole drilling experiments; however, this technique only infers relative forces. Conversion to absolute forces requires measurement of cellular viscoelastic properties. Here, we use passive microrheology of fluorescently labeled cell membranes to measure the viscoelastic properties of amnioserosa cells. These dynamic epithelial cells play an important mechanical role during two developmental stages: germ band retraction and dorsal closure. Passive microrheology in this system is confounded by active contractions in the cytoskeleton. Thus, the fruit fly embryos are transiently anesthetized with CO2, halting active cellular movements, leaving only passive Brownian motion. The power spectra of these fluctuations are well fit by a Lorentzian -- as expected for Brownian motion -- and allow us to extract cellular viscoelastic parameters at different developmental stages. These measured parameters inform previous hole-drilling experiments and provide inputs for quantitative computational models of fruit fly embryonic development.

  10. The sperm epigenome and potential implications for the developing embryo.

    PubMed

    Jenkins, Timothy G; Carrell, Douglas T

    2012-06-01

    Recent work in the field of male fertility has yielded significant increases in our understanding of the sperm epigenome and its potential role in embryonic development. These new findings have enabled a broad classification of a normal epigenetic state in the male gamete and have provided insight into the possible etiologies of some idiopathic male infertility cases. Histone retention and modification, protamine incorporation into the chromatin, DNA methylation, and spermatozoal RNA transcripts appear to play important roles in the epigenetic state of mature sperm. These epigenetic factors may reveal a historical record of spermatogenesis, portend future functions in embryogenesis, and help to elucidate mechanism of pluripotency. In contrast to the once held dogma regarding the importance of the paternal epigenome, the unique epigenetic landscape in sperm appears to serve more than the gamete itself and is likely influential in the developing embryo. In fact, growing evidence suggests that mature sperm provide appropriate epigenetic marks that drive specific genes toward activation and contribute to the pluripotent state of the embryonic cells. Although not definitive, the current literature provides evidence for the role of the sperm epigenome in the embryo. Future work must be focused on the characterization of epigenetic abnormalities commonly found in individuals with compromised fertility to further establish this role. Additionally, studies should target the effects of environment and aging on the sperm epigenetic program and subsequent fertility loss to determine the etiology of aberrant epigenetic profiles.

  11. In vitro development and chromosomal configuration of bovine somatic cloned embryos with nonenucleated metaphase II oocytes.

    PubMed

    Meng, Qinggang; Bai, Chunling; Liu, Ying; Wu, Xia; Bunch, Thomas D; Li, Guang-Peng

    2010-08-01

    This study was designed to examine the effects of the presence of oocyte nuclei on the donor cell nuclear remodeling, including premature chromosome condensation (PCC) and DNA configuration, and subsequent embryo development. The results showed that: (1) the presence of oocyte MII spindles was more likely to induce donor cell PCC. (2) The positional relationship between the fused donor cell and the oocyte metaphase spindle had an effect on oocyte PB2 extrusion. When the fused donor cell was widely separated from the MII spindle, 94.4% of the reconstructed oocytes expelled a PB2. When the donor cell was fused adjacently to the MII spindle, almost all of the reconstructed oocytes did not expel the PB2; the majority (67.9%) formed a very large M-phase spindle in which the oocyte and the donor cell chromosomes merged. (3) After activation, the oocyte and donor nuclei exhibited a variety of pronuclear patterns and asynchronous development. (4) The embryos reconstituted with nonenucleated oocytes resulted in a similar cleavage rate as observed in the control embryos reconstituted with enucleated oocytes. Blastocyst developmental rates were no different between nonenucleated and enucleated cloned embryos; however, the development rates from early to hatching blastocysts significantly decreased in the nonenucleation group compared to enucleation controls (0 vs. 23.1%; 27.5 vs. 67.8%), regardless with either cumulus cells or fibroblasts as donor cells. (5) All nonenucleated oocyte-derived blastocysts contained mixed polyploidy with a variety of compositions that included 2n/4n, 2n/6n, 2n/8n, and 2n/4n/8n. (6) Nuclear transfer preceding the oocyte enucleation experiment indicated that prolonged presence of oocyte nuclei induced abnormal DNA configuration and reduced in vitro development of transferred somatic nuclei, but short time presence of oocyte nuclei did not affect the in vitro development of cloned embryos. We conclude that oocyte MII spindles induce donor cell PCC

  12. Development of the stapes and associated structures in human embryos.

    PubMed

    Rodríguez-Vázquez, J F

    2005-08-01

    The objective of this study was to clarify the development of the stapes in humans and its relationship with the cartilage of the second branchial arch. The study was carried out in 25 human embryos between 6 and 28 mm crown-rump length. The stapes develops at the cranial end of the second branchial arch through an independent anlage of the cartilage of this arch. Between the stapedial anlage and the cranial end of the Reichert's cartilage there is a formation called the interhyale, the internal segment of which gives rise to the tendon of the stapedial muscle. The stapedial anlage is a unique formation with two distinct parts: the superior part that will comprise the base and the inferior part that will be crossed by the stapedial artery during embryonic development and will constitute the limbs and the head of the stapes. According to the results, the otic capsule is not involved in formation of the base of the stapes.

  13. The effect of different zwitterionic buffers and PBS used for out-of-incubator procedures during standard in vitro embryo production on development, morphology and gene expression of bovine embryos.

    PubMed

    Palasz, A T; Breña, P Beltrán; De la Fuente, J; Gutiérrez-Adán, A

    2008-12-01

    The effect of the zwitterionic buffers HEPES, TES and MOPS and of PBS used for out-of-incubator procedures during standard in vitro embryo production on bovine oocytes and embryo development, morphology and on the expression patterns of eight selected genes: Fgf-4, Lama1, Ube2a, Gsta4, Il6, Sod1, Prss11 and Hspb1, was evaluated. All buffers were prepared at a concentration of 10 mM in TALP medium, with the exception of PBS. The total time of oocyte/embryo exposure to each buffer was approximately 41 min. The cleavage rates and number of embryos that developed to > or =8 cells at day 4 were no different among the buffers tested, however, more blastocysts developed at day 7, 8 and 9 in HEPES and MOPS treatments than in PBS and TES (P<0.05). No difference between buffers in total and apoptotic cell number was found. Except for Hspb1 and Ube2a genes, the levels of expression of the six remaining transcripts were higher in in vivo than in in vitro embryos irrespective of buffer used (P<0.05). In addition, higher expression of Hspb1 and lower expression of Ube2a and Lama1 were observed in PBS and TES than in MOPS and HEPES treatments (P<0.05). Expression of Fgf-4 and Gsta4 in the in vitro embryos was lower in PBS than in the remaining three buffers (P<0.05) and the level of expression of the Il6 gene was not affected by any buffer tested but was lower in in vitro than in in vivo derived embryos. Expression of both Sod1 and Prss11 genes in MOPS were at the level of the in vivo embryos. These results showed that the choice of buffer and short exposure time of approximately 41 min, affects mRNA expression of in vitro produced bovine embryos.

  14. Metabolic and Transcriptional Reprogramming in Developing Soybean (Glycine max) Embryos

    PubMed Central

    Collakova, Eva; Aghamirzaie, Delasa; Fang, Yihui; Klumas, Curtis; Tabataba, Farzaneh; Kakumanu, Akshay; Myers, Elijah; Heath, Lenwood S.; Grene, Ruth

    2013-01-01

    Soybean (Glycine max) seeds are an important source of seed storage compounds, including protein, oil, and sugar used for food, feed, chemical, and biofuel production. We assessed detailed temporal transcriptional and metabolic changes in developing soybean embryos to gain a systems biology view of developmental and metabolic changes and to identify potential targets for metabolic engineering. Two major developmental and metabolic transitions were captured enabling identification of potential metabolic engineering targets specific to seed filling and to desiccation. The first transition involved a switch between different types of metabolism in dividing and elongating cells. The second transition involved the onset of maturation and desiccation tolerance during seed filling and a switch from photoheterotrophic to heterotrophic metabolism. Clustering analyses of metabolite and transcript data revealed clusters of functionally related metabolites and transcripts active in these different developmental and metabolic programs. The gene clusters provide a resource to generate predictions about the associations and interactions of unknown regulators with their targets based on “guilt-by-association” relationships. The inferred regulators also represent potential targets for future metabolic engineering of relevant pathways and steps in central carbon and nitrogen metabolism in soybean embryos and drought and desiccation tolerance in plants. PMID:24957996

  15. Transcriptional profiling of canola developing embryo and identification of the important roles of BnDof5.6 in embryo development and fatty acids synthesis.

    PubMed

    Deng, Wei; Yan, Fang; Zhang, Xiaolan; Tang, Yuwei; Yuan, Yujin

    2015-08-01

    Canola is an important vegetable oil crop globally, and the understanding of the molecular mechanism underlying fatty acids biosynthesis during seed embryo development is an important research goal. Here we report the transcriptional profiling analysis of developing canola embryos using RNA-sequencing (RNA-Seq) method. RNA-Seq analysis generated 58,579,451 sequence reads aligned with 32,243 genes. It was found that a total of 55 differential expression genes (DEGs) encoding 28 enzymes function in carbon flow to fatty acids of storage TAG. Most of the DEGs encoding above enzymes showed similar expression pattern, indicating the DEGs are cooperatively involved in carbon flow into fatty acids. In addition, 41 DEGs associated with signal transductions, transport and metabolic processing of auxin, gibberellin, abscisic acid, cytokinin and salicylic acids were found in the RNA-Seq database, which indicates the important roles of the phytohormones in controlling embryo development and fatty acids synthesis. 122 DEGs encoding transcriptional factor family members were found in developing canola embryos. Furthermore, BnDOF5.6, a zinc finger transcriptional factor gene, found in RNA-Seq database was down-regulated in developing canola embryos. The transgenic plants displayed reduced embryo sizes, decreased fatty acids contents and altered seed fatty acids composition in canola. Down-regulated of BnDof5.6 also changed the expression levels of genes involved in fatty acids synthesis and desaturation. Our results indicate that BnDof5.6 is required for embryo development and fatty acids synthesis in canola. Overall this study presents new information on the global expression patterns of genes during embryo development and will expand our understanding of the complex molecular mechanism of carbon flow into fatty acids and embryo development in canola.

  16. Effects of downregulating GLIS1 transcript on preimplantation development and gene expression of bovine embryos.

    PubMed

    Takahashi, Kazuki; Sakurai, Nobuyuki; Emura, Natsuko; Hashizume, Tsutomu; Sawai, Ken

    2015-01-01

    Krüppel-like protein Gli-similar 1 (GLIS1) is known as a direct reprogramming factor for the generation of induced pluripotent stem cells. The objective of this study was to investigate the role of GLIS1 in the preimplantation development of bovine embryos. GLIS1 transcripts in in vitro-matured oocytes and 1-cell to 4-cell stage embryos were detected, but they were either absent or at trace levels at the 8-cell to blastocyst stages. We attempted GLIS1 downregulation of bovine early embryos by RNA interference and evaluated developmental competency and gene transcripts, which are involved in zygotic gene activation (ZGA) in GLIS1-downregulated embryos. Injection of specific siRNA resulted in a distinct decrease in GLIS1 transcript in bovine embryos at the 4-cell stage. Although the bovine embryos injected with GLIS1-siRNA could develop to the 16-cell stage, these embryos had difficulty in developing beyond the 32-cell stage. Gene transcripts of PDHA1 and HSPA8, which are transcribed after ZGA, showed lower level in GLIS1 downregulated embryos. It is possible that GLIS1-downregulated embryos fail to initiate ZGA. Our results indicated that GLIS1 is an important factor for the preimplantation development of bovine embryos.

  17. Effects of downregulating GLIS1 transcript on preimplantation development and gene expression of bovine embryos

    PubMed Central

    TAKAHASHI, Kazuki; SAKURAI, Nobuyuki; EMURA, Natsuko; HASHIZUME, Tsutomu; SAWAI, Ken

    2015-01-01

    Krüppel-like protein Gli-similar 1 (GLIS1) is known as a direct reprogramming factor for the generation of induced pluripotent stem cells. The objective of this study was to investigate the role of GLIS1 in the preimplantation development of bovine embryos. GLIS1 transcripts in in vitro-matured oocytes and 1-cell to 4-cell stage embryos were detected, but they were either absent or at trace levels at the 8-cell to blastocyst stages. We attempted GLIS1 downregulation of bovine early embryos by RNA interference and evaluated developmental competency and gene transcripts, which are involved in zygotic gene activation (ZGA) in GLIS1-downregulated embryos. Injection of specific siRNA resulted in a distinct decrease in GLIS1 transcript in bovine embryos at the 4-cell stage. Although the bovine embryos injected with GLIS1-siRNA could develop to the 16-cell stage, these embryos had difficulty in developing beyond the 32-cell stage. Gene transcripts of PDHA1 and HSPA8, which are transcribed after ZGA, showed lower level in GLIS1 downregulated embryos. It is possible that GLIS1-downregulated embryos fail to initiate ZGA. Our results indicated that GLIS1 is an important factor for the preimplantation development of bovine embryos. PMID:26074126

  18. Gene expression profiling of the developing mouse kidney and embryo.

    PubMed

    Shaw, Lisa; Johnson, Penny A; Kimber, Susan J

    2010-02-01

    The metanephros is formed from the reciprocal inductive interaction of two precursor tissues, the metanephric mesenchyme (MM) and the ureteric bud (UB). The UB induces MM to condense and differentiate forming the glomerulus and renal tubules, whilst the MM induces the UB to differentiate into the collecting tubules of the mature nephron. Uninduced MM is considered the progenitor cell population of the developing metanephros because of its potential to differentiate into more renal cell types than the UB. Previous studies have identified the phenotype of renal precursor cells; however, expression of candidate marker genes have not been analysed in other tissues of the murine embryo. We have assayed up to 19 candidate genes in eight embryonic tissues at five gestation stages of the mouse embryo to identify markers definitively expressed by renal cells during metanephric induction and markers developmentally regulated during kidney maturation. We then analysed their expression in other developing tissues. Results show Dcn, Hoxc9, Mest, Wt1 and Ywhaq were expressed at moderate to high levels during the window of metanephric specification and early differentiation (E10.5-E12.5 dpc), and Hoxc9, Ren1 and Wt1 expression was characteristic of mature renal cells. We demonstrated Cd24a, Cdh11, Mest, Scd2 and Sim2 were regulated during brain development, and Scd2, Cd24a and Sip1 expression was enriched in developing liver. These markers may be useful negative markers of kidney development. Use of a combination of highly expressed and negative markers may aid in the identification and removal of non-renal cells from heterogeneous populations of differentiating stem cells.

  19. Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence?

    PubMed

    Shoukir, Y; Chardonnens, D; Campana, A; Sakkas, D

    1998-06-01

    The success of intracytoplasmic sperm injection (ICSI) warrants further study on the role of paternal factors in early human embryogenesis. To investigate whether poor sperm parameters can influence embryo development, we examined the development of ICSI-fertilized embryos to the blastocyst stage. We present results of blastocyst development from supernumerary ICSI embryos after co-culture on monkey kidney epithelial cells. In addition, we compare the development of supernumerary embryos to the blastocyst stage after ICSI and in-vitro fertilization (IVF). Of 168 supernumerary ICSI embryos, 45 (26.8%) developed to blastocysts. Sperm concentration and morphology did not influence blastocyst development. In contrast, blastocysts arose from spermatozoa that had a significantly higher (P = 0.015) forward progressive motility compared with spermatozoa from those patients who failed to produce blastocysts (42.7% versus 28.2%, respectively). Overall the rate of embryo development to the blastocyst stage after ICSI was lower (26.8%) than that after IVF (47.3%). When the rate of blastocyst development was calculated for patients with three or more supernumerary embryos, it remained significantly higher for the IVF patients than for the ICSI patients (45.6% versus 30.0%). There was no significant difference in the mean cell number and quality of the supernumerary embryos between the IVF and ICSI patients. This study confirms previous reports that have postulated that abnormal spermatozoa may manifest a negative paternal effect on preimplantation embryo development.

  20. Effects of growth hormone on nuclear maturation of ovine oocytes and subsequent embryo development.

    PubMed

    Shirazi, A; Shams-Esfandabadi, N; Ahmadi, E; Heidari, B

    2010-06-01

    The objective of this study was to determine the effect of the presence of recombinant ovine growth hormone either alone or together with follicle stimulating hormone (FSH) during ovine oocyte in vitro maturation (IVM) on nuclear maturation and subsequent embryo development. Moreover, the effect of growth hormine (GH) on embryo development whether influenced by the presence of foetal bovine serum (FBS) was assessed. The abattoir-derived oocytes were randomly divided into four treatment groups and cultured in maturation medium supplemented with: (i) 0.05 IU/ml FSH; (ii) 300 ng/ml roGH; (iii) FSH + roGH; and (iv) no FSH and GH (control). The percentages of germinal vesicle-stage oocytes in GH-treated group after 8 h of culture was significantly higher than the FSH and FSH + GH groups and lower than control (22.4%, 8.7%, 9.1%, and 32% respectively). The percentage of MII-stage oocytes was significantly increased in the presence of GH after 16 and 24 h of culture compared to the control (44.7% and 83.1% vs 32.6% and 73.6% respectively). There was no significant synergism between GH and FSH in terms of nuclear maturation. The blastocyst rates in serum-supplemented groups were enhanced by the presence of FSH and GH compared to the control (35.4% and 31.3 vs 11.4% respectively). Compared with either GH or FSH alone, the subsequent embryo development (blastocyst rate), however, was negatively influenced by co-presence of both hormones (22.8%). In contrast, the corresponding values were not affected in the absence of serum. In conclusion, GH had positive effect on nuclear maturation of sheep oocytes. Moreover, the pattern of the effect of GH on embryo development was influenced by the presence of FBS during IVM.

  1. Identification of the glycerol kinase gene and its role in diapause embryo restart and early embryo development of Artemia sinica.

    PubMed

    Cheng, Cheng; Yao, Feng; Chu, Bing; Li, Xuejie; Liu, Yan; Wu, Yang; Mei, Yanli; Wang, Peisheng; Hou, Lin; Zou, Xiangyang

    2014-03-01

    Glycerol kinase (GK) catalyzes the rate-limiting step in glycerol utilization by transferring a phosphate from ATP to glycerol, yielding glycerol 3-phosphate, which is an important intermediate for both energy metabolism and glycerolipid production. Artemia sinica has an unusual diapause process under stress conditions of high salinity, low temperature and lack of food. In the process, diapause embryos of A. sinica (brine shrimp) accumulate high concentrations of glycerol as a cryoprotectant to prevent low temperature damage to embryos. Upon embryo restart, glycerol is converted into glucose and other carbohydrates. Therefore, GK plays an important role in the diapause embryo restart process. However, the role of GK in diapause termination of embryo development in A. sinica remains unknown. In the present study, a 2096 bp full-length cDNA of gk from A. sinica (As-gk) was obtained, encoding putative 551 amino acids, 60.6 kDa protein. As a crucial enzyme in glycerol uptake and metabolism, GK has been conserved structurally and functionally during evolution. The expression pattern of As-gk was investigated by quantitative real-time PCR and Western blotting. Expression locations of As-gk were analyzed using in situ hybridization. As-gk was widely distributed in the early embryo and several main parts of Artemia after differentiation. The expression of As-GK was also induced by stresses such as cold exposure and high salinity. This initial research into the expression pattern and stress response of GK in Artemia provides a sound basis for further understanding of the function and regulation of genes in early embryonic development in A. sinica and the stress response.

  2. Dual Positive Regulation of Embryo Implantation by Endocrine and Immune Systems--Step-by-Step Maternal Recognition of the Developing Embryo.

    PubMed

    Fujiwara, Hiroshi; Araki, Yoshihiko; Imakawa, Kazuhiko; Saito, Shigeru; Daikoku, Takiko; Shigeta, Minoru; Kanzaki, Hideharu; Mori, Takahide

    2016-03-01

    In humans, HCG secreted from the implanting embryo stimulates progesterone production of the corpus luteum to maintain embryo implantation. Along with this endocrine system, current evidence suggests that the maternal immune system positively contributes to the embryo implantation. In mice, immune cells that have been sensitized with seminal fluid and then the developing embryo induce endometrial differentiation and promote embryo implantation. After hatching, HCG activates regulatory T and B cells through LH/HCG receptors and then stimulates uterine NK cells and monocytes through sugar chain receptors, to promote and maintain pregnancy. In accordance with the above, the intrauterine administration of HCG-treated PBMC was demonstrated to improve implantation rates in women with repeated implantation failures. These findings suggest that the maternal immune system undergoes functional changes by recognizing the developing embryos in a stepwise manner even from a pre-fertilization stage and facilitates embryo implantation in cooperation with the endocrine system.

  3. Developing Effective Affective Assessment Practices

    ERIC Educational Resources Information Center

    Glennon, William; Hart, Aaron; Foley, John T.

    2015-01-01

    Physical educators generally understand the importance of the affective domain for student growth and development. However, many teachers struggle with assessing affective behaviors in a way that can be documented and reported. The five-step process outlined in this article can assist teachers in developing an effective way to assess the affective…

  4. Development of a pronuclear DNA microinjection technique for production of green fluorescent protein-expressing bubaline (Bubalus bubalis) embryos.

    PubMed

    Verma, V; Gautam, S K; Palta, P; Manik, R S; Singla, S K; Chauhan, M S

    2008-04-01

    Oocytes from abattoir-derived bubaline (Bubalus bubalis) ovaries were subjected to IVM and IVF; the objective was to develop a pronuclear DNA microinjection technique to produce embryos expressing green fluorescent protein (GFP). The largest proportion (61.2%) of zygotes in which one (1 PN) or two pronuclei (2 PN) were visible was when centrifugation (14,000 x g for 15 min) was done 16 h after insemination. Centrifugation had no adverse effects on cleavage rate, development to morulae/blastocysts, and total cell number of embryos. Piercing the pronuclear but not the plasma membrane reduced (P<0.05) cleavage rate (44.0% vs. 51.0%), without affecting subsequent development. Following microinjection of a GFP-DNA construct, cleavage rate (55.9, 38.9, and 30.9%) and proportion of cleaved embryos that developed to morulae (39.9, 25.6, and 15.5%) and blastocyst stages (22.4, 13.4, and 2.8%) were higher (P<0.05) for non-injected controls than for those injected with buffer alone, which, in turn, were higher (P<0.05) than for those injected with buffer containing 5 microg/mL DNA. The cleavage rate (39.2% vs. 34.8%) and proportion of cleaved embryos that developed to morulae/blastocysts (37.5% vs. 10.9%) were higher (P<0.05) for microinjected zygotes with 2 PN than for those with 1 PN. The cleavage rate and the proportion of cleaved embryos that developed to morulae and blastocysts were higher (P<0.05) following culture of microinjected zygotes in mCR2aa medium (40.7, 32.7, and 9.1%, respectively) compared to those for mSOFaa (33.3, 26.0, and 6.5%, respectively) or after culture in TCM-199+co-culture with buffalo oviductal epithelial cells (31.2, 25.0, and 4.5%, respectively). The proportion of embryos expressing GFP was higher (P<0.01) for 2 PN than for 1 PN zygotes (15.9% vs. 13.7%). Thirty-five embryos expressed GFP; the proportion of mosaic embryos (62.8%) was higher (P<0.01) than of embryos in which all blastomeres expressed GFP (37.2%); eight and two of those embryos

  5. [Study on the effect of alcohol on embryonic development by using in vitro post-implantation rat whole embryo culture].

    PubMed

    Qu, W; Zhang, B; Wu, D; Wu, W

    2000-01-30

    In order to explore the effects of drinking alcohol during pregnancy on embryonic development and its mechanisms, a post-implantation whole embryo culture(WEC) technique was used. The 9.5 day rat embryos were explanted in rat serum medium(immediately centrifugal serum, ICS) with alcohol(0.0.4.1.0, 2.00 and 4.00 g/L), and cultured for 48 hours. The index of embryo development and morphological scores induced by alcohol were observed. The result showed that alcohol had obviously effects on the development and growth of embryos with a dose-response relationship. Embryonic development of 0.4 g/L group was not significantly different from the control group, whereas 1.0 g/L group could interfere with the development score of mid-brain, forebrain, neurotube, and visceral yolk sac(VYS) circle obviously. All scores of the 2.00 g/L group were significantly lower than that of control group (P < 0.05). Moreover, the rate of embryo lethality and teratogenecity were obvious increased. It is concluded that alcohol has developmental toxicity and teratogenicity. The target organ affected by alcohol is brain. The effects of alcohol on the developmental differentiation of visceral yolk sac and DNA synthesis are probably related to its developmental abnormalities.

  6. Beneficial effect of two culture systems with small groups of embryos on the development and quality of in vitro-produced bovine embryos.

    PubMed

    Cebrian-Serrano, A; Salvador, I; Silvestre, M A

    2014-02-01

    Currently, in vitro-produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one-third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU-IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF-ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500 μl medium) and three smaller groups [five embryos/50 μl medium without (control) or with EGF-ITS (EGF-ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF-ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF-ITS improved the embryo quality when smaller groups of embryos were cultured.

  7. Early cardiac development: a view from stem cells to embryos

    PubMed Central

    Van Vliet, Patrick; Wu, Sean M.; Zaffran, Stéphane; Pucéat, Michel

    2012-01-01

    From the 1920s, early cardiac development has been studied in chick and, later, in mouse embryos in order to understand the first cell fate decisions that drive specification and determination of the endocardium, myocardium, and epicardium. More recently, mouse and human embryonic stem cells (ESCs) have demonstrated faithful recapitulation of early cardiogenesis and have contributed significantly to this research over the past few decades. Derived almost 15 years ago, human ESCs have provided a unique developmental model for understanding the genetic and epigenetic regulation of early human cardiogenesis. Here, we review the biological concepts underlying cell fate decisions during early cardiogenesis in model organisms and ESCs. We draw upon both pioneering and recent studies and highlight the continued role for in vitro stem cells in cardiac developmental biology. PMID:22893679

  8. 104 FACTORS AFFECTING PREGNANCY RATES AND EMBRYO/FETAL LOSSES IN RECIPIENTS RECEIVING IN VITRO-PRODUCED EMBRYOS BY FIXED-TIME EMBRYO TRANSFER.

    PubMed

    Tribulo, A; Cedeño, A; Bernal, B; Andrada, S; Barajas, J L; Ortega, J; Oviedo, J M; Tribulo, H; Tribulo, R; Mapletoft, R J; Bó, G A

    2016-01-01

    A retrospective analysis evaluated pregnancy rates and embryo losses with in vitro-produced embryos in a commercial embryo transfer program on 15 different beef farms. Recipients were beef cows and heifers (n=1841) that were synchronized with 5 different protocols and transferred at a fixed-time (FTET). Recipients were examined by ultrasonography on Day 0, and those with a corpus luteum (CL) or a follicle ≥8mm in diameter and with body condition score 2 to 4 (1 to 5 scale) were synchronized. The synchronization treatments were as follows. (T1) Recipients received an intravaginal device with 0.5g of progesterone plus 2mg of oestradiol benzoate on Day 0; device removal, plus 500μg of cloprostenol (prostaglandin F2α), 400IU of eCG, and 0.5mg of oestradiol cypionate on Day 8; and FTET on Day 17. (T2) This treatment was similar to T1 but 1mg of oestradiol cypionate was injected at device removal instead of 0.5mg of oestradiol cypionate. (T3) This treatment was similar to T1 except that animals were tail-painted on Day 8 and observed on Day 10. Those with the tail-paint intact on Day 10 received 100μg of gonadorelin (gonadotropin-releasing hormone) and all recipients were FTET on Day 17. (T4) Recipients received a progesterone device on Day 0; device removal, prostaglandin F2α, and eCG on Day 5; gonadotropin-releasing hormone on Day 8; and FTET on Day 15. (T5) Recipients received a progesterone device and 2mg of oestradiol benzoate on Day 0; device removal, prostaglandin F2α, and eCG on Day 6; gonadotropin-releasing hormone on Day 9; and FTET on Day 16. On the day of FTET all recipients with CL ≥18mm in diameter (G1), ≥16 and <18mm in diameter (G2), and ≥14mm and <16mm in diameter (G3) received in vitro-produced fresh embryos. Pregnancy was diagnosed by ultrasonography at 30 and 60 days of gestation, and data were analysed by logistic regression. The overall proportion of recipients synchronized that were FTET was 80.8% (1487/1841), with a 30-day pregnancy

  9. Effects of β radiation on amphibian embryos (Pleurodeles waltlii) and capacities of regulation during development

    NASA Astrophysics Data System (ADS)

    Gallien, Cl. L.; Lenfant-Guyot, M.; Labrousse, J. P.

    The eukariotic cells of complex organisms possessing abundant and sophisticated genetic information, advanced metabolism and very diversified structures are particularly sensitive to the effects of radiation. One may note, however, that all cells of an organism which has been totally radiated may not be affected in the same way; this leaves room, particularly in embryonic organisms during development, for fairly broad possibilities of regulation. We have undertaken analysis of one aspect of these phenomena on a particularly favorable biological model: the embryo of the salamander Pleurodeles waltlii.

  10. CULTURING RABIES (HYDROPHOBIA) VIRUSES IN A DEVELOPING CHICKEN EMBRYO

    DTIC Science & Technology

    This report states that 19 successive passages of the rabies virus strain 83 through the brain of a chicken embryo and 6 passages through the yolk...sac are feasible. In the process of cultivation of the rabies virus in the organism of chicken embryo there was a variation-fixation of it; shortening

  11. Timing of human preimplantation embryonic development is confounded by embryo origin

    PubMed Central

    Kirkegaard, K.; Sundvall, L.; Erlandsen, M.; Hindkjær, J.J.; Knudsen, U.B.; Ingerslev, H.J.

    2016-01-01

    STUDY QUESTION To what extent do patient- and treatment-related factors explain the variation in morphokinetic parameters proposed as embryo viability markers? SUMMARY ANSWER Up to 31% of the observed variation in timing of embryo development can be explained by embryo origin, but no single factor elicits a systematic influence. WHAT IS KNOWN ALREADY Several studies report that culture conditions, patient characteristics and treatment influence timing of embryo development, which have promoted the perception that each clinic must develop individual models. Most of the studies have, however, treated embryos from one patient as independent observations, and only very few studies that evaluate the influence from patient- and treatment-related factors on timing of development or time-lapse parameters as predictors of viability have controlled for confounding, which implies a high risk of overestimating the statistical significance of potential correlations. STUDY DESIGN, SIZE, DURATION Infertile patients were prospectively recruited to a cohort study at a hospital fertility clinic from February 2011 to May 2013. Patients aged <38 years without endometriosis were eligible if ≥8 oocytes were retrieved. Patients were included only once. All embryos were monitored for 6 days in a time-lapse incubator. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 1507 embryos from 243 patients were included. The influence of fertilization method, BMI, maternal age, FSH dose and number of previous cycles on timing of t2-t5, duration of the 2- and 3-cell stage, and development of a blastocoel (tEB) and full blastocoel (tFB) was tested in multivariate, multilevel linear regression analysis. Predictive parameters for live birth were tested in a logistic regression analysis for 223 single transferred blastocysts, where time-lapse parameters were investigated along with patient and embryo characteristics. MAIN RESULTS AND THE ROLE OF CHANCE Moderate intra-class correlation coefficients

  12. Further Development and Validation of the Frog Embryo Teratogenesis Assay-Xenopus (FETAX).

    DTIC Science & Technology

    1992-11-23

    13. ABSTRACT (Maximum 200 words) The Frog Embryo Teratogenesis Assay Xenopus (FETAX) is a 96 hour whole embryo developmental toxicity screening assay...It was designed to detect xenobiotics that selectively impair embryo development, growth and survival (developmental toxicants )at concentrations far...developmental endpoints or risk missing toxic effects on a weak link in the life cycle of vertebrates. The purpose of the contract was to establish the

  13. Potentiality and human embryos.

    PubMed

    Lizza, John P

    2007-09-01

    Consideration of the potentiality of human embryos to develop characteristics of personhood, such as intellect and will, has figured prominently in arguments against abortion and the use of human embryos for research. In particular, such consideration was the basis for the call of the US President's Council on Bioethics for a moratorium on stem cell research on human embryos. In this paper, I critique the concept of potentiality invoked by the Council and offer an alternative account. In contrast to the Council's view that an embryo's potentiality is determined by definition and is not affected by external conditions that may prevent certain possibilities from ever being realized, I propose an empirically grounded account of potentiality that involves an assessment of the physical and decisional conditions that may restrict an embryo's possibilities. In my view, some human embryos lack the potentiality to become a person that other human embryos have. Assuming for the sake of argument that the potential to become a person gives a being special moral status, it follows that some human embryos lack this status. This argument is then used to support Gene Outka's suggestion that it is morally permissible to experiment on 'spare' frozen embryos that are destined to be destroyed.

  14. Transparent testa16 plays multiple roles in plant development and is involved in lipid synthesis and embryo development in canola.

    PubMed

    Deng, Wei; Chen, Guanqun; Peng, Fred; Truksa, Martin; Snyder, Crystal L; Weselake, Randall J

    2012-10-01

    Transparent Testa16 (TT16), a transcript regulator belonging to the B(sister) MADS box proteins, regulates proper endothelial differentiation and proanthocyanidin accumulation in the seed coat. Our understanding of its other physiological roles, however, is limited. In this study, the physiological and developmental roles of TT16 in an important oil crop, canola (Brassica napus), were dissected by a loss-of-function approach. RNA interference (RNAi)-mediated down-regulation of tt16 in canola caused dwarf phenotypes with a decrease in the number of inflorescences, flowers, siliques, and seeds. Fluorescence microscopy revealed that tt16 deficiency affects pollen tube guidance, resulting in reduced fertility and negatively impacting embryo and seed development. Moreover, Bntt16 RNAi plants had reduced oil content and altered fatty acid composition. Transmission electron microscopy showed that the seeds of the RNAi plants had fewer oil bodies than the nontransgenic plants. In addition, tt16 RNAi transgenic lines were more sensitive to auxin. Further analysis by microarray showed that tt16 down-regulation alters the expression of genes involved in gynoecium and embryo development, lipid metabolism, auxin transport, and signal transduction. The broad regulatory function of TT16 at the transcriptional level may explain the altered phenotypes observed in the transgenic lines. Overall, the results uncovered important biological roles of TT16 in plant development, especially in fatty acid synthesis and embryo development.

  15. Male Killing Spiroplasma Preferentially Disrupts Neural Development in the Drosophila melanogaster Embryo

    PubMed Central

    Martin, Jennifer; Chong, Trisha; Ferree, Patrick M.

    2013-01-01

    Male killing bacteria such as Spiroplasma are widespread pathogens of numerous arthropods including Drosophila melanogaster. These maternally transmitted bacteria can bias host sex ratios toward the female sex in order to ‘selfishly’ enhance bacterial transmission. However, little is known about the specific means by which these pathogens disrupt host development in order to kill males. Here we show that a male-killing Spiroplasma strain severely disrupts nervous tissue development in male but not female D. melanogaster embryos. The neuroblasts, or neuron progenitors, form properly and their daughter cells differentiate into neurons of the ventral nerve chord. However, the neurons fail to pack together properly and they produce highly abnormal axons. In contrast, non-neural tissue, such as mesoderm, and body segmentation appear normal during this time, although the entire male embryo becomes highly abnormal during later stages. Finally, we found that Spiroplasma is altogether absent from the neural tissue but localizes within the gut and the epithelium immediately surrounding the neural tissue, suggesting that the bacterium secretes a toxin that affects neural tissue development across tissue boundaries. Together these findings demonstrate the unique ability of this insect pathogen to preferentially affect development of a specific embryonic tissue to induce male killing. PMID:24236124

  16. Impact of ketorolac administration around ovarian stimulation on in vivo and in vitro fertilization and subsequent embryo development.

    PubMed

    Jee, Byung Chul; Youm, Hye Won; Lee, Jae Ho; Kim, Jee Hyun; Suh, Chang Suk; Kim, Seok Hyun

    2013-05-01

    We performed this study to investigate the effect of ketorolac (a non-steroidal anti-inflammatory drug) administration around ovarian stimulation on in vivo and in vitro fertilization process. Sixty-four female mice (ICR) were injected with ketorolac (0, 7.5, 15 and 30 µg/d) for 3 d starting from the day of eCG treatment. In experiment 1, 41 mice were triggered by hCG and then mated; two-cell embryos were obtained and in vitro development up to blastocyst was observed. In experiment 2, 23 mice were triggered by hCG and mature oocytes were collected; in vitro fertilization rate and subsequent embryo development up to blastocyst was recorded. In experiment 1, the blastocyst-forming rates per in vivo fertilized two-cell embryo showed an inverse relationship with a dosage of ketorolac (97.6%, 64.2%, 35.4% and 25.9%). In experiment 2, degenerated oocytes were frequently observed in a dose-dependent manner (4.3%, 22.9%, 22.4% and 75.0%). Lower fertilization rates were noted in all the three ketorolac-treating groups; blastocyst-forming rate was significantly lower in 30-µg-treating group when compared with the control group. Administration of ketorolac around ovarian stimulation significantly affects the development of in vivo fertilized embryo in a dose-dependent manner. High-dose ketorolac could result in a poor oocyte quality and decreased embryo developmental competence.

  17. In vitro effects of relaxin on gene expression in porcine cumulus ooxyte complexes and developing embryos

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Relaxin hormone peptide is found in porcine follicular and utero-tubal fluids, but its possible actions during early embryo development are still undetermined. Here, we investigated the effects of porcine relaxin during oocyte maturation and embryo development, and gene expression in the pig. Immat...

  18. Elevated Progesterone Levels on the Day of Oocyte Maturation May Affect Top Quality Embryo IVF Cycles.

    PubMed

    Huang, Bo; Ren, Xinling; Wu, Li; Zhu, Lixia; Xu, Bei; Li, Yufeng; Ai, Jihui; Jin, Lei

    2016-01-01

    In contrast to the impact of elevated progesterone on endometrial receptivity, the data on whether increased progesterone levels affects the quality of embryos is still limited. This study retrospectively enrolled 4,236 fresh in vitro fertilization (IVF) cycles and sought to determine whether increased progesterone is associated with adverse outcomes with regard to top quality embryos (TQE). The results showed that the TQE rate significantly correlated with progesterone levels on the day of human chorionic gonadotropin (hCG) trigger (P = 0.009). Multivariate linear regression analysis of factors related to the TQE rate, in conventional IVF cycles, showed that the TQE rate was negatively associated with progesterone concentration on the day of hCG (OR was -1.658, 95% CI: -2.806 to -0.510, P = 0.005). When the serum progesterone level was within the interval 2.0-2.5 ng/ml, the TQE rate was significantly lower (P <0.05) than when the progesterone level was < 1.0 ng/ml; similar results were obtained for serum progesterone levels >2.5 ng/ml. Then, we choose a progesterone level at 1.5ng/ml, 2.0 ng/ml and 2.5 ng/ml as cut-off points to verify this result. We found that the TQE rate was significantly different (P <0.05) between serum progesterone levels < 2.0 ng/ml and >2.0 ng/ml. In conclusion, the results of this study clearly demonstrated a negative effect of elevated progesterone levels on the day of hCG trigger, on TQE rate, regardless of the basal FSH, the total gonadotropin, the age of the woman, or the time of ovarian stimulation. These data demonstrate that elevated progesterone levels (>2.0 ng/ml) before oocyte maturation were consistently detrimental to the oocyte.

  19. Glycerol-3-phosphate acyltransferase 4 is essential for the normal development of reproductive organs and the embryo in Brassica napus.

    PubMed

    Chen, Xue; Chen, Guanqun; Truksa, Martin; Snyder, Crystal L; Shah, Saleh; Weselake, Randall J

    2014-08-01

    The enzyme sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) is involved in the biosynthesis of plant lipid poly-esters. The present study further characterizes the enzymatic activities of three endoplasmic reticulum-bound GPAT4 isoforms of Brassica napus and examines their roles in the development of reproductive organs and the embryo. All three BnGPAT4 isoforms exhibited sn-2 acyltransferase and phosphatase activities with dicarboxylic acid-CoA as acyl donor. When non-substituted acyl-CoA was used as acyl donor, the rate of acylation was considerably lower and phosphatase activity was not manifested. RNA interference (RNAi)-mediated down-regulation of all GPAT4 homologues in B. napus under the control of the napin promoter caused abnormal development of several reproductive organs and reduced seed set. Microscopic examination and reciprocal crosses revealed that both pollen grains and developing embryo sacs of the B. napus gpat4 lines were affected. The gpat4 mature embryos showed decreased cutin content and altered monomer composition. The defective embryo development further affected the oil body morphology, oil content, and fatty acid composition in gpat4 seeds. These results suggest that GPAT4 has a critical role in the development of reproductive organs and the seed of B. napus.

  20. Exposure to mono-n-butyl phthalate disrupts the development of preimplantation embryos.

    PubMed

    Chu, Da-Peng; Tian, Shi; Sun, Da-Guang; Hao, Chan-Juan; Xia, Hong-Fei; Ma, Xu

    2013-01-01

    Dibutyl phthalate (DBP), a widely used phthalate, is known to cause many serious diseases, especially in the reproductive system. However, little is known about the effects of its metabolite, mono-n-butyl phthalate (MBP), on preimplantation embryo development. In the present study, we found that treatment of embryos with 10⁻³ M MBP impaired developmental competency, whereas exposure to 10⁻⁴ M MBP delayed the progression of preimplantation embryos to the blastocyst stage. Furthermore, reactive oxygen species (ROS) levels in embryos were significantly increased following treatment with 10⁻³ M MBP. In addition, 10⁻³ M MBP increased apoptosis via the release of cytochrome c, whereas immunofluorescent analysis revealed that exposure of preimplantation embryos to MBP concentration-dependently (10⁻⁵, 10⁻⁴ and 10⁻³ M) decreased DNA methylation. Together, the results indicate a possible relationship between MBP exposure and developmental failure in preimplantation embryos.

  1. The Development of Vestibular Connections in Rat Embryos in Microgravity

    NASA Technical Reports Server (NTRS)

    Bruce, Laura L.; Fritzsch, Bernd

    1997-01-01

    Existing experimental embryological data suggests that the vestibular system initially develops in a very rigid and genetically controlled manner. Nevertheless, gravity appears to be a critical factor in the normal development of the vestibular system that monitors position with respect to gravity (saccule and utricle). In fact several studies have shown that prenatal exposure to microgravity causes temporary deficits in gravity-dependent righting behaviors, and prolonged exposure to hypergravity from conception to weaning causes permanent deficits in gravity-dependent righting behaviors. Data on hypergravity and microgravity exposure suggest some changes in the otolith formation during development, in particular the size although these changes may actually vary with the species involved. In adults exposed to microgravity there is a change in the synaptic density in the otic sensory epithelia suggesting that some adaptation may occur there. However, effects have also been reported in the brainstem. Several studies have shown synaptic changes in the lateral vestibular nucleus and in the nodulus of the cerebellum after neonatal exposure to hypergravity. We report here that synaptogenesis in the medial vestibular nucleus is retarded in developing rat embryos that were exposed to microgravity from gestation days 9 to 19.

