Stage-dependent toxicity of bisphenol a on Rhinella arenarum (anura, bufonidae) embryos and larvae.

PubMed

Wolkowicz, Ianina R Hutler; Herkovits, Jorge; Pérez Coll, Cristina S

2014-02-01

The acute and chronic toxicity of bisphenol A (BPA) was evaluated on the common South American toad Rhinella arenarum embryos and larvae by means of continuous and pulse exposure treatments. Embryos were treated continuously from early blastula (S.4) up to complete operculum (S.25), during early larval stages and by means of 24 h pulse exposures of BPA in concentrations ranging between 1.25 and 40 mg L(-1) , in order to evaluate the susceptibility to this compound in different developmental stages. For lethal effects, S.25 was the most sensitive and gastrula was the most resistant to BPA. The Teratogenic Index for neurula, the most sensitive embryonic stage for sublethal effects was 4.7. The main morphological alterations during early stages were: delayed or arrested development, reduced body size, persistent yolk plug, microcephaly, axial/tail flexures, edemas, blisters, waving fin, underdeveloped gills, mouth malformations, and cellular dissociation. BPA caused a remarkable narcotic effect from gill circulation stage (S.20) onwards in all the organisms exposed after 3 h of treatment with 10 mg L(-1) BPA. After recovering, the embryos exhibited scarce response to stimuli, erratic or circular swimming, and spasmodic contractions from 5 mg L(-1) onwards. Our results highlight the lethal and sublethal effectsof BPA on R. arenarum embryos and larvae, in the last case both at structural and functional levels. Copyright © 2011 Wiley Periodicals, Inc., A Wiley Company.

Sensitivity of early mouse embryos to (/sup 3/H)thymidine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spindle, A.; Wu, K.; Pedersen, R.A.

1982-12-01

Effects of intranuclear radiation on the developmental capacity of early mouse embryos were studied by exposing embryos to (/sup 3/H)thymidine and counting the number of embryos forming blastocysts, trophoblast outgrowths, inner cell masses (ICMs), and two-layer ICMs (differentiated into primary endoderm and ectoderm). When embryos were cultured from the 2-cell stage for 8 days in the continuous presence of (/sup 3/H)thymidine, concentrations as low as 0.2 nCi/ml reduced the number of embryos forming two-layer ICMs. At 1 nCi/ml, the number of both ICMs and two-layer ICMs were reduced, and at 10 nCi/ml the number of embryos developing to all threemore » post-blastocyst endpoints was reduced. Blastocyst formation was not affected even at the highst concentration (/sup 3/H)thymidine and then cultured further in unlabelled medium, the effects were similar to those of 8-day exposure. When embryos were exposed to (/sup 3/H)thymidine for 24 h at various developmental stages, effects were less severe than when they were exposed continuously for 3 or 8 days, and the sensitivity of embryos differed between stages. The 24-h exposure of immunosurgically isolated ICMS to (/sup 3/H)thymidine revealed that the high sensitivity of the ICM to (/sup 3/H)thymidine persists through the late blastocyst stage and declines progressively thereafter. Autoradiography indicated that the change in radiosensitivity of embryos or ICMs is generally related to their ability to incorporate (/sup 3/H)thymidine into the DNA.« less

Forskolin improves the cryosurvival of in vivo-derived porcine embryos at very early stages using two vitrification methods.

PubMed

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

2013-04-01

This study was aimed to determine the effect of forskolin on the viability of in vivo-derived porcine embryos vitrified by the superfine open pulled straw (SOPS) or solid surface vitrification (SSV) methods at the 2-cell, 4-cell, and blastocyst stages. Zygotes, 2- to 4-cell embryos, and morulae were obtained from superovulated sows. After collection, embryos were cultured for 24h with 0 or 10 μM forskolin and then vitrified using the SOPS and SSV method, or not vitrified (fresh controls). Fresh and vitrified-warmed 2-cells, 4-cells, and blastocysts were cultured for additional 96 h, 72 h and 24 h, respectively. At the end of the culture, embryos were evaluated for progression to the blastocyst stage and total cell number. The vitrification method did not affect any of the parameters evaluated for any embryo stage. Forskolin increased (P<0.01) the blastocyst formation and the final developmental stage of vitrified 2- and 4-cell embryos. However, these embryos exhibited lower (P<0.003) blastocyst formation rates than their fresh counterparts. The total cell number and hatching rate were similar in both groups (vitrified and fresh) of 2- and 4-cell embryos. Vitrified blastocysts exhibited viabilities, final developmental stages, hatching rates, and total cell numbers that were similar to those of their fresh counterparts, regardless of the addition of forskolin. In conclusion, the SOPS and SSV methods are suitable for the cryopreservation of in vivo-derived 2- to 4-cell porcine embryos. Pre-treatment with forskolin for 24h before vitrification improves the cryotolerance of 2- and 4-cell porcine embryos. Copyright © 2013 Elsevier Inc. All rights reserved.

Efficient embryonic culture method for the Japanese striped snake, Elaphe quadrivirgata, and its early developmental stages.

PubMed

Matsubara, Yoshiyuki; Sakai, Atsushi; Kuroiwa, Atsushi; Suzuki, Takayuki

2014-10-01

The morphogenesis of snake embryos is an elusive yet fascinating research target for developmental biologists. However, few data exist on development of early snake embryo due to limited availability of pregnant snakes, and the need to harvest early stage embryos directly from pregnant snakes before oviposition without knowing the date of fertilization. We established an ex vivo culture method for early snake embryos using the Japanese striped snake, Elaphe quadrivirgata. This method, which we named "sausage-style (SS) culture", allows us to harvest snake embryos at specific stages for each experiment. Using this SS culture system, we calculated somite formation rate at early stages before oviposition. The average somite formation rate between 6/7 and 12/13 somite stages was 145.9 min, between 60/70 and 80/91 somite stages 42.4 min, and between 113-115 and 126/127 somite stages 71 min. Thus, somite formation rate that we observed during early snake embryogenesis was changed over time. We also describe a developmental staging series for E. quadrivirgata. This is the first report of a developmental series of early snake embryogenesis prior to oviposition by full-color images with high-resolution. We propose that the SS culture system is an easy method for treating early snake embryos ex vivo. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

Expression of voltage-activated calcium channels in the early zebrafish embryo.

PubMed

Sanhueza, Dayán; Montoya, Andro; Sierralta, Jimena; Kukuljan, Manuel

2009-05-01

Increases in cytosolic calcium concentrations regulate many cellular processes, including aspects of early development. Calcium release from intracellular stores and calcium entry through non-voltage-gated channels account for signalling in non-excitable cells, whereas voltage-gated calcium channels (CaV) are important in excitable cells. We report the expression of multiple transcripts of CaV, identified by its homology to other species, in the early embryo of the zebrafish, Danio rerio, at stages prior to the differentiation of excitable cells. CaV mRNAs and proteins were detected as early as the 2-cell stages, which indicate that they arise from both maternal and zygotic transcription. Exposure of embryos to pharmacological blockers of CaV does not perturb early development significantly, although late effects are appreciable. These results suggest that CaV may have a role in calcium homeostasis and control of cellular process during early embryonic development.

Microspore embryogenesis in wheat: new marker genes for early, middle and late stages of embryo development.

PubMed

Sánchez-Díaz, Rosa Angélica; Castillo, Ana María; Vallés, María Pilar

2013-09-01

Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a 'star-like' morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation.

Unraveling the association between genetic integrity and metabolic activity in pre-implantation stage embryos

PubMed Central

D’Souza, Fiona; Pudakalakatti, Shivanand M.; Uppangala, Shubhashree; Honguntikar, Sachin; Salian, Sujith Raj; Kalthur, Guruprasad; Pasricha, Renu; Appajigowda, Divya; Atreya, Hanudatta S.; Adiga, Satish Kumar

2016-01-01

Early development of certain mammalian embryos is protected by complex checkpoint systems to maintain the genomic integrity. Several metabolic pathways are modulated in response to genetic insults in mammalian cells. The present study investigated the relationship between the genetic integrity, embryo metabolites and developmental competence in preimplantation stage mouse embryos with the aim to identify early biomarkers which can predict embryonic genetic integrity using spent medium profiling by NMR spectroscopy. Embryos carrying induced DNA lesions (IDL) developed normally for the first 2.5 days, but began to exhibit a developmental delay at embryonic day 3.5(E3.5) though they were morphologically indistinguishable from control embryos. Analysis of metabolites in the spent medium on E3.5 revealed a significant association between pyruvate, lactate, glucose, proline, lysine, alanine, valine, isoleucine and thymine and the extent of genetic instability observed in the embryos on E4.5. Further analysis revealed an association of apoptosis and micronuclei frequency with P53 and Bax transcripts in IDL embryos on the E4.5 owing to delayed induction of chromosome instability. We conclude that estimation of metabolites on E3.5 in spent medium may serve as a biomarker to predict the genetic integrity in pre-implantation stage embryos which opens up new avenues to improve outcomes in clinical IVF programs. PMID:27853269

Immunolocalization and expression of Na(+)/K(+) -ATPase in embryos, early larval stages and adults of the freshwater shrimp Palaemonetes argentinus (Decapoda, Caridea, Palaemonidae).

PubMed

Ituarte, Romina Belén; Lignot, Jehan-Hervé; Charmantier, Guy; Spivak, Eduardo; Lorin-Nebel, Catherine

2016-06-01

The euryhaline shrimp Palaemonetes argentinus exemplifies an evolutionary transition from brackish to freshwater habitats that requires adequate osmoregulatory capacities. Hyperosmoregulation is functional at hatching and it likely begins during the embryonic phase allowing this species to develop entirely in fresh water. Here, we investigated the Na(+)/K(+)-ATPase α-subunit gene (nka-α) expression using quantitative real-time PCR and localized Na(+)/K(+)-ATPase (NKA) in ion-transporting epithelia through immunofluorescence microscopy. We reared shrimps from spawning to juvenile stages at two salinities (1, 15 ‰) and maintained adults for 3 weeks at three salinity treatments (1, 15, 25 ‰). nka-α gene expression was measured in: (1) embryos at an early (SI), intermediate (SII) and late (SIII) stage of embryonic development; (2) newly hatched larvae (Zoea I, ZI); and (3) isolated gill tissue of adults. The nka-α expression was low in SI and SII embryos and reached maximum levels prior to hatching (SIII), which were similar to expression levels detected in the ZI. The nka-α expression in SIII and ZI was highest at 15 ‰, whereas salinity did not affect expression in earlier embryos. In SIII, in ZI and in a later zoeal stage ZIV, NKA was localized in epithelial cells of pleurae, in the inner-side epithelium of branchiostegite and in the antennal glands. Gills appeared in the ZIV but NKA immunolabeling of the cells of the gill shaft occurred in a subsequent developmental larval stage, the decapodid. Extrabranchial organs constitute the main site of osmoregulation in early ontogenetic stages of this freshwater shrimp.

Effect of exogenous transforming growth factor β1 (TGF-β1) on early bovine embryo development.

PubMed

Barrera, Antonio D; García, Elina V; Miceli, Dora C

2018-06-08

SummaryDuring preimplantation development, embryos are exposed and have the capacity to respond to different growth factors present in the maternal environment. Among these factors, transforming growth factor β1 (TGF-β1) is a well known modulator of embryonic growth and development. However, its action during the first stages of development, when the embryo transits through the oviduct, has not been yet elucidated. The objective of the present study was to examine the effect of early exposure to exogenous TGF-β1 on embryo development and expression of pluripotency (OCT4, NANOG) and DNA methylation (DNMT1, DNMT3A, DNMT3B) genes in bovine embryos produced in vitro. First, gene expression analysis of TGF-β receptors confirmed a stage-specific expression pattern, showing greater mRNA abundance of TGFBR1 and TGFBR2 from the 2- to the 8-cell stage, before embryonic genome activation. Second, embryo culture for the first 48 h in serum-free CR1aa medium supplemented with 50 or 100 ng/ml recombinant TGF-β1 did not affect the cleavage and blastocyst rate (days 7 and 8). However, RT-qPCR analysis showed a significant increase in the relative abundance of NANOG and DNMT3A in the 8-cell stage embryos and expanded blastocysts (day 8) derived from TGF-β1 treated embryos. These results suggest an early action of exogenous TGF-β1 on the bovine embryo, highlighting the importance to provide a more comprehensive understanding of the role of TGF-β signalling during early embryogenesis.

Expression of renin–angiotensin system components in the early bovine embryo

PubMed Central

Pijacka, Wioletta; Hunter, Morag G; Broughton Pipkin, Fiona; Luck, Martin R

2012-01-01

The renin–angiotensin system (RAS), mainly associated with the regulation of blood pressure, has been recently investigated in female reproductive organs and the developing foetus. Angiotensin II (Ang II) influences oviductal gamete movements and foetal development, but there is no information about RAS in the early embryo. The aim of this study was to determine whether RAS components are present in the pre-implantation embryo, to determine how early they are expressed and to investigate their putative role at this stage of development. Bovine embryos produced in vitro were used for analysis of RAS transcripts (RT-PCR) and localisation of the receptors AGTR1 and AGTR2 (immunofluorescent labelling). We also investigated the effects of Ang II, Olmesartan (AGTR1 antagonist) and PD123319 (AGTR2 antagonist) on oocyte cleavage, embryo expansion and hatching. Pre-implanted embryos possessed AGTR1 and AGTR2 but not the other RAS components. Both receptors were present in the trophectoderm and in the inner cell mass of the blastocyst. AGTR1 was mainly localised in granular-like structures in the cytoplasm, suggesting its internalisation into clathrin-coated vesicles, and AGTR2 was found mainly in the nuclear membrane and in the mitotic spindle of dividing trophoblastic cells. Treating embryos with PD123319 increased the proportion of hatched embryos compared with the control. These results, the first on RAS in the early embryo, suggest that the pre-implanted embryo responds to Ang II from the mother rather than from the embryo itself. This may be a route by which the maternal RAS influences blastocyst hatching and early embryonic development. PMID:23781300

Measurement of in vivo stress resultants in neurulation-stage amphibian embryos.

PubMed

Benko, Richard; Brodland, G Wayne

2007-04-01

In order to obtain the first quantitative measurements of the in vivo stresses in early-stage amphibian embryos, we developed a novel instrument that uses a pair of parallel wires that are glued to the surface of an embryo normal to the direction in which the stress is to be determined. When a slit is made parallel to the wires and between them, tension in the surrounding tissue causes the slit to open. Under computer control, one of the wires is moved so as to restore the original wire spacing, and the steady-state closure force is determined from the degree of wire flexure. A cell-level finite element model is used to convert the wire bending force to an in-plane stress since the wire force is not proportional to the slit length. The device was used to measure stress resultants (force carried per unit of slit length) on the dorsal, ventral and lateral aspects of neurulation-stage axolotl (Ambystoma mexicanum) embryos. The resultants were anisotropic and varied with location and developmental stage, with values ranging from -0.17 mN/m to 1.92 mN/m. In general, the resultants could be decomposed into patterns associated with internal pressure in the embryo, bending of the embryo along its mid-sagittal plane and neural tube closure. The patterns of stress revealed by the experiments support a number of current theories about the mechanics of neurulation.

Development of the embryonic heat shock response and the impact of repeated thermal stress in early stage lake whitefish (Coregonus clupeaformis) embryos.

PubMed

Whitehouse, Lindy M; McDougall, Chance S; Stefanovic, Daniel I; Boreham, Douglas R; Somers, Christopher M; Wilson, Joanna Y; Manzon, Richard G

2017-10-01

Lake whitefish (Coregonus clupeaformis) embryos were exposed to thermal stress (TS) at different developmental stages to determine when the heat shock response (HSR) can be initiated and if it is altered by exposure to repeated TS. First, embryos were subject to one of three different TS temperatures (6, 9, or 12°C above control) at 4 points in development (21, 38, 60 and 70 days post-fertilisation (dpf)) for 2h followed by a 2h recovery to understand the ontogeny of the HSR. A second experiment explored the effects of repeated TS on the HSR in embryos from 15 to 75 dpf. Embryos were subjected to one of two TS regimes; +6°C TS for 1h every 6 days or +9°C TS for 1h every 6 days. Following a 2h recovery, a subset of embryos was sampled. Our results show that embryos could initiate a HSR via upregulation of heat shock protein 70 (hsp70) mRNA at all developmental ages studied, but that this response varied with age and was only observed with a TS of +9 or +12°C. In comparison, when embryos received multiple TS treatments, hsp70 was not induced in response to the 1h TS and 2h recovery, and a downregulation was observed at 39 dpf. Downregulation of hsp47 and hsp90α mRNA was also observed in early age embryos. Collectively, these data suggest that embryos are capable of initiating a HSR at early age and throughout embryogenesis, but that repeated TS can alter the HSR, and may result in either reduced responsiveness or a downregulation of inducible hsps. Our findings warrant further investigation into both the short- and long-term effects of repeated TS on lake whitefish development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Metabolic and mitochondrial dysfunction in early mouse embryos following maternal dietary protein intervention.

PubMed

Mitchell, Megan; Schulz, Samantha L; Armstrong, David T; Lane, Michelle

2009-04-01

Dietary supply of nutrients, both periconception and during pregnancy, influence the growth and development of the fetus and offspring and their health into adult life. Despite the importance of research efforts surrounding the developmental origins of health and disease hypothesis, the biological mechanisms involved remain elusive. Mitochondria are of major importance in the oocyte and early embryo, particularly as a source of ATP generation, and perturbations in their function have been related to reduced embryo quality. The present study examined embryo development following periconception exposure of females to a high-protein diet (HPD) or a low-protein diet (LPD) relative to a medium-protein diet (MPD; control), and we hypothesized that perturbed mitochondrial metabolism in the mouse embryo may be responsible for the impaired embryo and fetal development reported by others. Although the rate of development to the blastocyst stage did not differ between diets, both the HPD and LPD reduced the number of inner cell mass cells in the blastocyst-stage embryo. Furthermore, mitochondrial membrane potential was reduced and mitochondrial calcium levels increased in the 2-cell embryo. Embryos from HPD females had elevated levels of reactive oxygen species and ADP concentrations, indicative of metabolic stress and, potentially, the uncoupling of oxidative phosphorylation, whereas embryos from LPD females had reduced mitochondrial clustering around the nucleus, suggestive of an overall quietening of metabolism. Thus, although periconception dietary supply of different levels of protein is permissive of development, mitochondrial metabolism is altered in the early embryo, and the nature of the perturbation differs between HPD and LPD exposure.

Gene expression analysis in zebrafish embryos: a potential approach to predict effect concentrations in the fish early life stage test.

PubMed

Weil, Mirco; Scholz, Stefan; Zimmer, Michaela; Sacher, Frank; Duis, Karen

2009-09-01

Based on the hypothesis that analysis of gene expression could be used to predict chronic fish toxicity, the zebrafish (Danio rerio) embryo test (DarT), developed as a replacement method for the acute fish test, was expanded to a gene expression D. rerio embryo test (Gene-DarT). The effects of 14 substances on lethal and sublethal endpoints of the DarT and on expression of potential marker genes were investigated: the aryl hydrocarbon receptor 2, cytochrome P450 1A (cypla), heat shock protein 70, fizzy-related protein 1, the transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene family protein g (avian) 1 and NF-E2-p45-related factor, and heme oxygenase 1 (hmox1). After exposure of zebrafish embryos for 48 h, differential gene expression was evaluated using reverse transcriptase-polymerase chain reaction, gel electrophoresis, and densitometric analysis of the gels. All tested compounds significantly affected the expression of at least one potential marker gene, with cyp1a and hmox1 being most sensitive. Lowest-observed-effect concentrations (LOECs) for gene expression were below concentrations resulting in 10% lethal effects in the DarT. For 10 (3,4- and 3,5-dichloroaniline, 1,4-dichlorobenzene, 2,4-dinitrophenol, atrazine, parathion-ethyl, chlorotoluron, genistein, 4-nitroquinoline-1-oxide, and cadmium) out of the 14 tested substances, LOEC values derived with the Gene-DarT differ by a factor of less than 10 from LOEC values of fish early life stage tests with zebrafish. For pentachloroaniline and pentachlorobenzene, the Gene-DarT showed a 23- and 153-fold higher sensitivity, respectively, while for lindane, it showed a 13-fold lower sensitivity. For ivermectin, the Gene-DarT was by a factor of more than 1,000 less sensitive than the acute fish test. The results of the present study indicate that gene expression analysis in zebrafish embryos could principally be used to predict effect concentrations in the fish early life stage test.

Early embryonic survival and embryo development in two lines of rabbits divergently selected for uterine capacity.

PubMed

Peiró, R; Santacreu, M A; Climent, A; Blasco, A

2007-07-01

The aim of this work is to study early embryo survival and development in 2 lines divergently selected for high and low uterine capacity throughout 10 generations. A total of 162 female rabbits from the high line and 133 from the low line were slaughtered at 25, 48, or 62 h of gestation. There were no differences in ovulation rate and fertilization rate between lines in any of the 3 stages of gestation. Embryo survival, estimated as the number of normal embryos recovered at a constant ovulation rate, was similar in both lines at 25 and 48 h. Embryo survival was greater in the high line [D (posterior mean of the difference between the high and low lines) = 0.57 embryos] at 62 h of gestation. There was no difference in embryonic stage of development at 25 h, but at 48 and 62 h of gestation, the high line, compared with the low line, had a greater percentage of early morulae (83 vs. 72%) and compacted morulae (55 vs. 38%). Divergent selection for uterine capacity appeared to modify embryo development, at least from 48 h of gestation, and embryo survival from 62 h.

Bovine oocytes and early embryos express Staufen and ELAVL RNA-binding proteins.

PubMed

Calder, M D; Madan, P; Watson, A J

2008-05-01

RNA-binding proteins (RBP) influence RNA editing, localization, stability and translation and may contribute to oocyte developmental competence by regulating the stability and turnover of oogenetic mRNAs. The expression of Staufen 1 and 2 and ELAVL1, ELAVL2 RNA-binding proteins during cow early development was characterized. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, matured, inseminated and subjected to embryo culture in vitro. Oocyte or preimplantation embryo pools were processed for RT-PCR and whole-mount immunofluorescence analysis of mRNA expression and protein distribution. STAU1 and STAU2 and ELAVL1 mRNAs and proteins were detected throughout cow preimplantation development from the germinal vesicle (GV) oocyte to the blastocyst stage. ELAVL2 mRNAs were detectable from the GV to the morula stage, whereas ELAVL2 protein was in all stages examined and localized to both cytoplasm and nuclei. The findings provide a foundation for investigating the role of RBPs during mammalian oocyte maturation and early embryogenesis.

Stage selection and restricted oviposition period improves cryopreservation of dipteran embryos.

PubMed

Rajamohan, Arun; Rinehart, Joseph P; Leopold, Roger A

2015-04-01

Embryos of two dipteran species (Musca domestica and Lucilia sericata) were assessed for an effective sampling time that would result in the highest post-cryopreservation hatch rate, with a primary goal to define species-specific egg collection periods and the effects of manual stage selection on post cryopreservation yield. The effects of the time taken to collect eggs on, (a) the proportion of embryos reaching a specific developmental stage between 17 and 20 h of development, and (b) the post-cryopreservation hatch rate were assessed. Permeabilization treatment applied at any stage of embryonic development did not significantly reduce embryo viability. Eggs collected over longer durations significantly reduced the number of embryos available in a specific developmental stage amenable to cryopreservation. Hatch percentage after cryopreservation of the embryos of M. domestica collected over a 60 min period was 10.7 ± 8.7% compared to 31 ± 5% for the eggs collected for just 15 min. Similarly, percent hatch in L. sericata resulted in 17.0 ± 3.9 and <2% for 15 and 60 min samples, respectively. Significantly higher hatching rates were obtained for cryopreservation after manual selection of specific embryonic developmental stages from the dechorionated samples. Post-cryopreservation hatching rate for stage-selected M. domestica embryos was 86.5 ± 5.5% compared to 33.3 ± 4.5% for embryos staged only by an overall visual confirmation. In the case of L. sericata, the hatching percentage was 79.0 ± 11.1 for stage-selected embryos compared to 17.0 ± 3.9% without individual selection. Published by Elsevier Inc.

Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

PubMed

García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

2015-09-01

Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the

The first whole transcriptomic exploration of pre-oviposited early chicken embryos using single and bulked embryonic RNA-sequencing.

PubMed

Hwang, Young Sun; Seo, Minseok; Choi, Hee Jung; Kim, Sang Kyung; Kim, Heebal; Han, Jae Yong

2018-04-01

The chicken is a valuable model organism, especially in evolutionary and embryology research because its embryonic development occurs in the egg. However, despite its scientific importance, no transcriptome data have been generated for deciphering the early developmental stages of the chicken because of practical and technical constraints in accessing pre-oviposited embryos. Here, we determine the entire transcriptome of pre-oviposited avian embryos, including oocyte, zygote, and intrauterine embryos from Eyal-giladi and Kochav stage I (EGK.I) to EGK.X collected using a noninvasive approach for the first time. We also compare RNA-sequencing data obtained using a bulked embryo sequencing and single embryo/cell sequencing technique. The raw sequencing data were preprocessed with two genome builds, Galgal4 and Galgal5, and the expression of 17,108 and 26,102 genes was quantified in the respective builds. There were some differences between the two techniques, as well as between the two genome builds, and these were affected by the emergence of long intergenic noncoding RNA annotations. The first transcriptome datasets of pre-oviposited early chicken embryos based on bulked and single embryo sequencing techniques will serve as a valuable resource for investigating early avian embryogenesis, for comparative studies among vertebrates, and for novel gene annotation in the chicken genome.

Chronology of early embryonic development and embryo uterine migration in alpacas.

PubMed

Picha, Y; Tibary, A; Memon, M; Kasimanickam, R; Sumar, J

2013-03-01

The objectives were to: (1) describe the chronology of early embryonic development from ovulation to entry into the uterus; and (2) to determine the timing of embryo migration to the left uterine horn when ovulation occurred from the right ovary. The experiment was conducted in Peru. Females (n = 132) were randomly assigned to 15 experimental groups. All females were mated to an intact male, given 50 μg GnRH im (Cystorelin) and ovulation time determined by transrectal ultrasonography, conducted every 6 hours, starting 24 hours postmating. Animals were slaughtered at a specific intervals postovulation and reproductive tracts were recovered and subjected to oviductal and uterine flushing for females slaughtered between 1 and 6 days postovulation (dpo; Day 0 = ovulation) and uterine flushing for females slaughtered from 7 to 15 dpo for recovery of oocytes/embryos. Season of mating did not influence the interval from mating to ovulation (winter: 29 ± 6 hours vs. summer: 30 ± 6 hours; P = 0.49). Ovulation rates for females mated during winter and summer were 92% versus 100%, respectively (P = 0.05). Fertilization rates for winter and summer mated females were 72% and 82% (P = 0.29). Unfertilized ova were not retained in the uterine tube. All embryos collected were in the uterine tube ipsilateral to the side of ovulation between 1 and 5 dpo. Embryos reached the uterus on 6 dpo. Embryos began to elongate on 9 dpo; at this time, 83% of embryos derived from right-ovary ovulations were collected from the left uterine horn. Embryos occupied the entire uterine cavity by 10 dpo. In conclusion, we characterized early embryo development and location of embryo during its early developmental stages in alpaca. This was apparently the first report regarding chronology of embryo development and migration to the left horn in alpaca which merits further investigation regarding its role in maternal recognition of pregnancy. Copyright © 2013 Elsevier Inc. All rights reserved.

Demands for carbohydrates as major energy substrates depend on the preimplantation developmental stage in pig embryos: Differential use of fructose by parthenogenetic diploids before and after the 4-cell stage in the pig

PubMed Central

SHIBUTANI, Mihiro; LEE, Jibak; MIYANO, Takashi; MIYAKE, Masashi

2015-01-01

The embryo culture technique has been improving, but the detailed demands for energy substrates such as glucose, fructose, pyruvate and lactate of preimplantation embryos are still unclear. In the present study, the demands of pig preimplantation embryos at each different developmental stage were investigated by use of parthenogenetic diploids as a model of pig preimplantation embryos. Pig parthenogenetic diploids showed different use of glucose and fructose before and after the 4-cell stage. Although glucose supported the development of pig embryos throughout the preimplantation stages and even maintained the expansion and hatching of blastocysts, it suppressed development to the blastocyst stage when glucose coexisted with pyruvate and lactate from 4 h after activation, but not after 48 h (early 4-cell stage). Since ketohexokinase that metabolizes fructose was not expressed in 2-cell and 4-cell diploids, a medium that included only fructose as a major energy substrate did not support early cleavage of pig diploids beyond the 4-cell stage, and almost no diploids developed to the morula stage just as in a medium without carbohydrates. These results may explain the different suppressive effects on pig preimplantation development between glucose and fructose when pyruvate and lactate were present in a medium. In addition, 4-cell diploids that had been cultured in a medium with pyruvate and lactate developed to the expanded blastocyst stage without any carbohydrates as a major energy substrate. These results show that the demands for carbohydrates are different depending on the developmental stage in pig preimplantation embryos. PMID:25736264

PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models

PubMed Central

2011-01-01

Background PreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo. Methods Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy. Results PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control). Conclusions PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF

A multicenter prospective study to assess the effect of early cleavage on embryo quality, implantation, and live-birth rate.

PubMed

de los Santos, Maria José; Arroyo, Gemma; Busquet, Ana; Calderón, Gloria; Cuadros, Jorge; Hurtado de Mendoza, Maria Victoria; Moragas, Marta; Herrer, Raquel; Ortiz, Agueda; Pons, Carme; Ten, Jorge; Vilches, Miguel Angel; Figueroa, Maria José

2014-04-01

To investigate the impact of early cleavage (EC) on embryo quality, implantation, and live-birth rates. Prospective cross-sectional study. Multicenter study. Seven hundred embryo transfers and 1,028 early-stage human embryos. None. Implantation according to the presence of EC and embryo quality. The presence of EC is associated with embryo quality, especially in cycles with autologous oocytes. However, the use of EC as an additional criterion for selecting an embryo for transfer does not appear to significantly improve likelihood of implantation. Furthermore, embryos that presented EC had live-birth rates per implanted embryo similar to those that did not show any sign of cleavage. At least for conventional embryo culture and morphologic evaluations, the additional evaluation of EC in embryos may not be valuable to improve embryo implantation. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development.

PubMed

Liu, C; Liu, Y; Larsen, K; Hou, Y P; Callesen, H

2018-01-01

It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.

Cinemicrographic study of the cell movement in the primitive-streak-stage mouse embryo.

PubMed

Nakatsuji, N; Snow, M H; Wylie, C C

1986-07-01

Migration of the mesoderm cells in the primitive-streak-stage mouse embryo was directly studied by cinemicrography using whole embryo culture and Nomarski differential interference contrast optics. Relative transparency and small size of the early mouse embryos enabled direct observation of the individual cells and their cell processes. Seven-day-old mouse embryos were isolated and cultured in a small chamber in a medium consisting of 50% rat serum and 50% Dulbecco's modified minimum essential medium. The mesoderm cells move away from the primitive streak in both anterior and antimesometrial (distal) directions at a mean velocity of 46 micron h-1. They extend cell processes and constantly change cell shape. They do not translocate extensively as isolated single cells, but usually maintain attachment to other mesoderm cells. They show frequent cell division preceded by rounding up of the cell bodies, and accompanied by vigorous blebbing before and after cytokinesis. This study shows that it is possible to examine the motility of embryonic cells inside the mammalian embryo by direct observation if the embryo is small and transparent enough for the use of the Nomarski optics.

Paternal Diet-Induced Obesity Retards Early Mouse Embryo Development, Mitochondrial Activity and Pregnancy Health

PubMed Central

Binder, Natalie K.; Hannan, Natalie J.; Gardner, David K.

2012-01-01

Worldwide, 48% of adult males are overweight or obese. An association between infertility and excessive body weight is now accepted, although focus remains primarily on females. It has been shown that parental obesity results in compromised embryo development, disproportionate changes in embryo metabolism and reduced blastocyst cell number. The aim of this study was to determine whether paternal obesity has negative effects on the resultant embryo. Specifically, using in vitro fertilisation (IVF), we wanted to isolate the functional effects of obesity on sperm by examining the subsequent embryo both pre- and post-implantation. Epididymal sperm was collected from age matched normal and obese C57BL/6 mice and cryopreserved for subsequent IVF with oocytes collected from Swiss females (normal diet/weight). Obesity was induced in male mice by feeding a high fat diet of 22% fat for 10 weeks. Resultant embryos were cultured individually and development monitored using time-lapse microscopy. Paternal obesity resulted in a significant delay in preimplantation embryo development as early as syngamy (P<0.05). Metabolic parameters were measured across key developmental stages, demonstrating significant reduction in mitochondrial membrane potential (P<0.01). Blastocysts were stained to determine trophectoderm (TE) and inner cell mass (ICM) cell numbers, revealing significant differences in the ratio of cell allocation to TE and ICM lineages (P<0.01). Functional studies examining blastocyst attachment, growth and implantation demonstrated that blastocysts derived from sperm of obese males displayed significantly reduced outgrowth on fibronectin in vitro (P<0.05) and retarded fetal development in vivo following embryo transfer (P<0.05). Taken together, these data clearly demonstrate that paternal obesity has significant negative effects on the embryo at a variety of key early developmental stages, resulting in delayed development, reduced placental size and smaller offspring

4D atlas of the mouse embryo for precise morphological staging.

PubMed

Wong, Michael D; van Eede, Matthijs C; Spring, Shoshana; Jevtic, Stefan; Boughner, Julia C; Lerch, Jason P; Henkelman, R Mark

2015-10-15

After more than a century of research, the mouse remains the gold-standard model system, for it recapitulates human development and disease and is quickly and highly tractable to genetic manipulations. Fundamental to the power and success of using a mouse model is the ability to stage embryonic mouse development accurately. Past staging systems were limited by the technologies of the day, such that only surface features, visible with a light microscope, could be recognized and used to define stages. With the advent of high-throughput 3D imaging tools that capture embryo morphology in microscopic detail, we now present the first 4D atlas staging system for mouse embryonic development using optical projection tomography and image registration methods. By tracking 3D trajectories of every anatomical point in the mouse embryo from E11.5 to E14.0, we established the first 4D atlas compiled from ex vivo 3D mouse embryo reference images. The resulting 4D atlas comprises 51 interpolated 3D images in this gestational range, resulting in a temporal resolution of 72 min. From this 4D atlas, any mouse embryo image can be subsequently compared and staged at the global, voxel and/or structural level. Assigning an embryonic stage to each point in anatomy allows for unprecedented quantitative analysis of developmental asynchrony among different anatomical structures in the same mouse embryo. This comprehensive developmental data set offers developmental biologists a new, powerful staging system that can identify and compare differences in developmental timing in wild-type embryos and shows promise for localizing deviations in mutant development. © 2015. Published by The Company of Biologists Ltd.

The First Human Cloned Embryo.

ERIC Educational Resources Information Center

Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

2002-01-01

Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

EVALUATING THE EFFECTS OF FLY ASH EXPOSURE ON FISH EARLY LIFE STAGES: FATHEAD MINNOW EMBRYO-LARVAL TESTS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greeley Jr, Mark Stephen; Elmore, Logan R; McCracken, Kitty

2012-05-01

On December 22, 2008, a dike containing fly ash and bottom ash in an 84-acre complex of the Tennessee Valley Authority's (TVA) Kingston Steam Plant in East Tennessee failed and released a large quantity of ash into the adjacent Emory River. Ash deposits extended as far as 4 miles upstream (Emory River mile 6) of the Plant, and some ash was carried as far downstream as Tennessee River mile 564 ({approx}4 miles downstream of the Tennessee River confluence with the Clinch River). A byproduct of coal burning power plants, fly ash contains a variety of metals and other elements which,more » at sufficient concentrations and in specific forms, can be toxic to biological systems. The effects of fly ash contamination on exposed fish populations depend on the magnitude and duration of exposure, with the most significant risk considered to be the effects of specific ash constituents, especially selenium, on fish early life stages. Uptake by adult female fish of fly ash constituents through the food chain and subsequent maternal transfer of contaminants to the developing eggs is thought to be the primary route of selenium exposure to larval fish (Woock and others 1987, Coyle and others 1993, Lemly 1999, Moscatello and others 2006), but direct contact of the fertilized eggs and developing embryos to ash constituents in river water and sediments is also a potential risk factor (Woock and others 1987, Coyle and others 1993, Jezierska and others 2009). To address the risk of fly ash from the Kingston spill to the reproductive health of downstream fish populations, ORNL has undertaken a series of studies in collaboration with TVA including: (1) a field study of the bioaccumulation of fly ash constituents in fish ovaries and the reproductive condition of sentinel fish species in reaches of the Emory and Clinch Rivers affected by the fly ash spill; (2) laboratory tests of the potential toxicity of fly ash from the spill area on fish embryonic and larval development (reported in the

The influence of early embryo traits on human embryonic stem cell derivation efficiency.

PubMed

O'Leary, Thomas; Heindryckx, Björn; Lierman, Sylvie; Van der Jeught, Margot; Menten, Björn; Deforce, Dieter; Cornelissen, Ria; de Sousa Lopes, Susana Chuva; De Sutter, Petra

2011-05-01

Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

PubMed Central

Yamauchi, N; Kiessling, A A; Cooper, G M

1994-01-01

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos. Images PMID:7935384

A novel application of motion analysis for detecting stress responses in embryos at different stages of development.

PubMed

Tills, Oliver; Bitterli, Tabitha; Culverhouse, Phil; Spicer, John I; Rundle, Simon

2013-02-01

Motion analysis is one of the tools available to biologists to extract biologically relevant information from image datasets and has been applied to a diverse range of organisms. The application of motion analysis during early development presents a challenge, as embryos often exhibit complex, subtle and diverse movement patterns. A method of motion analysis able to holistically quantify complex embryonic movements could be a powerful tool for fields such as toxicology and developmental biology to investigate whole organism stress responses. Here we assessed whether motion analysis could be used to distinguish the effects of stressors on three early developmental stages of each of three species: (i) the zebrafish Danio rerio (stages 19 h, 21.5 h and 33 h exposed to 1.5% ethanol and a salinity of 5); (ii) the African clawed toad Xenopus laevis (stages 24, 32 and 34 exposed to a salinity of 20); and iii) the pond snail Radix balthica (stages E3, E4, E6, E9 and E11 exposed to salinities of 5, 10 and 15). Image sequences were analysed using Sparse Optic Flow and the resultant frame-to-frame motion parameters were analysed using Discrete Fourier Transform to quantify the distribution of energy at different frequencies. This spectral frequency dataset was then used to construct a Bray-Curtis similarity matrix and differences in movement patterns between embryos in this matrix were tested for using ANOSIM. Spectral frequency analysis of these motion parameters was able to distinguish stage-specific effects of environmental stressors in most cases, including Xenopus laevis at stages 24, 32 and 34 exposed to a salinity of 20, Danio rerio at 33 hpf exposed to 1.5% ethanol, and Radix balthica at stages E4, E9 and E11 exposed to salinities of 5, 10 and 15. This technique was better able to distinguish embryos exposed to stressors than analysis of manual quantification of movement and within species distinguished most of the developmental stages studied in the control

Embryo sac formation and early embryo development in Agave tequilana (Asparagaceae).

PubMed

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

2014-01-01

Agave tequilana is an angiosperm species that belongs to the family Asparagaceae (formerly Agavaceae). Even though there is information regarding to some aspects related to the megagametogenesis of A. tequilana, this is the first report describing the complete process of megasporogenesis, megagametogenesis, the early embryo and endosperm development process in detail. The objective of this work was to study and characterize all the above processes and the distinctive morphological changes of the micropylar and chalazal extremes after fertilization in this species. The agave plant material for the present study was collected from commercial plantations in the state of Jalisco, Mexico. Ovules and immature seeds, previously fixed in FAA and kept in ethanol 70%, were stained based on a tissue clarification technique by using a Mayer's-Hematoxylin solution. The tissue clarification technique was successfully used for the characterization of the megasporogenesis, megagametogenesis, mature embryo sac formation, the early embryo and endosperm development processes by studying intact cells. The embryo sac of A. tequilana was confirmed to be of the monosporic Polygonum-type and an helobial endosperm formation. Also, the time-lapse of the developmental processes studied was recorded.

Chromosome fragility at FRAXA in human cleavage stage embryos at risk for fragile X syndrome.

PubMed

Verdyck, Pieter; Berckmoes, Veerle; De Vos, Anick; Verpoest, Willem; Liebaers, Inge; Bonduelle, Maryse; De Rycke, Martine

2015-10-01

Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS. © 2015 Wiley Periodicals, Inc.

Maternal program of apoptosis activated shortly after midblastula transition by overexpression of S-adenosylmethionine decarboxylase in Xenopus early embryos.

PubMed

Shiokawa, K; Kai, M; Higo, T; Kaito, C; Yokoska, J; Yasuhiko, Y; Kajita, E; Nagano, M; Yamada, Y; Shibata, M; Muto, T; Shinga, J; Hara, H; Takayama, E; Fukamachi, H; Yaoita, Y; Igarashi, K

2000-06-01

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.

Refrigeration of rainbow trout gametes and embryos.

PubMed

Babiak, Igor; Dabrowski, Konrad

2003-12-01

Prolonged access to early embryos composed of undifferentiated, totipotent blastomeres is desirable in situations when multiple collections of gametes are not possible. The objective of the present study is to examine whether the refrigeration of rainbow trout Oncorhynchus mykiss gametes and early embryos would be a suitable, reliable, and efficient tool for prolonging the availability of early developmental stages up to the advanced blastula stage. The study was conducted continuously during fall, winter, and spring spawning seasons. In all, more than 500 experimental variants were performed involving individual samples from 26 females and 33 males derived from three strains. These strains represented three possible circumstances. In optimal one, gametes from good quality donors were obtained soon after ovulation. In the two non-optimal sources, either donors were of poor genetic quality or gametes were collected from a distant location and transported as unfertilized gametes. A highly significant effect of variability of individual sample quality on efficiency of gamete and embryo refrigeration was revealed. The source of gametes significantly affected viability of refrigerated oocytes and embryos, but not spermatozoa. On average, oocytes from optimal source retained full fertilization viability for seven days of chilled storage, significantly longer than from non-optimal sources. Spermatozoa, regardless of storage method, retained full fertilization ability for the first week of storage. Refrigeration of embryos at 1.4+/-0.4 degrees C significantly slowed the development. Two- week-old embryos were still in blastula stage. Average survival rate of embryos refrigerated for 10 days and then transferred to regular incubation temperatures of 9-14 degrees C was 92% in optimal and 51 and 71% in non-optimal source variants. No effect of gamete and embryo refrigeration on the occurrence of developmental abnormalities was observed. Cumulative refrigeration of oocytes and

The early Cambrian fossil embryo Pseudooides is a direct-developing cnidarian, not an early ecdysozoan.

PubMed

Duan, Baichuan; Dong, Xi-Ping; Porras, Luis; Vargas, Kelly; Cunningham, John A; Donoghue, Philip C J

2017-12-20

Early Cambrian Pseudooides prima has been described from embryonic and post-embryonic stages of development, exhibiting long germ-band development. There has been some debate about the pattern of segmentation, but this interpretation, as among the earliest records of ecdysozoans, has been generally accepted. Here, we show that the 'germ band' of P. prima embryos separates along its mid axis during development, with the transverse furrows between the 'somites' unfolding into the polar aperture of the ten-sided theca of Hexaconularia sichuanensis , conventionally interpreted as a scyphozoan cnidarian; co-occurring post-embryonic remains of ecdysozoans are unrelated. We recognize H. sichuanensis as a junior synonym of P. prima as a consequence of identifying these two form-taxa as distinct developmental stages of the same organism. Direct development in P. prima parallels the co-occuring olivooids Olivooides, and Quadrapyrgites and Bayesian phylogenetic analysis of a novel phenotype dataset indicates that, despite differences in their tetra-, penta- and pseudo-hexa-radial symmetry, these hexangulaconulariids comprise a clade of scyphozoan medusozoans, with Arthrochites and conulariids, that all exhibit direct development from embryo to thecate polyp. The affinity of hexangulaconulariids and olivooids to extant scyphozoan medusozoans indicates that the prevalence of tetraradial symmetry and indirect development are a vestige of a broader spectrum of body-plan symmetries and developmental modes that was manifest in their early Phanerozoic counterparts. © 2017 The Authors.

The early Cambrian fossil embryo Pseudooides is a direct-developing cnidarian, not an early ecdysozoan

PubMed Central

2017-01-01

Early Cambrian Pseudooides prima has been described from embryonic and post-embryonic stages of development, exhibiting long germ-band development. There has been some debate about the pattern of segmentation, but this interpretation, as among the earliest records of ecdysozoans, has been generally accepted. Here, we show that the ‘germ band’ of P. prima embryos separates along its mid axis during development, with the transverse furrows between the ‘somites’ unfolding into the polar aperture of the ten-sided theca of Hexaconularia sichuanensis, conventionally interpreted as a scyphozoan cnidarian; co-occurring post-embryonic remains of ecdysozoans are unrelated. We recognize H. sichuanensis as a junior synonym of P. prima as a consequence of identifying these two form-taxa as distinct developmental stages of the same organism. Direct development in P. prima parallels the co-occuring olivooids Olivooides, and Quadrapyrgites and Bayesian phylogenetic analysis of a novel phenotype dataset indicates that, despite differences in their tetra-, penta- and pseudo-hexa-radial symmetry, these hexangulaconulariids comprise a clade of scyphozoan medusozoans, with Arthrochites and conulariids, that all exhibit direct development from embryo to thecate polyp. The affinity of hexangulaconulariids and olivooids to extant scyphozoan medusozoans indicates that the prevalence of tetraradial symmetry and indirect development are a vestige of a broader spectrum of body-plan symmetries and developmental modes that was manifest in their early Phanerozoic counterparts. PMID:29237861

Stage specific effects of soluble copper and copper oxide nanoparticles during sea urchin embryo development and their relation to intracellular copper uptake.

PubMed

Torres-Duarte, Cristina; Ramos-Torres, Karla M; Rahimoff, René; Cherr, Gary N

2017-08-01

The effects of exposure to either soluble copper (copper sulfate) or copper oxide nanoparticles (nano-CuO) during specific early developmental stages of sea urchin embryos were analyzed. Soluble copper caused significant malformations in embryos (skeletal malformations, delayed development or gut malformations) when present at any given stage, while cleavage stage was the most sensitive to nano-CuO exposure causing skeletal malformations and decreased total antioxidant capacity. The stage specificity was linked to higher endocytic activity during the first hours of development that leads to higher accumulation of copper in specific cells critical for development. Results indicate that nano-CuO results in higher accumulation of copper inside of embryos and this intracellular copper is more persistent as compared to soluble copper. The possible implications later in development are discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Early maternal serum ß-human chorionic gonadotropin (ß-hCG) levels and sex-related growth difference of IVF embryos.

PubMed

Esh-Broder, Efrat; Oron, Galia; Son, Weon-Young; Holzer, Hananel; Tulandi, Togas

2015-10-01

Maternal serum ß-human chorionic gonadotropin (ß-hCG) represents the trophoblastic cell mass and is an indirect measurement of embryo development at early implantation stage. Studies in animals and human embryos detected sex-related growth differences (SRGD) in favour of male embryos during the pre-implantation period. The purpose of our study was to correlate SRGD and maternal serum ß-hCG at 16 days after embryo transfer. We retrospectively analysed all (fresh and frozen) non-donor, single embryo transfers (SET), elective and not elective, that were performed between December 2008 and December 2013. We included ß-hCG values from day 16 after oocyte collection of pregnancies resulting in live birth. Neonatal gender was retrieved from patient files. Male and female embryos were further grouped to cleavage and blastocyst stage transfers. Regression analysis for confounding variables included maternal age, maternal body mass index (BMI), use of micromanipulation (ICSI), embryo quality (grade), assisted hatching, day of transfer and fresh or frozen embryo transfer. Seven hundred eighty-six non-donor SETs resulted in live birth. After including only day 16 serum ß-hCG results, 525 SETs were analysed. Neonatal gender was available for 522 cases. Mean maternal serum ß-hCG levels were similar, 347 ± 191 IU/L in the male newborn group and 371 ± 200 IU/L in the female group. The difference between ß-hCG levels remained insignificant after adjusting for confounding variables. Early maternal ß-hCG levels after embryo transfers did not represent SRGD in our study.