  2. Local activation of uterine Toll-like receptor 2 and 2/6 decreases embryo implantation and affects uterine receptivity in mice.

    PubMed

    Sanchez-Lopez, Javier Arturo; Caballero, Ignacio; Montazeri, Mehrnaz; Maslehat, Nasim; Elliott, Sarah; Fernandez-Gonzalez, Raul; Calle, Alexandra; Gutierrez-Adan, Alfonso; Fazeli, Alireza

    2014-04-01

    Embryo implantation is a complex interaction between maternal endometrium and embryonic structures. Failure to implant is highly recurrent and impossible to diagnose. Inflammation and infections in the female reproductive tract are common causes of infertility, embryo loss, and preterm labor. The current work describes how the activation of endometrial Toll-like receptor (TLR) 2 and 2/6 reduces embryo implantation chances. We developed a morphometric index to evaluate the effects of the TLR 2/6 activation along the uterine horn (UH). TLR 2/6 ligation reduced the endometrial myometrial and glandular indexes and increased the luminal index. Furthermore, TLR 2/6 activation increased the proinflammatory cytokines such as interleukin (IL)-1beta and monocyte chemotactic protein (MCP)-1 in UH lavages in the preimplantation day and IL-1 receptor antagonist in the implantation day. The engagement of TLR 2/6 with its ligand in the UH during embryo transfer severely affected the rate of embryonic implantation (45.00% ± 6.49% vs. 16.69% ± 5.01%, P < 0.05, control vs. test, respectively). Furthermore, this interference with the embryo implantation process was verified using an in vitro model of human embryo implantation where trophoblast spheroids failed to adhere to a monolayer of TLR 2- and TLR 2/6-activated endometrial cells. The inhibition of TLR receptors 2 and 6 in the presence of their specific ligands restored the ability of the spheroids to bind to the endometrial cells. In conclusion, the activation of the innate immune system in the uterus at the time of implantation interfered with the endometrial receptivity and reduced the chances of implantation success.

  3. Ribonuclease J is required for chloroplast and embryo development in Arabidopsis.

    PubMed

    Chen, Hongyu; Zou, Wenxuan; Zhao, Jie

    2015-04-01

    Chloroplasts perform many essential metabolic functions and their proper development is critically important in embryogenesis. However, little is known about how chloroplasts function in embryogenesis and more relevant components need to be characterized. In this study, we show that Arabidopsis Ribonuclease J (RNase J) is required for chloroplast and embryo development. Mutation of AtRNJ led to albino ovules containing aborted embryos; the morphological development of rnj embryos was disturbed after the globular stage. Observation of ultrastructures indicated that these aborted embryos may result from impaired chloroplast development. Furthermore, by analyzing the molecular markers of cell fate decisions (STM, FIL, ML1, SCR, and WOX5) in rnj embryos, we found that this impairment of chloroplast development may lead to aberrant embryo patterning along the apical-basal axis, indicating that AtRNJ is important in initiating and maintaining the organization of shoot apical meristems (SAMs), cotyledons, and hypocotyls. Moreover, the transport and response of auxin in rnj embryos was found to be disrupted, suggesting that AtRNJ may be involved in auxin-mediated pathways during embryogenesis. Therefore, we speculate that RNJ plays a vital role in embryo morphogenesis and apical-basal pattern formation by regulating chloroplast development.

  4. Oocyte quality determines bovine embryo development after fertilisation with hydrogen peroxide-stressed spermatozoa.

    PubMed

    Rahman, Mohammad Bozlur; Vandaele, Leen; Rijsselaere, Tom; Zhandi, Mahdi; Maes, Dominiek; Shamsuddin, Mohammed; Van Soom, Ann

    2012-01-01

    Exposure of gametes to specific stressors at sublethal levels can enhance the gametes' subsequent performance in processes such as cryopreservation. In the present study, bull spermatozoa were subjected to H₂O₂ for 4 h at 100-, 200- and 500-μM levels; computer-assisted sperm analysis (CASA) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay were used for evaluation of subsequent sperm motility and DNA integrity, respectively. Exposure of spermatozoa to H₂O₂ did not affect sperm motility but DNA integrity was negatively affected by 500 μM H₂O₂ compared with mock-exposed spermatozoa, whereas both motility and DNA integrity were affected compared with untreated spermatozoa. Nevertheless, insemination of oocytes with spermatozoa exposed to 200 μM H₂O₂ increased fertilisation, cleavage and blastocyst rates (P < 0.05). Furthermore, the higher blastocyst yield after fertilisation of oocytes with spermatozoa exposed to 200 μM H₂O₂ was related to oocyte diameter, with large-medium oocytes yielding higher blastocyst rates, while small-diameter oocytes consistently failed to develop into blastocysts. In conclusion, the results indicate that exposure of spermatozoa to 200 μM H₂O₂ before sperm-oocyte interaction may enhance in vitro embryo production in cattle. However, this increased embryo production is largely dependent on the intrinsic quality of the oocytes.

  5. Migratory ability of gonadal germ cells (GGCs) isolated from Ciconia boyciana and Geronticus eremita embryos into the gonad of developing chicken embryos

    PubMed Central

    NAKAJIMA, Yuki; FUKUDA, Haruka; ONUMA, Manabu; MURATA, Koichi; UEDA, Miya; SUNAGA, Emi; SHIRAISHI, Toshirou; TAJIMA, Atsushi

    2016-01-01

    We conducted experiments to evaluate the ability of gonadal germ cells (GGCs), isolated from the embryonic gonads of Ciconia boyciana or Geronticus eremita, to migrate into the gonads of developing chicken embryos. Fluorescently labeled GGCs, isolated by the PBS (−) method, were transferred into the dorsal aorta of 2-day-old chicken embryos. Five days after transfer, fluorescent GGCs were detected in the gonads of recipient embryos. Our results indicate that GGCs from Ciconia boyciana and Geronticus eremita are capable of migrating into the gonads of developing chicken embryos. PMID:26922915

  6. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

    PubMed Central

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming. PMID:26894831

  7. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

    PubMed

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

  8. Development of the juxta-oral organ in rat embryo.

    PubMed

    Velasco, J R Mérida; De La Cuadra Blanco, C; Velasco, J A Mérida

    2012-05-01

    The aim of this work is to clarify the development and morphology of the juxta-oral organ (JOO) in rat embryos from Day (E)14 to 19. Furthermore, in the region of the JOO, an analysis was made of the expression of the monoclonal antibody HNK-1, which recognizes cranial neural-crest cells. In this study, we report that JOO develops from an epithelial condensation at the end of the transverse groove of the primitive mouth at E14. During E15, it invaginates and is disconnected from the oral epithelium. At E16, the JOO forms an solid epithelial cord with three parts (anterior, middle, and posterior) and is related to the masseter, temporal, medial pterygoid, and tensor veli palatini muscles. During E17-19, no significant changes were detected in their position. Both the mesenchyme caudal to the anlage of the JOO at E14, as well as the mesenchyme that surrounds the bud of the JOO at E15, expressed positivity for HNK-1. Our results suggest that the mesenchyme surrounding the JOO at E15 could emit some inductive signal for the JOO to reach its position at E16. This work shows for the first time that the cranial neural-crest-derived mesenchyme participates in the development of the JOO.

  9. Development of the stapes and associated structures in human embryos

    PubMed Central

    Rodríguez-Vázquez, JF

    2005-01-01

    The objective of this study was to clarify the development of the stapes in humans and its relationship with the cartilage of the second branchial arch. The study was carried out in 25 human embryos between 6 and 28 mm crown–rump length. The stapes develops at the cranial end of the second branchial arch through an independent anlage of the cartilage of this arch. Between the stapedial anlage and the cranial end of the Reichert's cartilage there is a formation called the interhyale, the internal segment of which gives rise to the tendon of the stapedial muscle. The stapedial anlage is a unique formation with two distinct parts: the superior part that will comprise the base and the inferior part that will be crossed by the stapedial artery during embryonic development and will constitute the limbs and the head of the stapes. According to the results, the otic capsule is not involved in formation of the base of the stapes. PMID:16050903

  10. Cadmium but not lead exposure affects Xenopus laevis fertilization and embryo cleavage.

    PubMed

    Slaby, Sylvain; Lemière, Sébastien; Hanotel, Julie; Lescuyer, Arlette; Demuynck, Sylvain; Bodart, Jean-François; Leprêtre, Alain; Marin, Matthieu

    2016-08-01

    Among the toxicological and ecotoxicological studies, few have investigated the effects on germ cells, gametes or embryos, while an impact at these stages will result in serious damage at a population level. Thus, it appeared essential to characterize consequences of environmental contaminant exposures at these stages. Therefore, we proposed to assess the effects of exposure to cadmium and lead ions, alone or in a binary mixture, on early stages of Xenopus laevis life cycle. Fertilization and cell division during segmentation were the studied endpoints. Cadmium ion exposures decreased in the fertilization rates in a concentration-dependent manner, targeting mainly the oocytes. Exposure to this metal ions induced also delays or blockages in the embryonic development. For lead ion exposure, no such effect was observed. For the exposure to the mixture of the two metal ions, concerning the fertilization success, we observed results similar to those obtained with the highest cadmium ion concentration.

  11. Embryogenesis in Polianthes tuberosa L var. Simple: from megasporogenesis to early embryo development.

    PubMed

    González-Gutiérrez, Alejandra G; Rodríguez-Garay, Benjamín

    2016-01-01

    The genus Polianthes belongs to the subfamily Agavoideae of the Asparagaceae family formerly known as Agavaceae. The genus is endemic to México and comprises about 15 species, among them is Polianthes tuberosa L. The aim of this work was to study and characterize the embryo sac and early embryo development of this species in order to generate basic knowledge for its use in taxonomy, in vitro fertilization and production of haploid plants and to complement studies already performed in other genera and species belonging to the Agavoideae sub-family. It was found that the normal development of the P. tuberosa var. Simple embryo sac follows a monosporic pattern of the Polygonum type and starts its development from the chalazal megaspore. At maturity, the embryo sac is of a pyriform shape with a chalazal haustorial tube where the antipodals are located, just below the hypostase, which connects the embryo sac with the nucellar tissue of the ovule. The central cell nucleus shows a high polarity, being located at the chalazal extreme of the embryo sac. The position of cells inside the P. tuberosa embryo sac may be useful for in depth studies about the double fertilization. Furthermore, it was possible to make a chronological description of the events that happen from fertilization and early embryo development to the initial development of the endosperm which was classified as of the helobial type.

  12. Effect of sericin on preimplantation development of bovine embryos cultured individually.

    PubMed

    Isobe, T; Ikebata, Y; Onitsuka, T; Wittayarat, M; Sato, Y; Taniguchi, M; Otoi, T

    2012-09-01

    The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P < 0.05) than those from embryos cultured with 0 or 1% sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 μm H(2)O(2)), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 μm H(2)O(2). However, the protective effect of sericin was not observed in development of embryos exposed to 50 μm H(2)O(2). When embryos were exposed to 100 μm H(2)O(2) during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P < 0.01). In conclusion, the addition of 0.5% sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress.

  13. Adaptive plasticity of killifish (Fundulus heteroclitus) embryos: dehydration-stimulated development and differential aquaporin-3 expression.

    PubMed

    Tingaud-Sequeira, Angèle; Zapater, Cinta; Chauvigné, François; Otero, David; Cerdà, Joan

    2009-04-01

    Embryos of the marine killifish Fundulus heteroclitus are adapted to survive aerially. However, it is unknown if they are able to control development under dehydration conditions. Here, we show that air-exposed blastula embryos under saturated relative humidity were able to stimulate development, and hence the time of hatching was advanced with respect to embryos continuously immersed in seawater. Embryos exposed to air at later developmental stages did not hatch until water was added, while development was not arrested. Air-exposed embryos avoided dehydration probably because of their thickened egg envelope, although it suffered significant evaporative water loss. The potential role of aquaporins as part of the embryo response to dehydration was investigated by cloning the aquaporin-0 (FhAqp0), -1a (FhAqp1a), and -3 (FhAqp3) cDNAs. Functional expression in Xenopus laevis oocytes showed that FhaAqp1a was a water-selective channel, whereas FhAqp3 was permeable to water, glycerol, and urea. Expression of fhaqp0 and fhaqp1a was prominent during organogenesis, and their mRNA levels were similar between water- and air-incubated embryos. However, fhaqp3 transcripts were highly and transiently accumulated during gastrulation, and the protein product was localized in the basolateral membrane of the enveloping epithelial cell layer and in the membrane of ingressing and migrating blastomers. Interestingly, both fhaqp3 transcripts and FhAqp3 polypeptides were downregulated in air-exposed embryos. These data demonstrate that killifish embryos respond adaptively to environmental desiccation by accelerating development and that embryos are able to transduce dehydration conditions into molecular responses. The reduced synthesis of FhAqp3 may be one of these mechanisms to regulate water and/or solute transport in the embryo.

  14. Suppression of the transcription factor MSX1 gene delays bovine preimplantation embryo development in vitro.

    PubMed

    Tesfaye, D; Regassa, A; Rings, F; Ghanem, N; Phatsara, C; Tholen, E; Herwig, R; Un, C; Schellander, K; Hoelker, M

    2010-05-01

    This study was conducted to investigate the effect of suppressing transcription factor gene MSX1 on the development of in vitro produced bovine oocytes and embryos, and identify its potential target genes regulated by this gene. Injection of long double-stranded RNA (LdsRNA) and small interfering RNA (siRNA) at germinal vesicle stage oocyte reduced MSX1 mRNA expression by 73 and 37% respectively at metaphase II stage compared with non-injected controls. Similarly, injection of the same anti-sense oligomers at zygote stage reduced MSX1 mRNA expression by 52 and 33% at 8-cell stage compared with non-injected controls. Protein expression was also reduced in LdsRNA- and siRNA-injected groups compared with non-injected controls at both stages. Blastocysts rates were 33, 28, 20 and 18% in non-injected control, scrambled RNA (scRNA), LdsRNA- and siRNA-injected groups respectively. Cleavage rates were also significantly reduced in Smartpool siRNA (SpsiRNA)-injected group (53.76%) compared with scRNA-injected group (57.76%) and non-injected control group (61%). Large-scale gene expression analysis showed that 135 genes were differentially regulated in SpsiRNA-injected group compared with non-injected controls, of which 54 and 81 were down- and up-regulated respectively due to suppression of MSX1. Additionally, sequence homology mapping and gene enrichment analysis with known human pathway information identified several functional modules that were affected due to suppression of MSX1. In conclusion, suppression of MSX1 affects oocyte maturation, embryo cleavage rate and the expression of several genes, suggesting its potential role in the development of bovine preimplantation embryos.

  15. Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development.

    PubMed

    Gonçalves, F S; Barretto, L S S; Arruda, R P; Perri, S H V; Mingoti, G Z

    2010-02-01

    Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 microm beta-mercaptoethanol (beta-ME) and 50 microm cysteamine (Cyst)] or a pro-oxidant (5 mm buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for beta-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.

  16. Poisonous plants: effects on embryo and fetal development.

    PubMed

    Panter, Kip E; Welch, Kevin D; Gardner, Dale R; Green, Benedict T

    2013-12-01

    Poisonous plant research in the United States began over 100 years ago as a result of livestock losses from toxic plants as settlers migrated westward with their flocks, herds, and families. Major losses were soon associated with poisonous plants, such as locoweeds, selenium accumulating plants, poison-hemlock, larkspurs, Veratrum, lupines, death camas, water hemlock, and others. Identification of plants associated with poisoning, chemistry of the plants, physiological effects, pathology, diagnosis, and prognosis, why animals eat the plants, and grazing management to mitigate losses became the overarching mission of the current Poisonous Plant Research Laboratory. Additionally, spin-off benefits resulting from the animal research have provided novel compounds, new techniques, and animal models to study human health conditions (biomedical research). The Poisonous Plant Research Laboratory has become an international leader of poisonous plant research as evidenced by the recent completion of the ninth International Symposium on Poisonous Plant Research held July 2013 in Hohhot, Inner Mongolia, China. In this article, we review plants that negatively impact embryo/fetal and neonatal growth and development, with emphasis on those plants that cause birth defects. Although this article focuses on the general aspects of selected groups of plants and their effects on the developing offspring, a companion paper in this volume reviews current understanding of the physiological, biochemical, and molecular mechanisms of toxicoses and teratogenesis.

  17. Carbon and nitrogen provisions alter the metabolic flux in developing soybean embryos

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybeans store approximately 40% of their biomass in the form of protein. Protein concentrations reflect the carbon and nitrogen levels received by the developing embryo. The relationship between carbon and nitrogen supply and seed composition during filling was examined through a series of embryo c...

  18. Polystyrene nanoparticles affect Xenopus laevis development

    NASA Astrophysics Data System (ADS)

    Tussellino, Margherita; Ronca, Raffaele; Formiggini, Fabio; Marco, Nadia De; Fusco, Sabato; Netti, Paolo Antonio; Carotenuto, Rosa

    2015-02-01

    Exposing living organisms to nanoparticulates is potentially hazardous, in particular when it takes place during embryogenesis. In this investigation, we have studied the effects of 50-nm-uncoated polystyrene nanoparticles (PSNPs) as a model to investigate the suitability of their possible future employments. We have used the standardized Frog Embryo Teratogenesis Assay- Xenopus test during the early stages of larval development of Xenopus laevis, and we have employed either contact exposure or microinjections. We found that the embryos mortality rate is dose dependent and that the survived embryos showed high percentage of malformations. They display disorders in pigmentation distribution, malformations of the head, gut and tail, edema in the anterior ventral region, and a shorter body length compared with sibling untreated embryos. Moreover, these embryos grow more slowly than the untreated embryos. Expressions of the mesoderm markers, bra (T-box Brachyury gene), myod1 (myogenic differentiation1), and of neural crest marker sox9 (sex SRY (determining region Y-box 9) transcription factor sox9), are modified. Confocal microscopy showed that the nanoparticles are localized in the cytoplasm, in the nucleus, and in the periphery of the digestive gut cells. Our data suggest that PSNPs are toxic and show a potential teratogenic effect for Xenopus larvae. We hypothesize that these effects may be due either to the amount of NPs that penetrate into the cells and/or to the "corona" effect caused by the interaction of PSNPs with cytoplasm components. The three endpoints of our study, i.e., mortality, malformations, and growth inhibition, suggest that the tests we used may be a powerful and flexible bioassay in evaluating pollutants in aquatic embryos.

  19. Modifications to the translational apparatus which affect the regulation of protein synthesis in sea urchin embryos

    SciTech Connect

    Scalise, F.W.

    1988-01-01

    Protein synthesis can be regulated at a number of cellular levels. I have examined how modifications to specific components of the protein synthetic machinery are involved in regulating the efficiency of initiation of translation during early sea urchin embryogenesis. It is demonstrated that Ca{sup 2+} concentrations exceeding 500 uM cause the inhibition of protein synthesis in cell-free translation lysates prepared from sea urchin embryos. Specific changes in the state of phosphorylation of at least 8 proteins occur during this Ca{sup 2+}-mediated repression of translation. Analysis of these proteins has indicated that, unlike mammalian systems, there is no detectable level of Ca{sup 2+}-dependent phosphorylation of the {alpha}subunit eIF-2. Two of the proteins which do become phosphorylated in response to Ca{sup 2+} are calmodulin and an isoelectric form of sea urchin eIF-4D. In addition, 2 proteins which share similarities with kinases involved in the regulation of protein synthesis in mammalian cells, also become phosphorylated. I have investigated the consequences of changes in eIF-4D during sea urchin embryogenesis because it has been proposed that a polyamine-mediated conversion of lysine to hypusine in this factor may enhance translational activity. It is demonstrated that ({sup 3}H) spermidine-derived radioactivity is incorporated into a number of proteins when sea urchin embryos are labeled in vivo, and that the pattern of individual proteins that become labeled changes over the course of the first 30 hr of development.

  20. Automated Microinjection of Recombinant BCL-X into Mouse Zygotes Enhances Embryo Development

    PubMed Central

    Gertsenstein, Marina; Perumalsamy, Alagammal; Lai, Ingrid; Chi, Maggie; Moley, Kelle H.; Greenblatt, Ellen; Jurisica, Igor; Casper, Robert F.; Sun, Yu; Jurisicova, Andrea

    2011-01-01

    Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality. PMID:21799744

  1. Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

    PubMed

    Liu, Xinyu; Fernandes, Roxanne; Gertsenstein, Marina; Perumalsamy, Alagammal; Lai, Ingrid; Chi, Maggie; Moley, Kelle H; Greenblatt, Ellen; Jurisica, Igor; Casper, Robert F; Sun, Yu; Jurisicova, Andrea

    2011-01-01

    Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.

  2. Growth and development of cultured carrot cells and embryos under spaceflight conditions

    NASA Technical Reports Server (NTRS)

    Krikorian, A. D.; Dutcher, F. R.; Quinn, C. E.; Steward, F. C.

    1981-01-01

    Morphogenetically competent proembryonic cells and well-developed somatic embryos of carrot at two levels of organization were exposed for 18.5 days to a hypogravity environment aboard the Soviet Biosatellite Cosmos 1129. It was confirmed that cultured totipotent cells of carrot can give rise to embryos with well-developed roots and minimally developed shoots. It was also shown that the space hypogravity environment could support the further growth of already-organized, later somatic embryonic stages and give rise to fully developed embryo-plantlets with roots and shoots.

  3. Integrated holographic system for all-optical manipulation of developing embryos

    PubMed Central

    Torres-Mapa, Maria Leilani; Antkowiak, Maciej; Cizmarova, Hana; Ferrier, David E. K.; Dholakia, Kishan; Gunn-Moore, Frank J.

    2011-01-01

    We demonstrate a system for the combined optical injection and trapping of developing embryos. A Ti:sapphire femtosecond laser in tandem with a spatial light modulator, is used to perform fast and accurate beam-steering and multiplexing. We show successful intracellular delivery of a range of impermeable molecules into individual blastomeres of the annelid Pomatoceros lamarckii embryo by optoinjection, even when the embryo is still enclosed in a chorion. We also demonstrate the ability of the femtosecond laser optoinjection to deliver materials into inner layers of cells in a well-developed embryo. By switching to the continuous wave mode of the Ti:sapphire laser, the same system can be employed to optically trap and orient the 60 μm sized P. lamarckii embryo whilst maintaining its viability. Hence, a complete all-optical manipulation platform is demonstrated paving the way towards single-cell genetic modification and cell lineage mapping in emerging developmental biology model species. PMID:21698019

  4. Induction of DNA-protein cross-links in developing embryos of the purple sea urchin, Strongylocentrotus purpuratus

    SciTech Connect

    Garman, G.D.; Cherr, G.N.; Anderson, S.L.

    1994-12-31

    Exposure to environmental agents during embryonic development may result in DNA-protein cross-linking (DPC), as has been demonstrated for mammalian cell lines. In the latter, formation of DPC`s upon exposure to a wide variety of agents, including some metals, has been observed. To determine whether DPCs could be detected in the sea urchin embryo during development, the authors adapted a mammalian cell assay utilizing potassium-SDS precipitation and a DNA fluorochrome to quantify relative amounts of free and protein-bound DNA. Sea urchin embryos exposed to a known DPC agent, nickel, through gastrulation exhibited a dose-dependent increase in DPCs, as well as an increase in developmental abnormalities. Morphological studies demonstrated that stage-specific exposure to Ni prior to gastrulation resulted in similar levels of abnormal pluteus larval development as compared to embryos exposed through gastrulation. Sea urchin embryos exhibit temporal differences in DNA transcription and gene expression during development, and these could be affected by modifications in DNA-protein interactions. Therefore, the authors are investigating the hypothesis that the similarities in morphological responses observed may relate to susceptibility of a critical stage of development.

  5. The Well-of-the-Well system: an efficient approach to improve embryo development.

    PubMed

    Vajta, Gábor; Korösi, Tamás; Du, Yutao; Nakata, Kumiko; Ieda, Shoko; Kuwayama, Masashige; Nagy, Zsolt Peter

    2008-07-01

    Transfer of human embryos at the blastocyst stage may offer considerable benefits including an increased implantation rate and a decreased risk of multiple pregnancies; however, blastocyst culture requires an efficient and reliable in-vitro embryo culture system. In this study, the effect of the Well-of-the-Well (WOW) system consisting of microwells formed on the bottom of the culture dish was tested in three mammalian species, including humans. The WOW system resulted in significant improvement when comparing the drops for culture of in-vitro-matured and parthenogenetically activated porcine oocytes, and in-vivo-derived mouse zygotes. In human embryos, using a sibling oocyte design, embryos cultured in WOW developed to the blastocyst stage in a significantly higher proportion than did embryos cultured traditionally (55% in WOW and 37% in conventional culture; P < 0.05). In a separate study, also in human, a total of 48 patients with a cumulative 214 unsuccessful previous IVF cycles were selected for the trials. In subsequent intracytoplasmic sperm injection cycles, oocytes/embryos were cultured individually in the WOW system or in microdrops. Transferable quality blastocyst development (48.9% of cultured zygotes) was observed in the WOW system. Ninety-four blastocysts transferred to 45 patients resulted in clinical pregnancy rates of 48.9%, including nine twin pregnancies, seven single pregnancies, five miscarriages and one ectopic pregnancy. The results indicate that the WOW system provides a promising alternative for microdrop culture of mammalian embryos, including human embryos.

  6. Melatonin effect on bovine embryo development in vitro in relation to oxygen concentration.

    PubMed

    Papis, Krzysztof; Poleszczuk, Olga; Wenta-Muchalska, Elzbieta; Modlinski, Jacek A

    2007-11-01

    Melatonin promotes mouse embryo development in vitro. An effect of melatonin on bovine embryo development is described here. Slaughterhouse derived oocytes were subjected to standard in vitro maturation and fertilization procedures. Presumptive zygotes were cultured for 2 days in CR1aaLA medium supplemented with melatonin (10(-4) m) or without melatonin (control). Culture was performed under two different gas atmospheres containing physiological (7%) or atmospheric (20%) oxygen concentrations (2x2 factorial analysis). After day 2, embryos from each treatment group developed to at least four-cell stage, were cultured without melatonin until day 10 at optimum 7% O2 atmosphere. Blastocyst formation rates of presumptive zygotes and of four-cell embryos were calculated for each group. Significant interactions between oxygen tension and the melatonin treatment were found. Out of four-cell embryos put into in vitro culture after initial incubation in medium containing melatonin, decreased blastocyst rate was observed in melatonin group (47.7%) compared with control (67.7%; P=0.0327) when lower oxygen concentration was applied. A beneficial effect of melatonin was observed in 20% O2: out of 61 embryos, 42 (68.9%) developed to the blastocyst stage after treatment in melatonin versus 32 of 63 (50.8%; P=0.0458) blastocysts that developed in control group. In conclusion, beneficial or harmful effects of melatonin on bovine embryo development in vitro were observed, depending on the oxygen tension during the treatment.

  7. Effects of diquat, an aquatic herbicide, on the development of mallard embryos

    USGS Publications Warehouse

    Sewalk, C.J.; Brewer, G.L.; Hoffman, D.J.

    2001-01-01

    Bipyridylium herbicides produce embryotoxic and teratogenic effects in dipteran, amphibian, avian, and mammalian organisms. Diquat dibromide, a bipyridylium compound, is commonly used as an aquatic herbicide. Mallard (Anas platyrhynchos) eggs were exposed to diquat by immersing the eggs for 10s in solutions of 0.88, 3.5, 7, 14, or 56 g/L on either the fourth or twenty-first day of incubation. Application of diquat on day 4 yielded an estimated LC50 of 19.5 g/L through 18 days of incubation, and 9.6 g/L through hatching. Body and organ weights, and bone lengths of hatchlings did not differ between control and treatment groups with the exception of a slight increase in brain weight in the 14 g/L group. Malformations in diquat-treated embryos included defects of the brain, eye, bill, limb, and pelvis; skeletal scoliosis; and incomplete ossification. Subcutaneous edema was also present. Significant manifestations of oxidative stress were apparent in hatchlings and included increased hepatic thiobarbituric acid reactive substances (TBARS) (lipid peroxidation) and decreased brain reduced glutathione (GSH). Brain protein-bound sulfhydryls (PBSH) increased. Diquat applied on day 21 of incubation yielded an estimated LC50 of 12.6 g/L through hatching. Exposure at this late stage of development did not produce deformities. Body and organ weights, and, bone lengths of hatchlings did not differ between control and treatment groups. Significant manifestations of oxidative stress in hatchlings included decreased brain GSH, increased oxidized glutathione (GSSG) and ratio of GSSG:GSH. This study suggests that concentrations of diquat commonly used for aquatic weed control, when based upon the expected dilution effect of average water depth of the application area, would probably have little impact on mallard embryos. However, concentrations applied above ground to weeds and cattails along the edge of waters and ditches could adversely affect the survival and development of mallard

  8. Toxicity and cardiac effects of carbaryl in early developing zebrafish (Danio rerio) embryos

    SciTech Connect

    Lin, C.C.; Hui, Michelle N.Y.; Cheng, S.H. E-mail: bhcheng@cityu.edu.hk

    2007-07-15

    Carbaryl, an acetylcholinesterase inhibitor, is known to be moderately toxic to adult zebrafish and has been reported to cause heart malformations and irregular heartbeat in medaka. We performed experiments to study the toxicity of carbaryl, specifically its effects on the heart, in early developing zebrafish embryos. LC50 and EC50 values for carbaryl at 28 h post-fertilization were 44.66 {mu}g/ml and 7.52 {mu}g/ml, respectively, and 10 {mu}g/ml carbaryl was used in subsequent experiments. After confirming acetylcholinesterase inhibition by carbaryl using an enzymatic method, we observed red blood cell accumulation, delayed hatching and pericardial edema, but not heart malformation as described in some previous reports. Our chronic exposure data also demonstrated carbaryl-induced bradycardia, which is a common effect of acetylcholinesterase inhibitors due to the accumulation of acetylcholine, in embryos from 1 day post-fertilization (dpf) to 5 dpf. The distance between the sinus venosus, the point where blood enters the atrium, and the bulbus arteriosus, the point where blood leaves the ventricle, indicated normal looping of the heart tube. Immunostaining of myosin heavy chains with the ventricle-specific antibody MF20 and the atrium-specific antibody S46 showed normal development of heart chambers. At the same time, acute exposure resulted in carbaryl-induced bradycardia. Heart rate dropped significantly after a 10-min exposure to 100 {mu}g/ml carbaryl but recovered when carbaryl was removed. The novel observation of carbaryl-induced bradycardia in 1- and 2-dpf embryos suggested that carbaryl affected cardiac function possibly through an alternative mechanism other than acetylcholinesterase inhibition such as inhibition of calcium ion channels, since acetylcholine receptors in zebrafish are not functional until 3 dpf. However, the exact nature of this mechanism is currently unknown, and thus further studies are required.

  9. Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

    NASA Technical Reports Server (NTRS)

    Vasilenko, A.; McDaniel, J. K.; Conger, B. V.

    2000-01-01

    Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell-division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division, divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all. Grant numbers: NAGW-3141, NAG10-0221.

  10. The association between microenvironmental reactive oxygen species and embryo development in assisted reproduction technology cycles.

    PubMed

    Lee, Tsung-Hsien; Lee, Maw-Sheng; Liu, Chung-Hsien; Tsao, Hui-Mei; Huang, Chun-Chia; Yang, Yu-Shih

    2012-07-01

    This study was designed to determine the relevance between the levels of reactive oxygen species (ROS) in microenvironment (follicular fluid or culture media) and the embryo development in IVF/ICSI cycles. A total of 466 follicles from 174 IVF/ICSI cycles were collected for this study. The ROS levels in monofollicular fluid and spent culture media were evaluated by chemiluminescence assay with luminol as a probe. The results demonstrated that it is in ICSI cycles that elevated ROS levels in follicular fluid were associated with day 3 poor embryo quality. The ROS levels in spent culture media were correlated with advanced degree of fragmentation. In addition, ROS levels in culture media, instead of in follicular fluid, were negatively correlated with implantation potential of embryos. The ROS levels in culture media may be viewed as an embryo metabolic marker and function as an adjuvant criterion for embryo selection.

  11. Toxicity of selenium to developing Xenopus laevis embryos.

    PubMed

    Browne, C L; Dumont, J N

    1979-07-01

    Se in the form of sodium selenite is toxic to Xenopus laevis embryos and tadpoles continuously exposed to concentrations above 1 ppm. Concentrations of 2 ppm and above result in severe developmental abnormalities and increased mortality. Uptake and loss of radioactive Se from water are rapid, but depuration is not complete indicating that some Se can remain bound by the organism. The facts that Se is toxic at low levels to Xenopus embryos and tadpoles, can cause developmental abnormalities, and accumulates in tissues suggest that increased release of Se compounds into the environment poses a potential threat to aquatic organisms.

  12. Telomere-interactive agents affect proliferation rates and induce chromosomal destabilization in sea urchin embryos.

    PubMed

    Izbicka, E; Nishioka, D; Marcell, V; Raymond, E; Davidson, K K; Lawrence, R A; Wheelhouse, R T; Hurley, L H; Wu, R S; Von Hoff, D D

    1999-08-01

    Cationic porphyrins, which interact with guanine quadruplex (G4) telomeric folds, inhibit telomerase activity in human tumor cells. In this study, we have further examined effects of porphyrins and other telomere- and telomerase-interactive agents on proliferation rates and chromosome stability in a novel in vivo model, developing sea urchin embryos. We studied two porphyrins: (i) TMPyP4, a potent telomerase inhibitor; and (ii) TMPyP2, an isomer of TMPyP4 and an inefficient telomerase inhibitor, azidothymine (AZT), the reverse transcriptase inhibitor, antisense phosphorothioate oligonucleotide to telomerase RNA (TAG6) and a control scrambled sequence (ODN). TMPyP4, AZT and TAG6 (but not TMPyP2 or ODN) decreased the rates of cell proliferation and increased the percentage of cells trapped in mitosis. Nuclear localization of TAG6, but not of ODN, was demonstrated with 5'-fluoresceinated analogs of TAG6 and ODN. Formation of elongated chromosomes incapable of separating in anaphase, induced by TMPyP4, AZT and TAG6, closely resembled phenotypes resulting from telomerase template mutation or dominant negative TRF2 allele. Our data suggest that G4-interactive agents exert their antiproliferative effects via chromosomal destabilization and warrant their further development as valuable anticancer tools.

  13. Micro-magnetic resonance imaging study of live quail embryos during embryonic development.

    PubMed

    Duce, Suzanne; Morrison, Fiona; Welten, Monique; Baggott, Glenn; Tickle, Cheryll

    2011-01-01

    Eggs containing live Japanese quail embryos were imaged using micro-magnetic resonance imaging (μMRI) at 24-h intervals from Day 0 to 8, the period during which the main body axis is being laid down and organogenesis is taking place. Considerable detail of non-embryonic structures such as the latebra was revealed at early stages but the embryo could only be visualized around Day 3. Three-dimensional (3D) changes in embryo length and volume were quantified and also changes in volume in the extra- and non-embryonic components. The embryo increased in length by 43% and nearly trebled in volume between Day 4 and Day 5. Although the amount of yolk remained fairly constant over the first 5 days, the amount of albumen decreases significantly and was replaced by extra-embryonic fluid (EEF). ¹H longitudinal (T₁) and transverse (T₂) relaxation times of different regions within the eggs were determined over the first 6 days of development. The T₂ measurements mirrored the changes in image intensity observed, which can be related to the aqueous protein concentrations. In addition, a comparison of the development of Day 0 to 3 quail embryos exposed to radiofrequency (rf) pulses, 7 T static magnetic fields and magnetic field gradients for an average of 7 h with the development of control embryos did not reveal any gross changes, thus confirming that μMRI is a suitable tool for following the development of live avian embryos over time from the earliest stages.

  14. Effect of a single dose of ethanol on developing skeletal muscle of chick embryos.

    PubMed

    Chaudhuri, Joydeep D

    2004-01-01

    Fetal alcohol syndrome is a condition occurring in some children of mothers who have consumed alcohol during pregnancy. Many of these affected children show retarded physical growth in the postnatal period despite adequate nutrition. On the basis of findings from studies with animals, it has been proposed that this is due to allometric retardation of growth of skeletal muscle, although the exact reasons for this are not known. The aim of the current study was to examine the structural changes in skeletal muscle in fetal alcohol syndrome in an attempt to understand the mechanisms of growth retardation in fetal alcohol syndrome. Chick embryos were exposed to single doses of 5%, 10%, and 15% ethanol, and the effects on the general growth and development, as well as on the skeletal muscle, of these chicks were studied. There was a significant retardation in crown rump length, head circumference, and body weight in ethanol-exposed chicks when these parameters were compared with findings for appropriate control groups. This retardation was associated with significant and proportionate reductions in the weights of skeletal muscles. Microscopic examination of skeletal muscle showed areas of neutrophil infiltration and necrosis, suggestive of muscle damage, in chicks exposed to 10% and 15% ethanol. Thus, findings of the current study demonstrate the direct toxic effects of a single dose of ethanol on developing embryos in general and skeletal muscle in particular. The pathologic changes seen in skeletal muscle could account for the failure in postnatal growth in fetal alcohol syndrome.

  15. Adaptive Transition of Aquaporin 5 Expression and Localization during Preimplantation Embryo Development by In Vitro Culture.

    PubMed

    Park, Jae-Won; Shin, Yun Kyung; Choen, Yong-Pil

    2014-09-01

    Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second midpreimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization.

  16. Adaptive Transition of Aquaporin 5 Expression and Localization during Preimplantation Embryo Development by In Vitro Culture

    PubMed Central

    Park, Jae-Won; Shin, Yun Kyung; Choen, Yong-Pil

    2014-01-01

    Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second midpreimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization. PMID:25949184

  17. Ex Ovo Model for Directly Visualizing Chick Embryo Development

    ERIC Educational Resources Information Center

    Dorrell, Michael I.; Marcacci, Michael; Bravo, Stephen; Kurz, Troy; Tremblay, Jacob; Rusing, Jack C.

    2012-01-01

    We describe a technique for removing and growing chick embryos in culture that utilizes relatively inexpensive materials and requires little space. It can be readily performed in class by university, high school, or junior high students, and teachers of any grade level should be able to set it up for their students. Students will be able to…

  18. CULTIVATION OF CHICKEN POX VIRUS IN DEVELOPING CHICK EMBRYOS

    DTIC Science & Technology

    The virus of chicken pox adapts readily and multiplies in the chorio- allantoic membranes of a chick embryo. A virus which has undergone several...passages on chorioallantoic membrane causes macroscopic changes in it. The chicken pox virus possesses a hemagglutinating capacity.

  19. Poisonous plants: Effects on embryo and fetal development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The impact of natural toxins from poisonous plants on the embryo, fetus, and neonate are dramatic and economically significant to livestock producers worldwide. In livestock, reproductive success is the single most important economic multiplier for livestock producers in the U.S. followed by carcas...

  20. Effects of hyaluronan, BSA, and serum on bovine embryo in vitro development, ultrastructure, and gene expression patterns.

    PubMed

    Palasz, A T; Rodriguez-Martinez, H; Beltran-Breña, P; Perez-Garnelo, S; Martinez, M F; Gutierrez-Adan, A; De la Fuente, J

    2006-12-01

    Effects of hyaluronan (HA), BSA, and FCS on in vitro development, ultrastructure, and mRNA transcription of four developmentally important genes: apoptosis (Bax), oxidative stress (SOX), growth factor (IGF-II), and cell-to-cell adhesion (Ecad) were examined. Two biological origin HA, Hylartil and Hyonate and one produced by fermentation (f-HA) MAP-5 were tested. Embryos were cultured in SOF medium with 0.4% BSA or with 0.4% BSA and 10% FCS. HA was added 96 hr post insemination (pi) to half of the embryos from each culture group. Embryo development was not affected by either HA preparation, however, hatching rates were higher in Hyalartil and MAP-5 than in control and Hyonate (P < 0.05). There was no effect of HA on number of blastocysts developed in SOF + BSA. However, more blastocysts developed in SOF + BSA + f-HA than in SOF + BSA + FCS or with BSA + FCS + f-HA. HA added to SOF + BSA, increased level of expression of epidermal growth factor (EGF)-II and decreased the levels of expression of BAX, SOX, and Ecad (P < 0.05). Presence of FCS increased the levels of SOX and decreased the level of IGF-II (P < 0.05) and the addition of f-HA to SOF containing FCS showed no effect on the level of transcription of any analyzed genes. The fine structure of embryos cultured with f-HA irrespective of protein sources used was clearly improved. In summary, f-HA added 96 hr pi to SOF supplemented with BSA but not FCS improved development, molecular composition and fine structure of bovine embryos.

  1. Gene expression patterns during somatic embryo development and germination in maize Hi II callus cultures.

    PubMed

    Che, Ping; Love, Tanzy M; Frame, Bronwyn R; Wang, Kan; Carriquiry, Alicia L; Howell, Stephen H

    2006-09-01

    Gene expression patterns were profiled during somatic embryogenesis in a regeneration-proficient maize hybrid line, Hi II, in an effort to identify genes that might be used as developmental markers or targets to optimize regeneration steps for recovering maize plants from tissue culture. Gene expression profiles were generated from embryogenic calli induced to undergo embryo maturation and germination. Over 1,000 genes in the 12,060 element arrays showed significant time variation during somatic embryo development. A substantial number of genes were downregulated during embryo maturation, largely histone and ribosomal protein genes, which may result from a slowdown in cell proliferation and growth during embryo maturation. The expression of these genes dramatically recovered at germination. Other genes up-regulated during embryo maturation included genes encoding hydrolytic enzymes (nucleases, glucosidases and proteases) and a few storage genes (an alpha-zein and caleosin), which are good candidates for developmental marker genes. Germination is accompanied by the up-regulation of a number of stress response and membrane transporter genes, and, as expected, greening is associated with the up-regulation of many genes encoding photosynthetic and chloroplast components. Thus, some, but not all genes typically associated with zygotic embryogenesis are significantly up or down-regulated during somatic embryogenesis in Hi II maize line regeneration. Although many genes varied in expression throughout somatic embryo development in this study, no statistically significant gene expression changes were detected between total embryogenic callus and callus enriched for transition stage somatic embryos.

  2. Impact of maternal malnutrition during the periconceptional period on mammalian preimplantation embryo development.

    PubMed

    Velazquez, M A

    2015-04-01

    During episodes of undernutrition and overnutrition the mammalian preimplantation embryo undergoes molecular and metabolic adaptations to cope with nutrient deficits or excesses. Maternal adaptations also take place to keep a nutritional microenvironment favorable for oocyte development and embryo formation. This maternal-embryo communication takes place via several nutritional mediators. Although adaptive responses to malnutrition by both the mother and the embryo may ensure blastocyst formation, the resultant quality of the embryo can be compromised, leading to early pregnancy failure. Still, studies have shown that, although early embryonic mortality can be induced during malnutrition, the preimplantation embryo possesses an enormous plasticity that allows it to implant and achieve a full-term pregnancy under nutritional stress, even in extreme cases of malnutrition. This developmental strategy, however, may come with a price, as shown by the adverse developmental programming induced by even subtle nutritional challenges exerted exclusively during folliculogenesis and the preimplantation period, resulting in offspring with a higher risk of developing deleterious phenotypes in adulthood. Overall, current evidence indicates that malnutrition during the periconceptional period can induce cellular and molecular alterations in preimplantation embryos with repercussions for fertility and postnatal health.