Par-aPKC-dependent and -independent mechanisms cooperatively control cell polarity, Hippo signaling, and cell positioning in 16-cell stage mouse embryos.

PubMed

Hirate, Yoshikazu; Hirahara, Shino; Inoue, Ken-Ichi; Kiyonari, Hiroshi; Niwa, Hiroshi; Sasaki, Hiroshi

2015-10-01

In preimplantation mouse embryos, the Hippo signaling pathway plays a central role in regulating the fates of the trophectoderm (TE) and the inner cell mass (ICM). In early blastocysts with more than 32 cells, the Par-aPKC system controls polarization of the outer cells along the apicobasal axis, and cell polarity suppresses Hippo signaling. Inactivation of Hippo signaling promotes nuclear accumulation of a coactivator protein, Yap, leading to induction of TE-specific genes. However, whether similar mechanisms operate at earlier stages is not known. Here, we show that slightly different mechanisms operate in 16-cell stage embryos. Similar to 32-cell stage embryos, disruption of the Par-aPKC system activated Hippo signaling and suppressed nuclear Yap and Cdx2 expression in the outer cells. However, unlike 32-cell stage embryos, 16-cell stage embryos with a disrupted Par-aPKC system maintained apical localization of phosphorylated Ezrin/Radixin/Moesin (p-ERM), and the effects on Yap and Cdx2 were weak. Furthermore, normal 16-cell stage embryos often contained apolar cells in the outer position. In these cells, the Hippo pathway was strongly activated and Yap was excluded from the nuclei, thus resembling inner cells. Dissociated blastomeres of 8-cell stage embryos form polar-apolar couplets, which exhibit different levels of nuclear Yap, and the polar cell engulfed the apolar cell. These results suggest that cell polarization at the 16-cell stage is regulated by both Par-aPKC-dependent and -independent mechanisms. Asymmetric cell division is involved in cell polarity control, and cell polarity regulates cell positioning and most likely controls Hippo signaling. © The Authors Development, Growth & Differentiation published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Society of Developmental Biologists.

Organophosphorus pesticides effect on early stages of the axolotl Ambystoma mexicanum (Amphibia: Caudata).

PubMed

Robles-Mendoza, C; García-Basilio, C; Cram-Heydrich, S; Hernández-Quiroz, M; Vanegas-Pérez, C

2009-02-01

Ambystoma mexicanum is an endemic salamander of Xochimilco, a wetland of the basin of Mexico valley. Nowadays, axolotl populations are decreasing due environmental stressors. Particularly, studies about organophosphorus pesticides (OPPs; i.e. chlorpyrifos and malathion) toxicity are of great importance due to their intensive use in agricultural activities in Xochimilco. Thus, the aim of this study was to evaluate under controlled conditions the toxicity of chlorpyrifos (CPF) and malathion (MLT) on embryos and larvae (stage 44 and 54) of A. mexicanum. Embryos and larvae were exposure 96h from 0.5 to 3mg CPFL(-1) and from 10 to 30mg MLTL(-1) in independent tests. Embryos at the end of this period were maintained 9d without pesticide in order to identify possible recuperation. Differences obtained in mortality, hatching success, development, morphological abnormalities, behaviour and activity, suggest that toxicity of CPF and MLT differs in embryos and larval stages. Embryos were less sensitive to OPPs acute exposure than axolotl larvae; additionally, toxicity of CPF in larval stages was greater than MLT. On the other hand, data obtained in axolotl embryos during the period of recuperation to CPF in particular as delay and inhibition of development, malformations and success of hatching, indicated that these responses turned out more sensitive than mortality. This study allowed to identify the toxicological potential of OPPs on early developmental stages of A. mexicanum and it is a valuable contribution for the future management of the axolotl wild population.

Method of Electroporation for the Early Chick Embryo

NASA Astrophysics Data System (ADS)

Hatakeyama, Jun; Shimamura, Kenji

Chick embryos have long been one of the favored model systems in the field of embryology and developmental biology. Recent advances in the gene manipulation technologies (Muramatsu et al., 1997; Nakamura et al., 2004) make this model system even more attractive for the developmental biologists (see review by Stern, 2005). Thanks to its two dimensional geometry, easiness in accessibility and observation, and well-established fate maps (e.g. Couly and Le Douarin, 1988; Garcia-Martinez et al., 1993; Hatada and Stern, 1994; Psychoyos and Stern, 1996; Sawada and Aoyama, 1999; Cobos et al., 2001; Lopez-Sanchez et al., 2001; Redkar et al., 2001; Fernandez-Garre et al., 2002; Kimura et al., 2006; Matsushita et al., 2008), it has great advantages especially for studies at the early embryonic stages, such as the processes of gastrulation, neural induction, left-right patterning, etc. For such purposes, a whole embryo culture system, originally invented by Dennis A. T. New (New, 1955), and its derivatives (Flamme, 1987; Sundin and Eichele, 1992; Stern, 1993; Chapman et al., 2001) have been widely used.

Effects of ionizing radiation on embryos of the tardigrade Milnesium cf. tardigradum at different stages of development.

PubMed

Beltrán-Pardo, Eliana; Jönsson, K Ingemar; Wojcik, Andrzej; Haghdoost, Siamak; Harms-Ringdahl, Mats; Bermúdez-Cruz, Rosa M; Bernal Villegas, Jaime E

2013-01-01

Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies have documented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differential effects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effect of gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from the eutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, and late stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses, while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival with higher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might have taken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and late stage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that the tolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this pattern are unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of response mechanisms to damaged DNA in the course of development.

Effects of Ionizing Radiation on Embryos of the Tardigrade Milnesium cf. tardigradum at Different Stages of Development

PubMed Central

Beltrán-Pardo, Eliana; Jönsson, K. Ingemar; Wojcik, Andrzej; Haghdoost, Siamak; Harms-Ringdahl, Mats; Bermúdez-Cruz, Rosa M.; Bernal Villegas, Jaime E.

2013-01-01

Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies have documented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differential effects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effect of gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from the eutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, and late stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses, while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival with higher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might have taken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and late stage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that the tolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this pattern are unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of response mechanisms to damaged DNA in the course of development. PMID:24039737

Identification and functional analysis of long non-coding RNAs in human and mouse early embryos based on single-cell transcriptome data

PubMed Central

Qiu, Jia-jun; Ren, Zhao-rui; Yan, Jing-bin

2016-01-01

Epigenetics regulations have an important role in fertilization and proper embryonic development, and several human diseases are associated with epigenetic modification disorders, such as Rett syndrome, Beckwith-Wiedemann syndrome and Angelman syndrome. However, the dynamics and functions of long non-coding RNAs (lncRNAs), one type of epigenetic regulators, in human pre-implantation development have not yet been demonstrated. In this study, a comprehensive analysis of human and mouse early-stage embryonic lncRNAs was performed based on public single-cell RNA sequencing data. Expression profile analysis revealed that lncRNAs are expressed in a developmental stage–specific manner during human early-stage embryonic development, whereas a more temporal-specific expression pattern was identified in mouse embryos. Weighted gene co-expression network analysis suggested that lncRNAs involved in human early-stage embryonic development are associated with several important functions and processes, such as oocyte maturation, zygotic genome activation and mitochondrial functions. We also found that the network of lncRNAs involved in zygotic genome activation was highly preservative between human and mouse embryos, whereas in other stages no strong correlation between human and mouse embryo was observed. This study provides insight into the molecular mechanism underlying lncRNA involvement in human pre-implantation embryonic development. PMID:27542205

Copper induces expression and methylation changes of early development genes in Crassostrea gigas embryos.

PubMed

Sussarellu, Rossana; Lebreton, Morgane; Rouxel, Julien; Akcha, Farida; Rivière, Guillaume

2018-03-01

Copper contamination is widespread along coastal areas and exerts adverse effects on marine organisms such as mollusks. In the Pacific oyster, copper induces severe developmental abnormalities during early life stages; however, the underlying molecular mechanisms are largely unknown. This study aims to better understand whether the embryotoxic effects of copper in Crassostrea gigas could be mediated by alterations in gene expression, and the putative role of DNA methylation, which is known to contribute to gene regulation in early embryo development. For that purpose, oyster embryos were exposed to 4 nominal copper concentrations (0.1, 1, 10 and 20 μg L -1 Cu 2+ ) during early development assays. Embryotoxicity was monitored through the oyster embryo-larval bioassay at the D-larva stage 24 h post fertilization (hpf) and genotoxicity at gastrulation 7 hpf. In parallel, the relative expression of 15 genes encoding putative homeotic, biomineralization and DNA methylation proteins was measured at three developmental stages (3 hpf morula stage, 7 hpf gastrula stage, 24 hpf D-larvae stage) using RT-qPCR. Global DNA content in methylcytosine and hydroxymethylcytosine were measured by HPLC and gene-specific DNA methylation levels were monitored using MeDIP-qPCR. A significant increase in larval abnormalities was observed from copper concentrations of 10 μg L -1 , while significant genotoxic effects were detected at 1 μg L -1 and above. All the selected genes presented a stage-dependent expression pattern, which was impaired for some homeobox and DNA methylation genes (Notochord, HOXA1, HOX2, Lox5, DNMT3b and CXXC-1) after copper exposure. While global DNA methylation (5-methylcytosine) at gastrula stage didn't show significant changes between experimental conditions, 5-hydroxymethylcytosine, its degradation product, decreased upon copper treatment. The DNA methylation of exons and the transcript levels were correlated in control samples for HOXA1 but such

Early embryo mortality in natural human reproduction: What the data say

PubMed Central

Jarvis, Gavin E.

2017-01-01

How many human embryos die between fertilisation and birth under natural conditions? It is widely accepted that natural human embryo mortality is high, particularly during the first weeks after fertilisation, with total prenatal losses of 70% and higher frequently claimed. However, the first external sign of pregnancy occurs two weeks after fertilisation with a missed menstrual period, and establishing the fate of embryos before this is challenging. Calculations are additionally hampered by a lack of data on the efficiency of fertilisation under natural conditions. Four distinct sources are used to justify quantitative claims regarding embryo loss: (i) a hypothesis published by Roberts & Lowe in The Lancet  is widely cited but has no practical quantitative value; (ii) life table analyses give consistent assessments of clinical pregnancy loss, but cannot illuminate losses at earlier stages of development; (iii) studies that measure human chorionic gonadotrophin (hCG) reveal losses in the second week of development and beyond, but not before; and (iv) the classic studies of Hertig and Rock offer the only direct insight into the fate of human embryos from fertilisation under natural conditions. Re-examination of Hertig’s data demonstrates that his estimates for fertilisation rate and early embryo loss are highly imprecise and casts doubt on the validity of his numerical analysis. A recent re-analysis of hCG study data concluded that approximately 40-60% of embryos may be lost between fertilisation and birth, although this will vary substantially between individual women. In conclusion, natural human embryo mortality is lower than often claimed and widely accepted. Estimates for total prenatal mortality of 70% or higher are exaggerated and not supported by the available data. PMID:28580126

40 CFR 797.1600 - Fish early life stage toxicity test.

Code of Federal Regulations, 2013 CFR

2013-07-01

... dilution water or the test solution. (4) “Control” an exposure of test organisms to dilution water only or... (treatment) concentrations of a test substance and one control are required to conduct an early life stage... trays or cups for each test concentration and control (i.e., 30 per embryo cup with 2 replicates); (C...

40 CFR 797.1600 - Fish early life stage toxicity test.

Code of Federal Regulations, 2010 CFR

2010-07-01

... dilution water or the test solution. (4) “Control” an exposure of test organisms to dilution water only or... (treatment) concentrations of a test substance and one control are required to conduct an early life stage... trays or cups for each test concentration and control (i.e., 30 per embryo cup with 2 replicates); (C...

40 CFR 797.1600 - Fish early life stage toxicity test.

Code of Federal Regulations, 2012 CFR

2012-07-01

... dilution water or the test solution. (4) “Control” an exposure of test organisms to dilution water only or... (treatment) concentrations of a test substance and one control are required to conduct an early life stage... trays or cups for each test concentration and control (i.e., 30 per embryo cup with 2 replicates); (C...

Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos

EPA Science Inventory

Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mous...

[Association of human chorionic gonadotropin level in embryo culture media with early embryo development].

PubMed

Wang, Haiying; Zhang, Renli; Han, Dong; Liu, Caixia; Cai, Jiajie; Bi, Yanling; Wen, Anmin; Quan, Song

2014-06-01

To investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development. Spent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed. In the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02). ELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.

Embryotoxicity of nitrophenols to the early life stages of zebrafish (Danio rerio).

PubMed

Ceylan, Zeynep; Şişman, Turgay; Yazıcı, Zehra; Altıkat, Aysun Özen

2016-08-01

The nitrophenols (NPs) are water-soluble compounds. These compounds pose a significant health threat since they are priority environmental pollutants. In this study, 2-Nitrophenol (2NP) and 2,4-dinitrophenol (DNP) were examined for embryo and early life stage toxicity in zebrafish (Danio rerio). Acute toxicity and teratogenicity of 2NP and DNP were tested for 4 days using zebrafish embryos. The typical lesions observed were no somite formation, incomplete eye and head development, tail curvature, weak pigmentation (≤48 hours postfertilization (hpf)), kyphosis, scoliosis, yolk sac deformity, and nonpigmentation (72 hpf). Also, embryo and larval mortality increased and hatching success decreased. The severity of abnormalities and mortalities were concentration- and compound-dependent. Of the compounds tested, 2,4-DNP was found to be highly toxic to the fish embryos following exposure. The median lethal concentrations and median effective concentrations for 2NP are 18.7 mg/L and 7.9 mg/L, respectively; the corresponding values for DNP are 9.65 mg/L and 3.05 mg/L for 48 h. The chorda deformity was the most sensitive endpoint measured. It is suggested that the embryotoxicity may be mediated by an oxidative phosphorylation uncoupling mechanism. This article is the first to describe the teratogenicity and embryotoxicity of two NPs to the early life stages of zebrafish. © The Author(s) 2014.

Proteomic analysis of early-stage embryos: implications for egg quality in hapuku (Polyprion oxygeneios).

PubMed

Kohn, Yair Y; Symonds, Jane E; Kleffmann, Torsten; Nakagawa, Shinichi; Lagisz, Malgorzata; Lokman, P Mark

2015-12-01

In order to develop biomarkers that may help predict the egg quality of captive hapuku (Polyprion oxygeneios) and provide potential avenues for its manipulation, the present study (1) sequenced the proteome of early-stage embryos using isobaric tag for relative and absolute quantification analysis, and (2) aimed to establish the predictive value of the abundance of identified proteins with regard to egg quality through regression analysis. Egg quality was determined for eight different egg batches by blastomere symmetry scores. In total, 121 proteins were identified and assigned to one of nine major groups according to their function/pathway. A mixed-effects model analysis revealed a decrease in relative protein abundance that correlated with (decreasing) egg quality in one major group (heat-shock proteins). No differences were found in the other protein groups. Linear regression analysis, performed for each identified protein separately, revealed seven proteins that showed a significant decrease in relative abundance with reduced blastomere symmetry: two correlates that have been named in other studies (vitellogenin, heat-shock protein-70) and a further five new candidate proteins (78 kDa glucose-regulated protein, elongation factor-2, GTP-binding nuclear protein Ran, iduronate 2-sulfatase and 6-phosphogluconate dehydrogenase). Notwithstanding issues associated with multiple statistical testing, we conclude that these proteins, and especially iduronate 2-sulfatase and the generic heat-shock protein group, could serve as biomarkers of egg quality in hapuku.

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.

Tissue morphodynamics shaping the early mouse embryo.

PubMed

Sutherland, Ann E

2016-07-01

Generation of the elongated vertebrate body plan from the initially radially symmetrical embryo requires comprehensive changes to tissue form. These shape changes are generated by specific underlying cell behaviors, coordinated in time and space. Major principles and also specifics are emerging, from studies in many model systems, of the cell and physical biology of how region-specific cell behaviors produce regional tissue morphogenesis, and how these, in turn, are integrated at the level of the embryo. New technical approaches have made it possible more recently, to examine the morphogenesis of the mouse embryo in depth, and to elucidate the underlying cellular mechanisms. This review focuses on recent advances in understanding the cellular basis for the early fundamental events that establish the basic form of the embryo. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stress Induces AMP-Dependent Loss of Potency Factors Id2 and Cdx2 in Early Embryos and Stem Cells

PubMed Central

Xie, Yufen; Awonuga, Awoniyi; Liu, Jian; Rings, Edmond; Puscheck, Elizabeth Ella

2013-01-01

The AMP-activated protein kinase (AMPK) mediates rapid, stress-induced loss of the inhibitor of differentiation (Id)2 in blastocysts and trophoblast stem cells (TSC), and a lasting differentiation in TSC. However, it is not known if AMPK regulates other potency factors or regulates them before the blastocyst stage. The caudal-related homeodomain protein (Cdx)2 is a regulatory gene for determining TSC, the earliest placental lineage in the preimplantation mouse embryo, but is expressed in the oocyte and in early cleavage stage embryos before TSC arise. We assayed the expression of putative potency-maintaining phosphorylated Cdx2 ser60 in the oocyte, two-cell stage embryo, blastocyst, and in TSC. We studied the loss of Cdx2 phospho ser60 expression induced by hyperosmolar stress and its underlying mechanisms. Hyperosmolar stress caused rapid loss of nuclear Cdx2 phospho ser60 and Id2 in the two-cell stage embryo by 0.5 h. Stress-induced Cdx2 phospho ser60 and Id2 loss is reversed by the AMPK inhibitor compound C and is induced by the AMPK agonist 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide in the absence of stress. In the two-cell stage embryo and TSC hyperosmolar, stress caused AMPK-mediated loss of Cdx2 phospho ser60 as detected by immunofluorescence and immunoblot. We propose that AMPK may be the master regulatory enzyme for mediating stress-induced loss of potency as AMPK is also required for stress-induced loss of Id2 in blastocysts and TSC. Since AMPK mediates potency loss in embryos and stem cells it will be important to measure, test mechanisms for, and manage the AMPK function to optimize the stem cell and embryo quality in vitro and in vivo. PMID:23316940

Cooling strategies for brazilian flounder Paralichthys orbignyanus embryos.

PubMed

Varela, A S; Cardoso, T F; Fernandes E Silva, E; Goularte, K L; Okamoto, M H; Sampaio, L A; Jardim, R D; Corcini, C D

Paralichthys orbignyanus is the species of the greatest potential for marine and estuarine fish farming in southern Brazil. Consequently, embryo cryopreservation becomes an important tool for increasing their production. To evaluate the effects of cooling protocols on the viability of embryos of P. orbignyanus at two stages of development (neurula and early differentiation of the tail). Control embryos were maintained at 23 degree C and treated embryos were cooled to 15 degree C, 10 degree C and 5 degree C at rapid, moderate and slow cooling rates. Then embryos were maintained at these different temperatures for 30, 60 and 90 min and the loss of viability assessed as hatching rates (HR) and morphologically normal larvae (MNL). The average HR for embryos following cooling was higher for those at the tail stage compared to the neurula stage (P<0.05). In both stages there was no statistical difference between the HR of control embryos and those exposed to rapid cooling. Also for tail stage embryos, there was no difference between MNL of control and rapidly cooled embryos. As first steps in the development of cryopreservation methods for P. orbignyanus embryos, the use of a rapid cooling and holding at 5 degree C for 30 min are recommended.

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, Yan; Liu, Chunying; Lu, Wenwen

The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysismore » revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.« less

Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos.

PubMed

Stowe, Heather M; Curry, Erin; Calcatera, Samantha M; Krisher, Rebecca L; Paczkowski, Melissa; Pratt, Scott L

2012-06-15

MicroRNA (miRNA) is a class of small, single-stranded ribonucleic acids that regulate gene expression post-transcriptionally and are involved in somatic cell, germ cell, and embryonic development. As the enzyme responsible for producing mature miRNA, Dicer is crucial to miRNA production. Characterization of Dicer and its expression at the nucleotide level, as well as the identification of miRNA expression in reproductive tissues, have yet to be reported for the domestic pig (Sus scrofa), a species important for disease modeling, biomedical research, and food production. In this study we determined the primary cDNA sequence of porcine Dicer (pDicer), confirmed its expression in porcine oocytes and early stage embryos, and evaluated the expression of specific miRNA during early embryonic development and between in vivo (IVO) and in vitro (IVF) produced embryos. Total cellular RNA (tcRNA) was isolated and subjected to end point RT-PCR, subcloning, and sequencing. The pDicer coding sequence was found to be highly conserved, and phylogenetic analysis showed that pDicer is more highly conserved to human Dicer (hDicer) than the mouse homolog. Expression of pDicer mRNA was detected in oocytes and in IVO produced blastocyst embryos. Two RT-PCR procedures were conducted to identify and quantitate miRNA expressed in metaphase II oocytes (MII) and embryos. RT-PCR array was conducted using primers designed for human miRNA, and 86 putative porcine miRNA in MII and early embryos were detected. Fewer miRNAs were detected in 8-cell (8C) embryos compared to MII and blastocysts (B) (P=0.026 and P<0.0001, respectively). Twenty-one miRNA (of 88 examined) were differentially expressed between MII and 8C, 8C and B, or MII and B. Transcripts targeted by the differentially expressed miRNA were enriched in gene ontology (GO) categories associated with cellular development and differentiation. Further, we evaluated the effects of IVF culture on the expression of specific miRNA at the

Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish

NASA Astrophysics Data System (ADS)

Lee, Kerry J.; Browning, Lauren M.; Nallathamby, Prakash D.; Osgood, Christopher J.; Xu, Xiao-Hong Nancy

2013-11-01

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 +/- 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c << 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target

Developmental Neurotoxicity of Methamidophos in the Embryo-Larval Stages of Zebrafish.

PubMed

He, Xiaowei; Gao, Jiawei; Dong, Tianyu; Chen, Minjian; Zhou, Kun; Chang, Chunxin; Luo, Jia; Wang, Chao; Wang, Shoulin; Chen, Daozhen; Zhou, Zuomin; Tian, Ying; Xia, Yankai; Wang, Xinru

2016-12-28

Methamidophos is a representative organophosphate insecticide. The knowledge of its developmental neurotoxicity is limited, especially for zebrafish in the early stages of their life. Four hour post-fertilization (hpf) zebrafish embryos were exposed to several environmentally relevant concentrations of methamidophos (0, 25, and 500 μg/L) for up to 72 hpf. Locomotor behavior was then studied in the zebrafish larvae at this timepoint. Acridine orange (AO) staining was carried out in the zebrafish larvae, and the mRNA levels of genes associated with neural development ( mbp and syn2a ) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The number of escape responders for mechanical stimulation was significantly decreased in exposed groups. AO staining showed noticeable signs of apoptosis mainly in the brain. In addition, the mRNA levels of mbp and syn2a were both significantly down-regulated in exposed groups. Our study provides the first evidence that methamidophos exposure can cause developmental neurotoxicity in the early stages of zebrafish life, which may be caused by the effect of methamidophos on neurodevelopmental genes and the activation of cell apoptosis in the brain.

Detection of Apoptosis in Early Life Stages as a Tool to Evaluate Chemical Control of Invasive Species

DTIC Science & Technology

2007-08-01

ERDC/TN ANSRP-07-2 August 2007 Detection of Apoptosis in Early Life Stages as a Tool to Evaluate Chemical Control of Invasive Species by J...4. TITLE AND SUBTITLE Detection of Apoptosis in Early Life Stages as a Tool to Evaluate Chemical Control of Invasive Species 5a. CONTRACT NUMBER 5b...heralding apoptosis . Data analysis. An apoptotic index (API) was established by calculating the percentage of embryos in each life stage with

Evaluation of whole-mount in situ hybridization as a tool for pathway-based toxicological research with early-life stage fathead minnows

EPA Science Inventory

Early-life stage fish can be more sensitive to chemical exposure than adult fish. Therefore, determining possible adverse outcome pathways (AOPs) for early-life stages is crucial. To determine chemical effects and/or mechanisms of action in exposed fish embryos and larvae, whole-...

Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis.

PubMed

Tosti, Elisabetta; Gallo, Alessandra; Silvestre, Francesco

2011-01-01

Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the post-vitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development. Copyright © 2011 Wiley Periodicals, Inc.

Fish egg injection as an alternative exposure route for early life stage toxicity studies: Description of two unique methods: Chapter 4

USGS Publications Warehouse

Walker, Mary K.; Zabel, Erik W.; Akerman, Gun; Balk, Lennart; Wright, Peggy J.; Tillitt, Donald E.

1996-01-01

In the environment, lipophilic contaminants such as halogenated aromatic hydrocarbons (HAHs, e.g., polychlorinated biphenyls, PCBs) and polycyclic aromatic hydrocarbons (PAHs, e.g., benzo[a]pyrene) readily bioaccumulate in fish, and the bioaccumulation of these lipophilic chemicals by adult fish may have significant consequences on the development and survival of their offspring. Halogenated and polycyclic aromatic hydrocarbons translocate from adult female body stores into eggs during oocyte maturation, and early life stages of fish are often more sensitive than adults to the toxicity of these chemicals. Thus, the presence of persistent, bioaccumulative contaminants in the environment may pose a risk to fish early life stage survival and ultimately reduce recruitment into the adult population.Typically, standard early life stage toxicity studies exposed embryos, larvae, and juveniles to graded concentrations of waterborne toxicants, and dose-response relationships are based on the concentrations of chemicals in the water. However, use of waterborne exposure to assess the toxicity of persistent, bioaccumulative contaminants, such as HAHs and PAHs, has two significant drawbacks. First, uptake of hydrophobic chemicals, such as HAHs and PAHs, into the developing embryo from water is not a significant route of exposure in the environment since concentrations of these chemicals freely dissolved in water are extremely low. Rather, maternal deposition into developing oocytes is the most significant source of these chemicals to the embryo. Second, the dose received by the target tissue, in this case the developing embryo, is the most accurate predictor of the toxic response, and since extrapolation from water concentrations of the chemical to egg concentrations is required, the exact dose received by the embryo can only be estimated, often with large uncertainty. Due to these drawbacks, it is important to develop an alternative exposure method that will directly expose the

Extra-embryonic tissue spreading directs early embryo morphogenesis in killifish

PubMed Central

Reig, Germán; Cerda, Mauricio; Sepúlveda, Néstor; Flores, Daniela; Castañeda, Victor; Tada, Masazumi; Härtel, Steffen; Concha, Miguel L.

2017-01-01

The spreading of mesenchymal-like cell layers is critical for embryo morphogenesis and tissue repair, yet we know little of this process in vivo. Here we take advantage of unique developmental features of the non-conventional annual killifish embryo to study the principles underlying tissue spreading in a simple cellular environment, devoid of patterning signals and major morphogenetic cell movements. Using in vivo experimentation and physical modelling we reveal that the extra-embryonic epithelial enveloping cell layer, thought mainly to provide protection to the embryo, directs cell migration and the spreading of embryonic tissue during early development. This function relies on the ability of embryonic cells to couple their autonomous random motility to non-autonomous signals arising from the expansion of the extra-embryonic epithelium, mediated by cell membrane adhesion and tension. Thus, we present a mechanism of extra-embryonic control of embryo morphogenesis that couples the mechanical properties of adjacent tissues in the early killifish embryo. PMID:28580937

A technique for sexing fully developed embryos and early-instar larvae of the gypsy moth

Treesearch

Gilbert Levesque

1963-01-01

Because variation in sex ratio is an important factor in the population dynamics of the gypsy moth (Porthetria dispar), it is necessary to have some means of determining the ratio of males to females in a population at the beginning of the larval period as well as in the later stages. For determining the sex of fully developed embryos and early-...

Optimized ex-ovo culturing of chick embryos to advanced stages of development.

PubMed

Cloney, Kellie; Franz-Odendaal, Tamara Anne

2015-01-24

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

IN VITRO CULTURE OF POSTIMPLANTATION HAMSTER EMBRYOS

EPA Science Inventory

In vitro culture of intact rat and mouse embryos has been described extensively, but information on the culture of other species is sparse. The present study examined some culture requirements of early somite stage hamster embryos and assessed the embryotoxic effects of sodium sa...

Dynamic Properties of Electrotonic Coupling between Cells of Early Xenopus Embryos

PubMed Central

DiCaprio, R. A.; French, A. S.; Sanders, E. J.

1974-01-01

Frequency response functions were measured between the cells of Xenopus laevis embryos during the first two cleavage stages. Linear systems theory was then used to produce electronic models which account for the electrical behavior of the systems. Coupling between the cells may be explained by models which have simple resistive elements joining each cell to its neighbors. The vitelline, or fertilization, membrane which surrounds the embryos has no detectable resistance to the passage of electric current. The electrical properties of the four-cell embryo can only be explained by the existence of individual junctions linking each pair of cells. This arrangement suggests that electrotonic coupling is important in the development of the embryos, at least until the four-cell stage. ImagesFIGURE 5FIGURE 14FIGURE 15 PMID:19431351

Intracellular pH in early Xenopus embryos: its effect on current flow between blastomeres.

PubMed Central

Turin, L; Warner, A E

1980-01-01

1. Electrophysiological techniques were used to monitor the flow of electric current from one cell to the next in Xenopus laevis embryos between the 4-cell and early blastula stages of development. Intracellular pH and blastocoel pH were determined using pH-sensitive micro-electrodes. 2. The resting intracellular pH was 7.74+/-0.02 (S.E. of mean, n = 29); there were no systematic differences between developmental stages. Blastocoel cavity pH was 8.4+/-0.06 (S.E. of mean, n = 10). The intracellular buffer value was 18 m-equiv. H+/pH unit per litre. 3. In embryos treated with bicarbonate buffered Holtfreter solution equilibrated with 100% CO2 the intracellular pH fell to 6.3+/-0.17 (S.D., n = 8). The membrane potential fell and the input resistance increased. The size of the effect on membrane potential and input resistance varied. 4. From the 32-cell stage onwards current flow from one cell to the next was abolished when the intracellular pH fell to below 6.5; the effect was rapid in onset and completely reversible. At cleavage stages of development lowering intracellular pH with CO2 had no effect on current flow from cell to cell. 5. The relationship between intracellular pH and current flow from cell to cell was sigmoid and covered between 0.2 and 0.4 pH units. The pH at which current flow was completely abolished ranged from 6.85 to 6.4. 6. Alterations in extraembryonic pH over the range 5.8-7.5 had no effect on any parameter measured. 7. We conclude that lowering the intracellular pH increases the resistance of both non-junctional junctional membranes. The data do not allow us to extract the pH junctional conductance relationship. 8. Variations in intracellular pH may provide a useful tool for the study of the functional role of direct cell to cell communication in both adult organs and early embryos. PMID:6770084

Wounded cells drive rapid epidermal repair in the early Drosophila embryo

PubMed Central

Fernandez-Gonzalez, Rodrigo; Zallen, Jennifer A.

2013-01-01

Epithelial tissues are protective barriers that display a remarkable ability to repair wounds. Wound repair is often associated with an accumulation of actin and nonmuscle myosin II around the wound, forming a purse string. The role of actomyosin networks in generating mechanical force during wound repair is not well understood. Here we investigate the mechanisms of force generation during wound repair in the epidermis of early and late Drosophila embryos. We find that wound closure is faster in early embryos, where, in addition to a purse string around the wound, actomyosin networks at the medial cortex of the wounded cells contribute to rapid wound repair. Laser ablation demonstrates that both medial and purse-string actomyosin networks generate contractile force. Quantitative analysis of protein localization dynamics during wound closure indicates that the rapid contraction of medial actomyosin structures during wound repair in early embryos involves disassembly of the actomyosin network. By contrast, actomyosin purse strings in late embryos contract more slowly in a mechanism that involves network condensation. We propose that the combined action of two force-generating structures—a medial actomyosin network and an actomyosin purse string—contributes to the increased efficiency of wound repair in the early embryo. PMID:23985320

Essential Role of Chromatin Remodeling Protein Bptf in Early Mouse Embryos and Embryonic Stem Cells

PubMed Central

Landry, Joseph; Sharov, Alexei A.; Piao, Yulan; Sharova, Lioudmila V.; Xiao, Hua; Southon, Eileen; Matta, Jennifer; Tessarollo, Lino; Zhang, Ying E.; Ko, Minoru S. H.; Kuehn, Michael R.; Yamaguchi, Terry P.; Wu, Carl

2008-01-01

We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf−/− embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf−/− embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo. PMID:18974875

Endogenous Nod-Factor-Like Signal Molecules Promote Early Somatic Embryo Development in Norway Spruce1

PubMed Central

Dyachok, Julia V.; Wiweger, Malgorzata; Kenne, Lennart; von Arnold, Sara

2002-01-01

Embryogenic cultures of Norway spruce (Picea abies) are composed of pro-embryogenic masses (PEMs) and somatic embryos of various developmental stages. Auxin is important for PEM formation and proliferation. In this report we show that depletion of auxin blocks PEM development and causes large-scale cell death. Extracts of the media conditioned by embryogenic cultures stimulate development of PEM aggregates in auxin-deficient cultures. Partial characterization of the conditioning factor shows that it is a lipophilic, low-molecular-weight molecule, which is sensitive to chitinase and contains GlcNAc residues. On the basis of this information, we propose that the factor is a lipophilic chitin oligosaccharide (LCO). The amount of LCO correlates to the developmental stages of PEMs and embryos, with the highest level in the media conditioned by developmentally blocked cultures. LCO is not present in nonembryogenic cultures. Cell death, induced by withdrawal of auxin, is suppressed by extra supply of endogenous LCO or Nod factor from Rhizobium sp. NGR234. The effect can be mimicked by a chitotetraose or chitinase from Streptomyces griseus. Taken together, our data suggest that endogenous LCO acts as a signal molecule stimulating PEM and early embryo development in Norway spruce. PMID:11842156

Comparative responses to endocrine disrupting compounds in early life stages of Atlantic salmon, Salmo salar.

PubMed

Duffy, T A; Iwanowicz, L R; McCormick, S D

2014-07-01

Atlantic salmon (Salmo salar) are endangered anadromous fish that may be exposed to feminizing endocrine disrupting compounds (EDCs) during early development, potentially altering physiological capacities, survival and fitness. To assess differential life stage sensitivity to common EDCs, we carried out short-term (4 day) exposures using three doses each of 17 α-ethinylestradiol (EE2), 17 β-estradiol (E2), and nonylphenol (NP) on four early life stages; embryos, yolk-sac larvae, feeding fry and 1 year old smolts. Differential response was compared using vitellogenin (Vtg, a precursor egg protein) gene transcription. Smolts were also examined for impacts on plasma Vtg, cortisol, thyroid hormones (T4/T3) and hepatosomatic index (HSI). Compound-related mortality was not observed in any life stage, but Vtg mRNA was elevated in a dose-dependent manner in yolk-sac larvae, fry and smolts but not in embryos. The estrogens EE2 and E2 were consistently stronger inducers of Vtg than NP. Embryos responded significantly to the highest concentration of EE2 only, while older life stages responded to the highest doses of all three compounds, as well as intermediate doses of EE2 and E2. Maximal transcription was greater for fry among the three earliest life stages, suggesting fry may be the most responsive life stage in early development. Smolt plasma Vtg was also significantly increased, and this response was observed at lower doses of each compound than was detected by gene transcription suggesting plasma Vtg is a more sensitive indicator at this life stage. HSI was increased at the highest doses of EE2 and E2, and plasma T3 was decreased at the highest dose of EE2. Our results indicate that all life stages are potentially sensitive to endocrine disruption by estrogenic compounds and that physiological responses were altered over a short window of exposure, indicating the potential for these compounds to impact fish in the wild. Copyright © 2014 Elsevier B.V. All rights

Nucleoli from two-cell embryos support the development of enucleolated germinal vesicle oocytes in the pig.

PubMed

Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

2012-11-01

Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.

Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre-Implantation Stage.

PubMed

Camargo, L S A; Paludo, F; Pereira, M M; Wohlres-Viana, S; Gioso, M M; Carvalho, B C; Quintao, C C R; Viana, J H M

2016-02-01

Oocyte has been considered the major contributor for embryo thermo-tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo-tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo-sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post-insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat-shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat-shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock. © 2015 Blackwell Verlag GmbH.

Strain preservation of experimental animals: vitrification of two-cell stage embryos for multiple mouse strains.

PubMed

Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

2015-04-01

Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Redundant roles of Sox17 and Sox18 in early cardiovascular development of mouse embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamoto, Youhei; Hara, Kenshiro; Kanai-Azuma, Masami

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 posteriormore » 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.« less

Cryotop vitrification as compared to conventional slow freezing for human embryos at the cleavage stage: survival and outcomes.

PubMed

Lin, Tseng-Kai; Su, Jin-Tsung; Lee, Fa-Kung; Lin, Yu-Ru; Lo, Hsiao-Ching

2010-09-01

This study was conducted to compare the efficacy of cryotop vitrification of human cleavage-stage embryos to that of conventional slow freezing of these embryos with respect to survival. A second objective was to compare the two cryopreservation techniques with respect to outcomes for a cohort of women. Cleavage-stage embryos from 102 patients were cryopreserved either by vitrification (57 patients) or by traditional slow freezing (45 patients). After thawing, rates of embryo survival, implantation, and clinical pregnancy were determined. Survival of embryos was significantly higher with the vitrification procedure as compared to traditional slow freezing [287/298 (96.3%) vs. 294/446 (65.9%); p < 0.05). Rates of implantation and clinical pregnancy were also significantly higher using vitrification procedure as compared to the slow freezing procedure (24.3% vs. 7.1% and 35.6% vs. 15.6% respectively, p < 0.05). As compared to conventional slow freezing, cryopreservation of human cleavage-stage embryo using vitrification results in higher rates of embryo survival, implantation, and clinical pregnancy. Vitrification therefore represents the superior cryopreservation technique for cleavage-stage embryos. Copyright © 2010 Taiwan Association of Obstetric & Gynecology. Published by Elsevier B.V. All rights reserved.

Exceptional preservation of tiny embryos documents seed dormancy in early angiosperms.

PubMed

Friis, Else Marie; Crane, Peter R; Pedersen, Kaj Raunsgaard; Stampanoni, Marco; Marone, Federica

2015-12-24

The rapid diversification of angiosperms through the Early Cretaceous period, between about 130-100 million years ago, initiated fundamental changes in the composition of terrestrial vegetation and is increasingly well understood on the basis of a wealth of palaeobotanical discoveries over the past four decades and their integration with improved knowledge of living angiosperms. Prevailing hypotheses, based on evidence both from living and from fossil plants, emphasize that the earliest angiosperms were plants of small stature with rapid life cycles that exploited disturbed habitats in open, or perhaps understorey, conditions. However, direct palaeontogical data relevant to understanding the seed biology and germination ecology of Early Cretaceous angiosperms are sparse. Here we report the discovery of embryos and their associated nutrient storage tissues in exceptionally well-preserved angiosperm seeds from the Early Cretaceous. Synchrotron radiation X-ray tomographic microscopy of the fossil embryos from many taxa reveals that all were tiny at the time of dispersal. These results support hypotheses based on extant plants that tiny embryos and seed dormancy are basic for angiosperms as a whole. The minute size of the fossil embryos, and the modest nutrient storage tissues dictated by the overall small seed size, is also consistent with the interpretation that many early angiosperms were opportunistic, early successional colonizers of disturbance-prone habitats.

Effect of Culture Conditions on Viability of Mouse and Rat Embryos Developed in Vitro

PubMed Central

Popova, Elena; Bader, Michael; Krivokharchenko, Alexander

2011-01-01

Currently in vitro culture of mouse preimplantation embryos has become a very important technique to investigate different mechanisms of early embryogenesis. However, there is a big difference in the preimplantation development between mammalian species. Despite close relatedness to mice, in vitro cultivation of rat preimplantation embryos is still delicate and needs further investigation and optimizations. In this study we have compared the in vitro developmental potential of mouse and rat embryos cultured at different culture conditions in parallel experiments. Interestingly, mouse zygotes developed in vitro until blastocyst stage even in inadequate medium without any phosphates and with low osmolarity which was formulated especially for cultivation of rat embryos. Rat parthenotes and zygotes developed in M16 medium formulated for mouse embryos only till 2-cell stage and further development is blocked completely at this stage. Moreover, developmental ability of rat embryos in vitro was significantly lower in comparison with mouse even in special rat mR1ECM medium. Mouse and rat embryos at 2-cell stage obtained in vivo developed until blastocyst stages significantly more efficiently compared to zygotes. Culture of mouse zygotes in glass capillaries resulted in a significantly higher rate of morula and blastocyst development compared with dishes. The Well-of-the-Well system resulted in a significant improvement when compared with dishes for the culture of rat zygotes only until morula stage. Reduced oxygen tension increased the developmental rate of rat but not mouse zygotes until blastocyst stage. This study demonstrates that development of early preimplantation embryos is altered by different culture conditions and show strong differences even between two related species such as mice and rats. Therefore, for understanding the fundamental mechanisms of early mammalian development it is very important to use embryos of various species. PMID:24710194

Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish.

PubMed

Lee, Kerry J; Browning, Lauren M; Nallathamby, Prakash D; Osgood, Christopher J; Xu, Xiao-Hong Nancy

2013-12-07

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 ± 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c < 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target

A method of microinjection: delivering monoclonal antibody 1223 into sea urchin embryos.

PubMed

Cho, J W

1999-08-31

In this paper, a simpler method of microinjecting sea urchin embryos without using the conventional microinjection chamber designed by Kiehart is reported. A trough was made on a surface of 0.6% agarose gel dissolved in artificial sea water. Approximately fifty hatched embryos could be loaded in the trough and, consequently, swimming embryos were trapped in the trough. Monoclonal antibody (mAB) 1223 which blocks spiculogenesis in vitro was delivered into the blastocoels of sea urchin embryos to test whether this antibody inhibits spiculogenesis in vivo and also, whether this new technique is effective for the microinjection of the sea urchin embryos. The embryos were injected with mAB1223 at the hatched blastula, early mesenchyme blastula and early gastrula stages, and 63%, 90% and 97% of the embryos did not form spicules at the late gastrula stage, respectively. Therefore, mAB1223 was shown to also block spiculogenesis in vivo. From the fact that spiculogenesis occurred at a lower rate when mAB1223 was injected at the hatched blastula stage than at later stages, it may be speculated that endogenous proteases degraded the injected antibodies. Using this technique, extracellular events in the blastocoel or the function of certain molecules expressed in blastocoel can be easily investigated in vivo.

[Effect of human oviductal embryotrophic factors on gene expression of mouse preimplantation embryos].

PubMed

Yao, Yuan-Qing; Lee, Kai-Fai; Xu, Jia-Seng; Ho, Pak-Chung; Yeung, Shu-Biu

2007-09-01

To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.

Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos

PubMed Central

Geens, Mieke; Mateizel, Ileana; Sermon, Karen; De Rycke, Martine; Spits, Claudia; Cauffman, Greet; Devroey, Paul; Tournaye, Herman; Liebaers, Inge; Van de Velde, Hilde

2009-01-01

BACKGROUND Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT–PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent. PMID:19633307

Use of infrared imaging to predict the developmental stages and differences in chicken embryos exposed to different photoperiods

NASA Astrophysics Data System (ADS)

Frederick, Rebecca A.; Hsieh, Sheng-Jen; Palomares, Benjamin Giron

2012-06-01

Monitoring development of chicken embryos allows determination of when an egg is not developing and when eggs are close to hatching for more efficient production. Research has been conducted on the effects of temperature fluctuations and light exposure on embryo development; similarities between chicken and mammal embryos; and the use of MRI, tomography, and ultrasound to view specific areas and processes within the embryo. However, there has been little exploration of the use of infrared imaging as a non-destructive method for analyzing and predicting embryonic development. In this study, we built an automated loading system for image acquisition. Pilot experiments were conducted to determine the overall scanning time and scanning frequency. A batch of fertilized eggs was scanned each day as the embryos continued to grow. The captured images were analyzed and categorized into three stages: Stage 1 (days 1 to 7), Stage 2 (days 8 to 14), and Stage 3 (days 15 to 21). The temperature data abstracted from the captured images were divided into two groups. Group 1, consisting of two-thirds of the data, was used to construct a model. Group 2, consisting of one-third of the data, was used to evaluate the predictive accuracy of the model. A three-layer artificial neural network model was developed to predict embryo development stage given a temperature profile. Results suggest that the neural network model is sufficient to predict embryo development stage with good accuracy of 75%. Accuracy can likely be improved if more data sets for each development stage are available.

The effect of tramadol hydrochloride on early life stages of fish.

PubMed

Sehonova, Pavla; Plhalova, Lucie; Blahova, Jana; Berankova, Petra; Doubkova, Veronika; Prokes, Miroslav; Tichy, Frantisek; Vecerek, Vladimir; Svobodova, Zdenka

2016-06-01

The aim of this study was to perform the fish embryo acute toxicity test (FET) on zebrafish (Danio rerio) and the early-life stage toxicity test on common carp (Cyprinus carpio) with tramadol hydrochloride. The FET was performed using the method inspired by the OECD guideline 236. Newly fertilized zebrafish eggs were exposed to tramadol hydrochloride at concentrations of 10; 50; 100 and 200μg/l for a period of 144h. An embryo-larval toxicity test on C. carpio was performed according to OECD guideline 210 also with tramadol hydrochloride at concentrations 10; 50; 100 and 200μg/l for a period of 32 days. Hatching was significantly influenced in both acute and subchronic toxicity assays. Subchronic exposure also influenced early ontogeny, both morphometric and condition characteristics and caused changes in antioxidant enzyme activity. The LOEC value was found to be 10μg/l tramadol hydrochloride. Copyright © 2016 Elsevier B.V. All rights reserved.

Early developmental gene regulation in Strongylocentrotus purpuratus embryos in response to elevated CO₂ seawater conditions.

PubMed

Hammond, LaTisha M; Hofmann, Gretchen E

2012-07-15

Ocean acidification, or the increased uptake of CO(2) by the ocean due to elevated atmospheric CO(2) concentrations, may variably impact marine early life history stages, as they may be especially susceptible to changes in ocean chemistry. Investigating the regulatory mechanisms of early development in an environmental context, or ecological development, will contribute to increased understanding of potential organismal responses to such rapid, large-scale environmental changes. We examined transcript-level responses to elevated seawater CO(2) during gastrulation and the initiation of spiculogenesis, two crucial developmental processes in the purple sea urchin, Strongylocentrotus purpuratus. Embryos were reared at the current, accepted oceanic CO(2) concentration of 380 microatmospheres (μatm), and at the elevated levels of 1000 and 1350 μatm, simulating predictions for oceans and upwelling regions, respectively. The seven genes of interest comprised a subset of pathways in the primary mesenchyme cell gene regulatory network (PMC GRN) shown to be necessary for the regulation and execution of gastrulation and spiculogenesis. Of the seven genes, qPCR analysis indicated that elevated CO(2) concentrations only had a significant but subtle effect on two genes, one important for early embryo patterning, Wnt8, and the other an integral component in spiculogenesis and biomineralization, SM30b. Protein levels of another spicule matrix component, SM50, demonstrated significant variable responses to elevated CO(2). These data link the regulation of crucial early developmental processes with the environment that these embryos would be developing within, situating the study of organismal responses to ocean acidification in a developmental context.

Early Cambrian Pentamerous Cubozoan Embryos from South China

PubMed Central

Han, Jian; Kubota, Shin; Li, Guoxiang; Yao, Xiaoyong; Yang, Xiaoguang; Shu, Degan; Li, Yong; Kinoshita, Shunichi; Sasaki, Osamu; Komiya, Tsuyoshi; Yan, Gang

2013-01-01

Background Extant cubozoans are voracious predators characterized by their square shape, four evenly spaced outstretched tentacles and well-developed eyes. A few cubozoan fossils are known from the Middle Cambrian Marjum Formation of Utah and the well-known Carboniferous Mazon Creek Formation of Illinois. Undisputed cubozoan fossils were previously unknown from the early Cambrian; by that time probably all representatives of the living marine phyla, especially those of basal animals, should have evolved. Methods Microscopic fossils were recovered from a phosphatic limestone in the Lower Cambrian Kuanchuanpu Formation of South China using traditional acetic-acid maceration. Seven of the pre-hatched pentamerous cubozoan embryos, each of which bears five pairs of subumbrellar tentacle buds, were analyzed in detail through computed microtomography (Micro-CT) and scanning electron microscopy (SEM) without coating. Results The figured microscopic fossils are unequivocal pre-hatching embryos based on their spherical fertilization envelope and the enclosed soft-tissue that has preserved key anatomical features arranged in perfect pentaradial symmetry, allowing detailed comparison with modern cnidarians, especially medusozoans. A combination of features, such as the claustrum, gonad-lamella, suspensorium and velarium suspended by the frenula, occur exclusively in the gastrovascular system of extant cubozoans, indicating a cubozoan affinity for these fossils. Additionally, the interior anatomy of these embryonic cubozoan fossils unprecedentedly exhibits the development of many new septum-derived lamellae and well-partitioned gastric pockets unknown in living cubozoans, implying that ancestral cubozoans had already evolved highly specialized structures displaying unexpected complexity at the dawn of the Cambrian. The well-developed endodermic lamellae and gastric pockets developed in the late embryonic stages of these cubozoan fossils are comparable with extant pelagic

Clinical significance of intercellular contact at the four-cell stage of human embryos, and the use of abnormal cleavage patterns to identify embryos with low implantation potential: a time-lapse study.

PubMed

Liu, Yanhe; Chapple, Vincent; Feenan, Katie; Roberts, Peter; Matson, Phillip

2015-06-01

To investigate the clinical significance of intercellular contact point (ICCP) in four-cell stage human embryos and the effectiveness of morphology and abnormal cleavage patterns in identifying embryos with low implantation potential. Retrospective cohort study. Private IVF center. A total of 223 consecutive IVF and intracytoplasmic sperm injection treatment cycles, with all resulting embryos cultured in the Embryoscope, and a subset of 207 cycles analyzed for ICCP number where good-quality four-cell embryos were available on day 2 (n = 373 IVF and n = 392 intracytoplasmic sperm injection embryos). None. Morphologic score on day 3, embryo morphokinetic parameters, incidence of abnormal biological events, and known implantation results. Of 765 good-quality four-cell embryos, 89 (11.6%) failed to achieve six ICCPs; 166 of 765 (21.7%) initially had fewer than six ICCPs but were able to establish six ICCPs before subsequent division. Embryos with fewer than six ICCPs at the end of four-cell stage had a lower implantation rate (5.0% vs. 38.5%), with lower embryology performance in both conventional and morphokinetic assessments, compared with embryos achieving six ICCPs by the end of four-cell stage. Deselecting embryos with poor morphology, direct cleavage, reverse cleavage, and fewer than six ICCPs at the four-cell stage led to a significantly improved implantation rate (33.6% vs. 22.4%). Embryos with fewer than six ICCPs at the end of the four-cell stage show compromised subsequent development and reduced implantation potential. Deselection of embryos with poor morphology and abnormal cleavage revealed via time-lapse imaging could provide the basis of a qualitative algorithm for embryo selection. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

A 3D reconstruction of pancreas development in the human embryos during embryonic period (Carnegie stages 15-23).

PubMed

Radi, M; Gaubert, J; Cristol-Gaubert, R; Baecker, V; Travo, P; Prudhomme, M; Godlewski, G; Prat-Pradal, D

2010-01-01

The goal in this paper was to rebuild a three dimensional (3D) reconstruction of the dorsal and ventral pancreatic buds, in the human embryos, at Carnegie stages 15-23. The early development of the pancreas is studied by tissue observation and reconstruction by a computer-assisted method, using a light micrograph images from consecutive serial sagittal sections (diameter 7 microm) of ten human embryos ranging from Carnegie stages 15-23, CRL 7-27 mm, fixed, dehydrated and embedded in paraffin, were stained alternately with haematoxylin-eosin or Heindenhain'Azan. The images were digitalized by Canon Camera 350 EOS D. The serial views were aligned automatically by software, manual alignment was performed, the data were analysed following segmentation and threshold. The two buds were clearly identified at stage 15. In stage 16, both pancreatic buds were in final position, and begin to merge in stage 17. From stage 18 to the stage 23, surrounding connective tissue differentiated. In the stage 23, the morphology of the pancreas was definitive. The superior portion of the anterior face of the pancreas's head was arising from the dorsal bud. The rest of the head including the uncinate process emanated from the ventral bud. The 3D computer-assisted reconstruction of the human pancreas visualized the relationships between the two pancreatic buds. This explains the disposition and the modality of the components fusion. This embryologic development permits a better understanding of congenital abnormalities.

Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells.

PubMed

Guo, Fan; Li, Lin; Li, Jingyun; Wu, Xinglong; Hu, Boqiang; Zhu, Ping; Wen, Lu; Tang, Fuchou

2017-08-01

Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA

Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells

PubMed Central

Guo, Fan; Li, Lin; Li, Jingyun; Wu, Xinglong; Hu, Boqiang; Zhu, Ping; Wen, Lu; Tang, Fuchou

2017-01-01

Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA

Semiautomated analysis of embryoscope images: Using localized variance of image intensity to detect embryo developmental stages.

PubMed

Mölder, Anna; Drury, Sarah; Costen, Nicholas; Hartshorne, Geraldine M; Czanner, Silvester

2015-02-01

Embryo selection in in vitro fertilization (IVF) treatment has traditionally been done manually using microscopy at intermittent time points during embryo development. Novel technique has made it possible to monitor embryos using time lapse for long periods of time and together with the reduced cost of data storage, this has opened the door to long-term time-lapse monitoring, and large amounts of image material is now routinely gathered. However, the analysis is still to a large extent performed manually, and images are mostly used as qualitative reference. To make full use of the increased amount of microscopic image material, (semi)automated computer-aided tools are needed. An additional benefit of automation is the establishment of standardization tools for embryo selection and transfer, making decisions more transparent and less subjective. Another is the possibility to gather and analyze data in a high-throughput manner, gathering data from multiple clinics and increasing our knowledge of early human embryo development. In this study, the extraction of data to automatically select and track spatio-temporal events and features from sets of embryo images has been achieved using localized variance based on the distribution of image grey scale levels. A retrospective cohort study was performed using time-lapse imaging data derived from 39 human embryos from seven couples, covering the time from fertilization up to 6.3 days. The profile of localized variance has been used to characterize syngamy, mitotic division and stages of cleavage, compaction, and blastocoel formation. Prior to analysis, focal plane and embryo location were automatically detected, limiting precomputational user interaction to a calibration step and usable for automatic detection of region of interest (ROI) regardless of the method of analysis. The results were validated against the opinion of clinical experts. © 2015 International Society for Advancement of Cytometry. © 2015 International

Acute toxicity and histopathological effects of naproxen in zebrafish (Danio rerio) early life stages.

PubMed

Li, Qian; Wang, Peipei; Chen, Ling; Gao, Hongwen; Wu, Lingling

2016-09-01

Zebrafish (Danio rerio) embryos and larvae were selected to investigate the potential risk and aquatic toxicity of a widely used pharmaceutical, naproxen. The acute toxicity of naproxen to embryos and larvae was measured, respectively. The histopathology was investigated in the liver of zebrafish larvae after 8-day embryo-larvae exposure to naproxen. The values of 96-h LC50 were 115.2 mg/L for embryos and 147.6 mg/L for larvae, indicating that zebrafish embryos were more sensitive than larvae to naproxen exposure. Large suites of symptoms were induced in zebrafish (D. rerio) early life stages by different dosages of naproxen, including hatching inhibition, lower heart rate, and morphological abnormalities. The most sensitive sub-lethal effect caused by naproxen was pericardial edema, the 72-h EC50 values of which for embryos and larvae were 98.3 and 149.0 mg/L, respectively. In addition, naproxen-treated zebrafish larvae exhibited histopathological liver damage, including swollen hepatocytes, vacuolar degeneration, and nuclei pycnosis. The results indicated that naproxen is a potential threat to aquatic organisms.

MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo.

PubMed

Pernaute, Barbara; Spruce, Thomas; Rodriguez, Tristan A; Manzanares, Miguel

2011-02-15

At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

Birth of normal infants after transfer of embryos that were twice vitrified/warmed at cleavage stages: report of two cases.

PubMed

Valle, Marcello; Guimarães, Fernando; Cavagnoli, Melissa; Sampaio, Marcos; Geber, Selmo

2012-12-01

The role of cryopreservation in assisted reproductive technology programs has increased within the last years allowing the transfer of a limited number of embryos and the storage of the remaining for future use. The reduction in the number of transferred embryos decreases the frequency of multiple pregnancy rates and of ovarian hyperstimulation syndrome while the cumulative pregnancy rate can be maximized. Moreover, as not all embryos will survive the warming process more cleavage stage embryos are warmed to improve selection for transfer. Therefore, surplus good quality cleavage stage embryos and/or blastocysts must be re-vitrified for further transfer to achieve pregnancy. To our knowledge, there have been no reports demonstrating that human embryos can be successfully vitrified/warmed twice at the cleavage stage. Thus we report two successful pregnancies and deliveries of healthy babies after transfer of embryos that were twice vitrified/warmed at 2-4 cells stage. Copyright © 2012 Elsevier Inc. All rights reserved.

The Potential Role of As-sumo-1 in the Embryonic Diapause Process and Early Embryo Development of Artemia sinica

PubMed Central

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

2014-01-01

During embryonic development of Artemia sinica, environmental stresses induce the embryo diapause phenomenon, required to resist apoptosis and regulate cell cycle activity. The small ubiquitin-related modifier-1 (SUMO), a reversible post-translational protein modifier, plays an important role in embryo development. SUMO regulates multiple cellular processes, including development and other biological processes. The molecular mechanism of diapause, diapause termination and the role of As-sumo-1 in this processes and in early embryo development of Artemia sinica still remains unknown. In this study, the complete cDNA sequences of the sumo-1 homolog, sumo ligase homolog, caspase-1 homolog and cyclin B homolog from Artemia sinica were cloned. The mRNA expression patterns of As-sumo-1, sumo ligase, caspase-1, cyclin B and the location of As-sumo-1 were investigated. SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E proteins were analyzed during different developmental stages of the embryo of A. sinica. Small interfering RNA (siRNA) was used to verify the function of sumo-1 in A. sinica. The full-length cDNA of As-sumo-1 was 476 bp, encoding a 92 amino acid protein. The As-caspases-1 cDNA was 966 bp, encoding a 245 amino-acid protein. The As-sumo ligase cDNA was 1556 bp encoding, a 343 amino acid protein, and the cyclin B cDNA was 739 bp, encoding a 133 amino acid protein. The expressions of As-sumo-1, As-caspase-1 and As-cyclin B were highest at the 10 h stage of embryonic development, and As-sumo ligase showed its highest expression at 0 h. The expression of As-SUMO-1 showed no tissue or organ specificity. Western blotting showed high expression of As-SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E at the 10 h stage. The siRNA caused abnormal development of the embryo, with increased malformation and mortality. As-SUMO-1 is a crucial regulation and modification protein resumption of embryonic diapause and early embryo development of A. sinica. PMID:24404204

PITX2 and NODAL expression during axis formation in the early rabbit embryo.

PubMed

Plöger, Ruben; Viebahn, Christoph

2018-04-26

Attaining molecular and morphological axial polarity during gastrulation is a fundamental early requirement for normal development of the embryo. In mammals, the first morphological sign of the anterior-posterior axis appears anteriorly in the form of the anterior marginal crescent (or anterior visceral endoderm) while in the avian the first such sign is the Koller's sickle at the posterior pole of the embryonic disc. Despite this inverse mode of axis formation many genes and molecular pathways involved in various steps of this process seem to be evolutionary conserved amongst amniotes, the nodal gene being a well-known example with its functional involvement prior and during gastrulation. The pitx2 gene, however, is a new candidate described in the chick as an early marker for anterior-posterior polarity and as regulator of axis formation including twinning. To find out whether pitx2 has retained its inductive and early marker function during the evolution of mammals, this study analyzes pitx2 and nodal expression at parallel stages during formation of the anterior-posterior polarity in the early rabbit embryo using whole-mount in situ hybridization and serial light-microscopical sections. At a late pre-gastrulation stage a localized reduction of nodal expression presages the position of the anterior pole of the embryonic disc and thus serves as the earliest molecular marker of anterior-posterior polarity known so far. pitx2 is expressed in a polarized manner in the anterior marginal crescent and in the posterior half of the embryonic disc during further development only while nodal expression in the anterior segment of the posterior pitx2 expression domain helps to define the so-called anterior streak domain (ASD), a novel progenitor region of the anterior half of the primitive streak. The expression patterns of both genes thus serve as signs of a conserved involvement in early axis formation in amniotes and, possibly, in twinning in mammals as well. Copyright �

Lethal effect of dehydroleucodine (DhL) on amphibian Bufo arenarum embryos.

PubMed

Moreno, Liliana Elizabeth; Juárez, Américo Osvaldo; Pelzer, Lilian Eugenia

2012-03-01

The dehydroleucodine is a sesquiterpene lactone isolated from Artemisia douglasiana Besser which is used in popular medicine. Toxicity tests using embryos of amphibian have been widely used in order to predict toxic effects of different compounds. However, to our knowledge, there are not studies focussed on the toxic effects of dehydroleucodine on Bufo arenarum, which is an anuran widely distributed in South America. The effect of dehydroleucodine on the survival of embryos was evaluated in an acute test during the early life stage of B. arenarum embryos. Lethality and the degree of adverse effects were dehydroleucodine dose-dependent. Overall, amphibian early life stages appeared to be more susceptible to the embryotoxicity associated with exposure to dehydroleucodine, especially at concentration greater that 3mM. This increased susceptibility may result from the relatively high rate of cellular differentiation and morphogenesis that occurs at this early stage of development. Copyright © 2012 Elsevier Ltd. All rights reserved.

Early life sensory ability-ventilatory responses of thornback ray embryos (Raja clavata) to predator-type electric fields.

PubMed

Ball, Rachel Emma; Oliver, Matthew Kenneth; Gill, Andrew Bruce

2016-07-01

Predator avoidance is fundamental for survival and it can be particularly challenging for prey animals if physical movement away from a predatory threat is restricted. Many sharks and rays begin life within an egg capsule that is attached to the sea bed. The vulnerability of this sedentary life stage is exacerbated in skates (Rajidae) as the compulsory ventilatory activity of embryos makes them conspicuous to potential predators. Embryos can reduce this risk by mediating ventilatory activity if they detect the presence of a predator using an acute electrosense. To determine how early in embryonic life predator elicited behavioral responses can occur, the reactions of three different age groups (1/3 developed, 2/3 developed, and near hatching) of embryonic thornback rays Raja clavata were tested using predator-type electric field stimuli. Egg capsules were exposed to continuous or intermittent stimuli in order to assess varying predator-type encounter scenarios on the ventilatory behavior of different developmental stages. All embryos reacted with a "freeze response" following initial electric field (E-field) exposure, ceasing ventilatory behavior in response to predator presence, demonstrating electroreceptive functionality for the first time at the earliest possible stage in ontogeny. This ability coincided with the onset of egg ventilatory behavior and may represent an effective means to enhance survival. A continuous application of stimuli over time revealed that embryos can adapt their behavior and resume normal activity, whereas when presented intermittently, the E-field resulted in a significant reduction in overall ventilatory activity across all ages. Recovery from stimuli was significantly quicker in older embryos, potentially indicative of the trade-off between avoiding predation and adequate respiration. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 721-729, 2016. © 2015 Wiley Periodicals, Inc.

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.

Role of glucose in mouse preimplantation embryo development.

PubMed

Martin, K L; Leese, H J

1995-04-01

Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca x C57BL/6) were cultured from the one- and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D + L-lactate, in the presence and absence of 1 mM glucose (M16 + G and M16 - G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16 - G were transferred to M16 + G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16 + G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16 - G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 +/- 2.5 [28] vs. 105 +/- 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16 - G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16 - G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16 + G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster.

PubMed

de Vega-Bartol, José J; Simões, Marta; Lorenz, W Walter; Rodrigues, Andreia S; Alba, Rob; Dean, Jeffrey F D; Miguel, Célia M

2013-08-30

It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in

Making lineage decisions with biological noise: Lessons from the early mouse embryo.

PubMed

Simon, Claire S; Hadjantonakis, Anna-Katerina; Schröter, Christian

2018-04-30

Understanding how individual cells make fate decisions that lead to the faithful formation and homeostatic maintenance of tissues is a fundamental goal of contemporary developmental and stem cell biology. Seemingly uniform populations of stem cells and multipotent progenitors display a surprising degree of heterogeneity, primarily originating from the inherent stochastic nature of molecular processes underlying gene expression. Despite this heterogeneity, lineage decisions result in tissues of a defined size and with consistent proportions of differentiated cell types. Using the early mouse embryo as a model we review recent developments that have allowed the quantification of molecular intercellular heterogeneity during cell differentiation. We first discuss the relationship between these heterogeneities and developmental cellular potential. We then review recent theoretical approaches that formalize the mechanisms underlying fate decisions in the inner cell mass of the blastocyst stage embryo. These models build on our extensive knowledge of the genetic control of fate decisions in this system and will become essential tools for a rigorous understanding of the connection between noisy molecular processes and reproducible outcomes at the multicellular level. We conclude by suggesting that cell-to-cell communication provides a mechanism to exploit and buffer intercellular variability in a self-organized process that culminates in the reproducible formation of the mature mammalian blastocyst stage embryo that is ready for implantation into the maternal uterus. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Gene Expression and Transcriptional Hierarchies > Quantitative Methods and Models. © 2018 Wiley Periodicals, Inc.

Radiation induced abnormalities in early in vitro mouse embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirkpatrick, J.F.

1973-08-01

Female mice were superovulated and mated, and the two-cell embryos were collected and cultured in vitro. The embryos were exposed to x-irradiation (0 to 491 rads) during the two-cell stage before the appearance of the next cleavage plate, placed in new unirradiated culture medium and observed during subsequent development. Morphological abnormalities, which occurred as a result of irradiation, included fragmentation, disintegration, granlation, incomplete cleavage, cleavage cessation, nuclear degeneration and pycnosis and cytoplasmic vacuolization. There was no damage to the zona pellucida. The types of abnormalities indicate an agreement with the results of previous in vivo studies. A distinct correlation existedmore » between morphological abnormalities and embryo death. The greatest number of abnormalities resulted within five hours following irradiation, but increased through 20 hours post-exposure. At doses above 300 rads, the magnitude of damage was greater in the in vitro embryos than that shown in previous in vivo studies. (auth)« less

Maternal hCG concentrations in early IVF pregnancies: associations with number of cells in the Day 2 embryo and oocytes retrieved.

PubMed

Tanbo, T G; Eskild, A

2015-12-01

Do number of cells in the transferred cleavage stage embryo and number of oocytes retrieved for IVF influence maternal hCG concentrations in early pregnancies? Compared with transfer of a 2-cell embryo, transfer of a 4-cell embryo results in higher hCG concentrations on Day 12 after transfer, and more than 20 oocytes retrieved were associated with low hCG concentrations. Maternal hCG concentration in very early pregnancy varies considerably among women, but is likely to be an indicator of time since implantation of the embryo into the endometrium, in addition to number and function of trophoblast cells. We followed 1047 pregnancies after IVF/ICSI from oocyte retrieval until Day 12 after embryo transfer. Women were recruited in Norway during the years 2005-2013. Successful pregnancies after transfer of one single embryo that had been cultured for 2 days were included. Maternal hCG was quantified on Day 12 after embryo transfer by chemiluminescence immunoassay, which measures intact hCG and the free β-hCG chain. Information on a successful pregnancy, defined as birth after >16 weeks, was obtained by linkage to the Medical Birth Registry of Norway. Transfer of a 4-cell embryo resulted in higher maternal hCG concentrations compared with transfer of a 2-cell embryo (134.8 versus 87.8 IU/l, P < 0.05). A high number of oocytes retrieved (>20) was associated with low hCG concentrations (P < 0.05). The factors studied explain a limited part of the total variation of hCG concentrations in early pregnancy. Although embryo transfer was performed at the same time after fertilization, we do not know the exact time of implantation. A further limitation to our study is that the number of pregnancies after transfer of a 2-cell embryo was small (27 cases). Number of cells in the transferred embryo and number of oocytes retrieved may influence the conditions and timing for embryo implantation in different ways and thereby influence maternal hCG concentrations. Such knowledge may be

A transcriptional blueprint for a spiral-cleaving embryo.

PubMed

Chou, Hsien-Chao; Pruitt, Margaret M; Bastin, Benjamin R; Schneider, Stephan Q

2016-08-05

The spiral cleavage mode of early development is utilized in over one-third of all animal phyla and generates embryonic cells of different size, position, and fate through a conserved set of stereotypic and invariant asymmetric cell divisions. Despite the widespread use of spiral cleavage, regulatory and molecular features for any spiral-cleaving embryo are largely uncharted. To address this gap we use RNA-sequencing on the spiralian model Platynereis dumerilii to capture and quantify the first complete genome-wide transcriptional landscape of early spiral cleavage. RNA-sequencing datasets from seven stages in early Platynereis development, from the zygote to the protrochophore, are described here including the de novo assembly and annotation of ~17,200 Platynereis genes. Depth and quality of the RNA-sequencing datasets allow the identification of the temporal onset and level of transcription for each annotated gene, even if the expression is restricted to a single cell. Over 4000 transcripts are maternally contributed and cleared by the end of the early spiral cleavage phase. Small early waves of zygotic expression are followed by major waves of thousands of genes, demarcating the maternal to zygotic transition shortly after the completion of spiral cleavages in this annelid species. Our comprehensive stage-specific transcriptional analysis of early embryonic stages in Platynereis elucidates the regulatory genome during early spiral embryogenesis and defines the maternal to zygotic transition in Platynereis embryos. This transcriptome assembly provides the first systems-level view of the transcriptional and regulatory landscape for a spiral-cleaving embryo.

Properties of Barley Seed Chitinases and Release of Embryo-Associated Isoforms during Early Stages of Imbibition 1

PubMed Central

Swegle, Mark; Kramer, Karl J.; Muthukrishnan, Subbaratnam

1992-01-01

Barley (Hordeum vulgare L.) seeds contain at least five proteins with chitinase (CH) activity. Two of these (CH1 and CH2) are found primarily in the aleurone and endosperm tissues, and the other three (CH3, CH4, and CH5) are enriched in the embryo. From the bran fraction, three of these CHs (CH1, CH2, and CH3) were purified to apparent homogeneity. These three CHs have apparent molecular masses of 27, 34, and 35 kilodaltons and isoelectric points of 9.3, 9.2, and 8.7, respectively. CH2 and CH3 have amino terminal sequences resembling a portion of the chitin-binding domain of lectins and other plant defense proteins. CH1 lacks this domain. All three CHs exhibit antifungal activity and inhibit the mycelial growth of some species of trichoderma and Fusarium in vitro. During the early period of imbibition by seeds, two of the embryo-associated CHs are selectively released into the surrounding aqueous medium. ImagesFigure 1Figure 2Figure 3Figure 4 PMID:16668964

Cytoskeletal changes in oocytes and early embryos during in vitro fertilization process in mice.

PubMed

Gumus, E; Bulut, H E; Kaloglu, C

2010-02-01

The cytoskeleton plays crucial roles in the development and fertilization of germ cells and in the early embryo development. The growth, maturation and fertilization of oocytes require an active movement and a correct localization of cellular organelles. This is performed by the re-organization of microtubules and actin filaments. Therefore, the aim of the present study was to determine the changes in cytoskeleton during in vitro fertilization process using appropriate immunofluorescence techniques. While the chromatin content was found to be scattered throughout the nucleus during the oocyte maturation period, it was seen only around nucleolus following the completion of the maturation. Microtubules, during oocyte maturation, were regularly distributed throughout the ooplasm which was then localized in the subcortical region of oocytes. Similarly microfilaments were scattered throughout the ooplasm during the oocyte maturation period whereas they were seen in the subcortical region around the polar body and above the meiotic spindle throughout the late developmental stages. In conclusion, those changes occurred in microtubules and microfilaments might be closely related to the re-organization of the genetic material during the oocyte maturation and early embryo development.

Gene Expression in Pre-MBT Embryos and Activation of Maternally-Inherited Program of Apoptosis to be Executed at around MBT as a Fail-Safe Mechanism in Xenopus Early Embryogenesis

PubMed Central

Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Uchiyama, Hiroaki; Kuroyanagi, Shinsaku; Takai, Jun-Ichi; Takahashi, Senji; Kajitani, Masayuki; Kaito, Chikara; Sekimizu, Kazuhisa; Takayama, Eiji; Igarashi, Kazuei; Hara, Hiroshi

2008-01-01

S-adenosylmethionine decarboxylase (SAMDC) is an enzyme which converts S-adenosylmethione (SAM), a methyl donor, to decarboxylated SAM (dcSAM), an aminopropyl donor for polyamine biosynthesis. In our studies on gene expression control in Xenopus early embryogenesis, we cloned the mRNA for Xenopus SAMDC, and overexpressed the enzyme by microinjecting its mRNA into Xenopus fertilized eggs. In the mRNA-injected embryos, the level of SAMDC was enormously increased, the SAM was exhausted, and protein synthesis was greatly inhibited, but cellular polyamine content did not change appreciably. SAMDC-overexpressed embryos cleaved and developed normally up to the early blastula stage, but at the midblastula stage, or the stage of midblastula transition (MBT), all the embryos were dissociated into cells, and destroyed due to execution of apoptosis. During cleavage SAMDC-overexpressed embryos transcribed caspase-8 gene, and this was followed by activation of caspase-9. When we overexpressed p53 mRNA in fertilized eggs, similar apoptosis took place at MBT, but in this case, transcription of caspase-8 did not occur, however activation of caspase-9 took place. Apoptosis induced by SAMDC-overexpression was completely suppressed by Bcl-2, whereas apoptosis induced by p53 overexpression or treatments with other toxic agents was only partially rescued. When we injected SAMDC mRNA into only one blastomere of 8- to 32-celled embryos, descendant cells of the mRNA-injected blastomere were segregated into the blastocoel and underwent apoptosis within the blastocoel, although such embryos continued to develop and became tadpoles with various extents of anomaly, reflecting the developmental fate of the eliminated cells. Thus, embryonic cells appear to check themselves at MBT and if physiologically severely-damaged cells occur, they are eliminated from the embryo by activation and execution of the maternally-inherited program of apoptosis. We assume that the apoptosis executed at MBT is a �

Embryo density and medium volume effects on early murine embryo development.

PubMed

Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C

1992-10-01

One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

Influence of storage time on vitrified human cleavage-stage embryos froze in open system.

PubMed

Li, Wei; Zhao, Wanqiu; Xue, Xia; Zhang, Silin; Zhang, Xin; Shi, Juanzi

2017-02-01

During in vitro fertilization, rapid growth of vitrification and liquid nitrogen storage of embryos have been well characterized. However, the effect of storage time on vitrified cleavage-stage embryos in an open system is poorly understood. To investigate the influence of storage time on the survival and pregnancy outcomes of vitrified human cleavage-stage embryos froze and stored in an open system. A retrospective study of 786 vitrified-warmed cycles of 735 patients was performed from January 2013 to October 2013. The cycles were divided into five groups according to storage time: 1-3 months, 4-6 months, 7-12 months, 13-24 and 25-60 months. The clinical outcomes of cycles with different storage time were analyzed. There were no significant differences of the survival rate, clinical pregnancy outcomes, birth rate, gestational weeks and singleton birthweights at various storage times. For vitrified embryos froze and stored in an open system, the storage time would not influence the survival rate and pregnancy outcomes by storage time up to 5 years.

Patterns of protein synthesis in oocytes and early embryos of Rana esculenta complex.

PubMed

Chen, P S; Stumm-Zollinger, E

1986-01-01

We have used isotopic labelling and both one-and two-dimensional electrophoretic procedures to analyse the protien synthesis patterns in oocytes and early embryos of three phenotypes of the European green frogs. The results demonstrated that protein patterns of Rana ridibunda and R. esculenta are identical, but that they differ from those of R. lessonae. Progeny of the lethal cross R. esculenta × R. esculenta showed a distinct delay in the appearance of stage-specific proteins during early embryogenesis. The heat-shock response of R. ridibunda and R. esculenta oocytes was found to be identical, but different from that of Xenopus laevis. The implications of these findings, with respect to hybridogenesis in R. esculenta complex and variations in the regulations of heat shock genes in different amphibian species, are discussed.

Heavy Rare Earth Elements Affect Sphaerechinus granularis Sea Urchin Early Life Stages by Multiple Toxicity Endpoints.

PubMed

Gravina, Maria; Pagano, Giovanni; Oral, Rahime; Guida, Marco; Toscanesi, Maria; Siciliano, Antonietta; Di Nunzio, Aldo; Burić, Petra; Lyons, Daniel M; Thomas, Philippe J; Trifuoggi, Marco

2018-05-01

Heavy rare earth elements (HREEs) were tested for adverse effects to early life stages of the sea urchin Sphaerechinus granularis. Embryos were exposed to analytically measured HREE concentrations ranging from 10 -7 to 10 -5  M. No significant developmental defect (DD) increases were observed in embryos exposed to 10 -7  M HREEs, whereas 10 -5  M HREEs resulted in significant DD increase up to 96% for HoCl 3 versus 14% in controls. Embryos exposed to 10 -6  M HREEs showed the highest DD frequency in embryos exposed to 10 -6  M DyCl 3 and HoCl 3 . Cytogenetic analysis of HREE-exposed embryos revealed a significant decrease in mitotic activity, with increased mitotic aberrations. When S. granularis sperm were exposed to HREEs, the offspring of sperm exposed to 10 -5  M GdCl 3 and LuCl 3 showed significant DD increases. The results warrant investigations on HREEs in other test systems, and on REE-containing complex mixtures.

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster

PubMed Central

2013-01-01

Background It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Results Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning

Early Embryo Development in Fucus distichus Is Auxin Sensitive1

PubMed Central

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 [3H]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. PMID:12226509

Loss of maternal CTCF is associated with peri-implantation lethality of Ctcf null embryos.

PubMed

Moore, James M; Rabaia, Natalia A; Smith, Leslie E; Fagerlie, Sara; Gurley, Kay; Loukinov, Dmitry; Disteche, Christine M; Collins, Steven J; Kemp, Christopher J; Lobanenkov, Victor V; Filippova, Galina N

2012-01-01

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5-E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16-32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.

Telomere lengthening early in development.

PubMed

Liu, Lin; Bailey, Susan M; Okuka, Maja; Muñoz, Purificación; Li, Chao; Zhou, Lingjun; Wu, Chao; Czerwiec, Eva; Sandler, Laurel; Seyfang, Andreas; Blasco, Maria A; Keefe, David L

2007-12-01

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.

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.

Single-cell transcriptome of early embryos and cultured embryonic stem cells of cynomolgus monkeys

PubMed Central

Nakamura, Tomonori; Yabuta, Yukihiro; Okamoto, Ikuhiro; Sasaki, Kotaro; Iwatani, Chizuru; Tsuchiya, Hideaki; Saitou, Mitinori

2017-01-01

In mammals, the development of pluripotency and specification of primordial germ cells (PGCs) have been studied predominantly using mice as a model organism. However, divergences among mammalian species for such processes have begun to be recognized. Between humans and mice, pre-implantation development appears relatively similar, but the manner and morphology of post-implantation development are significantly different. Nevertheless, the embryogenesis just after implantation in primates, including the specification of PGCs, has been unexplored due to the difficulties in analyzing the embryos at relevant developmental stages. Here, we present a comprehensive single-cell transcriptome dataset of pre- and early post-implantation embryo cells, PGCs and embryonic stem cells (ESCs) of cynomolgus monkeys as a model of higher primates. The identities of each transcriptome were also validated rigorously by other way such as immunofluorescent analysis. The information reported here will serve as a foundation for our understanding of a wide range of processes in the developmental biology of primates, including humans. PMID:28649393

Role of nucleation-promoting factors in mouse early embryo development.

PubMed

Wang, Qiao-Chu; Liu, Jun; Wang, Fei; Duan, Xing; Dai, Xiao-Xin; Wang, Teng; Liu, Hong-Lin; Cui, Xiang-Shun; Sun, Shao-Chen; Kim, Nam-Hyung

2013-06-01

During mitosis nucleation-promoting factors (NPFs) bind to the Arp2/3 complex and activate actin assembly. JMY and WAVE2 are two critical members of the NPFs. Previous studies have demonstrated that NPFs promote multiple processes such as cell migration and cytokinesis. However, the role of NPFs in development of mammalian embryos is still unknown. Results of the present study show that the NPFs JMY and WAVE2 are critical for cytokinesis during development of mouse embryos. Both JMY and WAVE2 are expressed in mouse embryos. After injection of JMY or WAVE2 siRNA, all embryos failed to develop to the morula or blastocyst stages. Moreover, using fluorescence intensity analysis, we found that the expression of actin decreased, and multiple nuclei were observed within a single cell indicating that NPFs-induced actin reduction caused the failure of cell division. In addition, injection of JMY and WAVE2 siRNA also caused ARP2 degradation, indicating that involvement of NPFs in development of mouse embryos is mainly through regulation of ARP2/3-induced actin assembly. Taken together, these data suggested that WAVE2 and JMY are involved in development of mouse embryos, and their regulation may be through a NPFs-Arp2/3-actin pathway.

Cumulative Effects of Nutrient Enrichment and Elevated Temperature Compromise the Early Life History Stages of the Coral Acropora tenuis

PubMed Central

Noonan, Sam H. C.; Willis, Bette L.; Fabricius, Katharina E.; Negri, Andrew P.

2016-01-01

Inshore coral reefs are experiencing the combined pressures of excess nutrient availability associated with coastal activities and warming seawater temperatures. Both pressures are known to have detrimental effects on the early life history stages of hard corals, but studies of their combined effects on early demographic stages are lacking. We conducted a series of experiments to test the combined effects of nutrient enrichment (three levels) and elevated seawater temperature (up to five levels) on early life history stages of the inshore coral Acropora tenuis, a common species in the Indo-Pacific and Red Sea. Gamete fertilization, larval survivorship and larval settlement were all significantly reduced as temperature increased, but only fertilization was further affected by simultaneous nutrient enrichment. Combined high temperatures and nutrient enrichment affected fertilization in an additive manner, whereas embryo abnormalities increased synergistically. Higher than normal temperatures (32°C) increased coral juvenile growth rates 1.6-fold, but mortality also increased by 50%. The co-occurrence of nutrient enrichment with high temperatures reduced juvenile mortality to 36%, ameliorating temperature stress (antagonistic interaction). Overall, the types of effect (additive vs synergistic or antagonistic) and their magnitude varied among life stages. Gamete and embryo stages were more affected by temperature stress and, in some cases, also by nutrient enrichment than juveniles. The data suggest that coastal runoff events might exacerbate the impacts of warming temperatures on fertilization if these events co-occur during corals spawning. The cumulative impacts of simultaneous exposure to nutrient enrichment and elevated temperatures over all early life history stages increases the likelihood for failure of larval supply and recruitment for this coral species. Our results suggest that improving the water quality of river discharges into coastal areas might help to

Cinematographic analysis of bovine embryo development in serum-free oviduct-conditioned medium.

PubMed

Grisart, B; Massip, A; Dessy, F

1994-07-01

Development of bovine embryos produced in vitro from the one-cell to the blastocyst stage in serum-free oviduct-conditioned medium was investigated for 8 days consecutively by time-lapse cinematography. Three movies were analysed (130 embryos). The following observations were made. (1) Development under cine-recording conditions was similar to that in a classical incubator. (2) The highest proportion of embryos at the two-cell, three-four-cell, five-eight-cell, 9-16-cell, morula and blastocyst stages were recorded at 34, 46, 61, 115, 149 and 192 h after insemination, respectively. Cleavage asynchrony between blastomeres within individual embryos started at the two-cell stage. (3) The duration of the first three cell cycles was 35 h, 14 h and 11-62 h, respectively. (4) Detailed analysis of 13 embryos revealed that developmental arrest ('Lag-phase') occurred at the four-cell (1 of 13), five-cell (2 of 13), six-cell (3 of 13), seven-cell (3 of 13) or eight-cell stage (4 of 13); this phase lasted about 59 h. Embryos arrested at the eight-cell stage developed into morula-blastocysts (3 of 4) at a higher rate than did those arrested at earlier stages (2 of 9). (5) The faster the embryos cleaved into early stages (two-cell, three-four-cell and five-eight-cell), the higher the probability that they developed into morula-blastocyst: 70% of the embryos reaching the two-cell stage before 30-31 h after insemination developed into morula-blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of cryopreservation on perinatal outcome after blastocyst- vs cleavage-stage embryo transfer: systematic review and meta-analysis.

PubMed

Alviggi, C; Conforti, A; Carbone, I F; Borrelli, R; de Placido, G; Guerriero, S

2018-01-01

To compare the perinatal outcomes of singleton pregnancies resulting from blastocyst- vs cleavage-stage embryo transfer and to assess whether they differ between fresh and frozen embryo transfer cycles. A systematic review of the literature was carried out using the Scopus, MEDLINE and ISI Web of Science databases with no time restriction. We included only peer-reviewed articles involving humans, in which perinatal outcomes of singleton pregnancies after blastocyst-stage embryo transfer were compared with those after cleavage-stage embryo transfer. Primary outcomes were preterm birth before 37 weeks and low birth weight (< 2500 g). Secondary outcomes were very preterm birth before 32 weeks, very low birth weight (< 1500 g), small-for-gestational-age (SGA), large-for-gestational-age (LGA), perinatal mortality and congenital anomaly. A meta-analysis was performed using a random-effects model. Three subgroups were evaluated: fresh only, frozen only and fresh plus frozen embryo transfer cycles. From a total of 3928 articles identified, 14 were selected for qualitative/quantitative analysis. Significantly higher incidences of preterm birth < 37 weeks (11 studies, n = 106 629 participants; risk ratio (RR), 1.15 (95% CI, 1.05 - 1.25); P = 0.002) and very preterm birth < 32 weeks (seven studies, n = 103 742; RR, 1.16 (95% CI, 1.02-1.31); P = 0.03) were observed after blastocyst- than after cleavage-stage embryo transfer in fresh cycles. However, the risk of preterm and very preterm birth was similar after blastocyst- and cleavage-stage transfers in frozen and fresh plus frozen cycles. Overall effect size analysis revealed fewer SGA deliveries after blastocyst- compared with cleavage-stage transfer in fresh cycles but a similar number in frozen cycles. Conversely, more LGA deliveries were observed after blastocyst- compared with cleavage-stage transfer in frozen cycles (two studies, n = 39 044; RR, 1.18 (95% CI, 1

Toxic effects of magnesium oxide nanoparticles on early developmental and larval stages of zebrafish (Danio rerio).

PubMed

Ghobadian, Mehdi; Nabiuni, Mohammad; Parivar, Kazem; Fathi, Mojtaba; Pazooki, Jamileh

2015-12-01

Magnesium oxide nanoparticles (MgONPs) are used in medicine, manufacturing and food industries. Because of their extensive application in our daily lives, environmental exposure to these nanoparticles is inevitable. The present study examined the effects of MgONPs on zebrafish (Danio rerio) early developmental stages. The results showed that, at different concentrations, MgONPs induced cellular apoptosis and intracellular reactive oxygen species. The hatching rate and survival of embryos decreased in a dose dependent manner. The 96-h LC50 value of MgONPs on zebrafish survival was 428 mg/l and the 48-h EC50 value of MgONPs on zebrafish embryo hatching rate was 175 mg/l. Moreover different types of malformation were observed in exposed embryos. The results demonstrate the toxic effects of MgONPs on zebrafish embryos and emphasize the need for further studies. Copyright © 2015 Elsevier Inc. All rights reserved.

The effects of levetiracetam on neural tube development in the early stage of chick embryos.

PubMed

Guvenc, Yahya; Dalgic, Ali; Billur, Deniz; Karaoglu, Derya; Aydin, Sevim; Daglioglu, Ergun; Ozdol, Cagatay; Nacar, Osman Arikan; Yildirim, Ali Erdem; Belen, Deniz

2013-01-01

This study aimed to investigate the effects of a new generation antiepileptic agent, levetiracetam, on the neural tube development in a chick embryo model that corresponds to the first month of vertebral development in mammals. Forty-five Atabey® breed fertilized chicken eggs with no specific pathogens were randomly divided into 5 groups. All of the eggs were incubated at 37.8±2°C and 60±5 % relative humidity in an incubator. Group A was control group. The other eggs were applied physiological saline and drugs at a volume of 10 μL by the in ovo method at the 28th hour of the incubation period. Group B was given distilled water; Group C, physiological saline; Group D, Levetiracetam (L8668) at a dose equivalent to the treatment dose for humans (10 mg/ kg), and Group E, Levetiracetam (L8668) at a dose of 10 times the treatment dose. The embryos in all of the groups were removed from the shells at the 48th hour and morphologically and histologically evaluated. Of the 45 embryos incubated, neural tubes of 41 were closed and the embryos displayed normal development. Levetiracetam, at a dose equivalent to human treatment dose and 10 times the treatment dose, was shown not to cause neural tube defects in chick embryos.

Progesterone is critical for the development of mouse embryos.

PubMed

Zhang, Cong; Murphy, Bruce D

2014-08-01

Infertility affects approximately 10-15 % of reproductive-aged couples, and embryo loss due to preimplantation death is common to many mammals. Previous studies showed that a complex series of interactive molecular events are associated with this process, especially hormones (progesterone and estrogens) and growth factors, and are important for the cleavage and differentiation of the blastocysts. Yet, the mechanism of preimplantation embryo development is unclear. Using conditional knockout mice (CKO), we showed the development of blastocyst is tightly controlled by the level of progesterone (P4); furthermore, we found that the time when P4 should increase is also crucial for the formation of blastocysts. In CKO mice whose Lrh1 (liver receptor homolog 1) is deleted under the expression of Cre recombinase driven by progesterone receptor promoter, which reduced P4 synthesis, few of their embryos can reach blastocyst stage. When these CKO mice were supplied with P4 in the afternoon of dpc 1 (day post copulation), most of the embryos can form blastocysts; when CKO mice were supplied with P4 from the morning of dpc1, one-third of the embryos can reach blastocyst stage; however, the supplement of P4 in the morning of dpc 2 made very few of the embryos become blastocysts. We conclude that early exposure to P4 is essential for timely progression of early embryogenesis in the mouse.

Ocean Warming Enhances Malformations, Premature Hatching, Metabolic Suppression and Oxidative Stress in the Early Life Stages of a Keystone Squid

PubMed Central

Rosa, Rui; Pimentel, Marta S.; Boavida-Portugal, Joana; Teixeira, Tatiana; Trübenbach, Katja; Diniz, Mário

2012-01-01

Background The knowledge about the capacity of organisms’ early life stages to adapt to elevated temperatures is very limited but crucial to understand how marine biota will respond to global warming. Here we provide a comprehensive and integrated view of biological responses to future warming during the early ontogeny of a keystone invertebrate, the squid Loligo vulgaris. Methodology/Principal Findings Recently-spawned egg masses were collected and reared until hatching at present day and projected near future (+2°C) temperatures, to investigate the ability of early stages to undergo thermal acclimation, namely phenotypic altering of morphological, behavioural, biochemical and physiological features. Our findings showed that under the projected near-future warming, the abiotic conditions inside the eggs promoted metabolic suppression, which was followed by premature hatching. Concomitantly, the less developed newborns showed greater incidence of malformations. After hatching, the metabolic burst associated with the transition from an encapsulated embryo to a planktonic stage increased linearly with temperature. However, the greater exposure to environmental stress by the hatchlings seemed to be compensated by physiological mechanisms that reduce the negative effects on fitness. Heat shock proteins (HSP70/HSC70) and antioxidant enzymes activities constituted an integrated stress response to ocean warming in hatchlings (but not in embryos). Conclusions/Significance The stressful abiotic conditions inside eggs are expected to be aggravated under the projected near-future ocean warming, with deleterious effects on embryo survival and growth. Greater feeding challenges and the lower thermal tolerance limits of the hatchlings are strictly connected to high metabolic demands associated with the planktonic life strategy. Yet, we found some evidence that, in the future, the early stages might support higher energy demands by adjusting some cellular functional properties

Ocean Acidification Effects on the Early Life-Stages of Commercially Important Flatfish of the Northeast USA

NASA Astrophysics Data System (ADS)

Chambers, R. C.; Habeck, E. A.; Candelmo, A. C.; Poach, M.; Wieczorek, D.; Phelan, B.; Caldarone, E.; Cooper, K. R.

2012-12-01

The limited available evidence about effects on finfish of high CO2 levels and acidification of our oceans suggests that effects will differ across fish species, be subtle, and interact with other stressors. A carefully planned, experimental framework was developed to cast an extensive yet strategic inferential net. Three key elements of our approach are the use of 1) multiple marine finfish species of relevance to the northeastern USA that differ in their ecologies including spawning season and habitat of early life-stages; 2) a wide yet realistic range of environmental conditions (i.e., concurrent manipulation of CO2 levels and water temperatures), and 3) a diverse set of response variables related to fish sensitivity to elevated CO2 levels, water temperatures, and their interactions. The response variable set reflects fish condition, fitness, and likelihood of recruitment, and includes measures of viability, physiology, histopathology, growth, development, and behavior expressed during fish early life-stages (i.e., gametes, embryos, and larvae). Early life-stages were chosen due to the anticipation of their vulnerability to acid-base challenges in their environment. To date, factorial experiments have been implemented on summer flounder (Paralichthys dentatus) and winter flounder (Pseudopleuronectes americanus). Initial results reveal survival of summer flounder embryos is compromised by pH < 7.7 (CO2 > 790 ppm). These results were similar across offspring groups (i.e., embryos from different parents). Winter flounder are larger at hatching when exposed to high CO2 levels in the coolest environment implemented in our experiments (range 4 to 10 ○C). Further responses of advanced larvae of both flounder species are currently being assessed for evidence of other whole body, component organ, and biochemical impairment. This study will aid researchers and resource managers in identifying species types, life-stages, and biotic responses that are most sensitive to

Comparative responses to endocrine disrupting compounds in early life stages of Atlantic salmon, Salmo salar

USGS Publications Warehouse

Duffy, Tara A.; Iwanowicz, Luke R.; McCormick, Stephen D.

2014-01-01

Atlantic salmon (Salmo salar) are endangered anadromous fish that may be exposed to feminizing endocrine disrupting compounds (EDCs) during early development, potentially altering physiological capacities, survival and fitness. To assess differential life stage sensitivity to common EDCs, we carried out short-term (four day) exposures using three doses each of 17α-ethinylestradiol (EE2), 17β-estradiol (E2), and nonylphenol (NP) on four early life stages; embryos, yolk-sac larvae, feeding fry and one year old smolts. Differential response was compared using vitellogenin (Vtg, a precursor egg protein) gene transcription. Smolts were also examined for impacts on plasma Vtg, cortisol, thyroid hormones (T4/T3) and hepatosomatic index (HSI). Compound-related mortality was not observed in any life stage, but Vtg mRNA was elevated in a dose-dependent manner in yolk-sac larvae, fry and smolts but not in embyos. The estrogens EE2 and E2 were consistently stronger inducers of Vtg than NP. Embryos responded significantly to the highest concentration of EE2 only, while older life stages responded to the highest doses of all three compounds, as well as intermediate doses of EE2 and E2. Maximal transcription was greater for fry among the three earliest life stages, suggesting fry may be the most responsive life stage in early development. Smolt plasma Vtg was also significantly increased, and this response was observed at lower doses of each compound than was detected by gene transcription suggesting this is a more sensitive indicator at this life stage. HSI was increased at the highest doses of EE2 and E2 and plasma T3 decreased at the highest dose of EE2. Our results indicate that all life stages after hatching are potentially sensitive to endocrine disruption by estrogenic compounds and that physiological responses were altered over a short window of exposure, indicating the potential for these compounds to impact fish in the wild.

The role of Mixer in patterning the early Xenopus embryo.

PubMed

Kofron, Matt; Wylie, Chris; Heasman, Janet

2004-05-01

The transcription factor VegT, is required in early Xenopus embryos for the formation of both the mesoderm and endoderm germ layers. Inherited as a maternal mRNA localized only in vegetal cells, VegT activates the transcription of a large number of transcription factors, as well as signaling ligands that induce cells in the vegetal mass to form endoderm, and the marginal zone to form mesoderm. It is important now to understand the extent to which transcription factors downstream of VegT play individual, or overlapping, roles in the specification and patterning of the endoderm and mesoderm. In addition, it is important to understand the mechanism that specifies the boundary between endoderm and mesoderm. One of the downstream targets of VegT, the homeodomain protein Mixer, is expressed at high levels at the mesoderm/endoderm boundary at the late blastula stage. We therefore examined its functions by blocking its translation using morpholino oligos. In Mixer-depleted embryos, the expression of many signaling ligands and transcription factors was affected. In particular, we found that the expression of several genes, including several normally expressed in mesoderm, was upregulated. Functional assays of Mixer-depleted vegetal cells showed that they have increased mesoderm-inducing activity. This demonstrates that Mixer plays an essential role in controlling the amount of mesoderm induction by the vegetal cells.