  3. The differential transcription network between embryo and endosperm in the early developing maize seed.

    PubMed

    Lu, Xiaoduo; Chen, Dijun; Shu, Defeng; Zhang, Zhao; Wang, Weixuan; Klukas, Christian; Chen, Ling-ling; Fan, Yunliu; Chen, Ming; Zhang, Chunyi

    2013-05-01

    Transcriptome analysis of early-developing maize (Zea mays) seed was conducted using Illumina sequencing. We mapped 11,074,508 and 11,495,788 paired-end reads from endosperm and embryo, respectively, at 9 d after pollination to define gene structure and alternative splicing events as well as transcriptional regulators of gene expression to quantify transcript abundance in both embryo and endosperm. We identified a large number of novel transcribed regions that did not fall within maize annotated regions, and many of the novel transcribed regions were tissue-specifically expressed. We found that 50.7% (8,556 of 16,878) of multiexonic genes were alternatively spliced, and some transcript isoforms were specifically expressed either in endosperm or in embryo. In addition, a total of 46 trans-splicing events, with nine intrachromosomal events and 37 interchromosomal events, were found in our data set. Many metabolic activities were specifically assigned to endosperm and embryo, such as starch biosynthesis in endosperm and lipid biosynthesis in embryo. Finally, a number of transcription factors and imprinting genes were found to be specifically expressed in embryo or endosperm. This data set will aid in understanding how embryo/endosperm development in maize is differentially regulated.

  4. Hybrid embryos of Vicia faba develop enhanced sink strength, which is established during early development.

    PubMed

    Meitzel, Tobias; Radchuk, Ruslana; Nunes-Nesi, Adriano; Fernie, Alisdair R; Link, Wolfgang; Weschke, Winfriede; Weber, Hans

    2011-02-01

    Selfed and crossed seeds of two homozygous Vicia faba lines served as models for the analysis of the physiological and molecular mechanisms underlying embryo heterosis. Profiles of transcripts, metabolites and seed contents of developing embryos were analysed to compare the means of reciprocally crossed and selfed seeds growing on the same mother plants. The mean weight of mature hybrid seeds was demonstrably higher, revealing mid-parent heterosis. Hybrid embryos exhibited a prolonged early phase of development and delayed onset of storage activity. Accordingly, transcript profiling indicates stimulation of cell proliferation, an effect, which is potentially mediated by activation of auxin functions within a framework of growth-related transcription factors. At the transcript level, activated cell proliferation increased assimilate uptake activity and thereby seed sink strength. This situation might finally lead to the increased size of the hybrid seeds. We conclude that hybrid seeds are characterised by accelerated growth during early development, which increases storage capacity and leads to higher metabolic fluxes. These needs are, at least partially, met by increased assimilate uptake capacity. The stimulated growth of hybrid seeds shifted metabolite profiles and potentially depleted available pools. Such metabolic shifts are most likely secondary effects resulting from the higher storage capacity of hybrid seeds, a heterotic feature, which is itself established very early in seed development.

  5. Cleavage kinetics analysis of human embryos predicts development to blastocyst and implantation.

    PubMed

    Dal Canto, Mariabeatrice; Coticchio, Giovanni; Mignini Renzini, Mario; De Ponti, Elena; Novara, Paola Vittoria; Brambillasca, Fausta; Comi, Ruggero; Fadini, Rubens

    2012-11-01

    Cleavage kinetics of human embryos is indicative of ability to develop to blastocyst and implant. Recent advances in time-lapse microscopy have opened new and important research opportunities. In this study involving infertile couples requiring standard IVF/intracytoplasmic sperm injection treatment, zygotes were cultured by integrated embryo-culture time-lapse microscopy to analyse cleavage times from the 2- to the 8-cell stages in relation to the ability to develop to blastocyst, expand and implant. In comparison to embryos arresting after 8-cell stage, times of cleavage to 7- and 8-cell stages of embryos developing to blastocyst were shorter (56.5 ± 8.1 versus 58.8 ± 10.4h, P=0.03 and 61.0 ± 9.4 versus 65.2 ± 13.0 h, P=0.0008, respectively). In embryos developing to blastocyst, absence of blastocoele expansion on day 5 was associated with progressive cleavage delay. Implanting embryos developed to 8-cell stage in a shorter period compared with those unable to implant (54.9 ± 5.2 and 58.0 ± 7.2h, respectively, P=0.035). In conclusion, cleavage from 2- to 8-cell stage occurs progressively earlier in embryos with the ability to develop to blastocyst, expand and implant. Conventional observation times on days 2 and 3 are inappropriate for accurate embryo evaluation. The speed at which human embryos cleave is known to be suggestive of their ability to develop in vitro to the blastocyst stage and implant after transfer into the uterus. Recent advances in time-lapse microscopy, which allows acquisition of images every 15-20 min, have opened new and important research opportunities. In a retrospective study involving infertile couples requiring standard IVF or intracytoplasmic sperm injection treatment, fertilized oocytes were cultured by an integrated embryo-culture time-lapse microscopy system in order to perform an analysis of cleavage times from the 2- to the 8-cell stage in relation to the ability to develop to blastocyst, expand and implant. In comparison to

  6. High frequency ultrasound imaging of the growth and development of the normal chick embryo.

    PubMed

    Schellpfeffer, Michael A; Bolender, David L; Kolesari, Gary L

    2007-05-01

    The purpose of this study is to delineate with high frequency ultrasound imaging the normal growth and development of the chick embryo throughout its incubation period. White Leghorn chick embryos were imaged through an opening in the egg air cell from incubation day 0-19 (Hamburger & Hamilton stage 1-45) using a 13 MHz clinical high frequency linear small parts transducer. Multiple anatomic growth parameters were measured. Normal growth was confirmed with Hamburger and Hamilton staging. A timeline was constructed showing when each anatomic growth parameter could be visualized. Means and standard deviations of each parameter were plotted against incubation days studied to create nomograms and numerical tables of normal growth and development of the chick embryo. With this set of data, abnormal growth and development of the chick embryo can now be assessed.

  7. [Effect of krypton-containing gas mixture on Japanese quail embryo development].

    PubMed

    Kussmaul', A R; Gur'eva, T S; Dadasheva, O A; Pavlov, N B; Pavlov, B N

    2008-01-01

    Investigated were effects of gas mixture with up to 3.0 kgs/cm2 of krypton on the embryonic development of domesticated Japanese quail (Coturnix coturnix japonica dom.). Results demonstrated absence of a serious krypton effect on Japanese quail embryos. Development of embryos proceeded in due course; morphometrically the experimental embryos were essentially similar to controls. It should be noted that despite exposure to acute hypoxic hypoxia during the initial 12 hours of development in the krypton-containing gas mixture, viability of quail embryos was high enough which can be ascribed to the krypton protective action. Besides, an additional experiment showed that krypton partial pressure of 5-5.5 kgs/cm2 produces the narcotic effect on adult Japanese quails.

  8. The development and expression of pluripotency genes in embryos derived from nuclear transfer and in vitro fertilization.

    PubMed

    Ma, Li-Bing; He, Xiao-Ying; Wang, Feng-Mei; Cao, Jun-Wei; Cheng, Teng

    2014-11-01

    Somatic cell nuclear transfer can be used to produce embryonic stem (ES) cells, cloned animals, and can even increase the population size of endangered animals. However, the application of this technique is limited by the low developmental rate of cloned embryos, a situation that may result from abnormal expression of some zygotic genes. In this study, sheep-sheep intra-species cloned embryos, goat-sheep inter-species cloned embryos, or sheep in vitro fertilized embryos were constructed and cultured in vitro and the developmental ability and expression of three pluripotency genes, SSEA-1, Nanog and Oct4, were examined. The results showed firstly that the developmental ability of in vitro fertilized embryos was significantly higher than that of cloned embryos. In addition, the percentage of intra-species cloned embryos that developed to morula or blastocyst stages was also significantly higher than that of the inter-species cloned embryos. Secondly, all three types of embryos expressed SSEA-1 at the 8-cell and morula stages. At the 8-cell stage, a higher percentage of in vitro fertilized embryos expressed SSEA-1 than occurred for cloned embryos. However, at the morula stage, all detected embryos could express SSEA-1. Thirdly, the three types of embryos expressed Oct4 mRNA at the morula and blastocyst stages, and embryos at the blastocyst stage expressed Nanog mRNA. The rate of expression of Oct4 and Nanog mRNA at these developmental stages was higher in in vitro fertilized embryos than in cloned embryos. These results indicated that, during early development, the failure to reactivate some pluripotency genes maybe is a reason for the low cloning efficiency found with cloned embryos.

  9. Soybean roots retain the seed urease isozyme synthesized during embryo development. [Glycine max (L. ) Merr

    SciTech Connect

    Torisky, R.S.; Polacco, J.C. )

    1990-10-01

    Roots of young soybean (Glycine max (L.) Merr.) plants (up to 25 days old) contain two distinct urease isozymes, which are separable by hydroxyapatite chromatography. These two urease species (URE1 and URE2) differ in: (a) electrophoretic mobility in native gels, (b) pH dependence, and (c) recognition by a monoclonal antibody specific for the seed (embryo-specific) urease. By these parameters root URE1 urease is similar to the abundant embryo-specific urease isozyme, while root URE2 resembles the ubiquitous urease which has previously been found in all soybean tissues examined (leaf, embryo, seed coat, and cultured cells). The embryo-specific and ubiquitous urease isozymes are products of the Eu1 and Eu4 structural genes, respectively. Roots of the eu1-sun/eu1-sun genotype, which lacks the embryo-specific urease (i.e. seed urease-null), contain no URE1 urease activity. Roots of eu4/eu4, which lacks ubiquitous urease, lack the URE2 (leaflike) urease activity. From these genetic and biochemical criteria, then, we conclude that URE1 and URE2 are the embryo-specific and ubiquitous ureases, respectively. Adventitious roots generated from cuttings of any urease genotype lack URE1 activity. In seedling roots the seedlike (URE1) activity declines during development. Roots of 3-week-old plants contain 5% of the total URE1 activity of the radicle of 4-day-old seedlings, which, in turn, has approximately the same urease activity level as the dormant embryonic axis. The embryo-specific urease incorporates label from ({sup 35}S)methionine during embryo development but not during germination, indicating that there is no de novo synthesis of the embryo-specific (URE1) urease in the germinating root.

  10. Histone deacetylase inhibitor improves the development and acetylation levels of cat-cow interspecies cloned embryos.

    PubMed

    Wittayarat, Manita; Sato, Yoko; Do, Lanh Thi Kim; Morita, Yasuhiro; Chatdarong, Kaywalee; Techakumphu, Mongkol; Taniguchi, Masayasu; Otoi, Takeshige

    2013-08-01

    Abnormal epigenetic reprogramming, such as histone acetylation, might cause low efficiency of interspecies somatic cell nuclear transfer (iSCNT). This study was conducted to evaluate the effects of trichostatin A (TSA) on the developmental competence and histone acetylation of iSCNT embryos reconstructed from cat somatic cells and bovine cytoplasm. The iSCNT cat and parthenogenetic bovine embryos were treated with various concentrations of TSA (0, 25, 50, or 100 nM) for 24 h, respectively, following fusion and activation. Treatment with 50 nM TSA produced significantly higher rates of cleavage and blastocyst formation (84.3% and 4.6%, respectively) of iSCNT embryos than the rates of non-TSA-treated iSCNT embryos (63.8% and 0%, respectively). Similarly, the treatment of 50 nM TSA increased the blastocyst formation rate of parthenogenetic bovine embryos. The acetylation levels of histone H3 lysine 9 (H3K9) in the iSCNT embryos with the treatment of 50 nM TSA were similar to those of in vitro-fertilized embryos and significantly higher (p<0.05) than those of non-TSA-treated iSCNT embryos (control), irrespective of the embryonic development stage (two-cell, four-cell, and eight-cell stages). These results indicated that the treatment of 50 nM TSA postfusion was beneficial for development to the blastocyst stage of iSCNT cat embryos and correlated with the increasing levels of acetylation at H3K9.

  11. BPA exposure during in vitro oocyte maturation results in dose-dependent alterations to embryo development rates, apoptosis rate, sex ratio and gene expression.

    PubMed

    Ferris, Jacqueline; Mahboubi, Kiana; MacLusky, Neil; King, W Allan; Favetta, Laura A

    2016-01-01

    Alterations in the oocyte's environment can negatively affect embryo development. Oocyte quality, which can determine embryonic viability, is easily perturbed, thus factors affecting normal oocyte maturation are a concern. Bisphenol A (BPA) is an endocrine disrupting chemical that elicits a variety of reproductive effects. BPA has previously been found to disrupt meiosis, however the embryonic effects in mammals are not well documented. Here, bovine oocytes were matured in vitro with and without BPA treatment. Resulting embryos exhibited decreased embryonic development rates, increased apoptosis, and a skewed sex ratio. Gene expression in blastocysts was not altered, whereas treatment with 15ng/mL BPA resulted in increased expression of several of the genes studies, however this increase was largely due to a vehicle effect. BPA exposure during oocyte maturation in vitro can therefore, in a dose-dependent way, decrease oocyte and embryo quality and developmental potential and affect gene expression of developmentally important transcripts.

  12. Presumed monozygotic twins develop following transfer of an in vitro-produced equine embryo

    PubMed Central

    ROBERTS, Melissa Ann; LONDON, Kelly; CAMPOS-CHILLÓN, Lino Fernando; ALTERMATT, Joy Lynn

    2015-01-01

    ABSTRACT An equine embryo produced by intracytoplasmic sperm injection (ICSI) was trans-cervically transferred to a recipient mare and pregnancy was confirmed via ultrasound examination on days 11, 12 and 15. On days 20 and 22, a single embryonic proper with a heartbeat was observed. On day 29, two embryos proper appeared during ultrasound examination, each possessing a heartbeat. Subsequent examinations on days 35 and 39 revealed continued viability and development of both embryos proper. On day 49, demise of both fetuses was present. Although no DNA analysis or post-partum examinations were performed, it is presumed that the fetuses were monozygotic twins based on membrane classification by ultrasound imaging as well as development occurring after the transfer of a single in vitro-produced embryo. PMID:26435682

  13. In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

    PubMed

    Kim, Dong-Hoon; No, Jin-Gu; Choi, Mi-Kyung; Yeom, Dong-Hyeon; Kim, Dong-Kyo; Yang, Byoung-Chul; Yoo, Jae Gyu; Kim, Min Kyu; Kim, Hong-Tea

    2015-01-01

    The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.

  14. Rapid assimilation of yolk enhances growth and development of lizard embryos from a cold environment.

    PubMed

    Storm, Melissa A; Angilletta, Michael J

    2007-10-01

    Selection for rapid growth and development in cold environments results in a geographic pattern known as countergradient variation. The eastern fence lizard, Sceloporus undulatus, exhibits countergradient variation in embryonic growth and development along latitudinal clines. To identify the proximate causes of countergradient variation, we compared the energy budgets of embryos from a cold environment (Virginia) and a warm environment (South Carolina) during development at a realistic thermal cycle. The difference in mean egg size between populations was controlled by removing yolk from large eggs and performing a sham manipulation on other eggs. Respiration was measured every 4 days throughout 48 days of incubation. After this period, eggs were dissected and the energy contents of embryos and yolk were determined by calorimetry. As expected from previous experiments, embryos from Virginia reached a more advanced stage of development and deposited more energy within tissues than embryos from South Carolina. The greater absorption of yolk by embryos from Virginia was associated with a higher rate of respiration. Assimilation of yolk by rapidly growing embryos could reduce growth or survival after hatching. Such costs might explain the maintenance of countergradient variation in S. undulatus.

  15. CLE19 expressed in the embryo regulates both cotyledon establishment and endosperm development in Arabidopsis

    PubMed Central

    Xu, Ting-Ting; Ren, Shi-Chao; Song, Xiu-Fen; Liu, Chun-Ming

    2015-01-01

    Embryo and endosperm development are two well co-ordinated developmental processes in seed formation; however, signals involved in embryo and endosperm interactions remain poorly understood. It has been shown before that CLAVATA3/ESR-RELATED 19 (CLE19) peptide is able to trigger root meristem consumption in a CLV2-dependent manner. In this study, the role of CLE19 in Arabidopsis seed development was explored using antagonistic peptide technology. CLE19 is expressed in the epidermal layers of the cotyledon primordia, hypocotyl, and root cap in the embryo. Transgenic plants carrying an antagonistic CLE19 G6T construct expressed under the control of CLE19 regulatory elements exhibited a dominant seed abortion phenotype, with defective cotyledon establishment in embryos and delayed nuclear proliferation and cellularization in endosperms. Ectopic expression of CLE19 G6T in Arabidopsis under the control of an endosperm-specific ALE1 promoter led to a similar defect in cotyledon establishment in embryos but without an evident effect on endosperm development. We therefore propose that CLE19 may act as a mobile peptide co-ordinating embryo and endosperm development. PMID:26071532

  16. Plasticity in Dnmt3L-dependent and -independent modes of de novo methylation in the developing mouse embryo.

    PubMed

    Guenatri, Mounia; Duffié, Rachel; Iranzo, Julian; Fauque, Patricia; Bourc'his, Déborah

    2013-02-01

    A stimulatory DNA methyltransferase co-factor, Dnmt3L, has evolved in mammals to assist the process of de novo methylation, as genetically demonstrated in the germline. The function of Dnmt3L in the early embryo remains unresolved. By combining developmental and genetic approaches, we find that mouse embryos begin development with a maternal store of Dnmt3L, which is rapidly degraded and does not participate in embryonic de novo methylation. A zygotic-specific promoter of Dnmt3l is activated following gametic methylation loss and the potential recruitment of pluripotency factors just before implantation. Importantly, we find that zygotic Dnmt3L deficiency slows down the rate of de novo methylation in the embryo by affecting methylation density at some, but not all, genomic sequences. Dnmt3L is not strictly required, however, as methylation patterns are eventually established in its absence, in the context of increased Dnmt3A protein availability. This study proves that the postimplantation embryo is more plastic than the germline in terms of DNA methylation mechanistic choices and, importantly, that de novo methylation can be achieved in vivo without Dnmt3L.

  17. Rethinking In Vitro Embryo Culture: New Developments in Culture Platforms and Potential to Improve Assisted Reproductive Technologies1

    PubMed Central

    Smith, Gary D.; Takayama, Shuichi; Swain, Jason E.

    2011-01-01

    ABSTRACT The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future. PMID:21998170

  18. Onset of Buccal Pumping in Catshark Embryos: How Breathing Develops in the Egg Capsule

    PubMed Central

    Tomita, Taketeru; Nakamura, Masaru; Sato, Keiichi; Takaoka, Hiroko; Toda, Minoru; Kawauchi, Junro; Nakaya, Kazuhiro

    2014-01-01

    Respiration in fishes involves buccal pumping, which is characterized by the generation of nearly continuous water flow over the gills because of the rhythmic expansion/compression of the pharyngeal cavity. This mechanism is achieved by the functions of the vascular, skeletal, and muscular systems. However, the process by which the embryo establishes the mechanism remains a mystery. Morphological and kinematical observations on captive cloudy catsharks, Scyliorhinus torazame, have suggested that the embryo starts buccal pumping just before the respiratory slits open on the egg capsule. During the pre-opening period, the embryo acquires oxygen mainly via the external gill filaments. After slit opening, respiration of the embryo involves buccal pumping to pass water over the “internal gills.” The onset of buccal pumping accompanies four morphological changes: (1) regression of the external gill filaments, (2) development of blood vessels within the “internal gills,” (3) completion of the development of hyoid skeletal and muscular elements, and (4) development of the oral valve. A previous study showed that buccal pumping allows the embryo to actively regulate oxygen intake by changing the pumping frequency. Thus, establishment of buccal pumping in the egg capsule is probably important for embryo survival in the unstable oxygen environment of the egg capsule after slit opening. PMID:25329313

  19. Onset of buccal pumping in catshark embryos: how breathing develops in the egg capsule.

    PubMed

    Tomita, Taketeru; Nakamura, Masaru; Sato, Keiichi; Takaoka, Hiroko; Toda, Minoru; Kawauchi, Junro; Nakaya, Kazuhiro

    2014-01-01

    Respiration in fishes involves buccal pumping, which is characterized by the generation of nearly continuous water flow over the gills because of the rhythmic expansion/compression of the pharyngeal cavity. This mechanism is achieved by the functions of the vascular, skeletal, and muscular systems. However, the process by which the embryo establishes the mechanism remains a mystery. Morphological and kinematical observations on captive cloudy catsharks, Scyliorhinus torazame, have suggested that the embryo starts buccal pumping just before the respiratory slits open on the egg capsule. During the pre-opening period, the embryo acquires oxygen mainly via the external gill filaments. After slit opening, respiration of the embryo involves buccal pumping to pass water over the "internal gills." The onset of buccal pumping accompanies four morphological changes: (1) regression of the external gill filaments, (2) development of blood vessels within the "internal gills," (3) completion of the development of hyoid skeletal and muscular elements, and (4) development of the oral valve. A previous study showed that buccal pumping allows the embryo to actively regulate oxygen intake by changing the pumping frequency. Thus, establishment of buccal pumping in the egg capsule is probably important for embryo survival in the unstable oxygen environment of the egg capsule after slit opening.

  20. Osmotic measurements in whole megagametophytes and embryos of loblolly pine (Pinus taeda) during seed development.

    PubMed

    Pullman, Gerald S; Johnson, Shannon

    2009-06-01

    Water potential (Psi) and osmotic potential (Psis) were measured weekly through the sequence of seed development in megagametophytes of loblolly pine (Pinus taeda L.). A Wescor 5500XRS vapor pressure osmometer, modified with a cycle hold switch, was used to measure Psi for whole megagametophytes containing embryos. The Psi measurements for megagametophytes with embryos removed were also attempted but readings were distorted due to cell lysates from the cut surfaces. Six seasonal sets of megagametophyte Psi profiles were generated. Megagametophytes from most of the trees examined showed a consistent Psi pattern: low measurements of -1.0 to -0.75 MPa during early embryo development in late June to early July when embryo Stages 1-2 occur; an increase for one to several weeks to levels of -0.5 to -0.75 MPa, beginning at Stages 3-5 when apical dome formation occurs; followed by a steady drop from -0.85 to -1.7 to -2.0 MPa from Stage 6 onward from late August until just before cone seed release. The Psis was measured for supernatant from centrifuged frozen-thawed megagametophyte tissue (embryos removed). Megagametophyte Psis profiles were similar for seeds analyzed from two trees and resembled Psi observations starting low, rising around Stages 4-7 and then undergoing a major reduction indicating a strong solute accumulation beginning at Stages 7-9.1. Somatic embryos stop growth prematurely in vitro at Stages 8-9.1. The major change in the accumulation of megagametophyte solutes at Stages 8-9.1 correlates with the halt in somatic embryo maturation and suggests that identifying, quantifying and using the major natural soluble compounds that accumulate during mid- to late-stage seed development may be important to improve conifer somatic embryo maturation.

  1. Time-lapse microscopy and image analysis in basic and clinical embryo development research.

    PubMed

    Wong, C; Chen, A A; Behr, B; Shen, S

    2013-02-01

    Mammalian preimplantation embryo development is a complex process in which the exact timing and sequence of events are as essential as the accurate execution of the events themselves. Time-lapse microscopy (TLM) is an ideal tool to study this process since the ability to capture images over time provides a combination of morphological, dynamic and quantitative information about developmental events. Here, we systematically review the application of TLM in basic and clinical embryo research. We identified all relevant preimplantation embryo TLM studies published in English up to May 2012 using PubMed and Google Scholar. We then analysed the technical challenges involved in embryo TLM studies and how these challenges may be overcome with technological innovations. Finally, we reviewed the different types of TLM embryo studies, with a special focus on how TLM can benefit clinical assisted reproduction. Although new parameters predictive of embryo development potential may be discovered and used clinically to potentially increase the success rate of IVF, adopting TLM to routine clinical practice will require innovations in both optics and image analysis. Combined with such innovations, TLM may provide embryologists and clinicians with an important tool for making critical decisions in assisted reproduction. In this review, we perform a literature search of all published early embryo development studies that used time-lapse microscopy (TLM). From the literature, we discuss the benefits of TLM over traditional time-point analysis, as well as the technical difficulties and solutions involved in implementing TLM for embryo studies. We further discuss research that has successfully derived non-invasive markers that may increase the success rate of assisted reproductive technologies, primarily IVF. Most notably, we extend our discussion to highlight important considerations for the practical use of TLM in research and clinical settings.

  2. Development of a bovine luteal cell in vitro culture system suitable for co-culture with early embryos.

    PubMed

    Batista, M; Torres, A; Diniz, P; Mateus, L; Lopes-da-Costa, L

    2012-10-01

    The cross talk between the corpus luteum (CL) and the early embryo, potentially relevant to pregnancy establishment, is difficult to evaluate in the in vivo bovine model. In vitro co-culture of bovine luteal cells and early embryos (days 2-8 post in vitro fertilization) may allow the deciphering of this poorly understood cross talk. However, early embryos and somatic cells require different in vitro culture conditions. The objective of this study was to develop a bovine luteal cell in vitro culture system suitable for co-culture with early embryos in order to evaluate their putative steroidogenic and prostanoid interactions. The corpora lutea of the different stages of the estrous cycle (early, mid, and late) were recovered postmortem and enriched luteal cell populations were obtained. In experiments 1 and 2, the effects of CL stage, culture medium (TCM, DMEM-F12, or SOF), serum concentration (5 or 10%), atmosphere oxygen tension (5 or 20%), and refreshment of the medium on the ability of luteal cells to produce progesterone (P(4)) were evaluated. The production of P(4) was significantly increased in early CL cultures, and luteal cells adapted well to simple media (SOF), low serum concentrations (5%), and oxygen tensions (5%). In experiment 3, previous luteal cell cryopreservation did not affect the production of P(4), PGF(2α), and PGE(2) compared to fresh cell cultures. This enables the use of pools of frozen-thawed cells to decrease the variation in cell function associated with primary cell cultures. In experiment 4, mineral oil overlaying culture wells resulted in a 50-fold decrease of the P(4) quantified in the medium, but had no effect on PGF(2α) and PGE(2) quantification. In conclusion, a luteal cell in vitro culture system suitable for the 5-d-long co-culture with early embryos was developed.

  3. An environmentally relevant organochlorine mixture impairs sperm function and embryo development in the porcine model.

    PubMed

    Campagna, Céline; Guillemette, Christine; Paradis, René; Sirard, Marc-André; Ayotte, Pierre; Bailey, Janice L

    2002-07-01

    We evaluated the effects of an environmentally relevant mixture of more than 15 organochlorines on the development of pig oocytes and sperm during in vitro fertilization (IVF). Oocytes were cocultured with sperm in IVF medium containing increasing concentrations of an organochlorine mixture, similar to that found in women of highly exposed populations. Exposure to the organochlorine mixture diminished oocyte penetration rates and polyspermy in a linear manner. The mixture did not affect rates of cleavage nor development to multicell embryos. However, rates of development to the blastocyst stage were lower at the highest concentration at which oocyte penetration was observed. The same experiment was performed using oocytes that were preexposed during in vitro maturation. This greater exposure to the mixture also reduced penetration in a dose-response manner and affected polyspermy. Frozen-thawed pig sperm were also cultured in IVF medium containing the same organochlorine concentrations. Sperm motility parameters were immediately reduced in a dose-dependent manner by the organochlorines, followed by diminished viability 2 h later. From these results, it appears that reduced sperm quality would account for decreases in fertilization, polyspermy, and blastocyst formation. These results suggest that exposing porcine oocytes and sperm to an environmentally pertinent organochlorine mixture in vitro disrupts the oocyte block to polyspermy, sperm fertility, and further embryonic development, and supports recent concerns that such pollutants harm reproductive health in humans and other species.

  4. Effects of fluoride on development and growth of Rana chensinensis embryos and larvae.

    PubMed

    Chai, Lihong; Dong, Suiming; Zhao, Hongfeng; Deng, Hongzhang; Wang, Hongyuan

    2016-04-01

    The present study examined the adverse effects of fluoride exposure on embryos and larvae of Rana chensinensis. Survival, morphological abnormalities, growth and development, time to metamorphosis and size at metamorphic climax of R. chensinensis were examined. Our results showed that embryos malformation occurred in all fluoride treatments. Morphological abnormalities of embryos are characterized by axial flexures, the extrusion of fin axis, edema, and ruffled dorsal and ventral fin. Additionally, 4.1mg F(-)/L and above could significantly inhibit embryos growth and development. On day 15, total length and weight of tadpole were significantly lower in 19.6 and 42.4 mg F(-)/L treatments compared to control. However, significant reductions in total length and weight were observed only at 42.4 mg F(-)/L on day 30. Moreover, significant metamorphic delay and decrease in the size at metamorphic climax were found in larvae exposed to 42.4 mg F(-)/L. Taken together, embryos of R. chensinensis are more vulnerable to fluoride exposure than their tadpoles. Our results suggested that the presence of high concentrations fluoride might increase mortality risk and a reduction in juvenile recruitment in the field by increasing embryos malformation, delaying metamorphosis and decreasing size at metamorphosis.

  5. Low cost labeling with highlighter ink efficiently visualizes developing blood vessels in avian and mouse embryos.

    PubMed

    Takase, Yuta; Tadokoro, Ryosuke; Takahashi, Yoshiko

    2013-12-01

    To understand how blood vessels form to establish the intricate network during vertebrate development, it is helpful if one can visualize the vasculature in embryos. We here describe a novel labeling method using highlighter ink, easily obtained in stationery stores with a low cost, to visualize embryo-wide vasculatures in avian and mice. We tested 50 different highlighters for fluorescent microscopy with filter sets equipped in a standard fluorescent microscope. The yellow and violet inks yielded fluorescent signals specifically detected by the filters used for green fluorescent protein (GFP) and red fluorescent protein (RFP) detections, respectively. When the ink solution was infused into chicken/quail and mouse embryos, vasculatures including large vessels and capillaries were labeled both in living and fixed embryos. Ink-infused embryos were further subjected to histological sections, and double stained with antibodies including QH-1 (quail), α smooth muscle actin (αSMA), and PECAM-1 (mouse), revealing that the endothelial cells were specifically labeled by the infused highlighter ink. Highlighter-labeled signals were detected with a resolution comparable to or higher than signals of fluorescein isothiocyanate (FITC)-lectin and Rhodamine-dextran, conventionally used for angiography. Furthermore, macroconfocal microscopic analyses with ink-infused embryos visualized fine vascular structures of both embryo proper and extra-embryonic plexus in a Z-stack image of 2400 μm thick with a markedly high resolution. Together, the low cost highlighter ink serves as an alternative reagent useful for visualization of blood vessels in developing avian and mouse embryos and possibly in other animals.

  6. Insulin-Related Peptide 5 is Involved in Regulating Embryo Development and Biochemical Composition in Pea Aphid with Wing Polyphenism

    PubMed Central

    Guo, Shan-Shan; Zhang, Meng; Liu, Tong-Xian

    2016-01-01

    In aphids there is a fecundity-dispersal trade-off between wingless and winged morphs. Recent research on the molecular mechanism of wing morphs associated with dispersal reveals that insulin receptors in the insulin signaling (IS) pathway regulate alternation of wing morphs in planthoppers. However, little is known about whether genes in the IS pathway are involved in developmental regulation in aphid nymphs with different wing morphs. In this study, we show that expression of the insulin-related peptide 5 gene (Apirp5) affects biochemical composition and embryo development of wingless pea aphids, Acyrthosiphon pisum. After comparing expression levels of major genes in the IS pathway between third instar winged and wingless nymphs, we found that Apirp5 showed higher expression in head and thorax in the wingless nymphs than in the winged nymphs. Although microinjection treatment affects physical performance in aphids, nymphs with RNA interference of Apirp5 had less weight, smaller embryos, and higher carbohydrate and protein contents compared to the control group. Comparison between winged and wingless nymphs showed a similar trend. These results indicate that Apirp5 is involved in embryo development and metabolic regulation in wing dimorphic pea aphid. PMID:26903881

  7. Pouch brooding marsupial frogs transfer nutrients to developing embryos.

    PubMed

    Warne, Robin W; Catenazzi, Alessandro

    2016-10-01

    Marsupial frogs have a unique reproductive mode in which females carry eggs enclosed in a sealed dorsal brood pouch. While most anurans are considered to be oviparous with lecithotrophic eggs, the extensively vascularized membrane of the brood pouch in marsupial frogs suggests potential opportunities for nutrient transfer. We tested for matrotrophy in the live-bearing Gastrotheca excubitor (Hemiphractidae), through feeding insects labelled with a (13)C-fatty acid and a (15)N-amino acid to brooding marsupial frogs. We observed significant increases of δ(13)C and δ(15)N in both maternal pouch tissues and embryos, suggesting nutrient transfer. Embryo dry mass also increased with developmental stage, providing further direct evidence for matrotrophy. These results suggest that in addition to gas exchange, the vascularized brood pouch membrane of G. excubitor also enables maternal nutrient transfer. This finding revealed a suspected but untested trait in the evolution of parental care in marsupial frogs, in contrast to previous work on Gastrotheca species that release tadpoles, and suggests greater complexity in reproductive and provisioning modes than previously thought.

  8. Development, molecular composition and freeze tolerance of bovine embryos cultured in TCM-199 supplemented with hyaluronan.

    PubMed

    Palasz, A T; Breña, P Beltrán; Martinez, Marcelo F; Perez-Garnelo, S S; Ramirez, M A; Gutiérrez-Adán, A; De la Fuente, J

    2008-02-01

    Hyaluronan (HA) is glycosaminoglycan that is present from the start of embryonic development and its role and concentration increases with embryo development. The objective of this study was to evaluate if the presence of HA in TCM-199 culture medium had an effect on the development and quality of bovine embryos. There was no effect of HA on the total number of zygotes developing to blastocysts on day 7, however more expanded and hatched blastocyst stages were observed on days 8 and 9 in the group supplemented with HA (p<0.05). Following freeze/thawing, significantly more (p<0.05) embryos cultured in medium supplemented with HA hatched than those cultured in TCM-199 alone or those with BSA. Medium supplemented with HA and BSA significantly increased the level of expression of glucose metabolism Glut-1 gene and embryo compaction Cx43 gene (p<0.05), and had no effect on Glut-5 and IGF-II expression. In addition, HA presence in culture decreased the level of expression of apoptosis Bax and oxidative stress SOX genes (p<0.05). There was significant difference in total number of nuclei between TCM-199 medium only and the remaining media containing BSA or HA plus BSA, between which there was no difference. In summary, our results indicate that the addition of high molecular weight HA to TCM-199 medium that contains BSA on day 4 of culture improved embryo development to hatching and hatched blastocysts and the quality of produced embryos, which were superior to embryos cultured without HA addition.

  9. Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development.

    PubMed

    Wang, S; Panter, K E; Gaffield, W; Evans, R C; Bunch, T D

    2005-02-01

    alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.

  10. Does the Presence of Blood in the Catheter or the Degree of Difficulty of Embryo Transfer Affect Live Birth?

    PubMed

    Plowden, Torie C; Hill, Micah J; Miles, Shana M; Hoyt, Benjamin; Yauger, Belinda; Segars, James H; Csokmay, John M; Chason, Rebecca J

    2016-09-21

    The technique used for embryo transfer (ET) can affect implantation. Prior research that evaluated the effect of postprocedural blood of the transfer catheter tip have yielded mixed results, and it is unclear whether this is actually a marker of difficulty of the transfer. Our objective was to estimate the effect of blood at the time of ET and the difficulty of ET on live birth rates (LBR). This retrospective cohort study utilized generalized estimating equations (GEEs) with nesting for repeated cycles for all analyses. Univariate modeling was performed and a final multivariate (adjusted) GEE model accounted for all significant confounders. Embryo transfers were subjectively graded (easy, medium, or hard) by a physician at the time of transfer. The presence of blood at ET was associated with more difficult ETs, retained embryos, and presence of mucous in the catheter. In the univariate analysis, ET with blood was not associated with live birth, while the degree of difficulty for ET had a negative impact on LBR. In the final multivariate GEE model, which accounts for repeated cycles from a patient, the only factors associated with an increased LBR were the degree of difficulty of the ET, female age, and blastocyst transfer. After controlling for confounding variables, the presence of blood in the transfer catheter was not associated with the likelihood of pregnancy and thus was not an independent predictor of cycle outcome. This indicates that the difficulty of the transfer itself was a strong negative predictor of pregnancy.

  11. Raman spectroscopic imaging of the whole Ciona intestinalis embryo during development.

    PubMed

    Nakamura, Mitsuru J; Hotta, Kohji; Oka, Kotaro

    2013-01-01

    Intracellular composition and the distribution of bio-molecules play central roles in the specification of cell fates and morphogenesis during embryogenesis. Consequently, investigation of changes in the expression and distribution of bio-molecules, especially mRNAs and proteins, is an important challenge in developmental biology. Raman spectroscopic imaging, a non-invasive and label-free technique, allows simultaneous imaging of the intracellular composition and distribution of multiple bio-molecules. In this study, we explored the application of Raman spectroscopic imaging in the whole Ciona intestinalis embryo during development. Analysis of Raman spectra scattered from C. intestinalis embryos revealed a number of localized patterns of high Raman intensity within the embryo. Based on the observed distribution of bio-molecules, we succeeded in identifying the location and structure of differentiated muscle and endoderm within the whole embryo, up to the tailbud stage, in a label-free manner. Furthermore, during cell differentiation, we detected significant differences in cell state between muscle/endoderm daughter cells and daughter cells with other fates that had divided from the same mother cells; this was achieved by focusing on the Raman intensity of single Raman bands at 1002 or 1526 cm(-1), respectively. This study reports the first application of Raman spectroscopic imaging to the study of identifying and characterizing differentiating tissues in a whole chordate embryo. Our results suggest that Raman spectroscopic imaging is a feasible label-free technique for investigating the developmental process of the whole embryo of C. intestinalis.

  12. Does slow embryo development predict a high aneuploidy rate on trophectoderm biopsy?

    PubMed

    Piccolomini, Mariana M; Nicolielo, Mariana; Bonetti, Tatiana C S; Motta, Eduardo L A; Serafini, Paulo C; Alegretti, Jose Roberto

    2016-09-01

    The aneuploidy rates in expanded blastocysts biopsied on days 5 and 6 development were assessed in women undergoing IVF followed by array comparative genomic hybridization. This study included 1171 expanded blastocysts from 465 patients. Among the 465 patients, 215 and 141 underwent embryo biopsy on day 5 and day 6 (46.2% and 30.3%, respectively), and 109 underwent biopsy on both days 5 and 6 (23.4%). The cycles of 206 women were cancelled because only aneuploidy embryos were present (44.3%). The aneuploid embryos were classified according to the type as single, double or complex aneuploidy. No differences were observed in the distributions of these three categories according to the day of the biopsy. The aneuploidy rate was also evaluated according to maternal age, and was found to be higher in older patients; however, no differences in this rate were detected between embryos biopsied on days 5 and 6 according to maternal age. Biopsy was carried out when blastocysts reached the expanded stage. The embryos biopsied on day 6 had a higher rate of aneuploidy (69.9%) than those biopsied on day 5 (61.4%); however, the euploid embryos transferred had similar chances for successful and healthy gestation.

  13. Influences of textured substrates on the heart rate of developing zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Chen, Chia-Yun; Chen, Chia-Yuan

    2013-07-01

    Identification of the effects of different textured substrates on zebrafish (Danio rerio) embryos provides insights into the influence of external stimuli on normal cardiovascular functions in the developmental stages of the embryos. This knowledge can be used in numerous genetic studies using zebrafish as an animal model as well as in bioanalytical assays using digital microfluidics. In this study, zebrafish embryos were systematically positioned and in vivo imaged on four types of silicon substrates. These substrates exhibited surface textures and surface wettability that were well modulated by wet chemical etching. The heart rate of the developing embryos significantly increased by 9.1% upon exposure to textured Si substrates with nanostructured surfaces compared with bare Si substrates. Modulation of surface wettability in the tested substrates also responded to the increase in the heart rate of the embryo; however, the effect of surface wettability on heart rate was slight compared with the effect of texture. In-depth experimental and statistical investigations of heart rate under the effects of substrate textures imply a pathway through which the inner mass of the embryo reacts to external stimuli. These findings contribute to zebrafish-related studies and suggest other factors to consider in the design of nanostructure-based microfluidics and other biomedical devices.

  14. Influences of textured substrates on the heart rate of developing zebrafish embryos.

    PubMed

    Chen, Chia-Yun; Chen, Chia-Yuan

    2013-07-05

    Identification of the effects of different textured substrates on zebrafish (Danio rerio) embryos provides insights into the influence of external stimuli on normal cardiovascular functions in the developmental stages of the embryos. This knowledge can be used in numerous genetic studies using zebrafish as an animal model as well as in bioanalytical assays using digital microfluidics. In this study, zebrafish embryos were systematically positioned and in vivo imaged on four types of silicon substrates. These substrates exhibited surface textures and surface wettability that were well modulated by wet chemical etching. The heart rate of the developing embryos significantly increased by 9.1% upon exposure to textured Si substrates with nanostructured surfaces compared with bare Si substrates. Modulation of surface wettability in the tested substrates also responded to the increase in the heart rate of the embryo; however, the effect of surface wettability on heart rate was slight compared with the effect of texture. In-depth experimental and statistical investigations of heart rate under the effects of substrate textures imply a pathway through which the inner mass of the embryo reacts to external stimuli. These findings contribute to zebrafish-related studies and suggest other factors to consider in the design of nanostructure-based microfluidics and other biomedical devices.

  15. The first wave of B lymphopoiesis develops independently of stem cells in the murine embryo.

    PubMed

    Yoshimoto, Momoko

    2015-12-01

    In the developing mouse embryo, there are several waves of hematopoiesis. Primitive and definitive erythromyeloid lineages appear prior to hematopoietic stem cell (HSC) emergence, and these waves are considered to be transient and support embryonic homeostasis until HSC-derived hematopoiesis is established. However, recent evidence strongly suggests that HSC-independent immune cells, such as tissue macrophages and some innate lymphoid cells, develop in the mouse embryo and persist into postnatal life. Innate type B-1 cells have also been reported to emerge from hemogenic endothelial cells in the extraembryonic yolk sac and para-aortic splanchnopleura, and continue to develop in the fetal liver, even in HSC-deficient mouse embryos. Here, this review discusses B-1 cell development in the context of the layered immune system hypothesis of B lymphopoiesis and the emergence of B-1 cells independent of HSCs.

  16. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

    NASA Astrophysics Data System (ADS)

    Sun, Liangliang; Bertke, Michelle M.; Champion, Matthew M.; Zhu, Guijie; Huber, Paul W.; Dovichi, Norman J.