Morphological embryo selection: an elective single embryo transfer proposal.

PubMed

Déniz, Francisco Parera; Encinas, Carlos; Fuente, Jorge La

2018-03-01

To describe a patient selection method for elective single embryo transfer (eSET), emphasizing inclusion criteria and results. This retrospective study included all cases seen in a private clinic between June 2011 and December 2016, in La Paz, Bolivia (3600 meters above sea level). Elective single embryo transfer was the method of choice in 34 IVF/ICSI cycles, all in the blastocyst stage. Gardner's blastocyst classification criteria were used. Between the two stages of the study (July 2015), each embryo grade implantation rate was recalculated, which led to the expansion of the inclusion criteria. The clinical pregnancy rate of the 34 cases in the first transfer group was 55.9% (19/34). Twin or multiple pregnancies did not occur. The cumulative pregnancy rate to date is 64% [(19+3)/34]. The first stage comprised 2.56% (12/468) of the patients offered elective single embryo transfers; the implantation rate was 58.3% (7/12). In the second stage, 14.29% (22/154) of the patients were eligible, and the implantation rate was 54.55% (12/22). The implementation of an eSET program based on in-depth morphological embryo assessment combined with the calculation of the implantation potential of each embryo grade led to acceptable clinical outcomes and fewer multiple pregnancies in patients transferred two embryos. Each clinic should be aware of the implantation rates of each embryo grade in its own setting.

Morphological embryo selection: an elective single embryo transfer proposal

PubMed Central

Déniz, Francisco Parera; Encinas, Carlos; Fuente, Jorge La

2018-01-01

Objective To describe a patient selection method for elective single embryo transfer (eSET), emphasizing inclusion criteria and results. Methods This retrospective study included all cases seen in a private clinic between June 2011 and December 2016, in La Paz, Bolivia (3600 meters above sea level). Elective single embryo transfer was the method of choice in 34 IVF/ICSI cycles, all in the blastocyst stage. Gardner's blastocyst classification criteria were used. Between the two stages of the study (July 2015), each embryo grade implantation rate was recalculated, which led to the expansion of the inclusion criteria. Results The clinical pregnancy rate of the 34 cases in the first transfer group was 55.9% (19/34). Twin or multiple pregnancies did not occur. The cumulative pregnancy rate to date is 64% [(19+3)/34]. The first stage comprised 2.56% (12/468) of the patients offered elective single embryo transfers; the implantation rate was 58.3% (7/12). In the second stage, 14.29% (22/154) of the patients were eligible, and the implantation rate was 54.55% (12/22). Conclusion The implementation of an eSET program based on in-depth morphological embryo assessment combined with the calculation of the implantation potential of each embryo grade led to acceptable clinical outcomes and fewer multiple pregnancies in patients transferred two embryos. Each clinic should be aware of the implantation rates of each embryo grade in its own setting. PMID:29338137

Early somatic embryo induction events in alfalfa callus cultures. [Medicago sativa

DOE Office of Scientific and Technical Information (OSTI.GOV)

El-Bakry, A.A.; Hildebrand, D.F.

1987-04-01

High and low regenerating alfalfa Medicago sativa L. cv Regen S full sibs were isolated from a callus culture screen on modified Blaydes medium. The average number of embryos per ovary were thirty and zero for the high and low genotypes respectively after six weeks in culture. Proembryonic cell masses (4-8 celled) were observed after 4-5 days in culture and maximum meristematic activity was at 6-7 days in culture, for the high regenerating genotypes. Well formed globular embryos, both epidermal and subepidermal in origin, were observed after 2 weeks is culture. Samples in culture for 3, 6 and 14 daysmore » from the high and low regenerating genotypes were radiolabeled in vivo with /sup 35/S-methionine and run both on one and two dimension gels. The results will be discussed in relation to differences in proteins between the high and low regenerating genotypes at the stage of maximum meristematic activity (day 6) and differences occurring relative to the appearance of globular stage embryos (day 14) will be presented.« less

DYZ1 copy number variation, Y chromosome polymorphism and early recurrent spontaneous abortion/early embryo growth arrest.

PubMed

Yan, Junhao; Fan, Lingling; Zhao, Yueran; You, Li; Wang, Laicheng; Zhao, Han; Li, Yuan; Chen, Zi-Jiang

2011-12-01

To find the association between recurrent spontaneous abortion (RSA)/early embryo growth arrest and Y chromosome polymorphism. Peripheral blood samples of the male patients of big Y chromosome, small Y chromosome and other male patients whose partners suffered from unexplained RSA/early embryo growth arrest were collected. PCR and real-time fluorescent quantitative PCR were used to test the deletion and the copy number variation of DYZ1 region in Y chromosome of the patients. A total of 79 big Y chromosome patients (48 of whose partners suffered from RSA or early embryo growth arrest), 7 small Y chromosome patients, 106 other male patients whose partners had suffered from unexplained RSA or early embryo growth arrest, and 100 normal male controls were enrolled. There was no fraction deletion of DYZ1 detected both in big Y patients and in normal men. Of RSA patients, 1 case showed deletion of 266bp from the gene locus 25-290bp, and 2 cases showed deletion of 773bp from 1347 to 2119bp. Of only 7 small Y chromosome patients, 2 cases showed deletion of 266bp from 25 to 290bp, and 4 cases showed deletion of 773bp from 1347 to 2119bp and 275bp from 3128 to 3420bp. The mean of DYZ1 copies was 3900 in normal control men; the mean in big Y patients was 5571, in RSA patients was 2655, and in small Y patients was 1059. All of the others were significantly different (P<0.01) compared with normal control men, which meant that DYZ1 copy number in normal control men was less than that of big Y chromosome patients, and was more than that of unexplained early RSA patients and small Y patients. The integrity and copy number variation of DYZ1 are closely related to the Y chromosome length under microscope. The cause of RSA/early embryo growth arrest in some couples may be the increase (big Y patients) or decrease of DYZ1 copy number in the husbands' Y chromosome. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Effect of thiacloprid on early life stages of common carp (Cyprinus carpio).

PubMed

Velisek, Josef; Stara, Alzbeta

2018-03-01

Toxicity of the neonicotinoid pesticide thiacloprid to embryos and larvae of common carp Cyprinus carpio was assessed under laboratory conditions. Carp embryos and larvae were exposed to thiacloprid at four concentrations: 4.5 (environmental concentration), 45, 225, and 450 μg/L for 35 days and compared to a non-treated control group. The effects on lipid peroxidation, antioxidant enzyme levels (superoxide dismutase, glutathione reductase, catalase, glutathione S-transferase, and reduced glutathione), mortality, growth, development, and histopathogenesis were assessed. Thiacloprid at tested concentrations were not associated with negative effects on hatching, behaviour, embryo viability, or Fulton's condition factor. Carp exposed to 45 μg/L thiacloprid showed lower weight and length compared to controls. Thiacloprid at 225 and 450 μg/L delayed development and led to lower weight and length of carp. Significantly lower superoxide dismutase and glutathione reductase activity was observed at all tested concentrations of thiacloprid compared with the control group. Chronic thiacloprid exposure of early-life stages of carp affected ontogeny and growth rate, and inhibited antioxidant capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cryotolerance of Day 2 or Day 6 in vitro produced ovine embryos after vitrification by Cryotop or Spatula methods.

PubMed

Dos Santos Neto, P C; Vilariño, M; Barrera, N; Cuadro, F; Crispo, M; Menchaca, A

2015-02-01

This study was conducted to evaluate the cryotolerance of in vitro produced ovine embryos submitted to vitrification at different developmental stages using two methods of minimum volume and rapid cooling rate. Embryos were vitrified at early stage (2 to 8-cells) on Day 2 or at advanced stage (morulae and blastocysts) on Day 6 after in vitro fertilization. Vitrification procedure consisted of the Cryotop (Day 2, n=165; Day 6, n=174) or the Spatula method (Day 2, n=165; Day 6, n=175). Non vitrified embryos were maintained in in vitro culture as a control group (n=408). Embryo survival was determined at 3h and 24h after warming, development and hatching rates were evaluated on Day 6 and Day 8 after fertilization, and total cell number was determined on expanded blastocysts. Embryo survival at 24h after warming increased as the developmental stage progressed (P<0.05) and was not affected by the vitrification method. The ability for hatching of survived embryos was not affected by the stage of the embryos at vitrification or by the vitrification method. Thus, the proportion of hatching from vitrified embryos was determined by the survival rate and was lower for Day 2 than Day 6 vitrified embryos. The percentage of blastocysts on Day 8 was lower for the embryos vitrified on Day 2 than Day 6 (P<0.05), and was lower for both days of vitrification than for non-vitrified embryos (P<0.05). No interaction of embryo stage by vitrification method was found (P=NS) and no significant difference was found in the blastocyst cell number among vitrified and non-vitrified embryos. In conclusion, both methods using minimum volume and ultra-rapid cooling rate allow acceptable survival and development rates in Day 2 and Day 6 in vitro produced embryos in sheep. Even though early stage embryos showed lower cryotolerance, those embryos that survive the vitrification-warming process show high development and hatching rates, similar to vitrification of morulae or blastocysts. Copyright �

Effects of a commonly used glyphosate-based herbicide formulation on early developmental stages of two anuran species.

PubMed

Wagner, Norman; Müller, Hendrik; Viertel, Bruno

2017-01-01

Environmental contamination, especially due to the increasing use of pesticides, is suggested to be one out of six main reasons for the global amphibian decline. Adverse effects of glyphosate-based herbicides on amphibians have been already discussed in several studies with different conclusions, especially regarding sublethal effects at environmentally relevant concentrations. Therefore, we studied the acute toxic effects (mortality, growth, and morphological changes) of the commonly used glyphosate-based herbicide formulation Roundup® UltraMax on early aquatic developmental stages of two anuran species with different larval types (obligate vs. facultative filtrating suspension feeders), the African clawed frog (Xenopus laevis) and the Mediterranean painted frog (Discoglossus pictus). While X. laevis is an established anuran model organism in amphibian toxicological studies, we aim to establish D. pictus as another model for species with facultative filtrating larvae. A special focus of the present study lies on malformations in X. laevis embryos, which were investigated using histological preparations. In general, embryos and larvae of X. laevis reacted more sensitive concerning lethal effects compared to early developmental stages of D. pictus. It was suggested, that especially the different morphology of their filter apparatus and the higher volume of water pumped through the buccopharynx of X. laevis larvae lead to higher exposure to the formulation. The test substance induced similar lethal effects in D. pictus larvae as it does in the teleost standard test organism used in pesticide approval, the rainbow trout (Oncorhynchus mykiss), whereas embryos of both species are apparently more tolerant and, conversely, X. laevis larvae about two times more sensitive. In both species, early larvae always reacted significantly more sensitive than embryos. Exposure to the test substance increased malformation rates in embryos of both species in a concentration

Climatic factors affecting quantity and quality grade of in vivo derived embryos of cattle.

PubMed

Chinchilla-Vargas, Josué; Jahnke, Marianna M; Dohlman, Tyler M; Rothschild, Max F; Gunn, Patrick J

2018-05-01

The present study investigated the effects of climatic variables on the quality grade and quantity of in vivo derived cattle embryos in the Midwestern United States. Climatic information included greatest and least daily temperature, average daily wind speed and average temperature-humidity index for each of the 765 records. The response variables included the number of ovarian structures, viable embryos, quality grade 1 embryos, quality grade 2 embryos, quality grade 3 embryos, freezable embryos (sum of quality grade 1 and quality grade 2 embryos), transferable embryos (sum of quality grade 1-3 embryos), degenerate embryos and unfertilized ova. Measures for variables among the breeds of donors and sires grouped by geographical origin were compared. A negative effect of greater temperatures during the early embryonic development stage tended (P < 0.10) to be associated with a decrease in the quality of embryos recovered. Interestingly, the greater the Temperature-Humidity Index (THI) during the early ovarian antral follicular development stage 40-45 days prior to ovulation was associated with a tendency for greater numbers of total number of freezable and transferable embryos recovered per uterine flushing (P < 0.10). Increased wind speed at the early antral follicular phase 40-45 days prior to ovulation was associated with an increase in the percentage of quality grade 1 embryos recovered (P < 0.05). Wind speed during the estrous synchronization period was also associated with a lesser number of embryos recovered (P < 0.05). This retrospective study confirms that climatic variables have significant effects on the in vivo production of cattle embryos and that wind speed should be considered in future analyses of factors affecting embryo quality. Copyright © 2018 Elsevier B.V. All rights reserved.

Esrrb-Cre Excises loxP-Flanked Alleles in Early Four-Cell Embryos

PubMed Central

Kim, Suyeon; Shaffer, Benjamin; Simerly, Calvin R.; Richard Chaillet, J.; Barak, Yaacov

2015-01-01

Among transgenic mice with ubiquitous Cre recombinase activity, all strains to date excise loxP-flanked (floxed) alleles, either at or before the zygote stage or at nondescript stages of development. This manuscript describes a new mouse strain, in which Cre recombinase, integrated into the Esrrb locus, efficiently excises floxed alleles in pre-implantation embryos at the onset of the four-cell stage. By enabling inactivation of genes only after the embryo has undergone two cleavages, this strain should facilitate in vivo studies of genes with essential gametic or zygotic functions. In addition, this study describes a new, highly pluripotent hybrid C57BL/6J × 129S1/SvImJ mouse embryonic stem cell line, HYB12, in which this knock-in and additional targeted alleles have been generated. PMID:26663459

Precision matters for position decoding in the early fly embryo

NASA Astrophysics Data System (ADS)

Petkova, Mariela D.; Tkacik, Gasper; Wieschaus, Eric F.; Bialek, William; Gregor, Thomas

Genetic networks can determine cell fates in multicellular organisms with precision that often reaches the physical limits of the system. However, it is unclear how the organism uses this precision and whether it has biological content. Here we address this question in the developing fly embryo, in which a genetic network of patterning genes reaches 1% precision in positioning cells along the embryo axis. The network consists of three interconnected layers: an input layer of maternal gradients, a processing layer of gap genes, and an output layer of pair-rule genes with seven-striped patterns. From measurements of gap gene protein expression in hundreds of wild-type embryos we construct a ``decoder'', which is a look-up table that determines cellular positions from the concentration means, variances and co-variances. When we apply the decoder to measurements in mutant embryos lacking various combinations of the maternal inputs, we predict quantitative changes in the output layer such as missing, altered or displaced stripes. We confirm these predictions by measuring pair-rule expression in the mutant embryos. Our results thereby show that the precision of the patterning network is biologically meaningful and a necessary feature for decoding cell positions in the early fly embryo.

Mitotic wavefronts mediated by mechanical signaling in early Drosophila embryos

NASA Astrophysics Data System (ADS)

Kang, Louis; Idema, Timon; Liu, Andrea; Lubensky, Tom

2013-03-01

Mitosis in the early Drosophila embryo demonstrates spatial and temporal correlations in the form of wavefronts that travel across the embryo in each cell cycle. This coordinated phenomenon requires a signaling mechanism, which we suggest is mechanical in origin. We have constructed a theoretical model that supports nonlinear wavefront propagation in a mechanically-excitable medium. Previously, we have shown that this model captures quantitatively the wavefront speed as it varies with cell cycle number, for reasonable values of the elastic moduli and damping coefficient of the medium. Now we show that our model also captures the displacements of cell nuclei in the embryo in response to the traveling wavefront. This new result further supports that mechanical signaling may play an important role in mediating mitotic wavefronts.

Expression profiling of the mouse early embryo: Reflections and Perspectives

PubMed Central

Ko, Minoru S. H.

2008-01-01

Laboratory mouse plays important role in our understanding of early mammalian development and provides invaluable model for human early embryos, which are difficult to study for ethical and technical reasons. Comprehensive collection of cDNA clones, their sequences, and complete genome sequence information, which have been accumulated over last two decades, have provided even more advantages to mouse models. Here the progress in global gene expression profiling in early mouse embryos and, to some extent, stem cells are reviewed and the future directions and challenges are discussed. The discussions include the restatement of global gene expression profiles as snapshot of cellular status, and subsequent distinction between the differentiation state and physiological state of the cells. The discussions then extend to the biological problems that can be addressed only through global expression profiling, which include: bird’s-eye view of global gene expression changes, molecular index for developmental potency, cell lineage trajectory, microarray-guided cell manipulation, and the possibility of delineating gene regulatory cascades and networks. PMID:16739220

INCREASED APOPTOSIS IN ORGANOGENESIS-STAGED MOUSE EMBRYOS INDUCED BY DISINFECTION BY-PRODUCTS

EPA Science Inventory

Increased apoptosis in organogenesis-staged mouse embryos induced by disinfection by-products. Sid Hunter1,2, Ellen Rogers1 and Keith Ward2, 1 Developmental Biology Branch, Reproductive Toxicology Division, NHEERL, US EPA, RTP, NC; 2 Curriculum in Toxicology, UNC Chapel Hill, Cha...

Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients

PubMed Central

2012-01-01

Objectives This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. Study design We compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Results Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05) and implantation rate (31.8% vs 31.2%, p>0.05) in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05). Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8%) and implantation rate (47%) compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively). For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p<0.05). However, the cumulative multiple pregnancy rates showed no significant difference between the two groups. Conclusions In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic

Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients.

PubMed

Tong, Guo Qing; Cao, Shan Ren; Wu, Xun; Zhang, Jun Qiang; Cui, Ji; Heng, Boon Chin; Ling, Xiu Feng

2012-10-05

This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. We compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05) and implantation rate (31.8% vs 31.2%, p>0.05) in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05). Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8%) and implantation rate (47%) compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively). For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p<0.05). However, the cumulative multiple pregnancy rates showed no significant difference between the two groups. In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic Chinese patients.

Timing of The First Zygotic Cleavage Affects Post-Vitrification Viability of Murine Embryos Produced In Vivo

PubMed Central

Jusof, Wan-Hafizah Wan; Khan, Nor-Ashikin Mohamed Noor; Rajikin, Mohd Hamim; Satar, Nuraliza Abdul; Mustafa, Mohd-Fazirul; Jusoh, Norhazlin; Dasiman, Razif

2015-01-01

Background Timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early (EC) have been shown to exhibit higher develop- mental viability compared to those that cleaved at a later period (LC). However, the vi- ability of EC embryos in comparison to LC embryos after vitrification is unknown. The present study aims to investigate the post-vitrification developmental viability of murine EC versus LC embryos. Materials and Methods In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test. Results A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001). Conclusion Vitrified EC embryos are more vitrification tolerant than LC embryos. Prese- lection of EC embryos may be used as a tool for selection of embryos that exhibit higher developmental competence after vitrification. PMID:26246881

Effect of chronic copper and pentachlorophenol exposure to early life stages of Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fort, D.J.; Stover, E.L.

1995-12-31

An evaluation of the effects of low-level copper and pentachlorophenol exposure on various early life stages of the South African clawed frog, Xenopus laevis was performed using stage-specific and long-term continuous exposures. Stage-specific exposure experiments were conducted such that separate subsets of embryos and larvae from the same clutch were exposed to two toxicants, copper and pentachlorophenol, from 0 d to 4 d (standard Frog Embryo Teratagenesis Assay Xenopus [FETAX]), 4 d to 8 d, 8 d to 12 d, and 12 d to 16 d. Results from two separate concentration-response experiments indicated that sensitivity to either toxicant increased inmore » each successive time period. Continuous exposure studies conducted for 60 to 75 days indicated that copper, but not pentachlorophenol induced reduction deficiency malformations of the hind limb at concentrations as low as 0.05 mg/L. Pentachlorophenol concentrations as low as 0.5/{micro}g/L inhibited tail resorption. However, copper did not adversely affect the process of tail resorption. These results indicated that studies evaluating longer-term developmental processes are important in ecological hazard evaluation.« less

[Effect of TGF-beta1 on embryo implantation and development in mice in vitro].

PubMed

Luo, Shan; Yin, Hai-ning; Li, Shang-wei

2010-03-01

To investigate the role of TGF-beta1 in embryo implantation and development in vitro in mice. Mouse embryos at 2-cell stage were cultured in the media of M16 with exposure to different levels of TGF-beta1 (0, 1, 10 and 50 ng/mL). The percentage of embryos reaching fixed stages (early blastocyst, expanding blastocyst and hatched blastocyst) was monitored 68 h and 92 h after the culture. The expanding blastocys cultured for 68 h in M16 without TGF-beta1 and those with 10 ng/mL of TGF-beta1 were transferred to pseudopregnant mice. On the 6th day post transfer, the successful rates of implantation were counted. The level of IL-10/IFN-gamma in the serum and maternal-fetus interface of the mice was detected by ELISA on the 6th day post transfer. TGF-beta1 improved embryo growth in vitro. TGF-beta1 at a level of 10 ng/mL had the maximum impact, with 15.6%, 68.09%, 1.42% of embryos reaching early, expanding, and hatched stage, respectively, 68 h after culture, and 6.38%, 28.37%, 53.19% of embryos reaching early, expanding, and hatched stage, respectively, 92 h after culture. The promoting effect declined when TGF-beta1 reached 50 ng/mL. The successful rate of implantation of embryos cultured in M16 with TGF-beta1 was significantly higher than those cultured in M16 without TGF-beta1 (35. 2% vs. 17.19%, P < 0.05). The embryos cultured in M16 with TGF-beta1 had significantly lower level of IFN-gamma in the maternal-fetus interface than those cultured in M16 without TGF-beta1 [(30.89 +/- 11.31) pg/mL vs. (43.23 +/- 18. 09) pg/mL, P < 0.053. TGF-beta1 at an appropriate dose improves embryo implantation in mice in vitro. The mechanism may involve the improvement of the quality of embryos and their development, and decrease of IFN-gamma synthesis in maternal-fetal interface, a chemical that could cause Th2 bias.

Stage-dependent teratogenic and lethal effects exerted by ultraviolet B radiation on Rhinella (Bufo) arenarum embryos.

PubMed

Castañaga, Luis A; Asorey, Cynthia M; Sandoval, María T; Pérez-Coll, Cristina S; Argibay, Teresa I; Herkovits, Jorge

2009-02-01

The adverse effects of ultraviolet B radiation from 547.2 to 30,096 J/m2 on morphogenesis, cell differentiation, and lethality of amphibian embryos at six developmental stages were evaluated from 24 up to 168 h postexposure. The ultraviolet B radiation lethal dose 10, 50, and 90 values were obtained for all developmental stages evaluated. The lethal dose 50 values, considered as the dose causing lethality in the 50% of the organisms exposed, in J/m2 at 168 h postexposure, ranged from 2,307 to 18,930; gill circulation and blastula were the most susceptible and resistant stages, respectively. Ultraviolet B radiation caused malformations in all developmental stages but was significantly more teratogenic at the gill circulation and complete operculum stages. Moreover, at the gill circulation stage, even the lowest dose (547.2 J/m2) resulted in malformations to 100% of embryos. The most common malformations were persistent yolk plug, bifid spine, reduced body size, delayed development, asymmetry, microcephaly and anencephaly, tail and body flexures toward the irradiated side, agenesia or partial gill development, abnormal pigment distribution, and hypermotility. The stage-dependent susceptibility to ultraviolet B radiation during amphibian embryogenesis could be explained in the framework of evoecotoxicology, considering ontogenic features as biomarkers of environmental signatures of living forms ancestors during the evolutionary process. The stage-dependent susceptibility to ultraviolet B radiation on Rhinella (Bufo) arenarum embryos for both lethal and teratogenic effects could contribute to a better understanding of the role of the increased ultraviolet B radiation on worldwide amphibian populations decline.

Glucocorticoid teratogenesis in mouse whole embryo culture.

PubMed

Pratt, R M; Perry, E L; Chapman, L M; Goulding, E H

1984-08-01

Glucocorticoids, such as triamcinolone acetonide (TAC-A) and triamcinolone hexacetonide (TAC-HA), are potent inducers of cleft palate in vivo in various mouse strains when administered on day 11 of gestation, whereas they are poor or ineffective inducers of cleft lip when given on day 7. The purpose of the present study was to determine whether glucocorticoids are capable of interfering with early embryonic development in culture. CD-1 mouse embryos were cultured for 48 hours starting either on day 8 (plug day 0) with the embryo inside the yolk sac, or on day 10 with the embryo exteriorized from its functional yolk sac. At the end of the culture period, embryos were examined grossly for malformations and biochemically for altered DNA and protein levels. With the day 8 cultures, TAC-A produced a dose-dependent inhibition of growth along with malformations consisting of cardiac irregularities, abnormal rotation, and irregular neural tube closure. With the day 10 cultures, these malformations were not observed, presumably due to the advanced stage of development when the embryos were exposed to TAC-A; however, TAC-A did produce growth inhibition along with cleft lip. When TAC-HA was administered in vivo to pregnant donor females on day 7, in combination with TAC-A added on day 10 to the culture medium, there was a dramatic increase in the frequency of cleft lip along with other alterations in craniofacial appearance. Our results demonstrate that glucocorticoids are capable of directly affecting embryonic growth and development during the early stages of organogenesis.

Estrogen receptor-mediated effects of a xenoestrogen, bisphenol A, on preimplantation mouse embryos.

PubMed

Takai, Y; Tsutsumi, O; Ikezuki, Y; Hiroi, H; Osuga, Y; Momoeda, M; Yano, T; Taketani, Y

2000-04-21

The effects of bisphenol A, a xenoestrogen widely used in industry and dentistry, were studied in early preimplantation mouse embryos. Two-cell mouse embryos were cultured with 100 pM to 100 microM bisphenol A with or without 100 nM tamoxifen and evaluated at 24-h intervals for their development to eight-cell and blastocyst stages. At 72 h, blastocysts were cultured for another 48 h without bisphenol A, and surface areas of trophoblast spread were measured. At 24 h, more embryos exposed to 3 nM bisphenol A than to controls had reached the eight-cell stage. At 48 h, more embryos exposed to 1 nM and 3 nM bisphenol A than to controls had become blastocysts. At 100 microM, bisphenol A decreased frequency of development to blastocysts. Tamoxifen counteracted both stimulatory and inhibitory effects of bisphenol A on blastocyst formation. Although bisphenol A did not alter blastocyst morphology or cell number, early exposure to 100 microM bisphenol A increased subsequent trophoblast areas. These findings suggest that bisphenol A may not only effect early embryonic development via estrogen receptors even at low, environmentally relevant doses, but also exert some late effects on subsequent development of these embryos. Copyright 2000 Academic Press.

Transcriptome Analysis of Honeybee (Apis Mellifera) Haploid and Diploid Embryos Reveals Early Zygotic Transcription during Cleavage

PubMed Central

Pires, Camilla Valente; Freitas, Flávia Cristina de Paula; Cristino, Alexandre S.; Dearden, Peter K.; Simões, Zilá Luz Paulino

2016-01-01

In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development. PMID:26751956

Impacts of 17beta-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio).

PubMed

Brion, F; Tyler, C R; Palazzi, X; Laillet, B; Porcher, J M; Garric, J; Flammarion, P

2004-06-24

Zebrafish (Danio rerio) were exposed for 3 weeks to low concentrations of estradiol including environmentally relevant concentrations (5, 25 and 100 ng/l), encompassing either their embryo-larvae (from fertilization to 21 day post-fertilization (dpf)), juvenile (from 21 to 42 dpf) or adult life stages (>200 dpf) with a view to investigating the most sensitive life stage of the zebrafish to 17beta-estradiol (E2). At all sampling points, whole-body vitellogenin concentrations and gonadal development were analyzed in order to investigate the effects of estrogen exposure on these endpoint in the zebrafish. In the adult stage, additional endpoints were measured including secondary sexual characteristics (manifestation of the uro-genital papillae (UGP) in males), gonadal growth (the gonado-somatic index (GSI)) and sex ratio. For all the different life stage exposures, reproductive performance of the F0 generation was assessed (egg production) and survival and development of the F1 embryo-larvae. Exposure to low concentrations of E2 resulted in vitellogenin induction whatever the life stage exposed but these effects were reversible after depuration. The effective concentration for vitellogenin induction in zebrafish early life stages was 100 ng E2/l, and in adult male zebrafish the effective concentration for vitellogenin induction (between 5 and 25 ng/l) was lower than for the early life stage fish. Exposure to E2 prior to (from fertilization to 21 dpf) and during the time of sex differentiation (from 21 to 42 dpf) also caused disruptions in the process of sexual differentiation (resulting in formation of a retrogonadal cavity in presumptive male, germ cell development and leading to a significant change of the sex ratio towards the female sex at the dose of 100 ng E2/l for the fish exposure as embryo-larvae) and altered patterns of egg production in the subsequent adults. Exposure of adult fish to E2 resulted in a modification of the secondary sexual characteristic in

Behavioral thermoregulation by turtle embryos

PubMed Central

Du, Wei-Guo; Zhao, Bo; Chen, Ye; Shine, Richard

2011-01-01

Mobile ectothermic animals can control their body temperatures by selecting specific thermal conditions in the environment, but embryos—trapped within an immobile egg and lacking locomotor structures—have been assumed to lack that ability. Falsifying that assumption, our experimental studies show that even early stage turtle embryos move within the egg to exploit small-scale spatial thermal heterogeneity. Behavioral thermoregulation is not restricted to posthatching life and instead may be an important tactic in every life-history stage. PMID:21606350

Development of Monozygotic Twin Mouse Embryos from the Time of Blastomere Separation at the Two-Cell Stage to Blastocyst1

PubMed Central

Katayama, Mika; Ellersieck, Mark R.; Roberts, R. Michael

2010-01-01

The development of blastomeres separated from two-cell stage murine embryos has been compared. Blastomeres were removed from the zona pellucida (ZP) and cultured individually; the twin embryos were compared during their progression to blastocyst in terms of development rate, cell number, morphology, conformation at the four-cell stage, and CDX2 and POU5F1 (also known as OCT4) expression. In general, twin embryos, whether obtained from superovulated or normally bred dams, displayed comparable cell numbers as they advanced. They formed morulae and blastocysts more or less synchronously with each other and with control embryos, although possessing about half of the latter's cell number. Despite this apparent synchrony, the majority of twin blastocysts differed in terms of their relative complements of POU5F1+/CDX2− cells, which represent inner cell mass (ICM), and POU5F1+/CDX2+ cells, which identify trophectoderm (TE). Many, but not all, exhibited a disproportionately small ICM. By contrast, demiembryos retained within their ZP and created by randomly damaging one of the two blastomeres in two-cell stage embryos exhibited a more normal ratio of ICM to TE cells at blastocyst and significantly less variance in ICM cell number. One possible explanation is that ZP-free demiembryos only infrequently adopt the same conformation as their partners, including the favorable tetrahedral form, at the four-cell stage, suggesting that such embryos exhibit a high degree of plasticity with regard to the orientation of their first two cleavage planes and that a significant number likely deviate from paths that provide an optimal geometric progression to blastocyst. These data could explain the difficulty of creating monozygotic twins from two-cell stage embryos. PMID:20181620

Endometrium as an early sensor of in vitro embryo manipulation technologies

PubMed Central

Mansouri-Attia, Nadéra; Sandra, Olivier; Aubert, Julie; Degrelle, Séverine; Everts, Robin E.; Giraud-Delville, Corinne; Heyman, Yvan; Galio, Laurent; Hue, Isabelle; Yang, Xiangzhong; Tian, X. Cindy; Lewin, Harris A.; Renard, Jean-Paul

2009-01-01

Implantation is crucial for placental development that will subsequently impact fetal growth and pregnancy success with consequences on postnatal health. We postulated that the pattern of genes expressed by the endometrium when the embryo becomes attached to the mother uterus could account for the final outcome of a pregnancy. As a model, we used the bovine species where the embryo becomes progressively and permanently attached to the endometrium from day 20 of gestation onwards. At that stage, we compared the endometrial genes profiles in the presence of an in vivo fertilized embryo (AI) with the endometrial patterns obtained in the presence of nuclear transfer (SCNT) or in vitro fertilized embryos (IVF), both displaying lower and different potentials for term development. Our data provide evidence that the endometrium can be considered as a biological sensor able to fine-tune its physiology in response to the presence of embryos whose development will become altered much later after the implantation process. Compared with AI, numerous biological functions and several canonical pathways with a major impact on metabolism and immune function were found to be significantly altered in the endometrium of SCNT pregnancies at implantation, whereas the differences were less pronounced with IVF embryos. Determining the limits of the endometrial plasticity at the onset of implantation should bring new insights on the contribution of the maternal environment to the development of an embryo and the success of pregnancy. PMID:19297625

Lethal and teratogenic effects of phenol on Bufo arenarum embryos.

PubMed

Paisio, Cintia Elizabeth; Agostini, Elizabeth; González, Paola Solange; Bertuzzi, Mabel Lucía

2009-08-15

Phenol and their derivatives are used in several industries and they have a high potential toxicity for animal and plant species. They were found in variable concentrations, as high as 1000 mg/L, in industrial wastewater and, they are often discharged into the environment. Amphibian embryos are useful indicators of environmental pollution. However, to our knowledge, there are not studies focussed on the toxic effects of phenol on Bufo arenarum, which is an anuran widely distributed in South America. Therefore, the effect of phenol on the survival and morphogenesis of these amphibian embryos was evaluated by means of AMPHITOX test. Embryos at 25 stage of development (acute test) and embryos at 2-4 blastomers stage (early life stage test), were exposed to phenol solutions in concentrations ranging from 25 to 250 mg/L, which were frequently found in the environment. Mortality and malformations were registered each 24h. LC(50), LC(99), NOEC, TC(50) and TI(50) values were 183.70, 250, 60, 113 mg/L and 1.62, respectively, at 96 h of treatment. Mortality and the percentage of malformations increased with increasing phenol concentrations. Teratogenic effects more frequently produced by phenol were: axial flexure, persistent yolk plug and different abnormalities which caused death of blastulae. Moreover, other malformations were registered, such as irregular form, acephalism, edema, axial shortening and underdevelopment of gills, among others. Larvae of B. arenarum, at early embryonic stages (blastulae), showed higher sensitivity to phenol than tadpoles at stage 25. Results confirm high susceptibility of amphibians to phenol and that environmental concentrations of this pollutant might be harmful to these populations.

Susceptibility of early life stages of Xenopus laevis to cadmium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herkovits, J.; Perez-Coll, C.S.; Cardellini, P.

1997-02-01

The susceptibility of Xenopus laevis to cadmium during different stages of development was evaluated by exposing embryos to cadmium concentrations ranging from 0.1 to 10 mg Cd{sup 2+}/L for 24, 48, and 72 h and assessing lethality and malformations. Susceptibility increased from the two blastomeres stage (stage 2) to stage 40, in which the 24-h LC100 was 1.13 mg Cd{sup 2+}/L, and resistance increased from this stage onward. Malformations occurred at all developmental stages evaluated, the most common being reduced size, incurvated axis, underdeveloped or abnormally developed fin, microcephaly, and microphtalmy. Scanning electron microscopy revealed changes in the ectodermal surfacemore » ranging from slightly vaulted cells to a severe reduction in the number of ciliated cells as the concentration of cadmium increased. The intraspecific variation evaluated in embryos (from four sets of parents) at seven developmental stages, expressed as the coefficient of variation of the LC100, ranged from 10 to 112% and reflects the capacity of Xenopus laevis to adapt to changing environmental conditions at different embryonic stages.« less

Developmental and physiological challenges of octopus (Octopus vulgaris) early life stages under ocean warming.

PubMed

Repolho, Tiago; Baptista, Miguel; Pimentel, Marta S; Dionísio, Gisela; Trübenbach, Katja; Lopes, Vanessa M; Lopes, Ana Rita; Calado, Ricardo; Diniz, Mário; Rosa, Rui

2014-01-01

The ability to understand and predict the effects of ocean warming (under realistic scenarios) on marine biota is of paramount importance, especially at the most vulnerable early life stages. Here we investigated the impact of predicted environmental warming (+3 °C) on the development, metabolism, heat shock response and antioxidant defense mechanisms of the early stages of the common octopus, Octopus vulgaris. As expected, warming shortened embryonic developmental time by 13 days, from 38 days at 18 °C to 25 days at 21 °C. Concomitantly, survival decreased significantly (~29.9 %). Size at hatching varied inversely with temperature, and the percentage of smaller premature paralarvae increased drastically, from 0 % at 18 °C to 17.8 % at 21 °C. The metabolic costs of the transition from an encapsulated embryo to a free planktonic form increased significantly with warming, and HSP70 concentrations and glutathione S-transferase activity levels were significantly magnified from late embryonic to paralarval stages. Yet, despite the presence of effective antioxidant defense mechanisms, ocean warming led to an augmentation of malondialdehyde levels (an indicative of enhanced ROS action), a process considered to be one of the most frequent cellular injury mechanisms. Thus, the present study provides clues about how the magnitude and rate of ocean warming will challenge the buffering capacities of octopus embryos and hatchlings' physiology. The prediction and understanding of the biochemical and physiological responses to warmer temperatures (under realistic scenarios) is crucial for the management of highly commercial and ecologically important species, such as O. vulgaris.

Central cell-derived peptides regulate early embryo patterning in flowering plants.

PubMed

Costa, Liliana M; Marshall, Eleanor; Tesfaye, Mesfin; Silverstein, Kevin A T; Mori, Masashi; Umetsu, Yoshitaka; Otterbach, Sophie L; Papareddy, Ranjith; Dickinson, Hugh G; Boutiller, Kim; VandenBosch, Kathryn A; Ohki, Shinya; Gutierrez-Marcos, José F

2014-04-11

Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning in Arabidopsis. EMBRYO SURROUNDING FACTOR 1 (ESF1) peptides accumulate before fertilization in central cell gametes and thereafter in embryo-surrounding endosperm cells. Biochemical and structural analyses revealed cleavage of ESF1 propeptides to form biologically active mature peptides. Further, these peptides act in a non-cell-autonomous manner and synergistically with the receptor-like kinase SHORT SUSPENSOR to promote suspensor elongation through the YODA mitogen-activated protein kinase pathway. Our findings demonstrate that the second female gamete and its sexually derived endosperm regulate early embryonic patterning in flowering plants.

Morphological and histomorphological structures of testes and ovaries in early developmental stages of the silkworm, Bombyx mori.

PubMed

Sakai, Hiroki; Kirino, Yohei; Katsuma, Susumu; Aoki, Fugaku; Suzuki, Masataka G

2016-01-01

The gonad develops as a testis in male or an ovary in female. In the silkworm, B. mori , little is known about testis and ovary in the embryonic stages and early larval stages. In this study, we performed morphological and histomorphological observations of ovaries and testes from the late embryonic stage to the 1st instar larval stage. Results obtained with lack of accurate information on sex of examined individuals may be misleading, thus we performed phenotypic observations of gonads by utilizing sex-limited strain that enables us to easily discriminate female embryos from male ones based on those egg colors. In testis, four testicular follicles were clearly observed in the testis at the first instar larval stage, and boundary layers were formed between the testicular follicles. At the late embryonic stage, the testis consisted of four testicular follicles, while the boundary layers were still obscure. In ovary, four ovarioles were easily recognizable in the ovary at the first instar larval stage, and boundary layers were formed between the ovarioles. However, in the late embryonic stage, it was quite difficult to identify four ovarioles. Morphological characteristics were almost similar between testis and ovary in early developmental stages. Our present study demonstrates that the most reliable difference between testis and ovary in early developmental stages is the attaching point of the duct. Formation and development of the duct may be sensitive to the sex-determining signal and display sexual dimorphism in early embryonic stages.

Fossilized embryos are widespread but the record is temporally and taxonomically biased

USGS Publications Warehouse

Donoghue, P.C.J.; Kouchinsky, A.; Waloszek, Dieter; Bengtson, S.; Dong, X.-P.; Val'Kov, A.K.; Cunningham, J.A.; Repetski, J.E.

2006-01-01

We report new discoveries of embryos and egg capsules from the Lower Cambrian of Siberia, Middle Cambrian of Australia and Lower Ordovician of North America. Together with existing records, embryos have now been recorded from four of the seven continents. However, the new discoveries highlight secular and systematic biases in the fossil record of embryonic stages. The temporal window within which the embryos and egg capsules are found is of relatively short duration; it ends in the Early Ordovician and is roughly coincident with that of typical "Orsten"-type faunas. The reduced occurrence of such fossils has been attributed to reducing levels of phosphate in marine waters during the early Paleozoic, but may also be owing to the increasing depth of sediment mixing by infaunal metazoans. Furthermore, most records younger than the earliest Cambrian are of a single kind - large eggs and embryos of the priapulid-like scalidophoran Markuelia. We explore alternative explanations for the low taxonomic diversity of embryos recovered thus far, including sampling, size, anatomy, ecology, and environment, concluding that the preponderance of Markuelia embryos is due to its precocious development of cuticle at an embryonic stage, predisposing it to preservation through action as a substrate on which microbially mediated precipitation of authigenic calcium phosphate may occur. The fossil record of embryos may be limited to a late Neoproterozoic to early Ordovician snapshot that is subject to dramatic systematic bias. Together, these biases must be considered seriously in attempts to use the fossil record to arbitrate between hypotheses of developmental and life history evolution implicated in the origin of metazoan clades. ?? 2006 Blackwell Publishing Ltd.

Germ layer differentiation during early hindgut and cloaca formation in rabbit and pig embryos

PubMed Central

Hassoun, Romia; Schwartz, Peter; Rath, Detlef; Viebahn, Christoph; Männer, Jörg

2010-01-01

Relative to recent advances in understanding molecular requirements for endoderm differentiation, the dynamics of germ layer morphology and the topographical distribution of molecular factors involved in endoderm formation at the caudal pole of the embryonic disc are still poorly defined. To discover common principles of mammalian germ layer development, pig and rabbit embryos at late gastrulation and early neurulation stages were analysed as species with a human-like embryonic disc morphology, using correlative light and electron microscopy. Close intercellular contact but no direct structural evidence of endoderm formation such as mesenchymal–epithelial transition between posterior primitive streak mesoderm and the emerging posterior endoderm were found. However, a two-step process closely related to posterior germ layer differentiation emerged for the formation of the cloacal membrane: (i) a continuous mesoderm layer and numerous patches of electron-dense flocculent extracellular matrix mark the prospective region of cloacal membrane formation; and (ii) mesoderm cells and all extracellular matrix including the basement membrane are lost locally and close intercellular contact between the endoderm and ectoderm is established. The latter process involves single cells at first and then gradually spreads to form a longitudinally oriented seam-like cloacal membrane. These gradual changes were found from gastrulation to early somite stages in the pig, whereas they were found from early somite to mid-somite stages in the rabbit; in both species cloacal membrane formation is complete prior to secondary neurulation. The results highlight the structural requirements for endoderm formation during development of the hindgut and suggest new mechanisms for the pathogenesis of common urogenital and anorectal malformations. PMID:20874819

The Digestive Tract and Derived Primordia Differentiate by Following a Precise Timeline in Human Embryos Between Carnegie Stages 11 and 13.

PubMed

Ueno, Saki; Yamada, Shigehito; Uwabe, Chigako; Männer, Jörg; Shiraki, Naoto; Takakuwa, Tetsuya

2016-04-01

The precise mechanisms through which the digestive tract develops during the somite stage remain undefined. In this study, we examined the morphology and precise timeline of differentiation of digestive tract-derived primordia in human somite-stage embryos. We selected 37 human embryos at Carnegie Stage (CS) 11-CS13 (28-33 days after fertilization) and three-dimensionally analyzed the morphology and positioning of the digestive tract and derived primordia in all samples, using images reconstructed from histological serial sections. The digestive tract was initially formed by a narrowing of the yolk sac, and then several derived primordia such as the pharynx, lung, stomach, liver, and dorsal pancreas primordia differentiated during CS12 (21-29 somites) and CS13 (≥ 30 somites). The differentiation of four pairs of pharyngeal pouches was complete in all CS13 embryos. The respiratory primordium was recognized in ≥ 26-somite embryos and it flattened and then branched at CS13. The trachea formed and then elongated in ≥ 35-somite embryos. The stomach adopted a spindle shape in all ≥ 34-somite embryos, and the liver bud was recognized in ≥ 27-somite embryos. The dorsal pancreas appeared as definitive buddings in all but three CS13 embryos, and around these buddings, the small intestine bent in ≥ 33-somite embryos. In ≥ 35-somite embryos, the small intestine rotated around the cranial-caudal axis and had begun to form a primitive intestinal loop, which led to umbilical herniation. These data indicate that the digestive tract and derived primordia differentiate by following a precise timeline and exhibit limited individual variations. © 2016 Wiley Periodicals, Inc.

[Specification of cell destiny in early Caenorhabditis elegans embryo].

PubMed

Schierenberg, E

1997-02-01

Embryogenesis of the nematode Caenorhabditis elegans has been described completely on a cell-by-cell basis and found to be essentially invariant. With this knowledge in hands, micromanipulated embryos and mutants have been analyzed for cell lineage defects and the distribution of specific gene products. The results challenge the classical view of cell-autonomous development in nematodes and indicate that the early embryo of C. elegans is a highly dynamic system. A network of inductive events between neighboring cells is being revealed, which is necessary to assign different developmental programs to blastomeres. In those cases where molecules involved in these cell-cell interactions have been identified, homologies to cell surface receptors, ligands and transcription factors found in other systems have become obvious.

Impact of motorboats on fish embryos depends on engine type

PubMed Central

Jain-Schlaepfer, Sofia; Fakan, Eric; Rummer, Jodie L; Simpson, Stephen D; McCormick, Mark I

2018-01-01

Abstract Human generated noise is changing the natural underwater soundscapes worldwide. The most pervasive sources of underwater anthropogenic noise are motorboats, which have been found to negatively affect several aspects of fish biology. However, few studies have examined the effects of noise on early life stages, especially the embryonic stage, despite embryo health being critical to larval survival and recruitment. Here, we used a novel setup to monitor heart rates of embryos from the staghorn damselfish (Amblyglyphidodon curacao) in shallow reef conditions, allowing us to examine the effects of in situ boat noise in context with real-world exposure. We found that the heart rate of embryos increased in the presence of boat noise, which can be associated with the stress response. Additionally, we found 2-stroke outboard-powered boats had more than twice the effect on embryo heart rates than did 4-stroke powered boats, showing an increase in mean individual heart rate of 1.9% and 4.6%, respectively. To our knowledge this is the first evidence suggesting boat noise elicits a stress response in fish embryo and highlights the need to explore the ecological ramifications of boat noise stress during the embryo stage. Also, knowing the response of marine organisms caused by the sound emissions of particular engine types provides an important tool for reef managers to mitigate noise pollution. PMID:29593871

Impact of motorboats on fish embryos depends on engine type.

PubMed

Jain-Schlaepfer, Sofia; Fakan, Eric; Rummer, Jodie L; Simpson, Stephen D; McCormick, Mark I

2018-01-01

Human generated noise is changing the natural underwater soundscapes worldwide. The most pervasive sources of underwater anthropogenic noise are motorboats, which have been found to negatively affect several aspects of fish biology. However, few studies have examined the effects of noise on early life stages, especially the embryonic stage, despite embryo health being critical to larval survival and recruitment. Here, we used a novel setup to monitor heart rates of embryos from the staghorn damselfish ( Amblyglyphidodon curacao ) in shallow reef conditions, allowing us to examine the effects of in situ boat noise in context with real-world exposure. We found that the heart rate of embryos increased in the presence of boat noise, which can be associated with the stress response. Additionally, we found 2-stroke outboard-powered boats had more than twice the effect on embryo heart rates than did 4-stroke powered boats, showing an increase in mean individual heart rate of 1.9% and 4.6%, respectively. To our knowledge this is the first evidence suggesting boat noise elicits a stress response in fish embryo and highlights the need to explore the ecological ramifications of boat noise stress during the embryo stage. Also, knowing the response of marine organisms caused by the sound emissions of particular engine types provides an important tool for reef managers to mitigate noise pollution.

Early bovine embryos regulate oviduct epithelial cell gene expression during in vitro co-culture.

PubMed

Schmaltz-Panneau, Barbara; Cordova, Amanda; Dhorne-Pollet, Sophie; Hennequet-Antier, Christelle; Uzbekova, Sveltlana; Martinot, Emmanuelle; Doret, Sarah; Martin, Patrice; Mermillod, Pascal; Locatelli, Yann

2014-10-01

In mammals, the oviduct may participate to the regulation of early embryo development. In vitro co-culture of early bovine embryos with bovine oviduct epithelial cells (BOEC) has been largely used to mimic the maternal environment. However, the mechanisms of BOEC action have not been clearly elucidated yet. The aim of this study was to determine the response of BOEC cultures to the presence of developing bovine embryos. A 21,581-element bovine oligonucleotide array was used compare the gene expression profiles of confluent BOEC cultured for 8 days with or without embryos. This study revealed 34 differentially expressed genes (DEG). Of these 34 genes, IFI6, ISG15, MX1, IFI27, IFI44, RSAD2, IFITM1, EPSTI1, USP18, IFIT5, and STAT1 expression increased to the greatest extent due to the presence of embryos with a major impact on antiviral and immune response. Among the mRNAs at least 25 are already described as induced by interferons. In addition, transcript levels of new candidate genes involved in the regulation of transcription, modulation of the maternal immune system and endometrial remodeling were found to be increased. We selected 7 genes and confirmed their differential expression by quantitative RT-PCR. The immunofluorescence imaging of cellular localization of STAT1 protein in BOEC showed a nuclear translocation in the presence of embryos, suggesting the activation of interferon signaling pathway. This first systematic study of BOEC transcriptome changes in response to the presence of embryos in cattle provides some evidences that these cells are able to adapt their transcriptomic profile in response to embryo signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

Sildenafil citrate (Viagra) impairs fertilization and early embryo development in mice.

PubMed

Glenn, David R J; McClure, Neil; Cosby, S Louise; Stevenson, Michael; Lewis, Sheena E M

2009-03-01

To determine the effects of sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor known to affect sperm function, on fertilization and early embryo cleavage. This acute mammal study included male and female mice assigned randomly, the females sacrificed after mating and their oocytes/embryos evaluated at four time periods after treatment. Academic research environment. Male and female CBAB(6) mice. Female mice were injected intraperitoneally with 5 IU gonadotropin (hCG) to stimulate follicular growth and induce ovulation. They were each caged with a male that had been gavaged with sildenafil citrate (0.06 mg/0.05 mL) and allowed to mate. After 12, 36, 60, and 84 h, females were killed, their oviducts were dissected out, and retrieved embryos were assessed for blastomere number and quality. Fertilization rates and numbers of embryos were evaluated after treatment. Fertilization rates (day 1) were markedly reduced (-33%) in matings where the male had taken sildenafil citrate. Over days 2-4, the numbers of embryos developing in the treated group were significantly fewer than in the control group. There was also a trend for impaired cleavage rates within those embryos, although this did not reach significance. The impairments to fertility caused by sildenafil citrate have important implications for infertility centers and for couples who are using this drug precoitally while attempting to conceive.

Recognition of the CDEI motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo.

PubMed Central

Blangy, A; Léopold, P; Vidal, F; Rassoulzadegan, M; Cuzin, F

1991-01-01

We have reported previously (1) two unexpected consequences of the microinjection into fertilized mouse eggs of a recombinant plasmid designated p12B1, carrying a 343 bp insert of non-repetitive mouse DNA. Injected at very low concentrations, this plasmid could be established as an extrachromosomal genetic element. When injected in greater concentration, an early arrest of embryonic development resulted. In the present work, we have studied this toxic effect in more detail by microinjecting short synthetic oligonucleotides with sequences from the mouse insert. Lethality was associated with the nucleotide sequence GTCACATG, identical with the CDEl element of yeast centromeres. Development of injected embryos was arrested between the one-cell and the early morula stages, with abnormal structures and DNA contents. Electrophoretic mobility shift and DNAse foot-printing assays demonstrated the binding of mouse nuclear protein(s) to the CDEl-like box. Base changes within the CDEl sequence prevented both the toxic effects in embryos and the formation of protein complex in vitro, suggesting that protein binding at such sites in chromosomal DNA plays an important role in early development. Images PMID:1766880

Functional analysis of lysosomes during mouse preimplantation embryo development.

PubMed

Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Ohta, Yuki; Wada, Ayako; Ishida, Yuka; Kito, Seiji; Nishikawa, Tetsu; Minami, Naojiro; Sato, Ken; Kokubo, Toshiaki

2013-01-01

Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.

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. Copyright © 2016 Elsevier Inc. All rights reserved.

Embryo selection using time-lapse analysis (Early Embryo Viability Assessment) in conjunction with standard morphology: a prospective two-center pilot study.

PubMed

Kieslinger, Dorit C; De Gheselle, Stefanie; Lambalk, Cornelis B; De Sutter, Petra; Kostelijk, E Hanna; Twisk, Jos W R; van Rijswijk, Joukje; Van den Abbeel, Etienne; Vergouw, Carlijn G

2016-11-01

Does prospective embryo selection using the results from the Eava Test (Early Embryo Viability Assessment) in combination with standard morphology increase the pregnancy rate of IVF and ICSI patients compared to embryo selection based on morphology only? Embryo selection using the Eeva Test plus standard morphology on Day 3 results in comparable pregnancy rates as conventional morphological embryo selection. Time-lapse monitoring of embryo development may represent a superior way to culture and select embryos in vitro. The Eeva Test records the development of each embryo with a cell-tracking system and predicts the likelihood (High, Medium or Low) that an embryo will form a blastocyst based on an automated analysis of early cell division timings. This trial was designed as a prospective, observational, two-center pilot study with a propensity matched control group. The analysis involved 280 of 302 enrolled patients who were included in the Eeva Test group in 2013 and 560 control patients who were treated in the years 2011-2013. The majority of transfers (98%) were single embryo transfers. Two academic hospitals (VUmc Amsterdam and UZ Gent) enrolled patients <41 years old, with <3 previous attempts and ≥5 normally fertilized eggs. Propensity matching was used to identify a propensity matched control group from a cohort of 1777 patients based on age, cycle number, oocyte number and number of fertilized oocytes. There was no difference in patient baseline characteristics between the two groups. The ongoing pregnancy rate (OPR) of patients enrolled in the Eeva Test group (34.3%; 96/280) did not differ significantly from the OPR in the propensity matched control group (34.6%, 194/560; P = 0.92). However, significantly less top quality embryos (eight-cell embryos with ≤25% fragmentation) were transferred in the Eeva Test group compared to the propensity matched control group (70.4% vs. 82.3%; P < 0.001). The transfer of Eeva High and Medium embryos resulted in a

Acute toxicity of polybrominated diphenyl ethers (PBDEs) for turbot (Psetta maxima) early life stages (ELS).

PubMed

Mhadhbi, Lazhar; Fumega, José; Boumaiza, Moncef; Beiras, Ricardo

2012-03-01

The environmental presence of polybrominated diphenyl ethers (PBDEs), among which BDE-47 and BDE-99 are particularly abundant, makes toxicity data necessary to assess the hazard risk posed by PBDE to aquatic organisms. This study examines the effects of BDE-47 and BDE-99 on embryo-larval stages of the marine flatfish turbot. The turbot embryos were exposed at nominal concentrations of BDE-47 and BDE-99 for 6 days. Selected dose levels were relevant for investigating sublethal and lethal effects. Both tested compounds caused lethal toxicity as well as non-lethal malformations during embryo development. We found a high toxic potency of BDE-47 compared to BDE-99 (LC₅₀ values for embryos and larvae, respectively, BDE-47: 27.35 and 14.13 μg L⁻¹; BDE-99: 38.28 and 29.64 μg L⁻¹). The present study shows high sensitivity of fish early life stages (ELS) to PBDE compounds. Based on environmental concentrations of dissolved PBDEs from various aquatic ecosystems, waterborne BDE-47 and BDE-99 pose little risk of acute toxicity to marine fish at relevant environmental concentrations. Turbot fish ELS proved to be an excellent model for the study of ecotoxicity of contaminants in seawater. The results demonstrate harmful effects of PBDE on turbot ELS at concentrations in the range of parts per billion units. In the perspective of risk assessment, ELS endpoints provide rapid, cost-effective and ecologically relevant information, and links should be sought between these short-term tests and effects of long-term exposures in more realistic scenarios.

Embryonic lethality is not sufficient to explain hourglass-like conservation of vertebrate embryos.

PubMed

Uchida, Yui; Uesaka, Masahiro; Yamamoto, Takayoshi; Takeda, Hiroyuki; Irie, Naoki

2018-01-01

Understanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that gene expression profiles of mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods. While the hourglass-like divergence of developmental processes has been demonstrated in a variety of animal groups such as vertebrates, arthropods, and nematodes, the exact mechanism leading to this mid-embryonic conservation remains to be clarified. One possibility is that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Here, we tested this "mid-embryonic lethality hypothesis" by measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations. By subjecting zebrafish ( Danio rerio ), African clawed frog ( Xenopus laevis ), and chicken ( Gallus gallus ) embryos to transient perturbations, namely heat shock and inhibitor treatments during three developmental periods [early (represented by blastula and gastrula), pharyngula, and late], we found that the early stages showed the highest rate of lethal phenotypes in all three species. This result was corroborated by perturbation with genetic mutations. By tracking the survival rate of wild-type embryos and embryos with genetic mutations induced by UV irradiation in zebrafish and African clawed frogs, we found that the highest decrease in survival rate was at the early stages particularly around gastrulation in both these species. In opposition to the "mid-embryonic lethality hypothesis," our results consistently showed that the stage with the highest lethality was not around the

Effects of Phos-Chek® G75-F and Silv-Ex® on red-winged blackbird (Agelaius phoeniceus) embryos

USGS Publications Warehouse

Buscemi, D.M.; Hoffman, D.J.; Vyas, N.B.; Spann, J.W.; Kuenzel, W.J.

2007-01-01

Effects of field application levels of wildfire control chemicals, Phos-Chek® G75-F (PC) and Silv-Ex® (SE), were examined on red-winged blackbird (Agelaius phoeniceus) embryos. Embryos were more sensitive to PC and SE when eggs were immersed for 10 s at an early developmental stage (days 3–5 of incubation) than at a later stage (days 6–9 of incubation). The LC50 (concentration causing 50% mortality) for early stage embryos exposed to PC was 213.3 g/L (slope = 1.6; 95% confidence interval [CI] = 129.1–326.1). The no observed effect concentration (NOEC) was below 135 g PC/L, which caused a significant increase in embryonic mortality and represents the lowest field coverage level of 1 gal/100 feet2. The LC50 for early stage embryos exposed to SE was 19.8 g/L (slope = 1.5; 95% CI = 11.7–52.2). Significant mortality was observed at 10 g SE/L and marginal at 7.5 g SE/L with an apparent NOEC around 5 g SE/L. Neither chemical resulted in apparent developmental malformations.

Effect of low-level copper and pentachlorophenol exposure on various early life stages of Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fort, D.J.; Stover, E.L.

1996-12-31

An evaluation of the effects of low-level copper and pentachlorophenol exposure on various early life stages of the South African clawed frog, Xenopus laevis, was performed using stage-specific and long-term continuous exposures. Stage-specific exposure experiments were conducted such that separate subsets of embryos and larvae from the same clutch were exposed to two toxicants, copper and pentachlorphenol, from 0 d to 4 d (standard Frog Embryo Teratogenesis Assay--Xenopus [FETAX]), 4 d to 8 d, 8 d to 12 d, and 12 d to 16 d. Results from two separate concentration-response experiments indicated that sensitivity to either toxicant increased in eachmore » successive time period. Longer-term exposure studies conducted for 60 to 75 days indicated that copper, but not pentachlorophenol induced reduction deficiency malformations of the hind limb at concentrations as low as 0.05 mg/L. Pentachlorophenol concentrations as low as 0.5 {micro}g/L inhibited tail resorption. However, copper did not adversely affect the process of tail resorption. These results indicated that studies evaluating longer-term developmental processes are important in ecological hazard evaluation.« less

The presence of MWCNTs reduces developmental toxicity of PFOS in early life stage of zebrafish.

PubMed

Wang, Shutao; Zhuang, Changlu; Du, Jia; Wu, Chuan; You, Hong

2017-03-01

Both carbon nanotubes (CNTs) and perfluorooctane sulfonate (PFOS) are used widely. There is considerable concern regarding their ecotoxicity. CNTs might interact with PFOS in water and result in different impacts compared with those after single exposures. To our knowledge, the developmental toxicity of PFOS in the presence of multi-walled carbon nanotubes (MWCNTs) in the early life stage of zebrafish (from 3 h post fertilization (hpf) to 96 hpf) was investigated for the first time in this study. The embryos and larvae were exposed to PFOS (0.2, 0.4, 0.8, and 1.6 mg/L), MWCNTs (50 mg/L), and a mixture of both. Compared with PFOS exposure, the adverse effects induced by PFOS on the hatching rate of zebrafish embryos and the heart rate and body length of zebrafish larvae were reduced in the presence of MWCNTs, and mortality and malformation were also alleviated. In addition, zebrafish larvae exposed to PFOS showed decreased activities of superoxide dismutase, catalase, and glutathione peroxidase, as well as decreased levels of reactive oxygen species and malondialdehyde, in the presence of MWCNTs, indicating that oxidative stress and lipid peroxidation was relieved. Thus, the presence of MWCNTs reduces the developmental toxicity of PFOS in the early life stage of zebrafish. Copyright © 2016 Elsevier Ltd. All rights reserved.

Changes in Oscillatory Dynamics in the Cell Cycle of Early Xenopus laevis Embryos

PubMed Central

Tsai, Tony Y.-C.; Theriot, Julie A.; Ferrell, James E.

2014-01-01

During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development. PMID:24523664

Oxidative Damage to Rhesus Macaque Spermatozoa Results in Mitotic Arrest and Transcript Abundance Changes in Early Embryos1

PubMed Central

Burruel, Victoria; Klooster, Katie L.; Chitwood, James; Ross, Pablo J.; Meyers, Stuart A.

2013-01-01

ABSTRACT Our objective was to determine whether oxidative damage of rhesus macaque sperm induced by reactive oxygen species (ROS) in vitro would affect embryo development following intracytoplasmic sperm injection (ICSI) of metaphase II (MII) oocytes. Fresh rhesus macaque spermatozoa were treated with ROS as follows: 1 mM xanthine and 0.1 U/ml xanthine oxidase (XXO) at 37°C and 5% CO2 in air for 2.25 h. Sperm were then assessed for motility, viability, and lipid peroxidation. Motile ROS-treated and control sperm were used for ICSI of MII oocytes. Embryo culture was evaluated for 3 days for development to the eight-cell stage. Embryos were fixed and stained for signs of cytoplasmic and nuclear abnormalities. Gene expression was analyzed by RNA-Seq in two-cell embryos from control and treated groups. Exposure of sperm to XXO resulted in increased lipid peroxidation and decreased sperm motility. ICSI of MII oocytes with motile sperm induced similar rates of fertilization and cleavage between treatments. Development to four- and eight-cell stage was significantly lower for embryos generated with ROS-treated sperm than for controls. All embryos produced from ROS-treated sperm demonstrated permanent embryonic arrest and varying degrees of degeneration and nuclear fragmentation, changes that are suggestive of prolonged senescence or apoptotic cell death. RNA-Seq analysis of two-cell embryos showed changes in transcript abundance resulting from sperm treatment with ROS. Differentially expressed genes were enriched for processes associated with cytoskeletal organization, cell adhesion, and protein phosphorylation. ROS-induced damage to sperm adversely affects embryo development by contributing to mitotic arrest after ICSI of MII rhesus oocytes. Changes in transcript abundance in embryos destined for mitotic arrest is evident at the two-cell stage of development. PMID:23904511

Effects of heat stress on bovine preimplantation embryos produced in vitro

PubMed Central

SAKATANI, Miki

2017-01-01

Summer heat stress decreases the pregnancy rate in cattle and has been thought to be associated with the early embryonic death caused by the elevation of maternal body temperature. In vitro cultures have been widely used for the evaluation of effects of heat stress on oocytes, fertilization, preimplantation, and embryonic development. Susceptibility to heat stress is present in developmental stages from oocytes to cleavage-stage (before embryonic gene activation, EGA) embryos, leading to a consequent decrease in developmental competence. On the other hand, advanced-stage embryos such as morula or blastocysts have acquired thermotolerance. The mechanism for the developmental stage-dependent change in thermotolerance is considered to be the accumulation of antioxidants in embryos in response to heat-inducible production of reactive oxygen species. The supplementation of antioxidants to the culture media has been known to neutralize the detrimental effects of heat stress. Besides, EGA could be involved in acquisition of thermotolerance in later stages of embryos. Morulae or blastocysts can repair heat-induced unfolded proteins or prevent DNA damage occurring in processes such as apoptosis. Therefore, embryo transfer (ET) that can bypass the heat-sensitive stage could be a good solution to improve the pregnancy rate under heat stress. However, frozen-thawed ET could not improve the pregnancy rate as expected. Frozen-thawed blastocysts were more sensitive to heat stress and showed less proliferation upon heat exposure, compared to fresh blastocysts. Therefore, further research is required to improve the reduction in pregnancy rates due to summer heat stress. PMID:28496018

Effects of heat stress on bovine preimplantation embryos produced in vitro.

PubMed

Sakatani, Miki

2017-08-19

Summer heat stress decreases the pregnancy rate in cattle and has been thought to be associated with the early embryonic death caused by the elevation of maternal body temperature. In vitro cultures have been widely used for the evaluation of effects of heat stress on oocytes, fertilization, preimplantation, and embryonic development. Susceptibility to heat stress is present in developmental stages from oocytes to cleavage-stage (before embryonic gene activation, EGA) embryos, leading to a consequent decrease in developmental competence. On the other hand, advanced-stage embryos such as morula or blastocysts have acquired thermotolerance. The mechanism for the developmental stage-dependent change in thermotolerance is considered to be the accumulation of antioxidants in embryos in response to heat-inducible production of reactive oxygen species. The supplementation of antioxidants to the culture media has been known to neutralize the detrimental effects of heat stress. Besides, EGA could be involved in acquisition of thermotolerance in later stages of embryos. Morulae or blastocysts can repair heat-induced unfolded proteins or prevent DNA damage occurring in processes such as apoptosis. Therefore, embryo transfer (ET) that can bypass the heat-sensitive stage could be a good solution to improve the pregnancy rate under heat stress. However, frozen-thawed ET could not improve the pregnancy rate as expected. Frozen-thawed blastocysts were more sensitive to heat stress and showed less proliferation upon heat exposure, compared to fresh blastocysts. Therefore, further research is required to improve the reduction in pregnancy rates due to summer heat stress.

Comparison of survival rate of cleavage stage embryos produced from in vitro maturation cycles after slow freezing and after vitrification.

PubMed

Son, Weon-Young; Chung, Jin-Tae; Gidoni, Yariv; Holzer, Hananel; Levin, Dan; Chian, Ri-Cheng; Tan, Seang Lin

2009-09-01

Significantly more embryos survived the vitrification procedure compared to slow freezing (85.5% vs. 61.8%) in cleavage-stage human embryos produced from in vitro maturation cycles, suggesting that vitrification is more efficient than slow freezing for cryopreservation.

Enhance beef cattle improvement by embryo biotechnologies.

PubMed

Wu, B; Zan, L

2012-10-01

Embryo biotechnology has become one of the prominent high businesses worldwide. This technology has evolved through three major changes, that is, traditional embryo transfer (in vivo embryo production by donor superovulation), in vitro embryo production by ovum pick up with in vitro fertilization and notably current cloning technique by somatic cell nuclear transfer and transgenic animal production. Embryo biotechnology has widely been used in dairy and beef cattle industry and commercial bovine embryo transfer has become a large international business. Currently, many developed biotechnologies during the period from early oocyte stage to pre-implantation embryos can be used to create new animal breeds and accelerate genetic progression. Based on recent advances in embryo biotechnologies and authors current studies, this review will focus on a description of the application of this technology to beef cattle improvement and discuss how to use this technology to accelerate beef cattle breeding and production. The main topics of this presentation include the following: (i) how to increase calf production numbers from gametes including sperm and oocyte; (ii) multiple ovulation and embryo transfer breeding schemes; (iii) in vitro fertilization and intracytoplasm sperm injection in bovine; (iv) pronuclear development and transgenic animals; (v) sex selection from sperm and embryos; (vi) cloning and androgenesis; (vii) blastocyst development and embryonic stem cells; (viii) preservation of beef cattle genetic resources; and (ix) conclusions. © 2011 Blackwell Verlag GmbH.

Maternally derived trypsin may have multiple functions in the early development of turbot (Scopthalmus maximus).

PubMed

Chi, Liang; Liu, Qinghua; Xu, Shihong; Xiao, Zhizhong; Ma, Daoyuan; Li, Jun

2015-10-01

Trypsin is an important serine protease that is considered to be involved in digestion of protein in teleost fish. Nevertheless, studies on trypsin/trypsinogen in fish embryos are very limited. In this study, the trypsinogen of turbot (Scophthalmus maximus) (tTG) was identified and the expression patterns and activity of trypsinogen/trypsin were investigated. The results showed that the tTG mRNA was evenly distributed in the oocytes and was also expressed along the yolk periphery in early embryos. At later embryo stages and 1 days after hatching (dph), the tTG mRNA concentrated at the alimentary tract and head. Quantitative expression analysis showed that the tTG transcripts decreased after fertilization until the gastrula stage, then increased with the embryo and larvae development. This result was also confirmed by the specific activity analysis of trypsin and in-situ-hybridization (ISH). All of the results indicated that tTG in early embryo stages was maternally derived and expressed by itself after gastrula stages. Additionally, location of tTG mRNA in embryos and larvae was investigated; we considered that trypsin may have multiple functions during the embryo development process. Based on our results regarding trypsinogen in embryos and early development, we concluded that the trypsin/trypsinogen in turbot embryos was inherited from a maternal source and we suggested that trypsin in early development has multiple functions in the process of development. Copyright © 2015 Elsevier Inc. All rights reserved.

Parental contributions to early embryo development: influences of urinary phthalate and phthalate alternatives among couples undergoing IVF treatment.

PubMed

Wu, Haotian; Ashcraft, Lisa; Whitcomb, Brian W; Rahil, Tayyab; Tougias, Ellen; Sites, Cynthia K; Pilsner, J Richard

2017-01-01

Are preconception urinary concentrations of phthalates and phthalate alternatives associated with diminished early stage embryo quality in couples undergoing IVF? Male, but not female, urinary concentrations of select metabolites of phthalates and phthalate alternatives are associated with diminished blastocyst quality. Although phthalates are endocrine disrupting compounds associated with adverse reproductive health, they are in widespread use across the world. Male and female preconception exposures to select phthalates have been previously associated with adverse reproductive outcomes in both the general population and in those undergoing IVF. This prospective cohort included 50 subfertile couples undergoing IVF in western Massachusetts. This study includes the first 50 couples recruited from the Baystate Medical Center's Fertility Center in Springfield, MA, as part of the Sperm Environmental Epigenetics and Development Study (SEEDS). Relevant data from both partners, including embryo quality at the cleavage (Day 3) and blastocyst (Day 5) stages, were collected by clinic personnel during the normal course of an IVF cycle. A spot urine sample was collected from both male and female partners on the same day as semen sample procurement and oocyte retrieval. Concentrations of 17 urinary metabolite were quantified by liquid chromatography mass spectrometry and normalized via specific gravity. Generalized estimating equations were used to estimate odds ratios (OR) and 95% CI, with urinary phthalates and phthalate alternatives fitted as continuous variables and embryo quality as a binary variable. The 50 couples contributed 761 oocytes, of which 423 progressed to the cleavage stage, 261 were high-quality cleavage stage embryos, 137 were transferrable quality blastocysts and 47 were high-quality blastocysts. At the cleavage stage, male urinary monoethyl phthalate concentrations were positively associated with high-quality cleavage stage embryos (OR = 1.20, 95% CI 1

Effects of 5-Fluorodeoxyuridine and Related Halogenated Pyrimidines on the Sand-Dollar Embryo

PubMed Central

Karnofsky, David A.; Basch, Ross S.

1960-01-01

The embryo of the sand-dollar (Echinarachnius parma) was exposed to various concentrations of fluorinated pyrimidines immediately after fertilization. FUDR (5-fluorodeoxyuridine) was most active, and a concentration of 2 to 4 mγ/10 cc. (0.8 to 1.6 x 10-6 m.eq./liter) blocked development at the early blastula stage. Larger doses interrupted development at the same stage. This effect was prevented by thymidine (TDR) and thymine (T); and these pyrimidines protected against many times the minimal lethal concentration of FUDR. TDR was active as a protective agent if added just before early blastula formation. The other fluorinated pyrimidines, 5-fluorouracil (FU), 5-fluorouridine (FUR), 5-fluorocytidine (FCR), 5-fluorodeoxycytidine (FCDR), and 5-fluoroorotic acid (FO), were also studied. These drugs produced effects on embryonic development similar to those seen with FUDR. The effective concentrations, however, varied greatly. T and TDR provided protection against these drugs, but in most cases they were not so effective as against FUDR. 5-Bromodeoxyurdine (BrUDR), beginning at the early blastula stage, caused a random pattern of embryonic death up to the pluteus stage. This drug has been shown to be incorporated into bacterial DNA. BrUDR protected embryos against the early lethal effects of FUDR presumably acting as a thymidine substitute, but the embryos died subsequently in a pattern similar to that seen with BrUDR alone. FUDR and BrUDR appear to inhibit the formation and alter the structure of DNA, respectively, distinctive effects whch may provide a means for studying the role of DNA in embryonic development. PMID:14404541

Effects of 5-fluorodeoxyuridine and related halogenated pyrimidines on the sand-dollar embryo.

PubMed

KARNOFSKY, D A; BASCH, R S

1960-02-01

The embryo of the sand-dollar (Echinarachnius parma) was exposed to various concentrations of fluorinated pyrimidines immediately after fertilization. FUDR (5-fluorodeoxyuridine) was most active, and a concentration of 2 to 4 mgamma/10 cc. (0.8 to 1.6 x 10(-6) m.eq./liter) blocked development at the early blastula stage. Larger doses interrupted development at the same stage. This effect was prevented by thymidine (TDR) and thymine (T); and these pyrimidines protected against many times the minimal lethal concentration of FUDR. TDR was active as a protective agent if added just before early blastula formation. The other fluorinated pyrimidines, 5-fluorouracil (FU), 5-fluorouridine (FUR), 5-fluorocytidine (FCR), 5-fluorodeoxycytidine (FCDR), and 5-fluoroorotic acid (FO), were also studied. These drugs produced effects on embryonic development similar to those seen with FUDR. The effective concentrations, however, varied greatly. T and TDR provided protection against these drugs, but in most cases they were not so effective as against FUDR. 5-Bromodeoxyurdine (BrUDR), beginning at the early blastula stage, caused a random pattern of embryonic death up to the pluteus stage. This drug has been shown to be incorporated into bacterial DNA. BrUDR protected embryos against the early lethal effects of FUDR presumably acting as a thymidine substitute, but the embryos died subsequently in a pattern similar to that seen with BrUDR alone. FUDR and BrUDR appear to inhibit the formation and alter the structure of DNA, respectively, distinctive effects whch may provide a means for studying the role of DNA in embryonic development.

Effect of progesterone supplementation in the first week post conception on embryo survival in beef heifers.

PubMed

Beltman, M E; Lonergan, P; Diskin, M G; Roche, J F; Crowe, M A

2009-04-15

Progesterone is essential for establishment and maintenance of pregnancy in mammals. The objective of this study was to examine the effect of elevating progesterone during the different physiological stages of early embryo development on embryo survival. Estrus was synchronized in cross-bred beef heifers (n=197, approximately 2-years old) and they were inseminated 12-18h after estrus onset (=Day 0). Inseminated heifers were randomly assigned to 1 of 3 treatments: (1) Control, n=69; (2) progesterone supplementation using a Controlled Internal Drug Release Device (CIDR) from Day 3 to 6.5, n=64; or (3) progesterone supplementation using a CIDR from Day 4.5 to 8, n=64. Body condition (BCS) and locomotion scores (scale of 1-5) were recorded for all animals. Animals with a locomotion score >/=4 (very lame) were excluded. Embryo survival rate was determined at slaughter on Day 25. Conceptus length and weight were recorded and the corpus luteum (CL) of all pregnant animals was dissected and weighed. Supplementation with exogenous progesterone increased (P<0.05) peripheral progesterone concentrations, but did not affect embryo survival rate compared with controls. Mean CL weight, conceptus length and conceptus weight were not different between treatments. There was a positive relationship (P<0.04) between the increase in progesterone concentrations from Days 3 to 6.5 and embryo survival rate in treated heifers and a similar trend existed between the increase from Days 4.5 to 8 (P<0.06). There was also a positive relationship (P<0.05) between the progesterone concentration on Day 6.5 and the embryo survival rate in treated heifers. A direct correlation was seen between locomotion score and embryo survival rate, with higher (P<0.05) early embryo survival rates in heifers with a lower locomotion score. In conclusion, supplementation with progesterone at different stages of early embryo development increased peripheral progesterone concentration and resulted in a positive

The mRNA-bound proteome of the early fly embryo

PubMed Central

Wessels, Hans-Hermann; Imami, Koshi; Baltz, Alexander G.; Kolinski, Marcin; Beldovskaya, Anastasia; Selbach, Matthias; Small, Stephen; Ohler, Uwe; Landthaler, Markus

2016-01-01

Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first 2 h of Drosophila melanogaster embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 exhibited previously uncharacterized RNA-binding activity. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first 2 h of embryonic development. Integrating our resource with in situ hybridization data from existing databases showed that mRNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation. PMID:27197210

Are there symplastic connections between the endosperm and embryo in some angiosperms?--a lesson from the Crassulaceae family.

PubMed

Kozieradzka-Kiszkurno, Małgorzata; Płachno, Bartosz Jan

2012-10-01

It is believed that there is symplastic isolation between the embryo (new sporophyte) and the endosperm (maternal-parental origin tissue, which nourishes the embryo) in angiosperms. However, in embryological literature there are rare examples in which plasmodesmata between the embryo suspensor and endosperm cells have been recorded (three species from Fabaceae). This study was undertaken in order to test the hypothesis that plasmodesmata between the embryo suspensor and the endosperm are not so rare but also occur in other angiosperm families; in order to check this, we used the Crassulaceae family because embryogenesis in Crassulaceae has been studied extensively at an ultrastructure level recently and also we tread members of this family as model for suspensor physiology and function studies. These plasmodesmata even occurred between the basal cell of the two-celled proembryo and endosperm cells. The plasmodesmata were simple at this stage of development. During the development of the embryo proper and the suspensor, the structure of plasmodesmata changes. They were branched and connected with electron-dense material. Our results suggest that in Crassulaceae with plasmodesmata between the endosperm and suspensor, symplastic connectivity at this cell-cell boundary is still reduced or blocked at a very early stage of embryo development (before the globular stage). The occurrence of plasmodesmata between the embryo suspensor and endosperm cells suggests possible symplastic transport between these different organs, at least at a very early stage of embryo development. However, whether this transport actually occurs needs to be proven experimentally. A broader analysis of plants from various families would show whether the occurrence of plasmodesmata between the embryo suspensor and the endosperm are typical embryological characteristics and if this is useful in discussions about angiosperm systematic and evolution.

Excess Imidacloprid Exposure Causes the Heart Tube Malformation of Chick Embryos.

PubMed

Gao, Lin-Rui; Li, Shuai; Zhang, Jing; Liang, Chang; Chen, En-Ni; Zhang, Shi-Yao; Chuai, Manli; Bao, Yong-Ping; Wang, Guang; Yang, Xuesong

2016-11-30

As a neonicotinoid pesticide, imidacloprid is widely used to control sucking insects on agricultural planting and fleas on domestic animals. However, the extent to which imidacloprid exposure has an influence on cardiogensis in early embryogenesis is still poorly understood. In vertebrates, the heart is the first organ to be formed. In this study, to address whether imidacloprid exposure affects early heart development, the early chick embryo has been used as an experimental model because of its accessibility at its early developmental stage. The results demonstrate that exposure of the early chick embryo to imidacloprid caused malformation of heart tube. Furthermore, the data reveal that down-regulation of GATA4, NKX2.5, and BMP4 and up-regulation of Wnt3a led to aberrant cardiomyocyte differentiation. In addition, imidacloprid exposure interfered with basement membrane breakdown, E-cadherin/laminin expression, and mesoderm formation during the epithelial-mesenchymal transition (EMT) in gastrula chick embryos. Finally, the DiI-labeled cell migration trajectory indicated that imidacloprid restricted the cell migration of cardiac progenitors to primary heart field in gastrula chick embryos. A similar observation was also obtained from the cell migration assay of scratch wounds in vitro. Additionally, imidacloprid exposure negatively affected the cytoskeleton structure and expression of corresponding adhesion molecules. Taken together, these results reveal that the improper EMT, cardiac progenitor migration, and differentiation are responsible for imidacloprid exposure-induced malformation of heart tube during chick embryo development.

Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors.

PubMed

Kimber, S J; Sneddon, S F; Bloor, D J; El-Bareg, A M; Hawkhead, J A; Metcalfe, A D; Houghton, F D; Leese, H J; Rutherford, A; Lieberman, B A; Brison, D R

2008-05-01

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

AmphiFoxQ2, a novel winged helix/forkhead gene, exclusively marks the anterior end of the amphioxus embryo

NASA Technical Reports Server (NTRS)

Yu, Jr-Kai; Holland, Nicholas D.; Holland, Linda Z.

2003-01-01

A full-length FoxQ-related gene (AmphiFoxQ2) was isolated from amphioxus. Expression is first detectable in the animal/anterior hemisphere at the mid blastula stage. The midpoint of this expression domain coincides with the anterior pole of the embryo and is offset dorsally by about 20 degrees from the animal pole. During the gastrula stage, expression is limited to the anterior ectoderm. By the early neurula stage, expression remains in the anterior ectoderm and also appears in the adjacent anterior mesendoderm. By the early larval stages, expression is detectable in the anteriormost ectoderm and in the rostral tip of the notochord. AmphiFoxQ2 is never expressed anywhere except at the anterior tip of amphioxus embryos and larvae. This is the first gene known that exclusively marks the anterior pole of chordate embryos. It may, therefore, play an important role in establishing and/or maintaining the anterior/posterior axis.

Transcriptome profiles of embryos before and after cleavage in Eriocheir sinensis: identification of developmental genes at the earliest stages

NASA Astrophysics Data System (ADS)

Hui, Min; Cui, Zhaoxia; Liu, Yuan; Song, Chengwen

2017-07-01

In crab, embryogenesis is a complicated developmental program marked by a series of critical events. RNA-Sequencing technology offers developmental biologists a way to identify many more developmental genes than ever before. Here, we present a comprehensive analysis of the transcriptomes of Eriocheir sinensis oosperms (Os) and embryos at the 2-4 cell stage (Cs), which are separated by a cleavage event. A total of 18 923 unigenes were identified, and 403 genes matched with gene ontology (GO) terms related to developmental processes. In total, 432 differentially expressed genes (DEGs) were detected between the two stages. Nine DEGs were specifically expressed at only one stage. These DEGs may be relevant to stage-specific molecular events during development. A number of DEGs related to `hedgehog signaling pathway', `Wnt signaling pathway' `germplasm', `nervous system', `sensory perception' and `segment polarity' were identified as being up-regulated at the Cs stage. The results suggest that these embryonic developmental events begin before the early cleavage event in crabs, and that many of the genes expressed in the two transcriptomes might be maternal genes. Our study provides ample information for further research on the molecular mechanisms underlying crab development.

Live births after polar body biopsy and frozen-thawed cleavage stage embryo transfer: case report

PubMed Central

Guimarães, Fernando; Roque, Matheus; Valle, Marcello; Kostolias, Alessandra; de Azevedo, Rodrigo A; Martinhago, Ciro D; Sampaio, Marcos; Geber, Selmo

2016-01-01

Pre-implantation genetic diagnosis (PGD) or screening (PGS) technology, has emerged and developed in the past few years, benefiting couples as it allows the selection and transfer of healthy embryos during IVF treatments. These techniques can be performed in oocytes (polar-body biopsy) or embryos (blastomere or trophectoderm biopsy). In this case report, we describe the first two live births to be published in Brazil after a polar-body (PB) biopsy. In case 1, a 42-year-old was submitted to PB biopsy with PGS due to advanced maternal age and poor ovarian reserve. Five MII oocytes underwent first and second polar body biopsy and four cleavage embryos were cryopreserved. The PGS analysis resulted in two euploid embryos (next generation sequence). A frozen-thawed embryo transfer (FET) was performed after endometrial priming and a healthy baby was delivered after a cesarean section (37 weeks, female, 3390g, 47.5 cm). In case 2, a 40-year old patient with balanced translocation and poor ovarian response was submitted to PB biopsy. Two MII oocytes underwent first and second polar body biopsy and two embryos were cryopreserved in cleavage stage. The analysis resulted in one euploid embryo that was transferred after endometrial priming. A preterm healthy baby (34 weeks, female, 2100g, 40 cm) was delivered via cesarean section. In conclusion, although the blastocyst biopsy is the norm when performing PGS/PGD during IVF treatments, other alternatives (as PB biopsy) should be considered in some specific situations. PMID:28050963

Live births after polar body biopsy and frozen-thawed cleavage stage embryo transfer: case report.

PubMed

Guimarães, Fernando; Roque, Matheus; Valle, Marcello; Kostolias, Alessandra; Azevedo, Rodrigo A de; Martinhago, Ciro D; Sampaio, Marcos; Geber, Selmo

2016-12-01

Pre-implantation genetic diagnosis (PGD) or screening (PGS) technology, has emerged and developed in the past few years, benefiting couples as it allows the selection and transfer of healthy embryos during IVF treatments. These techniques can be performed in oocytes (polar-body biopsy) or embryos (blastomere or trophectoderm biopsy). In this case report, we describe the first two live births to be published in Brazil after a polar-body (PB) biopsy. In case 1, a 42-year-old was submitted to PB biopsy with PGS due to advanced maternal age and poor ovarian reserve. Five MII oocytes underwent first and second polar body biopsy and four cleavage embryos were cryopreserved. The PGS analysis resulted in two euploid embryos (next generation sequence). A frozen-thawed embryo transfer (FET) was performed after endometrial priming and a healthy baby was delivered after a cesarean section (37 weeks, female, 3390g, 47.5 cm). In case 2, a 40-year old patient with balanced translocation and poor ovarian response was submitted to PB biopsy. Two MII oocytes underwent first and second polar body biopsy and two embryos were cryopreserved in cleavage stage. The analysis resulted in one euploid embryo that was transferred after endometrial priming. A preterm healthy baby (34 weeks, female, 2100g, 40 cm) was delivered via cesarean section. In conclusion, although the blastocyst biopsy is the norm when performing PGS/PGD during IVF treatments, other alternatives (as PB biopsy) should be considered in some specific situations.

The Armadillo Repeat Gene ZAK IXIK Promotes Arabidopsis Early Embryo and Endosperm Development through a Distinctive Gametophytic Maternal Effect[C][W][OA

PubMed Central

Ngo, Quy A.; Baroux, Celia; Guthörl, Daniela; Mozerov, Peter; Collinge, Margaret A.; Sundaresan, Venkatesan; Grossniklaus, Ueli

2012-01-01

The proper balance of parental genomic contributions to the fertilized embryo and endosperm is essential for their normal growth and development. The characterization of many gametophytic maternal effect (GME) mutants affecting seed development indicates that there are certain classes of genes with a predominant maternal contribution. We present a detailed analysis of the GME mutant zak ixik (zix), which displays delayed and arrested growth at the earliest stages of embryo and endosperm development. ZIX encodes an Armadillo repeat (Arm) protein highly conserved across eukaryotes. Expression studies revealed that ZIX manifests a GME through preferential maternal expression in the early embryo and endosperm. This parent-of-origin–dependent expression is regulated by neither the histone and DNA methylation nor the DNA demethylation pathways known to regulate some other GME mutants. The ZIX protein is localized in the cytoplasm and nucleus of cells in reproductive tissues and actively dividing root zones. The maternal ZIX allele is required for the maternal expression of MINISEED3. Collectively, our results reveal a reproductive function of plant Arm proteins in promoting early seed growth, which is achieved through a distinct GME of ZIX that involves mechanisms for maternal allele-specific expression that are independent of the well-established pathways. PMID:23064319

Efficient Term Development of Vitrified Ferret Embryos Using a Novel Pipette Chamber Technique1

PubMed Central

Sun, Xingshen; Li, Ziyi; Yi, Yaling; Chen, Juan; Leno, Gregory H.; Engelhardt, John F.

2008-01-01

Development of an efficient cryopreservation technique for the domestic ferret is key for the long-term maintenance of valuable genetic specimens of this species and for the conservation of related endangered species. Unfortunately, current cryopreservation procedures, such as slow-rate freezing and vitrification with open pulled straws, are inefficient. In this report, we describe a pipette tip-based vitrification method that significantly improves the development of thawed ferret embryos following embryo transfer (ET). Ferret embryos at the morula (MR), compact morula (CM), and early blastocyst (EB) stages were vitrified using an Eppendorf microloader pipette tip as the chamber vessel. The rate of in vitro development was significantly (P < 0.05) higher among embryos vitrified at the CM (93.6%) and EB (100%) stages relative to those vitrified at the MR stages (58.7%). No significant developmental differences were observed when comparing CM and EB vitrified embryos with nonvitrified control CM (100%) and EB (100%) embryos. In addition, few differences in the ultrastructure of intracellular lipid droplets or in microfilament structure were observed between control embryos and embryos vitrified at any developmental stage. Vitrified-thawed CM/EB embryos cultured for 2 or 16 h before ET resulted in live birth rates of 71.3% and 77.4%, respectively. These rates were not significantly different from the control live birth rate (79.2%). However, culture for 32 h (25%) or 48 h (7.8%) after vitrification significantly reduced the rate of live births. These data indicate that the pipette chamber vitrification technique significantly improves the live birth rate of transferred ferret embryos relative to current state-of-the-art methods.. PMID:18633142

Neural differentiation of caudal cell mass (secondary neurulation) in chick embryos: Hamburger and Hamilton Stages 16-45.

PubMed

Yang, Hee-Jin; Wang, Kyu-Chang; Chi, Je G; Lee, Myung-Sook; Lee, Yun-Jin; Kim, Seung-Ki; Cho, Byung-Kyu

2003-04-14

In an attempt to understand the events in the secondary neurulation in embryonic stage, we investigated morphological changes in the tail bud of normal developing chick embryos. Hamburger and Hamilton stage 16-45 embryos were harvested and processed for light microscopic studies. The secondary neural tube is formed by aggregation of the caudal cell mass. Cells are arranged into a cord-like mass (medullary cord), which is continuous with the primary neural tube. Multiple small cavities develop in the medullary cord, and these cavities coalesce into one single lumen. The process of coalescence is completed by stage 35, and the whole neural tube is transformed into one tube with a single continuous lumen. At this stage, the terminal portion of the neural tube is bulged dorsally. Thereafter, the caudal portion of the neural tube regresses, and the proximal portion develops into normal spinal cord. Transient occlusion of the central canal was observed at stage 40 in one sample. The sequence of events elucidated in this study can be used as base-line data for experiments concerning congenital malformations involving secondary neurulation.

Optical coherence microscopy as a novel, non-invasive method for the 4D live imaging of early mammalian embryos.

PubMed

Karnowski, Karol; Ajduk, Anna; Wieloch, Bartosz; Tamborski, Szymon; Krawiec, Krzysztof; Wojtkowski, Maciej; Szkulmowski, Maciej

2017-06-23

Imaging of living cells based on traditional fluorescence and confocal laser scanning microscopy has delivered an enormous amount of information critical for understanding biological processes in single cells. However, the requirement for a high numerical aperture and fluorescent markers still limits researchers' ability to visualize the cellular architecture without causing short- and long-term photodamage. Optical coherence microscopy (OCM) is a promising alternative that circumvents the technical limitations of fluorescence imaging techniques and provides unique access to fundamental aspects of early embryonic development, without the requirement for sample pre-processing or labeling. In the present paper, we utilized the internal motion of cytoplasm, as well as custom scanning and signal processing protocols, to effectively reduce the speckle noise typical for standard OCM and enable high-resolution intracellular time-lapse imaging. To test our imaging system we used mouse and pig oocytes and embryos and visualized them through fertilization and the first embryonic division, as well as at selected stages of oogenesis and preimplantation development. Because all morphological and morphokinetic properties recorded by OCM are believed to be biomarkers of oocyte/embryo quality, OCM may represent a new chapter in imaging-based preimplantation embryo diagnostics.

Effects of gamma radiation on the early developmental stages of Zebrafish (Danio rerio).

PubMed

Praveen Kumar, M K; Shyama, S K; Kashif, Shamim; Dubey, S K; Avelyno, D'costa; Sonaye, B H; Kadam Samit, B; Chaubey, R C

2017-08-01

The zebrafish is gaining importance as a popular vertebrate model organism and is widely employed in ecotoxicological studies, especially for the biomonitoring of pollution in water bodies. There is limited data on the genetic mechanisms governing the adverse health effects in regards to an early developmental exposure to gamma radiation. In the present study zebrafish (Danio rerio) embryos were exposed to 1, 2.5, 5, 7.5 and 10Gy of gamma radiation at 3h post fertilization (hpf). Different developmental toxicity endpoints were investigated. Further, expression of genes associated with the development and DNA damage i.e. (sox2 sox19a and p53) were evaluated using Quantitative PCR (qPCR). The significant changes in the expression of sox2 sox19a and p53 genes were observed. This data was supported the developmental defects observed in the zebrafish embryo exposed to gamma radiation such as i.e. increased DNA damage, decreased hatching rate, increase in median hatching time, decreased body length, increased mortality rate, increased morphological deformities. Further, study shows that the potential ecotoxicological threat of gamma radiation on the early developmental stages of zebrafish. Further, it revealed that the above parameters can be used as predictive biomarkers of gamma radiation exposure. Copyright © 2017. Published by Elsevier Inc.

[Calcium in the developing skeletal muscles of the chick embryo].

PubMed

Samosudova, N V; Enenko, S O; Larin, Iu S; Shungskaia, V E

1982-07-01

The osmium-pyroantimonate technique was used for the ultrastructural study of Ca2+-localization in two types of chick embryo skeletal muscles: m. pectoralis and m. soleus. In 8- and 12-day old embryos the pyroantimonate precipitate was found on plasmalemma, condensed chromatine and ribosomes and in N-lines of I-band. During myogenesis (15-, 21-day old embryos) the calcium precipitate is redistributed from the above mentioned sites to terminal cisternae and N-line of I-band. It is proposed that calcium of N-lines may be involved in the glycogenolysis, its association with the muscle contraction occurring particularly at early developmental stages.

Improving the quality of miniature pig somatic cell nuclear transfer blastocysts: aggregation of SCNT embryos at the four-cell stage.

PubMed

Terashita, Y; Sugimura, S; Kudo, Y; Amano, R; Hiradate, Y; Sato, E

2011-04-01

Miniature pigs share many similar characteristics such as anatomy, physiology and body size with humans and are expected to become important animal models for therapeutic cloning using embryonic stem cells (ESCs) derived by somatic cell nuclear transfer (SCNT). In the present study, we observed that miniature pig SCNT blastocysts possessed a lower total number of nuclei and a lower percentage of POU5F1-positive cells than those possessed by in vitro fertilized (IVF) blastocysts. To overcome these problems, we evaluated the applicability of aggregating miniature pig SCNT embryos at the four-cell stage. We showed that (i) aggregation of two or three miniature pig SCNT embryos at the four-cell stage improves the total number of nuclei and the percentage of POU5F1-positive cells in blastocysts, and (ii) IVF blastocysts with low cell numbers induced by the removal of two blastomeres at the four-cell stage did not exhibit a decrease in the percentage of POU5F1-positive cells. These results suggest that the aggregation of miniature pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of miniature pig SCNT blastocysts and indicating that improvement in the percentage of POU5F1-positive cells in aggregated SCNT embryos is not simply the consequence of increased cell numbers. © 2010 Blackwell Verlag GmbH.