    2014-03-01

    While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development.

  17. Errors in development of fetuses and placentas from in vitro-produced bovine embryos.

    PubMed

    Farin, Peter W; Piedrahita, Jorge A; Farin, Charlotte E

    2006-01-07

    In vitro systems for oocyte maturation, fertilization and embryo culture [in vitro production (IVP)] have the potential for more wide-spread use in creative breeding programs for dairy and beef cattle. However, one negative consequence of both IVP and somatic cell nuclear transfer (SCNT) in cattle and other species is that embryos, fetuses, placentas, and offspring can differ significantly in morphology and developmental competence compared with those from embryos produced in vivo. Fetuses and placentas derived from IVP and SCNT embryos may fall within the normal range of development, may have obvious abnormalities such as increased fetal and placental weights, or may have subtle abnormalities such as aberrant development of fetal skeletal muscle, placental blood vessels, and altered metabolism. Failures in physiologic and/or genetic mechanisms essential for proper fetal growth and survival outside of the uterus contribute significantly to pregnancy and neonatal losses. Oversized fetuses are at increased risk of death during parturition and the adverse consequences of severe dystocia may compromise the dam. Collectively, these abnormalities have been referred to as 'large offspring syndrome' or 'large calf syndrome'. Abnormal phenotypes resulting from IVP and SCNT embryos are stochastic in occurrence and they have not been consistently linked to aberrant expression of single genes or specific pathophysiology. Thus, reliable methods of early diagnosis of the condition are not yet available. The objective of this paper is to examine abnormal development of fetuses and placentas resulting from embryos produced using in vitro systems. The term 'abnormal offspring syndrome (AOS)' is introduced and a classification system of developmental outcomes is proposed to facilitate research efforts on the mechanisms of the various abnormal phenotypes. We also discuss potential genetic and physiologic mechanisms that may contribute to abnormal phenotypes following transfer of IVP

  18. Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos

    PubMed Central

    Kawamura, Kazuhiro; Kawamura, Nanami; Mulders, Sabine M.; Gelpke, Maarten D. Sollewijn; Hsueh, Aaron J. W.

    2005-01-01

    Optimal development of fertilized eggs into preimplantation embryos is essential for reproduction. Although mammalian oocytes ovulated after luteinizing hormone (LH) stimulation can be fertilized and promoted into early embryos in vitro, little is known about ovarian factors important for the conditioning of eggs for early embryo development. Because LH interacts only with ovarian somatic cells, its potential regulation of oocyte functions is presumably mediated by local paracrine factors. We performed DNA microarray analyses of ovarian transcripts and identified brain-derived neurotrophic factor (BDNF) secreted by granulosa and cumulus cells as an ovarian factor stimulated by the preovulatory LH surge. Ovarian BDNF acts on TrkB receptors expressed exclusively in oocytes to enhance first polar body extrusion of oocytes and to promote the in vitro development of zygotes into preimplantation embryos. Furthermore, in vivo treatment with a Trk receptor inhibitor suppressed first polar body extrusion and the progression of zygotes into blastocysts. Thus, ovarian BDNF is important to nuclear and cytoplasmic maturation of the oocyte, which is essential for successful oocyte development into preimplantation embryos. Treatment with BDNF could condition the cultured oocytes for optimal progression into the totipotent blastocysts. PMID:15967989

  19. ZINC INFLUENCES THE IN VITRO DEVELOPMENT OF PERI-IMPLANTATION MOUSE EMBRYOS

    EPA Science Inventory

    Background: For humans, it is estimated that over 70% of concepti are lost during early development. In culture, mouse peri-implantation embryos can mimic development from the blastocyst to the egg cylinder stage of development, a period during which implantation occurs in viv...

  20. The phosphorylated pathway of serine biosynthesis is essential both for male gametophyte and embryo development and for root growth in Arabidopsis.

    PubMed

    Cascales-Miñana, Borja; Muñoz-Bertomeu, Jesús; Flores-Tornero, María; Anoman, Armand Djoro; Pertusa, José; Alaiz, Manuel; Osorio, Sonia; Fernie, Alisdair R; Segura, Juan; Ros, Roc

    2013-06-01

    This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular development. The expression of PSP1 in the tapetum at critical stages of microspore development suggests that PSP1 activity in this cell layer is essential in pollen development. In addition to embryo death and male sterility, conditional psp1 mutants displayed a short-root phenotype, which was reverted in the presence of Ser. A metabolomic study demonstrated that the PPSB plays a crucial role in plant metabolism by affecting glycolysis, the tricarboxylic acid cycle, and the biosynthesis of amino acids. We provide evidence of the crucial role of the PPSB in embryo, pollen, and root development and suggest that this pathway is an important link connecting primary metabolism with development.

  1. The Phosphorylated Pathway of Serine Biosynthesis Is Essential Both for Male Gametophyte and Embryo Development and for Root Growth in Arabidopsis[W

    PubMed Central

    Cascales-Miñana, Borja; Muñoz-Bertomeu, Jesús; Flores-Tornero, María; Anoman, Armand Djoro; Pertusa, José; Alaiz, Manuel; Osorio, Sonia; Fernie, Alisdair R.; Segura, Juan; Ros, Roc

    2013-01-01

    This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular development. The expression of PSP1 in the tapetum at critical stages of microspore development suggests that PSP1 activity in this cell layer is essential in pollen development. In addition to embryo death and male sterility, conditional psp1 mutants displayed a short-root phenotype, which was reverted in the presence of Ser. A metabolomic study demonstrated that the PPSB plays a crucial role in plant metabolism by affecting glycolysis, the tricarboxylic acid cycle, and the biosynthesis of amino acids. We provide evidence of the crucial role of the PPSB in embryo, pollen, and root development and suggest that this pathway is an important link connecting primary metabolism with development. PMID:23771893

  2. Patterning in time and space: HoxB cluster gene expression in the developing chick embryo.

    PubMed

    Gouveia, Analuce; Marcelino, Hugo M; Gonçalves, Lisa; Palmeirim, Isabel; Andrade, Raquel P

    2015-01-01

    The developing embryo is a paradigmatic model to study molecular mechanisms of time control in Biology. Hox genes are key players in the specification of tissue identity during embryo development and their expression is under strict temporal regulation. However, the molecular mechanisms underlying timely Hox activation in the early embryo remain unknown. This is hindered by the lack of a rigorous temporal framework of sequential Hox expression within a single cluster. Herein, a thorough characterization of HoxB cluster gene expression was performed over time and space in the early chick embryo. Clear temporal collinearity of HoxB cluster gene expression activation was observed. Spatial collinearity of HoxB expression was evidenced in different stages of development and in multiple tissues. Using embryo explant cultures we showed that HoxB2 is cyclically expressed in the rostral presomitic mesoderm with the same periodicity as somite formation, suggesting a link between timely tissue specification and somite formation. We foresee that the molecular framework herein provided will facilitate experimental approaches aimed at identifying the regulatory mechanisms underlying Hox expression in Time and Space.

  3. Localization and expression of histone H2A variants during mouse oogenesis and preimplantation embryo development.

    PubMed

    Wu, B J; Dong, F L; Ma, X S; Wang, X G; Lin, F; Liu, H L

    2014-08-07

    Epigenetic modifications of the genome, such as histone H2A variants, ensure appropriate gene activation or silencing during oogenesis and preimplantation embryo development. We examined global localization and expression of the histone H2A variants, including H2A.Bbd, H2A.Z and H2A.X, during mouse oogenesis and preimplantation embryo development. Immunocytochemistry with specific antibodies against various histone H2A variants showed their localization and changes during oogenesis and preimplantation development. H2A.Bbd and H2A.Z were almost absent from nuclei of growing oocytes (except 5-day oocyte), whereas H2A.X was deposited in nuclei throughout oogenesis and in preimplantation embryos. In germinal vesicle (GV) oocyte chromatin, H2A.Bbd was detected as a weak signal, whereas no fluorescent signal was detected in GV breakdown (GVBD) or metaphase II (MII) oocytes; H2A.Z showed intense signals in chromatin of GV, GVBD and MII oocytes. H2A. Bbd showed very weak signals in both pronucleus and 2-cell embryo nuclei, but intense signals were detected in nuclei from 4-cell embryo to blastula. The H2A.Z signal was absent from pronucleus to morula chromatin, whereas a fluorescent signal was detected in blastula nuclei. Our results suggest that histone H2A variants are probably involved in reprogramming of genomes during oocyte meiosis or after fertilization.

  4. Peptidylarginine deiminase 1-catalyzed histone citrullination is essential for early embryo development

    PubMed Central

    Zhang, Xiaoqian; Liu, Xiaoqiu; Zhang, Mei; Li, Tingting; Muth, Aaron; Thompson, Paul R.; Coonrod, Scott A.; Zhang, Xuesen

    2016-01-01

    Peptidylarginine deiminase (PADI) enzymes are increasingly being associated with the regulation of chromatin structure and gene activity via histone citrullination. As one of the PADI family members, PADI1 has been mainly reported to be expressed in the epidermis and uterus, where the protein in keratinocytes is thought to promote differentiation by citrullinating filament proteins. However, the roles of PADI1 in preimplantation development have not been addressed. Using a PADI1-specific inhibitor and Padi1-morpholino knockdown, we found that citrullination of histone tails at H4R3 and H3R2/8/17 were markedly reduced in the 2- and 4-cell embryos. Consistent with this observation, early embryo development was also arrested at the 4-cell stage upon depletion of PADI1 or inhibition of PADI1 enzyme activity. Additionally, by employing 5-ethynyl uridine (EU) incorporation analysis, ablation of PADI1 function led to a dramatic decrease in overall transcriptional activity, correlating well with the reduced levels of phosphorylation of RNA Pol II at Ser2 observed at 2- or 4-cell stage of embryos under Padi1 knockdown or inhibiting PADI1. Thus, our data reveal a novel function of PADI1 during early embryo development transitions by catalyzing histone tail citrullination, which facilitates early embryo genome transactivation. PMID:27929094

  5. Embryo development inside female salamander (Ambystoma jeffersonianum-laterale) prior to egg laying.

    PubMed

    Charney, Noah D; Castorino, John J; Dobro, Megan J; Steely, Sarah L

    2014-01-01

    The length of embryo retention prior to oviposition is a critical evolutionary trait. In all oviparous salamanders, which include the vast majority of species in the order, fertilization is thought to occur at the time of egg laying. Embryos then enter the first cleavage stage several hours after being deposited. This pattern holds for previously studied individuals in the Ambystoma jeffersonianum-laterale complex. Here, we document an instance in which a female Ambystoma jeffersonianum-laterale was carrying embryos internally that had already reached stage 10 of development. Development likely began several days prior to the start of migration to the breeding pond. This is the first such record for any egg-laying salamander, and suggests a degree of plasticity in the timing of fertilization and development not previously recognized. Further work is needed to ascertain the prevalence, mechanics, and evolutionary significance of this phenomenon.

  6. Hematocrit and blood osmolality in developing chicken embryos (Gallus gallus): in vivo and in vitro regulation.

    PubMed

    Andrewartha, Sarah J; Tazawa, Hiroshi; Burggren, Warren W

    2011-12-15

    Hematocrit (Hct) regulation is a complex process involving potentially many factors. How such regulation develops in vertebrate embryos is still poorly understood. Thus, we investigated the role of blood pH in the regulation of Hct across developmental time in chicken embryos. We hypothesized that blood pH alterations in vitro (i.e., in a test tube) would affect Hct far more than in vivo because of in vivo compensatory regulatory processes for Hct. Large changes in Hct (through mean corpuscular volume (MCV)) and blood osmolality (Osm) occur when the blood was exposed to varying ambient temperatures (T(a)'s) and P(CO2) in vitro alongside an experimentally induced blood pH change from ~7.3 to 8.2. However, homeostatic regulatory mechanisms apparently limited these alterations in vivo. Changes in blood pH in vitro were accompanied by hydration or dehydration of red blood cells depending on embryonic age, resulting in changes in Hct that also were specific to developmental stage, due likely to initial blood gas and [HCO(3)(-)](v) values. Significant linear relationships between Hct and pH (Hct/ΔpH=-21.4%/(pH unit)), Hct and [HCO(3)(-)] (ΔHct/Δ[HCO(3)(-)]=1.6%/(mEq L(-1))) and the mean buffer value (Δ[HCO(3)(-)]/ΔpH=-13.4 (mEq L(-1))/(pH unit)) demonstrate that both pH and [HCO(3)(-)] likely play a role in the regulation of Hct through MCV at least in vitro. Low T(a) (24°C) resulted in relatively large changes in pH with small changes in Hct and Osm in vitro with increased T(a) (42°C) conversely resulting in larger changes in both Hct and Osm. In vivo exposure to altered T(a) caused age-dependent changes in Hct, demonstrating a trend towards increased Hct at higher T(a). Further, exposing embryos to a gas mixture where P(CO2) = 5.1 kPa for >4 h period at T(a) of 37 or 42°C also did not elicit a change in Hct or Osm. Presumably, homeostatic mechanisms ensured that in vivo Hct was stable during a 4-6 h temperature and/or hypercapnic stress. Thus, although blood p

  7. Nucleolar development and allocation of key nucleolar proteins require de novo transcription in bovine embryos.

    PubMed

    Svarcova, Olga; Laurincik, Jozef; Avery, Birthe; Mlyncek, Milos; Niemann, Heiner; Maddox-Hyttel, Poul

    2007-11-01

    The goal of the present study was to investigate whether key nucleolar proteins involved in ribosomal RNA (rRNA) transcription and processing are transcribed de novo or from maternally inherited messenger RNAs (mRNA) in bovine embryos, and to which extent de novo transcription of these proteins mRNA is required for the development of functional nucleoli during the major activation of the embryonic genome. Immunofluorescence for localization of key nucleolar proteins, autoradiography for detection of transcriptional activity, and transmission electron microscopy were applied to in vitro produced bovine embryos cultured from the 2-cell stage with or without (control groups) alpha-amanitin, which blocks the RNA polymerases II and III transcription and, thus the synthesis of mRNA. In the control groups, weak autoradiographic labeling was initially observed in the periphery of few nuclei at the 4-cell and the early 8-cell stage, and the entire nucleoplasm as well as nucleolus precursor bodies (NBBs) were prominently labelled in all late 8-cell stages. The NPBs displayed initial transformation into fibrillo-granular nucleoli. In the alpha-amanitin group, lack of autoradiographic labeling was seen at all developmental stages and disintegrated NPBs stage were found at the late 8-cell. Our immunofluorescence data indicate that RNA polymerase I, UBF, topoisomerase I and fibrillarin are transcribed de novo whereas nucleolin and nucleophosmin are maternally inherited as demonstrated by alpha -amanitin inhibition. However, localization of these two proteins to the nucleolar compartments was negatively affected by the alpha-amanitin treatment. Consequently, functional nucleoli were not established.

  8. Influence of lactation on metabolic characteristics and embryo development in postpartum Holstein dairy cows.

    PubMed

    Maillo, V; Rizos, D; Besenfelder, U; Havlicek, V; Kelly, A K; Garrett, M; Lonergan, P

    2012-07-01

    The aim of this study was to examine the direct effect of lactation on the ability of the reproductive tract of postpartum dairy cows to support early embryo development. Twenty-one primiparous Holstein heifers were used. Immediately after calving, half of the cows were dried off (i.e., never milked), and the other half entered the milking herd and were milked twice daily. Jugular blood samples were taken twice per week from 15 d before calving to approximately 100 d postpartum to measure nonesterified fatty acids, β-hydroxybutyrate, glucose, insulin, and insulin-like growth factor-I. At the same time, body weight and body condition score were recorded for each cow. At approximately 60 d postpartum (experiment 1), approximately 65 two- to four-cell embryos, produced by in vitro maturation and fertilization, were endoscopically transferred to the oviduct ipsilateral to the corpus luteum of all cows on d 2 of the estrous cycle. Five days later (d 7), the oviduct and uterus were flushed nonsurgically and the number of embryos developing to the blastocyst stage was recorded. At approximately 90 d postpartum (experiment 2), the estrous cycles of the same cows were resynchronized and 15 to 20 in vitro-produced blastocysts were transferred to the uterus of each recipient on d 7. All cows were slaughtered on d 14 to assess embryo survival and dimensions. Body weight and body condition score were significantly different between groups for the entire postpartum period of the study. Concentrations of nonesterified fatty acids and β-hydroxybutyrate were higher and concentrations of glucose, insulin, and insulin-like growth factor-I were lower in lactating compared with nonlactating cows. Embryo recovery rates from lactating and dry cows were similar. In experiment 1, fewer embryos developed to the blastocyst stage in the lactating cows compared with the nonlactating cows. In experiment 2, embryo survival and conceptus dimensions were not different between lactating and

  9. In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.

    PubMed

    Isobe, Tomohiro; Ikebata, Yoshihisa; Do, Lanh Thi Kim; Tanihara, Fuminori; Taniguchi, Masayasu; Otoi, Takeshige

    2015-07-01

    The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients.

  10. Effects of copper sulfate on growth, development, and escape behavior in Epidalea calamita embryos and larvae.

    PubMed

    García-Muñoz, E; Guerrero, F; Parra, G

    2009-04-01

    Epidalea calamita embryos at Gosner stages 3 and 19, and larvae at Gosner stage 25, were exposed to different copper sulfate concentrations, ranging from 0.05 to 0.40 mg Cu L(-1), in 96-h acute toxicity tests. Embryonic and larval mortality, development, growth, and larval escape behavior were evaluated. LC(50) at 96 h obtained at Gosner stages 3, 19, and 25 were 0.22, 0.08, and 0.11 mg Cu L(-1), respectively. Embryonic and larval developments were delayed after 96 h of copper sulfate exposure. Growth was also affected and individuals in control treatments grew to twice the size of those exposed to copper concentrations over 0.2 mg Cu L(-1) during the experiments initiated at Gosner stage 19. Escape behavior was altered after 96 h of copper sulfate exposure; larvae showed shorter distances moved and abnormal displacement types. However, after 4 days of recovery process, most of the larvae showed normal escape behavior. For amphibians that develop in temporary wetlands, increased development time, lower size, and altered escape behavior might have repercussions on the number of individuals that can successfully complete metamorphosis and, consequently, on recruitment.

  11. Plastic Hatching Timing by Red-Eyed Treefrog Embryos Interacts with Larval Predator Identity and Sublethal Predation to Affect Prey Morphology but Not Performance

    PubMed Central

    Touchon, Justin C.; Wojdak, Jeremy M.

    2014-01-01

    Many animals respond to predation risk by altering their morphology, behavior, or life-history. We know a great deal about the cues prey respond to and the changes to prey that can be induced by predation risk, but less is known about how plastic responses to predators may be affected by separate plastic responses occurring earlier in life, particularly during the embryonic period. Embryos of a broad array of taxa can respond to egg- or larval-stage risks by altering hatching timing, which may alter the way organisms respond to future predators. Using the red-eyed treefrog (Agalychnis callidryas), a model for understanding the effects of plasticity across life-stages, we assessed how the combined effects of induced variation in the timing of embryo hatching and variation in the larval predator community impacted tadpole morphology, pigmentation and swimming performance. We found that A. callidryas tadpoles developed deeper tail muscles and fins and darker pigmentation in response to fish predators, either when alone or in diverse community with other predators. Tadpoles altered morphology much less so to dragonfly naiads or water bugs. Interestingly, morphological responses to predators were also affected by induced differences in hatching age, with early and late-hatched tadpoles exhibiting different allometric relationships between tail height and body length in different predator environments. Beyond induced morphological changes, fish predators often damaged tadpoles’ tails without killing them (i.e., sublethal predation), but these tadpoles swam equally quickly to those with fully intact tails. This was due to the fact that tadpoles with more damaged tails increased tail beats to achieve equal swimming speed. This study demonstrates that plastic phenotypic responses to predation risk can be influenced by a complex combination of responses to both the embryo and larval environments, but also that prey performance can be highly resilient to sublethal predation

  12. Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

    PubMed Central

    Hagedorn, Elliott J.; Cillis, Jennifer L.; Curley, Caitlyn R.; Patch, Taylor C.; Li, Brian; Blaser, Bradley W.; Riquelme, Raquel; Zon, Leonard I.; Shah, Dhvanit I.

    2016-01-01

    Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development. PMID:27341538

  13. Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae.

    PubMed

    Hagedorn, Elliott J; Cillis, Jennifer L; Curley, Caitlyn R; Patch, Taylor C; Li, Brian; Blaser, Bradley W; Riquelme, Raquel; Zon, Leonard I; Shah, Dhvanit I

    2016-06-11

    Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been  used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

  14. Expression of DMP1 in the developing mouse tongue embryo.

    PubMed

    Murata, Hidetaka; Sunohara, Msataka; Sato, Iwao

    2015-07-01

    Dentin matrix protein 1 (DMP-1) is an important factor in the mineralization of hard tissues. However, it has many other functions in addition to the regulation of mineralized tissues. We analyzed the expression and localization of DMP-1 by immunohistochemical staining and in situ hybridization in the developing mouse tongue during embryonic days 12.5 (E12.5), E14.5, E17.5, and E18.5. We also detected the mRNA abundance of tongue morphogenesis markers such as FGF6, TGF-β1, Collagen I, osteocalcin, chondromodulin 1, tenomodulin, Vascular endothelial growth factor (VEGF), caspase-3, and Aifm from embryonic stages by real-time RT-PCR. The antisense probe for DMP-1 was detected in a few mesenchymal cells surrounding blood vessels at E12.5, and faint localization was seen at E18.5 in the embryonic mouse tongue by in situ hybridization. The DMP-1 and osteocalcin abundance levels gradually increased compared with the other tongue markers from E12.5 to E18.5 (p<0.001). Cluster analyses identified the following distinct clusters for mRNA abundance in the tongue: cluster 1, E12.5; cluster 2, E14.5 and E17.5; and cluster 3, E18.5. The positive correlation between DMP-1 and osteocalcin (Pearson's r=0.685; p<0.05) and negative correlation between DMP-1 and Caspase-3 (Pearson's r=-0.632; p<0.05) were analyzed. These data suggested that DMP-1 potentially influences osteocalcin and Caspase-3 during mouse tongue development and morphogenesis. DMP-1 also affects the angiogenic marker VEGF in specific stages and areas, terminating the differentiation of the tongue from other developing tissues. We conclude that DMP-1 may be involved in regulating the temporal expression at embryonic stages in the mouse tongue.

  15. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    SciTech Connect

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue; Pang, Daxin; Ouyang, Hongsheng

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  16. Variable breeding phenology affects the exposure of amphibian embryos to ultraviolet radiation

    USGS Publications Warehouse

    Corn, P.S.; Muths, E.

    2002-01-01

    Reduced water depth in dry years has been proposed to interact with ultraviolet-B (UV-B) radiation and pathogenic fungus to cause episodes of high mortality of amphibian embryos. Observations of breeding phenology of boreal chorus frogs (Pseudacris maculata) in Colorado from 1986-2001 show that dry years result in earlier breeding. The earliest and latest dates of maximum calling activity by males were 20 May and 16 June, and the date of maximum calling was strongly related to the amount of snow accumulation during the winter. Surface UV-B flux, estimated from satellite-based measurements, was positively related to date of maximum calling. In dry years, surface UV-B during calling was reduced by an amount similar to that attributed to reduced depth. Although there was a significant trend of increasing UV-B from 1978-2001 on the average date (2 June) of maximum calling activity, there was no relationship between year and surface UV-B at actual dates of maximum calling. Exposure to extreme temperatures is an alternative explanation for increased mortality of amphibian embryos in shallow water.

  17. Variable breeding phenology affects the exposure of amphibian embryos to ultraviolet radiation

    USGS Publications Warehouse

    Corn, P.S.; Muths, E.

    2002-01-01

    Reduced water depth in dry years has been proposed to interact with ultraviolet- B (UV-B) radiation and a pathogenic fungus to cause episodes of high mortality of amphibian embryos. Observations of breeding phenology of boreal chorus frogs (Pseudacris maculata) in Colorado from 1986 to 2001 show that dry years result in earlier breeding. The earliest and latest dates of maximum calling activity by males were 20 May and 16 June, and the date of maximum calling was strongly related to the amount of snow accumulation during the winter. Surface UV-B flux, estimated from satellite-based measurements, was positively related to date of maximum calling. In dry years, surface UV-B during calling was reduced by an amount similar to that attributed to reduced depth. Although there was a significant trend of increasing UV-B from 1978 to 2001 on the average date (2 June) of maximum calling activity, there was no relationship between year and surface UV-B at actual dates of maximum calling. Exposure to extreme temperatures is an alternative explanation for increased mortality of amphibian embryos in shallow water.

  18. Regional Localization of Suspensor mRNAs during Early Embryo Development

    PubMed Central

    Weterings, Koen; Apuya, Nestor R.; Bi, Yuping; Fischer, Robert L.; Harada, John J.; Goldberg, Robert B.

    2001-01-01

    We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/β-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote. PMID:11701878

  19. Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis.

    PubMed

    Crispo, M; Vilariño, M; dos Santos-Neto, P C; Núñez-Olivera, R; Cuadro, F; Barrera, N; Mulet, A P; Nguyen, T H; Anegón, I; Menchaca, A

    2015-02-01

    Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4%; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3%, 46/117) and IVF embryos (39.6%, 44/111). Pregnancy rate was detected in 50.0% (6/12) of recipient ewes with TG embryos, in 46.7% (7/15) with IVF embryos, and in 65.0% (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9%) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs.

  20. Intracapsular algae provide fixed carbon to developing embryos of the salamander Ambystoma maculatum.

    PubMed

    Graham, Erin R; Fay, Scott A; Davey, Adam; Sanders, Robert W

    2013-02-01

    Each spring, North American spotted salamander (Ambystoma maculatum) females each lay hundreds of eggs in shallow pools of water. Eggs are surrounded by jelly layers and are deposited as large gelatinous masses. Following deposition, masses are penetrated by a mutualistic green alga, Oophila amblystomatis, which enters individual egg capsules, proliferates and aggregates near the salamander embryo, providing oxygen that enhances development. We examined the effects of population density of intracapsular O. amblystomatis on A. maculatum embryos and show that larger algal populations promote faster embryonic growth and development. Also, we show that carbon fixed by O. amblystomatis is transferred to the embryos, providing the first evidence of direct translocation of photosynthate from a symbiont to a vertebrate host.

  1. Elective single-embryo transfer: persuasive communication strategies can affect choice in a young British population.

    PubMed

    van den Akker, O B A; Purewal, S

    2011-12-01

    This study tested the effectiveness of the framing effect and fear appeals to inform young people about the risks of multiple births and the option of selecting elective single-embryo transfer (eSET). A non-patient student sample (age (mean±SD) 23±5.5 years; n=321) were randomly allocated to one of seven groups: (1) framing effect: (1a) gain and (1b) loss frame; (2) fear appeal: (2a) high, (2b) medium and (2c) low fear; or (3) a control group: (3a) education and (3b) non-education. The primary outcome measure was the Attitudes towards Single Embryo Transfer questionnaire, before exposure to the messages (time 1) and immediately afterwards (time 2). Results revealed participants in the high fear, medium fear and gain condition demonstrated the most positive and significant differences (P<0.001 to P<0.05) in their knowledge, hypothetical intentions and modest changes in attitudes towards eSET than the low fear, loss frame and education and non-education messages. The results demonstrate that the use of complex persuasive communication techniques on a student population to promote immediate and hypothetical eSET preferences is more successful at promoting eSET than merely reporting educational content. Future research should investigate its application in a clinical population. A multiple pregnancy is a health risk to both infant and mother following IVF treatment. The aims of this study were to test the effectiveness of two persuasive communication techniques (the framing effect and fear appeals) to inform young people about the risks of multiple births and the hypothetical option of selecting elective single-embryo transfer (eSET) (i.e., only one embryo is transferred to the uterus using IVF treatment). A total of 321 non-patient student sample (mean age 23) were randomly allocated to read a message from one of seven groups: (1) framing effect: (1a) gain and (1b) loss frame; (2) fear appeal: (2a) high, (2b) medium and (2c) low fear; or (3) a control group

  2. Alteration of development and gene expression induced by in ovo-nanoinjection of 3-hydroxybenzo[c]phenanthrene into Japanese medaka (Oryzias latipes) embryos.

    PubMed

    Chen, Kun; Tsutsumi, Yuki; Yoshitake, Shuhei; Qiu, Xuchun; Xu, Hai; Hashiguchi, Yasuyuki; Honda, Masato; Tashiro, Kosuke; Nakayama, Kei; Hano, Takeshi; Suzuki, Nobuo; Hayakawa, Kazuichi; Shimasaki, Yohei; Oshima, Yuji

    2017-01-01

    Benzo[c]phenanthrene (BcP) is a highly toxic polycyclic aromatic hydrocarbon (PAHs) found throughout the environment. In fish, it is metabolized to 3-hydroxybenzo[c]phenanthrene (3-OHBcP). In the present study, we observed the effects of 1nM 3-OHBcP on the development and gene expression of Japanese medaka (Oryzias latipes) embryos. Embryos were nanoinjected with the chemical after fertilization. Survival, developmental stage, and heart rate of the embryos were observed, and gene expression differences were quantified by messenger RNA sequencing (mRNA-Seq). The exposure to 1nM 3-OHBcP accelerated the development of medaka embryos on the 1st, 4th, and 6th days post fertilization (dpf), and increased heart rates significantly on the 5th dpf. Physical development differences of exposed medaka embryos were consistent with the gene expression profiles of the mRNA-Seq results for the 3rd dpf, which show that the expression of 780 genes differed significantly between the solvent control and 1nM 3-OHBcP exposure groups. The obvious expression changes in the exposure group were found for genes involved in organ formation (eye, muscle, heart), energy supply (ATPase and ATP synthase), and stress-response (heat shock protein genes). The acceleration of development and increased heart rate, which were consistent with the changes in mRNA expression, suggested that 3-OHBcP affects the development of medaka embryos. The observation on the developmental stages and heart beat, in ovo-nanoinjection and mRNA-Seq may be efficient tools to evaluate the effects of chemicals on embryos.

  3. Increase in membrane thickness during development compensates for eggshell thinning due to calcium uptake by the embryo in falcons

    NASA Astrophysics Data System (ADS)

    Castilla, Aurora M.; van Dongen, Stefan; Herrel, Anthony; Francesch, Amadeu; Martínez de Aragón, Juan; Malone, Jim; José Negro, Juan

    2010-02-01

    We compared membrane thickness of fully developed eggs with those of non-developed eggs in different endangered falcon taxa. To our knowledge, membrane thickness variation during development has never been examined before in falcons or any other wild bird. Yet, the egg membrane constitutes an important protective barrier for the developing embryo. Because eggshell thinning is a general process that occurs during bird development, caused by calcium uptake by the embryo, eggs are expected to be less protected and vulnerable to breakage near the end of development. Thus, egg membranes could play an important protective role in the later stages of development by getting relatively thicker. We used linear mixed models to explore the variation in membrane thickness ( n = 378 eggs) in relation to developmental stage, taxon, female age, mass and identity (73 females), egg-laying sequence (105 clutches) and the study zone. Our results are consistent with the prediction that egg membranes are thicker in fully developed eggs than in non-developed eggs, suggesting that the increase in membrane thickness during development may compensate for eggshell thinning. In addition, our data shown that thicker membranes are associated with larger, heavier and relatively wider eggs, as well as with eggs that had thinner eggshells. Egg-laying sequence, female age and the study zone did not explain the observed variation of membrane thickness in the falcon taxa studied. As we provide quantitative data on membrane thickness variation during development in falcons not subjected to contamination or food limitation (i.e. bred under captive conditions), our data may be used as a reference for studies on eggs from natural populations. Considering the large variation in membrane thickness and the multiple factors affecting on it and its importance in the protection of the embryo, we encourage other researchers to include measurements on membranes in studies exploring eggshell thickness variation.

  4. Increase in membrane thickness during development compensates for eggshell thinning due to calcium uptake by the embryo in falcons.

    PubMed

    Castilla, Aurora M; Van Dongen, Stefan; Herrel, Anthony; Francesch, Amadeu; Martínez de Aragón, Juan; Malone, Jim; Negro, Juan José

    2010-02-01

    We compared membrane thickness of fully developed eggs with those of non-developed eggs in different endangered falcon taxa. To our knowledge, membrane thickness variation during development has never been examined before in falcons or any other wild bird. Yet, the egg membrane constitutes an important protective barrier for the developing embryo. Because eggshell thinning is a general process that occurs during bird development, caused by calcium uptake by the embryo, eggs are expected to be less protected and vulnerable to breakage near the end of development. Thus, egg membranes could play an important protective role in the later stages of development by getting relatively thicker. We used linear mixed models to explore the variation in membrane thickness (n = 378 eggs) in relation to developmental stage, taxon, female age, mass and identity (73 females), egg-laying sequence (105 clutches) and the study zone. Our results are consistent with the prediction that egg membranes are thicker in fully developed eggs than in non-developed eggs, suggesting that the increase in membrane thickness during development may compensate for eggshell thinning. In addition, our data shown that thicker membranes are associated with larger, heavier and relatively wider eggs, as well as with eggs that had thinner eggshells. Egg-laying sequence, female age and the study zone did not explain the observed variation of membrane thickness in the falcon taxa studied. As we provide quantitative data on membrane thickness variation during development in falcons not subjected to contamination or food limitation (i.e. bred under captive conditions), our data may be used as a reference for studies on eggs from natural populations. Considering the large variation in membrane thickness and the multiple factors affecting on it and its importance in the protection of the embryo, we encourage other researchers to include measurements on membranes in studies exploring eggshell thickness variation.

  5. Visualizing Compound Distribution during Zebrafish Embryo Development: The Effects of Lipophilicity and DMSO.

    PubMed

    de Koning, Coco; Beekhuijzen, Manon; Tobor-Kapłon, Marysia; de Vries-Buitenweg, Selinda; Schoutsen, Dick; Leeijen, Nico; van de Waart, Beppy; Emmen, Harry

    2015-12-01

    The predictability of the zebrafish embryo model is highly influenced by internal exposure of the embryo/larva. As compound uptake is likely to be influenced by factors such as lipophilicity, solvent use, and chorion presence, this article focuses on investigating their effects on compound distribution within the zebrafish embryo. To visualize compound uptake and distribution, zebrafish embryos were exposed for 96 hr, starting at 4 hr postfertilization, to water-soluble dyes: Schiff's reagent (logP -4.63), Giemsa stain (logP -0.77), Van Gierson stain (logP 1.64), Cresyl fast violet (logP 3.5), Eosine Y (logP 4.8), Sudan III (logP 7.5), and Oil red O (logP 9.81), with and without 1% dimethyl-sulfoxide (DMSO). Three additional compounds were used to analytically determine the uptake and distribution: Acyclovir (logP -1.56), Zidovudine (logP 0.05), and Metoprolol Tartrate Salt (logP 1.8). Examinations were performed every 24 hr. Both methods (visualization and specific analysis) showed that exposure to higher logP values results in higher compound uptake. Specific analysis showed that for lipophilic compounds >90% of compound is taken up by the embryo. For hydrophilic compounds, >90% of compound within the complete egg could not be associated to embryo or chorion and is probably distributed into the perivitelline space. Overall, internal exposure analyses on at least two occasions (i.e., before and after hatching) is crucial for interpretation of zebrafish embryotoxicity data, especially for compounds with extreme logP values. DMSO did not affect exposure when examined with the visualization method, however, this method might be not sensitive enough to draw hard conclusions.

  6. Diploid oocyte formation and tetraploid embryo development induced by cytochalasin B in bovine.

    PubMed

    Bai, Chunling; Liu, Hui; Liu, Ying; Wu, Xia; Cheng, Lei; Bou, Shorgan; Li, Guang-Peng

    2011-02-01

    Tetraploid embryos are a useful model for postimplantation development of polyploidy cells, and tetraploid cells are an advantage in studies for chimeras yielding offspring completely derived from embryo stem cells or induced pluripotent cells. This study was designed to investigate the effects of cytochalasin B (CB) on bovine oocyte meiosis, and to induce the formation of diploid oocytes and tetraploid embryos. The results showed that: (1) incubation of oocytes in CB at ≥2.0 μg/mL concentrations for 24 h significantly decreased oocyte maturation and the matured oocytes' haploid composition. Over 50% of the CB-treated oocytes did not expel PB1 (non-PB1), and most of the non-PB1 oocytes contained 2n (60) chromosomes. (2) Pretreatment of oocytes with CB at concentrations of 7.5 and 15 μg/mL for 10 h significantly decreased oocyte maturation. Posttreatment of oocytes with CB resulted in most of the oocytes containing 2n chromosomes. (3) The parthenogenetic blastocysts (25-28%) derived from the non-PB1 oocytes of posttreatment group was significantly higher than that from pretreatment, whole period treatment, and the control oocytes (12-16%). (4) Cytogenetic analysis of the embryos derived from CB-treated non-PB1 oocytes resulted in 74% of the one-cell stage embryos being 4n = 120 chromosomes, 82% of two-cell stage embryos contained 4n chromosomes in each blastomere, and 75% of the blastocysts were tetraploidy (4n = 120). (6) The stopped uncleaved one-cell embryos showed an amazing phenomenon of over 15% of them containing extra chromosomes, which suggested multiple DNA duplication occurred within 40 h after activation. In conclusion, CB inhibits PB1 extrusion, disfigures spindle structure, decreases oocyte maturation, and results in formation of diploid (2n or 4c) oocytes. The diploid oocytes resulted in a higher development of tetraploid embryos, which would be a unique approach for the production of tetraploid embryos in bovine.

  7. Perifusion culture system for bovine embryos: improvement of embryo development by use of bovine oviduct epithelial cells, an antioxidant and polyvinyl alcohol.

    PubMed

    Lim, J M; Reggio, B C; Godke, R A; Hansel, W

    1997-01-01

    Three experiments were conducted in an attempt to improve a continuous flow-perifusion system capable of maintaining embryo development for long periods of time. Bovine embryos (8-16 cells) obtained from static co-culture with cumulus cells in a serum-free medium were perifused in an ACUSYST-S cell culture incubator. Culture chambers of the incubator consisted of a 0.2-mL unit (Chamber 1) connected to a 1.5-mL unit (Chamber 2), with the outflow from Chamber 1 routed to the inlet to Chamber 2. A bovine embryo culture medium supplemented with 3 mg mL-1 bovine serum albumin (BSA) and 25 mM HEPES was used as a perifusion culture medium (PCM). Embryos were perifused in Chamber 2 for 24, 48 and 72 h and further co-cultured in a static system up to 216 h after insemination. In Experiment 1, conditioning PCM with frozen-thawed bovine oviduct epithelial cells (BOEC) placed in Chamber 1 enhanced (P < 0.05) blastocyst formation of embryos in Chamber 2, after 24, 48 and 72 h of perifusion culture. The proportion of blastocysts was not further increased by placing BOEC in Chamber 2 along with the embryos. In Experiment 2, embryos were perifused with PCM conditioned with BOEC in Chamber 1 for 48 h or 72 h. A higher proportion of perifused embryos developed to the blastocyst stage after addition of 25 U mL-1 or 50 U mL-1 of superoxide dismutase (SOD) to PCM than in its absence. However, blastocyst formation of embryos perifused for 72 h was not increased after addition of 50 U mL-1 SOD compared with its absence. In Experiment 3, the proportions of morulae and blastocysts were not decreased by replacement of 3 mg mL-1 BSA with 1 mg mL-1 polyvinyl alcohol (PVA) in a BOEC-conditioned medium containing 50 U mL-1 SOD after perifusion for 48 h. In conclusion, PCM conditioning with BOEC and addition of an antioxidant to the perifusion medium improved the developmental capacity of perifused embryos. PVA is an adequate replacement for BSA in the perifusion medium.

  8. Semi-viviparous embryo development and dehydrin expression in the mangrove Rhizophora mucronata Lam.

    PubMed Central

    Ismail, Flora AbdulRahman; Nitsch, Lisette M. C.; Wolters-Arts, Mieke M. C.; Derksen, Jan W. M.

    2010-01-01

    Rhizophora mucronata Lam. is a tropical mangrove with semi-viviparous (cotyledon body protrusion before shedding), non-quiescent and non-desiccating (recalcitrant) seeds. As recalcitrance has been thought to relate to the absence of desiccation-related proteins such as dehydrins, we for the first time systematically described and classified embryogenesis in R. mucronata and assessed the presence of dehydrin-like proteins. Embryogenesis largely follows the classic pattern till stage eight, the torpedo stage, with the formation of a cotyledonary body. Ovule and embryo express radical adaptations to semi-vivipary in the saline environment: (1) A large, highly vacuolated and persistent endosperm without noticeable food reserves that envelopes the developing embryo. (2) Absence of vascular tissue connections between embryo and maternal tissue, but, instead, transfer layers in between endosperm and integument and endosperm and embryo. Dehydrin-like proteins (55–65 kDa) were detected by the Western analysis, in the ovules till stage 10 when the integuments are dehisced. An additional 50 kDa band was detected at stages 6–8. Together these results suggest a continuous flow of water with nutrients from the integument via the endosperm to the embryo, circumventing the vascular route and probably suppressing the initially induced dehydrin expression. PMID:20084524

  9. Survival and development of horseshoe crab (Limulus polyphemus) embryos and larvae in hypersaline conditions.

    PubMed

    Ehlinger, Gretchen S; Tankersley, Richard A

    2004-04-01

    The horseshoe crab Limulus polyphemus spawns in the mid- to upper intertidal zone where females deposit eggs in nests below the sediment surface. Although adult crabs generally inhabit subtidal regions of estuaries with salinities from 5 to 34 ppt, developing embryos and larvae within nests are often exposed to more extreme conditions of salinity and temperature during summer spawning periods. To test whether these conditions have a negative impact on early development and survival, we determined development time, survival, and molt cycle duration for L. polyphemus embryos and larvae raised at 20 combinations of salinity (range: 30-60 ppt) and temperature (range: 25-40 degrees C). Additionally, the effect of hyperosmotic and hypoosmotic shock on the osmolarity of the perivitelline fluid of embryos was determined at salinities between 5 and 90 ppt. The embryos completed their development and molted at salinities below 60 ppt, yet failed to develop at temperatures of 35 degrees C or higher. Larval survival was high at salinities of 10-70 ppt but declined significantly at more extreme salinities (i.e., 5, 80, and 90 ppt). Perivitelline fluid remained nearly isoosmotic over the range of salinities tested. Results indicate that temperature and salinity influence the rate of crab development, but only the extremes of these conditions have an effect on survival.

  10. Loblolly pine (Pinus taeda) female gametophyte and embryo pH changes during seed development.

    PubMed

    Pullman, Gerald S; Johnson, Shannon

    2009-06-01

    Stage-specific measurements of female gametophyte (FG) and embryo pH (hydrogen ion concentration) were made through the sequence of loblolly pine (Pinus taeda L.) seed development. The FG tissue from two open-pollinated trees showed similar pH profiles starting at 5.5 shortly after fertilization, increasing to about 6.1 at stage 7, levelling off at 6.3-6.5 towards the end of development and dropping to 6.0 just before cone opening. Measurements of the chalazal end were 0.05-0.2 pH units less than the micropylar end through early-to-mid-development. In contrast, embryo pH maintained a nearly constant value near 7.0 through development. Profiles of pH through seed development were similar whether portrayed by date or stage of embryo present in the seed. The pH profiles assisted in the development of improved embryogenic tissue initiation techniques. When post-autoclaving maturation medium pH was raised from about 5.3 in control medium to 5.7 or 5.5-5.7 with 2(n-morpholino)ethanesulphonic acid, cotyledonary embryo yields increased.