Starch Turnover and Metabolism during Flower and Early Embryo Development1[CC-BY

PubMed Central

Pazmino, Diana; Gagliardini, Valeria

2016-01-01

The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed. PMID:27794100

Immunogold detection of glycoprotein antigens in sea urchin embryos.

PubMed

Benson, N C; Benson, S C; Wilt, F

1989-01-01

Four developmental stages of sea urchin embryos were labeled with colloidal gold in an attempt to elucidate the intracellular trafficking patterns within the cells that produce the glycoprotein matrix of the embryonic spicule. The primary mesenchyme cells (PMCs) form a syncytium and secrete an organic matrix on which calcium carbonate is laid down to form an endoskeletal spicule. The organic matrix has been isolated and characterized as glycoprotein consisting of four major bands. Polyclonal antibodies to these glycoproteins were used to label embryos from the mesenchyme blastula, early gastrula, late gastrula, and plutei stages of development. The label is concentrated in the Golgi complex and associated vesicles, in secretory vesicles, and in the organic matrix. The density of the labeling increases as development proceeds.

Ectodermal Wnt6 is an early negative regulator of limb chondrogenesis in the chicken embryo

PubMed Central

2010-01-01

Background Pattern formation of the limb skeleton is regulated by a complex interplay of signaling centers located in the ectodermal sheath and mesenchymal core of the limb anlagen, which results, in the forelimb, in the coordinate array of humerus, radius, ulna, carpals, metacarpals and digits. Much less understood is why skeletal elements form only in the central mesenchyme of the limb, whereas muscle anlagen develop in the peripheral mesenchyme ensheathing the chondrogenic center. Classical studies have suggested a role of the limb ectoderm as a negative regulator of limb chondrogenesis. Results In this paper, we investigated the molecular nature of the inhibitory influence of the ectoderm on limb chondrogenesis in the avian embryo in vivo. We show that ectoderm ablation in the early limb bud leads to increased and ectopic expression of early chondrogenic marker genes like Sox9 and Collagen II, indicating that the limb ectoderm inhibits limb chondrogenesis at an early stage of the chondrogenic cascade. To investigate the molecular nature of the inhibitory influence of the ectoderm, we ectopically expressed Wnt6, which is presently the only known Wnt expressed throughout the avian limb ectoderm, and found that Wnt6 overexpression leads to reduced expression of the early chondrogenic marker genes Sox9 and Collagen II. Conclusion Our results suggest that the inhibitory influence of the ectoderm on limb chondrogenesis acts on an early stage of chondrogenesis upsteam of Sox9 and Collagen II. We identify Wnt6 as a candidate mediator of ectodermal chondrogenic inhibition in vivo. We propose a model of Wnt-mediated centripetal patterning of the limb by the surface ectoderm. PMID:20334703

Early-stage aeolian protodune development and migration

NASA Astrophysics Data System (ADS)

Nield, J. M.; Baddock, M. C.; Wiggs, G.

2017-12-01

Early-stage bedforms, or protodunes, can be observed to form on sandy beaches, desert gravels or superimposed on the surfaces of larger dunes and can develop topography of 0.1 m or more over several hours. These protodunes are the precursors to embryo and eventually mature dunes, and so it is important to understand how feedbacks between flow, transport and form contribute to this development sequence. Whilst theory and conceptual models have offered some explanation for protodune existence and development, we know surprisingly little about how these bedforms initiate and migrate because it is difficult to measure small changes in form (millimetres; seconds) on highly active surfaces of limited topographic expression. Here, we employ terrestrial laser scanning (TLS) to measure morphological change at the high frequency and spatial resolution (sub-millimetre) required to gain new insights into protodune behaviour. Along with TLS derived saltation and surface moisture, additional sediment flux and windspeed measurements help to elucidate how the protodune topography interacts with airflow and sand transport. We focus on a number of coastal bedforms in various development stages including a 0.06 m high protodune which grew vertically by 0.005 m in two hours with the switch from erosion to deposition identified to occur at a point 0.07 m upwind of the crest. This growth was associated with a reduction in time-averaged sediment flux of 18% over the crestal region. We also observed a decline in lower stoss slope steepness (by 3°) and a steepening of the lee slope, indicating a reshaping of initial protodune form towards the morphology of a more mature dune. Our findings highlight the crucial role of form-flow feedbacks, even on very small bedforms, in driving early-stage bedform growth and development, and show how the use of high resolution TLS to measure both surface topography and grains moving above the surface, can offer new insights into a long standing deficiency

Transcriptome analyses of rhesus monkey preimplantation embryos reveal a reduced capacity for DNA double-strand break repair in primate oocytes and early embryos

PubMed Central

Wang, Xinyi; Liu, Denghui; He, Dajian; Suo, Shengbao; Xia, Xian; He, Xiechao; Han, Jing-Dong J.; Zheng, Ping

2017-01-01

Preimplantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA), and cell-fate commitment. The molecular basis of these processes remains obscure in primates in which there is a high rate of embryo wastage. Thus, understanding the factors involved in genome reprogramming and ZGA might help reproductive success during this susceptible period of early development and generate induced pluripotent stem cells with greater efficiency. Moreover, explaining the molecular basis responsible for embryo wastage in primates will greatly expand our knowledge of species evolution. By using RNA-seq in single and pooled oocytes and embryos, we defined the transcriptome throughout preimplantation development in rhesus monkey. In comparison to archival human and mouse data, we found that the transcriptome dynamics of monkey oocytes and embryos were very similar to those of human but very different from those of mouse. We identified several classes of maternal and zygotic genes, whose expression peaks were highly correlated with the time frames of genome reprogramming, ZGA, and cell-fate commitment, respectively. Importantly, comparison of the ZGA-related network modules among the three species revealed less robust surveillance of genomic instability in primate oocytes and embryos than in rodents, particularly in the pathways of DNA damage signaling and homology-directed DNA double-strand break repair. This study highlights the utility of monkey models to better understand the molecular basis for genome reprogramming, ZGA, and genomic stability surveillance in human early embryogenesis and may provide insights for improved homologous recombination-mediated gene editing in monkey. PMID:28223401

Effects of embryo size at transfer (whole versus demi) and early pregnancy progesterone supplementation on embryo growth and pregnancy-specific protein bovine concentrations in recipient dairy heifers.

PubMed

Lopes-da-Costa, L; Chagas e Silva, J; Deloche, M C; Jeanguyot, N; Humblot, P; Horta, A E M

2011-08-01

The objectives of this study were to evaluate embryonic size and survival, plasma progesterone (P4) and pregnancy-specific protein bovine (PSPB) concentrations in early pregnancies (n = 99) following the transfer of one whole (n = 66) or one demi (n = 33) embryo to recipient virgin dairy heifers. The experiment was designed to evaluate the fixed effects of embryo size at transfer (whole or demi embryo) on Day 7 of the estrous cycle (Day 0 = estrus) and P4 supplementation between Days 7 to 19 through an intravaginal device (yes or no) on plasma P4 and PSPB concentrations and on embryo measurements. Plasma P4 concentrations were measured by RIA on Days 0, 7, 14, 19, 21, 25, 35, 42, 49, 56 and 63 of pregnancy and, PSPB concentrations were measured by ELISA on Days 7, 21, 25, 35, 42, 49, 56 and 63. The presence of an embryonic vesicle was detected on Day 25, embryonic/fetal movements and heartbeat were evaluated on Days 42 and 63 and embryo measurements [crown-rump length (CRL) and width at mid body] were obtained on Day 42 through ultrasonography. In non-supplemented pregnancies, Day 42 whole embryos had higher (P < 0.05) CRL and width than demi embryos, but the difference averaged only 1 to 2 mm. In P4 supplemented pregnancies, whole and demi embryos attained a similar size on Day 42 of pregnancy. Embryo size at transfer, early exogenous P4 supplementation and their interactions had no effects (P > 0.05) on plasma P4 concentrations. However, the post-hoc LSD evaluation showed that plasma P4 concentrations on Day 25 were higher (P < 0.001) in whole than in demi embryo derived pregnancies and, that exogenous P4 supplementation increased (P < 0.05) plasma P4 concentrations on Day 19 of pregnancy. The plasma PSPB detection rate on Days 7 to 63 of pregnancy was similar in pregnancies resulting from the transfer of whole and demi embryos. From a total of 93 recipients remaining pregnant until Day 63, plasma PSPB was constantly undetectable on Day 7, was detected in 4% of

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...

The Chromosomal Constitution of Embryos Arising from Monopronuclear Oocytes in Programmes of Assisted Reproduction

PubMed Central

2014-01-01

The assessment of oocytes showing only one pronucleus during assisted reproduction is associated with uncertainty. A compilation of data on the genetic constitution of different developmental stages shows that affected oocytes are able to develop into haploid, diploid, and mosaic embryos with more or less complex chromosomal compositions. In the majority of cases (~80%), haploidy appears to be caused by gynogenesis, whereas parthenogenesis or androgenesis is less common. Most of the diploid embryos result from a fertilization event involving asynchronous formation of the two pronuclei or pronuclear fusion at a very early stage. Uniparental diploidy may sometimes occur if one pronucleus fails to develop and the other pronucleus already contains a diploid genome or alternatively a haploid genome undergoes endoreduplication. In general, the chance of obtaining a biparental diploid embryo appears higher after conventional in vitro fertilization than after intracytoplasmic sperm injection. If a transfer of embryos obtained from monopronuclear oocytes is envisaged, it should be tried to culture them up to the blastocyst since most haploid embryos are not able to reach this stage. Comprehensive counselling of patients on potential risks is advisable before transfer and a preimplantation genetic diagnosis could be offered if available. PMID:25763399

Embryonic cardiac morphometry in Carnegie stages 15-23, from the Complutense University of Madrid Institute of Embryology Human Embryo Collection.

PubMed

Arráez-Aybar, L A; Turrero-Nogués, A; Marantos-Gamarra, D G

2008-01-01

We performed a morphometric study of cardiac development on human embryos to complement the scarce data on human embryonic cardiac morphometry and to attempt to establish, from these, algorithms describing cardiac growth during the second month of gestation. Thirty human embryos from Carnegie stages 15-23 were included in the study. Shrinkage and compression effects from fixation and inclusion in paraffin were considered in our calculations. Growth of the cardiac (whole heart) volume and volume of ventricular myocardium through the Carnegie stages were analysed by ANOVA. Linear correlation was used to describe the relationship between the ventricular myocardium and cardiac volumes. Comparisons of models were carried out through the R2 statistic. The relationship volume of ventricular myocardium versus cardiac volume is expressed by the equation: cardiac volume = 0.6266 + 2.4778 volume of ventricular myocardium. The relationship cardiac volume versus crown-rump length is expressed by the equation: cardiac volume = 1.3 e(0.126 CR length), where e is the base of natural logarithms. At a clinical level, these results can contribute towards the establishment of a normogram for cardiac development, useful for the design of strategies for early diagnosis of congenital heart disease. They can also help in the study of embryogenesis, for example in the discussion of ventricular trabeculation. Copyright 2007 S. Karger AG, Basel.

Evaluation of Stage-Dependent Genotoxic Effect of Roundup® (Glyphosate) on Caiman latirostris Embryos.

PubMed

Burella, Pamela Mariana; Simoniello, Maria Fernanda; Poletta, Gisela Laura

2017-01-01

The agricultural expansion over the past decades, along with the associated increase in the use of pesticides, represents a high risk for many wild species. Caiman latirostris is a South American caiman with many features that make it highly vulnerable to pesticide exposure. Considering previous finding on the genotoxicity of the glyphosate-based formulation Roundup ® in this species, the aim of this study was to evaluate the possible stage-dependent effect of this compound on C. latirostris embryos through the Comet assay (CA), micronuclei (MN), and nuclear abnormalities (NA) tests. Caiman eggs were exposed to three effective concentrations of Roundup® (750, 1250, 1750 µg/egg) in three different stages of the incubation period (total duration 70 ± 3 days at 31 ± 2 °C) of approximately 23 days each. A statistically significant difference in DNA damage determined by the CA was found between groups exposed to different concentrations of RU (p < 0.05) and the negative control, but no difference was observed among the three stages of exposure within any treatment (p > 0.05). There was no differences in the MN or NA frequencies between the different groups and the negative control (p > 0.05), nor among the different stages within each treatment. The results obtained in this study indicate that RU produce DNA damage on C. latirostris embryos independently of the developmental stage where the exposure occurs, implying an important risk for the species during all its period of development, when pesticide application is at maximum rate.

PXD101 significantly improves nuclear reprogramming and the in vitro developmental competence of porcine SCNT embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Jun-Xue; Kang, Jin-Dan; Li, Suo

2015-01-02

Highlights: • First explored that the effects of PXD101 on the development of SCNT embryos in vitro. • 0.5 μM PXD101 treated for 24 h improved the development of porcine SCNT embryos. • Level of AcH3K9 was significantly higher than control group at early stages. - Abstract: In this study, we investigated the effects of the histone deacetylase inhibitor PXD101 (belinostat) on the preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos and their expression of the epigenetic markers histone H3 acetylated at lysine 9 (AcH3K9). We compared the in vitro developmental competence of SCNT embryos treated with variousmore » concentrations of PXD101 for 24 h. Treatment with 0.5 μM PXD101 significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3% vs. 11.5%, P < 0.05). We tested the in vitro developmental competence of SCNT embryos treated with 0.5 μM PXD101 for various amounts of times following activation. Treatment for 24 h significantly improved the development of porcine SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7% vs. 10.6%, P < 0.05). PXD101-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and four fetuses developed. PXD101 treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9 in embryos at the pseudo-pronuclear and 2-cell stages. At these stages, the fluorescence intensities of immunostaining for AcH3K9 were significantly higher in PXD101-treated embryos than in control untreated embryos. In conclusion, this study demonstrates that PXD101 can significantly improve the in vitro and in vivo developmental competence of porcine SCNT embryos and can enhance their nuclear reprogramming.« less

The Effect of Levetiracetam on Closure of the Midline in Early Chicken Embryos.

PubMed

Ozgural, Onur; Armagan, Ercan; Bozkurt, Melih; Eroglu, Umit; Kahilogullari, Gokmen; Unlu, Agahan

2015-01-01

Genetic predisposition and some environmental factors play an important role in the development of neural tube defects. Levetiracetam is a new drug that has been approved in the treatment of partial seizures. We aimed in this study to determine the effect of levetiracetam on chick embryos. One hundred and sixty fertile non-pathogenic Super Nick eggs were incubated for 24 hours and were divided into four groups of 40 eggs each. Levetiracetam was administered via the sub-blastodermic route. The eggs were incubated for another 24 hours. All eggs were opened at the 48th hour, and the embryos were evaluated morphologically and histopathologically. The effects of levetiracetam on the embryo were correlated with the dose of levetiracetam. In the light of the results, it was determined that the use of increasing doses of levetiracetam led to defects of midline closure in early chicken embryos. Levetiracetam, a new antiepileptic drug that is effective especially on calcium ion concentration, leads to defects in midline closure in embryos in a dose-dependent manner. Further studies are needed to show the mechanism of embryonic damage and the mechanisms of its teratogenous effects associated with genetic and environmental factors.

Developmental toxicity of PAH mixtures in fish early life stages. Part II: adverse effects in Japanese medaka.

PubMed

Le Bihanic, Florane; Clérandeau, Christelle; Le Menach, Karyn; Morin, Bénédicte; Budzinski, Hélène; Cousin, Xavier; Cachot, Jérôme

2014-12-01

In aquatic environments, polycyclic aromatic hydrocarbons (PAHs) mostly occur as complex mixtures, for which risk assessment remains problematic. To better understand the effects of PAH mixture toxicity on fish early life stages, this study compared the developmental toxicity of three PAH complex mixtures. These mixtures were extracted from a PAH-contaminated sediment (Seine estuary, France) and two oils (Arabian Light and Erika). For each fraction, artificial sediment was spiked at three different environmental concentrations roughly equivalent to 0.5, 4, and 10 μg total PAH g(-1) dw. Japanese medaka embryos were incubated on these PAH-spiked sediments throughout their development, right up until hatching. Several endpoints were recorded at different developmental stages, including acute endpoints, morphological abnormalities, larvae locomotion, and genotoxicity (comet and micronucleus assays). The three PAH fractions delayed hatching, induced developmental abnormalities, disrupted larvae swimming activity, and damaged DNA at environmental concentrations. Differences in toxicity levels, likely related to differences in PAH proportions, were highlighted between fractions. The Arabian Light and Erika petrogenic fractions, containing a high proportion of alkylated PAHs and low molecular weight PAHs, were more toxic to Japanese medaka early life stages than the pyrolytic fraction. This was not supported by the toxic equivalency approach, which appeared unsuitable for assessing the toxicity of the three PAH fractions to fish early life stages. This study highlights the potential risks posed by environmental mixtures of alkylated and low molecular weight PAHs to early stages of fish development.

Deep cytoplasmic rearrangements in ventralized Xenopus embryos

NASA Technical Reports Server (NTRS)

Brown, E. E.; Denegre, J. M.; Danilchik, M. V.

1993-01-01

Following fertilization in Xenopus, dramatic rearrangements of the egg cytoplasm relocalize maternally synthesized egg components. During the first cell cycle the vegetal yolk mass rotates relative to the egg surface, toward the sperm entry point (SEP) (J. P. Vincent, G. F. Oster, and J. C. Gerhart, 1986, Dev. Biol. 113, 484-500), while concomitant deep cytoplasmic rearrangements occur in the animal hemisphere (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). In this paper we examine the role of vegetal yolk mass rotation in producing the animal cytoplasmic rearrangements. We inhibited rotation by uv-irradiating embryos during the first cell cycle, a treatment that yields an extremely ventralized phenotype. Both uv-irradiated embryos and unirradiated control embryos show cytoplasmic rearrangements in the animal hemisphere during the first cell cycle. Cytoplasmic rearrangements on the SEP side of the embryo associated with the path of the sperm pronucleus, plus a swirl on the anti-SEP (dorsal) side, are seen, whether or not yolk mass rotation has occurred. This result suggests a role for the expanding sperm aster in directing animal hemisphere cytoplasmic movements. In unirradiated control embryos the anti-SEP (dorsal) swirl is larger than that in uv-irradiated embryos and often extends into the vegetal hemisphere, consistent with the animal cytoplasm having been pulled dorsally and vegetally by the sliding vegetal yolk mass. Thus the yolk mass rotation may normally enhance the dorsalward cytoplasmic movement, begun by the sperm aster, enough to induce normal axis formation. We extended our observations of unirradiated control and uv-irradiated embryos through early cleavages. The vegetal extent of the anti-SEP (dorsal) swirl pattern seen in control embryos persists through the early cleavage period, such that labeled animal cytoplasm extends deep into dorsal third-tier blastomeres at the 32-cell stage. Significantly, in uv-irradiated embryos

Label free cell-tracking and division detection based on 2D time-lapse images for lineage analysis of early embryo development.

PubMed

Cicconet, Marcelo; Gutwein, Michelle; Gunsalus, Kristin C; Geiger, Davi

2014-08-01

In this paper we report a database and a series of techniques related to the problem of tracking cells, and detecting their divisions, in time-lapse movies of mammalian embryos. Our contributions are (1) a method for counting embryos in a well, and cropping each individual embryo across frames, to create individual movies for cell tracking; (2) a semi-automated method for cell tracking that works up to the 8-cell stage, along with a software implementation available to the public (this software was used to build the reported database); (3) an algorithm for automatic tracking up to the 4-cell stage, based on histograms of mirror symmetry coefficients captured using wavelets; (4) a cell-tracking database containing 100 annotated examples of mammalian embryos up to the 8-cell stage; and (5) statistical analysis of various timing distributions obtained from those examples. Copyright © 2014 Elsevier Ltd. All rights reserved.

Xmsx-1 modifies mesodermal tissue pattern along dorsoventral axis in Xenopus laevis embryo.

PubMed

Maeda, R; Kobayashi, A; Sekine, R; Lin, J J; Kung, H; Maéno, M

1997-07-01

This study analyzes the expression and the function of Xenopus msx-1 (Xmsx-1) in embryos, in relation to the ventralizing activity of bone morphogenetic protein-4 (BMP-4). Expression of Xmsx-1 was increased in UV-treated ventralized embryos and decreased in LiCl-treated dorsalized embryos at the neurula stage (stage 14). Whole-mount in situ hybridization analysis showed that Xmsx-1 is expressed in marginal zone and animal pole areas, laterally and ventrally, but not dorsally, at mid-gastrula (stage 11) and late-gastrula (stage 13) stages. Injection of BMP-4 RNA, but not activin RNA, induced Xmsx-1 expression in the dorsal marginal zone at the early gastrula stage (stage 10+), and introduction of a dominant negative form of BMP-4 receptor RNA suppressed Xmsx-1 expression in animal cap and ventral marginal zone explants at stage 14. Thus, Xmsx-1 is a target gene specifically regulated by BMP-4 signaling. Embryos injected with Xmsx-1 RNA in dorsal blastomeres at the 4-cell stage exhibited a ventralized phenotype, with microcephaly and swollen abdomen. Histological observation and immunostaining revealed that these embryos had a large block of muscle tissue in the dorsal mesodermal area instead of notochord. On the basis of molecular marker analysis, however, the injection of Xmsx-1 RNA did not induce the expression of alpha-globin, nor reduce cardiac alpha-actin in dorsal marginal zone explants. Furthermore, a significant amount of alpha-actin was induced and alpha-globin was turned off in the ventral marginal zone explants injected with Xmsx-1. These results indicated that Xmsx-1 is a target gene of BMP-4 signaling, but possesses a distinct activity on dorsal-ventral patterning of mesodermal tissues.

Kid depletion in mouse oocytes associated with multinucleated blastomere formation and inferior embryo development.

PubMed

Egashira, Akiyoshi; Yamauchi, Nobuhiko; Islam, Md Rashedul; Yamagami, Kazuki; Tanaka, Asami; Suyama, Hikaru; El-Sayed, El-Sharawy Mohamed; Tabata, Shoji; Kuramoto, Takashi

2016-08-01

This study investigated the knockdown (KD) of Kid on maturation developmental competence and multinucleation of mouse germinal vesicle (GV) oocytes after parthenogenetic activation. Data revealed that Kid messenger RNA (mRNA) was expressed in GV and MII stage oocyte and 1- and 2-cell embryos. Additionally, Kid mRNA expression in the Kid KD group decreased by nearly 46% compared to the control small interfering RNA (siRNA) groups. The rate of multinucleated embryos in the Kid KD group (52.4%) was significantly higher (P < 0.05) than the control siRNA group (4.7%). Finally, the developmental rates were significantly lower in the Kid siRNA group at > 4-cell stage (28.6% vs. 53.5%) and the blastocyst stage (2.4% vs. 23.3%) compared to the control siRNA groups. Suppression of Kid using siRNA caused multinucleation in early embryos with high frequency and it may increase 2- to 4-cell arrested embryos and reduce the developmental competence to blastocyst. © 2016 Japanese Society of Animal Science.

Editor's Highlight: Hydroxyurea Exposure Activates the P53 Signaling Pathway in Murine Organogenesis-Stage Embryos

PubMed Central

El Husseini, Nazem; Schlisser, Ava E.; Hales, Barbara F.

2016-01-01

Hydroxyurea, an anticancer agent and potent teratogen, induces oxidative stress and activates a DNA damage response pathway in the gestation day (GD) 9 mouse embryo. To delineate the stress response pathways activated by this drug, we investigated the effect of hydroxyurea exposure on the transcriptome of GD 9 embryos. Timed pregnant CD-1 mice were treated with saline or hydroxyurea (400 mg/kg or 600 mg/kg) on GD 9; embryonic gene and protein expression were examined 3 h later. Microarray analysis revealed that the expression of 1346 probe sets changed significantly in embryos exposed to hydroxyurea compared with controls; the P53 signaling pathway was highly affected. In addition, P53 related family members, P63 and P73, were predicted to be activated and had common and unique downstream targets. Western blot analysis revealed that active phospho-P53 was significantly increased in drug-exposed embryos; confocal microscopy showed that the translocation of phospho-P53 to the nucleus was widespread in the embryo. Furthermore, qRT-PCR showed that the expression of P53-regulated genes (Cdkn1A, Fas, and Trp53inp1) was significantly upregulated in hydroxyurea-exposed embryos; the concentration of the redox sensitive P53INP1 protein was also increased in a hydroxyurea dose-dependent fashion. Thus, hydroxyurea elicits a significant effect on the transcriptome of the organogenesis stage murine embryo, activating several key developmental signaling pathways related to DNA damage and oxidative stress. We propose that the P53 pathway plays a central role in the embryonic stress response and the developmental outcome after teratogen exposure. PMID:27208086

Automation and Optimization of Multipulse Laser Zona Drilling of Mouse Embryos During Embryo Biopsy.

PubMed

Wong, Christopher Yee; Mills, James K

2017-03-01

Laser zona drilling (LZD) is a required step in many embryonic surgical procedures, for example, assisted hatching and preimplantation genetic diagnosis. LZD involves the ablation of the zona pellucida (ZP) using a laser while minimizing potentially harmful thermal effects on critical internal cell structures. Develop a method for the automation and optimization of multipulse LZD, applied to cleavage-stage embryos. A two-stage optimization is used. The first stage uses computer vision algorithms to identify embryonic structures and determines the optimal ablation zone farthest away from critical structures such as blastomeres. The second stage combines a genetic algorithm with a previously reported thermal analysis of LZD to optimize the combination of laser pulse locations and pulse durations. The goal is to minimize the peak temperature experienced by the blastomeres while creating the desired opening in the ZP. A proof of concept of the proposed LZD automation and optimization method is demonstrated through experiments on mouse embryos with positive results, as adequately sized openings are created. Automation of LZD is feasible and is a viable step toward the automation of embryo biopsy procedures. LZD is a common but delicate procedure performed by human operators using subjective methods to gauge proper LZD procedure. Automation of LZD removes human error to increase the success rate of LZD. Although the proposed methods are developed for cleavage-stage embryos, the same methods may be applied to most types LZD procedures, embryos at different developmental stages, or nonembryonic cells.

Small Molecule Injection into Single-Cell C. elegans Embryos via Carbon-Reinforced Nanopipettes

PubMed Central

Morton, Diane G.; Fellman, Shanna M.; Chung, SueYeon; Soltani, Mohammad; Kevek, Joshua W.; McEuen, Paul M.; Kemphues, Kenneth J.; Wang, Michelle D.

2013-01-01

The introduction of chemical inhibitors into living cells at specific times in development is a useful method for investigating the roles of specific proteins or cytoskeletal components in developmental processes. Some embryos, such as those of Caenorhabditis elegans, however, possess a tough eggshell that makes introducing drugs and other molecules into embryonic cells challenging. We have developed a procedure using carbon-reinforced nanopipettes (CRNPs) to deliver molecules into C. elegans embryos with high temporal control. The use of CRNPs allows for cellular manipulation to occur just subsequent to meiosis II with minimal damage to the embryo. We have used our technique to replicate classical experiments using latrunculin A to inhibit microfilaments and assess its effects on early polarity establishment. Our injections of latrunculin A confirm the necessity of microfilaments in establishing anterior-posterior polarity at this early stage, even when microtubules remain intact. Further, we find that latrunculin A treatment does not prevent association of PAR-2 or PAR-6 with the cell cortex. Our experiments demonstrate the application of carbon-reinforced nanopipettes to the study of one temporally-confined developmental event. The use of CRNPs to introduce molecules into the embryo should be applicable to investigations at later developmental stages as well as other cells with tough outer coverings. PMID:24086620

Small molecule injection into single-cell C. elegans embryos via carbon-reinforced nanopipettes.

PubMed

Brennan, Lucy D; Roland, Thibault; Morton, Diane G; Fellman, Shanna M; Chung, SueYeon; Soltani, Mohammad; Kevek, Joshua W; McEuen, Paul M; Kemphues, Kenneth J; Wang, Michelle D

2013-01-01

The introduction of chemical inhibitors into living cells at specific times in development is a useful method for investigating the roles of specific proteins or cytoskeletal components in developmental processes. Some embryos, such as those of Caenorhabditis elegans, however, possess a tough eggshell that makes introducing drugs and other molecules into embryonic cells challenging. We have developed a procedure using carbon-reinforced nanopipettes (CRNPs) to deliver molecules into C. elegans embryos with high temporal control. The use of CRNPs allows for cellular manipulation to occur just subsequent to meiosis II with minimal damage to the embryo. We have used our technique to replicate classical experiments using latrunculin A to inhibit microfilaments and assess its effects on early polarity establishment. Our injections of latrunculin A confirm the necessity of microfilaments in establishing anterior-posterior polarity at this early stage, even when microtubules remain intact. Further, we find that latrunculin A treatment does not prevent association of PAR-2 or PAR-6 with the cell cortex. Our experiments demonstrate the application of carbon-reinforced nanopipettes to the study of one temporally-confined developmental event. The use of CRNPs to introduce molecules into the embryo should be applicable to investigations at later developmental stages as well as other cells with tough outer coverings.

Effects of Histone Deacetylase Inhibitor Oxamflatin on In Vitro Porcine Somatic Cell Nuclear Transfer Embryos

PubMed Central

Hou, Liming; Ma, Fanhua; Yang, Jinzeng; Riaz, Hasan; Wang, Yongliang; Wu, Wangjun; Xia, Xiaoliang; Ma, Zhiyuan; Zhou, Ying; Zhang, Lin; Ying, Wenqin; Xu, Dequan; Zuo, Bo; Ren, Zhuqing

2014-01-01

Abstract Low cloning efficiency is considered to be caused by the incomplete or aberrant epigenetic reprogramming of differentiated donor cells in somatic cell nuclear transfer (SCNT) embryos. Oxamflatin, a novel class of histone deacetylase inhibitor (HDACi), has been found to improve the in vitro and full-term developmental potential of SCNT embryos. In the present study, we studied the effects of oxamflatin treatment on in vitro porcine SCNT embryos. Our results indicated that the rate of in vitro blastocyst formation of SCNT embryos treated with 1 μM oxamflatin for 15 h postactivation was significantly higher than all other treatments. Treatment of oxamflatin decreased the relative histone deacetylase (HDAC) activity in cloned embryos and resulted in hyperacetylation levels of histone H3 at lysine 9 (AcH3K9) and histone H4 at lysine 5 (AcH4K5) at pronuclear, two-cell, and four-cell stages partly through downregulating HDAC1. The suppression of HDAC6 through oxamflatin increased the nonhistone acetylation level of α-tubulin during the mitotic cell cycle of early SCNT embryos. In addition, we demonstrated that oxamflatin downregulated DNA methyltransferase 1 (DNMT1) expression and global DNA methylation level (5-methylcytosine) in two-cell-stage porcine SCNT embryos. The pluripotency-related gene POU5F1 was found to be upregulated in the oxamflatin-treated group with a decreased DNA methylation tendency in its promoter regions. Treatment of oxamflatin did not change the locus-specific DNA methylation levels of Sus scrofa heterochromatic satellite DNA sequences at the blastocyst stage. Meanwhile, our findings suggest that treatment with HDACi may contribute to maintaining the stable status of cytoskeleton-associated elements, such as acetylated α-tubulin, which may be the crucial determinants of donor nuclear reprogramming in early SCNT embryos. In summary, oxamflatin treatment improves the developmental potential of porcine SCNT embryos in vitro. PMID

Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos.

PubMed

Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Takai, Jun-Ichi; Yoshida, Junki; Mishina, Takamichi; Fuchimukai, Kota; Ogasawara, Tsukasa; Kariya, Taro; Tashiro, Kosuke; Igarashi, Kazuei

2010-02-01

We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

Effects of in vitro fertilization and embryo culture on TRP53 and Bax expression in B6 mouse embryos.

PubMed

Chandrakanthan, Vashe; Li, Aiqing; Chami, Omar; O'Neill, Christopher

2006-11-21

In the mouse, embryo culture results in a characteristic phenotype of retarded embryo preimplantation development and reduced numbers of cells within embryos. The expression of TRP53 is central to the regulation of the cell's capacity to proliferate and survive. In this study we found that Trp53 mRNA is expressed throughout the preimplantation stage of development. Levels of TRP53 protein expression were low during the cleavage stages and increased at the morula and blastocyst stages in B6 embryos collected from the reproductive tract. Embryos collected at the zygote stage and cultured for 96 h also showed low levels of TRP53 expression at precompaction stages. There were higher levels of TRP53 in cultured morula and the level in cultured blastocysts was clearly increased above blastocysts collected directly from the uterus. Immunolocalization of TRP53 showed that its increased expression in cultured blastocysts corresponded with a marked accumulation of TRP53 within the nuclei of embryonic cells. This pattern of expression was enhanced in embryos produced by in vitro fertilization and subjected to culture. The TRP53 was transcriptionally active since culture also induced increased expression of Bax, yet this did not occur in embryos lacking Trp53 (Trp53-/-). The rate of development of Trp53-/- zygotes to the blastocyst stage was not different to wildtype controls when embryos were cultured in groups of ten but was significantly faster when cultured individually. The results show that zygote culture resulted in the accumulation of transcription activity of TRP53 in the resulting blastocysts. This accounts for the adverse effects of culture of embryos individually, but does not appear to be the sole cause of the retarded preimplantation stage growth phenotype associated with culture in vitro.

Expression and localization of components of the histone deacetylases multiprotein repressory complexes in the mouse preimplantation embryo.

PubMed

Kantor, Boris; Makedonski, Kirill; Shemer, Ruth; Razin, Aharon

2003-12-01

DNA methylation had been implicated in the assembly of multiprotein repressory complexes that affect chromatin architecture thereby rendering genes inactive. Proteins containing methyl binding domains (MBDs) are major components of these complexes. MBD3 is a component of the HDAC associated chromatin remodeling complex Mi2/NuRD. The addition of MBD2 to the Mi2/NuRD complex creates MeCP1, a complex that is known to inactivate methylated promoters. The undermethylated state of the mouse preimplantation embryo prompted us to investigate the known repressory complexes at this developmental stage. We found individual components of Mi2/NuRD: MBD3, Mi2, HDAC1 and HDAC2 to be expressed from a very early stage of embryo development and to localize in close proximity with each other and with constitutive heterochromatin by the blastula stage. Expression of MBD2, a component of MeCP1, starts in the blastula stage. Then it is also found to be in proximity with heterochromatin (based on DAPI staining) and with MBD3, Mi2 and HDAC1. In contrast, expression of MeCP2, an MBD containing component of a third repressory complex (MeCP2/Sin3A), is not seen in the preimplantation embryo. Our results suggest that both Mi2/NuRD and MeCP1 complexes are already present at the very early stages of embryo development, while a MeCP2 complex is added to the arsenal of repressory complexes post-implantation at a stage when DNA methylation takes place.

Supplementation with CTGF, SDF1, NGF, and HGF promotes ovine in vitro oocyte maturation and early embryo development.

PubMed

Wang, D H; Ren, J; Zhou, C J; Han, Z; Wang, L; Liang, C G

2018-05-17

The strategies for improving the in vitro maturation (IVM) of domestic animal oocytes focus on promoting nuclear and cytoplasmic maturation. The identification of paracrine factors and their supplementation in the culture medium represent effective approaches for oocyte maturation and embryo development. This study investigated the effects of paracrine factor supplementation including connective tissue growth factor (CTGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and stromal derived factor 1 (SDF1) on ovine oocytes and early parthenogenetic embryos using an in vitro culture system. First, we identified the optimal concentrations of CTGF (30 ng/mL), SDF1 (10 ng/mL), NGF (3 ng/mL), and HGF (100 ng/mL) for promoting oocyte maturation, which combined, induced nuclear maturation in 94.19% of oocytes. This combination also promoted cumulus cell expansion and inhibited oocyte/cumulus apoptosis, while enabling a larger proportion (33.04%) of embryos to develop into blastocysts than in the controls and prevented embryo apoptosis. These novel findings demonstrate that the paracrine factors CTGF, SDF1, NGF, and HGF facilitate ovine oocyte and early parthenogenetic embryo development in vitro. Thus, supplementation with these factors may help optimize the IVM of ovine oocytes and early parthenogenetic embryo development strategies. Copyright © 2018 Elsevier Inc. All rights reserved.

Pre-hatching embryo-dependent and -independent programming of endometrial function in cattle

PubMed Central

Sponchiado, Mariana; Gomes, Nathália Souza; Fontes, Patrícia Kubo; Martins, Thiago; del Collado, Maite; Pastore, Athos de Assumpção; Pugliesi, Guilherme; Nogueira, Marcelo Fábio Gouveia

2017-01-01

The bovine pre-implantation embryo secretes bioactive molecules from early development stages, but effects on endometrial function are reported to start only after elongation. Here, we interrogated spatially defined regions of the endometrium transcriptome for responses to a day 7 embryo in vivo. We hypothesize that exposure to an embryo changes the abundance of specific transcripts in the cranial region of the pregnant uterine horn. Endometrium was collected from the uterotubal junction (UTJ), anterior (IA), medial (IM) and posterior (IP) regions of the uterine horn ipsilateral to the CL 7 days after estrus from sham-inseminated (Con) or artificially inseminated, confirmed pregnant (Preg) cows. Abundance of 86 transcripts was evaluated by qPCR using a microfluidic platform. Abundance of 12 transcripts was modulated in the Preg endometrium, including classical interferon-stimulated genes (ISG15, MX1, MX2 and OAS1Y), prostaglandin biosynthesis genes (PTGES, HPGD and AKR1C4), water channel (AQP4) and a solute transporter (SLC1A4) and this was in the UTJ and IA mainly. Additionally, for 71 transcripts, abundance varied according to region of the reproductive tract. Regulation included downregulation of genes associated with proliferation (IGF1, IGF2, IGF1R and IGF2R) and extracellular matrix remodeling (MMP14, MMP19 and MMP2) and upregulation of anti-adhesive genes (MUC1) in the cranial regions of uterine horn. Physical proximity to the embryo provides paracrine regulation of endometrial function. Embryo-independent regulation of the endometrial transcriptome may support subsequent stages of embryo development, such as elongation and implantation. We speculate that successful early embryo-dependent and -independent programming fine-tune endometrial functions that are important for maintenance of pregnancy in cattle. PMID:28423001

Initial stages of calcium uptake and mineral deposition in sea urchin embryos

PubMed Central

Vidavsky, Netta; Addadi, Sefi; Mahamid, Julia; Shimoni, Eyal; Ben-Ezra, David; Shpigel, Muki; Weiner, Steve; Addadi, Lia

2014-01-01

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. We reconstructed various stages of the formation pathway of calcium carbonate from calcium ions in sea water to mineral deposition and integration into the forming spicules. Monitoring calcium uptake with the fluorescent dye calcein shows that calcium ions first penetrate the embryo and later are deposited intracellularly. Surprisingly, calcium carbonate deposits are distributed widely all over the embryo, including in the primary mesenchyme cells and in the surface epithelial cells. Using cryo-SEM, we show that the intracellular calcium carbonate deposits are contained in vesicles of diameter 0.5–1.5 μm. Using the newly developed airSEM, which allows direct correlation between fluorescence and energy dispersive spectroscopy, we confirmed the presence of solid calcium carbonate in the vesicles. This mineral phase appears as aggregates of 20–30-nm nanospheres, consistent with amorphous calcium carbonate. The aggregates finally are introduced into the spicule compartment, where they integrate into the growing spicule. PMID:24344263

Initial stages of calcium uptake and mineral deposition in sea urchin embryos.

PubMed

Vidavsky, Netta; Addadi, Sefi; Mahamid, Julia; Shimoni, Eyal; Ben-Ezra, David; Shpigel, Muki; Weiner, Steve; Addadi, Lia

2014-01-07

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. We reconstructed various stages of the formation pathway of calcium carbonate from calcium ions in sea water to mineral deposition and integration into the forming spicules. Monitoring calcium uptake with the fluorescent dye calcein shows that calcium ions first penetrate the embryo and later are deposited intracellularly. Surprisingly, calcium carbonate deposits are distributed widely all over the embryo, including in the primary mesenchyme cells and in the surface epithelial cells. Using cryo-SEM, we show that the intracellular calcium carbonate deposits are contained in vesicles of diameter 0.5-1.5 μm. Using the newly developed airSEM, which allows direct correlation between fluorescence and energy dispersive spectroscopy, we confirmed the presence of solid calcium carbonate in the vesicles. This mineral phase appears as aggregates of 20-30-nm nanospheres, consistent with amorphous calcium carbonate. The aggregates finally are introduced into the spicule compartment, where they integrate into the growing spicule.

Time-lapse evaluation of human embryo development in single versus sequential culture media--a sibling oocyte study.

PubMed

Ciray, Haydar Nadir; Aksoy, Turan; Goktas, Cihan; Ozturk, Bilgen; Bahceci, Mustafa

2012-09-01

To compare the dynamics of early development between embryos cultured in single and sequential media. Randomized, comparative study. Private IVF centre. A total of 446 metaphase II oocytes from 51 couples who underwent oocyte retrieval procedure for intracytoplasmic sperm injection. Forty-nine resulted in embryo transfer. Oocytes were split between single and sequential media produced by the same manufacturer and cultured in a time-lapse incubator. Morphokinetic parameters until the embryos reached the 5-cell stage (t5), utilization, clinical pregnancy and implantation rates. Embryos cultured in single media were advanced from the first mitosis cycle and reached 2- to 5-cell stages earlier. There was not any difference between the durations for cell cycle two (cc2 = t3-t2) and s2 (t4-t3). The utilization, clinical pregnancy and implantation rates did not differ between groups. The proportion of cryopreserved day 6 embryos to two pronuclei oocytes was significantly higher in sequential than in single media. Morphokinetics of embryo development vary between single and sequential culture media at least until the 5-cell stage. The overall clinical and embryological parameters remain similar regardless of the culture system.

Mouse Embryo Compaction.

PubMed

White, M D; Bissiere, S; Alvarez, Y D; Plachta, N

2016-01-01

Compaction is a critical first morphological event in the preimplantation development of the mammalian embryo. Characterized by the transformation of the embryo from a loose cluster of spherical cells into a tightly packed mass, compaction is a key step in the establishment of the first tissue-like structures of the embryo. Although early investigation of the mechanisms driving compaction implicated changes in cell-cell adhesion, recent work has identified essential roles for cortical tension and a compaction-specific class of filopodia. During the transition from 8 to 16 cells, as the embryo is compacting, it must also make fundamental decisions regarding cell position, polarity, and fate. Understanding how these and other processes are integrated with compaction requires further investigation. Emerging imaging-based techniques that enable quantitative analysis from the level of cell-cell interactions down to the level of individual regulatory molecules will provide a greater understanding of how compaction shapes the early mammalian embryo. © 2016 Elsevier Inc. All rights reserved.

(14)C METHANOL INCORPORATION INTO DNA AND SPECIFIC PROTEINS OF ORGANOGENESIS STAGE MOUSE EMBRYOS IN VITRO

EPA Science Inventory

Methanol (MeOH), a widely used industrial solvent and alternative motor fuel, has been shown to be mutagenic and teratogenic. We have demonstrated that methanol is teratogenic in mice in vivo and causes dysmorphogenesis in cultured organogenesis stage mouse embryos. Methanol is ...

A dysmorphology score system for assessing embryo abnormalities in rat whole embryo culture.

PubMed

Zhang, Cindy X; Danberry, Tracy; Jacobs, Mary Ann; Augustine-Rauch, Karen

2010-12-01

The rodent whole embryo culture (WEC) system is a well-established model for characterizing developmental toxicity of test compounds and conducting mechanistic studies. Laboratories have taken various approaches in describing type and severity of developmental findings of organogenesis-stage rodent embryos, but the Brown and Fabro morphological score system is commonly used as a quantitative approach. The associated score criteria is based upon developmental stage and growth parameters, where a series of embryonic structures are assessed and assigned respective scores relative to their gestational stage, with a Total Morphological Score (TMS) assigned to the embryo. This score system is beneficial because it assesses a series of stage-specific anatomical landmarks, facilitating harmonized evaluation across laboratories. Although the TMS provides a quantitative approach to assess growth and determine developmental delay, it is limited to its ability to identify and/or delineate subtle or structure-specific abnormalities. Because of this, the TMS may not be sufficiently sensitive for identifying compounds that induce structure or organ-selective effects. This study describes a distinct morphological score system called the "Dysmorphology Score System (DMS system)" that has been developed for assessing gestation day 11 (approximately 20-26 somite stage) rat embryos using numerical scores to differentiate normal from abnormal morphology and define the respective severity of dysmorphology of specific embryonic structures and organ systems. This method can also be used in scoring mouse embryos of the equivalent developmental stage. The DMS system enhances capabilities to rank-order compounds based upon teratogenic potency, conduct structure- relationships of chemicals, and develop statistical prediction models to support abbreviated developmental toxicity screens. © 2010 Wiley-Liss, Inc.

Moral uncertainty in bioethical argumentation: a new understanding of the pro-life view on early human embryos.

PubMed

Żuradzki, Tomasz

2014-12-01

In this article, I present a new interpretation of the pro-life view on the status of early human embryos. In my understanding, this position is based not on presumptions about the ontological status of embryos and their developmental capabilities but on the specific criteria of rational decisions under uncertainty and on a cautious response to the ambiguous status of embryos. This view, which uses the decision theory model of moral reasoning, promises to reconcile the uncertainty about the ontological status of embryos with the certainty about normative obligations. I will demonstrate that my interpretation of the pro-life view, although seeming to be stronger than the standard one, has limited scope and cannot be used to limit destructive research on human embryos.

Editor's Highlight: Hydroxyurea Exposure Activates the P53 Signaling Pathway in Murine Organogenesis-Stage Embryos.

PubMed

El Husseini, Nazem; Schlisser, Ava E; Hales, Barbara F

2016-08-01

Hydroxyurea, an anticancer agent and potent teratogen, induces oxidative stress and activates a DNA damage response pathway in the gestation day (GD) 9 mouse embryo. To delineate the stress response pathways activated by this drug, we investigated the effect of hydroxyurea exposure on the transcriptome of GD 9 embryos. Timed pregnant CD-1 mice were treated with saline or hydroxyurea (400 mg/kg or 600 mg/kg) on GD 9; embryonic gene and protein expression were examined 3 h later. Microarray analysis revealed that the expression of 1346 probe sets changed significantly in embryos exposed to hydroxyurea compared with controls; the P53 signaling pathway was highly affected. In addition, P53 related family members, P63 and P73, were predicted to be activated and had common and unique downstream targets. Western blot analysis revealed that active phospho-P53 was significantly increased in drug-exposed embryos; confocal microscopy showed that the translocation of phospho-P53 to the nucleus was widespread in the embryo. Furthermore, qRT-PCR showed that the expression of P53-regulated genes (Cdkn1A, Fas, and Trp53inp1) was significantly upregulated in hydroxyurea-exposed embryos; the concentration of the redox sensitive P53INP1 protein was also increased in a hydroxyurea dose-dependent fashion. Thus, hydroxyurea elicits a significant effect on the transcriptome of the organogenesis stage murine embryo, activating several key developmental signaling pathways related to DNA damage and oxidative stress. We propose that the P53 pathway plays a central role in the embryonic stress response and the developmental outcome after teratogen exposure. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived Embryo Development1[W

PubMed Central

Joosen, Ronny; Cordewener, Jan; Supena, Ence Darmo Jaya; Vorst, Oscar; Lammers, Michiel; Maliepaard, Chris; Zeilmaker, Tieme; Miki, Brian; America, Twan; Custers, Jan; Boutilier, Kim

2007-01-01

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants. PMID:17384159

Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption.

PubMed

Kurosawa, Hiroki; Utsunomiya, Hiroki; Shiga, Naomi; Takahashi, Aiko; Ihara, Motomasa; Ishibashi, Masumi; Nishimoto, Mitsuo; Watanabe, Zen; Abe, Hiroyuki; Kumagai, Jin; Terada, Yukihiro; Igarashi, Hideki; Takahashi, Toshifumi; Fukui, Atsushi; Suganuma, Ryota; Tachibana, Masahito; Yaegashi, Nobuo

2016-10-01

. Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality

Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

PubMed Central

Akagi, Jin; Khoshmanesh, Khashayar; Evans, Barbara; Hall, Chris J.; Crosier, Kathryn E.; Cooper, Jonathan M.; Crosier, Philip S.; Wlodkowic, Donald

2012-01-01

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale. PMID:22606275

Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses.