  11. Combined endosulfan and cypermethrin-induced toxicity to embryo-larval development of Rhinella arenarum.

    PubMed

    Svartz, Gabriela V; Aronzon, Carolina M; Pérez Coll, Cristina S

    2016-01-01

    The combined effects of two widely used pesticides, endosulfan and cypermethrin, on survival of embryo-larval development of the South American toad (Rhinella arenarum) were examined. The toxicity bioassays were performed according to the AMPHITOX test. Embryos and larvae were exposed to mixtures of these pesticides at equitoxic ratios from acute or chronic exposure to evaluate interaction effects. The results were analyzed using both Marking's additive index and combination index (CI)-isobologram methods. Acute (96-h) and intermediate (168-h) toxicity of endosulfan-cypermethrin mixtures remained almost constant for larvae and embryos, but when exposure duration was increased, there was a significant elevation in toxicity, obtaining chronic (240-h) no-observed-effect concentrations (NOEC) values of 0.045 and 0.16 mg/L for embryos and larvae, respectively. These are environmentally relevant concentrations that reflect a realistic risk of this pesticide mixture to this native amphibian species. The toxicity increment with the exposure duration was coincident with the central nervous system development on embryos reaching the larval period, the main target organ of these pesticides. The interactions of the pesticide mixtures at acute and chronic exposure were antagonistic for embryo development (CI > 1), and additive (CI = 1) for larvae, while chronic exposure interactions were synergistic (CI < 1) for both developmental periods. Data indicated that endosulfan-cypermethrin mixtures resulted in different interaction types depending on duration and developmental stage exposed. As a general pattern and considering conditions of overall developmental period and chronic exposure, this pesticide mixture usually applied in Argentine crop fields is synergistic with respect to toxicity for this native amphibian species.

  12. Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis.

    PubMed

    Bryant, Nicole; Lloyd, Johnny; Sweeney, Colleen; Myouga, Fumiyoshi; Meinke, David

    2011-04-01

    We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function.

  13. Genes and Conditions Controlling Mammalian Pre- and Post-implantation Embryo Development

    PubMed Central

    Anifandis, G.; Messini, C.I.; Dafopoulos, K.; Messinis, I.E.

    2015-01-01

    Embryo quality during the in vitro developmental period is of great clinical importance. Experimental genetic studies during this period have demonstrated the association between specific gene expression profiles and the production of healthy blastocysts. Although the quality of the oocyte may play a major role in embryo development, it has been well established that the post – fertilization period also has an important and crucial role in the determination of blastocyst quality. A variety of genes (such as OCT, SOX2, NANOG) and their related signaling pathways as well as transcription molecules (such as TGF-β, BMP) have been implicated in the pre- and post-implantation period. Furthermore, DNA methylation has been lately characterized as an epigenetic mark since it is one of the most important processes involved in the maintenance of genome stability. Physiological embryo development appears to depend upon the correct DNA methylation pattern. Due to the fact that soon after fertilization the zygote undergoes several morphogenetic and developmental events including activation of embryonic genome through the transition of the maternal genome, a diverse gene expression pattern may lead to clinically important conditions, such as apoptosis or the production of a chromosomically abnormal embryo. The present review focused on genes and their role during pre-implantation embryo development, giving emphasis on the various parameters that may alter gene expression or DNA methylation patterns. The pre-implantation embryos derived from in vitro culture systems (in vitro fertilization) and the possible effects on gene expression after the prolonged culture conditions are also discussed. PMID:25937812

  14. Environmental issues affecting CCT development

    SciTech Connect

    Reidy, M.

    1997-12-31

    While no final legislative schedule has been set for the new Congress, two issues with strong environmental ramifications which are likely to affect the coal industry seem to top the list of closely watched debates in Washington -- the Environmental Protection Agency`s proposed new ozone and particulate matter standards and utility restructuring. The paper discusses the background of the proposed standards, public comment, the Congressional review of regulations, other legislative options, and utility restructuring.

  15. EMAGE: a spatial database of gene expression patterns during mouse embryo development

    PubMed Central

    Christiansen, Jeffrey H.; Yang, Yiya; Venkataraman, Shanmugasundaram; Richardson, Lorna; Stevenson, Peter; Burton, Nicholas; Baldock, Richard A.; Davidson, Duncan R.

    2006-01-01

    EMAGE () is a freely available, curated database of gene expression patterns generated by in situ techniques in the developing mouse embryo. It is unique in that it contains standardized spatial representations of the sites of gene expression for each gene, denoted against a set of virtual reference embryo models. As such, the data can be interrogated in a novel and abstract manner by using space to define a query. Accompanying the spatial representations of gene expression patterns are text descriptions of the sites of expression, which also allows searching of the data by more conventional text-based methods. PMID:16381949

  16. EMAGE: a spatial database of gene expression patterns during mouse embryo development.

    PubMed

    Christiansen, Jeffrey H; Yang, Yiya; Venkataraman, Shanmugasundaram; Richardson, Lorna; Stevenson, Peter; Burton, Nicholas; Baldock, Richard A; Davidson, Duncan R

    2006-01-01

    EMAGE (http://genex.hgu.mrc.ac.uk/Emage/database) is a freely available, curated database of gene expression patterns generated by in situ techniques in the developing mouse embryo. It is unique in that it contains standardized spatial representations of the sites of gene expression for each gene, denoted against a set of virtual reference embryo models. As such, the data can be interrogated in a novel and abstract manner by using space to define a query. Accompanying the spatial representations of gene expression patterns are text descriptions of the sites of expression, which also allows searching of the data by more conventional text-based methods.

  17. Probing cell mechanics with subcellular laser dissection of actomyosin networks in the early developing Drosophila embryo.

    PubMed

    Rauzi, M; Lenne, P-F

    2015-01-01

    Laser dissection is a useful tool in developmental biology to probe mechanical forces from the subcellular to the tissue/embryo scale. During tissue morphogenesis, cells are equipped with networks of actomyosin that generate forces. Here we present a technique based on near-infrared (NIR) femtosecond (fs) pulsed laser dissection that allows subcellular ablation of actomyosin networks. This technique allows to selectively ablate actomyosin networks while preserving cell plasma membrane. The resulting relaxation of the remaining network after laser dissection is imaged and analyzed to deduce local forces responsible for tissue morphogenesis in the developing Drosophila embryo.

  18. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development

    PubMed Central

    Kwon, Jeongwoo; Jeong, Sung-min; Choi, Inchul; Kim, Nam-Hyung

    2016-01-01

    ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10) is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD) of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ), and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR), supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development. PMID:27043020

  19. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development.

    PubMed

    Kwon, Jeongwoo; Jeong, Sung-min; Choi, Inchul; Kim, Nam-Hyung

    2016-01-01

    ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10) is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD) of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ), and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR), supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development.

  20. The importance of pyridoxine for the impact of the dietary selenium sources on redox balance, embryo development, and reproductive performance in gilts.

    PubMed

    Dalto, Danyel Bueno; Audet, Isabelle; Lapointe, Jérôme; Matte, J Jacques

    2016-03-01

    This study aimed to determine the effects of dietary pyridoxine and selenium (Se) on embryo development, reproductive performance and redox system in gilts. Eighty-four gilts were fed one of five diets: CONT) basal diet; MSeB60) CONT+0.3mg/kg of Na-selenite; MSeB610) diet 2+10mg/kg of HCl-pyridoxine; OSeB60) CONT+0.3mg/kg of Se-enriched yeast; and OSeB610) diet 4+10mg/kg of HCl-pyridoxine. Blood samples were collected for long-term (each estrus and slaughter) and peri-estrus (fourth estrus d -4 to d +3) profiles. At slaughter (gestation d 30), organs and embryos were collected. For long-term and peri-estrus profiles, Se level and source affected (P<0.01) blood Se concentration whereas B6 level increased (P<0.01) erythrocyte pyridoxal-5-phosphate concentration. A B6 level (P<0.05) effect was observed on long-term plasma Se-dependent glutathione peroxidase (Se-GPX) activity whereas peri-estrus Se-GPX was minimum on d -1 (P<0.01). Selenium level increased sows' organs and embryo Se concentration (P<0.01). Selenium source tended to enhance embryo Se content (P=0.06). Within-litter embryo Se content was increased by B6 level (P<0.01). Selenium level tended to affect Se-GPX and total GPX activities in organs mitochondria (P=0.09 and 0.07, respectively). Selenium source affected kidney ATP synthesis (P=0.05). In conclusion, B6 level affected the Se-GPX activity on a long-term basis, whereas the basal level of Se was adequate during the peri-estrus period. Embryo quality was not improved by dietary Se, and B6 impaired within-litter homogeneity.

  1. Developing Xenopus embryos recover by compacting and expelling single wall carbon nanotubes.

    PubMed

    Holt, Brian D; Shawky, Joseph H; Dahl, Kris Noel; Davidson, Lance A; Islam, Mohammad F

    2016-04-01

    Single wall carbon nanotubes are high aspect ratio nanomaterials being developed for use in materials, technological and biological applications due to their high mechanical stiffness, optical properties and chemical inertness. Because of their prevalence, it is inevitable that biological systems will be exposed to nanotubes, yet studies of the effects of nanotubes on developing embryos have been inconclusive and are lacking for single wall carbon nanotubes exposed to the widely studied model organism Xenopus laevis (African clawed frog). Microinjection of experimental substances into the Xenopus embryo is a standard technique for toxicology studies and cellular lineage tracing. Here we report the surprising finding that superficial (12.5 ± 7.5 µm below the membrane) microinjection of nanotubes dispersed with Pluronic F127 into one- to two-cell Xenopus embryos resulted in the formation and expulsion of compacted, nanotube-filled, punctate masses, at the blastula to mid-gastrula developmental stages, which we call "boluses." Such expulsion of microinjected materials by Xenopus embryos has not been reported before and is dramatically different from the typical distribution of the materials throughout the progeny of the microinjected cells. Previous studies of microinjections of nanomaterials such as nanodiamonds, quantum dots or spherical nanoparticles report that nanomaterials often induce toxicity and remain localized within the embryos. In contrast, our results demonstrate an active recovery pathway for embryos after exposure to Pluronic F127-coated nanotubes, which we speculate is due to a combined effect of the membrane activity of the dispersing agent, Pluronic F127, and the large aspect ratio of nanotubes.

  2. In vitro development of bison embryos using interspecies somatic cell nuclear transfer.

    PubMed

    Seaby, R P; Alexander, B; King, W A; Mastromonaco, G F

    2013-12-01

    Interspecies somatic cell nuclear transfer (interspecies SCNT) has been explored in many domestic and non-domestic animal species. However, problems arise during the development of these embryos, which may be related to species-specific differences in nuclear-cytoplasmic communication. The objectives of this study were to investigate the possibility of producing bison embryos in vitro using interspecies SCNT and assess the developmental potential of these embryos. Treatment groups consisted of cattle in vitro fertilization (IVF) and cattle SCNT as controls and wood bison SCNT, plains bison SCNT and wisent SCNT as experimental groups. Cleavage and blastocyst rates were assessed, and blastocyst quality was determined using total cell number, apoptotic incidence and relative quantification of mitochondria-related genes NRF1, MT-CYB and TFAM. These results indicate that embryos can be produced by interspecies SCNT in all bison species/subspecies (13.34-33.54% blastocyst rates). Although increased incidence of apoptosis was observed in bison SCNT blastocysts compared to cattle SCNT controls (10.45-12.69 vs 8.76, respectively) that corresponded with significantly lower cell numbers (80-87 cells vs >100 cells, respectively), no major differences were observed in the expression of NRF1, MT-CYB and TFAM. This study is the first to report the production of bison embryos by interspecies SCNT. Blastocyst development in all three bison species/subspecies was greater than the rates obtained in previous studies by IVF, which supports the potential role of SCNT for in vitro embryo production in this species. Yet, further investigation of developmental competence and the factors influencing blastocyst quality and viability is required.

  3. Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression.

    PubMed

    Moro, L N; Hiriart, M I; Buemo, C; Jarazo, J; Sestelo, A; Veraguas, D; Rodriguez-Alvarez, L; Salamone, D F

    2015-07-01

    The aim of this study was to evaluate the capacity of domestic cat (Dc, Felis silvestris) oocytes to reprogram the nucleus of cheetah (Ch, Acinonyx jubatus) cells by interspecies SCNT (iSCNT), by using embryo aggregation. Dc oocytes were in vitro matured and subjected to zona pellucida free (ZP-free) SCNT or iSCNT, depending on whether the nucleus donor cell was of Dc or Ch respectively. ZP-free reconstructed embryos were then cultured in microwells individually (Dc1X and Ch1X groups) or in couples (Dc2X and Ch2X groups). Embryo aggregation improved in vitro development obtaining 27.4, 47.7, 16.7 and 28.3% of blastocyst rates in the Dc1X, Dc2X, Ch1X and Ch2X groups, respectively (P<0.05). Moreover, aggregation improved the morphological quality of blastocysts from the Dc2X over the Dc1X group. Gene expression analysis revealed that Ch1X and Ch2X blastocysts had significantly lower relative expression of OCT4, CDX2 and NANOG than the Dc1X, Dc2X and IVF control groups. The OCT4, NANOG, SOX2 and CDX2 genes were overexpressed in Dc1X blastocysts, but the relative expression of these four genes decreased in the Dc2X, reaching similar relative levels to those of Dc IVF blastocysts. In conclusion, Ch blastocysts were produced using Dc oocytes, but with lower relative expression of pluripotent and trophoblastic genes, indicating that nuclear reprogramming could be still incomplete. Despite this, embryo aggregation improved the development of Ch and Dc embryos, and normalized Dc gene expression, which suggests that this strategy could improve full-term developmental efficiency of cat and feline iSCNT embryos.

  4. Light enables a very high efficiency of carbon storage in developing embryos of rapeseed.

    PubMed

    Goffman, Fernando D; Alonso, Ana P; Schwender, Jörg; Shachar-Hill, Yair; Ohlrogge, John B

    2005-08-01

    The conversion of photosynthate to seed storage reserves is crucial to plant fitness and agricultural production, yet quantitative information about the efficiency of this process is lacking. To measure metabolic efficiency in developing seeds, rapeseed (Brassica napus) embryos were cultured in media in which all carbon sources were [U-14C]-labeled and their conversion into CO2, oil, protein, and other biomass was determined. The conversion efficiency of the supplied carbon into seed storage reserves was very high. When provided with 0, 50, or 150 micromol m(-2) s(-1) light, the proportion of carbon taken up by embryos that was recovered in biomass was 60% to 64%, 77% to 86%, and 85% to 95%, respectively. Light not only improved the efficiency of carbon storage, but also increased the growth rate, the proportion of 14C recovered in oil relative to protein, and the fixation of external 14CO2 into biomass. Embryos grown at 50 micromol m(-2) s(-1) in the presence of 5 microM 1,1-dimethyl-3-(3,4-dichlorophenyl) urea (an inhibitor of photosystem II) were reduced in total biomass and oil synthesis by 3.2-fold and 2.8-fold, respectively, to the levels observed in the dark. To explore if the reduced growth and carbon conversion efficiency in dark were related to oxygen supplied by photosystem II, embryos and siliques were cultured with increased oxygen. The carbon conversion efficiency of embryos remained unchanged when oxygen levels were increased 3-fold. Increasing the O2 levels surrounding siliques from 21% to 60% did not increase oil synthesis rates either at 1,000 micromol m(-2) s(-1) or in the dark. We conclude that light increases the growth, efficiency of carbon storage, and oil synthesis in developing rapeseed embryos primarily by providing reductant and/or ATP.

  5. The indeterminate gametophyte1 Gene of Maize Encodes a LOB Domain Protein Required for Embryo Sac and Leaf Development[W

    PubMed Central

    Evans, Matthew M.S.

    2007-01-01

    Angiosperm embryo sac development begins with a phase of free nuclear division followed by cellularization and differentiation of cell types. The indeterminate gametophyte1 (ig1) gene of maize (Zea mays) restricts the proliferative phase of female gametophyte development. ig1 mutant female gametophytes have a prolonged phase of free nuclear divisions leading to a variety of embryo sac abnormalities, including extra egg cells, extra polar nuclei, and extra synergids. Positional cloning of ig1 was performed based on the genome sequence of the orthologous region in rice. ig1 encodes a LATERAL ORGAN BOUNDARIES domain protein with high similarity to ASYMMETRIC LEAVES2 of Arabidopsis thaliana. A second mutant allele of ig1 was identified in a noncomplementation screen using active Mutator transposable element lines. Homozygous ig1 mutants have abnormal leaf morphology as well as abnormal embryo sac development. Affected leaves have disrupted abaxial–adaxial polarity and fail to repress the expression of meristem-specific knotted-like homeobox (knox) genes in leaf primordia, causing a proliferative, stem cell identity to persist in these cells. Despite the superficial similarity of ig1-O leaves and embryo sacs, ectopic knox gene expression cannot be detected in ig1-O embryo sacs. PMID:17209126

  6. Effects of Di-butyl Phthalate (DBP) on Developing Medaka Embryos

    ERIC Educational Resources Information Center

    Tang, Sherry

    2012-01-01

    Plasticizers are chemical additives that enhance plastic flexibility. They are ubiquitous environmental contaminants and are commonly found in river and lake waters (Fromme et al 2002). The present study aimed to investigate the effects of a water-soluble plasticizer, dibutyl phthalate (DBP) on developing Medaka ("Oryzias latipes") embryos. Three…

  7. Simulated Microgravity Influences Bovine Oocyte In Vitro Fertilization and Preimplantation Embryo Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to investigate whether in vitro fertilization and preimplantation embryos exposed to a simulated microgravity environment in vitro would improve, or be deleterious to, their fertilization and embryonic development. A Rotating Cell Culture System™ (RCCS) bioreactor with a Hi...

  8. N, N-Dimethylglycine decreases oxidative stress and improves in vitro development of bovine embryos

    PubMed Central

    TAKAHASHI, Toshikiyo; SASAKI, Kouya; SOMFAI, Tamas; NAGAI, Takashi; MANABE, Noboru; EDASHIGE, Keisuke

    2016-01-01

    The antioxidant effect of N, N-dimethylglycine (DMG) on in vitro-produced (IVP) bovine embryos was examined. After in vitro fertilization, presumptive zygotes were cultured with or without 0.1 μM DMG under different oxygen tensions. The percentage of embryos developing to the blastocyst stage was lowest under a 20% oxygen concentration without DMG, and it was significantly increased (P < 0.05) by applying a 5% oxygen concentration. Under the 20% oxygen concentration, supplementation of the medium with DMG significantly improved blastocyst development, which was nearly equal to that achieved under 5% oxygen without DMG. Furthermore, a tendentious increase (P = 0.06) in blastocyst cell numbers was observed when DMG was applied. In the second experiment, addition of H2O2 (0.5 mM) to the culture medium significantly (P < 0.01) reduced the percentage of embryos developing to the blastocyst stage. However, DMG supplementation prevented this reduction. In conclusion, DMG enhanced the in vitro development of IVP bovine embryos by acting as an antioxidant. PMID:26875568

  9. Phospholipid transfer activities in toad oocytes and developing embryos. [Bufo arenarum

    SciTech Connect

    Rusinol, A.; Salomon, R.A.; Bloj, B.

    1987-01-01

    The role of lipid transfer proteins during plasma membrane biogenesis was explored. Developing amphibia embryos were used because during their growth an active plasma membrane biosynthesis occurs together with negligible mitochondrial and endoplasmic reticulum proliferation. Sonicated vesicles, containing /sup 14/C-labeled phospholipids and /sup 3/H-labeled triolein, as donor particles and cross-linked erythrocyte ghosts as acceptor particles were used to measure phospholipid transfer activities in unfertilized oocytes and in developing embryos of the toad Bufo arenarum. Phosphatidylcholine transfer activity in pH 5.1 supernatant of unfertilized oocytes was 8-fold higher than the activity found in female toad liver supernatant, but dropped steadily after fertilization. After 20 hr of development, at the stage of late blastula, the phosphatidylcholine transfer activity had dropped 4-fold. Unfertilized oocyte supernatant exhibited phosphatidylinositol and phosphatidylethanolamine transfer activity also, but at the late blastula stage the former had dropped 18-fold and the latter was no longer detectable under our assay conditions. Our results show that fertilization does not trigger a phospholipid transport process catalyzed by lipid transfer proteins. Moreover, they imply that 75% of the phosphatidylcholine transfer activity and more than 95% of the phosphatidylinositol and phosphatidylethanolamine transfer activities present in pH 5.1 supernatants of unfertilized oocytes may not be essential for toad embryo development. Our findings do not rule out, however, that a phosphatidylcholine-specific lipid transfer protein could be required for embryo early growth.

  10. Pre-fertilization zona pellucida hardening by different cross-linkers affects IVF in pigs and cattle and improves embryo production in pigs.

    PubMed

    Canovas, Sebastian; Romar, Raquel; Grullon, Luis Alberto; Aviles, Manuel; Coy, Pilar

    2009-05-01

    Zona pellucida (ZP) hardening (resistance to proteolysis) has been classically identified as a post-fertilization event that contributes to the block to polyspermy. Di-(N-succinimidyl)-3,3'-dithiodipropionate (DSP), a permeable amine-reactive cross-linker, was recently shown to induce pre-fertilization ZP hardening and to improve porcine IVF productivity. The objectives of this study were to investigate i) how DSP affects pre-fertilization ZP hardening and IVF in cattle, ii) if a non-permeable amine-reactive cross-linker such as bis(sulfosuccinimidyl) suberate (BS3) affects ZP hardening and IVF in cattle and pigs, and iii) whether DSP or BS3, if improvement in IVF productivity was demonstrated in either species, affects in vitro embryo development. Bovine and porcine in vitro matured oocytes were incubated with the cross-linkers (0.06, 0.3, and 0.6 mg/ml) for 30 min. Then they were subjected to ZP digestion or IVF. In cattle, both DSP and BS3 induced ZP hardening and decreased the penetration rate, although monospermy, penetration, or male pronuclear formation was not affected. In pigs, BS3 treatment induced ZP hardening, decreased penetration and male pronuclear formation, and increased monospermy. IVF productivity only improved when porcine oocytes were exposed to DSP. When porcine zygotes derived from this treatment were further cultured in vitro, the cleavage and blastocyst formation rates increased. These results support the idea that mechanisms involved in the prevention of polyspermic fertilization in cattle and pigs have different efficiencies, and ZP hardening induced by DSP cross-linker may be useful for improving porcine embryo production.

  11. Is the zona pellucida an intrinsic source of signals activating maternal recognition of the developing mammalian embryo?

    PubMed

    Fujiwara, Hiroshi; Araki, Yoshihiko; Toshimori, Kiyotaka

    2009-07-01

    Mammalian mothers undergoing embryo implantation must specifically recognize the developing embryo in a species-restricted manner. We previously observed that immune cells derived from early pregnant mice could promote endometrial differentiation and embryo implantation in blastocyst-transferred pseudopregnant mice. Although the precise mechanism remains unknown, it is suggested that the maternal immune system undergoes functional changes after recognizing developing embryos from the very early stages of pregnancy. Since it is physically impossible for immune cells to directly interact with the developing embryo while it is surrounded by the zona pellucida (ZP), it is speculated that the embryo produces certain embryo- and species-specific soluble factor(s) in the oviduct before hatching. As a candidate for this factor, we have paid attention to the ZP that is normally protected from immunological attack during oogenesis in the ovarian follicle. ZP-specific glycoproteins are known to play important roles in the species- and oocyte-specific binding of sperm, and the ZP can also be considered an abundant store of oocyte- and species-specific glycoproteins. In contrast to unfertilized oocytes, developing embryos may degrade the ZP starting just after fertilization and proceeding until hatching using enzymes that are released from cortical granules or produced by the developing embryo. Accordingly, the developing embryo might provide ZP-degradation products including oligosaccharide chains to the immune system from the very early stages. Taken together, we propose here a novel hypothesis that these ZP-derivatives can act as an intrinsic signal from the developing embryo for maternal recognition by the immune system.

  12. Transmission and Scanning Electron Microscopy of the Accessory Cells and Chorion During Development of Ciona intestinalis Type B Embryos and the Impact of Their Removal on Cell Morphology.

    PubMed

    Thompson, Helen; Shimeld, Sebastian M

    2015-06-01

    Spawned ascidian oocytes are surrounded by a membrane called the chorion (or vitelline coat) and associated with two populations of maternally-supplied cells. Outside the chorion are follicle cells, which may affect the buoyancy of eggs. Inside the chorion are test cells, which during oogenesis provision the egg and which after fertilisation contribute to the larval tunic. The structure of maternal cells may vary between species. The model ascidian Ciona intestinalis has been recently split into two species, currently named type A and type B. The ultrastructure of extraembryonic cells and structures from type A embryos has been reported. Here we describe the ultrastructure of follicle and test cells from C. intestinalis type B embryos. Test cells are about 5 µm in diameter and line the inside of the chorion of developing embryos in a dense sheet. Follicle cells are large (> 100 µm long) and spike-shaped, with many large vesicles. Terminal electron dense granules are found towards the tips of spikes, adjacent to cytoplasm containing numerous small electron dense bodies connected by filaments. These are probably vesicles containing material for the terminal granules. Removal of maternal structures and cells just after fertilisation, as commonly used in many experiments manipulating C. intestinalis development, has been reported to affect embryonic patterning. We examined the impact of this on embryonic ectoderm cells by scanning electron microscopy. Cells of embryos that developed without maternal structures still developed cilia, but had indistinct cell boundaries and a more flattened appearance than those that developed within the chorion.

  13. Establishment of a chick embryo model for analyzing liver development and a search for candidate genes.

    PubMed

    Yokouchi, Yuji

    2005-08-01

    The liver plays a crucial role in metabolism. There is considerable interest in how the liver develops, as such knowledge could prove of importance in regenerative medicine. However, our understanding of liver development remains somewhat limited. We have developed a model system using the chick embryo that is cost effective and is easy to manipulate experimentally. We performed four fundamental studies: (i) construction of an atlas of the developing chick liver; (ii) identification of differentiation marker genes in the developing chick embryo; (iii) development of germ-layer specific electroporation; and (iv) establishment of organ culture from the developing chick liver. Using this system, we have been able to demonstrate the functions of candidate genes within a shorter period and in a more cost-effective manner. In parallel with the establishment of this system, we examined the expression patterns of genes known to be required for organ development in the developing chick embryo in order to identify genes also involved in liver development. To date, we have found sixteen genes that are expressed in the developing chick liver (GELD, genes expressed in liver development). This knowledge will be fundamental to the establishment of the basic technology for engineering liver tissue in the future.

  14. Improving metabolic health in obese male mice via diet and exercise restores embryo development and fetal growth.

    PubMed

    McPherson, Nicole O; Bakos, Hassan W; Owens, Julie A; Setchell, Brian P; Lane, Michelle

    2013-01-01

    Paternal obesity is now clearly associated with or causal of impaired embryo and fetal development and reduced pregnancy rates in humans and rodents. This appears to be a result of reduced blastocyst potential. Whether these adverse embryo and fetal outcomes can be ameliorated by interventions to reduce paternal obesity has not been established. Here, male mice fed a high fat diet (HFD) to induce obesity were used, to determine if early embryo and fetal development is improved by interventions of diet (CD) and/or exercise to reduce adiposity and improve metabolism. Exercise and to a lesser extent CD in obese males improved embryo development rates, with increased cell to cell contacts in the compacting embryo measured by E-cadherin in exercise interventions and subsequently, increased blastocyst trophectoderm (TE), inner cell mass (ICM) and epiblast cell numbers. Implantation rates and fetal development from resulting blastocysts were also improved by exercise in obese males. Additionally, all interventions to obese males increased fetal weight, with CD alone and exercise alone, also increasing fetal crown-rump length. Measures of embryo and fetal development correlated with paternal measures of glycaemia, insulin action and serum lipids regardless of paternal adiposity or intervention, suggesting a link between paternal metabolic health and subsequent embryo and fetal development. This is the first study to show that improvements to metabolic health of obese males through diet and exercise can improve embryo and fetal development, suggesting such interventions are likely to improve offspring health.

  15. Assessment of DNA damage during in vitro development of buffalo (Bubalus bubalis) embryos: effect of cysteamine.

    PubMed

    Mukherjee, A; Kumar, D; Singh, K P; Chauhan, M S; Singla, S K; Palta, P; Manik, R S

    2010-12-01

    Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 μM, respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8- to 16-cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8- to 16- (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2-cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8- to 16-cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2-cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H2O2 caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.

  16. Melatonin rescues cardiovascular dysfunction during hypoxic development in the chick embryo.

    PubMed

    Itani, Nozomi; Skeffington, Katie L; Beck, Christian; Niu, Youguo; Giussani, Dino A

    2016-01-01

    There is a search for rescue therapy against fetal origins of cardiovascular disease in pregnancy complicated by chronic fetal hypoxia, particularly following clinical diagnosis of fetal growth restriction (FGR). Melatonin protects the placenta in adverse pregnancy; however, whether melatonin protects the fetal heart and vasculature in hypoxic pregnancy independent of effects on the placenta is unknown. Whether melatonin can rescue fetal cardiovascular dysfunction when treatment commences following FGR diagnosis is also unknown. We isolated the effects of melatonin on the developing cardiovascular system of the chick embryo during hypoxic incubation. We tested the hypothesis that melatonin directly protects the fetal cardiovascular system in adverse development and that it can rescue dysfunction following FGR diagnosis. Chick embryos were incubated under normoxia or hypoxia (14% O2) from day 1 ± melatonin treatment (1 mg/kg/day) from day 13 of incubation (term ~21 days). Melatonin in hypoxic chick embryos rescued cardiac systolic dysfunction, impaired cardiac contractility and relaxability, increased cardiac sympathetic dominance, and endothelial dysfunction in peripheral circulations. The mechanisms involved included reduced oxidative stress, enhanced antioxidant capacity and restored vascular endothelial growth factor expression, and NO bioavailability. Melatonin treatment of the chick embryo starting at day 13 of incubation, equivalent to ca. 25 wk of gestation in human pregnancy, rescues early origins of cardiovascular dysfunction during hypoxic development. Melatonin may be a suitable antioxidant candidate for translation to human therapy to protect the fetal cardiovascular system in adverse pregnancy.

  17. Microdrop preparation factors influence culture-media osmolality, which can impair mouse embryo preimplantation development.

    PubMed

    Swain, J E; Cabrera, L; Xu, X; Smith, G D

    2012-02-01

    Because media osmolality can impact embryo development, the effect of conditions during microdrop preparation on osmolality was examined. Various sizes of microdrops were prepared under different laboratory conditions. Drops were pipetted directly onto a dish and covered by oil (standard method) or pipetted on the dish, overlaid with oil before removing the underlying media and replaced with fresh media (wash-drop method). Drops were made at 23°C or on a heated stage (37°C) and with or without airflow. Osmolality was assessed at 5 min and 24h. The biological impact of osmolality change was demonstrated by culturing 1-cell mouse embryos in media with varying osmolality. Reduced drop volume, increased temperature and standard method were associated with a significant increase in osmolality at both 5 min and 24h (P-values <0.001, <0.0001 and <0.0001, respectively). There was a significant interaction between airflow, decreased volume, increased temperature and standard method that caused a significant increase in osmolality (40mOsm/kg) compared with controls (P<0.04). There was no significant change in osmolality over time. Mouse embryo development was significantly reduced in media with elevated osmolality (>310mOsm/kg; P<0.05). Procedures in the IVF laboratory can alter osmolality and impact embryo development.

  18. Green fluorescent protein gene-transfected peafowl somatic cells participate in the development of chicken embryos.

    PubMed

    Xi, Yongmei; Nada, Yoich; Soh, Tomoki; Fujihara, Noboru; Hattori, Masa-Aki

    2004-02-01

    This study was performed to investigate whether the embryonic somatic cells are capable of reconstituting and participating in the embryonic development of chickens to produce chimeras. In order to track the migration behavior of the donor cells, a cell line, originally isolated from an Indian peafowl embryo, was fluorescent-labeled by transfection of the cells with enhanced Green Fluorescent Protein (GFP) and Neomycin resistant (Neo) genes prior to injection into the stage X blastoderm of White Leghorn chickens. The injection was performed with a medium in the presence of 1-5% polyethylene glycol. The development of putative chimeric embryos between the stages three and 24 was examined for GFP expression under fluorescent light. To trace the peafowl cells in the developing chicken embryos, both a species-specific genetic marker originating from the mitochondrial DNA cytochrome b (cyt b) gene and a DNA fragment of GFP gene were used. Of the 185 fertile eggs manipulated, 173 developed into embryos. Fifty-five of them showed positive GFP patches in extra-embryonic tissues, and 15 expressed GFP in intra-embryonic tissues such as those of the head, heart, and gonad. PCR analysis revealed that PCR fragments for the peafowl mitochondrial DNA cyt b and GFP genes were detected in the samples of the GFP positive extra- and intra-embryonic tissues of the chimeras. The present results provide evidence that fluorescent-labeled peafowl embryonic cells carrying GFP and Neo genes are able to participate in the development of chicken embryos to generate chimeras.

  19. Quantitative comparison of cerebral artery development in human embryos with other eutherians.

    PubMed

    Ashwell, Ken W S; Shulruf, Boaz

    2015-09-01

    The embryonic and early fetal human brain is known to undergo extraordinary expansion of its cellular population during embryonic and early fetal life, and is critically dependant on a steady supply of nutrients and oxygen for proper brain development. Quantitative analysis of the internal radius of the aorta and cerebral arteries in a range of eutherian mammals has been used to compare arterial flow to the developing human brain with that to the brains of non-human eutherians. Human embryos showed a much steeper rise of internal radius of the aorta with increasing body size than the embryos of non-human eutherians, but the thickness of the aorta rose at the same pace relative to body size in both humans and non-humans, suggesting that aortic pressure is similar in all eutherian embryos of a similar size. The sums of internal radii of both the internal carotids and vertebral arteries of human embryos raised to the fourth power were much lower at embryonic stages (less than 22 mm body length) than in non-human eutherians, were similar between humans and non-humans at 22-30 mm body length, and exceeded the non-humans at body lengths of more than 30 mm. The relative size of the internal calibre of the cerebral feeder arteries (internal carotid and vertebral) to the aorta did not change between embryonic and fetal sizes in either humans or non-humans. The findings suggest that the developing human brain may actually receive less blood flow at embryonic sizes (less than 22 mm body length) than do other mammalian embryos of a similar body size, but that internal carotid and vertebral flow is higher in human fetuses (body length greater than 30 mm) than in developing non-humans of the same body size. Increased flow to the developing human brain relative to non-humans is achieved by simultaneous increases in both aortic and cerebral feeder artery internal calibre.

  20. A role for biliverdin IXalpha in dorsal axis development of Xenopus laevis embryos.

    PubMed

    Falchuk, Kenneth H; Contin, Jennifer M; Dziedzic, T Scott; Feng, Zhongling; French, Thayer C; Heffron, Gregory J; Montorzi, Marcelo

    2002-01-08

    The determinants of Xenopus laevis embryos that act before their first cell division are mandatory for the formation of mRNas required to establish the dorsal axis. Although their chemical identities are unknown, a number of their properties have long been recognized. One of the determinants is present in the cytoplasm and is sensitive to UV light. Thus, exposing stage 1 embryos to either standard 254-nm or, as shown here, to 366-nm UV light during the 0.3-0.4 time fraction of their first cycle inactivates the cytoplasmic determinant. As a consequence, both types of irradiated embryos fail to express dorsal markers, e.g., goosecoid and chordin, without affecting formation of ventral markers, e.g., Vent-1. The developmental outcome is dorsal axis-deficient morphology. We report here that biliverdin IXalpha, a normal constituent of cytoplasmic yolk platelets, is photo-transformed by irradiation with either 254- or 366-nm UV light and that the transformation triggers the dorsal axis deficiency. When the 254- or 366-nm UV-irradiated embryos, fated to dorsal axis deficiency, are incubated solely with microM amounts of biliverdin, they recover and form the axis. In contrast, incubation with either in vitro photo-transformed biliverdin or biliverdin IXalpha dimethyl ester does not induce recovery. The results define an approach to produce dorsal axis-deficient embryos by photo-transforming its biliverdin by irradiation with 366-nm UV light and identify an unsuspected role for biliverdin IXalpha in X. laevis embryogenesis.

  1. Reading Enjoyment and Affective Development.

    ERIC Educational Resources Information Center

    Reporting on Reading, 1978

    1978-01-01

    The articles in this publication offer ideas for developing enjoyment of reading in children. Among the topics discussed are the following: the need for teachers and parents to build children's self-esteem through increasing their experiences of success, their expectations of success, and the value they place on reading; methods for increasing…

  2. Effects of industrial effluents, heavy metals, and organic solvents on mallard embryo development

    USGS Publications Warehouse

    Hoffman, D.J.; Eastin, W.C.

    1981-01-01

    Mallard eggs were externally exposed at 3 and 8 days of incubation to 7 different industrial effluents and to 7 different heavy metal, organic solvent, and petroleum solutions to screen for potential embryo-toxic effects. This route of exposure was chosen in order to simulate the transfer of pollutant from the plumage of aquatic birds to their eggs. Five of the effluents including mineral pigment, scouring effluent, sludge, and tannery effluent resulted in small but significant reductions in embryonic growth. Treatment with methyl mercury chloride solution of 50 ppm (Hg) impaired embryonic growth but much higher concentrations were required to affect survival and cause teratogenic effects. Oil used to suppress road dust was the most toxic of the pollutants tested and only 0.5 microliter/egg caused 60% mortality by 18 days of development. These findings, in combination with other studies suggest that petroleum pollutants, or effluents in combination with petroleum, may pose a hazard to birds' eggs when exposure is by this route.

  3. Stress response to cadmium and manganese in Paracentrotus lividus developing embryos is mediated by nitric oxide.

    PubMed

    Migliaccio, Oriana; Castellano, Immacolata; Romano, Giovanna; Palumbo, Anna

    2014-11-01

    Increasing concentrations of contaminants, often resulting from anthropogenic activities, have been reported to occur in the marine environment and affect marine organisms. Among these, the metal ions cadmium and manganese have been shown to induce developmental delay and abnormalities, mainly reflecting skeleton elongation perturbation, in the sea urchin Paracentrotus lividus, an established model for toxicological studies. Here, we provide evidence that the physiological messenger nitric oxide (NO), formed by l-arginine oxidation by NO synthase (NOS), mediates the stress response induced by cadmium and manganese in sea urchins. When NO levels were lowered by inhibiting NOS, the proportion of abnormal plutei increased. Quantitative expression of a panel of 19 genes involved in stress response, skeletogenesis, detoxification and multidrug efflux processes was followed at different developmental stages and under different conditions: metals alone, metals in the presence of NOS inhibitor, NO donor and NOS inhibitor alone. These data allowed the identification of different classes of genes whose metal-induced transcriptional expression was directly or indirectly mediated by NO. These results open new perspectives on the role of NO as a sensor of different stress agents in sea urchin developing embryos.

  4. Expression profiling identifies novel Hh/Gli-regulated genes in developing zebrafish embryos.

    PubMed

    Bergeron, Sadie A; Milla, Luis A; Villegas, Rosario; Shen, Meng-Chieh; Burgess, Shawn M; Allende, Miguel L; Karlstrom, Rolf O; Palma, Verónica

    2008-02-01

    The Hedgehog (Hh) signaling pathway plays critical instructional roles during embryonic development. Misregulation of Hh/Gli signaling is a major causative factor in human congenital disorders and in a variety of cancers. The zebrafish is a powerful genetic model for the study of Hh signaling during embryogenesis, as a large number of mutants that affect different components of the Hh/Gli signaling system have been identified. By performing global profiling of gene expression in different Hh/Gli gain- and loss-of-function scenarios we identified known (e.g., ptc1 and nkx2.2a) and novel Hh-regulated genes that are differentially expressed in embryos with altered Hh/Gli signaling function. By uncovering changes in tissue-specific gene expression, we revealed new embryological processes that are influenced by Hh signaling. We thus provide a comprehensive survey of Hh/Gli-regulated genes during embryogenesis and we identify new Hh-regulated genes that may be targets of misregulation during tumorigenesis.

  5. Development of chrysin loaded poloxamer micelles and toxicity evaluation in fish embryos.

    PubMed

    Sassa-Deepaeng, Tanongsak; Pikulkaew, Surachai; Okonogi, Siriporn

    Poloxamer micelles promise safety and efficacy for many water insoluble drugs. Chrysin has been reported to have anticancer, anti-inflammatory, antioxidant, and anti-aromatase activities but its water insoluble properties limit its pharmaceutical application. In the present study, chrysin loaded poloxamer micelles were developed. Two types of poloxamers, Pluronic F-68 and Pluronic F-127 were compared. It was found that chrysin loaded Pluronic F-68 micelles (CS-P68) and chrysin loaded Pluronic F-127 micelles (CS-P127) obviously increase the aqueous solubility of chrysin. The results also indicated that the type of polymer and ratio of drug to polymer affected size and desirable characteristics of the micelles. The micelle system of CS-P68 and CS-P127 formed at drug to polymer ratios of 1:4 and 1:2, respectively, was found to be the most suitable monodispersed system with a nanosize-range diameter. The in vivo study in zebrafish eggs indicates that the toxicity of CS-P68 and CS-P127 is a dose response. CS-P68 and CS-P127 at a drug dose of 10 ng/mL or less is safe for zebrafish embryo growth. The results of this study indicate enhanced water solubility of chrysin. Chrysin loaded poloxamer micelles are promising for further use in in vivo studies in mammalian animals and humans.

  6. Inactivation of a glycyl-tRNA synthetase leads to an arrest in plant embryo development.

    PubMed Central

    Uwer, U; Willmitzer, L; Altmann, T

    1998-01-01

    Embryo formation is the first patterning process during vegetative plant growth. Using transposons as insertional mutagens in Arabidopsis, we identified the mutant edd1 that shows embryo-defective development. The insertion mutation is lethal, arresting embryo growth between the globular and heart stages of embryonic development. The mutant phenotype cosegregates with a transposed Dissociation element. Sequences flanking the transposed element were isolated and used to isolate a full-length cDNA clone representing the wild-type EDD1 gene. Complementation of the mutant through Agrobacterium-mediated gene transfer of an EDD1 wild-type copy as well as loss of the transposon concomitant with phenotypic reversion demonstrated that the transposon had caused the mutation. Based on homology to Escherichia coli, the EDD1 gene is predicted to encode a novel glycyl-tRNA synthetase (GlyRS) that has not been identified previously in higher plants. An N-terminal portion of the plant protein is able to direct a marker protein into pea chloroplasts. Thus, the gene identified by the embryo-defective insertion mutation encodes a GlyRS homolog, probably acting within the plastidic compartment. PMID:9707529

  7. Cost of protein synthesis and energy allocation during development of antarctic sea urchin embryos and larvae.

    PubMed

    Pace, Douglas A; Manahan, Donal T

    2007-04-01

    Cold environments represent a substantial volume of the biosphere. To study developmental physiology in subzero seawater temperatures typically found in the Southern Ocean, rates and costs of protein synthesis were measured in embryos and larvae of Sterechinus neumayeri, the Antarctic sea urchin. Our analysis of the "cost of living" in extreme cold for this species shows (1) that cost of protein synthesis is strikingly low during development, at 0.41 +/- 0.05 J (mg protein synthesized)(-1) (n = 16); (2) that synthesis cost is fixed and independent of synthesis rate; and (3) that a low synthesis cost permits high rates of protein turnover at -1 degrees C, at rates comparable to those of temperate species of sea urchin embryos developing at 15 degrees C. With a low synthesis cost, even at the highest synthesis rates measured (gastrulae), the proportion of total metabolism accounted for by protein synthesis in the Antarctic sea urchin was 54%-a value similar to that of temperate sea urchin embryos. In the Antarctic sea urchin, up to 87% of metabolic rate can be accounted for by the combined energy costs of protein synthesis and the sodium pump. We conclude that, in Antarctic sea urchin embryos, high rates of protein synthesis can be supported in extreme-cold environments while still maintaining low rates of respiration.

  8. Morphometric and autoradiographic analysis of frontonasal development in the chick embryo

    SciTech Connect

    Patterson, S.B.; Minkoff, R.

    1985-05-01

    Dimensional changes in the nasal processes were measured in chick embryos from Hamburger and Hamilton (1951) stages 20 through 27.5. Transverse measurements in the frontonasal region of freshly fixed embryos were compared to frontal sections of the nasal region of comparably staged embryos. These observations were correlated with autoradiographic studies of cell movement employing an implant labeling technique. Morphometric analysis indicated that between stages 20 and 25 the separation of the nasal pit orifices increased coincidentally with rapid forebrain enlargement. Since the separation of the nasal pit fundi increased more rapidly, the orientation of the nasal pits changed. Autoradiographic studies indicated that lateral movement of medial nasal process mesenchyme into the base of the nasal groove and medial area at the base of the lateral nasal process had occurred. After stage 25, the separation of the nasal orifices declined dramatically, coincidental with rapid orbital enlargement. In contrast, the separation of the nasal pit fundi was maintained. It is proposed that nasal development of the chick embryo may be governed initially by forebrain enlargement and associated lateral movements of mesenchyme in the medial nasal processes, resulting in reorientation of the invaginating nasal placodes; subsequently, orbital enlargement and an associated medial redirection of growth of the lateral nasal processes assumes greater significance to the continued development of the frontonasal region.

  9. The role of embryo movement in the development of the furcula.

    PubMed

    Pollard, A S; Boyd, S; McGonnell, I M; Pitsillides, A A

    2017-03-01

    The pectoral girdle is a complex structure which varies in its morphology between species. A major component in birds is the furcula, which can be considered equivalent to a fusion of the paired clavicles found in many mammals, and the single interclavicle found in many reptiles. These elements are a remnant of the dermal skeleton and the only intramembranous bones in the trunk. Postnatally, the furcula plays important mechanical roles by stabilising the shoulder joint and acting as a mechanical spring during flight. In line with its mechanical role, previous studies indicate that, unlike many other intramembranous bones, furcula growth during development can be influenced by mechanical stimuli. This study investigated the response of individual aspects of furcula growth to both embryo immobilisation and hypermotility in the embryonic chicken. The impact of altered incubation temperature, which influences embryo motility, on crocodilian interclavicle development was also explored. We employed whole-mount bone and cartilage staining and 3D imaging by microCT to quantify the impact of rigid paralysis, flaccid paralysis and hypermobility on furcula growth in the chicken, and 3D microCT imaging to quantify the impact of reduced temperature (32-28 °C) and motility on interclavicle growth in the crocodile. This revealed that the growth rates of the clavicular and interclavicular components of the furcula differ during normal development. Total furcula area was reduced by total unloading produced by flaccid paralysis, but not by rigid paralysis which maintains static loading of embryonic bones. This suggests that dynamic loading, which is required for postnatal bone adaptation, is not a requirement for prenatal furcula growth. Embryo hypermotility also had no impact on furcula area or arm length. Furcula 3D shape did, however, differ between groups; this was marked in the interclavicular component of the furcula, the hypocleideum. Hypocleideum length was reduced by both

  10. Changes in the dielectric properties of medaka fish embryos during development, studied by electrorotation

    SciTech Connect

    Shirakashi, Ryo; Mischke, Miriam; Fischer, Peter; Memmel, Simon; Krohne, Georg; Fuhr, Guenter R.; Zimmermann, Heiko; Sukhorukov, Vladimir L.

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer Electrorotation offers a non-invasive tool for dielectric analysis of fish embryos. Black-Right-Pointing-Pointer The three-shell dielectric model matches the rotation spectra of medaka eggs. Black-Right-Pointing-Pointer The capacitance value suggests a double-membrane structure of yolk envelope. -- Abstract: The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during pre-hatching development by means of the electrorotation (ROT) technique. Due to their layered structure, medaka eggs exhibited up to three ROT peaks in the kHz-MHz frequency range. During development from blastula to early somite stage, ROT spectra varied only slightly. But as the embryo progressed to the late-somite stage, the ROT peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the ROT spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field ROT peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.

  11. Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis.

    PubMed

    Bai, Guang-Yu; Song, Si-Hang; Wang, Zhen-Dong; Shan, Zhi-Yan; Sun, Rui-Zhen; Liu, Chun-Jia; Wu, Yan-Shuang; Li, Tong; Lei, Lei

    2016-04-01

    Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study.

  12. Progress Towards the Development of a Fathead Minnow Embryo Test and Comparison to the Zebrafish Embryo Test for Assessing Acute Fish Toxicity

    EPA Science Inventory

    The Zebrafish Embryo Test (ZFET) for acute fish toxicity is a well developed method nearing adoption as an OECD Test Guideline. Early drafts of the test guideline (TG) envisioned a suite of potential test species to be covered including zebrafish, fathead minnow, Japanese Medaka...

  13. Information superhighway: Issues affecting development

    NASA Astrophysics Data System (ADS)

    1994-09-01

    Technological advances in the transmission of voice, video, and data are fostering fundamental changes in the telecommunications industry. For example, large local telephone companies plan to offer video services in competition with cable and broadcast television, while cable television companies plan to offer local telephone service over their wires in competition with the local telephone companies. The administration believes that these technological changes provide the opportunity to develop an 'Information Superhighway' that could provide every element of society with ready access to data, voice, and video communications. Concurrently, the Congress is considering sweeping changes to telecommunications regulations to keep pace with this dynamic industry. GAO prepared this report to serve as an overview of three key issues that decisionmakers may face as they deliberate telecommunications legislation; it focuses on three pivotal issues they face in formulating new telecommunications legislation: (1) managing the transition to a more competitive local telecommunications marketplace; (2) ensuring that all consumers have access to affordable telecommunications as competition develops; and (3) ensuring that the Information Superhighway provides adequate security, privacy, reliability, and interoperability.

  14. Microgravity in the STS-29 space shuttle discovery affected the vestibular system of chick embryos

    NASA Technical Reports Server (NTRS)

    Fermin, C. D.; Martin, D.; Jones, T.; Vellinger, J.; Deuser, M.; Hester, P.; Hullinger, R.

    1996-01-01

    Out of 32 embryos flown (16 @ E2 + 16 @ E9) for 5 days, 16 survived. All sixteen E2 were dead at landing. Eight were opened and eight were incubated at 1.0G. Autopsy showed that 4 E2 survived over 24 hours in space. Eight E14 hatched without anatomical malformations, and 8 E14 were fixed. The height of the macular epithelia was 31 mu m (mean) in control and 26 mu m in flight chicks. The cross-sectional area of macular nuclei of control was 17 mu m(2) for hair cells and 14 mu m(2) in supporting cells. In flight, cross-sectional area was 17 mu m(2) in hair cells and 15 mu m(2) in supporting cells (n=250). The shape factor of cartilage cells (1.0 = perfect circle) between control (mean = 0.70) and flight (mean = 0.72), and the area of cartilaginous cells between controls (mean = 9 mu m(2)) and flight (mean = 9 mu m(2)) did not differ (n=300). The nuclei of support cells were closer to the basement membrane in flight than in control chicks. The immunoreactivity of otoconia with anti keratan, fibronectin or chrondroitin sulfate was not different between flight and control ears. There were more afferent fibers inside the macular epithelia of flight (p<0.05) than control. Three of 8 flight animals had elevated vestibular thresholds (VT), with normal mean response amplitudes and latencies. Modified afferent innervation patterns requiring weeks to compensate are sufficient to elevate VT, and should be investigated further. Other reversible (sublethal) microgravity effects on sensory epithelia (vacuoles, swelling, etc) require quantification.

  15. Control of rice embryo development, shoot apical meristem maintenance, and grain yield by a novel cytochrome p450.

    PubMed

    Yang, Weibing; Gao, Mingjun; Yin, Xin; Liu, Jiyun; Xu, Yonghan; Zeng, Longjun; Li, Qun; Zhang, Shubiao; Wang, Junmin; Zhang, Xiaoming; He, Zuhua

    2013-11-01

    Angiosperm seeds usually consist of two major parts: the embryo and the endosperm. However, the molecular mechanism(s) underlying embryo and endosperm development remains largely unknown, particularly in rice, the model cereal. Here, we report the identification and functional characterization of the rice GIANT EMBRYO (GE) gene. Mutation of GE resulted in a large embryo in the seed, which was caused by excessive expansion of scutellum cells. Post-embryonic growth of ge seedling was severely inhibited due to defective shoot apical meristem (SAM) maintenance. Map-based cloning revealed that GE encodes a CYP78A subfamily P450 monooxygenase that is localized to the endoplasmic reticulum. GE is expressed predominantly in the scutellar epithelium, the interface region between embryo and endosperm. Overexpression of GE promoted cell proliferation and enhanced rice plant growth and grain yield, but reduced embryo size, suggesting that GE is critical for coordinating rice embryo and endosperm development. Moreover, transgenic Arabidopsis plants overexpressing AtCYP78A10, a GE homolog, also produced bigger seeds, implying a conserved role for the CYP78A subfamily of P450s in regulating seed development. Taken together, our results indicate that GE plays critical roles in regulating embryo development and SAM maintenance.

  16. Supplementation of culture medium with L-carnitine improves development and cryotolerance of bovine embryos produced in vitro.

    PubMed

    Takahashi, Toshikiyo; Inaba, Yasushi; Somfai, Tamas; Kaneda, Masahiro; Geshi, Masaya; Nagai, Takashi; Manabe, Noboru

    2013-01-01

    High lipid content in embryos is associated with low freezing tolerance. This study assessed the effects of exogenous L-carnitine, an enhancer of lipid metabolism, on the in vitro development and freezing survival of bovine embryos. Also, effects on metabolic activity, reactive oxygen species (ROS) and apoptosis were investigated. Supplementation of embryo culture medium with 1.518 mM or 3.030 mM L-carnitine significantly increased the rates of zygote development to the blastocyst stage and blastocyst cell numbers whereas 6.072 mM of this compound did not improve embryo development. Survival rates after slow freezing of blastocysts were significantly higher when embryos were cultured in the presence of 1.518 mM or 3.030 mM L-carnitine compared with the control. A lower density of lipid droplets was detected in L-carnitine-treated blastocysts compared with the control. L-carnitine significantly reduced ROS levels in 2-cell embryos but did not reduce ROS levels at later stages. The apoptotic cell rate was not different between control and L-carnitine-treated blastocysts. L-carnitine significantly increased ATP levels in 2-cell embryos but not at the 8-cell or blastocyst stages. L-carnitine increased the expression of metabolism-related ATP6 and COX1 genes in blastocysts. In conclusion, L-carnitine supplementation enhanced lipid metabolism in embryos resulting in improved development and cryotolerance of bovine blastocysts produced in vitro.

  17. Effect of insulin-transferrin-selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development.

    PubMed

    Guimarães, A L S; Pereira, S A; Diógenes, M N; Dode, M A N

    2016-12-01

    The aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal-Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1-3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

  18. The effects of manipulation medium, culture system and recipient cytoplast on in vitro development of intraspecies and intergeneric felid embryos.

    PubMed

    Imsoonthornruksa, Sumeth; Lorthongpanich, Chanchao; Sangmalee, Anawat; Srirattana, Kanokwan; Laowtammathron, Chuti; Tunwattana, Wanchai; Somsa, Wachiravit; Ketudat-Cairns, Mariena; Nagai, Takashi; Parnpai, Rangsun

    2011-06-01

    The aim of this study was to investigate if reconstructed felid embryos obtained by intraspecies or intergeneric cloning can develop in vitro. Fibroblast cells (f) from a domestic cat (DCf), marbled cat (MCf) and bovine (Bf) were used as donor cells, and oocytes (o) from domestic cats (DCo) and bovine (Bo) were used as recipient cytoplasts. There were two intraspecies (donor cell + recipient cytoplast: DCf + DCo and Bf + Bo) and three intergeneric (MCf + DCo, DCf + Bo and MCf + Bo) cloning groups in the study. In Experiment 1, the effects of manipulation media, modified TCM-199 (199H) or Emcare holding medium (EHM), on in vitro development of DCf + DCo embryos were investigated. The blastocyst formation rate (BFR) of the embryos manipulated in EHM (33.3%) was higher (P<0.05) compared with those manipulated in 199H (18.1%). In Experiment 2, DCf + DCo and MCf + DCo embryos were cocultured with or without domestic cat oviductal epithelium cells. Irrespective of coculture, the same BFR was obtained for DCf + DCo embryos (44.4 vs. 38.0%), while MCf + DCo embryos could not develop beyond the morula stage. In experiment 3, although the development of MCf + DCo and DCf + Bo embryos was arrested at the morula stage, 8.6% of MCf + Bo embryos were able to develop to the blastocyst stage. These results demonstrated that EHM was superior to 199H as an embryo manipulation medium and that the DCo and Bo could support the early embryonic development of intergeneric cloned marbled cat embryos up to the morula stage. However, postimplantation development still needs to be investigated.

  19. Supply of fatty acid is one limiting factor in the accumulation of triacylglycerol in developing embryos

    SciTech Connect

    Bao, X.; Ohlrogge, J.

    1999-08-01

    The metabolic factors that determine oil yield in seeds are still not well understood. To begin to examine the limits on triacylglycerol (TAG) production, developing Cuphea lanceolata, Ulmus carpinifolia, and Ulmus parvifolia embryos were incubated with factors whose availability might limit oil accumulation. The addition of glycerol or sucrose did not significantly influence the rate of TAG synthesis. However, the rate of {sup 14}C-TAG synthesis upon addition of 2.1 mM {sup 14}C-decanoic acid (10:0) was approximately four times higher than the in vivo rate of TAG accumulation in C. lanceolata and two times higher than the in vivo rate in U. carpinifolia and U. parvifolia. In C. lanceolata embryos, the highest rate of {sup 14}C-TAG synthesis (14.3 nmol h{sup {minus}1} embryo {sup {minus}1}) was achieved with the addition of 3.6 mM decanoic acid. {sup 14}C-Decanoic acid was incorporated equally well in all three acyl positions of TAG. The results suggest that C. lancelata, U. Carpinifolia, and U. parvifolia embryos have sufficient acyltransferase activities and glycerol-3-phosphate levels to support rates of TAG synthesis in excess of those found in vivo. Consequently, the amount of TAG synthesized in these oilseeds may be in part determined by the amount of fatty acid produced in plastids.

  20. Pollen Viability, Pistil Receptivity, and Embryo Development in Hybridization of Nelumbo nucifera Gaertn

    PubMed Central

    Wang, Yan-Li; Guan, Zhi-Yong; Chen, Fa-Di; Fang, Wei-Min; Teng, Nian-Jun

    2012-01-01

    Seed set is usually low and differs for different crosses of flower lotus (Nelumbo nucifera Gaertn.). The reasons remain unknown, and this has a negative impact on lotus breeding. To determine the causes, we carried out two crosses of flower lotus, that is, “Jinsenianhua” × “Qinhuaihuadeng” and “Qinhuaihuadeng” × “Jinsenianhua” and pollen viability, pistil receptivity, and embryo development were investigated. The pollen grains collected at 05:00-06:00 hrs had the highest viability, and the viabilities of “Jinsenianhua” and “Qinhuaihuadeng” were 20.6 and 15.7%, respectively. At 4 h after artificial pollination, the number of pollen grains germinating on each stigma reached a peak: 63.0 and 17.2 per stigma in “Jinsenianhua” × “Qinhuaihuadeng” and “Qinhuaihuadeng” × “Jinsenianhua”, respectively. At 1 d after artificial pollination, the percentages of normal embryos in the two crosses were 55.0 and 21.9%, respectively; however, at 11 d after pollination, the corresponding percentages were 20.8 and 11.2%. Seed sets of the two crosses were 17.9 and 8.0%, respectively. The results suggested that low pistil receptivity and embryo abortion caused low seed set in “Qinhuaihuadeng” × “Jinsenianhua”, whereas low fecundity of “Jinsenianhua” × “Qinhuaihuadeng” was mainly attributable to embryo abortion. PMID:22629182

  1. Redundant roles of Sox17 and Sox18 in early cardiovascular development of mouse embryos

    SciTech Connect

    Sakamoto, Youhei; Hara, Kenshiro; Kanai-Azuma, Masami; Matsui, Toshiyasu; Miura, Yutaroh; Tsunekawa, Naoki; Kurohmaru, Masamichi; Saijoh, Yukio; Koopman, Peter; Kanai, Yoshiakira . E-mail: aykanai@mail.ecc.u-tokyo.ac.jp

    2007-08-31

    Sox7, -17 and -18 constitute the Sox subgroup F (SoxF) of HMG box transcription factor genes, which all are co-expressed in developing vascular endothelial cells in mice. Here we characterized cardiovascular phenotypes of Sox17/Sox18-double and Sox17-single null embryos during early-somite stages. Whole-mount PECAM staining demonstrated the aberrant heart looping, enlarged cardinal vein and mild defects in anterior dorsal aorta formation in Sox17 single-null embryos. The Sox17/Sox18 double-null embryos showed more severe defects in formation of anterior dorsal aorta and head/cervical microvasculature, and in some cases, aberrant differentiation of endocardial cells and defective fusion of the endocardial tube. However, the posterior dorsal aorta and allantoic microvasculature was properly formed in all of the Sox17/Sox18 double-null embryos. The anomalies in both anterior dorsal aorta and head/cervical vasculature corresponded with the weak Sox7 expression sites. This suggests the region-specific redundant activities of three SoxF members along the anteroposterior axis of embryonic vascular network.

  2. Development of a general baseline toxicity QSAR model for the fish embryo acute toxicity test.

    PubMed

    Klüver, Nils; Vogs, Carolina; Altenburger, Rolf; Escher, Beate I; Scholz, Stefan

    2016-12-01

    Fish embryos have become a popular model in ecotoxicology and toxicology. The fish embryo acute toxicity test (FET) with the zebrafish embryo was recently adopted by the OECD as technical guideline TG 236 and a large database of concentrations causing 50% lethality (LC50) is available in the literature. Quantitative Structure-Activity Relationships (QSARs) of baseline toxicity (also called narcosis) are helpful to estimate the minimum toxicity of chemicals to be tested and to identify excess toxicity in existing data sets. Here, we analyzed an existing fish embryo toxicity database and established a QSAR for fish embryo LC50 using chemicals that were independently classified to act according to the non-specific mode of action of baseline toxicity. The octanol-water partition coefficient Kow is commonly applied to discriminate between non-polar and polar narcotics. Replacing the Kow by the liposome-water partition coefficient Klipw yielded a common QSAR for polar and non-polar baseline toxicants. This developed baseline toxicity QSAR was applied to compare the final mode of action (MOA) assignment of 132 chemicals. Further, we included the analysis of internal lethal concentration (ILC50) and chemical activity (La50) as complementary approaches to evaluate the robustness of the FET baseline toxicity. The analysis of the FET dataset revealed that specifically acting and reactive chemicals converged towards the baseline toxicity QSAR with increasing hydrophobicity. The developed FET baseline toxicity QSAR can be used to identify specifically acting or reactive compounds by determination of the toxic ratio and in combination with appropriate endpoints to infer the MOA for chemicals.

  3. Protective effects of melatonin on bovine sperm characteristics and subsequent in vitro embryo development.

    PubMed

    Pang, Yun-Wei; Sun, Ye-Qing; Jiang, Xiao-Long; Huang, Zi-Qiang; Zhao, Shan-Jiang; Du, Wei-Hua; Hao, Hai-Sheng; Zhao, Xue-Ming; Zhu, Hua-Bin

    2016-11-01

    We aimed to investigate the effect of melatonin on bovine frozen-thawed semen and its impact on fertilization outcome. Plasma membrane integrity, mitochondrial activity, acrosome integrity, and levels of intracellular reactive oxygen species (ROS) were measured in spermatozoa treated with different concentrations of melatonin. Melatonin-treated spermatozoa were then used for in vitro fertilization, followed by analysis of subsequent embryo development and the expression of apoptosis- and antioxidant-related genes. The results revealed that 10(-5) and 10(-3)  M melatonin led to higher plasma membrane integrity, mitochondrial activity, and acrosome integrity, and significantly decreased intracellular ROS levels (P < 0.05). The blastocyst development rate of in vitro-produced bovine embryos originating from 10(-3)  M melatonin-treated spermatozoa was significantly higher, while the incidence of apoptotic nuclei in blastocysts was markedly lower than for embryos from any other group (P < 0.05). CASP3 and BAX mRNA abundance were significantly reduced whereas BCL2, XIAP, and CAT transcript abundance were significantly increased in embryos produced from spermatozoa treated with 10(-3)  M melatonin; GPX4 expression, however, was comparable in all treatment groups. Thus, 10(-3)  M melatonin can improve the quality of bovine frozen-thawed semen. These beneficial effects appear to influence preimplantation embryos, given the correlation with its anti-apoptotic and anti-oxidative properties. Mol. Reprod. Dev. 83: 993-1002, 2016 © 2016 Wiley Periodicals, Inc.

  4. Development of segments and appendages in embryos of the desert scorpion Paruroctonus mesaensis (Scorpiones: Vaejovidae).

    PubMed

    Farley, R D

    2001-10-01

    The scanning electron microscope was used to study the changing features of scorpion embryos from the blastula through early stages in the development of appendages. The earliest scorpion fossils (Silurian period) have structures more advanced than the embryos herein, so the possibility is considered that these embryos still retain and display some features indicative of evolutionary patterns in adult pre-Silurian ancestors. The blastodisc stage is followed by a knob-like germinal center that gives rise to most of the embryo body. The germinal center elongates on the ventral surface of the spherical yolk mass. The broad cephalic lobe is first delineated from the following pedipalpal segment. The limbbuds for the pedipalps and anterior walking legs appear, as additional segments are added at a growth zone at the rear of the embryo body. Initially, in the cephalic lobe there are no limbbuds; then the cheliceral buds emerge from the posterior part of the lobe. The stomodeum appears first in the anterior half of the cephalic lobe, but an oral groove forms and the mouth is displaced posteriorly within the groove. This repositioning allows space anteriorly for invagination (semilunar grooves) of epithelium for the brain and medial eyes. The mouth is directed ventrally in all stages of this study. The widespread chelicerae are initially posterior to the mouth, but later move anterior and dorsal to it. Small limbbud bulges on mesosomal segments disappear later and never become protruding appendages. Metasomal segments are produced free from the yolk surface in a ventral flexure beneath the embryo body. The telson starts as two spherical lobes, but later elongates and tapers distally, not yet developing the sharp sting (aculeus) seen in Silurian and all subsequent scorpions. The walking legs are digitigrade, as in most fossil aquatic scorpions. Segments are delineated in the appendages; the chelicerae and pedipalps are divided distally for chela (claw) formation. Bilateral

  5. Analysis of global gene expression profiles to identify differentially expressed genes critical for embryo development in Brassica rapa.

    PubMed

    Zhang, Yu; Peng, Lifang; Wu, Ya; Shen, Yanyue; Wu, Xiaoming; Wang, Jianbo

    2014-11-01

    Embryo development represents a crucial developmental period in the life cycle of flowering plants. To gain insights into the genetic programs that control embryo development in Brassica rapa L., RNA sequencing technology was used to perform transcriptome profiling analysis of B. rapa developing embryos. The results generated 42,906,229 sequence reads aligned with 32,941 genes. In total, 27,760, 28,871, 28,384, and 25,653 genes were identified from embryos at globular, heart, early cotyledon, and mature developmental stages, respectively, and analysis between stages revealed a subset of stage-specific genes. We next investigated 9,884 differentially expressed genes with more than fivefold changes in expression and false discovery rate ≤ 0.001 from three adjacent-stage comparisons; 1,514, 3,831, and 6,633 genes were detected between globular and heart stage embryo libraries, heart stage and early cotyledon stage, and early cotyledon and mature stage, respectively. Large numbers of genes related to cellular process, metabolism process, response to stimulus, and biological process were expressed during the early and middle stages of embryo development. Fatty acid biosynthesis, biosynthesis of secondary metabolites, and photosynthesis-related genes were expressed predominantly in embryos at the middle stage. Genes for lipid metabolism and storage proteins were highly expressed in the middle and late stages of embryo development. We also identified 911 transcription factor genes that show differential expression across embryo developmental stages. These results increase our understanding of the complex molecular and cellular events during embryo development in B. rapa and provide a foundation for future studies on other oilseed crops.

  6. Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development

    PubMed Central

    O’Shields, Britton; McArthur, Andrew G.; Holowiecki, Andrew; Kamper, Martin; Tapley, Jeffrey; Jenny, Matthew J.

    2014-01-01

    The metal responsive element-binding transcription factor-1 (MTF-1) responds to changes in cellular zinc levels caused by zinc exposure or disruption of endogenous zinc homeostasis by heavy metals or oxygen-related stress. Here we report the functional characterization of a complete zebrafish MTF-1 in comparison with the previously identified isoform lacking the highly conserved cysteine-rich motif (Cys-X-Cys-Cys-X-Cys) found in all other vertebrate MTF-1 orthologues. In an effort to develop novel molecular tools, a constitutively nuclear dominant-negative MTF-1 (dnMTF-1) was generated as tool for inhibiting endogenous MTF-1 signaling. The in vivo efficacy of the dnMTF-1 was determined by microinjecting in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (1–2 cell stage) followed by transcriptomic profiling using an Agilent 4 × 44K array on 28- and 36-hpf embryos. A total of 594 and 560 probes were identified as differentially expressed at 28 hpf and 36 hpf, respectively, with interesting overlaps between timepoints. The main categories of genes affected by the inhibition of MTF-1 signaling were: nuclear receptors and genes involved in stress signaling, neurogenesis, muscle development and contraction, eye development, and metal homeostasis, including novel observations in iron and heme homeostasis. Finally, we investigate both the transcriptional activator and transcriptional repressor role of MTF-1 in potential novel target genes identified by transcriptomic profiling during early zebrafish development. PMID:24751692

  7. Messenger RNAs in metaphase II oocytes correlate with successful embryo development to the blastocyst stage.

    PubMed

    Biase, Fernando Henrique; Everts, Robin Edward; Oliveira, Rosane; Santos-Biase, Weruska Karyna Freitas; Fonseca Merighe, Giovana Krempel; Smith, Lawrence Charles; Martelli, Lúcia; Lewin, Harris; Meirelles, Flávio Vieira

    2014-02-01

    The mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8-16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: 'RNA processing', 'translation' and 'mRNA metabolic process'. Genes that are important to the molecular functions of 'RNA binding' and 'translation factor activity, RNA binding' were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

  8. P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

    PubMed Central

    Dahlberg, Olle; Shilkova, Olga; Tang, Min; Holmqvist, Per-Henrik; Mannervik, Mattias

    2015-01-01

    Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3’ end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos. PMID:25679530

  9. Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos.

    PubMed

    Alonso, Ana P; Goffman, Fernando D; Ohlrogge, John B; Shachar-Hill, Yair

    2007-10-01

    The efficiency with which developing sunflower embryos convert substrates into seed storage reserves was determined by labeling embryos with [U-(14)C6]glucose or [U-(14)C5]glutamine and measuring their conversion to CO2, oil, protein and other biomass compounds. The average carbon conversion efficiency was 50%, which contrasts with a value of over 80% previously observed in Brassica napus embryos (Goffman et al., 2005), in which light and the RuBisCO bypass pathway allow more efficient conversion of hexose to oil. Labeling levels after incubating sunflower embryos with [U-(14)C4]malate indicated that some carbon from malate enters the plastidic compartment and contributes to oil synthesis. To test this and to map the underlying pattern of metabolic fluxes, separate experiments were carried out in which embryos were labeled to isotopic steady state using [1-(13)C1]glucose, [2-(13)C1]glucose, or [U-(13)C5]glutamine. The resultant labeling in sugars, starch, fatty acids and amino acids was analyzed by NMR and GC-MS. The fluxes through intermediary metabolism were then quantified by computer-aided modeling. The resulting flux map accounted well for the labeling data, was in good agreement with the observed carbon efficiency, and was further validated by testing for agreement with gas exchange measurements. The map shows that the influx of malate into oil is low and that flux through futile cycles (wasting ATP) is low, which contrasts with the high rates previously determined for growing root tips and heterotrophic cell cultures.

  10. Vitamin D receptor signaling is required for heart development in zebrafish embryo.

    PubMed

    Kwon, Hye-Joo

    2016-02-12

    Vitamin D has been found to be associated with cardiovascular diseases. However, the role of vitamin D in heart development during embryonic period is largely unknown. Vitamin D induces its genomic effects through its nuclear receptor, the vitamin D receptor (VDR). The present study investigated the role of VDR on heart development by antisense-mediated knockdown approaches in zebrafish model system. In zebrafish embryos, two distinct VDR genes (vdra and vdrb) have been identified. Knockdown of vdra has little effect on heart development, whereas disrupting vdrb gene causes various cardiac phenotypes, characterized by pericardial edema, slower heart rate and laterality defects. Depletion of both vdra and vdrb (vdra/b) produce additive, but not synergistic effects. To determine whether atrioventricular (AV) cardiomyocytes are properly organized in these embryos, the expression of bmp4, which marks the developing AV boundary at 48 h post-fertilization, was examined. Notably, vdra/b-deficient embryos display ectopic expression of bmp4 towards the ventricle or throughout atrial and ventricular chambers. Taken together, these results suggest that VDR signaling plays an essential role in heart development.

  11. Real-time prediction of cell division timing in developing zebrafish embryo

    PubMed Central

    Kozawa, Satoshi; Akanuma, Takashi; Sato, Tetsuo; Sato, Yasuomi D.; Ikeda, Kazushi; Sato, Thomas N.

    2016-01-01

    Combination of live-imaging and live-manipulation of developing embryos in vivo provides a useful tool to study developmental processes. Identification and selection of target cells for an in vivo live-manipulation are generally performed by experience- and knowledge-based decision-making of the observer. Computer-assisted live-prediction method would be an additional approach to facilitate the identification and selection of the appropriate target cells. Herein we report such a method using developing zebrafish embryos. We choose V2 neural progenitor cells in developing zebrafish embryo as their successive shape changes can be visualized in real-time in vivo. We developed a relatively simple mathematical method of describing cellular geometry of V2 cells to predict cell division-timing based on their successively changing shapes in vivo. Using quantitatively measured 4D live-imaging data, features of V2 cell-shape at each time point prior to division were extracted and a statistical model capturing the successive changes of the V2 cell-shape was developed. By applying sequential Bayesian inference method to the model, we successfully predicted division-timing of randomly selected individual V2 cells while the cell behavior was being live-imaged. This system could assist pre-selecting target cells desirable for real-time manipulation–thus, presenting a new opportunity for in vivo experimental systems. PMID:27597656

  12. Real-time prediction of cell division timing in developing zebrafish embryo.

    PubMed

    Kozawa, Satoshi; Akanuma, Takashi; Sato, Tetsuo; Sato, Yasuomi D; Ikeda, Kazushi; Sato, Thomas N

    2016-09-06

    Combination of live-imaging and live-manipulation of developing embryos in vivo provides a useful tool to study developmental processes. Identification and selection of target cells for an in vivo live-manipulation are generally performed by experience- and knowledge-based decision-making of the observer. Computer-assisted live-prediction method would be an additional approach to facilitate the identification and selection of the appropriate target cells. Herein we report such a method using developing zebrafish embryos. We choose V2 neural progenitor cells in developing zebrafish embryo as their successive shape changes can be visualized in real-time in vivo. We developed a relatively simple mathematical method of describing cellular geometry of V2 cells to predict cell division-timing based on their successively changing shapes in vivo. Using quantitatively measured 4D live-imaging data, features of V2 cell-shape at each time point prior to division were extracted and a statistical model capturing the successive changes of the V2 cell-shape was developed. By applying sequential Bayesian inference method to the model, we successfully predicted division-timing of randomly selected individual V2 cells while the cell behavior was being live-imaged. This system could assist pre-selecting target cells desirable for real-time manipulation-thus, presenting a new opportunity for in vivo experimental systems.

  13. Real-time prediction of cell division timing in developing zebrafish embryo

    NASA Astrophysics Data System (ADS)

    Kozawa, Satoshi; Akanuma, Takashi; Sato, Tetsuo; Sato, Yasuomi D.; Ikeda, Kazushi; Sato, Thomas N.

    2016-09-01

    Combination of live-imaging and live-manipulation of developing embryos in vivo provides a useful tool to study developmental processes. Identification and selection of target cells for an in vivo live-manipulation are generally performed by experience- and knowledge-based decision-making of the observer. Computer-assisted live-prediction method would be an additional approach to facilitate the identification and selection of the appropriate target cells. Herein we report such a method using developing zebrafish embryos. We choose V2 neural progenitor cells in developing zebrafish embryo as their successive shape changes can be visualized in real-time in vivo. We developed a relatively simple mathematical method of describing cellular geometry of V2 cells to predict cell division-timing based on their successively changing shapes in vivo. Using quantitatively measured 4D live-imaging data, features of V2 cell-shape at each time point prior to division were extracted and a statistical model capturing the successive changes of the V2 cell-shape was developed. By applying sequential Bayesian inference method to the model, we successfully predicted division-timing of randomly selected individual V2 cells while the cell behavior was being live-imaged. This system could assist pre-selecting target cells desirable for real-time manipulation–thus, presenting a new opportunity for in vivo experimental systems.

  14. Full-term development of gaur-bovine interspecies somatic cell nuclear transfer embryos: effect of trichostatin A treatment.

    PubMed

    Srirattana, Kanokwan; Imsoonthornruksa, Sumeth; Laowtammathron, Chuti; Sangmalee, Anawat; Tunwattana, Wanchai; Thongprapai, Thamnoon; Chaimongkol, Chockchai; Ketudat-Cairns, Mariena; Parnpai, Rangsun

    2012-06-01

    Trichostatin A (TSA) has previously been used in somatic cell nuclear transfer (SCNT) to improve the cloning efficiency in several species, which led our team to investigate the effects of TSA on the full-term development of bovine SCNT and gaur-bovine interspecies SCNT (gaur iSCNT; gaur somatic cells as donors and bovine oocytes as recipients) embryos. Treatment with 50 nM TSA for 10 h after fusion had no positive effects on the rates of fusion, cleavage, or the development to eight-cell or morula stages in both bovine SCNT and gaur iSCNT embryos. However, TSA treatment significantly enhanced the blastocyst formation rate in bovine SCNT embryos (44 vs. 32-34% in the TSA-treated and TSA-untreated groups, respectively), but had no effects on gaur iSCNT embryos. The fresh blastocysts derived from bovine SCNT and gaur iSCNT embryos (fresh groups), as well as vitrified bovine SCNT blastocysts (vitrified group), were transferred to bovine recipients. We found that TSA treatment increased the pregnancy rates only in recipients receiving fresh bovine SCNT embryos. In recipients receiving TSA-treated bovine SCNT embryos, three cloned calves from the fresh group and twin cloned calves from the vitrified group were delivered; however, no calf was born from the TSA-untreated bovine SCNT embryos. In contrast, one gaur iSCNT calf was born from a recipient receiving blastocysts from the TSA-untreated group. In summary, TSA improved the preimplantation development and pregnancy rates of bovine SCNT embryos, but did not have any beneficial effect on gaur iSCNT embryos. However, one gaur iSCNT calf reached full-term development.

  15. The FRK1 mitogen-activated protein kinase kinase kinase (MAPKKK) from Solanum chacoense is involved in embryo sac and pollen development.

    PubMed

    Lafleur, Edith; Kapfer, Christelle; Joly, Valentin; Liu, Yang; Tebbji, Faiza; Daigle, Caroline; Gray-Mitsumune, Madoka; Cappadocia, Mario; Nantel, André; Matton, Daniel P

    2015-04-01

    The fertilization-related kinase 1 (ScFRK1), a nuclear-localized mitogen-activated protein kinase kinase kinase (MAPKKK) from the wild potato species Solanum chacoense, belongs to a small group of pMEKKs that do not possess an extended N- or C-terminal regulatory domain. Initially selected based on its highly specific expression profile following fertilization, in situ expression analyses revealed that the ScFRK1 gene is also expressed early on during female gametophyte development in the integument and megaspore mother cell and, later, in the synergid and egg cells of the embryo sac. ScFRK1 mRNAs are also detected in pollen mother cells. Transgenic plants with lower or barely detectable levels of ScFRK1 mRNAs lead to the production of small fruits with severely reduced seed set, resulting from a concomitant decline in the number of normal embryo sacs produced. Megagametogenesis and microgametogenesis were affected, as megaspores did not progress beyond the functional megaspore (FG1) stage and the microspore collapsed around the first pollen mitosis. As for other mutants that affect embryo sac development, pollen tube guidance was severely affected in the ScFRK1 transgenic lines. Gametophyte to sporophyte communication was also affected, as observed from a marked change in the transcriptomic profiles of the sporophytic tissues of the ovule. The ScFRK1 MAPKKK is thus involved in a signalling cascade that regulates both male and female gamete development.

  16. Effects of a fungicide formulation on embryo-larval development, metamorphosis, and gonadogenesis of the South American toad Rhinella arenarum.

    PubMed

    Svartz, Gabriela; Meijide, Fernando; Pérez Coll, Cristina

    2016-07-01

    Sublethal toxicity of the formulated fungicide Maxim(®) XL on embryonic, larval and juvenile development of Rhinella arenarum was evaluated by means of standardized bioassays. Maxim(®) XL, one of the most used fungicides in Argentina, is based on a mixture of two active ingredients: Fludioxonil and Metalaxyl-M. Maxim(®) XL exposure induced severe sublethal effects on the embryos, expressed as general underdevelopment, axial flexures, microcephaly, cellular dissociation, abnormal pigmentation, underdeveloped gills, marked edema and wavy tail. As the embryo development advanced, alterations in behavior as spasmodic contractions, general weakness and inanition were observed. Maxim(®) XL did not affect neither the time required to complete metamorphosis nor sex proportions, but gonadal development and differentiation were impaired. Gross gonadal analysis revealed a significant proportion of exposed individuals with underdevelopment of one or both gonads. Histological analysis confirmed that 18% and 10% of the individuals exposed to 0.25 and 2mg/L Maxim(®) XL, respectively, exhibited undifferentiated gonads characterized by a reduced number (or absence) of germ cells. Taking into account the risk evaluation performed by means of Hazard Quotients, this fungicide could be a threat to R. arenarum populations under chronic exposure. This study represents the first evidence of toxic effects exerted by Maxim(®) XL on amphibians. Finally, our findings highlight the properties of this fungicide that might jeopardize non-target living species exposed to it in agricultural environments.

  17. Effects of breeder age, broiler strain, and eggshell temperature on development and physiological status of embryos and hatchlings.

    PubMed

    Nangsuay, A; Meijerhof, R; van den Anker, I; Heetkamp, M J W; Morita, V De Souza; Kemp, B; van den Brand, H

    2016-07-01

    Breeder age and broiler strain can influence the availability of nutrients and oxygen, particularly through differences in yolk size and shell conductance. We hypothesized that these egg characteristics might affect embryonic responses to changes in eggshell temperature (EST). This study aimed to investigate the effect of breeder age, broiler strain, and EST on development and physiological status of embryos. A study was designed as a 2 × 2 × 2 factorial arrangement using 4 batches of 1,116 hatching eggs of 2 flock ages at 29 to 30 wk (young) and 54 to 55 wk (old) of Ross 308 and Cobb 500. EST of 37.8 (normal) or 38.9°C (high) was applied from incubation d 7 (E7) until hatching. The results showed that breeder age rather than broiler strain had an influence on yolk size (P = 0.043). The shell conductance was higher in Ross 308 than in Cobb 500 (P < 0.001). A high EST resulted in a higher yolk free body mass (YFBM) compared to the normal EST at E14 and E16, but at 3 h after hatch YFBM was lower when eggs were incubated at high EST compared to normal EST (all P < 0.001). Cobb 500 eggs yielded embryos with a lower YFBM at E14, E18, and 3 h after hatch (all P < 0.05) than Ross 308 eggs. Breeder age had no effect on YFBM, but the RSY weight was higher in embryos from the old flock compared to the young flock embryos at E14 and E16 (both P < 0.05). A 3-way interaction among breeder age, strain, and EST was found, especially for incubation duration, navel quality, and relative heart and stomach weights at 3 h after hatch (all P < 0.05). Based on the results obtained, we conclude that oxygen availability rather than nutrient availability determines embryonic development, and the egg characteristics affected embryonic responses to changes of EST, especially for variables related to chick quality.

  18. Proteomic analysis of the Gallus gallus embryo at stage-29 of development.

    PubMed

    Agudo, David; Agudo Garcillán, David; Gómez-Esquer, Francisco; Díaz-Gil, Gema; Martínez-Arribas, Fernando; Delcán, José; Schneider, José; Palomar, María Angustias; Linares, Rafael

    2005-12-01

    The chicken (Gallus gallus) is one of the primary models for embryological and developmental studies. In order to begin to understand the molecular mechanisms underlying the normal and abnormal development of the chicken, we used 2-DE to construct a whole-embryo proteome map. Proteins were separated by IEF on IPG strips, and by 11% SDS-PAGE) gels. Protein identification was performed by means of PMF with MALDI-TOF-MS. In all, 105 protein spots were identified, 35 of them implicated in embryo development, 10 related with some diseases, and 16, finally, being proteins that have never been identified, purified or characterized in the chicken before. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level under different physiological conditions.