PubMed

Morel, Alexandre; Teyssier, Caroline; Trontin, Jean-François; Eliášová, Kateřina; Pešek, Bedřich; Beaufour, Martine; Morabito, Domenico; Boizot, Nathalie; Le Metté, Claire; Belal-Bessai, Leila; Reymond, Isabelle; Harvengt, Luc; Cadene, Martine; Corbineau, Françoise; Vágner, Martin; Label, Philippe; Lelu-Walter, Marie-Anne

2014-09-01

Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets. © 2014 Scandinavian Plant Physiology Society.

Selective loss of mouse embryos due to the expression of transgenic major histocompatibility class I molecules early in embryogenesis.

PubMed

Aït-Azzouzene, D; Langkopf, A; Cohen, J; Bleux, C; Gendron, M C; Kanellopoulos-Langevin, C

1998-05-01

Among the numerous hypotheses proposed to explain the absence of fetal rejection by the mother in mammals, it has been suggested that regulation of expression of the polymorphic major histocompatibility complex (MHC) at the fetal-maternal interface plays a major role. In addition to a lack of MHC gene expression in the placenta throughout gestation, the absence of polymorphic MHC molecules on the early embryo, as well as their low level of expression after midgestation, could contribute to this important biologic phenomenon. In order to test this hypothesis, we have produced transgenic mice able to express polymorphic MHC class I molecules early in embryogenesis. We have placed the MHC class la gene H-2Kb under the control of a housekeeping gene promoter, the hydroxy-methyl-glutaryl coenzyme A reductase (HMG) gene minimal promoter. This construct has been tested for functionality after transfection into mouse fibroblast L cells. The analysis of three founder transgenic mice and their progeny suggested that fetoplacental units that could express the H-2Kb heavy chains are unable to survive in utero beyond midgestation. We have shown further that a much higher resorption rate, on days 11 to 13 of embryonic development, is observed among transgenic embryos developing from eggs microinjected at the one-cell stage with the pHMG-Kb construct than in control embryos. This lethality is not due to immune phenomena, since it is observed in histocompatible combinations between mother and fetus. These results are discussed in the context of what is currently known about the regulation of MHC expression at the fetal-maternal interface and in various transgenic mouse models.

[A comparative analysis of notochord formation in amphibian embryos].

PubMed

Novoselov, V V

1992-01-01

We studied the origin, structure, and development of the notochord in Pleurodeles waltlii (Urodela) and Xenopus laevis (Anura) embryos. The notochord rudiment is formed in both species at the early gastrula stage as a cluster of polarized chorda-mesoderm cells located along the sagittal plane of the embryo. In Pl. waltlii the notochord rudiment is separated from the gastrocoele roof as a result of contraction of apical cell surfaces. The contraction wave spreads forward and backward along craniocaudal axis, i.e., segregation of the notochord rudiment progresses in two directions simultaneously. Similar process takes place in X. laevis embryos; however, propagation of the contraction wave in the anterior part of the body somewhat differs from that in the posterior part. While the "anterior" contraction wave resembles that in Pl. waltlii embryos, progression of the wave in the posterior part of the body is distinguished by a closer association of the notochord rudiment with ectoderm and the presence of its delamination boundaries with the somite mesoderm.

Kinetics of fertilization and development, and sex ratio of bovine embryos produced using the semen of different bulls.

PubMed

Alomar, M; Tasiaux, H; Remacle, S; George, F; Paul, D; Donnay, I

2008-08-01

The between bulls variation in in vitro fertility and the shift of sex ratio towards male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the kinetics of fertilization, embryo development and the sex ratio of the resulting embryos using the frozen/thawed semen of four different bulls. In a first experiment, the kinetics of pronucleus (PN) formation was evaluated at 8, 12 and 18 h post-insemination (hpi). Based upon the pronuclei sizes and the distance between the two pronuclei, inseminated oocytes were classified in three PN stages. Differences between bulls were observed at each time point, but were more important at 12 hpi. At 8 and 12 hpi bull III showed a significantly faster PN evolution by comparison with the three other bulls (P<0.05), while at 18 hpi, the proportion of the three PN stages was similar to those of bulls I and IV, bull II being delayed. In a second experiment, the kinetics of in vitro embryo development was compared using time-lapse cinematography. The analysis of embryos reaching the blastocyst stage revealed significant differences in the mean time of first cleavage (range of 22.7-25.6h, P<0.05), while the lengths of the subsequent three cell cycles did not differ between bulls. The early mean time of first cleavage with bull III was associated with an early blastulation and a high blastocyst rate at Day 7, in opposition to what was observed with bull II showing a later timing of first cleavage (first cleavage 22.1 hpi versus 25.5 hpi; blastulation 140.4 hpi versus 152.5 hpi; D7 blastocyst rates: 31.3% versus 21.9%; P<0.05). In a third experiment, 65-76 Day 8 blastocysts per bull were sexed by PCR. Only blastocysts obtained with bull III showed a shift in sex ratio towards male embryos (76% male embryos; P<0.05). Such shift was already observed at the 2-cell and morula stages. In conclusion, the bull influences the kinetics of PN formation, of embryo development and the sex

Elucidating the origin of chromosomal aberrations in IVF embryos by preimplantation genetic analysis.

PubMed

Frumkin, Tsvia; Malcov, Mira; Yaron, Yuval; Ben-Yosef, Dalit

2008-01-30

Preimplantation genetic screening (PGS) has been proposed as a method for improving success rates in patients with repeated IVF failures. This approach is based on the hypothesis that such failures are the result of aneuploid embryos. It has been suggested that FISH analysis of blastomeres removed from preimplantation embryos represent the chromosomal constitution of the entire embryo. However, it is not yet clear whether it also represents the chromosomal constitution of the implanted embryo. PGS reanalysis on day 5 of embryos designated as "aneuploid" on day 3 may demonstrate a high rate of mosaicism for chromosomal aberration. Some of these mosaic embryos are capable of developing into normal embryos by "self-correction". Others, however, may accumulate additional chromosomal anomalies. It is therefore concluded that the chromosomal constitution of a preimplantation embryo may evolve during early cleavages. Meiotic and post zygotic mitotic errors may account for these chromosomal aberrations. This review will focus on elucidating the origin of chromosomal changes during preimplantation embryo development by studying their chromosomal constitution at different stages.

Sex Bias and Maternal Contribution to Gene Expression Divergence in Drosophila Blastoderm Embryos

PubMed Central

Paris, Mathilde; Villalta, Jacqueline E.; Eisen, Michael B.; Lott, Susan E.

2015-01-01

Early embryogenesis is a unique developmental stage where genetic control of development is handed off from mother to zygote. Yet the contribution of this transition to the evolution of gene expression is poorly understood. Here we study two aspects of gene expression specific to early embryogenesis in Drosophila: sex-biased gene expression prior to the onset of canonical X chromosomal dosage compensation, and the contribution of maternally supplied mRNAs. We sequenced mRNAs from individual unfertilized eggs and precisely staged and sexed blastoderm embryos, and compared levels between D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. First, we find that mRNA content is highly conserved for a given stage and that studies relying on pooled embryos likely systematically overstate the degree of gene expression divergence. Unlike studies done on larvae and adults where most species show a larger proportion of genes with male-biased expression, we find that transcripts in Drosophila embryos are largely female-biased in all species, likely due to incomplete dosage compensation prior to the activation of the canonical dosage compensation mechanism. The divergence of sex-biased gene expression across species is observed to be often due to lineage-specific decrease of expression; the most drastic example of which is the overall reduction of male expression from the neo-X chromosome in D. pseudoobscura, leading to a pervasive female-bias on this chromosome. We see no evidence for a faster evolution of expression on the X chromosome in embryos (no “faster-X” effect), unlike in adults, and contrary to a previous study on pooled non-sexed embryos. Finally, we find that most genes are conserved in regard to their maternal or zygotic origin of transcription, and present evidence that differences in maternal contribution to the blastoderm transcript pool may be due to species-specific divergence of transcript degradation rates. PMID:26485701

Alcohol exposure alters DNA methylation profiles in mouse embryos at early neurulation

PubMed Central

Liu, Yunlong; Balaraman, Yokesh; Wang, Guohua; Nephew, Kenneth P.; Zhou, Feng C.

2009-01-01

Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88 mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10 and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p < 0.01) changes in expression for 84 genes. Sequenom EpiTYPER DNA methylation analysis was used for validation of the MeDIP-chip data. Increased methylation of genes known to play a role in metabolism (Cyp4f13) and decreased methylation of genes associated with development (Nlgn3, Elavl2, Sox21 and Sim1), imprinting (Igf2r) and chromatin (Hist1h3d) was confirmed. In a mouse model for FASD, we show for the first time that alcohol exposure during early neurulation can induce aberrant changes in DNA methylation patterns with associated changes

Alcohol exposure alters DNA methylation profiles in mouse embryos at early neurulation.

PubMed

Liu, Yunlong; Balaraman, Yokesh; Wang, Guohua; Nephew, Kenneth P; Zhou, Feng C

2009-10-01

Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10, and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p<0.01) changes in expression for 84 genes. Sequenom EpiTYPER DNA methylation analysis was used for validation of the MeDIP-chip data. Increased methylation of genes known to play a role in metabolism (Cyp4f13) and decreased methylation of genes associated with development (Nlgn3, Elavl2, Sox21 and Sim1), imprinting (Igf2r) and chromatin (Hist1h3d) was confirmed. In a mouse model for FASD, we show for the first time that alcohol exposure during early neurulation can induce aberrant changes in DNA methylation patterns with associated changes in

Protein Phosphorylation during Coconut Zygotic Embryo Development1

PubMed Central

Islas-Flores, Ignacio; Oropeza, Carlos; Hernández-Sotomayor, S.M. Teresa

1998-01-01

Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [γ-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species. PMID:9733545

Expression pattern of Chlamys farreri sox2 in eggs, embryos and larvae of various stages

NASA Astrophysics Data System (ADS)

Liang, Shaoshuai; Ma, Xiaoshi; Han, Tiantian; Yang, Dandan; Zhang, Zhifeng

2015-08-01

The SOX2 protein is an important transcription factor functioning during the early development of animals. In this study, we isolated a full-length cDNA sequence of scallop Chlamys farreri sox2, Cf-sox2 which was 2194 bp in length with a 981 bp open reading frame encoding 327 amino acids. With real-time PCR analysis, it was detected that Cf-sox2 was expressed in unfertilized oocytes, fertilized eggs and all the tested embryos and larvae. The expression level increased significantly ( P < 0.01) in embryos from 2-cell to blastula, and then decreased significantly ( P < 0.01) and reached the minimum in umbo larva. Moreover, location of the Cf-sox2 expression was revealed using whole mount in situ hybridization technique. Positive hybridization signal could be detected in the central region of unfertilized oocytes and fertilized eggs, and then strong signals dispersed throughout the embryos from 2-cell to gastrula. During larval development, the signals were concentrated and strong signals were restricted to 4 regions of viscera mass in veliger larva. In umbo larva, weak signals could be detected in regions where presumptive visceral and pedal ganglia may be formed. The expression pattern of Cf-sox2 during embryogenesis was similar to that of mammal sox2, which implied that Cf-SOX2 may participate in the regulation of early development of C. farreri.

Effects of temperature on survival and development of early life stage Pacific and western brook lampreys

USGS Publications Warehouse

Meeuwig, M.H.; Bayer, J.M.; Seelye, J.G.

2005-01-01

We examined the effects of temperature (10, 14, 18, and 22??C) on survival and development of Pacific lampreys Lampetra tridentata and western brook lampreys L. richardsoni during embryological and early larval stages. The temperature for zero development was estimated for each species, and the response to temperature was measured as the proportion of individuals surviving to hatch, surviving to the larval stage, and exhibiting abnormalities at the larval stage (i.e., malformations of the body). The estimated temperature for zero development was 4.850C for Pacific lampreys and 4.97??C for western brook lampreys. Survival was greatest at 18??C, followed by 14, 10, and 22??C, significant differences being observed between 22??C and the other temperatures. Overall survival was significantly greater for western brook lampreys than for Pacific lampreys; however, the overall difference in proportion of individuals surviving was only 0.02. Overall survival significantly decreased from the time of hatch (proportion surviving = 0.85) to the larval stage (0.82; i.e., during the free-embryo stage). The proportion of individuals exhibiting abnormalities at the larval stage was greatest at 22??C, followed by 18, 10, and 14??C, significant differences being observed between 22??C and the other temperatures. These data provide baseline information on the thermal requirements of early life stage Pacific and western brook lampreys and will aid in assessment and prediction of suitable spawning and rearing habitats for these species.

The Effect of Vitrification and in vitro Culture on the Adenosine Triphosphate Content and Mitochondrial Distribution of Mouse Pre-Implantation Embryos

PubMed Central

Amoushahi, Mahboobeh; Salehnia, Mojdeh; HosseinKhani, Saman

2013-01-01

Background: The mitochondria are an important source of adenosine triphosphate (ATP) production in pre-implantation embryo. Therefore, the objective of this study was to investigate the effect of vitrification and in vitro culture of mouse embryos on their mitochondrial distribution and ATP content. Methods: The embryos at 2-PN, 4-cell and blastocyst stages were collected from the oviduct of stimulated pregnant mice and uterine horns. Then, the embryos were vitrified with the cryotop method using ethylene glycol and dimethylsulphoxide. After evaluating the survival rates of vitrified embryos, their development to hatching stages were assessed. The ATP content of collected in vivo and in vitro embryos at different stages was measured by luciferin-luciferase bioluminescence assay. The distribution of mitochondria was studied using Mito-tracker green staining under a fluorescent microscope. Results: The survival rates of vitrified embryos at 2-PN, 4-cell and early blastocyst stages were 84.3, 87.87 and 89.89%, respectively. The hatching rates in previous developmental stages in vitrified group were 57.44, 66.73 and 70.89% and in non-vitrified group were 66.32, 73.25 and 75.89%, respectively (P>0.05). The ATP content of in vivo or in vitro collected embryos was not significantly different in both vitrified and non-vitrified groups (P>0.05). Mitochondrial distribution of vitrified and non-vitrified 2-PN embryos was similar, but some clampings or large aggregation of mitochondria within the vitrified 4-cell embryos was prominent. Conclusions: Vitrification method did not affect the mouse embryo ATP content. Also, the cellular stress was not induced by this procedure and the safety of vitrification was shown. PMID:23748889

Culturing of avian embryos for time-lapse imaging.

PubMed

Rupp, Paul A; Rongish, Brenda J; Czirok, Andras; Little, Charles D

2003-02-01

Monitoring morphogenetic processes, at high resolution over time, has been a long-standing goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however, imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of development, birds are ideal for imaging, since the avian body plan is very similar to that of mammals. We have devised a culturing technique that allows for the acquisition of high-resolution differential interference contrast and epifluorescence images of developing avian embryos in a 4-D (3-D + time) system. The resulting information, from intact embryos, is derived from an area encompassing several millimeters, at micrometer resolution for up to 30 h.

Chicken Embryos as a Potential New Model for Early Onset Type I Diabetes

PubMed Central

Shi, Liheng; Ko, Michael L.; Huang, Cathy Chia-Yu; Park, So-Young; Hong, Min-Pyo; Ko, Gladys Y.-P.

2014-01-01

Diabetic retinopathy (DR) is the leading cause of blindness among the American working population. The purpose of this study is to establish a new diabetic animal model using a cone-dominant avian species to address the distorted color vision and altered cone pathway responses in prediabetic and early diabetic patients. Chicken embryos were injected with either streptozotocin (STZ), high concentration of glucose (high-glucose), or vehicle at embryonic day 11. Cataracts occurred in varying degrees in both STZ- and high glucose-induced diabetic chick embryos at E18. Streptozotocin-diabetic chicken embryos had decreased levels of blood insulin, glucose transporter 4 (Glut4), and phosphorylated protein kinase B (pAKT). In STZ-injected E20 embryos, the ERG amplitudes of both a- and b-waves were significantly decreased, the implicit time of the a-wave was delayed, while that of the b-wave was significantly increased. Photoreceptors cultured from STZ-injected E18 embryos had a significant decrease in L-type voltage-gated calcium channel (L-VGCC) currents, which was reflected in the decreased level of L-VGCCα1D subunit in the STZ-diabetic retinas. Through these independent lines of evidence, STZ-injection was able to induce pathological conditions in the chicken embryonic retina, and it is promising to use chickens as a potential new animal model for type I diabetes. PMID:25133191

Measuring the electric activity of chick embryos heart through 16 bit audio card monitored by the Goldwavetm software

NASA Astrophysics Data System (ADS)

Silva, Dilson; Cortez, Celia Martins

2015-12-01

In the present work we used a high-resolution, low-cost apparatus capable of detecting waves fit inside the sound bandwidth, and the software package GoldwaveTM for graphical display, processing and monitoring the signals, to study aspects of the electric heart activity of early avian embryos, specifically at the 18th Hamburger & Hamilton stage of the embryo development. The species used was the domestic chick (Gallus gallus), and we carried out 23 experiments in which cardiographic spectra of QRS complex waves representing the propagation of depolarization waves through ventricles was recorded using microprobes and reference electrodes directly on the embryos. The results show that technique using 16 bit audio card monitored by the GoldwaveTM software was efficient to study signal aspects of heart electric activity of early avian embryos.

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.

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

PubMed Central

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. PMID:25602523

Laboratory techniques for human embryos.

PubMed

Geber, Selmo; Sales, Liana; Sampaio, Marcos A C

2002-01-01

This review is concerned with laboratory techniques needed for assisted conception, particularly the handling of gametes and embryos. Such methods are being increasingly refined. Successive stages of fertilization and embryogenesis require especial care, and often involve the use of micromanipulative methods for intracytoplasmic sperm injection (ICSI) or preimplantation genetic diagnosis. Embryologists must take responsibility for gamete collection and preparation, and for deciding on the means of insemination or ICSI. Embryos must be assessed in culture, during the 1-cell, cleaving and morula/blastocyst stages, and classified according to quality. Co-culture methods may be necessary. The best embryos for transfer must be selected and loaded into the transfer catheter. Embryos not transferred must be cryopreserved, which demands the correct application of current methods of media preparation, seeding and the correct speed for cooling and warming. Before too long, methods of detecting abnormal embryos and avoiding their transfer may become widespread.

Comparison of effects of albendazole sulfoxide on in vitro produced bovine embryos and rat embryos.

PubMed

Piscopo, S E; Smoak, I W

1997-09-01

To evaluate and compare effects of albendazole sulfoxide (ABZSO) on rat embryos and bovine embryos produced in vitro. In vitro produced bovine embryos. Rat embryos recovered from naturally bred Sprague-Dawley rats. 4- and 8-cell bovine embryos were randomly allocated to ABZSO or vehicle control groups. After 48 hours, embryos were evaluated for cell number and blastomere morphology. Rat embryos of similar stages, flushed from the uterine tube on gestational day 2-5, were randomly allocated to treatment or control groups. After 24 hours, embryos were evaluated as described previously. 44% of control bovine embryos divided in culture (> or = 16-cell stage). Fifteen percent of the controls had morphologic abnormalities, including disparity in blastomere size and cytoplasmic vacuoles and stippling. Treated (> or = 1 microgram of ABZSO/ml) bovine embryos differed (P < 0.0001) from controls, with 4% development and 93% abnormal morphology. Forty-five percent of control rat embryos divided in culture. Treated (> or = 500 ng of ABZSO/ml) rat embryos differed (P < 0.0003) from controls with regard to ability to divide. There were no consistent morphologic abnormalities in rat embryos. In vitro produced bovine embryos were susceptible to ABZSO at a concentration > or = 1 microgram/ ml, resulting in decreased ability to divide and presence of gross morphologic abnormalities. Rat embryos produced in vivo and exposed in vitro to ABZSO at a concentration > or = 500 ng/ml had decreased ability to divide in culture. Despite severe effects of ABZSO (> or = 1 microgram/ml) on bovine embryo development in vitro, it is beyond the scope of this study to speculate whether a therapeutic dosage of albendazole (10 mg/kg of body weight) would result in necessary concentrations of ABZSO in vivo to disrupt embryogenesis.

A potential determinant role of adiponectin and receptors for the early embryo development in PCOS patients with obesity hinted by quantitative profiling.

PubMed

Zhang, Ning; Hao, Cuifang; Liu, Xiaoyan; Zhang, Shouxin; Zhang, Fengrong; Zhuang, Lili; Zhao, Dongmei

2017-02-01

To identify the quantitative profiling of adiponectin and its receptors (AdipoR1, AdipoR2, and T-cadherin) in cumulus cells (CCs) and to evaluate their roles in the early embryo development of polycystic ovary syndrome (PCOS) patients, in part, with obesity. Fifty-five subjects were divided into two groups according to the body mass index. Oocytes were further inseminated and only mature and normal fertilized oocytes (2PN) were included in this research. Real-time PCR and western blot were performed to identify adiponectin and its receptors in CCs. Adiponectin and receptors were ubiquitously expressed in CCs of PCOS and non-PCOS patients. The level of AdipoR2 in CCs from the oocytes yielding blastocyst after 5/6 days in vitro culture was markedly higher than in those from oocytes could not develop to blastocyst stage after Day 6, for non-obese or obese PCOS patients (0.1647 ± 0.0161 versus 0.0783 ± 0.0385, 0.1948 ± 0.0307 versus 0.1057 ± 0.0236, respectively, p < 0.05). In addition, only in patients with PCOS and concurrent obesity the AdipoR1 in CCs was considerably increased in CC-B + compared with CC-B - subgroup (0.5162 ± 0.0371 versus 0.2448 ± 0.0333, p < 0.01). The development of early embryo was associated with the up-regulation of AdipoR1 and AdipoR2 in PCOS patients. Our results suggested that adiponectin could positively modulate embryo development in humans. Further investigations should be carried out to unlock the crucial role that adiponectin plays in embryo development.

Disparities in reproductive outcomes according to the endometrial preparation protocol in frozen embryo transfer : The risk of early pregnancy loss in frozen embryo transfer cycles.

PubMed

Hatoum, I; Bellon, L; Swierkowski, N; Ouazana, M; Bouba, S; Fathallah, K; Paillusson, B; Bailly, M; Boitrelle, F; Alter, L; Bergère, M; Selva, J; Wainer, R

2018-03-01

The purpose of this study was to determine the effect of stimulated and artificial endometrial preparation protocols on reproductive outcomes in frozen embryo transfer (FET) cycles. We performed a retrospective study of 1926 FET cycles over a 3.5-year period in the Fertility Unit at a University Hospital. Stimulated and artificial protocols were used for endometrial preparation. The embryos for FET were obtained from either in vitro fertilization or intracytoplasmic sperm injection cycles. Live birth rate and early pregnancy loss rates were retrospectively compared. In artificial protocols, oral or vaginal administration of oestradiol 2 mg two or three times a day was followed by vaginal supplementation with progesterone 200 mg two or three times a day. In stimulated protocols, recombinant follicle-stimulating hormone was administered from day 4 onward. Vaginal ultrasound was used for endometrial and ovarian monitoring. A pregnancy test was performed 14 days after FET. If it was positive, oestradiol and progesterone were administered up until the 12th week of gestation in artificial cycles. We defined early pregnancy losses as biochemical pregnancies (preclinical losses) and miscarriages. Data on 865 artificial cycles (45% of the total) and 1061 stimulated cycles (55%) were collected. Early pregnancy loss rate was significantly lower for stimulated cycles (34.2%) than for artificial cycles (56.9%), and the live birth rate was significantly higher for stimulated cycles (59.7%) than for artificial cycles (29.1%). In frozen embryo transfer, artificial cycles were associated with more early pregnancy loss and lower live birth rate than stimulated cycles.

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.

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

PubMed Central

Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

2016-01-01

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

PubMed

Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Swann, Karl; Borri, Paola

2016-06-15

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. © 2016. Published by The Company of Biologists Ltd.

PGF₂α levels in Day 8 blood plasma are increased by the presence of one or more embryos in the uterus.

PubMed

Gomez, E; Martin, D; Carrocera, S; Muñoz, M

2015-08-01

In cattle, the detection of very early endometrial responses is considered to be hampered by the presence of only a single embryo. Therefore, we have previously developed a model of multiple embryo transfer to circumvent this hindrance. In this work, we analysed embryo-maternal interactions in the bovine uterus on day 8 of development while comparing the presence of multiple v. single embryos using embryo transfer and artificial insemination, respectively. Concentration of proteins (β-actin, NFkB, clusterin and immunoproteosome 20S β5i subunit-i20S), by western blot, and hexoses (glucose and fructose) were measured in paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus and were compared with UF obtained after artificial insemination. Prostaglandin (PG) F2 α and PGE2 concentrations were also analysed in blood plasma. The four proteins analysed and hexoses were unaffected by the presence of one or more embryos in the uterus. However, blood PGF2 α showed similar, significant increases with one or more embryos over cyclic animals; such changes were not observed in blood PGE2. Although multiple embryo transfer may appear to be non-physiological, we showed that the uterus, at the very early embryonic stages, does exhibit physiological reactions. Multiple embryo transfer can, therefore, be used for studies of very early embryo-maternal interactions in vivo in monotocous species.

Comparison of prehatch C-start responses in rainbow trout and lake trout embryos by means of a tactile stimulus test

USGS Publications Warehouse

Wright, P.J.; Noltie, Douglas B.; Tillitt, D.E.

2003-01-01

The C-start in teleost fishes, a type of startle response, mediates the ability to respond to abrupt, unexpected stimuli and is characterized by a short-latency, C-type fast start acceleration. In prehatch fish embryos, the C-start appears necessary for mechanical breakdown of the egg chorion and successful hatching by way of increased embryo movement and distribution of the hatching enzymes. In later stages, the C-start plays an important role in predator avoidance. Using tactile stimulation, we evaluated the C-start response in rainbow trout Oncorhynchus mykiss at 170 degree-days, when 6.6% of embryos exhibited C-starts, and lake trout Salvelinus namaycush embryos at 320 degree-days, when 23% of embryos exhibited C-starts. Triplicate groups of embryos were later tested at three developmental stages: early (220 and 360 degree-days for rainbow trout and lake trout, respectively), middle (260 and 480 degree-days, respectively), and late (320 and 560 degree-days, respectively). The proportion of trout embryos exhibiting C-start increased through time, such that 100% had responded by the late stage, just prior to hatching. C-starts could be obtained by repeated stimulation, and the relative activity of the embryos (based on the number of flexures per stimulus) also increased over time. Rainbow trout and lake trout showed very similar C-start responses at parallel developmental stages, and these patterns of response were similar to those reported in other fish species.

Localization and expression of peroxiredoxin II in the mouse ovary, oviduct, uterus, and preimplantation embryo.

PubMed

Wang, Shie; Huang, Weiquan; Shi, Hexiu; Lin, Cuiying; Xie, Meirong; Wang, Jianxin

2010-02-01

Peroxiredoxin (Prx) II belongs to a recently discovered family of peroxidases that play important roles in antioxidation and signal transduction. In this study, we aimed to study the localization and expression of Prx II in the mouse ovary, oviduct, and uterus, and preimplantation embryos. Immunohistochemical staining analysis showed that, in the ovary, Prx II was expressed in the oocyte cytoplasm of the primary follicle, the secondary follicle, and the premature follicle; Prx II was expressed in germinal vesicle-intact oocytes (GV oocytes) and metaphase II eggs (MII eggs), as well as at various stages in early embryos. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that the Prx II mRNA was expressed at a high level in GV eggs, slightly lower levels in MII eggs, and had no detectable expression in four-cell embryos and early blastocysts. In the oviduct, Prx II was expressed in the epithelia, while in the uterus Prx II was mainly distributed in the endometrial stroma. Taken together, our results suggest that Prx II plays a key antioxidation role in the maturation of oocytes and development of early embryos, thus providing crucial experimental evidence for further exploring the function of Prx II in the development of oocytes and preimplantation embryos. 2009 Wiley-Liss, Inc.

No significant regulation of bicoid mRNA by Pumilio or Nanos in the early Drosophila embryo.

PubMed

Wharton, Tammy H; Nomie, Krystle J; Wharton, Robin P

2018-01-01

Drosophila Pumilio (Pum) is a founding member of the conserved Puf domain class of RNA-binding translational regulators. Pum binds with high specificity, contacting eight nucleotides, one with each of the repeats in its RNA-binding domain. In general, Pum is thought to block translation in collaboration with Nanos (Nos), which exhibits no binding specificity in isolation but is recruited jointly to regulatory sequences containing a Pum binding site in the 3'-UTRs of target mRNAs. Unlike Pum, which is ubiquitous in the early embryo, Nos is tightly restricted to the posterior, ensuring that repression of its best-characterized target, maternal hunchback (hb) mRNA, takes place exclusively in the posterior. An exceptional case of Nos-independent regulation by Pum has been described-repression of maternal bicoid (bcd) mRNA at the anterior pole of the early embryo, dependent on both Pum and conserved Pum binding sites in the 3'-UTR of the mRNA. We have re-investigated regulation of bcd in the early embryo; our experiments reveal no evidence of a role for Pum or its conserved binding sites in regulation of the perdurance of bcd mRNA or protein. Instead, we find that Pum and Nos control the accumulation of bcd mRNA in testes.

Amino and fatty acid dynamics of octopus (Octopus vulgaris) early life stages under ocean warming.

PubMed

Lopes, Vanessa M; Faleiro, Filipa; Baptista, Miguel; Pimentel, Marta S; Paula, José R; Couto, Ana; Bandarra, Narcisa; Anacleto, Patrícia; Marques, António; Rosa, Rui

2016-01-01

The oceans are becoming warmer, and the higher temperatures are expected to have a major impact on marine life at different levels of biological organization, especially at the most vulnerable early life stages. Thus, we hypothesize that the future warmer scenarios (here +3 °C) will affect the biochemical composition (amino acid - AA, and fatty acid-FA) of octopod (Octopus vulgaris) embryos and recently-hatched pelagic paralarvae. The main essential amino acids found in octopus embryos were arginine, leucine and lysine; while aspartic and glutamic acids, and taurine were the main non-essential amino acids. Palmitic, eicosapentaenoic and docosahexaenoic acids were the main FAs found in octopus tissues. Relevant ontogenetic changes were observed, namely a steep decrease in the content of many AAs, and a selective retention of FAs, thus evidencing the protein-based metabolism of these cephalopods. Temperature per si did not elicit significant changes in the overall FA composition, but was responsible for a significant decrease in the content of several AAs, indicating increased embryonic consumption. Copyright © 2015 Elsevier Ltd. All rights reserved.

Role of the FGF and MEK signaling pathway in the ascidian embryo.

PubMed

Kim, G J; Nishida, H

2001-10-01

In the ascidian embryo, a fibroblast growth factor (FGF)-like signal from presumptive endoderm blastomeres between the 32-cell and early 64-cell stages induces the formation of notochord and mesenchyme cells. However, it has not been known whether endogenous FGF signaling is involved in the process. Here it is shown that 64-cell embryos exhibit a marked increase in endogenous extracellular signal-regulated kinase (ERK/MAPK) activity. The increase in ERK activity was reduced by treatment with an FGF receptor 1 inhibitor, SU5402, and a MEK (ERK kinase/MAPKK) inhibitor, U0126. Both drugs blocked the formation of notochord and mesenchyme when embryos were treated at the 32-cell stage, but not at the 2- or 110-cell stages. The dominant-negative form of Ras also suppressed notochord and mesenchyme formation. Both inhibitors suppressed induction by exogenous basic FGF. These results suggest that the FGF signaling cascade is indeed necessary for the formation of notochord and mesenchyme cells during ascidian embryogenesis. It is also shown that FGF signaling is required for formation of the secondary notochord, secondary muscle and neural tissues, and at least ERK activity is necessary for the formation of trunk lateral cells and posterior endoderm. Therefore, FGF and MEK signaling are required for the formation of various tissues in the ascidian embryo.

Maternal organism and embryo biosensoring: insights from ruminants.

PubMed

Sandra, Olivier; Constant, Fabienne; Vitorino Carvalho, Anais; Eozénou, Caroline; Valour, Damien; Mauffré, Vincent; Hue, Isabelle; Charpigny, Gilles

2015-04-01

In terms of contribution to pregnancy, the mother not only produces gametes, but also hosts gestation, whose progression in the uterus is conditioned by early events during implantation. In ruminants, this period is associated with elongation of the extra-embryonic tissues, gastrulation of the embryonic disk and cross-talk with the endometrium. Recent data have prompted the need for accurate staging of the bovine conceptus and shown that asynchrony between elongation and gastrulation processes may account for pregnancy failure. Data mining of endometrial gene signatures has allowed the identification of molecular pathways and new factors regulated by the conceptus (e.g. FOXL2, SOCS6). Interferon-tau has been recognised to be the major signal of pregnancy recognition, but prostaglandins and lysophospholipids have also been demonstrated to be critical players at the conceptus-endometrium interface. Interestingly, up-regulation of interferon-regulated gene expression has been identified in circulating immune cells during implantation, making these factors a potential source of non-invasive biomarkers for early pregnancy. Distinct endometrial responses have been shown to be elicited by embryos produced by artificial insemination, in vitro fertilisation or somatic cell nuclear transfer. These findings have led to the concept that endometrium is an early biosensor of embryo quality. This biological property first demonstrated in cattle has been recently extended and associated with embryo selection in humans. Hence, compromised or suboptimal endometrial quality can subtly or deeply affect embryo development, with visible and sometimes severe consequences for placentation, foetal development, pregnancy outcome and the long-term health of the offspring. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Studies on trypsin-like enzymes in sperm and early embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Penn, A.

1975-12-09

Results are reported from a study of acrosomal proteinase, a trypsin-like enzyme (TLE), found in the acrosome of all eutherian mammals studied to date. It has been implicated in the dissolution of a passage for the sperm through the zona pellucida of the egg, a step necessary for in vivo fertilization. A cytochemical procedure employing autoradiographic film as a gelatin substrate is described for in situ detection and localization of acrosomal proteolytic activity. A role for TLE in the early development of embryos is suggested. (CH)

Polypeptide profiles of human oocytes and preimplantation embryos.

PubMed

Capmany, G; Bolton, V N

1993-11-01

The polypeptides that direct fertilization and early development until activation of the embryonic genome occurs, at the 4-8 cell stage in the human, are exclusively maternal in origin, and are either synthesized during oogenesis or translated later from maternal mRNA. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and silver stain, we have visualized and compared the polypeptides present in different populations of human oocytes and cleavage stage embryos obtained after superovulation and insemination in vitro. Two polypeptide patterns were resolved, differing in the region of mol. wt 69 kDa. The distribution of these patterns showed no correlation with the ability of individual oocytes to achieve fertilization and develop normally to the 8-cell stage.

Embryo developmental events and the egg case of the Aleutian skate Bathyraja aleutica (Gilbert) and the Alaska skate Bathyraja parmifera (Bean).

PubMed

Hoff, G R

2009-02-01

Embryo development events were correlated with egg-case changes for the Aleutian skate Bathyraja aleutica and the Alaska skate Bathyraja parmifera. Yolk absorption underwent two phases: that of steady absorption during early development and that of rapid yolk absorption during the final development stages. Total length (L(T)) for 50% of the pre-hatching embryos egg-case jelly disappearance was 92.04 mm (range 81-102 mm) and 99.36 mm (range 81-100 mm) for B. aleutica and B. parmifera, respectively, allowing the inner chamber to open to seawater flow. The tail filament underwent three phases of growth: rapid elongation during early development (<100 mm embryo L(T)), stasis of tail filament length during the remainder of embryo development and rapid absorption soon after hatching. Complete tail filament development coincided with the disappearance of egg-case jelly. Clasper buds first developed at embryos >70 mm L(T) for both species and the sex ratio was 1:1 well before hatching. Egg cases that were devoid of an ova or developing embryo were c. 5.0 and 6.5% of the egg cases examined for B. aleutica and B. parmifera, respectively. Measurements showed that egg cases containing only egg jelly were smaller in both width and length than those possessing an ova. Embryo stages were punctuated with distinct events that correlated with egg case changes controlling the internal environment of the developing embryo.

Fish embryo toxicity of carbamazepine, diclofenac and metoprolol.

PubMed

van den Brandhof, Evert-Jan; Montforts, Mark

2010-11-01

Frequently measured pharmaceuticals in environmental samples were tested in fish embryo toxicity (FET) tests with Danio rerio, based on the draft OECD test protocol. In this FET test 2-h-old zebrafish embryos were exposed for 72 h to carbamazepine, diclofenac and metoprolol to observe effects on embryo mortality, gastrulation, somite formation, tail movement and detachment, pigmentation, heartbeat, malformation of head, otoliths and heart, scoliosis, deformity of yolk, and hatching success at 24, 48 and 72 h. We found specific effects on growth retardation above 30.6 mg/l for carbamazepine, on hatching, yolk sac and tail deformation above 1.5mg/l for diclofenac, and on scoliosis and growth retardation above 12.6 mg/l for metoprolol. Scoring all effect parameters, the 72-h-EC(50) values were: for carbamazepine 86.5mg/l, for diclofenac 5.3mg/l and for metoprolol 31.0mg/l (mean measured concentrations). In conclusion, our results for carbamazepine and metoprolol are in agreement with other findings for aquatic toxicity, and also fish embryos responded in much the same way as rat embryos did. For diclofenac, the FET test performs comparably to Early Life Stage testing. Copyright © 2010 Elsevier Inc. All rights reserved.

Human chorionic gonadotropin (hCG) in the secretome of cultured embryos: hyperglycosylated hCG and hCG-free beta subunit are potential markers for infertility management and treatment.

PubMed

Butler, Stephen A; Luttoo, Jameel; Freire, Maísa O T; Abban, Thomas K; Borrelli, Paola T A; Iles, Ray K

2013-09-01

Human chorionic gonadotropin (hCG) is produced by trophoblast cells throughout pregnancy, and gene expression studies have indicated that hCG-beta subunit (hCGβ) expression is active at the 2 blastomere stage. Here, we investigated the qualitative hCG output of developing embryos in culture and hCG isoforms expressed in the secretome as a novel sensitive method for detecting hCG. Culture media was collected from the culture plates of 118 embryos in culture (including controls and embryos at different stages of culture) from 16 patients undergoing routine fertility treatment. The hCGβ was detectable in media from 2 pronuclear (2PN) stage embryos through to the blastocyst stage. The hCGβ was absent in 1PN and arrested embryos as well as all media controls. Prior to hatching, hyperglycosylated hCG (hCGh) was observed selectively in 3PN embryos, but after hatching, along with hCG, became the dominant hCG molecule observed. We have reported at the 2PN stage the earliest evidence of hCGβ expression in embryos. There is a suggestion this may be indicative of quality in early embryos, and hCGh seen at the pronuclear stage may suggest triploid abnormality. The dominance of hCG, and hCGh expression, seen after blastocyst hatching may be indicative of potential implantation success. Thus, hCG isoforms have potential roles as biomarkers of embryo viability for embryo/blastocyst transfer.

The vertebrate phylotypic stage and an early bilaterian-related stage in mouse embryogenesis defined by genomic information.

PubMed

Irie, Naoki; Sehara-Fujisawa, Atsuko

2007-01-12

Embryos of taxonomically different vertebrates are thought to pass through a stage in which they resemble one another morphologically. This "vertebrate phylotypic stage" may represent the basic vertebrate body plan that was established in the common ancestor of vertebrates. However, much controversy remains about when the phylotypic stage appears, and whether it even exists. To overcome the limitations of studies based on morphological comparison, we explored a comprehensive quantitative method for defining the constrained stage using expressed sequence tag (EST) data, gene ontologies (GO), and available genomes of various animals. If strong developmental constraints occur during the phylotypic stage of vertebrate embryos, then genes conserved among vertebrates would be highly expressed at this stage. We established a novel method for evaluating the ancestral nature of mouse embryonic stages that does not depend on comparative morphology. The numerical "ancestor index" revealed that the mouse indeed has a highly conserved embryonic period at embryonic day 8.0-8.5, the time of appearance of the pharyngeal arch and somites. During this period, the mouse prominently expresses GO-determined developmental genes shared among vertebrates. Similar analyses revealed the existence of a bilaterian-related period, during which GO-determined developmental genes shared among bilaterians are markedly expressed at the cleavage-to-gastrulation period. The genes associated with the phylotypic stage identified by our method are essential in embryogenesis. Our results demonstrate that the mid-embryonic stage of the mouse is indeed highly constrained, supporting the existence of the phylotypic stage. Furthermore, this candidate stage is preceded by a putative bilaterian ancestor-related period. These results not only support the developmental hourglass model, but also highlight the hierarchical aspect of embryogenesis proposed by von Baer. Identification of conserved stages and tissues

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. Copyright © 2011 Elsevier Inc. All rights reserved.

Contraction and stress-dependent growth shape the forebrain of the early chicken embryo.

PubMed

Garcia, Kara E; Okamoto, Ruth J; Bayly, Philip V; Taber, Larry A

2017-01-01

During early vertebrate development, local constrictions, or sulci, form to divide the forebrain into the diencephalon, telencephalon, and optic vesicles. These partitions are maintained and exaggerated as the brain tube inflates, grows, and bends. Combining quantitative experiments on chick embryos with computational modeling, we investigated the biophysical mechanisms that drive these changes in brain shape. Chemical perturbations of contractility indicated that actomyosin contraction plays a major role in the creation of initial constrictions (Hamburger-Hamilton stages HH11-12), and fluorescent staining revealed that F-actin is circumferentially aligned at all constrictions. A finite element model based on these findings shows that the observed shape changes are consistent with circumferential contraction in these regions. To explain why sulci continue to deepen as the forebrain expands (HH12-20), we speculate that growth depends on wall stress. This idea was examined by including stress-dependent growth in a model with cerebrospinal fluid pressure and bending (cephalic flexure). The results given by the model agree with observed morphological changes that occur in the brain tube under normal and reduced eCSF pressure, quantitative measurements of relative sulcal depth versus time, and previously published patterns of cell proliferation. Taken together, our results support a biphasic mechanism for forebrain morphogenesis consisting of differential contractility (early) and stress-dependent growth (late). Copyright © 2016 Elsevier Ltd. All rights reserved.

Entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kao, K.R.; Elinson, R.P.

1988-05-01

The body plan of Xenopus laevis can be respecified by briefly exposing early cleavage stage embryos to lithium. Such embryos develop exaggerated dorsoanterior structures such as a radial eye and cement gland. In this paper, we demonstrate that the enhanced dorsoanterior phenotype results from an overcommitment of mesoderm to dorsoanterior mesoderm. Histological and immunohistochemical observations reveal that the embryos have a greatly enlarged notochord with very little muscle tissue. In addition, they develop a radial, beating heart, suggesting that lithium also specifies anterior mesoderm and pharyngeal endoderm. Randomly oriented diametrically opposed marginal zone grafts from lithium-treated embryos, when transplanted intomore » ultraviolet (uv)-irradiated axis-deficient hosts, rescue dorsal axial structures. These transplantation experiments demonstrate that the entire marginal zone of the early gastrula consists of presumptive dorsal mesoderm. Vital dye marking experiments also indicate that the entire marginal zone maps to the prominent proboscis that is composed of chordamesoderm and represents the long axis of the embryo. These results suggest that lithium respecifies the mesoderm of Xenopus laevis embryos so that it differentiates into the Spemann organizer. We suggest that the origin of the dorsoanterior enhanced phenotypes generated by lithium and the dorsoanterior deficient phenotypes generated by uv irradiation are due to relative quantities of organizer. Our evidence demonstrates the existence of a continuum of body plan phenotypes based on this premise.« less

Microcystin-LR affects the hypothalamic-pituitary-inter-renal (HPI) axis in early life stages (embryos and larvae) of zebrafish.

PubMed

Chen, Liang; Wang, Yeke; Giesy, John P; Chen, Feng; Shi, Ting; Chen, Jun; Xie, Ping

2018-05-22

Frequencies and durations of blooms of cyanobacteria are increasing. Some cyanobacteria can produce cyanotoxins including microcystins (MCs). MCs are the most common toxic products of hazardous algal blooms (HABs), with the greatest potential for exposure and to cause toxicity. Recently, MCs have been shown to disrupt endocrine functions. In this study, for the first time, effects of MC-LR on the hypothalamic-pituitary-inter-renal (HPI) axis during early embryonic development (embryos/larvae) of zebrafish (Danio rerio), were investigated. Embryos/larvae of zebrafish were exposed to 1, 10, 100, or 300 μg MC-LR/L during the period of 4-168 h post-fertilization (hpf). Exposure to 300 μg MC-LR/L resulted in significantly greater concentrations of whole-body cortisol than those in controls. Expressions of genes along the HPI axis and mineralocorticoid receptor (MR-) and glucocorticoid receptor (GR-) centered gene networks were evaluated by use of quantitative real-time PCR. Expression of mRNA for crh was significantly down-regulated by exposure to 300 μg MC-LR/L, while expressions of crhbp, crhr1, and crhr2 were significantly up-regulated, relative to controls. MC-LR caused significantly lesser levels of mRNA for steroidogenic genes including hmgra, star, and cyp17, but expression of mRNA for hsd20b was significantly greater than that of controls. Treatment with MC-LR also altered profiles of transcription of MR- and GR-centered gene networks, which might result in multiple responses. Taken together, these results demonstrated that MC-LR affected the corticosteroid-endocrine system of larvae of zebrafish. This study provided valuable insights into molecular mechanisms behind potential toxicity and endocrine disruption of MCs. Copyright © 2018 Elsevier Ltd. All rights reserved.

Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisition of thermotolerance

PubMed Central

2013-01-01

Background While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3’ tag digital gene expression (3’DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance. Results Using 3’DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT. Conclusions Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene

Cloning, expression pattern, and potential role of apoptosis inhibitor 5 in the termination of embryonic diapause and early embryo development of Artemia sinica.

PubMed

Zhang, Shuang; Yao, Feng; Jing, Ting; Zhang, Mengchen; Zhao, Wei; Zou, Xiangyang; Sui, Linlin; Hou, Lin

2017-09-10

During the embryonic development of Artemia sinica, the diapause phenomenon can be induced by high salinity or low temperature conditions. The diapause embryo at the gastrula stage is maintained under the threat of apoptosis to guarantee the embryo's normal development. In this process, apoptosis inhibitor proteins play vital roles in protecting embryos against apoptosis. Apoptosis inhibitor5 (API5) plays a pivotal role in regulating the cell cycle and preventing programmed cell death after growth factor starvation. In the present study, we cloned the full-length cDNA representing the api5 gene from A. sinica (As-api5), which encodes a 372-amino acid protein. In situ hybridization experiments revealed that As-api5 expression is not tissue or organ specific. Quantitative real-time PCR analyses of the developmental expression of As-api5 showed that it reached its highest level at 10h, after which its expression decreased. High salinity and low temperature treatments increased the expression of As-api5. Western blotting was used to assess the abundance of As-API5 and related proteins (As-CyclinA, As-CyclinE, As-E2F1, As-CDK2, As-APAF1, and As-Caspase9). Downregulation of As-api5 expression using a short interfering RNA resulted in increased mortality and embryo malformation of A. sinica. Taken together, the results indicated that API5 plays a crucial role in embryonic diapause termination and early embryo development of A. sinica. Copyright © 2017. Published by Elsevier B.V.

Computer ranking of the sequence of appearance of 100 features of the brain and related structures in staged human embryos during the first 5 weeks of development.

PubMed

O'Rahilly, R; Müller, F; Hutchins, G M; Moore, G W

1984-11-01

The sequence of events in the development of the brain in staged human embryos was investigated in much greater detail than in previous studies by listing 100 features in 165 embryos of the first 5 weeks. Using a computerized bubble-sort algorithm, individual embryos were ranked in ascending order of the features present. This procedure made feasible an appreciation of the slight variation found in the developmental features. The vast majority of features appeared during either one or two stages (about 2 or 3 days). In general, the soundness of the Carnegie system of embryonic staging was amply confirmed. The rhombencephalon was found to show increasing complexity around stage 13, and the postoptic portion of the diencephalon underwent considerable differentiation by stage 15. The need for similar investigations of other systems of the body is emphasized, and the importance of such studies in assessing the timing of congenital malformations and in clarifying syndromic clusters is suggested.