  19. Transcriptomic analysis of rice (Oryza sativa) developing embryos using the RNA-Seq technique.

    PubMed

    Xu, Hong; Gao, Yi; Wang, Jianbo

    2012-01-01

    Rice (Oryza sativa) is an excellent model monocot with a known genome sequence for studying embryogenesis. Here we report the transcriptome profiling analysis of rice developing embryos using RNA-Seq as an attempt to gain insight into the molecular and cellular events associated with rice embryogenesis. RNA-Seq analysis generated 17,755,890 sequence reads aligned with 27,190 genes, which provided abundant data for the analysis of rice embryogenesis. A total of 23,971, 23,732, and 23,592 genes were identified from embryos at three developmental stages (3-5, 7, and 14 DAP), while an analysis between stages allowed the identification of a subset of stage-specific genes. The number of genes expressed stage-specifically was 1,131, 1,443, and 1,223, respectively. In addition, we investigated transcriptomic changes during rice embryogenesis based on our RNA-Seq data. A total of 1,011 differentially expressed genes (DEGs) (log(2)Ratio ≥ 1, FDR ≤ 0.001) were identified; thus, the transcriptome of the developing rice embryos changed considerably. A total of 672 genes with significant changes in expression were detected between 3-5 and 7 DAP; 504 DEGs were identified between 7 and 14 DAP. A large number of genes related to metabolism, transcriptional regulation, nucleic acid replication/processing, and signal transduction were expressed predominantly in the early and middle stages of embryogenesis. Protein biosynthesis-related genes accumulated predominantly in embryos at the middle stage. Genes for starch/sucrose metabolism and protein modification were highly expressed in the middle and late stages of embryogenesis. In addition, we found that many transcription factor families may play important roles at different developmental stages, not only in embryo initiation but also in other developmental processes. These results will expand our understanding of the complex molecular and cellular events in rice embryogenesis and provide a foundation for future studies on embryo

  20. Increased basal cAMP-dependent protein kinase activity inhibits the formation of mesoderm-derived structures in the developing mouse embryo.

    PubMed

    Amieux, Paul S; Howe, Douglas G; Knickerbocker, Heidi; Lee, David C; Su, Thomas; Laszlo, George S; Idzerda, Rejean L; McKnight, G Stanley

    2002-07-26

    A targeted disruption of the RIalpha isoform of protein kinase A (PKA) was created by using homologous recombination in embryonic stem cells. Unlike the other regulatory and catalytic subunits of PKA, RIalpha is the only isoform that is essential for early embryonic development. RIalpha homozygous mutant embryos fail to develop a functional heart tube at E8.5 and are resorbed at approximately E10.5. Mutant embryos show significant growth retardation and developmental delay compared with wild type littermates from E7.5 to E10.5. The anterior-posterior axis of RIalpha mutants is well developed, with a prominent head structure but a reduced trunk. PKA activity measurements reveal an increased basal PKA activity in these embryos. Brachyury mRNA expression in the primitive streak of RIalpha mutants is significantly reduced, consistent with later deficits in axial, paraxial, and lateral plate mesodermal derivatives. This defect in the production and migration of mesoderm can be completely rescued by crossing RIalpha mutants to mice carrying a targeted disruption in the Calpha catalytic subunit, demonstrating that unregulated PKA activity rather than a specific loss of RIalpha is responsible for the phenotype. Primary embryonic fibroblasts from RIalpha mutant embryos display an abnormal cytoskeleton and an altered ability to migrate in cell culture. Our results demonstrate that unregulated PKA activity negatively affects growth factor-mediated mesoderm formation during early mouse development.

  1. Role of progesterone in embryo development in cattle.

    PubMed

    Lonergan, Pat; Forde, Niamh; Spencer, Thomas

    2016-01-01

    Progesterone (P4) from the corpus luteum is critical for the establishment and maintenance of pregnancy and plays a major role in regulating endometrial secretions essential for stimulating and mediating changes in conceptus growth and differentiation throughout early pregnancy in ruminants. Numerous studies have demonstrated an association between elevated systemic P4 and acceleration in conceptus elongation. A combination of in vivo and in vitro experiments found that the effects of P4 on conceptus elongation are indirect and mediated through P4-induced effects in the endometrium. Despite effects on elongation, data on the effects of post-insemination supplementation with P4 on pregnancy rates are conflicting. This review highlights the effects of P4 on conceptus development and examines strategies that have been undertaken to manipulate P4 concentrations to increase fertility.

  2. MicroRNA-34c expression in donor cells influences the early development of somatic cell nuclear transfer bovine embryos.

    PubMed

    Wang, Bo; Wang, Yongsheng; Zhang, Man; Du, Yue; Zhang, Yijun; Xing, Xupeng; Zhang, Lei; Su, JianMin; Zhang, Yong; Zheng, Yuemao

    2014-12-01

    The essence of the reprogramming activity of somatic cell nuclear transfer (SCNT) embryos is to produce normal fertilized embryos. However, reprogramming of somatic cells is not as efficient as the reprogramming of sperm. In this report, we describe the effect of an inducible, specific miR-34 microRNA expression in donor cells that enables a similar level of sperm:transgene expression on the early development of SCNT embryos. Our results showed that donor cells with doxycycline (dox)-induced miR-34c expression for the preparation of SCNT embryos resulted in altered developmental rates, histone modification (H3K9ac and H3K4me3), and extent of apoptosis. The cleavage rate and blastocyst formation of the induced nuclear transfer (NT) group were significantly increased. The immunofluorescence signal of H3K9ac in embryos in the induced NT group significantly increased in two-cell- and eight-cell-stage embryos; that of H3K4me3 increased significantly in eight-cell-stage embryos. Although significant differences in staining signals of apoptosis were not detected between groups, lower apoptosis levels were observed in the induced NT group. In conclusion, miR-34c expression induced by dox treatment enhances the developmental potential of SCNT embryos, modifies the epigenetic status, and changes blastocyst quality.

  3. Effect of superovulation induction on embryonic development on day 5 and subsequent development and survival after nonsurgical embryo transfer in pigs.

    PubMed

    Hazeleger, W; Bouwman, E G; Noordhuizen, J P; Kemp, B

    2000-03-15

    To evaluate the effects of eCG dosage on recovery and quality of Day 5 embryos and on subsequent development and survival after embryo transfer, batches of 5 to 10 donor sows were treated with 1000 or 1500 IU eCG. Recipients from the same batch were synchronously treated with 800 IU eCG. Ovulation was induced with 750 IU hCG (72 h after eCG) in donors and recipients. Donors were inseminated and embryos were collected at 162 h after hCG (120 h after ovulation). Ovulation rate was lower using 1000 IU eCG (28.5+/-11.7; n=48) than 1500 IU eCG (45.7+/-20.3; n=32; P<0.0001). Embryo recovery rate (82.9+/-16.9%) and percentage expanded blastocysts (56.2+/-31.4%) were similar (P>0.05). Expanded blastocysts from each group of sows were pooled into 2 groups within eCG treatment, containing embryos from normally ovulating sows (< or = 25 corpora lutea [CL]) or from superovulated sows (> 25 CL). Average diameter and number of cells of a random sample of the expanded blastocysts per pool were recorded. The average diameter of blastocysts (160.5+/-11.5 microm) was not affected by eCG dosage or ovulation rate (P>0.10). The average number of cells per embryo was higher in the 1000 IU eCG group (84.3+/-15.3) than in the 1500 IU eCG group (70.2+/-1.9; P<0.05) but was similar for normal and superovulated donors within each eCG group (P>0.10). Of the 4 groups, litters of 28 to 30 blastocysts were nonsurgically transferred to 27 synchronous recipients. Pregnant recipients were slaughtered on Day 37 after hCG treatment to evaluate embryonic development and survival. Pregnancy rate for the 1000 and 1500 IU eCG donor groups was 71% (10/14) and 46% (6/13; P>0.10), respectively. The number of implantations and fetuses for the 1000 IU eCG groups was 12.9+/-3.0 and 11.1+/-2.7, and 14.2+/-7.0 and 10.5+/-4.6, respectively, for the 1500 IU eCG groups (P>0.10). After post-priory categorizing the litters of blastocysts to below or above the average diameter (158 microm) of the transferred embryos

  4. EphrinB2 Affects Apical Constriction in Xenopus Embryos and is Regulated by ADAM10 and Flotillin-1

    PubMed Central

    Ji, Yon Ju; Hwang, Yoo-Seok; Mood, Kathleen; Cho, Hee-Jun; Lee, Hyun-Shik; Winterbottom, Emily; Cousin, Hèléne; Daar, Ira O.

    2014-01-01

    The Eph/ephrin signaling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development. Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis. Such a decrease in ephrinB2 protein levels is observed upon the loss of flotillin-1 scaffold protein, a newly identified ephrinB2-binding partner. This dramatic decline in ephrinB2 protein levels upon the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10. These findings indicate that flotillin-1 regulates ephrinB2 protein levels through ADAM10, and is required for appropriate neural tube morphogenesis in the Xenopus embryo. PMID:24662724

  5. EphrinB2 affects apical constriction in Xenopus embryos and is regulated by ADAM10 and flotillin-1

    NASA Astrophysics Data System (ADS)

    Ji, Yon Ju; Hwang, Yoo-Seok; Mood, Kathleen; Cho, Hee-Jun; Lee, Hyun-Shik; Winterbottom, Emily; Cousin, Hélène; Daar, Ira O.

    2014-03-01

    The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development. Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis. Such a decrease in ephrinB2 protein levels is observed on the loss of flotillin-1 scaffold protein, a newly identified ephrinB2-binding partner. This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10. These findings indicate that flotillin-1 regulates ephrinB2 protein levels through ADAM10, and is required for appropriate neural tube morphogenesis in the Xenopus embryo.

  6. Halogenated carbazoles induce cardiotoxicity in developing zebrafish (Danio rerio) embryos.

    PubMed

    Fang, Mingliang; Guo, Jiehong; Chen, Da; Li, An; Hinton, David E; Dong, Wu

    2016-10-01

    Halogenated carbazoles are increasingly identified as a novel class of environmental contaminants. However, no in vivo acute toxicity information on those compounds was available. In the present study, an in vivo zebrafish embryonic model (Danio rerio) was used to investigate the developmental toxicity of those halogenated carbazoles. The results suggested that acute toxicity was structure-dependent. Two of the 6 tested carbazoles, 2,7-dibromocarbazole (27-DBCZ) and 2,3,6,7-tetrachlorocarbazole, showed obvious developmental toxicity at nanomolar levels. The typical phenotypes were similar to dioxin-induced cardiotoxicity, including swollen yolk sac, pericardial sac edema, elongated and unlooped heart, and lower jaw shortening. During embryonic development 27-DBCZ also induced a unique pigmentation decrease. Gene expression and protein staining of cytochrome P4501A (CYP1A) showed that both halogenated carbazoles could induce CYP1A expression at the micromolar level and primarily in the heart area, which was similar to dioxin activity. Further, aryl hydrocarbon receptor-(AhR)2 gene knockdown with morpholino confirmed that the acute cardiotoxicity is AhR-dependent. In conclusion, the results demonstrate that halogenated carbazoles represent yet another class of persistent organic pollutants with dioxin-like activity in an in vivo animal model. Environ Toxicol Chem 2016;35:2523-2529. © 2016 SETAC.

  7. Androgenetic development of X- and Y-chromosome bearing haploid rainbow trout embryos.

    PubMed

    Michalik, Oliwia; Kowalski, Radosław K; Judycka, Sylwia; Rożyński, Rafał; Dobosz, Stefan; Ocalewicz, Konrad

    2016-09-01

    Haploid fish embryos are important in studies regarding role of the recessive traits during early ontogeny. In fish species with the male heterogamety, androgenetic haploid embryos might be also useful tool in studies concerning role of the sex chromosomes during an embryonic development. Morphologically differentiated X and Y chromosomes have been found in a limited number of fish species including rainbow trout (Oncorhynchus mykiss Walbaum 1792). To evaluate role of the sex chromosomes during rainbow trout embryonic development, survival of the androgenetic haploids in the presence of X or Y sex chromosomes has been examined. Androgenetic haploid rainbow trout were produced by fertilization of X-irradiated eggs with spermatozoa derived from the normal males (XY) and neomales, that is, sex-reversed females (XX) to produce X- and Y-bearing haploids, and all X-bearing haploids, respectively. Survival rates of the androgenetic progenies of normal males and neomales examined during embryogenesis and at hatching did not differ significantly. However, all haploids died within next few days after hatching. Cytogenetic analysis of the androgenetic embryos confirmed their haploid status. Moreover, apart from the intact paternal chromosomes, residues of the irradiated maternal chromosomes observed as chromosome fragments were identified in some of the haploids. Provided results suggested that rainbow trout X and Y chromosomes despite morphological and genetic differences are at the early stage of differentiation and still share genetic information responsible for the proper embryonic development.

  8. Caffeine interferes embryonic development through over-stimulating serotonergic system in chicken embryo.

    PubMed

    Li, Xiao-Di; He, Rong-Rong; Qin, Yang; Tsoi, Bun; Li, Yi-Fang; Ma, Zheng-Lai; Yang, Xuesong; Kurihara, Hiroshi

    2012-06-01

    The potential harmful effects of caffeine in pregnant women aroused public interests due to its possibility to jeopardize fetal development. Monoamine neurotransmitters are thought to regulate neural development processes through maternal-fetal interactions, which may have long term impact on mental and behavioral effects. The current study focuses on investigating the effects of caffeine on the monoamine neurotransmitter system using developmental chicken embryos. The ED(50) value of caffeine toxicity was 27.3 μmol/egg in chicken embryo. Administration of caffeine, with lower dosage than ED(50) (2.5, 5.0 and 10.0 μmol/egg), caused failure of neural tube closure. In addition, contents of 5-HT and its metabolite 5-HIAA were increased under dosage of 10.0 μmol/egg caffeine administration. Gene expression of TPH2 was also increased by caffeine treatment. Caffeine could result in defect of neural tube closure and induce disorder of serotonergic system development, which may increase teratogenic rate of embryos. Meanwhile, it is probably an underlying factor for inducing psychological and behavioral disorders in adult. Moreover, caffeine was found to be accumulated in the embryonic brain and not being metabolized, which may incur a magnification of adverse effects. This study may provide valuable data for further investigations on toxicology of caffeine during different stages of pregnancy.

  9. Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos

    PubMed Central

    2012-01-01

    Background Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein). Results The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2), the synthetic oestrogen 17α- ethinyloestradiol (EE2), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures). For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish). We also demonstrate that

  10. Effects of Trichostatin A and PXD101 on the In Vitro Development of Mouse Somatic Cell Nuclear Transfer Embryos.

    PubMed

    Qiu, Xiaoyan; You, Haihong; Xiao, Xiong; Li, Nan; Li, Yuemin

    2017-02-01

    The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with aberrant epigenetic nuclear reprogramming. It has been demonstrated that treatment with histone deacetylase inhibitors (HDACis) enhances developmental potential of SCNT embryos. Previous studies in many species revealed that treatment of SCNT embryos with trichostatin A (TSA)-an HDACi-significantly enhances the in vitro development of SCNT embryos. In this study, we compared two different SCNT protocols with TSA and investigate, for the first time, the effect of another new HDACi, PXD101 (belinostat), on in vitro development of mouse SCNT embryo. Rates of blastocyst development in mouse SCNT embryos treated with either 5 nM TSA during (6 hours) and after (4 hours) activation (39.1%) or with 50 nM PXD101 during (6 hours) and after (4 hours) activation (40.2%) were significantly higher than those of nontreated SCNT embryos (11.5%) and both treatments also significantly improved the subsequent establishment of NT-ESCs in comparison with the nontreated group (38.1% and 40.9% vs. 11.8%). In conclusion, we optimized the TSA concentration and treatment timing and, for the first time, investigated the effect of PXD101 on mouse development of SCNT embryos and establishment of NT-ESCs.

  11. Hormonal control of somatic embryo development from cultured cells of caraway: interactions of abscisic Acid, zeatin, and gibberellic Acid.

    PubMed

    Ammirato, P V

    1977-04-01

    The effects of abscisic acid, zeatin, and gibberellic acid on the development of somatic embryos from cultured cells of caraway (Carum carvi L.) were observed.Somatic embryos complete development on a basal medium without exogenous hormones, but some are subject to developmental abnormalities including malformed cotyledons and accessory embryos. Both zeatin and gibberellic acid, especially in combination, stimulate growth and increase the frequency of aberrant forms. Zeatin causes the formation of multiple shoots, leafy and abnormal cotyledons, and in the dark, enlarged hypocotyls; gibberellic acid effects root elongation, polycotyledony, and some callus formation. In contrast, abscisic acid, at concentrations which do not inhibit embryo maturation, selectively suppresses abnormal proliferations. With abscisic acid, and especially in the dark, a high percentage of embryos complete development with two fleshy cotyledons on unelongated axes free of accessory embryos.In the light, zeatin eliminates abscisic acid inhibition while gibberellic acid only partially counters its effect, promoting elongated radicles and green rather than white cotyledons. In the dark, zeatin in combination with abscisic acid stimulates extensive callusing. Gibberellic acid does not reverse the effects of abscisic acid but rather enhances them and can counter the disruptive effects of zeatin.The results demonstrate that the balance between abscisic acid on the one hand and zeatin and gibberellic acid on the other can effectively control somatic embryo development and either disrupt or ensure normal maturation.

  12. The cellular and molecular progression of mitochondrial dysfunction induced by 2,4-dinitrophenol in developing zebrafish embryos

    PubMed Central

    Bestman, Jennifer E.; Stackley, Krista D.; Rahn, Jennifer J.; Williamson, Tucker J.; Chan, Sherine S. L.

    2015-01-01

    The etiology of mitochondrial disease is poorly understood. Furthermore, treatment options are limited, and diagnostic methods often lack the sensitivity to detect disease in its early stages. Disrupted oxidative phosphorylation (OXPHOS) that inhibits ATP production is a common phenotype of mitochondrial disorders that can be induced in zebrafish by exposure to 2,4-dinitrophenol (DNP), a FDA-banned weight-loss agent and EPA-regulated environmental toxicant, traditionally used in research labs as an uncoupler of OXPHOS. Despite the DNP-induced OXPHOS inhibition we observed using in vivo respirometry, the development of the DNP-treated and control zebrafish were largely similar during the first half of embryogenesis. During this period, DNP-treated embryos induced gene expression of mitochondrial and nuclear genes that stimulated the production of new mitochondria and increased glycolysis to yield normal levels of ATP. DNP-treated embryos were incapable of sustaining this mitochondrial biogenic response past mid-embryogenesis, as shown by significantly lowered ATP production and ATP levels, decreased gene expression, and the onset of developmental defects. Examining neural tissues commonly affected by mitochondrial disease, we found that DNP exposure also inhibited motor neuron axon arbor outgrowth and the proper formation of the retina. We observed and quantified the molecular and physiological progression of mitochondrial dysfunction during development with this new model of OXPHOS dysfunction, which has great potential for use in diagnostics and therapies for mitochondrial disease. PMID:25771346

  13. The cellular and molecular progression of mitochondrial dysfunction induced by 2,4-dinitrophenol in developing zebrafish embryos.

    PubMed

    Bestman, Jennifer E; Stackley, Krista D; Rahn, Jennifer J; Williamson, Tucker J; Chan, Sherine S L

    2015-01-01

    The etiology of mitochondrial disease is poorly understood. Furthermore, treatment options are limited, and diagnostic methods often lack the sensitivity to detect disease in its early stages. Disrupted oxidative phosphorylation (OXPHOS) that inhibits ATP production is a common phenotype of mitochondrial disorders that can be induced in zebrafish by exposure to 2,4-dinitrophenol (DNP), a FDA-banned weight-loss agent and EPA-regulated environmental toxicant, traditionally used in research labs as an uncoupler of OXPHOS. Despite the DNP-induced OXPHOS inhibition we observed using in vivo respirometry, the development of the DNP-treated and control zebrafish were largely similar during the first half of embryogenesis. During this period, DNP-treated embryos induced gene expression of mitochondrial and nuclear genes that stimulated the production of new mitochondria and increased glycolysis to yield normal levels of ATP. DNP-treated embryos were incapable of sustaining this mitochondrial biogenic response past mid-embryogenesis, as shown by significantly lowered ATP production and ATP levels, decreased gene expression, and the onset of developmental defects. Examining neural tissues commonly affected by mitochondrial disease, we found that DNP exposure also inhibited motor neuron axon arbor outgrowth and the proper formation of the retina. We observed and quantified the molecular and physiological progression of mitochondrial dysfunction during development with this new model of OXPHOS dysfunction, which has great potential for use in diagnostics and therapies for mitochondrial disease.

  14. Insulin-like growth factor-2 regulates early neural and cardiovascular system development in zebrafish embryos.

    PubMed

    Hartnett, Lori; Glynn, Catherine; Nolan, Catherine M; Grealy, Maura; Byrnes, Lucy

    2010-01-01

    The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf-2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play anti-apoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf-2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome.

  15. Development of the endolymphatic sac in chick embryos, with reference to the degradation of otoconia

    NASA Technical Reports Server (NTRS)

    Yoshihara, T.; Kaname, H.; Narita, N.; Ishii, T.; Igarashi, M.; Fermin, C. D.

    1992-01-01

    The endolymphatic sac of chick embryos (from embryonic day 7 to 1-day-old chicks) was studied light- and electron-microscopically. At stage 30-31 (embryonic day 7-7.5), the epithelial cells of the endolymphatic sac were cuboidal to columnar in shape. Microvilli were relatively well developed. The intercellular space was wide. In the endolymphatic space of the endolymphatic sac, varying shapes and sizes of otoconia-like bodies were often observed. Intracytoplasmic phagosomes containing these bodies were rarely found. After stage 37 (embryonic day 11), otoconia-like bodies in the endolymphatic sac decreased in number and size. They were almost the same as the otoconia in the macular organs, ultrastructurally. These findings indicate that the endolymphatic sac of the chick embryos may possess the function of otoconial degradation and removal of calcium from otoconia.

  16. Probing tissue interaction with laser-based cauterization in the early developing Drosophila embryo.

    PubMed

    Rauzi, M

    2017-01-01

    Tissue morphogenesis is governed by mechanical forces generated by cell cytoskeletal networks. It has been shown that subcellular forces are responsible for cell shape changes. Nevertheless cells in a developing organism do not act in isolation: cells contact and adhere one another, and forces are transmitted from cell-to-cell throughout tissues. Understanding how forces are integrated at the tissue level and finally at the full animal scale is nowadays a major challenge that will allow shedding new light on how embryo morphogenesis takes place. In this chapter, I present a new laser-based technique to probe tissue coupling in a living Drosophila embryo. Such technique allows generating mechanical fix boundaries that can eventually impair or modulate cell flows and tissue displacements to probe tissue interaction.

  17. Neurotransmitter signaling pathways required for normal development in Xenopus laevis embryos: a pharmacological survey screen

    PubMed Central

    Sullivan, Kelly G.; Levin, Michael

    2016-01-01

    Neurotransmitters are not only involved in brain function but are also important signaling molecules for many diverse cell types. Neurotransmitters are widely conserved, from evolutionarily ancient organisms lacking nervous systems through man. Here, we report results from a loss- and gain-of-function survey, using pharmacologic modulators of several neurotransmitter pathways to examine possible roles in normal embryogenesis. Applying reagents targeting the glutamatergic, adrenergic, and dopaminergic pathways to embryos of Xenopus laevis from gastrulation to organogenesis stages, we observed and quantified numerous malformations including craniofacial defects, hyperpigmentation, muscle mispatterning, and miscoiling of the gut. These data implicate several key neurotransmitters in new embryonic patterning roles, reveal novel earlier stages for processes involved in eye development, suggest new targets for subsequent molecular-genetic investigation, and highlight the necessity for in-depth toxicology studies of psychoactive compounds to which human embryos might be exposed during pregnancy. PMID:27060969

  18. Differential toxicity of three PCB congeners in developing sea urchin embryos and implication of TEQ approach

    SciTech Connect

    Schweitzer, L.; Suffet, I.; Hose, J.; Bay, S.

    1995-12-31

    The relationship between body burden and toxicity of three individual PCB congeners in developing sea urchin embryos was investigated to evaluate the validity of current predictive models of PCB toxicity in an invertebrate system. The uptake and accumulation of radiolabeled PCB congeners from sea water was measured in the sea urchin embryo tissues and the relative toxicity determined. According to the toxic equivalents (TEQ) approach of assessing risk to mammals, congener 77, a nonortho-substituted congener, is predicted to be more toxic than the diortho-substituted congeners 47 and 153. Using a 72 hour embryo development assay, congener 47 was found to be at least four times as toxic as congener 77, with EC50s of 15.7 and > 72.5 mmol/kg, respectively. Congener 153, a hexachlorobiphenyl, was virtually nontoxic even at the highest dose used. Cytologic and cytogenetic anomalies were studied to find a possibly more sensitive endpoint and to suggest a mechanism of toxicity. The cytogenetic analysis revealed that the PCBs inhibited mitosis. At the highest doses, complete mitotic arrest was observed. Congener 77 was found to be at least two times more toxic than congener 153 but not as toxic as congener 47 using mitotic activity as the endpoint. Thus, the two endpoints of toxicity did not change the order in which the congeners are toxic, but established different EC50s. The relative toxicities of these congeners in this study contradict the structure-activity prediction of the mammalian-based TEQ approach.

  19. Guarding Embryo Development of Zebrafish by Shell Engineering: A Strategy to Shield Life from Ozone Depletion

    PubMed Central

    Wang, Ben; Liu, Peng; Tang, Yanyan; Pan, Haihua; Xu, Xurong; Tang, Ruikang

    2010-01-01

    Background The reduced concentration of stratospheric ozone results in an increased flux of biologically damaging mid-ultraviolet radiation (UVB, 280 to 320 nm) reaching earth surfaces. Environmentally relevant levels of UVB negatively impact various natural populations of marine organisms, which is ascribed to suppressed embryonic development by increased radiation. Methodology/Principal Findings Inspired by strategies in the living systems generated by evolution, we induce an extra UVB-adsorbed coat on the chorion (eggshell surrounding embryo) of zebrafish, during the blastula period. Short and long UV exposure experiments show that the artificial mineral-shell reduces the UV radiation effectively and the enclosed embryos become more robust. In contrast, the uncoated embryos cannot survive under the enhanced UVB condition. Conclusions We suggest that an engineered shell of functional materials onto biological units can be developed as a strategy to shield lives to counteract negative changes of global environment, or to provide extra protection for the living units in biological research. PMID:20376356

  20. Polymethoxy-1-alkenes from Aphanizomenon ovalisporum Inhibit Vertebrate Development in the Zebrafish (Danio rerio) Embryo Model

    PubMed Central

    Jaja-Chimedza, Asha; Gantar, Miroslav; Gibbs, Patrick D. L.; Schmale, Michael C.; Berry, John P.

    2012-01-01

    Cyanobacteria are recognized producers of a wide array of toxic or otherwise bioactive secondary metabolites. The present study utilized the zebrafish (Danio rerio) embryo as an aquatic animal model of vertebrate development to identify, purify and characterize lipophilic inhibitors of development (i.e., developmental toxins) from an isolate of the freshwater cyanobacterial species, Aphanizomenon ovalisporum.Bioassay-guided fractionation led to the purification, and subsequent chemical characterization, of an apparent homologous series of isotactic polymethoxy-1-alkenes (1–6), including three congeners (4–6) previously identified from the strain, and two variants previously identified from other species (2 and 3), as well as one apparently novel member of the series (1). Five of the PMAs in the series (1–5) were purified in sufficient quantity for comparative toxicological characterization, and toxicity in the zebrafish embryo model was found to generally correlate with relative chain length and/or methoxylation. Moreover, exposure of embryos to a combination of variants indicates an apparent synergistic interaction between the congeners. Although PMAs have been identified previously in cyanobacteria, this is the first report of their apparent toxicity. These results, along with the previously reported presence of the PMAs from several cyanobacterial species, suggest a possibly widespread distribution of the PMAs as toxic secondary metabolites and warrants further chemical and toxicological investigation. PMID:23170087

  1. Indole Alkaloids from Fischerella Inhibit Vertebrate Development in the Zebrafish (Danio rerio) Embryo Model

    PubMed Central

    Walton, Katherine; Gantar, Miroslav; Gibbs, Patrick D. L.; Schmale, Michael C.; Berry, John P.

    2014-01-01

    Cyanobacteria are recognized producers of toxic or otherwise bioactive metabolite associated, in particular, with so-called “harmful algal blooms” (HABs) and eutrophication of freshwater systems. In the present study, two apparently teratogenic indole alkaloids from a freshwater strain of the widespread cyanobacterial genus, Fischerella (Stigonemataceae), were isolated by bioassay-guided fractionation, specifically using the zebrafish (Danio rerio) embryo, as a model of vertebrate development. The two alkaloids include the previously known 12-epi-hapalindole H isonitrile (1), and a new nitrile-containing variant, 12-epi-ambiguine B nitrile (2). Although both compounds were toxic to developing embryos, the former compound was shown to be relatively more potent, and to correlate best with the observed embryo toxicity. Related indole alkaloids from Fischerella, and other genera in the Stigonemataceae, have been widely reported as antimicrobial compounds, specifically in association with apparent allelopathy. However, this is the first report of their vertebrate toxicity, and the observed teratogenicity of these alkaloids supports a possible contribution to the toxicity of this widespread cyanobacterial family, particularly in relation to freshwater HABs and eutrophication. PMID:25533520

  2. Effect of PMA-induced protein kinase C activation on development and apoptosis in early zebrafish embryos.

    PubMed

    Hrubik, Jelena; Glisic, Branka; Samardzija, Dragana; Stanic, Bojana; Pogrmic-Majkic, Kristina; Fa, Svetlana; Andric, Nebojsa

    2016-12-01

    Protein kinase C (PKC) isoforms have been implicated in several key steps during early development, but the consequences of xenobiotic-induced PKC activation during early embryogenesis are still unknown. In this study, zebrafish embryos were exposed to a range of phorbol 12-myristate 13-acetate (PMA) concentrations (0-200μg/L) at different time points after fertilization. Results showed that 200μgPMA/L caused development of yolk bags, cardiac edema, slow blood flow, pulsating blood flow, slow pulse, elongated heart, lack of tail fins, curved tail, and coagulation. PMA exposure decreased survival rate of the embryos starting within the first 24h and becoming more pronounced after prolonged exposure (96h). PMA increased the number of apoptotic cells in the brain region as demonstrated by acridine orange staining and caused up-regulation of caspase 9 (casp9) and p53 up-regulated modulator of apoptosis (puma) mRNA in whole embryos. PMA caused oxidative stress in the embryos as demonstrated by decreased mRNA expression of catalase and superoxide dismutase 2. Inhibition of Pkc with GF109203X improved overall survival rate, reduced apoptosis in the brain and decreased expression of casp9 and puma in the PMA-exposed embryos. However, Pkc inhibition neither prevented development of deformities nor reversed oxidative stress in the PMA-exposed embryos. These data suggest that direct over-activation of Pkc during early embryogenesis of zebrafish is associated with apoptosis and decreased survival rate of the embryos.

  3. The Periconceptional Environment and Cardiovascular Disease: Does In Vitro Embryo Culture and Transfer Influence Cardiovascular Development and Health?

    PubMed Central

    Padhee, Monalisa; Zhang, Song; Lie, Shervi; Wang, Kimberley C.; Botting, Kimberley J.; McMillen, I. Caroline; MacLaughlin, Severence M.; Morrison, Janna L.

    2015-01-01

    Assisted Reproductive Technologies (ARTs) have revolutionised reproductive medicine; however, reports assessing the effects of ARTs have raised concerns about the immediate and long-term health outcomes of the children conceived through ARTs. ARTs include manipulations during the periconceptional period, which coincides with an environmentally sensitive period of gamete/embryo development and as such may alter cardiovascular development and health of the offspring in postnatal life. In order to identify the association between ARTs and cardiovascular health outcomes, it is important to understand the events that occur during the periconceptional period and how they are affected by procedures involved in ARTs. This review will highlight the emerging evidence implicating adverse cardiovascular outcomes before and after birth in offspring conceived through ARTs in both human and animal studies. In addition, it will identify the potential underlying causes and molecular mechanisms responsible for the congenital and adult cardiovascular dysfunctions in offspring whom were conceived through ARTs. PMID:25699984

  4. Accumulation and embryotoxicity of polystyrene nanoparticles at early stage of development of sea urchin embryos Paracentrotus lividus.

    PubMed

    Della Torre, C; Bergami, E; Salvati, A; Faleri, C; Cirino, P; Dawson, K A; Corsi, I

    2014-10-21

    Nanoplastic debris, resulted from runoff and weathering breakdown of macro- and microplastics, represents an emerging concern for marine ecosystems. The aim of the present study was to investigate disposition and toxicity of polystyrene nanoparticles (NPs) in early development of sea urchin embryos (Paracentrotus lividus). NPs with two different surface charges where chosen, carboxylated (PS-COOH) and amine (PS-NH2) polystyrene, the latter being a less common variant, known to induce cell death in several in vitro cell systems. NPs stability in natural seawater (NSW) was measured while disposition and embryotoxicity were monitored within 48 h of postfertilization (hpf). Modulation of genes involved in cellular stress response (cas8, 14-3-3ε, p-38 MAPK, Abcb1, Abcc5) was investigated. PS-COOH forms microaggregates (PDI > 0.4) in NSW, whereas PS-NH2 results are better dispersed (89 ± 2 nm) initially, though they also aggregated partially with time. Their respectively anionic and cationic nature was confirmed by ζ-potential measurements. No embryotoxicity was observed for PS-COOH up to 50 μg mL(-1) whereas PS-NH2 caused severe developmental defects (EC50 3.85 μg mL(-1) 24 hpf and EC50 2.61 μg mL(-1) 48 hpf). PS-COOH accumulated inside embryo's digestive tract while PS-NH2 were more dispersed. Abcb1 gene resulted up-regulated at 48 hpf by PS-COOH whereas PS-NH2 induced cas8 gene at 24 hpf, suggesting an apoptotic pathway. In line with the results obtained with the same PS NPs in several human cell lines, also in sea urchin embryos, differences in surface charges and aggregation in seawater strongly affect their embryotoxicity.

  5. Effect of the volume of medium and number of oocytes during in vitro fertilization on embryo development in pigs.

    PubMed

    Gil, Maria Antonia; Abeydeera, Lalantha R; Day, Billy N; Vazquez, Juan M; Roca, Jordi; Martinez, Emilio A

    2003-09-01

    The present study was designed to determine the effect of the volume of medium (VM) and the number of oocytes (NOOC) during in vitro fertilization (IVF) on embryo development in pigs. Groups of 15, 30 and 50 in vitro matured oocytes were transferred to 2, 1 and 0.1 ml of modified Tris-buffered medium (mTBM) and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) in a 3 x 3 factorial experiment. A total of 2739 oocytes from four replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The efficiency of fertilization (EF: number of monospermic oocytes/total number of inseminated oocytes) and BF decreased (P<0.03) as the VM increased (EF: 45.9+/-2.2, 43.8+/-2.6 and 36.9+/-1.6% and BF: 29.4+/-2.7, 23.2+/-1.8 and 19.9+/-2.1% for VM 0.1, 1 and 2 ml, respectively). The BF, but not EF, was also affected (P<0.04) by NOOC (19.8+/-1.6, 28.1+/-2.3 and 24.6+/-2.9% for groups of 15, 30 and 50 oocytes, respectively). The effect of the interaction VM x NOOC on EF and BF was not significant. These results indicate that when 2000 spermatozoa/oocyte were used, a low volume of IVF medium (0.1 ml) and the number of oocytes during IVF (30-50) can improve the in vitro embryo production in pigs.

  6. The development of hindlimb motor activity studied in the isolated spinal cord of the chick embryo.

    PubMed

    O'Donovan, M J; Landmesser, L

    1987-10-01

    The development of hindlimb motor activity was studied in an isolated preparation of the chick spinal cord. The motor output from lumbosacral segments was characterized by recording the pattern of ventral root and muscle nerve discharge in 6-14-d-old embryos. In addition, the synaptic drive underlying motoneuron activity was monitored electrotonically from the ventral roots. Spontaneous motor activity consisted of recurring episodes of cyclical motoneuron discharge. During development, both the number of cycles in each episode and the intensity of discharge in each cycle progressively increased. Monophasic, positive ventral root potentials accompanied each cycle of motoneuron discharge. Prior to the innervation of hindlimb muscles at stage 26, ventral root discharge was barely detectable despite the presence of large ventral root potentials. Following hindlimb muscle innervation, each cycle of activity was initiated by a brief, intense discharge that coincided with the rising phase of the ventral root potential. In embryos older than stage 30, the initial discharge was followed, after a delay, by a more prolonged discharge. The duration of ventral root potentials was shortest in the stage 26 embryos, but was similar in embryos at stage 29 and older. The developmental changes in the coordination of antagonist activity were documented by recording the pattern of discharge in sartorius (flexor) and caudilioflexorius (extensor) muscle nerves between stage 30 and stage 36. At stage 30 both sets of motoneurons were coactivated during the brief discharge that initiated each cycle. By stage 31 a second discharge occurred in each cycle. The second discharge was delayed in flexor, but not in extensor, motoneurons, which led to an alternating pattern of activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Rotenone Decreases Hatching Success in Brine Shrimp Embryos by Blocking Development: Implications for Zooplankton Egg Banks

    PubMed Central

    Hutchison, Evan R.; Neumeyer, Courtney H.; Gunderson, Matthew D.

    2016-01-01

    While many zooplankton species recover quickly after the treatment of water resources with the piscicide, rotenone, some fail to reach pretreatment population density or, in rare cases, do not reappear at all. The variable impact of rotenone on zooplankton populations could stem from differences in the capacity of species to switch entirely to anaerobic catabolic pathways in the presence of rotenone, which blocks mitochondrial electron transport. Alternatively, variable responses among species could originate from differences in permeability of dormant life-stages to lipophilic chemicals like rotenone. The purpose of the present study was to determine the effects of rotenone on development, emergence and hatching of zooplankton embryos that lack both the anaerobic capacity to develop in the presence of rotenone and a permeability barrier to prevent the entry of rotenone during dormancy. Post-diapause embryos of the brine shrimp, Artemia franciscana, were employed as a model system, because they are permeable to lipophilic compounds when dechorionated and require aerobic conditions to support development. Early development in this species is also well characterized in the literature. Brine shrimp embryos were exposed to rotenone while development was either slowed by chilling or suspended by anoxia. Development, emergence and hatching were then observed in rotenone-free artificial seawater. The data presented demonstrate that rotenone freely diffuses across the embryonic cuticle in a matter of hours, and prevents development and emergence after brief exposures to ecologically relevant concentrations (0.025–0.5 mg L-1) of the piscicide. Neither the removal of rotenone from the environment, nor the removal of embryonic water with a hypertonic solution, are sufficient to reverse this block on development and emergence. These data indicate that rotenone could impair recruitment from egg banks for species of zooplankton that lack both an embryonic barrier to the entry

  8. Modeling the disruption of vascular development in a Virtual Embryo using ToxCast HTS Bioactivity Profiles

    EPA Science Inventory

    Blood vessel formation is an important aspect of embryo development and teratogenesis. Recent studies address the signaling networks responsible for vasculogenesis and how they might be targeted by certain chemicals. For example, disruption of vasculogenesis and angiogenesis path...

  9. Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

    USGS Publications Warehouse

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2011-01-01

    In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers. ?? Copyright Cambridge University Press 2010.

  10. Breast cancer 1 (BRCA1)-deficient embryos develop normally but are more susceptible to ethanol-initiated DNA damage and embryopathies.

    PubMed

    Shapiro, Aaron M; Miller-Pinsler, Lutfiya; Wells, Peter G

    2016-04-01

    The breast cancer 1 (brca1) gene is associated with breast and ovarian cancers, and heterozygous (+/-) brca1 knockout progeny develop normally, suggesting a negligible developmental impact. However, our results show BRCA1 plays a broader biological role in protecting the embryo from oxidative stress. Sox2-promoted Cre-expressing hemizygous males were mated with floxed brca1 females, and gestational day 8 +/- brca1 conditional knockout embryos with a 28% reduction in protein expression were exposed in culture to the reactive oxygen species (ROS)-initiating drug ethanol (EtOH). Untreated +/- brca1-deficient embryos developed normally, but when exposed to EtOH exhibited increased levels of oxidatively damaged DNA, measured as 8-oxo-2'-deoxyguanosine, γH2AX, which is a marker of DNA double strand breaks that can result from 8-oxo-2'-deoxyguanosine, formation, and embryopathies at EtOH concentrations that did not affect their brca1-normal littermates. These results reveal that even modest BRCA1 deficiencies render the embryo more susceptible to drug-enhanced ROS formation, and corroborate a role for DNA oxidation in the mechanism of EtOH teratogenesis.

  11. Breast cancer 1 (BRCA1)-deficient embryos develop normally but are more susceptible to ethanol-initiated DNA damage and embryopathies☆

    PubMed Central

    Shapiro, Aaron M.; Miller-Pinsler, Lutfiya; Wells, Peter G.

    2015-01-01

    The breast cancer 1 (brca1) gene is associated with breast and ovarian cancers, and heterozygous (+/−) brca1 knockout progeny develop normally, suggesting a negligible developmental impact. However, our results show BRCA1 plays a broader biological role in protecting the embryo from oxidative stress. Sox2-promoted Cre-expressing hemizygous males were mated with floxed brca1 females, and gestational day 8 +/− brca1 conditional knockout embryos with a 28% reduction in protein expression were exposed in culture to the reactive oxygen species (ROS)-initiating drug ethanol (EtOH). Untreated +/− brca1-deficient embryos developed normally, but when exposed to EtOH exhibited increased levels of oxidatively damaged DNA, measured as 8-oxo-2'-deoxyguanosine, γH2AX, which is a marker of DNA double strand breaks that can result from 8-oxo-2'-deoxyguanosine, formation, and embryopathies at EtOH concentrations that did not affect their brca1-normal littermates. These results reveal that even modest BRCA1 deficiencies render the embryo more susceptible to drug-enhanced ROS formation, and corroborate a role for DNA oxidation in the mechanism of EtOH teratogenesis. PMID:26629949

  12. Embryos, microscopes, and society.

    PubMed

    Maienschein, Jane

    2016-06-01

    Embryos have different meanings for different people and in different contexts. Seen under the microscope, the biological embryo starts out as one cell and then becomes a bunch of cells. Gradually these divide and differentiate to make up the embryo, which in humans becomes a fetus at eight weeks, and then eventually a baby. At least, that happens in those cases that carry through normally and successfully. Yet a popular public perception imagines the embryo as already a little person in the very earliest stages of development, as if it were predictably to become an adult. In actuality, cells can combine, pull apart, and recombine in a variety of ways and still produce embryos, whereas most embryos never develop into adults at all. Biological embryos and popular imaginations of embryos diverge. This paper looks at some of the historical reasons for and social implications of that divergence.

  13. The Chromatin Regulator Brpf1 Regulates Embryo Development and Cell Proliferation*

    PubMed Central

    You, Linya; Yan, Kezhi; Zou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-01-01

    With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs. PMID:25773539

  14. Zinc availability during germline development impacts embryo viability in Caenorhabditis elegans.

    PubMed

    Mendoza, Adelita D; Woodruff, Teresa K; Wignall, Sarah M; O'Halloran, Thomas V

    2017-01-01

    Zinc is an essential metal that serves as a cofactor in a variety of cellular processes, including meiotic maturation. Cellular control of zinc uptake, availability and efflux is closely linked to meiotic progression in rodent and primate reproduction where large fluctuations in zinc levels are critical at several steps in the oocyte-to-embryo transition. Despite these well-documented roles of zinc fluxes during meiosis, only a few of the genes encoding key zinc receptors, membrane-spanning transporters, and downstream signaling pathway factors have been identified to date. Furthermore, little is known about analogous roles for zinc fluxes in the context of a whole organism. Here, we evaluate whether zinc availability regulates germline development and oocyte viability in the nematode Caenorhabditis elegans, an experimentally flexible model organism. We find that similar to mammals, mild zinc limitation in C. elegans profoundly impacts the reproductive axis: the brood size is significantly reduced under conditions of zinc limitation where other physiological functions are not perturbed. Zinc limitation in this organism has a more pronounced impact on oocytes than sperm and this leads to the decrease in viable embryo production. Moreover, acute zinc limitation of isolated zygotes prevents extrusion of the second polar body during meiosis and leads to aneuploid embryos. Thus, the zinc-dependent steps in C. elegans gametogenesis roughly parallel those described in meiotic-to-mitotic transitions in mammals.

  15. When two obese parents are worse than one! Impacts on embryo and fetal development.

    PubMed

    McPherson, N O; Bell, V G; Zander-Fox, D L; Fullston, T; Wu, L L; Robker, R L; Lane, M

    2015-09-15

    The prevalence of overweight and obesity in reproductive-age adults is increasing worldwide. While the effects of either paternal or maternal obesity on gamete health and subsequent fertility and pregnancy have been reported independently, the combination of having both parents overweight/obese on fecundity and offspring health has received minimal attention. Using a 2 × 2 study design in rodents we established the relative contributions of paternal and maternal obesity on fetal and embryo development and whether combined paternal and maternal obesity had an additive effect. Here, we show that parental obesity reduces fetal and placental weights without altering pregnancy establishment and is not dependent on an in utero exposure to a high-fat diet. Interestingly combined parental obesity seemed to accumulate both the negative influences of paternal and maternal obesity had alone on embryo and fetal health rather than an amplification, manifested as reduced embryo developmental competency, reduced blastocyst cell numbers, impaired mitochondrial function, and alterations to active and repressive embryonic chromatin marks, resulting in aberrant placental gene expression and reduced fetal liver mtDNA copy numbers. Further understanding both the maternal cytoplasmic and paternal genetic interactions during this early developmental time frame will be vital for understanding how developmental programming is regulated and for the proposition of interventions to mitigate their effects.

  16. Embryo-fetal development toxicity of honokiol microemulsion intravenously administered to pregnant rats.

    PubMed

    Zhang, Qianqian; Ye, Xiangfeng; Wang, Lingzhi; Peng, Bangjie; Zhang, Yingxue; Bao, Jie; Li, Wanfang; Wei, Jinfeng; Wang, Aiping; Jin, Hongtao; Chen, Shizhong

    2016-02-01

    The aim of this study was to evaluate the embryo-fetal development toxicity of honokiol microemulsion. The drug was intravenously injected to pregnant SD rats at dose levels of 0, 200, 600 and 2000 μg/kg/day from day 6-15 of gestation. All the pregnant animals were observed for body weights and any abnormal changes and subjected to caesarean-section on gestation day (GD) 20; all fetuses obtained from caesarean-section were assessed by external inspection, visceral and skeletal examinations. No treatment-related external alterations as well as visceral and skeletal malformations were observed in honokiol microemulsion groups. There was no significant difference in the body weight gain of the pregnant rats, average number of corpora lutea, and the gravid uterus weight in the honokiol microemulsion groups compared with the vehicle control group. However, at a dose level of 2000 μg/kg/day, there was embryo-fetal developmental toxicity observed, including a decrease in the body length and tail length of fetuses. In conclusion, the no-observed-adverse-effect level (NOAEL) of honokiol microemulsion is 600 μg/kg/day, 75 times above the therapeutic dosage and it has embryo-fetal toxicity at a dose level of 2000 μg/kg/day, which is approximately 250 times above the therapeutic dosage.

  17. Seed Embryo Development Is Regulated via an AN3-MINI3 Gene Cascade

    PubMed Central

    Meng, Lai-Sheng; Wang, Yi-Bo; Loake, Gary J.; Jiang, Ji-Hong

    2016-01-01

    In agriculture, seed mass is one of the most important components related to seed yield. MINISEED3 (MINI3) which encodes the transcriptional activator WRKY10, is thought to be a pivotal regulator of seed mass. In Arabidopsis SHORT HYPOCOTYL UNDER BLUE1 (SHB1) associates with the promoter of MINI3, regulating embryo cell proliferation (both cell division and elongation), which, in turn, modulates seed mass. Furthermore, the recruitment of SHB1 via MINI3 to both its cognate promoter and that of IKU2 implies a two-step amplification for countering the low expression level of IKU2, which is thought to function as a molecular switch for seed cavity enlargement. However, it is largely unknown how embryo cell proliferation, which encompasses both cell division and elongation, is regulated by SHB1 and MINI3 function. Here, we show that a loss of function mutation within the transcriptional coactivator ANGUSTIFOLIA3 (AN3), increases seed mass. Further, AN3 associates with the MINI3 promoter in vivo. Genetic evidence indicates that the absence of MINI3 function suppresses the decrease of cell number observed in an3-4 mutants by regulating cell division and in turn inhibits increased cell size of the an3-4 line by controlling cell elongation. Thus, seed embryo development is modulated via an AN3-MINI3 gene cascade. This regulatory model provides a deeper understanding of seed mass regulation, which may in turn lead to increased crop yields. PMID:27857719

  18. Monitoring in-vitro bovine embryo development during the first days after fertilization (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Kandel, Mikhail E.; Rubessa, Marcello; Fernandes, Daniel; Nguyen, Tan H.; Wheeler, Matthew B.; Popescu, Gabriel

    2016-03-01

    Conventional label-based contrast enhancement techniques (e.g., fluorescence) frequently modify the genetic makeup of tagged cells, making them poor candidates for use in in-vitro fertilization applications. Instead, we choose a label-free form of contrast, based on interferometric imaging, sensitive to optical path length differences. Compared to, single HeLa cells, typical mammalian ova and embryos are more than an order of magnitude thicker. As a result, regions of large phase variation lead to phase wrapping and an overall reduction in signal intensity occurs due to multiple scattering. These effects manifest themselves in low-spatial frequencies (blurs), with the desired details buried in the background. We present a phase shifting interferometer that yields the derivative of the phase, a quantity whose value is particularly sensitive to local variations and fine details. We demonstrate that our new real-time imaging platform is valuable in measuring the multiday development of bovine embryos. Reconstructing the derivative of the image phase and amplitude, we characterize the motion of previously low-contrast structures, which are relevant for embryo viability tests.

  19. Carbon and Nitrogen Provisions Alter the Metabolic Flux in Developing Soybean Embryos1[W][OA

    PubMed Central

    Allen, Doug K.; Young, Jamey D.

    2013-01-01

    Soybean (Glycine max) seeds store significant amounts of their biomass as protein, levels of which reflect the carbon and nitrogen received by the developing embryo. The relationship between carbon and nitrogen supply during filling and seed composition was examined through a series of embryo-culturing experiments. Three distinct ratios of carbon to nitrogen supply were further explored through metabolic flux analysis. Labeling experiments utilizing [U-13C5]glutamine, [U-13C4]asparagine, and [1,2-13C2]glucose were performed to assess embryo metabolism under altered feeding conditions and to create corresponding flux maps. Additionally, [U-14C12]sucrose, [U-14C6]glucose, [U-14C5]glutamine, and [U-14C4]asparagine were used to monitor differences in carbon allocation. The analyses revealed that: (1) protein concentration as a percentage of total soybean embryo biomass coincided with the carbon-to-nitrogen ratio; (2) altered nitrogen supply did not dramatically impact relative amino acid or storage protein subunit profiles; and (3) glutamine supply contributed 10% to 23% of the carbon for biomass production, including 9% to 19% of carbon to fatty acid biosynthesis and 32% to 46% of carbon to amino acids. Seed metabolism accommodated different levels of protein biosynthesis while maintaining a consistent rate of dry weight accumulation. Flux through ATP-citrate lyase, combined with malic enzyme activity, contributed significantly to acetyl-coenzyme A production. These fluxes changed with plastidic pyruvate kinase to maintain a supply of pyruvate for amino and fatty acids. The flux maps were independently validated by nitrogen balancing and highlight the robustness of primary metabolism. PMID:23314943

  20. Influence of bovine sperm DNA fragmentation and oxidative stress on early embryo in vitro development outcome.

    PubMed

    Simões, Renata; Feitosa, Weber Beringui; Siqueira, Adriano Felipe Perez; Nichi, Marcilio; Paula-Lopes, Fabíola Freitas; Marques, Mariana Groke; Peres, Maria Angélica; Barnabe, Valquíria Hyppolito; Visintin, José Antônio; Assumpção, Mayra Elena Ortiz

    2013-01-01

    Sperm chromatin fragmentation may be caused by a number of factors, the most significant of which is reactive oxygen species. However, little is known about the effect of sperm oxidative stress (OS) on DNA integrity, fertilization, and embryonic development in cattle. Therefore, the goal of this study was to evaluate the influence of sperm OS susceptibility on the DNA fragmentation rate and in vitro embryo production (IVP) in a population of bulls. Groups of cryopreserved sperm samples were divided into four groups, based on their susceptibility to OS (G1, low OS; G2, average OS; G3, high OS; and G4, highest OS). Our results demonstrated that the sperm DNA integrity was compromised in response to increased OS susceptibility. Furthermore, semen samples with lower susceptibility to OS were also less susceptible to DNA damage (G1, 4.06%; G2, 6.09%; G3, 6.19%; and G4, 6.20%). In addition, embryo IVP provided evidence that the embryo cleavage rate decreased as the OS increased (G1, 70.18%; G2, 62.24%; G3, 55.85%; and G4, 50.93%), but no significant difference in the blastocyst rate or the number of blastomeres was observed among the groups. The groups with greater sensitivity to OS were also associated with a greater percentage of apoptotic cells (G1, 2.6%; G2, 2.76%; G3, 5.59%; and G4, 4.49%). In conclusion, we demonstrated that an increased susceptibility to OS compromises sperm DNA integrity and consequently reduces embryo quality.

  1. Role of Prolactin in the Recovered T-Cell Development of Early Partially Decapitated Chicken Embryo

    PubMed Central

    Moreno, J.; Varas, A.; Vicente, A.

    1998-01-01

    Although different experimental approaches have suggested certain regulation of the mammalian immune system by the neuroendocrine system, the precise factors involved in the process are largely unknown. In previous reports, we demonstrated important changes in the thymic development of chickens deprived of the major neuroendocrine centers by the removal of embryonic prosencephalon at 33-38 hr of incubation (DCx embryos) (Herradón et al., 1991; Moreno et al., 1995). In these embryos, there was a stopping of T-cell maturation that resulted in an accumulation of the most immature T-cell subsets (CD4-CD8- cells and CD4-CD81o cells) and, accordingly, in decreased numbers of DP (CD4+CD8+) thymocytes and mature CD3+TcRαβ + cells, but not CD3+TcRγδ lymphocytes. In the present work, we restore the thymic histology as well as the percentage of distinct T-cell subsets of DCx embryos by supplying recombinant chicken prolactin, grafting of embryonic pituitary gland, or making cephalic chick-quail chimeras. The recovery was not, however, whole and the percentage of CD3+TcRαβ thymocytes did not reach the normal values observed in 17-day-old control Sham-DCx embryos. The results are discussed on the basis of a key role for prolactin in chicken T-cell maturation. This hormone could regulate the transition of DN (CD4-CD8-) thymocytes to the DP (CD4+CD8+) cell compartment through its capacity for inducing IL-2 receptor expression on the former. PMID:9851358

  2. Oxidative Stress in Mouse Sperm Impairs Embryo Development, Fetal Growth and Alters Adiposity and Glucose Regulation in Female Offspring

    PubMed Central

    Lane, Michelle; McPherson, Nicole O.; Fullston, Tod; Spillane, Marni; Sandeman, Lauren; Kang, Wan Xian; Zander-Fox, Deirdre L.

    2014-01-01

    Paternal health cues are able to program the health of the next generation however the mechanism for this transmission is unknown. Reactive oxygen species (ROS) are increased in many paternal pathologies, some of which program offspring health, and are known to induce DNA damage and alter the methylation pattern of chromatin. We therefore investigated whether a chemically induced increase of ROS in sperm impairs embryo, pregnancy and offspring health. Mouse sperm was exposed to 1500 µM of hydrogen peroxide (H2O2), which induced oxidative damage, however did not affect sperm motility or the ability to bind and fertilize an oocyte. Sperm treated with H2O2 delayed on-time development of subsequent embryos, decreased the ratio of inner cell mass cells (ICM) in the resulting blastocyst and reduced implantation rates. Crown-rump length at day 18 of gestation was also reduced in offspring produced by H2O2 treated sperm. Female offspring from H2O2 treated sperm were smaller, became glucose intolerant and accumulated increased levels of adipose tissue compared to control female offspring. Interestingly male offspring phenotype was less severe with increases in fat depots only seen at 4 weeks of age, which was restored to that of control offspring later in life, demonstrating sex-specific impacts on offspring. This study implicates elevated sperm ROS concentrations, which are common to many paternal health pathologies, as a mediator of programming offspring for metabolic syndrome and obesity. PMID:25006800

  3. Role of ghrelin in fertilization, early embryo development, and implantation periods.

    PubMed

    Luque, Eugenia Mercedes; Torres, Pedro Javier; de Loredo, Nicolás; Vincenti, Laura María; Stutz, Graciela; Santillán, María Emilia; Ruiz, Rubén Daniel; de Cuneo, Marta Fiol; Martini, Ana Carolina

    2014-08-01

    In order to clarify the physiological role of ghrelin in gestation, we evaluated the effects of administration of exogenous ghrelin (2 or 4 nmol/animal per day) or its antagonist (6 nmol/animal per day of (d-Lys3)GHRP6) on fertilization, early embryo development, and implantation periods in mice. Three experiments were performed, treating female mice with ghrelin or its antagonist: i) starting from 1 week before copulation to 12 h after copulation, mice were killed at day 18 of gestation; ii) since ovulation induction until 80 h later, when we retrieved the embryos from oviducts/uterus, and iii) starting from days 3 to 7 of gestation (peri-implantation), mice were killed at day 18. In experiments 1 and 3, the antagonist and/or the highest dose of ghrelin significantly increased the percentage of atrophied fetuses and that of females exhibiting this finding or a higher amount of corpora lutea compared with fetuses (nCL/nF) (experiment 3: higher nCL/nF-atrophied fetuses: ghrelin 4, 71.4-71.4% and antagonist, 75.0-62.5% vs ghrelin 2, 46.2-15.4% and control, 10-0.0%; n=7-13 females/group; P<0.01). In experiment 2, the antagonist diminished the fertilization rate, and both, ghrelin and the antagonist, delayed embryo development (blastocysts: ghrelin 2, 62.5%; ghrelin 4, 50.6%; and antagonist, 61.0% vs control 78.4%; n=82-102 embryos/treatment; P<0.0001). In experiment 3, additionally, ghrelin (4 nmol/day) and the antagonist significantly diminished the weight gain of fetuses and dams during pregnancy. Our results indicate that not only hyperghrelinemia but also the inhibition of the endogenous ghrelin effects exerts negative effects on the fertilization, implantation, and embryo/fetal development periods, supporting the hypothesis that ghrelin (in 'adequate' concentrations) has a physiological role in early gestational events.

  4. Analysis of factors influencing morphokinetic characteristics of embryos in ART cycles.

    PubMed

    Gryshchenko, Mykola Grygorievich; Pravdyuk, Alexey Igorovich; Parashchyuk, Valentin Yurievich

    2014-10-01

    In this article, some factors were evaluated for their impact on embryo morphokinetics during assisted reproductive technology (ART) cycles. We detected significant differences in the fourth cell division time (t5) of embryos obtained after controlled ovarian stimulation in long GnRH agonists and GnRH antagonist protocols. We also found that higher gonadotropin dose may slow down the development of embryos. However, both male and female age, the number of oocytes and number of normal forms of sperm in the ejaculate did not affect the kinetic parameters of embryo development. Further research is needed to identify all the spectrum of factors, which can affect the rate of embryo development.

  5. Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro

    PubMed Central

    Salilew-Wondim, Dessie; Fournier, Eric; Hoelker, Michael; Saeed-Zidane, Mohammed; Tholen, Ernst; Looft, Christian; Neuhoff, Christiane; Besenfelder, Urban; Havlicek, Vita; Rings, Franca; Gagné, Dominic; Sirard, Marc-André; Robert, Claude; A. Shojaei Saadi, Habib; Gad, Ahmed; Schellander, Karl; Tesfaye, Dawit

    2015-01-01

    Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and

  6. Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo

    PubMed Central

    Timme-Laragy, Alicia R.; Goldstone, Jared V.; Imhoff, Barry R.; Stegeman, John J.; Hahn, Mark E.; Hansen, Jason M.

    2013-01-01

    Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous non-protein antioxidant defense molecule is the tri-peptide glutathione (γ-glutamyl-cysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0–5 days post-fertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione (GSH, GSSG) using HPLC, and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0–120 hours of zebrafish development (including mature oocytes, fertilization, mid-blastula transition, gastrulation, somitogenesis, pharyngula, pre-hatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12 hours post fertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12 h, and then oscillated around −190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (−220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study

  7. Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo.

    PubMed

    Timme-Laragy, Alicia R; Goldstone, Jared V; Imhoff, Barry R; Stegeman, John J; Hahn, Mark E; Hansen, Jason M

    2013-12-01

    Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous nonprotein antioxidant defense molecule is the tripeptide glutathione (γ-glutamylcysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0-5 days postfertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione using HPLC and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0-120 h of zebrafish development (including mature oocytes, fertilization, midblastula transition, gastrulation, somitogenesis, pharyngula, prehatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12h postfertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12h, and then oscillated around -190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (-220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding

  8. Addition of L-arginine to the fertilization medium enhances subsequent bovine embryo development rates.

    PubMed

    Santana, Priscila P B; da Silva, Bruno B; Silva, Thiago V G; Costa, Nathalia N; Cordeiro, Marcela S; Santos, Simone S D; Ohashi, Otávio M; Miranda, Moysés S

    2016-04-01

    Although L-Arginine (ARG) has been reported as a promising bovine sperm capacitation agent, its effects on embryo development are still poorly understood. Herein, we compared the effects of ARG and/or heparin (HEP) addition to the fertilization medium for bovine oocytes on sperm capacitation and embryo development. We chose 10 mM ARG based on blastocyst development rates in a titration experiment. Addition of ARG and/or HEP to the fertilization medium resulted in similar rates of blastocyst development (P > 0.05). However, when ARG, but not HEP, was combined with a nitric oxide (NO) synthase inhibitor (N-Nitro-L-ARG-methyl ester, 10 mM) blastocyst development was decreased (P < 0.05). To assess the effects on capacitation, bovine sperm were incubated for 0, 3, and 6 hours in fertilization medium containing ARG and/or HEP and/or N-Nitro-L-ARG-methyl esterand acrosomal exocytosis rates were evaluated using fluorescein isothiocyanate conjugated Pisum sativum lectin (FITC-PSA) staining and flow cytometry. With HEP, acrosomal exocytosis rates were highest by 3 hours of incubation; however, by 6 hours, rates were similar for HEP and/or ARG (P > 0.05) and higher than those in control media (P < 0.05). Although both ARG and HEP increased sperm NO production (P < 0.05), combination with L-NAME only precluded acrosomal exocytosis when ARG added alone in the medium (P > 0.05). These results suggest that although both ARG and HEP supported sperm capacitation, only the effects of the former were driven via NO production. Moreover, ARG was also as effective as HEP at improving blastocyst development rates. Therefore, ARG may be used as a low-cost alternative sperm capacitation agent for bovine in vitro embryo production.

  9. Effects of multi-well plate incubation on embryo-larval development in the fathead minnow (Pimephales promelas).

    PubMed

    Marentette, Julie R; Sullivan, Cheryl A; Lavalle, Christine; Shires, Kallie; Parrott, Joanne L

    2015-01-01

    Fathead minnow embryos and larvae are frequently used in toxicology, including short-term embryo-only tests which often use small volumes of test solution. The effect that such conditions may have on fathead minnow development has yet to be explicitly described. Here we compared rates of embryonic development in fathead minnow embryos reared under standard light and temperature conditions with a range of possible methods. All methods yielded excellent control survival. We demonstrated that fathead minnow embryos incubated in a range of small volumes in multi-well plates (500 μL to 2 mL per embryo) did not substantially vary in developmental rate, but flexed less frequently as embryos, hatched smaller, later and with larger yolk-sacs, and initiated feeding later than embryos reared in an excess of solution (20 mL per embryo) with or without supplemental aeration. Faster hatch and growth were promoted with an orbital shaker, but growth benefits were not sustained into the larval stage. Developmental differences persisted in larvae reared to 20 days post-fertilization when monitoring ceased, but growth differences did not magnify and in some measurements partially resolved. To our knowledge we are the first to report effects of incubation in multi-well plates in any fish taxa. As our data revealed that the eleutheroembryonic stage for fathead minnow may be prolonged in multi-well plates, this may allow the use of longer toxicity tests using fathead minnow embryos without conflicting with existing animal welfare legislation in many countries.

  10. A Tribolium castaneum whole-embryo culture protocol for studying the molecular mechanisms and morphogenetic movements involved in insect development.

    PubMed

    Macaya, Constanza C; Saavedra, Patricio E; Cepeda, Rodrigo E; Nuñez, Viviana A; Sarrazin, Andres F

    2016-01-01

    The development of the red flour beetle Tribolium castaneum is more representative of arthropods than the evolutionarily derived fly, Drosophila melanogaster. Thus, Tribolium is becoming an emerging organism model for studying the evolution of the mechanisms that control embryonic development in arthropods. In this regard, diverse genetic and molecular tools are currently available for Tribolium, as well as imaging and embryonic techniques. Recently, we developed a method for culturing embryos in order to study specific stages during Tribolium development. In this report, we present a detailed and "easy-to-follow" protocol for embryo handling and dissection, extending the use of whole-embryo culture to functional analysis by performing in vivo pharmacological manipulations. This experimental accessibility allowed us to study the relevance of microtubules in axis elongation, using nocodazole and taxol drugs to interfere with microtubule networks, followed by length measurement analysis. Additionally, we demonstrated that embryo handling had no effect on the development of Tribolium embryos, and we checked viability after dissection and bisection and during incubation using propidium iodide. The embryo culture protocol we describe here can be applied to study diverse developmental processes in Tribolium. We expect that this protocol can be adapted and applied to other arthropods.

  11. Maternal serum progesterone concentration and early conceptus development of bovine embryos produced in vivo or in vitro.

    PubMed

    Barnwell, C V; Farin, P W; Whisnant, C S; Alexander, J E; Farin, C E

    2015-07-01

    The hormone progesterone is essential for proper embryonic development. The objective of this study was to examine the relationship between recipient serum concentrations of progesterone, at the time of embryo transfer and at conceptus recovery, on conceptus development from in vivo- or in vitro-produced embryos. Embryos were produced in vivo by superovulation of Holstein cows (IVO; n = 17) or in vitro with either serum-containing (IVPS; n = 27) or serum-restricted medium (IVPSR; n = 34). Single grade I blastocysts from each embryo production system were transferred into heifers on day 7 of development. Conceptuses were recovered on day 17 of gestation and classified as complete, degenerated, or no conceptus. Compared with the IVO group, in vitro-produced embryos had more (P = 0.055) degenerated conceptuses (IVO, 0%; IVPS, 18.5%; and IVPSR, 20.6%). There were no differences in progesterone concentrations at the time of transfer when recipients received either male or female embryos (P > 0.05). Progesterone concentrations in recipients receiving in vivo-produced embryos were higher (P < 0.05; 3.74 ± 0.4 ng/mL; least-squares mean ± standard error of the mean) on day 7 compared with those receiving in vitro-produced embryos (IVPS, 2.4 ± 0.2; IVPSR, 2.58 ± 0.3 ng/mL). However, there was no difference in progesterone concentration on day 7 between treatment groups for heifers from which short conceptuses (≤194 mm) were recovered on day 17. In contrast, when longer (>194 mm) conceptuses were recovered on day 17, heifers receiving in vitro-produced embryos had lower (P = 0.05) serum concentrations of progesterone on day 7 compared with those receiving in vivo-produced embryos (IVPS, 2.2 ± 0.5; IVPSR, 2.3 ± 0.5; IVO, 3.9 ± 0.5 ng/mL). In conclusion, differences in autonomy may exist between in vitro- and in vivo-produced embryos during the period of conceptus elongation with in vitro-produced embryos relying more on intrinsic factors to influence elongation.

  12. Effects of ionizing radiation on the developing embryo and fetus: a review. Final report

    SciTech Connect

    Hoffman, D.A.; Felten, R.P.; Cyr, W.H.

    1981-08-01

    A general review of the literature dealing with effects of ionizing radiation on the developing embryo and fetus. Encompasses both experimental and epidemiological data based on the age of the organism at exposure, with major emphasis on exposure during pregnancy. An appendix presents this information in table format. This review consists of three main sections: experimental, genetic, and epidemiological. These sections are organized according to the age of the organism at the time of irradiation. Data are presented by period of development and endpoints observed. In addition to the individual section summaries, an overall summation is presented.

  13. Identical triplets and twins developed from isolated blastomeres of 8- and 16-cell mouse embryos supported with tetraploid blastomeres.

    PubMed

    Tarkowski, Andrzej K; Ozdzenski, Waclaw; Czolowska, Renata

    2005-01-01

    We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.

  14. Improved Development of Somatic Cell Cloned Mouse Embryos by Vitamin C and Latrunculin A

    PubMed Central

    Mallol, Anna; Santaló, Josep; Ibáñez, Elena

    2015-01-01

    Impaired development of embryos produced by somatic cell nuclear transfer (SCNT) is mostly associated with faulty reprogramming of the somatic nucleus to a totipotent state and can be improved by treatment with epigenetic modifiers. Here we report that addition of 100 μM vitamin C (VitC) to embryo culture medium for at least 16 h post-activation significantly increases mouse blastocyst formation and, when combined with the use of latrunculin A (LatA) during micromanipulation and activation procedures, also development to term. In spite of this, no significant effects on pluripotency (OCT4 and NANOG) or nuclear reprogramming markers (H3K14 acetylation, H3K9 methylation and DNA methylation and hydroxymethylation) could be detected. The use of LatA alone significantly improved in vitro development, but not full-term development. On the other hand, the simultaneous treatment of cloned embryos with VitC and the histone deacetylase inhibitor psammaplin A (PsA), in combination with the use of LatA, resulted in cloning efficiencies equivalent to those of VitC or PsA treatments alone, and the effects on pluripotency and nuclear reprogramming markers were less evident than when only the PsA treatment was applied. These results suggest that although both epigenetic modifiers improve cloning efficiencies, possibly through different mechanisms, they do not show an additive effect when combined. Improvement of SCNT efficiency is essential for its applications in reproductive and therapeutic cloning, and identification of molecules which increase this efficiency should facilitate studies on the mechanism of nuclear reprogramming and acquisition of totipotency. PMID:25749170

  15. Environmental Factors Affecting Preschoolers' Motor Development

    ERIC Educational Resources Information Center

    Venetsanou, Fotini; Kambas, Antonis

    2010-01-01

    The process of development occurs according to the pattern established by the genetic potential and also by the influence of environmental factors. The aim of the present study was to focus on the main environmental factors affecting motor development. The review of the literature revealed that family features, such as socioeconomic status,…

  16. Methionine requirements for the preimplantation bovine embryo.

    PubMed

    BONILLA, Luciano; LUCHINI, Daniel; DEVILLARD, Estelle; HANSEN, Peter J

    2010-10-01

    The early embryo's nutritional environment plays an important role in establishing its developmental potential. However, little is known about the specific nutrient requirements of the embryo. The objective of the present study was to determine requirements of the in vitro produced bovine embryo for the essential amino acid methionine. In addition to serving as a precursor for polypeptides, methionine plays roles in regulation of translation, DNA methylation, and antioxidant balance. In the first experiment, embryos were cultured in potassium simplex optimized medium - bovine embryo modification 2 containing 0, 35, 50, 100, 200 or 400 µmol/l L-methionine for 8 days. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 than other groups but was similar for embryos cultured with 35-400 µmol/l. Neither total cell number, allocation of cells to trophectoderm or inner cell mass, or frequency of apoptosis was affected by methionine concentration. In the second experiment, embryos were cultured with 0, 7, 14, 21, 28 or 35 µmol/l methionine. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 but was not different between embryos cultured with 7-35 µmol/l methionine. However, the proportion of blastocysts that were expanded, hatching or hatched on Day 7 was reduced at lower concentrations of methionine (7 and 14). DNA methylation of blastocyst nuclei was unaffected by methionine concentration but intracellular glutathione content was higher for embryos cultured without methionine. In conclusion, the methionine requirement for preimplantation development is between 14 and 21 µmol/l. These concentrations are lower or similar to those found in the reproductive tract and suggest that methionine deficiency is not a common cause of

  17. Effect of embryo density on in vitro developmental characteristics of bovine preimplantative embryos with respect to micro and macroenvironments.

    PubMed

    Hoelker, M; Rings, F; Lund, Q; Phatsara, C; Schellander, K; Tesfaye, D

    2010-10-01

    To overcome developmental problems as a consequence of single embryo culture, the Well of the Well (WOW) culture system has been developed. In this study, we aimed to examine the effect of embryo densities with respect to both microenvironment and macroenvironment on developmental rates and embryo quality to get a deeper insight into developmentally important mechanisms. WOW diameter and depth significantly affected developmental rates (p < 0.05). WOWs with diameter of 500 μm reached significantly higher blastocyst rates (32.5 vs 21.1% vs 20.3%) compared to embryos cultured in WOWs of 300 μm diameter or plain cultured controls. Embryos cultured in WOWs with 700 μm depth reached significant higher developmental rates compared with embryos cultured in WOWs of 300 μm depth and control embryos (30.6 vs 22.6% vs 20.3%). Correlation of the embryo per WOW volume with developmental rates was higher (r(2) = 0.92, p = 0.0004) than correlation of WOW diameter or WOW depth with developmental rates. However, the embryo per WOW volume did not affect differential cell counts. An embryo per culture dish volume of 1 : 30 μl was identified to be optimal when the embryo per WOW volume was 1 : 0.27 μl increasing developmental rates up to the level of mass embryo production. Giving the opportunity to track each embryo over the complete culture period while keeping high developmental rates with normal mitotic dynamics, the results of this work will provide benefit for the single culture of embryos in human assisted reproduction, mammalian embryos with high economic interest as well as for scientific purpose.

  18. Effects of silver nanoparticles on pregnant dams and embryo-fetal development in rats.

    PubMed

    Yu, Wook-Joon; Son, Jung-Mo; Lee, Jinsoo; Kim, Sung-Hwan; Lee, In-Chul; Baek, Hyung-Seon; Shin, In-Sik; Moon, Changjong; Kim, Sung-Ho; Kim, Jong-Choon

    2014-08-01

    Although the potential risk of silver nanoparticles (AgNPs) to humans has recently increased due to widespread application, the potential effects of AgNPs on embryo-fetal development have not yet been determined. This study investigated the potential effects of AgNPs on pregnant dams and embryo-fetal development after maternal exposure on gestational days (GD) 6-19 in rats. AgNPs were administered to pregnant rats by gavage at concentrations of 0, 100, 300, and 1000 mg/kg/day. All dams were subjected to Cesarean section on GD 20 and the fetuses were examined for signs of embryotoxic and teratogenic effects. Examinations of hepatic oxidant/antioxidant balance and serum biochemistry were also added to the routine developmental toxicity study. Treatment with AgNPs caused a decrease in catalase and glutathione reductase activities at ≥100 mg/kg/day and a reduction in glutathione content at 1000 mg/kg/day in maternal liver tissues. However, no treatment-related deaths or clinical signs were observed in any of the animals treated with AgNPs. No treatment-related differences in maternal body weight, food consumption, gross findings, serum biochemistry, organ weight, gestation index, fetal deaths, fetal and placental weights, sex ratio, or morphological alterations were observed between the groups. The results show that repeated oral doses of AgNPs during pregnancy caused oxidative stress in hepatic tissues at ≥100 mg/kg/day, but did not cause developmental toxicity at doses of up to 1000 mg/kg/day. The no-observed-adverse-effect level of AgNPs is considered to be <100 mg/kg/day for dams and 1000 mg/kg/day for embryo-fetal development.

  19. Differential toxicity of three polychlorinated biphenyl congeners in developing sea urchin embryos

    SciTech Connect

    Schweitzer, L.E.; Suffet, I.H.; Hose, J.E.; Bay, S.M.

    1997-07-01

    The relationship between body burden and toxicity of three individual polychlorinated biphenyl (PCB) congeners in developing sea urchin embryos was investigated to evaluate the validity of current predictive models of PCB toxicity in an invertebrate system. Body burdens of radiolabeled PCB congeners (IUPAC-47, 77, and 153) accumulated from a seawater were used to determine median effective concentrations (EC50s) for developmental and cytogenetic effects following a 72-h exposure. Congener 47, a di-ortho-substituted tetrachlorobiphenyl, was found to be at least four times more toxic than congener 77, a non-ortho-substituted (coplanar) tetrachlorobiphenyl, with EC50s of 47 and >218 mmol/kg, respectively, using an embryo development assay. This result contradicts the structure-activity prediction of the mammalian-based toxic equivalents (TEQs) approach, demonstrating the need for an ecotoxicologic model. Congener 153, a di-ortho-substituted hexachlorobiphenyl, was virtually nontoxic in terms of developmental effects at the highest dose achievable at its limit of water solubility. Cytogenetic analysis was a more sensitive method for assessing toxicity than the embryo development assay. Dose-response relationships were established with mitotic activity being the most sensitive endpoint because the PCBs appeared to inhibit mitosis. At the highest doses, complete mitotic arrest was observed. Congener 77 was found to be at least two times more toxic than congener 153 but not as toxic as congener 47 using mitotic activity as the endpoint for toxicity. Thus, the developmental and cytogenetic endpoints ranked the toxicity of the congeners similarly, but established different EC50s.

  20. Effects of granulosa cell mitochondria transfer on the early development of bovine embryos in vitro.

    PubMed

    Hua, Song; Zhang, Yong; Li, Xiang-Chen; Ma, Li-Bing; Cao, Jun-Wei; Dai, Jin-Po; Li, Rong

    2007-01-01

    The objective of this study was to determine the effect of exogenous mitochondria obtained from granulosa cells on the development of bovine embryos in vitro. We classified cumulus oocyte complexes (COCs) as good (G)- and poor (P)-quality oocytes based on cytoplasmic appearance and cumulus characteristics, and assessed mtDNA copy numbers in the G and P oocytes with real-time polymerase chain reaction (PCR). The mitochondria were isolated by fractionation and suspended in mitochondria injection buffer (MIB). Part one of the experiment consisted of the following treatments: (1) G-oocytes + sperm, (2) P-oocytes + mitochondria + MIB + sperm, (3) P-oocytes + MIB + sperm, and (4) P-oocytes + sperm. In part 2, oocytes were parthenogenetically activated. The treatments were: (1) G-oocytes, (2) P-oocytes + mitochondria + MIB, (3) P-oocytes + MIB, and (4) P-oocytes alone. The results indicated a significant difference in mtDNA copy number between G (361 113 +/- 147 114) and P (198 293 +/- 174 178) oocytes (p < 0.01). The rates of morula, blastocyst, and hatched blastocysts derived from P-oocytes + mitochondria were similar to those of G-oocytes, but significantly higher than P-oocytes without exogenous mitochondria in both the ICSI and parthenogenetic activation experiments. We found no difference in blastomere numbers between G-oocytes and P-oocytes + mitochondria in either experiment, but blastomere numbers in these two groups were significantly higher than in P-oocyte groups without exogenous mitochondria. These data suggest that mtDNA content is very important for early embryo development. Furthermore, the transfer of mitochondria from the same breed may improve embryo quality during preimplantation development.

  1. Comparative toxicity of metal oxide nanoparticles (CuO, ZnO and TiO2) to developing zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Vicario-Parés, Unai; Castañaga, Luis; Lacave, Jose Maria; Oron, Miriam; Reip, Paul; Berhanu, Deborah; Valsami-Jones, Eugenia; Cajaraville, Miren P.; Orbea, Amaia

    2014-08-01

    Increasing use of nanomaterials is resulting in their release into the environment, making necessary to determine the toxicity of these materials. With this aim, the effects of CuO, ZnO and TiO2 nanoparticles (NPs) on zebrafish development were assessed in comparison with the effects caused by the ionic forms (for copper and zinc), bulk counterparts and the stabilizer used for rutile TiO2 NPs. None of the NPs caused significant embryo mortality. CuO NPs were the most toxic affecting hatching and increasing malformation prevalence (≥1 mg Cu/L), followed by ZnO NPs that affected hatching at ≥5 mg Zn/L and stabilized TiO2 NPs that caused mortality and decreased hatching at 100 mg Ti/L. Exposure to the stabilizer alone provoked the same effect. Thus, toxicity of the TiO2 NP suspension can be linked to the surfactant. For all the endpoints, the greatest effects were exerted by the ionic forms, followed by the NPs and finally by the bulk compounds. By autometallography, metal-bearing deposits were observed in embryos exposed to CuO and ZnO NPs, being more abundant in the case of embryos exposed to CuO NPs. The largest and most abundant metal-bearing deposits were detected in embryos exposed to ionic copper. In conclusion, metal oxide NPs affected zebrafish development altering hatching and increasing the prevalence of malformations. Thus, the use and release of metal oxide NPs to the environment may pose a risk to aquatic organisms as a result of the toxicity caused by NPs themselves or by the additives used in their production.

  2. Effects of environmental oxygen on development and respiration of Australian lungfish (Neoceratodus forsteri) embryos.

    PubMed

    Mueller, Casey A; Joss, Jean M P; Seymour, Roger S

    2011-10-01

    The effects of oxygen partial pressure ([Formula: see text]) on development and respiration were investigated in the eggs of the Australian lungfish, Neoceratodus forsteri. At 20°C, embryonic survival and development was optimal at 15 and 20.9 kPa. Development was slowed at 5 and 10 kPa and embryos did not survive 2 kPa. At lower [Formula: see text], the rate of oxygen consumption also decreased. Embryos responded to hypoxia by hatching at an earlier age and stage of development, and hatching wet and dry gut-free masses were reduced. The role of oxygen conductance ([Formula: see text]) in gas exchange was also examined under selected environmental [Formula: see text] and temperatures. The breakdown of the vitelline membrane changed capsule geometry, allowed water to be absorbed into the perivitelline space and increased capsule [Formula: see text]. This occurred at embryonic stage 32 under all treatments and was largely independent of both [Formula: see text] and temperature (15, 20 and 25°C), demonstrating that capsule [Formula: see text] cannot adaptively respond to altered environmental conditions. The membrane breakdown increased capsule diffusive [Formula: see text] and stabilised perivitelline [Formula: see text], but reduced the convective [Formula: see text] of the perivitelline fluid, as the large perivitelline volume and inadequate convective current resulted in a [Formula: see text] gradient within the egg prior to hatch.

  3. Early development of Drosophila embryos requires Smc5/6 function during oogenesis.

    PubMed

    Tran, Martin; Tsarouhas, Vasilios; Kegel, Andreas

    2016-07-15

    Mutations in structural maintenance of chromosomes (Smc) proteins are frequently associated with chromosomal abnormalities commonly observed in developmental disorders. However, the role of Smc proteins in development still remains elusive. To investigate Smc5/6 function during early embryogenesis we examined smc5 and smc6 mutants of the fruit fly Drosophila melanogaster using a combination of reverse genetics and microscopy approaches. Smc5/6 exhibited a maternally contributed function in maintaining chromosome stability during early embryo development, which manifested as female subfertility in its absence. Loss of Smc5/6 caused an arrest and a considerable delay in embryo development accompanied by fragmented nuclei and increased anaphase-bridge formation, respectively. Surprisingly, early embryonic arrest was attributable to the absence of Smc5/6 during oogenesis, which resulted in insufficient repair of pre-meiotic and meiotic DNA double-strand breaks. Thus, our findings contribute to the understanding of Smc proteins in higher eukaryotic development by highlighting a maternal function in chromosome maintenance and a link between oogenesis and early embryogenesis.

  4. Early development of Drosophila embryos requires Smc5/6 function during oogenesis

    PubMed Central

    Tran, Martin; Tsarouhas, Vasilios

    2016-01-01

    ABSTRACT Mutations in structural maintenance of chromosomes (Smc) proteins are frequently associated with chromosomal abnormalities commonly observed in developmental disorders. However, the role of Smc proteins in development still remains elusive. To investigate Smc5/6 function during early embryogenesis we examined smc5 and smc6 mutants of the fruit fly Drosophila melanogaster using a combination of reverse genetics and microscopy approaches. Smc5/6 exhibited a maternally contributed function in maintaining chromosome stability during early embryo development, which manifested as female subfertility in its absence. Loss of Smc5/6 caused an arrest and a considerable delay in embryo development accompanied by fragmented nuclei and increased anaphase-bridge formation, respectively. Surprisingly, early embryonic arrest was attributable to the absence of Smc5/6 during oogenesis, which resulted in insufficient repair of pre-meiotic and meiotic DNA double-strand breaks. Thus, our findings contribute to the understanding of Smc proteins in higher eukaryotic development by highlighting a maternal function in chromosome maintenance and a link between oogenesis and early embryogenesis. PMID:27288507

  5. Effects of Madagascar yam extracts, Dioscorea antaly, on embryo-larval development of medaka fish, Oryzias latipes.

    PubMed

    Rakotobe, Lolona; Berkal, Miassa; Huet, Hélène; Djediat, Chakib; Jeannoda, Victor; Bodo, Bernard; Mambu, Lengo; Crespeau, François; Edery, Marc

    2010-01-01

    The yams edible starchy tubers, are of cultural, economic and nutritional importance in tropical and subtropical regions. The present study concerns the analysis at different levels of Dioscorea antaly toxicity to medaka embryo-larval development. The incubation of medaka fish embryos in a medium containing Dioscorea antaly extract resulted in a dose dependent reduction in survival rate. Survival rates were reduced up to 100% with extract concentrations of 4mg mL(-1). The LD(50) was estimated to be 0.86mg mL(-1)Dioscorea antaly. Anatomopathological studies did not show any caustic effects, irritation to mouth, throat or intestinal tract in surviving embryos but rather an inflammatory reaction in the liver. The data presented in this paper thus extends the use of medaka embryos as a valuable model to analyze the effects of food toxins.

  6. A Genetic Screen for Mutations