Genome-Scale Transcriptomic Insights into Early-Stage Fruit Development in Woodland Strawberry Fragaria vesca[C][W

PubMed Central

Kang, Chunying; Darwish, Omar; Geretz, Aviva; Shahan, Rachel; Alkharouf, Nadim; Liu, Zhongchi

2013-01-01

Fragaria vesca, a diploid woodland strawberry with a small and sequenced genome, is an excellent model for studying fruit development. The strawberry fruit is unique in that the edible flesh is actually enlarged receptacle tissue. The true fruit are the numerous dry achenes dotting the receptacle’s surface. Auxin produced from the achene is essential for the receptacle fruit set, a paradigm for studying crosstalk between hormone signaling and development. To investigate the molecular mechanism underlying strawberry fruit set, next-generation sequencing was employed to profile early-stage fruit development with five fruit tissue types and five developmental stages from floral anthesis to enlarged fruits. This two-dimensional data set provides a systems-level view of molecular events with precise spatial and temporal resolution. The data suggest that the endosperm and seed coat may play a more prominent role than the embryo in auxin and gibberellin biosynthesis for fruit set. A model is proposed to illustrate how hormonal signals produced in the endosperm and seed coat coordinate seed, ovary wall, and receptacle fruit development. The comprehensive fruit transcriptome data set provides a wealth of genomic resources for the strawberry and Rosaceae communities as well as unprecedented molecular insight into fruit set and early stage fruit development. PMID:23898027

Early embryonic expression of FGF4/6/9 gene and its role in the induction of mesenchyme and notochord in Ciona savignyi embryos.

PubMed

Imai, Kaoru S; Satoh, Nori; Satou, Yutaka

2002-04-01

In early Ciona savignyi embryos, nuclear localization of beta-catenin is the first step of endodermal cell specification, and triggers the activation of various target genes. A cDNA for Cs-FGF4/6/9, a gene activated downstream of beta-catenin signaling, was isolated and shown to encode an FGF protein with features of both FGF4/6 and FGF9/20. The early embryonic expression of Cs-FGF4/6/9 was transient and the transcript was seen in endodermal cells at the 16- and 32-cell stages, in notochord and muscle cells at the 64-cell stage, and in nerve cord and muscle cells at the 110-cell stage; the gene was then expressed again in cells of the nervous system after neurulation. When the gene function was suppressed with a specific antisense morpholino oligo, the differentiation of mesenchyme cells was completely blocked, and the fate of presumptive mesenchyme cells appeared to change into that of muscle cells. The inhibition of mesenchyme differentiation was abrogated by coinjection of the morpholino oligo and synthetic Cs-FGF4/6/9 mRNA. Downregulation of beta-catenin nuclear localization resulted in the absence of mesenchyme cell differentiation due to failure of the formation of signal-producing endodermal cells. Injection of synthetic Cs-FGF4/6/9 mRNA in beta-catenin-downregulated embryos evoked mesenchyme cell differentiation. These results strongly suggest that Cs-FGF4/6/9 produced by endodermal cells acts an inductive signal for the differentiation of mesenchyme cells. On the other hand, the role of Cs-FGF4/6/9 in the induction of notochord cells is partial; the initial process of the induction was inhibited by Cs-FGF4/6/9 morpholino oligo, but notochord-specific genes were expressed later to form a partial notochord.

Lethal and Sublethal Effects of the Herbicide Atrazine in the Early Stages of Development of Physalaemus gracilis (Anura: Leptodactylidae).

PubMed

Rutkoski, Camila F; Macagnan, Natani; Kolcenti, Cassiane; Vanzetto, Guilherme V; Sturza, Paola F; Hartmann, Paulo A; Hartmann, Marilia T

2018-05-01

Water sources used as reproductive sites by crying frog, Physalaemus gracilis, are extensively associated with agroecosystems in which the herbicide atrazine is employed. To evaluate the lethal and sublethal effects of atrazine commercial formulation, acute and chronic toxicity tests were performed in the embryonic phase and the beginning of the larval phase of P. gracilis. Tests were started on stage 19 of Gosner (Herpetologica 16:183-190, 1960) and performed in 24-well cell culture plates. Acute tests had a duration of 96 h with embryo mortality monitoring every 24 h. Chronic assays contemplated the transition from the embryonic to larval stages and lasted 168 h. Every 24 h the embryos/larvae were observed for mortality, mobility, and malformations. The LC50 of atrazine determined for P. gracilis embryos was 229.34 mg L -1 . The sublethal concentrations did not affect the development of the larvae but were observed effects on mobility and malformations, such as spasmodic contractions, reduced mobility, malformations in mouth and intestine, and edema arising. From 1 mg L -1 atrazine, the exposed larvae began to have changes in mobility and malformations. The atrazine commercial formulation has caused early life effects of P. gracilis that may compromise the survival of this species but at higher concentrations than recorded in the environment, so P. gracilis can be considered tolerant to this herbicide at environmentally relevant concentrations.

Development of the arterial pattern in the upper limb of staged human embryos: normal development and anatomic variations

PubMed Central

RODRÍGUEZ-NIEDENFÜHR, M.; BURTON, G. J.; DEU, J.; SAÑUDO, J. R.

2001-01-01

A total of 112 human embryos (224 upper limbs) between stages 12 and 23 of development were examined. It was observed that formation of the arterial system in the upper limb takes place as a dual process. An initial capillary plexus appears from the dorsal aorta during stage 12 and develops at the same rate as the limb. At stage 13, the capillary plexus begins a maturation process involving the enlargement and differentiation of selected parts. This remodelling process starts in the aorta and continues in a proximal to distal sequence. By stage 15 the differentiation has reached the subclavian and axillary arteries, by stage 17 it has reached the brachial artery as far as the elbow, by stage 18 it has reached the forearm arteries except for the distal part of the radial, and finally by stage 21 the whole arterial pattern is present in its definitive morphology. This differentiation process parallels the development of the skeletal system chronologically. A number of arterial variations were observed, and classified as follows: superficial brachial (7.7%), accessory brachial (0.6%), brachioradial (14%), superficial brachioulnar (4.7%), superficial brachioulnoradial (0.7%), palmar pattern of the median (18.7%) and superficial brachiomedian (0.7%) arteries. They were observed in embryos belonging to stages 17–23 and were not related to a specific stage of development. Statistical comparison with the rates of variations reported in adults did not show significant differences. It is suggested that the variations arise through the persistence, enlargement and differentiation of parts of the initial network which would normally remain as capillaries or even regress. PMID:11693301

Ammonia and urea handling by early life stages of fishes.

PubMed

Zimmer, Alex M; Wright, Patricia A; Wood, Chris M

2017-11-01

Nitrogen metabolism in fishes has been a focus of comparative physiologists for nearly a century. In this Review, we focus specifically on early life stages of fishes, which have received considerable attention in more recent work. Nitrogen metabolism and excretion in early life differs fundamentally from that of juvenile and adult fishes because of (1) the presence of a chorion capsule in embryos that imposes a limitation on effective ammonia excretion, (2) an amino acid-based metabolism that generates a substantial ammonia load, and (3) the lack of a functional gill, which is the primary site of nitrogen excretion in juvenile and adult fishes. Recent findings have shed considerable light on the mechanisms by which these constraints are overcome in early life. Perhaps most importantly, the discovery of Rhesus (Rh) glycoproteins as ammonia transporters and their expression in ion-transporting cells on the skin of larval fishes has transformed our understanding of ammonia excretion by fishes in general. The emergence of larval zebrafish as a model species, together with genetic knockdown techniques, has similarly advanced our understanding of ammonia and urea metabolism and excretion by larval fishes. It has also now been demonstrated that ammonia excretion is one of the primary functions of the developing gill in rainbow trout larvae, leading to new hypotheses regarding the physiological demands driving gill development in larval fishes. Here, we highlight and discuss the dramatic changes in nitrogen handling that occur over early life development in fishes. © 2017. Published by The Company of Biologists Ltd.

Analysis of compaction initiation in human embryos by using time-lapse cinematography.

PubMed

Iwata, Kyoko; Yumoto, Keitaro; Sugishima, Minako; Mizoguchi, Chizuru; Kai, Yoshiteru; Iba, Yumiko; Mio, Yasuyuki

2014-04-01

To analyze the initiation of compaction in human embryos in vitro by using time-lapse cinematography (TLC), with the goal of determining the precise timing of compaction and clarifying the morphological changes underlying the compaction process. One hundred and fifteen embryos donated by couples with no further need for embryo-transfer were used in this study. Donated embryos were thawed and processed, and then their morphological behavior during the initiation of compaction was dynamically observed via time-lapse cinematography (TLC) for 5 days. Although the initiation of compaction occurred throughout the period from the 4-cell to 16-cell stage, 99 (86.1 %) embryos initiated compaction at the 8-cell stage or later, with initiation at the 8-cell stage being most frequent (22.6 %). Of these 99 embryos, 49.5 % developed into good-quality blastocysts. In contrast, of the 16 (13.9 %) embryos that initiated compaction prior to the 8-cell stage, only 18.8 % developed into good-quality blastocysts. Embryos that initiated compaction before the 8-cell stage showed significantly higher numbers of multinucleated blastomeres, due to asynchronism in nuclear division at the third mitotic division resulting from cytokinetic failure. The initiation of compaction primarily occurs at the third mitotic division or later in human embryos. Embryos that initiate compaction before the 8-cell stage are usually associated with aberrant embryonic development (i.e., cytokinetic failure accompanied by karyokinesis).

MicroRNA Signaling in Embryo Development

PubMed Central

Gross, Nicole; Khatib, Hasan

2017-01-01

Expression of microRNAs (miRNAs) is essential for embryonic development and serves important roles in gametogenesis. miRNAs are secreted into the extracellular environment by the embryo during the preimplantation stage of development. Several cell types secrete miRNAs into biological fluids in the extracellular environment. These fluid-derived miRNAs have been shown to circulate the body. Stable transport is dependent on proper packaging of the miRNAs into extracellular vesicles (EVs), including exosomes. These vesicles, which also contain RNA, DNA and proteins, are on the forefront of research on cell-to-cell communication. Interestingly, EVs have been identified in many reproductive fluids, such as uterine fluid, where their miRNA content is proposed to serve as a mechanism of crosstalk between the mother and conceptus. Here, we review the role of miRNAs in molecular signaling and discuss their transport during early embryo development and implantation. PMID:28906477

Inter- and intra-specific differences in the formation of O-xylosyl derivatives of zeatin and dihydrozeatin in Phaseolus embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mok, D.W.S.; Mok, M.C.

1987-08-01

The metabolism of /sup 14/C-zeatin was examined in embryos of P. coccineus cvs. Scarlet Runner (SR) and Desiree (Des). The O-xylosyl derivatives of zeatin and ribosylzeatin previously isolated from P. vulgaris embryos were found in both cultivars. In addition, two new metabolites were isolated from SR embryos and were identified by enzymatic degradation and GC-MS analyses as O-xylosyldihydrozeatin and its ribonucleaside. O-xylosylation of zeatin was also detected in P. acutifolius, but not in P. lunatus embryos. Both the formation of O-xylosyl derivatives and side chain reduction seem to be genotype specific. Moreover, they are most pronounced at early stages ofmore » embryo development.« less

Assessing Aromatic-Hydrocarbon Toxicity to Fish Early Life Stages Using Passive-Dosing Methods and Target-Lipid and Chemical-Activity Models.

PubMed

Butler, Josh D; Parkerton, Thomas F; Redman, Aaron D; Letinski, Daniel J; Cooper, Keith R

2016-08-02

Aromatic hydrocarbons (AH) are known to impair fish early life stages (ELS). However, poorly defined exposures often confound ELS-test interpretation. Passive dosing (PD) overcomes these challenges by delivering consistent, controlled exposures. The objectives of this study were to apply PD to obtain 5 d acute embryo lethality and developmental data and 30 d chronic embryo-larval survival and growth-effects data using zebrafish with different AHs; to analyze study and literature toxicity data using target-lipid (TLM) and chemical-activity (CA) models; and to extend PD to a mixture and test the assumption of AH additivity. PD maintained targeted exposures over a concentration range of 6 orders of magnitude. AH toxicity increased with log Kow up to pyrene (5.2). Pericardial edema was the most sensitive sublethal effect that often preceded embryo mortality, although some AHs did not produce developmental effects at concentrations causing mortality. Cumulative embryo-larval mortality was more sensitive than larval growth, with acute-to-chronic ratios of <10. More-hydrophobic AHs did not exhibit toxicity at aqueous saturation. The relationship and utility of the TLM-CA models for characterizing fish ELS toxicity is discussed. Application of these models indicated that concentration addition provided a conservative basis for predicting ELS effects for the mixture investigated.

Stage-specific regulation of four HD-ZIP III transcription factors during polar pattern formation in Larix leptolepis somatic embryos.

PubMed

Li, Shui-gen; Li, Wan-feng; Han, Su-ying; Yang, Wen-hua; Qi, Li-wang

2013-06-15

Polar auxin transport provides a developmental signal for cell fate specification during somatic embryogenesis. Some members of the HD-ZIP III transcription factors participate in regulation of auxin transport, but little is known about this regulation in somatic embryogenesis. Here, four HD-ZIP III homologues from Larix leptolepis were identified and designated LaHDZ31, 32, 33 and 34. The occurrence of a miR165/166 target sequence in all four cDNA sequences indicated that they might be targets of miR165/166. Identification of the cleavage products of LaHDZ31 and LaHDZ32 in vivo confirmed that they were regulated by miRNA. Their mRNA accumulation patterns during somatic embryogenesis and the effects of 1-N-naphthylphthalamic acid (NPA) on their transcript levels and somatic embryo maturation were investigated. The results showed that the four genes had higher transcript levels at mature stages than at the proliferation stage, and that NPA treatment down-regulated the mRNA abundance of LaHDZ31, 32 and 33 at cotyledonary embryo stages, but had no effect on the mRNA abundance of LaHDZ34. We concluded that these four members of Larix HD-ZIP III family might participate in polar auxin transport and the development of somatic embryos, providing new insights into the regulatory mechanisms of somatic embryogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

PubMed

Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

2012-12-01

In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

Toxicity of naproxen sodium and its mixture with tramadol hydrochloride on fish early life stages.

PubMed

Sehonova, Pavla; Plhalova, Lucie; Blahova, Jana; Doubkova, Veronika; Prokes, Miroslav; Tichy, Frantisek; Fiorino, Emma; Faggio, Caterina; Svobodova, Zdenka

2017-12-01

Pharmaceuticals occur in water bodies as a consequence of their incomplete removal during waste water treatment processes. The occurence of pharmaceuticals in surface waters as well as their possible impact on aquatic vertebrates have received considerable attention in recent years. However, there is still a lack of informations on the chronic effects of widely used drugs as well as their possible mixture toxicity on non-target aquatic vertebrates as well as their possible mixture toxicity. The aim of this study was to assess the effects of naproxen sodium on early life stages of fish and evaluate its mixture toxicity with tramadol hydrochloride, which was assessed in our earlier study as a single substance. Two embryo-larval toxicity tests with common carp (Cyprinus carpio) were performed according to the OECD guideline 210 (Fish, Early-life Stage Toxicity Test) in order to assess the subchronic toxicity of naproxen sodium and tramadol hydrochlorid-naproxen sodium mixture at the concentrations of 10; 50; 100 and 200 μg/L. These experiments were conducted for 32 days. The subchronic exposure to naproxen sodium and naproxen sodium and tramadol hydrochloride mixture had a strong effect on the early life stages of common carp. Hatching, developmental rate, morphology, histopathology and, in the case of the naproxen sodium and tramadol hydrochloride mixture, mortality were influenced. The bioindicators of oxidative stress were also influenced. The LOEC was determined at 10 μg/L for both naproxen sodium and naproxen sodium and tramadol hydrochloride mixture. Copyright © 2017 Elsevier Ltd. All rights reserved.

Developmental bias in cleavage-stage mouse blastomeres

PubMed Central

Tabansky, Inna; Lenarcic, Alan; Draft, Ryan W.; Loulier, Karine; Keskin, Derin B; Rosains, Jacqueline; Rivera-Feliciano, José; Lichtman, Jeff W.; Livet, Jean; Stern, Joel NH; Sanes, Joshua R.; Eggan, Kevin

2012-01-01

Summary Introduction The cleavage stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, as preimplantation genetic diagnosis (PGD) requires removal of blastomeres from the early human embryo. To determine if blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, non-invasive combinatorial fluorescent labeling method for embryonic lineage tracing. Results When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into post-implantation stages, and therefore has relevance for subsequent development. Discussion The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues. PMID:23177476

Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein.

PubMed

Chen, Linan; Dumelie, Jason G; Li, Xiao; Cheng, Matthew Hk; Yang, Zhiyong; Laver, John D; Siddiqui, Najeeb U; Westwood, J Timothy; Morris, Quaid; Lipshitz, Howard D; Smibert, Craig A

2014-01-07

Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug's target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.

Glutathione and abscisic acid supplementation influences somatic embryo maturation and hormone endogenous levels during somatic embryogenesis in Podocarpus lambertii Klotzsch ex Endl.

PubMed

Fraga, Hugo Pacheco de Freitas; Vieira, Leila do Nascimento; Puttkammer, Catarina Corrêa; Dos Santos, Henrique Pessoa; Garighan, Julio de Andrade; Guerra, Miguel Pedro

2016-12-01

Here we propose a protocol for embryogenic cultures induction, proliferation and maturation for the Brazilian conifer Podocarpus lambertii, and investigated the effect of abscisic acid (ABA) and glutathione (GSH) supplementation on the maturation phase. ABA, zeatin (Z) and salicylic acid (SA) endogenous levels were quantified. Number of somatic embryos obtained in ABA-supplemented treatment was significant higher than in ABA-free treatment, showing the relevance of ABA supplementation during somatic embryos maturation. Histological analysis showed the stereotyped sequence of developmental stages in conifer somatic embryos, reaching the late torpedo-staged embryo. GSH supplementation in maturation culture medium improved the somatic embryos number and morphological features. GSH 0mM and GSH 0.1mM treatments correlated with a decreased ABA endogenous level during maturation, while GSH 0.5mM treatment showed constant levels. All treatments resulted in decreased Z endogenous levels, supporting the concept that cytokinins are important during the initial cell division but not for the later stages of embryo development. The lowest SA levels found in GSH 0.5mM treatment were coincident with early embryonic development, and this treatment resulted in the highest development of somatic embryos. Thus, a correlation between lower SA levels and improved somatic embryo formation can be hypothesized. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Characterization of the altered gene expression profile in early porcine embryos generated from parthenogenesis and somatic cell chromatin transfer.

PubMed

Zhou, Chi; Dobrinsky, John; Tsoi, Stephen; Foxcroft, George R; Dixon, Walter T; Stothard, Paul; Verstegen, John; Dyck, Michael K

2014-01-01

The in vitro production of early porcine embryos is of particular scientific and economic interest. In general, embryos produced from in vitro Assisted Reproductive Technologies (ART) manipulations, such as somatic cell chromatin transfer (CT) and parthenogenetic activation (PA), are less developmentally competent than in vivo-derived embryos. The mechanisms underlying the deficiencies of embryos generated from PA and CT have not been completely understood. To characterize the altered genes and gene networks in embryos generated from CT and PA, comparative transcriptomic analyses of in vivo (IVV) expanded blastocysts (XB), IVV hatched blastocyst (HB), PA XB, PA HB, and CT HB were performed using a custom microarray platform enriched for genes expressed during early embryonic development. Differential expressions of 1492 and 103 genes were identified in PA and CT HB, respectively, in comparison with IVV HB. The "eIF2 signalling", "mitochondrial dysfunction", "regulation of eIF4 and p70S6K signalling", "protein ubiquitination", and "mTOR signalling" pathways were down-regulated in PA HB. Dysregulation of notch signalling-associated genes were observed in both PA and CT HB. TP53 was predicted to be activated in both PA and CT HB, as 136 and 23 regulation targets of TP53 showed significant differential expression in PA and CT HB, respectively, in comparison with IVV HB. In addition, dysregulations of several critical pluripotency, trophoblast development, and implantation-associated genes (NANOG, GATA2, KRT8, LGMN, and DPP4) were observed in PA HB during the blastocyst hatching process. The critical genes that were observed to be dysregulated in CT and PA embryos could be indicative of underlying developmental deficiencies of embryos produced from these technologies.

Moving and fusion of the pancreatic buds in the rat embryos during the embryonic period (carnegie stages 13-17) by a three-dimensional computer-assisted reconstruction.

PubMed

Godlewski, Guilhem; Gaubert, Jacques; Cristol-Gaubert, Renée; Radi, Maïada; Baecker, Volker; Travo, Pierre; Prudhomme, Michel; Prat-Pradal, Dominique

2011-10-01

The purpose of the present study was to illustrate the modality of rotation of ventral and dorsal pancreatic buds by three-dimensional (3D) reconstructions in the rat embryos, during the Carnegie stages 13-17. Serial sections of thirty rat embryos stages 13-17, were observed. The embryos were fixed in Bouin's solution, dehydrated, and paraffin embedded. The sections, 7 μm thick, were cut in longitudinal or transverse planes and were stained alternately by hematoxylin-eosin or Heindenhain' azan. The images were digitalized by Canon Camera 350 EOS D. The 3D reconstruction was performed by computer using Cell Image Analyser software. The two pancreatic buds ventral and dorsal, were clearly identified at stage 13, in anterior and posterior position, respectively, in relation to the duodenum. In stage 15, the duodenum started its rotation of 90° clockwise. The ventral bud moved 90° from the midline to the right. In stage 16, the ventral pancreas continued its rotation until 180° in posterior position behind the duodenum. In stage 17, the two pancreatic buds were related closely to the ventral part of the portal vein. The two buds began to merge. The anterior face of the pancreas's head was arising from the dorsal pancreatic bud. The rest of the head including the omental tuberosity and the uncinate process emanated from the ventral pancreatic bud. The use of 3D reconstruction of the pancreas of rat embryos illustrates the modality of the two pancreatic buds rotation and fusion. This method explains the final position of the pancreas.

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.

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.

γ-BUTYROBETAINE AS A SPECIFIC ANTAGONIST FOR CARNITINE IN THE DEVELOPMENT OF THE EARLY CHICK EMBRYO

PubMed Central

Ito, Toshio; Fraenkel, G.

1957-01-01

The effect of γ-butyrobetaine alone and with the addition of carnitine on the development of the early excised chick embryo has been studied. γ-Butyrobetaine in appropriate amounts exerts an inhibitory effect which can be relieved or annulled by the inclusion of appropriate amounts of carnitine. This has been interpreted as a metabolite-antimetabolite relationship, in which the normal metabolite, carnitine, is antagonized by the structurally closely related γ-butyrobetaine, and is regarded as evidence of an important role of carnitine in the metabolism of the developing chick embryo. PMID:13475691

Impact of CdSe/ZnS quantum dots on the development of zebrafish embryos

NASA Astrophysics Data System (ADS)

Lei, Yong; Xiao, Qi; Huang, Shan; Xu, Wansu; Zhang, Zhe; He, Zhike; Liu, Yi; Deng, Fengjiao

2011-12-01

Due to their unique fluorescent characteristics, quantum dots (QDs) have been successfully applied in the fields of biotechnology and medicine, but there is very limited information regarding their biodistribution and chronic toxicity in vivo. In this article, the biological behavior and toxic effects of mercaptoacetic acid-CdSe/ZnS QDs (MAA-QDs) in developing zebrafish embryos were investigated by in vivo tests. The MAA-QDs were introduced into zebrafish through microinjection at early stage. The results showed that the MAA-QDs at certain concentrations influenced the survival of zebrafish embryos, but treated embryos without developmental defects were also observed. MAA-QDs injected into the cytoplasm at the one-cell stage were allocated to progeny blastoderm cells during proliferation and almost never entered the yolk. The formation of notochord and primordial germ cells with normal morphologies was detected in the treated embryos by whole-mount in situ hybridization. Furthermore, traces of the element cadmium were mainly discovered in the tissue of liver and kidney of 3-month-old-treated zebrafish by quantitative assessment with inductively coupled plasma mass spectrometry. Thus, we hypothesized that low concentration MAA-QDs have chronic toxicities when they were delivered into zebrafish organs.

Cloning of noggin gene from hydra and analysis of its functional conservation using Xenopus laevis embryos.

PubMed

Chandramore, Kalpana; Ito, Yuzuro; Takahashi, Shuji; Asashima, Makoto; Ghaskadbi, Surendra

2010-01-01

Hydra, a member of phylum Cnidaria that arose early in evolution, is endowed with a defined axis, organized nervous system, and active behavior. It is a powerful model system for the elucidation of evolution of developmental mechanisms in animals. Here, we describe the identification and cloning of noggin-like gene from hydra. Noggin is a secreted protein involved at multiple stages of vertebrate embryonic development including neural induction and is known to exert its effects by inhibiting the bone morphogenetic protein (BMP)-signaling pathway. Sequence analysis revealed that hydra Noggin shows considerable similarity with its orthologs at the amino acid level. When microinjected in the early Xenopus embryos, hydra noggin mRNA induced a secondary axis in 100% of the injected embryos, demonstrating functional conservation of hydra noggin in vertebrates. This was further confirmed by the partial rescue of Xenopus embryos by hydra noggin mRNA from UV-induced ventralization. By using animal cap assay in Xenopus embryos, we demonstrate that these effects of hydra noggin in Xenopus embryos are because of inhibition of BMP signaling by Noggin. Our data indicate that BMP/Noggin antagonism predates the bilaterian divergence and is conserved during the evolution.

The study of early human embryos using interactive 3-dimensional computer reconstructions.

PubMed

Scarborough, J; Aiton, J F; McLachlan, J C; Smart, S D; Whiten, S C

1997-07-01

Tracings of serial histological sections from 4 human embryos at different Carnegie stages were used to create 3-dimensional (3D) computer models of the developing heart. The models were constructed using commercially available software developed for graphic design and the production of computer generated virtual reality environments. They are available as interactive objects which can be downloaded via the World Wide Web. This simple method of 3D reconstruction offers significant advantages for understanding important events in morphological sciences.

Exploring uptake and biodistribution of polystyrene (nano)particles in zebrafish embryos at different developmental stages.

PubMed

van Pomeren, M; Brun, N R; Peijnenburg, W J G M; Vijver, M G

2017-09-01

In ecotoxicology, it is continuously questioned whether (nano)particle exposure results in particle uptake and subsequent biodistribution or if particles adsorb to the epithelial layer only. To contribute to answering this question, we investigated different uptake routes in zebrafish embryos and how they affect particle uptake into organs and within whole organisms. This is addressed by exposing three different life stages of the zebrafish embryo in order to cover the following exposure routes: via chorion and dermal exposure; dermal exposure; oral and dermal exposure. How different nanoparticle sizes affect uptake routes was assessed by using polystyrene particles of 25, 50, 250 and 700nm. In our experimental study, we showed that particle uptake in biota is restricted to oral exposure, whereas the dermal route resulted in adsorption to the epidermis and gills only. Ingestion followed by biodistribution was observed for the tested particles of 25 and 50nm. The particles spread through the body and eventually accumulated in specific organs and tissues such as the eyes. Particles larger than 50nm were predominantly adsorbed onto the intestinal tract and outer epidermis of zebrafish embryos. Embryos exposed to particles via both epidermis and intestine showed highest uptake and eventually accumulated particles in the eye, whereas uptake of particles via the chorion and epidermis resulted in marginal uptake. Organ uptake and internal distribution should be monitored more closely to provide more in depth information of the toxicity of particles. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Human developmental anatomy: microscopic magnetic resonance imaging (μMRI) of four human embryos (from Carnegie Stage 10 to 20).

PubMed

Lhuaire, Martin; Martinez, Agathe; Kaplan, Hervé; Nuzillard, Jean-Marc; Renard, Yohann; Tonnelet, Romain; Braun, Marc; Avisse, Claude; Labrousse, Marc

2014-12-01

Technological advances in the field of biological imaging now allow multi-modal studies of human embryo anatomy. The aim of this study was to assess the high magnetic field μMRI feasibility in the study of small human embryos (less than 21mm crown-rump) as a new tool for the study of human descriptive embryology and to determine better sequence characteristics to obtain higher spatial resolution and higher signal/noise ratio. Morphological study of four human embryos belonging to the historical collection of the Department of Anatomy in the Faculty of Medicine of Reims was undertaken by μMRI. These embryos had, successively, crown-rump lengths of 3mm (Carnegie Stage, CS 10), 12mm (CS 16), 17mm (CS 18) and 21mm (CS 20). Acquisition of images was performed using a vertical nuclear magnetic resonance spectrometer, a Bruker Avance III, 500MHz, 11.7T equipped for imaging. All images were acquired using 2D (transverse, sagittal and coronal) and 3D sequences, either T1-weighted or T2-weighted. Spatial resolution between 24 and 70μm/pixel allowed clear visualization of all anatomical structures of the embryos. The study of human embryos μMRI has already been reported in the literature and a few atlases exist for educational purposes. However, to our knowledge, descriptive or morphological studies of human developmental anatomy based on data collected these few μMRI studies of human embryos are rare. This morphological noninvasive imaging method coupled with other techniques already reported seems to offer new perspectives to descriptive studies of human embryology.

High stability of nuclear microsatellite loci during the early stages of somatic embryogenesis in Norway spruce.

PubMed

Helmersson, Andreas; von Arnold, Sara; Burg, Kornel; Bozhkov, Peter V

2004-10-01

Somatic embryos of Norway spruce (Picea abies (L.) Karst.) differentiate from proembryogenic masses (PEMs), which are subject to autodestruction through programmed cell death. In PEMs, somatic embryo formation and activation of programmed cell death are interrelated processes. We sought to determine if activation of programmed cell death in PEMs is caused by genetic aberrations during somatic embryogenesis. Based on the finding that withdrawal of auxin and cytokinin induces programmed cell death in PEMs, 1-week-old cell suspensions were cultured in medium either with or without auxin and cytokinin and then transferred to maturation medium containing abscisic acid. We analyzed the stability of three nuclear simple sequence repeat (SSR) microsatellite markers at successive stages of somatic embryogenesis in two cell lines. There were no mutations at the SSR loci at any of the successive developmental stages from PEMs to cotyledonary embryos, irrespective of whether or not the proliferation medium in which cell suspensions had been cultured contained auxin or cytokinin. The morphologies of plants regenerated from the cultures were similar, although withdrawal of auxin and cytokinin significantly stimulated the yield of both embryos and plants. We conclude, therefore, that the high genetic stability of somatic embryos in Norway spruce is unaffected by the induction of programmed cell death caused by withdrawal of auxin and cytokinin.

Developmental anatomy of the liver from computerized three-dimensional reconstructions of four human embryos (from Carnegie stage 14 to 23).

PubMed

Lhuaire, Martin; Tonnelet, Romain; Renard, Yohann; Piardi, Tullio; Sommacale, Daniele; Duparc, Fabrice; Braun, Marc; Labrousse, Marc

2015-07-01

Some aspects of human embryogenesis and organogenesis remain unclear, especially concerning the development of the liver and its vasculature. The purpose of this study was to investigate, from a descriptive standpoint, the evolutionary morphogenesis of the human liver and its vasculature by computerized three-dimensional reconstructions of human embryos. Serial histological sections of four human embryos at successive stages of development belonging to three prestigious French historical collections were digitized and reconstructed in 3D using software commonly used in medical radiology. Manual segmentation of the hepatic anatomical regions of interest was performed section by section. In this study, human liver organogenesis was examined at Carnegie stages 14, 18, 21 and 23. Using a descriptive and an analytical method, we showed that these stages correspond to the implementation of the large hepatic vascular patterns (the portal system, the hepatic artery and the hepatic venous system) and the biliary system. To our knowledge, our work is the first descriptive morphological study using 3D computerized reconstructions from serial histological sections of the embryonic development of the human liver between Carnegie stages 14 and 23. Copyright © 2015 Elsevier GmbH. All rights reserved.

Immunocytochemical localisation of the nucleolar protein fibrillarin and RNA polymerase I during mouse early embryogenesis.

PubMed

Cuadros-Fernández, J M; Esponda, P

1996-02-01

We have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA polymerase I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar precursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.

[Effect of phytohemagglutinin (PHA) from Yunnan white kidney bean on development of mouse embryos].

PubMed

Zhang, Lifen; Wang, Changmei; Yang, Mingjie; Zhang, Tian; Wang, Minkang

2011-06-01

To study the effect of different concentration of phytohemagglutinin (PHA) on mouse embryo development. In experiment 1, crude and purified PHA extracted from Yunnan white kidney bean with different concentration were added into M16 culture medium, the final concentration of PHA were: 50, 100, 200, 500, 1 000, 2 000 and 5 000 mg x L(-1) respectively. 2-cell stage embryos were collected and cultured in PHA containing or control medium for 72-96 h and their development were recorded. In experiment 2, different stage of embryos from 1-cell to blastocyst were treated by different concentrations of PHA same as experiment 1 and 10 000 mg x L(-1) in culture medium for 24 h before washing and cultured in M16 + PVA without PHA to blastocyst or hatching blastocyst stage. Low concentrations PHA at 50-100 mg x L(-1) promoted embryo development and increased the number of blastocyst stage embryos. In contrast, high concentrations of PHA (> 1 000 mg x L(-1)) blocked the embryos development from 1-cell to blastocyst stage and showed apoptosis morphology or death. Depending on the concentrations, PHA from white kidney bean shown promotion or inhibition on mouse embryo development. 1-cell stage embryo shown more sensitive to PHA treatment than that of later stage embryos. Pretreatment 24 h in PHA containing medium can influence the further development of embryos. Low concentrations of PHA is benefit to embryo development, but high concentrations of PHA (> 1 000 mg x L(-1)) will block of the development of embryos.

Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos.

PubMed

Rezazadeh Valojerdi, Mojtaba; Eftekhari-Yazdi, Poopak; Karimian, Leila; Hassani, Fatemeh; Movaghar, Bahar

2009-06-01

The objective of this retrospective study was to evaluate the efficacy of vitrification and slow freezing for the cryopreservation of human cleavage stage embryos in terms of post-warming survival rate, post-warming embryo morphology and clinical outcomes. The embryos of 305 patients at cleavage stages were cryopreserved either with vitrification (153 patients) or slow-freezing (152 patients) methods. After warming; the survival rate, post-warmed embryo morphology, clinical pregnancy and implantation rates were evaluated and compared between the two groups. In the vitrification group versus slow freezing group, the survival rate (96.9% vs. 82.8%) and the post-warmed excellent morphology with all blastomeres intact (91.8% vs. 56.2%) were higher with an odds ratio of 6.607 (95% confidence interval; 4.184-10.434) and 8.769 (95% confidence interval; 6.460-11.904), respectively. In this group, the clinical pregnancy rate (40.5% vs. 21.4%) and the implantation rate (16.6% vs. 6.8%) were also higher with an odds ratio of 2.427 (95%confidence interval; 1.461-4.033) and 2.726 (95% confidence interval; 1.837-4.046), respectively. Vitrification in contrast to slow freezing is an efficient method for cryopreservation of human cleavage stage embryos. Vitrification provides a higher survival rate, minimal deleterious effects on post-warming embryo morphology and it can improve clinical outcomes.

Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation.

PubMed

Velasquez, Alejandra E; Castro, Fidel O; Veraguas, Daniel; Cox, Jose F; Lara, Evelyn; Briones, Mario; Rodriguez-Alvarez, Lleretny

2016-02-01

Embryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7-9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.

Relationships between oxygen consumption rate, viability, and subsequent development of in vivo-derived porcine embryos.

PubMed

Sakagami, N; Nishida, K; Akiyama, K; Abe, H; Hoshi, H; Suzuki, C; Yoshioka, K

2015-01-01

Oxygen consumption rate of in vivo-derived porcine embryos was measured, and its value as an objective method for the assessment of embryo quality was evaluated. Embryos were surgically collected 5 or 6 days after artificial insemination (AI), and oxygen consumption rate of embryos was measured using an embryo respirometer. The average oxygen consumption rate (F × 10(14)/mol s(-1)) of the embryos that developed to the compacted morula stage on Day 5 (Day 0 = the day of artificial insemination) was 0.58 ± 0.03 (mean ± standard error of the mean). The Day-6 embryos had consumption rates of 0.56 ± 0.13, 0.87 ± 0.06, and 1.13 ± 0.07 at the early blastocyst, blastocyst, and expanded blastocyst stages, respectively, showing a gradual increase as the embryos developed. Just after collection, the average oxygen consumption rates of embryos that hatched and of those that did not hatch after culture were 0.60 ± 0.04 and 0.50 ± 0.04 for Day 5 (P = 0.08) and 1.05 ± 0.09 and 0.77 ± 0.05 for Day 6 (P < 0.05), respectively. The value and probability of discrimination by measuring the oxygen consumption rates of embryos to predict their hatching ability after culture were 0.56 and 63.6% for Day-5 embryos and 0.91 and 68.4% for Day-6 blastocysts, respectively. When Day-5 embryos were classified based on the oxygen consumption rate and then transferred non-surgically to recipient sows, three of the seven sows, to which embryos having a high oxygen consumption rate (≥ 0.59) were transferred, became pregnant and farrowed a total of 20 piglets. However, none of the four sows, to which embryos having low oxygen consumption rate (< 0.59) were transferred, became pregnant. These results suggest that the viability of in vivo-derived porcine embryos and subsequent development can be estimated by measuring the oxygen consumption rate. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of complement depletion by cobra venom factor on fowlpox virus infection in chickens and chicken embryos.

PubMed Central

Ohta, H; Yoshikawa, Y; Kai, C; Yamanouchi, K; Taniguchi, H; Komine, K; Ishijima, Y; Okada, H

1986-01-01

The course of infection with an attenuated strain of fowlpox virus (FPV), which is known to induce antibody-independent activation of complement via the alternative pathway, was investigated in 1- to 3-day-old chickens and 14-day-old chicken embryos by treatment with cobra venom factor (CVF). CVF was found to inhibit complement activity transiently via the alternative pathway but not via the classical pathway. In chickens treated with CVF, virus growth in the skin was enhanced, and pock lesions tended to disseminate, leading to fatal infection in some birds. Histologically, an acute inflammation at an early stage of infection (within 3 days) was inhibited, and virus content in the pock lesion was increased. In chicken embryos with immature immune capacities, CVF treatment caused changes in pock morphology from clear pocks to diffuse ones, an increase in virus content in the pock, and inhibition of cell infiltration. Thus, FPV infection was aggravated in both CVF-treated chickens and chicken embryos. These results are discussed in relation to roles of complement in the elimination of virus at an early stage of FPV infection. Images PMID:3003397

Pregnancy rates for embryos transferred from early postpartum beef cows into recipients with normal estrous cycles.

PubMed

Schrick, F N; Inskeep, E K; Butcher, R L

1993-09-01

Survival rate of embryos from first ovulations of postpartum cows with SHORT (6.9 +/- 0.3 days; n = 35) or NORMAL (17.1 +/- 0.3 days; n = 42) luteal phases and quality of the embryos on Day 6 were compared. At 19 to 23 days postpartum, cows were allotted to receive a norgestomet implant for 9 days (normal luteal phase) or to serve as untreated controls (short luteal phase). Calves were weaned 7 days after initiation of treatment to induce behavioral estrus in cows for mating. In 25 cows, growth of the ovulatory follicle was monitored by ultrasonography. On Day 6 after estrus, embryos were recovered nonsurgically, and live embryos were transferred into recipient cows exhibiting normal estrous cycles. The medium used to flush the embryos from the uterus of each donor cow was assayed for prostaglandin F2 alpha (PGF2 alpha). Days from calf removal to estrus and size of ovulatory follicles at ovulation (4.1 +/- 0.3 days and 16.7 +/- 0.7 mm, respectively) did not differ between NORMAL and SHORT cows. Interval from detection of the ovulatory follicle to ovulation was longer in NORMAL (10 +/- 0.7 days) than in SHORT cows (8 +/- 0.6 days; p < 0.05). Rates of recovery of an embryo or ovum (64%), rates of fertilization (65%), and quality or stage of development of Day 6 embryos did not differ between SHORT and NORMAL cows. Overall pregnancy rate from recovered oocytes was 13% for SHORT and 32% for NORMAL cows (p = 0.06); survival of fertilized oocytes was 23% for SHORT and 47% for NORMAL cows (p = 0.08).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of repeated light-dark shifts on uterine receptivity and early gestation in mice undergoing embryo transfer.

PubMed

Goldstein, Cathy A; O'Brien, Louise M; Bergin, Ingrid L; Saunders, Thomas L

2018-04-01

Female shift workers are at increased risk for negative reproductive outcomes, and animal evidence suggests that manipulation of the light-dark cycle is detrimental to early gestation in female mice. Specifically, failure of implantation may be responsible for these findings. The objective of this study was to better delineate which reproductive processes are vulnerable to detrimental effects of maternal circadian disturbance. We exposed mice undergoing embryo transfer to repetitive phase advances of the photoperiod. Embryos were derived from donor sperm and eggs from mice living in normal light-dark conditions to isolate the effects of photoperiod disruption on uterine receptivity and early gestation. Twenty-eight mice receiving embryo transfer underwent an experimental light-dark condition (advance of lights on and lights off by 6 hours every 4 days). Twenty-eight mice remained in a normal light-dark condition. Animals lived in their assigned light-dark condition beginning 2 weeks prior to embryo transfer and ending the day of uterine necropsy (post-coitus day 14.5). Wilcoxon-Mann-Whitney test demonstrated no significant differences between control and experimental light-dark conditions in pups (Z=0.10, p=.92), resorptions (Z=0.20, p=.84), or implantations (Z=-0.34, p=.73). Pup and placental weights were similar between groups. In this investigation, uterine receptivity and maintenance of early gestation were preserved despite recurrent phase advances in photoperiod. This finding, in the context of the current literature, suggests that the negative effects of circadian disruption are mediated by reproductive processes upstream of implantation.

Cell wall invertase as a regulator in determining sequential development of endosperm and embryo through glucose signaling early in seed development.

PubMed

Wang, Lu; Liao, Shengjin; Ruan, Yong-Ling

2013-01-01

Seed development depends on coordination among embryo, endosperm and seed coat. Endosperm undergoes nuclear division soon after fertilization, whereas embryo remains quiescent for a while. Such a developmental sequence is of great importance for proper seed development. However, the underlying mechanism remains unclear. Recent results on the cellular domain- and stage-specific expression of invertase genes in cotton and Arabidopsis revealed that cell wall invertase may positively and specifically regulate nuclear division of endosperm after fertilization, thereby playing a role in determining the sequential development of endosperm and embryo, probably through glucose signaling.

Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein

PubMed Central

2014-01-01

Background Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. Results To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug’s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Conclusions Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism. PMID:24393533

Nucleolus Precursor Bodies and Ribosome Biogenesis in Early Mammalian Embryos: Old Theories and New Discoveries.

PubMed

Fulka, Helena; Aoki, Fugaku

2016-06-01

In mammals, mature oocytes and early preimplantation embryos contain transcriptionally inactive structures termed nucleolus precursor bodies instead of the typical fibrillo-granular nucleoli. These nuclear organelles are essential and strictly of maternal origin. If they are removed from oocytes, the resulting embryos are unable to replace them and consequently fail to develop. Historically, nucleolus precursor bodies have been perceived as a passive repository site of nucleolar proteins that are required for embryos to form fully functional nucleoli. Recent results, however, contradict this long-standing dogma and show that these organelles are dispensable for nucleologenesis and ribosome biogenesis. In this article, we discuss the possible roles of nucleolus precursor bodies and propose how they might be involved in embryogenesis. Furthermore, we argue that these organelles are essential only shortly after fertilization and suggest that they might actively participate in centromeric chromatin establishment. © 2016 by the Society for the Study of Reproduction, Inc.

Methods for imaging individual cilia in living echinoid embryos.

PubMed

Morris, Robert L; Pope, Hans W; Sholi, Adam N; Williams, Leah M; Ettinger, Chelsea R; Beacham, Gwendolyn M; Shintaku, Tatsushi; Abbott, Zachary D; Doherty, Elyse M

2015-01-01

The embryos of echinoids (sea urchins and sand dollars) serve as excellent models for studying cilia differentiation and stages of the cilia life cycle including ciliogenic initiation, growth, maintenance, and retraction. Early in echinoid development, uniform motile cilia form on all cells simultaneously but then rapidly differentiate into multiple cilia types that differ in morphology, motility, and signaling sensitivity. Metal ion treatments that shift germ layer boundaries and thereby "animalize" or "vegetalize" embryos can be used to enrich for low-abundance cilia types rendering those specialized cilia and the differentiation processes they exhibit much easier to study. The experimental advantages of having robust cilia growth and differentiation is tempered by the challenge of restraining ciliated embryos well enough to view the process of ciliogenesis live. We have developed four observation chambers as modifications of the Kiehart chamber for long-term light microscopic imaging of ciliated echinoid embryos. One of these systems employs paramagnetic beads to render ciliated larvae magnetic so they can be gently and reversibly trapped directly under the objective lens. With this magnetic trapping system, the larva can be positioned and repositioned until they achieve the orientation with the clearest view of any cilia of interest. These methods of gentle embryo restraint allow normal embryo development and the normal ciliogenic cycle and ciliary differentiation processes to continue in direct view. Sequential image series can then be collected and analyzed to quantitatively study the wide spectrum of cilia behaviors and properties that arise in developing echinoid embryos. Copyright © 2015. Published by Elsevier Inc.

Laser Fusion of Mouse Embryonic Cells and Intra-Embryonic Fusion of Blastomeres without Affecting the Embryo Integrity

PubMed Central

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. PMID:23227157

Time-lapse cinematography of dynamic changes occurring during in vitro development of human embryos.

PubMed

Mio, Yasuyuki; Maeda, Kazuo

2008-12-01

The purpose of this study was to clarify developmental changes of early human embryos by using time-lapse cinematography (TLC). For human ova, fertilization and cleavage, development of the blastocyst, and hatching, as well as consequent changes were repeatedly photographed at intervals of 5-6 days by using an inverse microscope under stabilized temperature and pH. Photographs were taken at 30 frames per second and the movies were studied. Cinematography has increased our understanding of the morphologic mechanisms of fertilization, development, and behavior of early human embryos, and has identified the increased risk of monozygotic twin pregnancy based on prolonged incubation in vitro to the blastocyst stage. Using TLC, we observed the fertilization of an ovum by a single spermatozoon, followed by early cleavages, formation of the morula, blastocyst hatching, changes in the embryonic plates, and the development of monozygotic twins from the incubated blastocysts.

Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

USGS Publications Warehouse

Nel-Themaat, L.; Gomez, M.C.; Pope, C.E.; Lopez, M.; Wirtu, G.; Jenkins, J.A.; Cole, A.; Dresser, B.L.; Bondioli, K.R.; Godke, R.A.

2008-01-01

The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p < 0.0001), than for eland IgNT embryos (0.5 and 0%, respectively). DNA synthesis was not observed in either bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had ???8 cells at 84 hpa, while 32% of the bovine NT embryos had ???8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had ???8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further. ?? 2008 Mary Ann Liebert, Inc.

Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors.

PubMed Central

Paria, B C; Dey, S K

1990-01-01

We have established a model that shows cooperative interaction among preimplantation embryos and the role of growth factors on their development and growth. Two-cell mouse embryos cultured singly in 25-microliters microdrops had inferior development to blastocysts and lower cell numbers per blastocyst compared with those cultured in groups of 5 or 10. The inferior development of singly cultured embryos was markedly improved by addition of epidermal growth factor (EGF) or transforming growth factor alpha or beta 1 (TGF-alpha or TGF-beta 1) to the culture medium. The stage of embryonic development, primarily affected by these treatments, was between eight-cell/morula and blastocyst. Furthermore, blastocysts developed from eight-cell embryos cultured in groups or singly in the presence of EGF showed a higher incidence of zona hatching compared with those cultured singly in the absence of EGF. Detection of EGF receptors on the embryonic cell surface at eight-cell/morula and blastocyst stages suggests beneficial effects of EGF or TGF-alpha on preimplantation embryo development and blastocyst functions. Insulin-like growth factor I (IGF-I) had no influence on embryo development. To further document the cooperative interactions among embryos, the volume of the culture medium was doubled to 50 microliters. This increase in culture volume was even more detrimental to the development of singly cultured embryos. However, this detrimental effect was significantly reversed by EGF and reversed even more markedly by a combination of EGF and TGF-beta 1 but not by TGF-beta 1 alone. Although TGF-beta 1 plus IGF-I caused a modest improvement of embryo development, the response was not as great as shown by EGF alone. Furthermore, IGF-I had no additive effect on EGF-induced embryonic development. The study presents clear evidence that specific growth factors of embryonic and/or reproductive tract origin participate in preimplantation embryo development and blastocyst functions in an

Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice.