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.

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.

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

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.

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.

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

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.

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.

The early-stage diagnosis of albinic embryos by applying optical coherence tomography

NASA Astrophysics Data System (ADS)

Yang, Bor-Wen; Wang, Shih-Yuan; Wang, Yu-Yen; Cai, Jyun-Jhang; Chang, Chung-Hao

2013-09-01

Albinism is a kind of congenital disease of abnormal metabolism. Poecilia reticulata (guppy fish) is chosen as the model to study the development of albinic embryos as it is albinic, ovoviviparous and with short life period. This study proposed an imaging method for penetrative embryo investigation using optical coherence tomography. By imaging through guppy mother’s reproduction purse, we found the embryo’s eyes were the early-developed albinism features. As human’s ocular albinism typically appear at about four weeks old, it is the time to determine if an embryo will grow into an albino.

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.

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.

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.

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.

[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.

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.

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

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

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.

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

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

The effect of flurbiprofen on the development of anencephaly in early stage chicken embryos.

PubMed

Özeren, Ersin; Er, Uygur; Güvenç, Yahya; Demirci, Adnan; Arıkök, Ata Türker; Şenveli, Engin; Ergün, Rüçhan Behzat

2015-04-01

The study investigated the effect of flurbiprofen on the development of anencephaly in early stage chicken embryos. We looked at four groups with a total of 36 embryos. There was a control group, a normal saline group, a normal-dose group and a high-dose group with ten, ten, eight and eight eggs with embryo respectively. Two embryos in the control group, studied with light microscopy at 48 h, were consistent with 28-29 hours' incubation in the Hamburger-Hamilton System. They had open neural tubes. The other embryos in this group were considered normal. One embryo in the normal saline group was on the occlusion stage at 48 h. One embryo showed an open neural tube. They were compatible with 28-29 hours' incubation in the Hamburger-Hamilton system. The remaining eight embryos showed normal development. In the normal dose group, one embryo showed underdevelopment of the embryonic disc and the embryo was dead. In four embryos, the neural tubes were open. One cranial malformation was found that was complicated with anencephaly in one embryo. In two embryos the neural tubes were closed, as they showed normal development, and they reached their expected stages according to the Hamburger-Hamilton classification. There was no malformation or growth retardation. Four experimental embryos were anencephalic in the high dose group, and three embryos had open neural tubes. One embryo exhibited both anencephaly and a neural tube closure defect. None of the embryos in this group showed normal development. Even the usual therapeutic doses of flurbiprofen increased the risk of neural tube defect. Flurbiprofen was found to significantly increase the risk of anencephaly. The provision of improved technical materials and studies with larger sample sizes will reveal the stage of morphological disruption during the development of embryos.

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

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.

Experimenting with embryos: can philosophy help?

PubMed

Heyd, David

1996-10-01

Beyond the well-known ethical issues involved in medical experimentation on human subjects, experimenting with embryos raises unique and particularly hard problems. Besides the psychological obstacles connected with the fear of "playing God" and the awe with which we hold the process of the creation of human beings, there are three philosophical problems which are the main subject of the article: 1. The logical problem of circularity: the morality of experimenting on embryos is dependent on the status of the embryo, which in turn is partly decided by experimentation; 2. The metaphysical problem: experiments are justified by the benefits they bring to human subjects; but it is doubtful whether an early embryo is a "subject" and whether coming into being is a "benefit"; 3. The moral problem: the standard constraint on medical experiments is that they benefit either the individual subject or at least members of a relevantly defined group of patients suffering from the same syndrome. But embryo experimentation is often associated with potential cure to people of a completely different category (like geriatric patients). Finally, the article discusses the limits of the force of philosophical arguments in the formation of actual policies for regulating such practices as experimenting with embryos. The widely-shared fourteen-day limit is shown to be a sound practical compromise despite the difficulties in justifying it philosophically.

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

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.

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

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.

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.

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.

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

Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.

PubMed

Flores, Luis E; Hildebrandt, Thomas B; Kühl, Anja A; Drews, Barbara

2014-05-10

Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9-11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert's membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart

Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy

PubMed Central

2014-01-01

Background Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. Methods In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. Results The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9–11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert’s membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and

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.

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

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

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.

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

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

PubMed Central

Murray, Jessica R.; Stanciauskas, Monika E.; Aralere, Tejas S.; Saha, Margaret S.

2014-01-01

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and memory and learning4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access. PMID:24999108

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.

Ovarian stimulation and embryo banking for fertility preservation in a woman with severe mixed connective tissue disease: Is it safe?

PubMed

Sioulas, Vasileios D; Gracia, Clarisa R

2012-03-01

To report the first case of using assisted reproductive technologies (ART) for fertility preservation in a patient with mixed connective tissue disease (MCTD), secondary pulmonary hypertension (PH) and antiphospholipid syndrome (APS). Case-report and review of the literature. Academic infertility practice and tertiary care center. A 25-year-old woman with MCTD, complicated with PH and APS, who was scheduled for gonadotoxic therapy Controlled ovarian hyperstimulation, egg retrieval, embryo banking. Successful ART cycle leading to embryo banking without worsening her underlying disease. Following successful embryo cryopreservation, the patient experienced respiratory failure and other severe complications, resulting in a prolonged hospital stay. Controlled ovarian hyperstimulation for embryo banking in women with MCTD, PH and APS may pose a risk for potentially catastrophic complications. A multidisciplinary approach to these patients is necessary to optimize the outcomes of such procedures. More data are needed regarding the safety of fertility preservation technologies in patients with complex medical diseases.

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

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.

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.

Characterization of somatic embryo attached structures in Feijoa sellowiana Berg. (Myrtaceae).

PubMed

Correia, Sandra M; Canhoto, Jorge M

2010-06-01

The presence of an attached organ to somatic embryos of angiosperms connecting the embryo to the supporting tissue has been a subject of controversy. This study shows that 67% of the morphologically normal somatic embryos of Feijoa sellowiana possess this type of organ and that its formation was not affected by culture media composition. Histological and ultrastructural analysis indicated that the attached structures of somatic embryos displayed a great morphological diversity ranging from a few cells to massive and columnar structures. This contrast with the simple suspensors observed in zygotic embryos which were only formed by five cells. As well as the suspensor of zygotic embryos, somatic embryo attached structures undergo a process of degeneration in later stages of embryo development. Other characteristic shared by zygotic suspensors and somatic embryo attached structures was the presence of thick cell walls surrounding the cells. Elongated thin filaments were often associated with the structures attached to somatic embryos, whereas in other cases, tubular cells containing starch grains connected the embryo to the supporting tissue. These characteristics associated with the presence of plasmodesmata in the cells of the attached structures seem to indicate a role on embryo nutrition. However, cell proliferation in the attached structures resulting into new somatic embryos may also suggest a more complex relationship between the embryo and the structures connecting it to the supporting tissue.

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)

Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

PubMed Central

Baurand, Pierre-Emmanuel; Pedrini-Martha, Veronika; de Vaufleury, Annette; Niederwanger, Michael; Capelli, Nicolas; Scheifler, Renaud; Dallinger, Reinhard

2015-01-01

The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene. PMID:25706953

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)

Notch and Delta mRNAs in early-stage and mid-stage Drosophila embryos exhibit complementary patterns of protein producing potentials

PubMed Central

Shepherd, Andrew; Wesley, Uma; Wesley, Cedric

2010-01-01

Notch and Delta proteins generate Notch signaling that specifies cell fates during animal development. There is an intriguing phenomenon in Drosophila embryogenesis that has not received much attention and whose significance to embryogenesis is unknown. Notch and Delta mRNAs expressed in early-stage embryos are shorter than their counterparts in mid-stage embryos. We show here that the difference in sizes is due to mRNA 3′ processing at alternate polyadenylation sites. While the early-stage Notch mRNA has a lower protein-producing potential than the mid-stage Notch mRNA, the early-stage Delta mRNA has a higher protein-producing potential than the mid-stage Delta mRNA. Our data can explain the complementary patterns of Notch and Delta protein levels in early-stage and mid-stage embryos. Our data also raise the possibility that the manner and regulation of Notch signaling change in the course of embryogenesis and that this change is effected by 3′ UTR and mRNA 3′ processing factors. PMID:20201103

Feminists on the inalienability of human embryos.

PubMed

McLeod, Carolyn; Baylis, Francoise

2006-01-01

The feminist literature against the commodification of embryos in human embryo research includes an argument to the effect that embryos are "intimately connected" to persons, or morally inalienable from them. We explore why embryos might be inalienable to persons and why feminists might find this view appealing. But, ultimately, as feminists, we reject this view because it is inconsistent with full respect for women's reproductive autonomy and with a feminist conception of persons as relational, embodied beings. Overall, feminists should avoid claims about embryos' being inalienable to persons in arguments for or against the commodification of human embryos.

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.

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

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.

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.

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

Embryo apoptosis identification: Oocyte grade or cleavage stage?

PubMed Central

Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

2015-01-01

Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

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

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.

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in early Xenopus embryos induces cell dissociation and inhibits transition from the blastula to gastrula stage.

PubMed

Shibata, M; Shinga, J; Yasuhiko, Y; Kai, M; Miura, K; Shimogori, T; Kashiwagi, K; Igarashi, K; Shiokawa, K

1998-07-01

Xenopus early embryos contain relatively low levels of S-adenosyl-methionine decarboxylase (SAMDC) and its mRNA. When SAMDC mRNA was injected into Xenopus embryos, it was preserved until the blastula stage and induced a large increase in SAMDC activity. The SAMDC-overexpressed embryos developed normally until the blastula stage but at the early gastrula stage cells which received the mRNA, dissociated autonomously and stopped synthesizing protein. In a hypotonic medium, the dissociated cells, and hence whole embryos, autolyzed. However, in isotonic media dissociated cells did not autolyze, although they did not divide and their DNA and RNA synthesis activity was greatly inhibited. The effects of SAMDC overexpression were abolished by coinjection of ethylglyoxal-bis(guanylhydrazone) (EGBG), a specific inhibitor of SAMDC. In SAMDC-overexpressed embryos the level of putrescine decreased and that of spermidine increased, though to limited extents, resulting in a considerable decrease in the putrescine/spermidine ratio. However, direct injection of spermidine did not mimic the effect of SAMDC overexpression, and putrescine coinjected with SAMDC mRNA to maintain the normal putrescine/spermidine ratio did not rescue the embryos. Conversely, the level of S-adenosylmethionine (SAM) greatly decreased and coinjection of SAM, which restored the level of SAM, rescued the embryos. We concluded that in SAMDC-overexpressed embryos a SAM-deficient state was induced and this caused cell dissociation and inhibition of transition from the blastula to gastrula stage. We suggest that the SAM-deficient embryos obtained in the present study provide a unique system for studying the cellular control mechanism underlying the blastula-gastrula transition.

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.

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

[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.

The Power of Well-Connected Arguments: Early Sensitivity to the Connective "because"

ERIC Educational Resources Information Center

Bernard, Stephane; Mercier, Hugo; Clement, Fabrice

2012-01-01

Connectives, such as "because," are routinely used by parents when addressing their children, yet we do not know to what extent children are sensitive to their use. Given children's early developing abilities to evaluate testimony and produce arguments containing connectives, it was hypothesized that young children would show an appropriate…

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.

Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice.

PubMed

Wu, Yibo; Lv, Zhuo; Yang, Yang; Dong, Guoying; Yu, Yang; Cui, Yiqiang; Tong, Man; Wang, Liu; Zhou, Zuomin; Zhu, Hui; Zhou, Qi; Sha, Jiahao

2014-05-01

Blastomere biopsy is used in preimplantation genetic diagnosis; however, the long-term implications on the offspring are poorly characterized. We previously reported a high risk of memory defects in adult biopsied mice. Here, we assessed nervous function of aged biopsied mice and further investigated the mechanism of neural impairment after biopsy. We found that aged biopsied mice had poorer spatial learning ability, increased neuron degeneration, and altered expression of proteins involved in neural degeneration or dysfunction in the brain compared to aged control mice. Furthermore, the MeDIP assay indicated a genome-wide low methylation in the brains of adult biopsied mice when compared to the controls, and most of the genes containing differentially methylated loci in promoter regions were associated with neural disorders. When we further compared the genomic DNA methylation profiles of 7.5-days postconception (dpc) embryos between the biopsy and control group, we found the whole genome low methylation in the biopsied group, suggesting that blastomere biopsy was an obstacle to de novo methylation during early embryo development. Further analysis on mRNA profiles of 4.5-dpc embryos indicated that reduced expression of de novo methylation genes in biopsied embryos may impact de novo methylation. In conclusion, we demonstrate an abnormal neural development and function in mice generated after blastomere biopsy. The impaired epigenetic reprogramming during early embryo development may be the latent mechanism contributing to the impairment of the nervous system in the biopsied mice, which results in a hypomethylation status in their brains.

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

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.

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.

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

DOE PAGES

Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz; ...

2017-03-22

Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

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

Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz

Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less

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

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo.

PubMed

Gandhi, Shashank; Piacentino, Michael L; Vieceli, Felipe M; Bronner, Marianne E

2017-12-01

The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Shorter Exposures to Harder X-Rays Trigger Early Apoptotic Events in Xenopus laevis Embryos

PubMed Central

Dong, JiaJia; Mury, Sean P.; Drahos, Karen E.; Moscovitch, Marko

2010-01-01

Background A long-standing conventional view of radiation-induced apoptosis is that increased exposure results in augmented apoptosis in a biological system, with a threshold below which radiation doses do not cause any significant increase in cell death. The consequences of this belief impact the extent to which malignant diseases and non-malignant conditions are therapeutically treated and how radiation is used in combination with other therapies. Our research challenges the current dogma of dose-dependent induction of apoptosis and establishes a new parallel paradigm to the photoelectric effect in biological systems. Methodology/Principal Findings We explored how the energy of individual X-ray photons and exposure time, both factors that determine the total dose, influence the occurrence of cell death in early Xenopus embryo. Three different experimental scenarios were analyzed and morphological and biochemical hallmarks of apoptosis were evaluated. Initially, we examined cell death events in embryos exposed to increasing incident energies when the exposure time was preset. Then, we evaluated the embryo's response when the exposure time was augmented while the energy value remained constant. Lastly, we studied the incidence of apoptosis in embryos exposed to an equal total dose of radiation that resulted from increasing the incoming energy while lowering the exposure time. Conclusions/Significance Overall, our data establish that the energy of the incident photon is a major contributor to the outcome of the biological system. In particular, for embryos exposed under identical conditions and delivered the same absorbed dose of radiation, the response is significantly increased when shorter bursts of more energetic photons are used. These results suggest that biological organisms display properties similar to the photoelectric effect in physical systems and provide new insights into how radiation-mediated apoptosis should be understood and utilized for therapeutic

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.

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.

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 �

Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

PubMed Central

Petrek, Jiri; Zitka, Ondrej; Adam, Vojtech; Bartusek, Karel; Anjum, Naser A.; Pereira, Eduarda; Havel, Ladislav; Kizek, Rene

2015-01-01

Background Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters. Methodology/Principal Findings The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we

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

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-...

Effect of women’s age on embryo morphology, cleavage rate and competence—A multicenter cohort study

PubMed Central

Grøndahl, Marie Louise; Christiansen, Sofie Lindgren; Kesmodel, Ulrik Schiøler; Agerholm, Inge Errebo; Lemmen, Josephine Gabriela; Lundstrøm, Peter; Bogstad, Jeanette; Raaschou-Jensen, Morten; Ladelund, Steen

2017-01-01

This multicenter cohort study on embryo assessment and outcome data from 11,744 IVF/ICSI cycles with 104,830 oocytes and 42,074 embryos, presents the effect of women’s age on oocyte, zygote, embryo morphology and cleavage parameters, as well as cycle outcome measures corrected for confounding factors as center, partner’s age and referral diagnosis. Cycle outcome data confirmed the well-known effect of women’s age. Oocyte nuclear maturation and proportion of 2 pro-nuclear (2PN) zygotes were not affected by age, while a significant increase in 3PN zygotes was observed in both IVF and ICSI (p<0.0001) with increasing age. Maternal age had no effect on cleavage parameters or on the morphology of the embryo day 2 post insemination. Interestingly, initial hCG value after single embryo transfer followed by ongoing pregnancy was increased with age in both IVF (p = 0.007) and ICSI (p = 0.001) cycles. For the first time, we show that a woman’s age does impose a significant footprint on early embryo morphological development (3PN). In addition, the developmentally competent embryos were associated with increased initial hCG values as the age of the women increased. Further studies are needed to elucidate, if this increase in initial hCG value with advancing maternal age is connected to the embryo or the uterus. PMID:28422964

Centrosome movement in the early divisions of Caenorhabditis elegans: A cortical site determining centrosome position

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

Hyman, A.A.

1989-09-01

In Caenorhabditis elegans embryos, early blastomeres of the P cell lineage divide successively on the same axis. This axis is a consequence of the specific rotational movement of the pair of centrosomes and nucleus. A laser has been used to perturb the centrosome movements that determine the pattern of early embryonic divisions. The results support a previously proposed model in which a centrosome rotates towards its correct position by shortening of connections, possibly microtubules, between a centrosome and a defined site on the cortex of the embryo.

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

Fish embryos on land: terrestrial embryo deposition lowers oxygen uptake without altering growth or survival in the amphibious fish Kryptolebias marmoratus.

PubMed

Wells, Michael W; Turko, Andy J; Wright, Patricia A

2015-10-01

Few teleost fishes incubate embryos out of water, but the oxygen-rich terrestrial environment could provide advantages for early growth and development. We tested the hypothesis that embryonic oxygen uptake is limited in aquatic environments relative to air using the self-fertilizing amphibious mangrove rivulus, Kryptolebias marmoratus, which typically inhabits hypoxic, water-filled crab burrows. We found that adult mangrove rivulus released twice as many embryos in terrestrial versus aquatic environments and that air-reared embryos had accelerated developmental rates. Surprisingly, air-reared embryos consumed 44% less oxygen and possessed larger yolk reserves, but attained the same mass, length and chorion thickness. Water-reared embryos moved their opercula ∼2.5 more times per minute compared with air-reared embryos at 7 days post-release, which probably contributed to the higher rates of oxygen uptake and yolk utilization we observed. Genetically identical air- and water-reared embryos from the same parent were raised to maturity, but the embryonic environment did not affect growth, reproduction or emersion ability in adults. Therefore, although aspects of early development were plastic, these early differences were not sustained into adulthood. Kryptolebias marmoratus embryos hatched out of water when exposed to aerial hypoxia. We conclude that exposure to a terrestrial environment reduces the energetic costs of development partly by reducing the necessity of embryonic movements to dispel stagnant boundary layers. Terrestrial incubation of young would be especially beneficial to amphibious fishes that occupy aquatic habitats of poor water quality, assuming low terrestrial predation and desiccation risks. © 2015. Published by The Company of Biologists Ltd.

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

Time-lapse culture with morphokinetic embryo selection improves pregnancy and live birth chances and reduces early pregnancy loss: a meta-analysis.

PubMed

Pribenszky, Csaba; Nilselid, Anna-Maria; Montag, Markus

2017-11-01

Embryo evaluation and selection is fundamental in clinical IVF. Time-lapse follow-up of embryo development comprises undisturbed culture and the application of the visual information to support embryo evaluation. A meta-analysis of randomized controlled trials was carried out to study whether time-lapse monitoring with the prospective use of a morphokinetic algorithm for selection of embryos improves overall clinical outcome (pregnancy, early pregnancy loss, stillbirth and live birth rate) compared with embryo selection based on single time-point morphology in IVF cycles. The meta-analysis of five randomized controlled trials (n = 1637) showed that the application of time-lapse monitoring was associated with a significantly higher ongoing clinical pregnancy rate (51.0% versus 39.9%), with a pooled odds ratio of 1.542 (P < 0.001), significantly lower early pregnancy loss (15.3% versus 21.3%; OR: 0.662; P = 0.019) and a significantly increased live birth rate (44.2% versus 31.3%; OR 1.668; P = 0.009). Difference in stillbirth was not significant between groups (4.7% versus 2.4%). Quality of the evidence was moderate to low owing to inconsistencies across the studies. Selective application and variability were also limitations. Although time-lapse is shown to significantly improve overall clinical outcome, further high-quality evidence is needed before universal conclusions can be drawn. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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

Selection and dynamics of embryonic stem cell integration into early mouse embryos

PubMed Central

Alexandrova, Stoyana; Kalkan, Tuzer; Humphreys, Peter; Riddell, Andrew; Scognamiglio, Roberta; Trumpp, Andreas; Nichols, Jennifer

2016-01-01

The process by which pluripotent cells incorporate into host embryos is of interest to investigate cell potency and cell fate decisions. Previous studies suggest that only a minority of the embryonic stem cell (ESC) inoculum contributes to the adult chimaera. How incoming cells are chosen for integration or elimination remains unclear. By comparing a heterogeneous mix of undifferentiated and differentiating ESCs (serum/LIF) with more homogeneous undifferentiated culture (2i/LIF), we examine the role of cellular heterogeneity in this process. Time-lapse ex vivo imaging revealed a drastic elimination of serum/LIF ESCs during early development in comparison with 2i/LIF ESCs. Using a fluorescent reporter for naive pluripotency (Rex1-GFP), we established that the acutely eliminated serum/LIF ESCs had started to differentiate. The rejected cells were apparently killed by apoptosis. We conclude that a selection process exists by which unwanted differentiating cells are eliminated from the embryo. However, occasional Rex1− cells were able to integrate. Upregulation of Rex1 occurred in a proportion of these cells, reflecting the potential of the embryonic environment to expedite diversion from differentiation priming to enhance the developing embryonic epiblast. PMID:26586221

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.

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

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

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

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...

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.

Characterisation of the dynamic behaviour of lipid droplets in the early mouse embryo using adaptive harmonic generation microscopy.

PubMed

Watanabe, Tomoko; Thayil, Anisha; Jesacher, Alexander; Grieve, Kate; Debarre, Delphine; Wilson, Tony; Booth, Martin; Srinivas, Shankar

2010-06-03

Lipid droplets (LD) are organelles with an important role in normal metabolism and disease. The lipid content of embryos has a major impact on viability and development. LD in Drosophila embryos and cultured cell lines have been shown to move and fuse in a microtubule dependent manner. Due to limitations in current imaging technology, little is known about the behaviour of LD in the mammalian embryo. Harmonic generation microscopy (HGM) allows one to image LD without the use of exogenous labels. Adaptive optics can be used to correct aberrations that would otherwise degrade the quality and information content of images. We have built a harmonic generation microscope with adaptive optics to characterise early mouse embryogenesis. At fertilization, LD are small and uniformly distributed, but in the implanting blastocyst, LD are larger and enriched in the invading giant cells of the trophectoderm. Time-lapse studies reveal that LD move continuously and collide but do not fuse, instead forming aggregates that subsequently behave as single units. Using specific inhibitors, we show that the velocity and dynamic behaviour of LD is dependent not only on microtubules as in other systems, but also on microfilaments. We explore the limits within which HGM can be used to study living embryos without compromising viability and make the counterintuitive finding that 16 J of energy delivered continuously over a period of minutes can be less deleterious than an order of magnitude lower energy delivered dis-continuously over a period of hours. LD in pre-implantation mouse embryos show a previously unappreciated complexity of behaviour that is dependent not only on microtubules, but also microfilaments. Unlike LD in other systems, LD in the mouse embryo do not fuse but form aggregates. This study establishes HGM with adaptive optics as a powerful tool for the study of LD biology and provides insights into the photo-toxic effects of imaging embryos.

HNK-1 immunoreactivity during early morphogenesis of the head region in a nonmodel vertebrate, crocodile embryo

NASA Astrophysics Data System (ADS)

Kundrát, Martin

2008-11-01

The present study examines HNK-1 immunoidentification of a population of the neural crest (NC) during early head morphogenesis in the nonmodel vertebrate, the crocodile ( Crocodylus niloticus) embryos. Although HNK-1 is not an exclusive NC marker among vertebrates, temporospatial immunoreactive patterns found in the crocodile are almost consistent with NC patterns derived from gene expression studies known in birds (the closest living relatives of crocodiles) and mammals. In contrast to birds, the HNK-1 epitope is immunoreactive in NC cells at the neural fold level in crocodile embryos and therefore provides sufficient base to assess early migratory events of the cephalic NC. I found that crocodile NC forms three classic migratory pathways in the head: mandibular, hyoid, and branchial. Further, I demonstrate that, besides this classic phenotype, there is also a forebrain-derived migratory population, which consolidates into a premandibular stream in the crocodile. In contrast to the closely related chick model, crocodilian premandibular and mandibular NC cells arise from the open neural tube suggesting that species-specific heterochronic behavior of NC may be involved in the formation of different vertebrate facial phenotypes.

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

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

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

Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos.

PubMed

Tao, T; Reichelt, B; Niemann, H

1995-07-01

Pig morulae, early blastocysts and blastocysts were microsurgically bisected to produce zona-free demi-embryos or remained nonbisected with or without zona pellucida, and the presence of inner cell mass cells was determined using a differential fluorochrome staining technique. After 24 h of in vitro culture, all demi-embryos were classified into three categories, based on morphological criteria: 1, excellent; 2, fair; and 3, degenerated. The average number of total cells and inner cell mass cells in intact embryos cultured without zona pellucida for 24 h was higher (P < 0.05) than that for those with zona pellucida in morulae and early blastocysts. The percentage of demi-embryos without inner cell mass cells in these different morphological categories was 18.7%, 22.2% and 29.8% for morulae, respectively; 3.8%, 16.7% and 30.8% for early blastocysts, respectively; and 3.7%, 32.0% and 36.4% for blastocysts, respectively. The percentage of demi-embryos without inner cell mass cells was lower (P < 0.01) in demi-embryos classified in category 1 compared with category 3 in early blastocysts and in category 1 compared with categories 2 and 3 in blastocysts. Significant differences in the total number of cells and the number of inner cell mass cells were apparent among the three morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts. The ratio of total cells to inner cell mass cells was similar among intact pig embryos and the different morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts, with the exception of that between demi-blastocysts of category 1 and the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Functional Connectivity of the Amygdala in Early Childhood Onset Depression

PubMed Central

Luking, Katherine R.; Repovs, Grega; Belden, Andy C.; Gaffrey, Michael S.; Botteron, Kelly N.; Luby, Joan L.; Barch, Deanna M.

2011-01-01

Objective Adult major depressive disorder (MDD) is associated with reduced cortico-limbic functional connectivity thought to indicate decreased top-down control of emotion. However, it is unclear whether such connectivity alterations are also present in early childhood onset MDD. Method Fifty-one children ages 7–11 years, prospectively studied since preschool age, completed resting state fMRI and were assigned to four groups: 1) C-MDD (N=13) personal history of early childhood onset MDD; 2) M-MDD (N=11) a maternal history of affective disorders; 3) CM-MDD (N=13) both maternal and early childhood onset MDD or 4) CON (N=14) without either a personal or maternal history. We used seed-based resting state functional connectivity (rsfcMRI) analysis in an independent sample of adults to identify networks showing both positive (e.g., limbic regions) and negative (e.g., dorsal frontal/parietal regions) connectivity with the amygdala. These regions were then used in ROI based analyses of our child sample. Results We found a significant interaction between maternal affective disorder history and the child's MDD history for both positive and negative rsfcMRI networks. Specifically, when copared to CON, we found reduced connectivity between the amygdala and the “Negative Network” in children with C-MDD, M-MDD and CM-MDD. Children with either C-MDD or a maternal history of MDD (but not CM-MDD) displayed reduced connectivity between the amygdala and the “Positive Network”. Conclusions Our finding of an attenuated relationship between the amygdala, a region affected in MDD and involved in emotion processing, and cognitive control regions is consistent with a hypothesis of altered regulation of emotional processing in C-MDD suggesting developmental continuity of this alteration into early childhood. PMID:21961777

Ethanol impedes embryo transport and impairs oviduct epithelium.

PubMed

Xu, Tonghui; Yang, Qiuhong; Liu, Ruoxi; Wang, Wenfu; Wang, Shuanglian; Liu, Chuanyong; Li, Jingxin

2016-05-16

Most studies have demonstrated that alcohol consumption is associated with decreased fertility. The aim of this study was to investigate the effects of alcohol on pre-implantation embryo transport and/or early embryo development in the oviduct. We reported here that ethanol concentration-dependently suppressed the spontaneous motility of isolated human oviduct strips (EC50 50±6mM), which was largely attenuated in the present of L-NAME, a classical nitric oxide synthase(NOS) competitive inhibitor. Notably, either acute or chronic alcohol intake delayed egg transport and retarded early development of the embryo in the mouse oviduct, which was largely rescued by co-administration of L-NAME in a acute alcohol intake group but not in chronic alcohol intake group. It is worth mentioning that the oviductal epithelium destruction was verified by scanning electron microscope (SEM) observations in chronic alcohol intake group. In conclusion, alcohol intake delayed egg transport and retarded early development of the embryo in the oviduct by suppressing the spontaneous motility of oviduct and/or impairing oviductal epithelium. These findings suggested that alcohol abuse increases the incident of ectopic pregnancy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The effects of the early uterine environment on the subsequent development of embryo and fetus.

PubMed

Barnes, F L

2000-01-15

Synchrony between the embryo and the uterine endometrium is essential for the establishment of pregnancy and birth in people and livestock. When asynchronous conditions occur a variety of complication result that include failure of the embryo to implant, early embryonic mortality, retarded development and growth, and accelerated development and growth. These complications all appear to be induced within the first week of embryo development and not withstanding the immediate endpoint of large or small size at birth, may alter the course of development throughout the life of the animal. Progesterone appears to play a causative role in establishing the abnormal growth of the fetus by decelerating or accelerating embryonic development. This may act through increasing the transport of blood born growth factors into the uterine lumen or by stimulating the release of growth factors from the endometrium directly. It can not be ruled out that progesterone mediated abundance of, or absence of, appropriate nutrition may bring about the same lifelong outcome. In vitro culture situations that include serum and/or co-culture can also bring about these abnormalities of growth. It is hypothesized that exposure to growth factors "out of phase" may result in an irreversible induction of abnormal development. The described abnormalities that occur in sheep and cattle have not yet been described for children resulting from IVF.

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

P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

PubMed Central

Dahlberg, Olle; Shilkova, Olga; Tang, Min; Holmqvist, Per-Henrik; Mannervik, Mattias

2015-01-01

Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3’ end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos. PMID:25679530

[Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].

PubMed

Jiang, Hua; Feng, You-Ji; Xie, Yi; Han, Jin-Lan; Wang, Zack; Chen, Tong

2008-10-14

To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo. Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed. The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.

The immunologic and antioxidant effects of L-phenylalanine on the uterine implantation of mice embryos during early pregnancy.

PubMed

Dong, Yulan; Bai, Yongping; Liu, Guanhui; Wang, Zixu; Cao, Jing; Chen, Yaoxing; Yang, Hongliang

2014-10-01

L-phenylalanine (L-PHE) is a synthetic precursor of catecholamines. Because it cannot be synthesised by an organism, it must be absorbed from the environment. Despite the wide use of L-PHE, whether L-PHE has a negative impact on embryo implantation and development is poorly understood. This study attempted to determine the roles of L-PHE in embryo implantation and development and in the immune response and antioxidant status of the uterus in early pregnancy mice injected intraperitoneally with 320 mg/kg L-PHE. The embryo number of treated mice decreased by 57.6%, and the size of their embryos was reduced by 2.8% (P⟩0.05) along the long diameter and 11.9% (P⟨0.05) along the short diameter at E9 compared with control mice. In addition, L-PHE significantly suppressed B lymphocyte proliferation. L-PHE increased IL-2 secretion but decreased the IL-4 concentration, thereby up-regulating the ratio of IL-2/IL-4 to 1.37-8.45. An analysis of the oxidant and antioxidant status showed that, compared with the control mice, the level of superoxide dismutase activity decreased by 21.54-39.94% and the glutathione peroxidase activity decreased by 15.27-18.96% among the L-PHE-treated mice at E1-E9. However, the malonaldehyde content increased by 14.29%-90.11% among the L-PHE-treated mice. Therefore, L-PHE impaired embryo implantation by disrupting cytokine-based immunity and oxidative stress in the uterus.

Transcriptome analysis of PCOS arrested 2-cell embryos.

PubMed

Lu, Cuiling; Chi, Hongbin; Wang, Yapeng; Feng, Xue; Wang, Lina; Huang, Shuo; Yan, Liying; Lin, Shengli; Liu, Ping; Qiao, Jie

2018-06-18

In an attempt to explore the early developmental arrest in embryos from polycystic ovarian syndrome (PCOS) patients, we sequenced the transcriptome profiles of PCOS arrested 2-cell embryos, non-PCOS arrested 2-cell embryos and non-arrested 2-cell embryos using single-cell RNA-Seq technique. Differential expression analysis was performed using the DEGSeq R package. Gene Ontology (GO) enrichment was analyzed using the GOseq R package. Data revealed 62 differentially expressed genes between non-PCOS arrested and PCOS arrested embryos and 2217 differentially expressed genes between PCOS arrested and non-arrested 2-cell embryos. A total of 49 differently expressed genes (DEGs) were annotated with GO terms in the up-regulated genes between PCOS arrested and non-PCOS arrested embryos after GO enrichment. A total of 29 DEGs were annotated with GO terms in the down-regulated genes between PCOS arrested and non-arrested 2-cell embryos after GO enrichment. These data can provide a reference for screening specific genes involved in the arrest of PCOS embryos.

Spatial distribution of the capacity to initiate a secondary embryo in the 32-cell embryo of Xenopus laevis.

PubMed

Kageura, H

1990-12-01

To examine the spatial distribution of dorsal determinants in the early embryos of Xenopus laevis, individual cells from the 32-cell embryo were transplanted into the same tier of the ventral side of a synchronous recipient. Their abilities to initiate a secondary embryo were measured by the incidence of secondary embryos and by the length of the secondary axis relative to the primary embryo. The ability was found to be localized in all cells (A1, B1, C1, and D1) of the dorsal most column and in the vegetal cells (C2 and D2) of the dorsolateral column. Transplanted C1 (subequatorial) cells caused the highest incidence of a secondary embryo and the average relative length of the secondary embryo was also greatest. Effectiveness decreased in the order: D1, B1, D2, C2, and A1. When these results were compared with Dale and Slack's fate map of the 32-cell embryo, it was concluded that the distribution of dorsal determinants is unique and does not coincide with the prospective regions for any tissues, though it is somewhat similar to the prospective region of dorsal endoderm or notochord. From these results it seems that dorsal determinants do not determine a particular tissue in an embryo but rather the "dorsal" region of an embryo.

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.

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

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.

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.

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

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

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

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

2007-07-15

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

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.

Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos

PubMed Central

Gupta, Prabuddha; Martin, René; Knölker, Hans-Joachim; Nihalani, Deepak; Kumar Sinha, Deepak

2017-01-01

Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1–8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis. PMID:28678859

Mapping thalamocortical functional connectivity in chronic and early stages of psychotic disorders

PubMed Central

Woodward, Neil D.; Heckers, Stephan

2015-01-01

Objective There is considerable evidence that the thalamus is abnormal in psychotic disorders. Resting-state fMRI (RS-fMRI) has revealed an intriguing pattern of thalamic dysconnectivity in psychosis characterized by reduced prefrontal cortex (PFC) connectivity and increased somatomotor-thalamic connectivity. However, critical knowledge gaps remain with respect to the onset, anatomical specificity, and clinical correlates of thalamic dysconnectivity in psychosis. Method RS-fMRI was collected on 105 healthy subjects and 148 individuals with psychosis, including 53 early stage psychosis patients. Using all 253 subjects, the thalamus was parceled into functional regions-of-interest (ROIs) on the basis of connectivity with six a-priori defined cortical ROIs covering most of the cortical mantle. Functional connectivity between each cortical ROI and its corresponding thalamic ROI was quantified and compared across groups. Significant differences in the ROI-to-ROI analysis were followed up with voxel-wise seed-based analyses to further localize thalamic dysconnectivity. Results ROI analysis revealed reduced PFC-thalamic connectivity and increased somatomotor-thalamic connectivity in both chronic and early stages psychosis patients. PFC hypo-connectivity and motor cortex hyper-connectivity correlated in patients suggesting they result from a common pathophysiological mechanism. Seed-based analyses revealed thalamic hypo-connectivity in psychosis localized to dorsolateral PFC, medial PFC, and cerebellar areas of the well-described ‘executive control’ network. Across all subjects, thalamic connectivity with areas of the fronto-parietal network correlated with cognitive functioning, including verbal learning and memory. Conclusions Thalamocortical dysconnectivity is present in both chronic and early stages of psychosis, includes reduced thalamic connectivity with the executive control network, and is related to cognitive impairment. PMID:26248537

Functional Connectivity of the Amygdala in Early-Childhood-Onset Depression

ERIC Educational Resources Information Center

Luking, Katherine R.; Repovs, Grega; Belden, Andy C.; Gaffrey, Michael S.; Botteron, Kelly N.; Luby, Joan L.; Barch, Deanna M.

2011-01-01

Objective: Adult major depressive disorder (MDD) is associated with reduced cortico-limbic functional connectivity thought to indicate decreased top-down control of emotion. However, it is unclear whether such connectivity alterations are also present in early-childhood-onset MDD. Method: A total of 51 children 7 through 11 years of age who had…

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 �

Fostering Connections to Nature -- Strategies for Community College Early Childhood Teachers

ERIC Educational Resources Information Center

Murphy, Debra

2017-01-01

How can early childhood teacher educators at the community college level create opportunities for their students to explore and relate to the natural world? This article discusses three learning opportunities in an early childhood associate-degree program that foster connections between preservice and inservice early childhood teachers and nature…

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.

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

PubMed

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

2013-01-01

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

Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

PubMed

Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

2013-10-01

approach we are proposing may provide a novel, non-invasive, objective tool for embryo quality grading. The correlation between a high mtDNA concentration and the fragmentation rate of embryos is suggestive that fragments are mainly anuclear cytoplasmatic debris arising during cleavage. Therefore, blastomere shaping as an early event during in vitro development may play a homeostatic role and be related to embryo competence. This project was funded by Merck Serono (Grant for Fertility Innovation 2011). The sponsor had no role in study design, data collection, data analysis, data interpretation and writing of the paper. Authors declare no conflicts of interest. ClinicalTrials.gov Identifier: NCT01397136.

Types of neural cells in the spinal ganglia of human embryos and early fetuses.

PubMed

Olszewska, B; Woźniak, W; Gardner, E; O'Rahilly, R

1979-01-01

Spinal ganglial of human embryos and fetuses ranging in C.-R. length from 15 to 74 mm and in age from 6 1/2 to 11 postovulatory weeks were studied by light and electron microscopy. A sequence of events in differentiation and maturation enabled five types of cells to be distinguished: 1. apolar, undifferentiated neuroblasts are the main cells at 6 1/2 to 7 1/2 weeks; 2. early bipolar neuroblasts (strictly speaking, types 2 to 5 are immature neurons) predominate at the end of the embryonic period proper (8 postovulatory weeks); 3. intermediate bipolar neuroblasts are characteristic of the early fetal period; 4. late bipolar neuroblasts, in which two proceses arise separately from one pole of the cell, appear at about 10 postovulatory weeks; 5. unipolar neuroblasts are found within another week and, by that time, cells of types 1 and 2 are no longer present.

The evolution of porcine embryo in vitro production.

PubMed

Grupen, Christopher G

2014-01-01

The in vitro production of porcine embryos has presented numerous challenges to researchers over the past four decades. Some of the problems encountered were specific to porcine gametes and embryos and needed the concerted efforts of many to overcome. Gradually, porcine embryo in vitro production systems became more reliable and acceptable rates of blastocyst formation were achieved. Despite the significant improvements, the problem of polyspermic fertilization has still not been adequately resolved and the embryo in vitro culture conditions are still considered to be suboptimal. Whereas early studies focused on increasing our understanding of the reproductive processes involved, the technology evolved to the point where in vitro-matured oocytes and in vitro-produced embryos could be used as research material for developing associated reproductive technologies, such as SCNT and embryo cryopreservation. Today, the in vitro procedures used to mature oocytes and culture embryos are integral to the production of transgenic pigs by SCNT. This review discusses the major achievements, advances, and knowledge gained from porcine embryo in vitro production studies and highlights the future research perspectives of this important technology. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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.

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

Sex determination of duck embryos: observations on syrinx development

USGS Publications Warehouse

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

2013-01-01

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

The C. elegans homolog of Drosophila Lethal giant larvae functions redundantly with PAR-2 to maintain polarity in the early embryo.

PubMed

Beatty, Alexander; Morton, Diane; Kemphues, Kenneth

2010-12-01

Polarity is essential for generating cell diversity. The one-cell C. elegans embryo serves as a model for studying the establishment and maintenance of polarity. In the early embryo, a myosin II-dependent contraction of the cortical meshwork asymmetrically distributes the highly conserved PDZ proteins PAR-3 and PAR-6, as well as an atypical protein kinase C (PKC-3), to the anterior. The RING-finger protein PAR-2 becomes enriched on the posterior cortex and prevents these three proteins from returning to the posterior. In addition to the PAR proteins, other proteins are required for polarity in many metazoans. One example is the conserved Drosophila tumor-suppressor protein Lethal giant larvae (Lgl). In Drosophila and mammals, Lgl contributes to the maintenance of cell polarity and plays a role in asymmetric cell division. We have found that the C. elegans homolog of Lgl, LGL-1, has a role in polarity but is not essential. It localizes asymmetrically to the posterior of the early embryo in a PKC-3-dependent manner, and functions redundantly with PAR-2 to maintain polarity. Furthermore, overexpression of LGL-1 is sufficient to rescue loss of PAR-2 function. LGL-1 negatively regulates the accumulation of myosin (NMY-2) on the posterior cortex, representing a possible mechanism by which LGL-1 might contribute to polarity maintenance.

5Alpha-Reduced Steroids Are Major Metabolites in the Early Equine Embryo Proper and Its Membranes.

PubMed

Raeside, James I; Christie, Heather L; Betteridge, Keith J

2015-09-01

Steroid production and metabolism by early conceptuses are very important for the establishment and maintenance of pregnancy in horses. Our earlier work suggested the possible formation of 5alpha-reduced steroids in equine conceptuses. We have now demonstrated the formation of 5alpha-reduced metabolites of androstenedione, testosterone, and progesterone by the embryo and its membranes. A total of 44 conceptuses were collected from 26 mares between 20 and 31 days of pregnancy. Tissues from the embryo proper and from the separated components of the conceptus (bilaminar and trilaminar trophoblast, allantois) were incubated with tritium-labeled substrates. 5Alpha-reduced metabolites (5alpha-dihydro- and 3beta,5alpha-tetrahydro- steroids) as radiolabeled products were identified from a series of chromatographic steps using four solvent systems for high-performance liquid chromatography. Use of a 5alpha-reductase inhibitor confirmed the metabolites were indeed 5alpha-reduced steroids. For the embryo, the only products from androstenedione were 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione, with no evidence of more polar metabolites; there was some 3beta,5alpha-tetrahydrotestosterone but no 5alpha-dihydrotestosterone from testosterone, and formation of androstenedione was followed by the production of 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione. The major 5alpha-reduced product from progesterone was 3beta,5alpha-tetrahydroprogesterone, with lesser amounts of 5alpha-dihydroprogesterone. For the membranes, reductions to tetrahydro, 5alpha-reduced steroids were prominent in most instances, but also present were considerable amounts of products more polar than the substrates. The well-recognized activity of some 5alpha-reduced steroids--for example, 5alpha-dihydrotestosterone in male sexual differentiation--provokes interest in their even earlier appearance, as seen in this study, and suggests a possible role for them in

Dynamic Imaging of Mouse Embryos and Cardiodynamics in Static Culture.

PubMed

Lopez, Andrew L; Larina, Irina V

2018-01-01

The heart is a dynamic organ that quickly undergoes morphological and mechanical changes through early embryonic development. Characterizing these early moments is important for our understanding of proper embryonic development and the treatment of heart disease. Traditionally, tomographic imaging modalities and fluorescence-based microscopy are excellent approaches to visualize structural features and gene expression patterns, respectively, and connect aberrant gene programs to pathological phenotypes. However, these approaches usually require static samples or fluorescent markers, which can limit how much information we can derive from the dynamic and mechanical changes that regulate heart development. Optical coherence tomography (OCT) is unique in this circumstance because it allows for the acquisition of three-dimensional structural and four-dimensional (3D + time) functional images of living mouse embryos without fixation or contrast reagents. In this chapter, we focus on how OCT can visualize heart morphology at different stages of development and provide cardiodynamic information to reveal mechanical properties of the developing heart.

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.

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.

Early-Course Unmedicated Schizophrenia Patients Exhibit Elevated Prefrontal Connectivity Associated with Longitudinal Change

PubMed Central

Anticevic, Alan; Hu, Xinyu; Xiao, Yuan; Hu, Junmei; Li, Fei; Bi, Feng; Cole, Michael W.; Savic, Aleksandar; Yang, Genevieve J.; Repovs, Grega; Murray, John D.; Wang, Xiao-Jing; Huang, Xiaoqi; Lui, Su; Krystal, John H.

2015-01-01

Strong evidence implicates prefrontal cortex (PFC) as a major source of functional impairment in severe mental illness such as schizophrenia. Numerous schizophrenia studies report deficits in PFC structure, activation, and functional connectivity in patients with chronic illness, suggesting that deficient PFC functional connectivity occurs in this disorder. However, the PFC functional connectivity patterns during illness onset and its longitudinal progression remain uncharacterized. Emerging evidence suggests that early-course schizophrenia involves increased PFC glutamate, which might elevate PFC functional connectivity. To test this hypothesis, we examined 129 non-medicated, human subjects diagnosed with early-course schizophrenia and 106 matched healthy human subjects using both whole-brain data-driven and hypothesis-driven PFC analyses of resting-state fMRI. We identified increased PFC connectivity in early-course patients, predictive of symptoms and diagnostic classification, but less evidence for “hypoconnectivity.” At the whole-brain level, we observed “hyperconnectivity” around areas centered on the default system, with modest overlap with PFC-specific effects. The PFC hyperconnectivity normalized for a subset of the sample followed longitudinally (n = 25), which also predicted immediate symptom improvement. Biologically informed computational modeling implicates altered overall connection strength in schizophrenia. The initial hyperconnectivity, which may decrease longitudinally, could have prognostic and therapeutic implications. PMID:25568120

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.

Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program.

PubMed

Herrera, C; Morikawa, M I; Bello, M B; von Meyeren, M; Centeno, J Eusebio; Dufourq, P; Martinez, M M; Llorente, J

2014-03-15

Preimplantation genetic diagnosis (PGD) allows identifying genetic traits in early embryos. Because in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied (i) the effect on pregnancy rates of holding biopsied embryos for 7 to 10 hours in holding medium at 32 °C before transfer, (ii) the effect on pregnancy rates of using embryos of different sizes for biopsy, and (iii) the efficiency of amplification by heating biopsies before polymerase chain reaction. Equine embryos were classified by size (≤300, 300-1000, and >1000 μm), biopsied, and transferred 1 to 2 or 7 to 10 hours after flushing. Some of the biopsy samples obtained were incubated for 10 minutes at 95 °C and the rest remained untreated. Pregnancy rates were recorded at 25 days of gestation; fetal gender was determined using ultrasonography and compared with PGD results. Holding biopsied embryos for 7 to 10 hours before transfer produced pregnancy rates similar to those for biopsied embryos transferred within 2 hours (63% and 57%, respectively). These results did not differ from pregnancy rates of nonbiopsied embryos undergoing the same holding times (50% for 7-10 hours and 63% for 1-2 hours). Pregnancy rates for biopsied and nonbiopsied embryos did not differ between size groups or between biopsied and nonbiopsied embryos within the same size group (P > 0.05). Incubating biopsy samples for 10 minutes at 95 °C before polymerase chain reaction significantly increased the diagnosis rate (78.5% vs. 45.5% for treated and nontreated biopsy samples respectively). Gender determination using incubated biopsy samples matched the

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

NASA Astrophysics Data System (ADS)

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

2015-11-01

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

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

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.

Undernutrition affects embryo quality of superovulated ewes.

PubMed

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

2015-02-01

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

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

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.

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.

Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos

PubMed Central

Tavernier, Nicolas; Noatynska, Anna; Panbianco, Costanza; Martino, Lisa; Van Hove, Lucie; Schwager, Françoise; Léger, Thibaut

2015-01-01

The molecular mechanisms governing mitotic entry during animal development are incompletely understood. Here, we show that the mitotic kinase CDK-1 phosphorylates Suppressor of Par-Two 1 (SPAT-1)/Bora to regulate its interaction with PLK-1 and to trigger mitotic entry in early Caenorhabditis elegans embryos. Embryos expressing a SPAT-1 version that is nonphosphorylatable by CDK-1 and that is defective in PLK-1 binding in vitro present delays in mitotic entry, mimicking embryos lacking SPAT-1 or PLK-1 functions. We further show that phospho–SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A. Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans. Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis. We propose the existence of a positive feedback loop that connects Cdk1 and Plk1 activation to ensure a robust control of mitotic entry and cell division timing. PMID:25753036

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.

Size-dependent regulation of dorsal-ventral patterning in the early Drosophila embryo

PubMed Central

Garcia, Mayra; Nahmad, Marcos; Reeves, Gregory T.; Stathopoulos, Angelike

2013-01-01

How natural variation in embryo size affects patterning of the Drosophila embryo dorsal-ventral (DV) axis is not known. Here we examined quantitatively the relationship between nuclear distribution of the Dorsal transcription factor, boundary positions for several target genes, and DV axis length. Data were obtained from embryos of a wild-type background as well as from mutant lines inbred to size select embryos of smaller or larger sizes. Our data show that the width of the nuclear Dorsal gradient correlates with DV axis length. In turn, for some genes expressed along the DV axis, the boundary positions correlate closely with nuclear Dorsal levels and with DV axis length; while the expression pattern of others is relatively constant and independent of the width of the Dorsal gradient. In particular, the patterns of snail (sna) and ventral nervous-system defective (vnd) correlate with nuclear Dorsal levels and exhibit scaling to DV length; while the pattern of intermediate neuroblasts defective (ind) remains relatively constant with respect to changes in Dorsal and DV length. However, in mutants that exhibit an abnormal expansion of the Dorsal gradient which fails to scale to DV length, only sna follows the Dorsal distribution and exhibits overexpansion; in contrast, vnd and ind do not overexpand suggesting some additional mechanism acts to refine the dorsal boundaries of these two genes. Thus, our results argue against the idea that the Dorsal gradient works as a global system of relative coordinates along the DV axis and suggest that individual targets respond to changes in embryo size in a gene-specific manner. PMID:23800450

In vitro and in vivo effects of ulipristal acetate on fertilization and early embryo development in mice.

PubMed

Gómez-Elías, Matías D; Munuce, María J; Bahamondes, Luis; Cuasnicú, Patricia S; Cohen, Débora J

2016-01-01

Does ulipristal acetate (UPA), a selective progesterone receptor modulator used for emergency contraception (EC), interfere with fertilization or early embryo development in vitro and in vivo? At doses similar to those used for EC, UPA does not affect mouse gamete transport, fertilization or embryo development. UPA acts as an emergency contraceptive mainly by inhibiting or delaying ovulation. However, there is little information regarding its effects on post-ovulatory events preceding implantation. This was an in vitro and in vivo experimental study involving the use of mouse gametes and embryos from at least three animals in each set of experiments. For in vitro fertilization experiments, mouse epididymal spermatozoa capacitated in the presence of different concentrations of UPA (0-1000 ng/ml) were used to inseminate cumulus-intact or cumulus-free eggs in the presence or absence of UPA during gamete co-incubation, and the percentage of fertilized eggs was determined. For in vivo fertilization experiments, superovulated females caged with proven fertile males were injected with UPA (40 mg/kg) or vehicle just before or just after mating and the percentage of fertilized eggs recovered from the ampulla was determined. To investigate the effect of UPA on embryo development, zygotes were recovered from mated females, cultured in the presence of UPA (1000 ng/ml) for 4 days and the progression of embryo development was monitored daily. In vitro studies revealed that the presence of UPA during capacitation and/or gamete co-incubation does not affect fertilization. Whereas the in vivo administration of UPA at the same time as hCG injection produced a decrease in the number of eggs ovulated compared with controls (vehicle injected animals, P < 0.05), no effects on fertilization were observed when UPA was administered shortly before or after mating. No differences were observed in either the percentage of cleaved embryos or the cleavage speed when UPA was present during in

Comprehensive embryo testing. Experts' opinions regarding future directions: an expert panel study on comprehensive embryo testing.

PubMed

Hens, Kristien; Dondorp, Wybo J; Geraedts, Joep P M; de Wert, Guido M

2013-05-01

four Western Europe countries. As willingness to participate in this study may be connected with expectations regarding the pace and direction of future developments, selection bias cannot be excluded. The introduction of comprehensive screening techniques in embryo testing calls for further ethical reflection that is grounded in empirical work. Specifically, there is a need for studies querying the opinions of infertile couples undergoing IVF/PGS regarding the desirability of embryo screening beyond aneuploidy. This research was supported by the CSG, Centre for Society and Life Sciences (project number: 70.1.074). The authors declare no conflict of interest. N/A.

Early-course unmedicated schizophrenia patients exhibit elevated prefrontal connectivity associated with longitudinal change.

PubMed

Anticevic, Alan; Hu, Xinyu; Xiao, Yuan; Hu, Junmei; Li, Fei; Bi, Feng; Cole, Michael W; Savic, Aleksandar; Yang, Genevieve J; Repovs, Grega; Murray, John D; Wang, Xiao-Jing; Huang, Xiaoqi; Lui, Su; Krystal, John H; Gong, Qiyong

2015-01-07

Strong evidence implicates prefrontal cortex (PFC) as a major source of functional impairment in severe mental illness such as schizophrenia. Numerous schizophrenia studies report deficits in PFC structure, activation, and functional connectivity in patients with chronic illness, suggesting that deficient PFC functional connectivity occurs in this disorder. However, the PFC functional connectivity patterns during illness onset and its longitudinal progression remain uncharacterized. Emerging evidence suggests that early-course schizophrenia involves increased PFC glutamate, which might elevate PFC functional connectivity. To test this hypothesis, we examined 129 non-medicated, human subjects diagnosed with early-course schizophrenia and 106 matched healthy human subjects using both whole-brain data-driven and hypothesis-driven PFC analyses of resting-state fMRI. We identified increased PFC connectivity in early-course patients, predictive of symptoms and diagnostic classification, but less evidence for "hypoconnectivity." At the whole-brain level, we observed "hyperconnectivity" around areas centered on the default system, with modest overlap with PFC-specific effects. The PFC hyperconnectivity normalized for a subset of the sample followed longitudinally (n = 25), which also predicted immediate symptom improvement. Biologically informed computational modeling implicates altered overall connection strength in schizophrenia. The initial hyperconnectivity, which may decrease longitudinally, could have prognostic and therapeutic implications. Copyright © 2015 the authors 0270-6474/15/350267-20$15.00/0.

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

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.

The current status and future of commercial embryo transfer in cattle.

PubMed

Hasler, John F

2003-12-15

A commercially viable cattle embryo transfer (ET) industry was established in North America during the early 1970s, approximately 80 years after the first successful embryo transfer was reported in a mammal. Initially, techniques for recovering and transferring cattle embryos were exclusively surgical. However, by the late 1970s, most embryos were recovered and transferred nonsurgically. Successful cryopreservation of embryos was widespread by the early 1980s, followed by the introduction of embryo splitting, in vitro procedures, direct transfer of frozen embryos and sexing of embryos. The wide spread adoption of ethylene glycol as a cryoprotectant has simplified the thaw-transfer procedures for frozen embryos. The number of embryos recovered annually has not grown appreciably over the last 10 years in North America and Europe; however, there has been significant growth of commercial ET in South America. Within North America, ET activity has been relatively constant in Holstein cattle, whereas there has been a large ET increase in the Angus breed and a concomitant ET decrease in some other beef breeds. Although a number of new technologies have been adopted within the ET industry in the last decade, the basic procedure of superovulation of donor cattle has undergone little improvement over the last 20 years. The export-import of frozen cattle embryos has become a well-established industry, governed by specific health regulations. The international movement of embryos is subject to sudden and dramatic disturbances, as exemplified by the 2001 outbreak of foot and mouth disease in Great Britain. It is probable that there will be an increased influence of animal rights issues on the ET industry in the future. Several companies in North America are currently commercially producing cloned cattle. The sexing of bovine semen with the use of flow cytometry is extremely accurate and moderate pregnancy rates in heifers have been achieved in field trials, but sexed semen

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.

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.

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

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

Multimodal MR-imaging reveals large-scale structural and functional connectivity changes in profound early blindness

PubMed Central

Bauer, Corinna M.; Hirsch, Gabriella V.; Zajac, Lauren; Koo, Bang-Bon; Collignon, Olivier

2017-01-01

In the setting of profound ocular blindness, numerous lines of evidence demonstrate the existence of dramatic anatomical and functional changes within the brain. However, previous studies based on a variety of distinct measures have often provided inconsistent findings. To help reconcile this issue, we used a multimodal magnetic resonance (MR)-based imaging approach to provide complementary structural and functional information regarding this neuroplastic reorganization. This included gray matter structural morphometry, high angular resolution diffusion imaging (HARDI) of white matter connectivity and integrity, and resting state functional connectivity MRI (rsfcMRI) analysis. When comparing the brains of early blind individuals to sighted controls, we found evidence of co-occurring decreases in cortical volume and cortical thickness within visual processing areas of the occipital and temporal cortices respectively. Increases in cortical volume in the early blind were evident within regions of parietal cortex. Investigating white matter connections using HARDI revealed patterns of increased and decreased connectivity when comparing both groups. In the blind, increased white matter connectivity (indexed by increased fiber number) was predominantly left-lateralized, including between frontal and temporal areas implicated with language processing. Decreases in structural connectivity were evident involving frontal and somatosensory regions as well as between occipital and cingulate cortices. Differences in white matter integrity (as indexed by quantitative anisotropy, or QA) were also in general agreement with observed pattern changes in the number of white matter fibers. Analysis of resting state sequences showed evidence of both increased and decreased functional connectivity in the blind compared to sighted controls. Specifically, increased connectivity was evident between temporal and inferior frontal areas. Decreases in functional connectivity were observed

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

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.

Developmental consequences of cryopreservation of mammalian oocytes and embryos.

PubMed

Smith, Gary D; Silva E Silva, Cristine Ane

2004-08-01

During the last three decades, significant advances have been made in successful cryopreservation of mammalian preimplantation embryos, and more recently oocytes. The ability to cryopreserve, thaw, and establish pregnancies with supernumerary preimplantation embryos has become an important tool in fertility treatment. Human oocyte cryopreservation has practical application in preserving fertility for individuals at risk of compromised egg quality due to cancer treatments or advanced maternal age. While oocyte/embryo cryopreservation success has increased over time, there is still room for improvement. Oocytes and embryos are susceptible to cryo-damage, which collectively entails cellular damage caused by mechanical, chemical, or thermal forces during the freeze-thaw process. Basic studies focused on understanding cellular structures, their composition, and more importantly their functions, in normal cell developments will continue to be critical in assessing, understanding, and correcting oocyte/embryo cryo-damage. This review will delineate many of the oocyte/embryo intracellular and extracellular structures that are or may be compromised during cryopreservation. A global theme presented throughout this review is that many structural components of the oocyte/embryo also have essential functional roles in development. Compromising these cellular structures, and thus their cellular homeostatic functions, can deleteriously influence initial cryo-survival or compromise subsequent normal development through effects on the oocyte and/or early embryo.

Viability of bovine demi- and quarter-embryos after transfer.

PubMed

Bredbacka, P; Huhtinen, M; Aalto, J; Rainio, V

1992-07-01

The viability of bovine demi- and quarter-embryos was investigated. Early compacting morulae were nonsurgically flushed from superovulated donor cows and were bisected by two microneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6/13) for heifers and 33.3% (4/12) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8/13) for heifers and 8.3% (1/12) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos.

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.

Cognitive Enhancement Therapy Improves Resting-State Functional Connectivity in Early Course Schizophrenia

PubMed Central

Eack, Shaun M.; Newhill, Christina E.; Keshavan, Matcheri S.

2016-01-01

Objective Cognitive remediation is emerging as an effective psychosocial intervention for addressing untreated cognitive and functional impairments in persons with schizophrenia, and might achieve its benefits through neuroplastic changes in brain connectivity. This study seeks to examine the effects of cognitive enhancement therapy (CET) on fronto-temporal brain connectivity in a randomized controlled trial with individuals in the early course of schizophrenia. Method Stabilized, early course outpatients with schizophrenia or schizoaffective disorder (N = 41) were randomly assigned to CET (n = 25) or an active enriched supportive therapy (EST) control (n = 16) and treated for 2 years. Functional MRI data were collected annually, and pseudo resting-state functional connectivity analysis was used to examine differential changes in fronto-temporal connectivity between those treated with CET compared with EST. Results Individuals receiving CET evidenced significantly less functional connectivity loss between the resting-state network and the left dorsolateral prefrontal cortex as well as significantly increased connectivity with the right insular cortex compared to EST (all corrected p < .01). These neural networks are involved in emotion processing and problem-solving. Increased connectivity with the right insula significantly mediated CET effects on improved emotion perception (z′ = −1.96, p = .021), and increased connectivity with the left dorsolateral prefrontal cortex mediated CET-related improvements in emotion regulation (z′ = −1.71, p = .052). Conclusions These findings provide preliminary evidence that CET, a psychosocial cognitive remediation intervention, may enhance connectivity between frontal and temporal brain regions implicated in problem-solving and emotion processing in service of cognitive enhancement in schizophrenia. PMID:27713804

Early miscarriage rate in lean polycystic ovary syndrome women after euploid embryo transfer - a matched-pair study.

PubMed

Luo, Lu; Gu, Fang; Jie, Huying; Ding, Chenhui; Zhao, Qiang; Wang, Qiong; Zhou, Canquan

2017-11-01

The early miscarriage rate is reported to be higher in patients with polycystic ovary syndrome (PCOS) compared with non-PCOS patients. However, whether PCOS is an independent risk factor for early miscarriage is still controversial; to what extent embryonic aneuploidy accounts for miscarriages of PCOS is still unknown. In this 1:3 matched-pair study, 67 lean PCOS patients and 201 controls matched for age, body mass index (BMI) and embryo scores undergoing a single euploid blastocyst transfer in vitrified-warmed cycles were analysed. Clinical pregnancy, early miscarriage and live birth rates were compared. Logistic regression analysis was performed to further evaluate the factors associated with early miscarriage and live birth. Clinical pregnancy rates were 50.7% in PCOS and 55.2% in control groups. Early miscarriage rate was significantly (P = 0.029) increased in the PCOS group compared with controls; non-PCOS patients had a significantly higher live birth rate than PCOS patients, P < 0.001. Further regression analyses showed that PCOS was significantly associated with a higher risk of early miscarriage and decreased chance of live birth. In conclusion, PCOS in women undergoing pre-implantation genetic diagnosis may, independently from BMI and karyotype, increase the risk of miscarriage. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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

Inactivation of the Huntington's disease gene (Hdh) impairs anterior streak formation and early patterning of the mouse embryo

PubMed Central

Woda, Juliana M; Calzonetti, Teresa; Hilditch-Maguire, Paige; Duyao, Mabel P; Conlon, Ronald A; MacDonald, Marcy E

2005-01-01

Background Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdhex4/5 huntingtin deficient embryos. Results In the absence of huntingtin, expression of nutritive genes appears normal but E7.0–7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified. Conclusion Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease. PMID:16109169

Inactivation of the Huntington's disease gene (Hdh) impairs anterior streak formation and early patterning of the mouse embryo.

PubMed

Woda, Juliana M; Calzonetti, Teresa; Hilditch-Maguire, Paige; Duyao, Mabel P; Conlon, Ronald A; MacDonald, Marcy E

2005-08-18

Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdh(ex4/5) huntingtin deficient embryos. In the absence of huntingtin, expression of nutritive genes appears normal but E7.0-7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified. Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease.

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

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.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

PubMed

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

2016-01-01

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

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.

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

Culture of Cells from Amphibian Embryos.

ERIC Educational Resources Information Center

Stanisstreet, Martin

1983-01-01

Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

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.

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts.

PubMed

Skrzyszowska, M; Smorag, Z; Katska, L

1997-09-01

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (embryo configuration, connected by a very thin cell bridge (figure eight in shape). To separate the parts of the embryo, the cell bridge was cut using a glass microneedle. During the separation only a few cells were damaged. As a result of the procedure 4 20 (20.0%), 48 144 (33.3%) and 3 40 (7.5%) middle, late and expanded blastocysts hatched according to the pattern described. The developed procedure could be considered as a non-invasive alternative to conventional embryo splitting.

The first cell-fate decisions in the mouse embryo: destiny is a matter of both chance and choice.

PubMed

Zernicka-Goetz, Magdalena

2006-08-01

Development of the early mouse embryo has always been classified as regulative, meaning that when parts or blastomeres of the embryo are isolated they change their developmental fate and can even reconstruct the whole. However, regulative development does not mean that, in situ, these parts or blastomeres are equivalent; it does not mean that the early mammalian embryo is a ball of identical cells without any bias. Regulative development simply means that whatever bias the regions of the embryo might have they still remain flexible and can respond to experimental interference by changes of fate. This realization -- that regulative development and patterning can co-exist -- has led to a renaissance of interest in the first days of development of the mouse embryo, and several laboratories have provided evidence for some early bias. Now the challenge is to gain some understanding of the molecular basis of this bias.

Live imaging of rat embryos with Doppler swept-source optical coherence tomography

NASA Astrophysics Data System (ADS)

Larina, Irina V.; Furushima, Kenryo; Dickinson, Mary E.; Behringer, Richard R.; Larin, Kirill V.

2009-09-01

The rat has long been considered an excellent system to study mammalian embryonic cardiovascular physiology, but has lacked the extensive genetic tools available in the mouse to be able to create single gene mutations. However, the recent establishment of rat embryonic stem cell lines facilitates the generation of new models in the rat embryo to link changes in physiology with altered gene function to define the underlying mechanisms behind congenital cardiovascular birth defects. Along with the ability to create new rat genotypes there is a strong need for tools to analyze phenotypes with high spatial and temporal resolution. Doppler OCT has been previously used for 3-D structural analysis and blood flow imaging in other model species. We use Doppler swept-source OCT for live imaging of early postimplantation rat embryos. Structural imaging is used for 3-D reconstruction of embryo morphology and dynamic imaging of the beating heart and vessels, while Doppler-mode imaging is used to visualize blood flow. We demonstrate that Doppler swept-source OCT can provide essential information about the dynamics of early rat embryos and serve as a basis for a wide range of studies on functional evaluation of rat embryo physiology.

Live imaging of rat embryos with Doppler swept-source optical coherence tomography

PubMed Central

Larina, Irina V.; Furushima, Kenryo; Dickinson, Mary E.; Behringer, Richard R.; Larin, Kirill V.

2009-01-01

The rat has long been considered an excellent system to study mammalian embryonic cardiovascular physiology, but has lacked the extensive genetic tools available in the mouse to be able to create single gene mutations. However, the recent establishment of rat embryonic stem cell lines facilitates the generation of new models in the rat embryo to link changes in physiology with altered gene function to define the underlying mechanisms behind congenital cardiovascular birth defects. Along with the ability to create new rat genotypes there is a strong need for tools to analyze phenotypes with high spatial and temporal resolution. Doppler OCT has been previously used for 3-D structural analysis and blood flow imaging in other model species. We use Doppler swept-source OCT for live imaging of early postimplantation rat embryos. Structural imaging is used for 3-D reconstruction of embryo morphology and dynamic imaging of the beating heart and vessels, while Doppler-mode imaging is used to visualize blood flow. We demonstrate that Doppler swept-source OCT can provide essential information about the dynamics of early rat embryos and serve as a basis for a wide range of studies on functional evaluation of rat embryo physiology. PMID:19895102

Use of the Coelomic Grafting Technique for Prolonged ex utero Cultivation of Late Preprimitive Streak-Stage Rabbit Embryos.

PubMed

Püschel, Bernd; Männer, Jörg

2016-01-01

Due to its morphological similarity with the early human embryo, the pregastrulation-stage rabbit may represent an appropriate mammalian model for studying processes involved in early human development. The usability of mammalian embryos for experimental studies depends on the availability of whole embryo culture methods facilitating prolonged ex utero development. While currently used culture methods yield high success rates for embryos from primitive streak stages onward, the success rate of extended cultivation of preprimitive streak-stage mammalian embryos is low for all previously established methods and for all studied species. This limits the usability of preprimitive streak-stage rabbit embryos in experimental embryology. We have tested whether the extraembryonic coelom of 4-day-old chick embryos may be used for prolonged ex utero culture of preprimitive streak-stage rabbit embryos (stage 2, 6.2 days post coitum). We found that, within this environment, stage 2 rabbit blastocysts can be cultured at decreasing success rates (55% after 1 day, 35% after 2 days, 15% after 3 days) up to a maximum of 72 h. Grafted blastocysts can continue development from the onset of gastrulation to early organogenesis and thereby form all structures characterizing age-matched controls (e.g. neural tube, somites, beating heart). Compared to normal controls, successfully cultured embryos developed at a slower rate and finally showed some structural and gross morphological anomalies. The method presented here was originally developed for whole embryo culture of mouse embryos by Gluecksohn-Schoenheimer in 1941. It is a simple and inexpensive method that may represent a useful extension to presently available ex utero culture systems for rabbit embryos. © 2016 S. Karger AG, Basel.

Effects of growth hormone on the ultrastructure of bovine preimplantation embryos.

PubMed

Kölle, Sabine; Stojkovic, Miodrag; Reese, Sven; Reichenbach, Horst-Dieter; Wolf, Eckhard; Sinowatz, Fred

2004-07-01

Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.

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

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

PubMed Central

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

2013-01-01

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

Quantifying Three-Dimensional Morphology and RNA from Individual Embryos

PubMed Central

Green, Rebecca M.; Leach, Courtney L.; Hoehn, Natasha; Marcucio, Ralph S.; Hallgrímsson, Benedikt

2017-01-01

Quantitative analysis of morphogenesis aids our understanding of developmental processes by providing a method to link changes in shape with cellular and molecular processes. Over the last decade many methods have been developed for 3D imaging of embryos using microCT scanning to quantify the shape of embryos during development. These methods generally involve a powerful, cross-linking fixative such as paraformaldehyde to limit shrinkage during the CT scan. However, the extended time frames that these embryos are incubated in such fixatives prevent use of the tissues for molecular analysis after microCT scanning. This is a significant problem because it limits the ability to correlate variation in molecular data with morphology at the level of individual embryos. Here, we outline a novel method that allows RNA, DNA or protein isolation following CT scan while also allowing imaging of different tissue layers within the developing embryo. We show shape differences early in craniofacial development (E11.5) between common mouse genetic backgrounds, and demonstrate that we are able to generate RNA from these embryos after CT scanning that is suitable for downstream RT-PCR and RNAseq analyses. PMID:28152580

A modified culture method significantly improves the development of mouse somatic cell nuclear transfer embryos.

PubMed

Dai, Xiangpeng; Hao, Jie; Zhou, Qi

2009-08-01

Many strategies have been established to improve the efficiency of somatic cell nuclear transfer (SCNT), but relatively few focused on improving culture conditions. The effect of different culture media on preimplantation development of mouse nuclear transfer embryos was investigated. A modified sequential media method, named D media (M16/KSOM and CZB-EG/KSOM), was successfully established that significantly improves SCNT embryo development. Our result demonstrated that while lacking any adverse effect on in vivo fertilized embryos, the D media dramatically improves the blastocyst development of SCNT embryos compared with other commonly used media, including KSOM, M16, CZB, and alphaMEM. Specifically, the rate of blastocyst formation was 62.3% for D1 (M16/KSOM) versus 10-30% for the other media. An analysis of media components indicated that removing EDTA and glutamine from the media can be beneficial for early SCNT embryo development. Our results suggest that in vitro culture environment plays an important role in somatic cell reprogramming, and D media represent the most efficient culture method reported to date to support mouse SCNT early embryo development in vitro.

Endometrial signals improve embryo outcome: functional role of vascular endothelial growth factor isoforms on embryo development and implantation in mice.

PubMed

Binder, N K; Evans, J; Gardner, D K; Salamonsen, L A; Hannan, N J

2014-10-10

Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation? VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development. Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth. Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment). Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and

White Matter Structural Connectivity and Episodic Memory in Early Childhood

PubMed Central

Ngo, Chi T.; Alm, Kylie H.; Metoki, Athanasia; Hampton, William; Riggins, Tracy; Newcombe, Nora S.; Olson, Ingrid R.

2018-01-01

Episodic memory undergoes dramatic improvement in early childhood; the reason for this is poorly understood. In adults, episodic memory relies on a distributed neural network. Key brain regions that supporting these processes include the hippocampus, portions of the parietal cortex, and portions of prefrontal cortex, each of which shows different developmental profiles. Here we asked whether developmental differences in the axonal pathways connecting these regions may account for the robust gains in episodic memory in young children. Using diffusion weighted imaging, we examined whether white matter connectivity between brain regions implicated in episodic memory differed with age, and were associated with memory performance differences in 4- and 6-year-old children. Results revealed that white matter connecting the hippocampus to the inferior parietal lobule significantly predicted children’s performance on episodic memory tasks. In contrast, variation in the white matter connecting the hippocampus to the medial prefrontal cortex did not relate to memory performance. These findings suggest that structural connectivity between the hippocampus and lateral parietal regions is relevant to the development of episodic memory PMID:29175538

Loss of Bmal1 decreases oocyte fertilization, early embryo development and implantation potential in female mice.

PubMed

Xu, Jian; Li, Yan; Wang, Yizi; Xu, Yanwen; Zhou, Canquan

2016-10-01

Biological clock genes expressed in reproductive tissues play important roles in maintaining the normal functions of reproductive system. However, disruption of female circadian rhythm on oocyte fertilization, preimplantation embryo development and blastocyst implantation potential is still unclear. In this study, ovulation, in vivo and in vitro oocyte fertilization, embryo development, implantation and intracellular reactive oxygen species (ROS) levels in ovary and oviduct were studied in female Bmal1+/+ and Bmal1-/- mice. The number of naturally ovulated oocyte in Bmal1-/- mice decreased (5.2 ± 0.8 vs 7.8 ± 0.8, P < 0.001), with an increasing abnormal oocyte ratio (20.4 ± 3.5 vs 11.7 ± 2.0%, P = 0.001) after superovulation. Significantly lower fertilization rate and obtained blastocyst number were observed in Bmal1-/- female mice either mated with wild-type in vivo or fertilized by sperm from wild-type male mice in vitro (all P < 0.05). Interestingly, in vitro fertilization rate of oocytes derived from Bmal1-/- increased significantly compared with in vivo study (P < 0.01). After transferring blastocysts derived from Bmal1+/+ and Bmal1-/- female mice to pseudopregnant mice, the implantation sites of the latter decreased 5 days later (8.0 ± 0.8 vs 5.3 ± 1.0, P = 0.005). The intracellular ROS levels in the ovary on proestrus day and in the oviduct on metestrus day increased significantly in Bmal1-/- mice compared with that of Bmal1+/+ mice. Deletion of the core biological clock gene Bmal1 significantly decreases oocyte fertilization rate, early embryo development and implantation potential in female mice, and these may be possibly caused by excess ROS levels generated in ovary and oviduct.

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.

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.

Three-dimensional imaging of the brain cavities in human embryos.

PubMed

Blaas, H G; Eik-Nes, S H; Kiserud, T; Berg, S; Angelsen, B; Olstad, B

1995-04-01

A system for high-resolution three-dimensional imaging of small structures has been developed, based on the Vingmed CFM-800 annular array sector scanner with a 7.5-MHz transducer attached to a PC-based TomTec Echo-Scan unit. A stepper motor rotates the transducer 180 degrees and the complete three-dimensional scan consists of 132 two-dimensional images, video-grabbed and scan-converted into a regular volumetric data set by the TomTec unit. Three normal pregnancies with embryos of gestational age 7, 9 and 10 weeks received a transvaginal examination with special attention to the embryonic/fetal brain. In all three cases, it was possible to obtain high-resolution images of the brain cavities. At 7 weeks, both hemispheres and their connection to the third ventricle were delineated. The isthmus rhombencephali could be visualized. At 9 weeks, the continuous development of the brain cavities could be followed and at 11 weeks the dominating size of the hemispheres could be depicted. It is concluded that present ultrasound technology has reached a stage where structures of only a few millimeters can be imaged in vivo in three-dimensions with a quality that resembles the plaster figures used in embryonic laboratories. The method can become an important tool in future embryological research and also in the detection of early developmental disorders of the embryo.

Short communication: expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos.

PubMed

Laporta, J; Driver, A; Khatib, H

2011-08-01

Early embryo loss is a major contributing factor to cow infertility and that 70 to 80% of this loss occurs between d 8 and 16 postfertilization. However, little is known about the molecular mechanisms and the nature of genes involved in normal and abnormal embryonic development. Moreover, information is limited on the contributions of the genomes of dams and of embryos to the development and survival of preimplantation embryos. We hypothesized that proper gene expression level in the developing embryo is essential for embryo survival and pregnancy success. As such, the characterization of expression profiles in early embryos could lead to a better understanding of the mechanisms involved in normal and abnormal embryo development. To test this hypothesis, 2 d-8 embryo populations (degenerate embryos and blastocysts) that differed in morphology and developmental status were investigated. Expression levels of POU1F1 pathway genes were estimated in 4 sets of biological replicate pools of degenerate embryos and blastocysts. The OPN and STAT5A genes were found to be upregulated in degenerate embryos compared with blastocysts, whereas STAT5B showed similar expression levels in both embryo groups. Analysis of splice variants of OPN and STAT5A revealed expression patterns different from the total expression values of these genes. As such, measuring expression of individual transcripts should be considered in gene expression studies. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo apoptosis.

PubMed

Ji, Qianqian; Zhu, Kongju; Liu, Zhiguo; Song, Zhenwei; Huang, Yuankai; Zhao, Haijing; Chen, Yaosheng; He, Zuyong; Mo, Delin; Cong, Peiqing

2013-03-15

Trichostain A (TSA), an inhibitor of histone deacetylases, improved developmental competence of SCNT embryos in many species, apparently by improved epigenetic reprogramming. The objective of the present study was to determine the effects of TSA-induced apoptosis in cloned porcine embryos. At various developmental stages, a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were used to detect apoptosis, and real-time polymerase chain reaction was used to assess expression of genes related to apoptosis and pluripotency. In this study, TSA significantly induced apoptosis (in a dose-dependent manner) at the one-, two-, and four-cell stages. However, in blastocyst stage embryos, TSA decreased the apoptotic index (P < 0.05). Expression levels of Caspase 3 were higher in TSA-treated versus control embryos at the two-cell stage (not statistically significant). The expression ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of antiapoptotic potential, was higher in TSA-treated groups at the one-, two-, and four-cell and blastocyst stages. Furthermore, expression levels of pluripotency-related genes, namely, Oct4 and Nanog, were elevated at the morula stage (P < 0.05) in TSA treatment groups. We concluded that inducing apoptosis might be a mechanism by which TSA promotes development of reconstructed embryos. At the initial stage of apoptosis induction, abnormal cells were removed, thereby enhancing proliferation of healthy cells and improving embryo quality. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

[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.

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.

[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.

Time to take human embryo culture seriously.

PubMed

Sunde, Arne; Brison, Daniel; Dumoulin, John; Harper, Joyce; Lundin, Kersti; Magli, M Cristina; Van den Abbeel, Etienne; Veiga, Anna

2016-10-01

Is it important that end-users know the composition of human embryo culture media? We argue that there is as strong case for full transparency concerning the composition of embryo culture media intended for human use. Published data suggest that the composition of embryo culture media may influence the phenotype of the offspring. A review of the literature was carried out. Data concerning the potential effects on embryo development of culture media were assessed and recommendations for users made. The safety of ART procedures, especially with respect to the health of the offspring, is of major importance. There are reports from the literature indicating a possible effect of culture conditions, including culture media, on embryo and fetal development. Since the introduction of commercially available culture media, there has been a rapid development of different formulations, often not fully documented, disclosed or justified. There is now evidence that the environment the early embryo is exposed to can cause reprogramming of embryonic growth leading to alterations in fetal growth trajectory, birthweight, childhood growth and long-term disease including Type II diabetes and cardiovascular problems. The mechanism for this is likely to be epigenetic changes during the preimplantation period of development. In the present paper the ESHRE working group on culture media summarizes the present knowledge of potential effects on embryo development related to culture media, and makes recommendations. There is still a need for large prospective randomized trials to further elucidate the link between the composition of embryo culture media used and the phenotype of the offspring. We do not presently know if the phenotypic changes induced by in vitro embryo culture represent a problem for long-term health of the offspring. Published data indicate that there is a strong case for demanding full transparency concerning the compositions of and the scientific rationale behind the

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.

Successful Object Encoding Induces Increased Directed Connectivity in Presymptomatic Early-Onset Alzheimer’s Disease

PubMed Central

Ochoa, John Fredy; Alonso, Joan Francesc; Duque, Jon Edinson; Tobón, Carlos Andrés; Mañanas, Miguel Angel; Lopera, Francisco; Hernández, Alher Mauricio

2016-01-01

Background: Recent studies report increases in neural activity in brain regions critical to episodic memory at preclinical stages of Alzheimer’s disease (AD). Although electroencephalography (EEG) is widely used in AD studies, given its non-invasiveness and low cost, there is a need to translate the findings in other neuroimaging methods to EEG. Objective: To examine how the previous findings using functional magnetic resonance imaging (fMRI) at preclinical stage in presenilin-1 E280A mutation carriers could be assessed and extended, using EEG and a connectivity approach. Methods: EEG signals were acquired during resting and encoding in 30 normal cognitive young subjects, from an autosomal dominant early-onset AD kindred from Antioquia, Colombia. Regions of the brain previously reported as hyperactive were used for connectivity analysis. Results: Mutation carriers exhibited increasing connectivity at analyzed regions. Among them, the right precuneus exhibited the highest changes in connectivity. Conclusion: Increased connectivity in hyperactive cerebral regions is seen in individuals, genetically-determined to develop AD, at preclinical stage. The use of a connectivity approach and a widely available neuroimaging technique opens the possibility to increase the use of EEG in early detection of preclinical AD. PMID:27792014

White matter structural connectivity and episodic memory in early childhood.

PubMed

Ngo, Chi T; Alm, Kylie H; Metoki, Athanasia; Hampton, William; Riggins, Tracy; Newcombe, Nora S; Olson, Ingrid R

2017-12-01

Episodic memory undergoes dramatic improvement in early childhood; the reason for this is poorly understood. In adults, episodic memory relies on a distributed neural network. Key brain regions that supporting these processes include the hippocampus, portions of the parietal cortex, and portions of prefrontal cortex, each of which shows different developmental profiles. Here we asked whether developmental differences in the axonal pathways connecting these regions may account for the robust gains in episodic memory in young children. Using diffusion weighted imaging, we examined whether white matter connectivity between brain regions implicated in episodic memory differed with age, and were associated with memory performance differences in 4- and 6-year-old children. Results revealed that white matter connecting the hippocampus to the inferior parietal lobule significantly predicted children's performance on episodic memory tasks. In contrast, variation in the white matter connecting the hippocampus to the medial prefrontal cortex did not relate to memory performance. These findings suggest that structural connectivity between the hippocampus and lateral parietal regions is relevant to the development of episodic memory. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Stronger net posterior cortical forces and asymmetric microtubule arrays produce simultaneous centration and rotation of the pronuclear complex in the early Caenorhabditis elegans embryo

PubMed Central

Coffman, Valerie C.; McDermott, Matthew B. A.; Shtylla, Blerta; Dawes, Adriana T.

2016-01-01

Positioning of microtubule-organizing centers (MTOCs) incorporates biochemical and mechanical cues for proper alignment of the mitotic spindle and cell division site. Current experimental and theoretical studies in the early Caenorhabditis elegans embryo assume remarkable changes in the origin and polarity of forces acting on the MTOCs. These changes must occur over a few minutes, between initial centration and rotation of the pronuclear complex and entry into mitosis, and the models do not replicate in vivo timing of centration and rotation. Here we propose a model that incorporates asymmetry in the microtubule arrays generated by each MTOC, which we demonstrate with in vivo measurements, and a similar asymmetric force profile to that required for posterior-directed spindle displacement during mitosis. We find that these asymmetries are capable of and important for recapitulating the simultaneous centration and rotation of the pronuclear complex observed in vivo. The combination of theoretical and experimental evidence provided here offers a unified framework for the spatial organization and forces needed for pronuclear centration, rotation, and spindle displacement in the early C. elegans embryo. PMID:27733624

Brachyury expression in tailless Molgulid ascidian embryos.

PubMed

Takada, Norio; York, Jonathan; Davis, J Muse; Schumpert, Brenda; Yasuo, Hitoyoshi; Satoh, Nori; Swalla, Billie J

2002-01-01

The T-box transcription factor gene Brachyury is important for the differentiation of notochord in all chordates, including the ascidians Halocynthia roretzi and Ciona intestinalis. We isolated Brachyury from molgulid ascidians, which have evolved tailless larvae multiple times independently, and found the genes appear functional by cDNA sequence analyses. We then compared the expression of Mocu-Bra in tailed Molgula oculata embryos to two tailless species, Molgula occulta (Mocc-Bra) and Molgula tectiformis (Mt-Bra). Here we show that both tailless species express Brachyury in the notochord lineage during embryogenesis. Initial expression of Mocu-Bra is normal in tailed M. oculata embryos; 10 precursor notochord cells divide twice to result in 40 notochord cells that converge and extend to make a notochord down the center of the tail. In contrast, in tailless Molgula occulta, Mocc-Bra expression disappears prematurely, and there is only one round of division, resulting in 20 cells in the final notochord lineage that never converge or extend. In M. occulta x M. oculata hybrid embryos, expression of Mocu-Bra is prolonged, and the embryos form a tail with 20 notochord cells that converge and extend normally. However, in Molgula tectiformis, a different tailless ascidian, Mt-Bra was expressed only in the 10 notochord precursor cells, which never divide, converge, or extend. In summary, neither Brachyury function nor the early establishment of the notochord lineage appears to be impaired in tailless embryos. In light of these results, we are continuing to investigate how and why notochord development is lost in tailless molgulid ascidian embryos.

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.

Critical role of mTOR, PPARγ and PPARδ signaling in regulating early pregnancy decidual function, embryo viability and feto-placental growth.

PubMed

Roberti, Sabrina L; Higa, Romina; White, Verónica; Powell, Theresa L; Jansson, Thomas; Jawerbaum, Alicia

2018-06-01

What are the consequences of inhibiting mTOR, the mechanistic target of rapamycin (mTOR), and the peroxisome proliferator activated receptor gamma (PPARγ) and PPARδ pathways in the early post-implantation period on decidual function, embryo viability and feto-placental growth in the rat? mTOR inhibition from Days 7 to 9 of pregnancy in rats caused decidual PPARγ and PPARδ upregulation on Day 9 of pregnancy and resulted in embryo resorption by Day 14 of pregnancy. PPARγ and PPARδ inhibition differentially affected decidual mTOR signaling and levels of target proteins relevant to lipid histotrophic nutrition and led to reduced feto-placental weights on Day 14 of pregnancy. Although mTOR, PPARγ and PPARδ are nutrient sensors important during implantation, the role of these signaling pathways in decidual function and how they interact in the early post-implantation period are unknown. Perilipin 2 (PLIN2) and fatty acid binding protein 4 (FABP4), two adipogenic proteins involved in lipid histotrophic nutrition, are targets of mTOR and PPAR signaling pathways in a variety of tissues. Rapamycin (mTOR inhibitor, 0.75 mg/kg, sc), T0070907 (PPARγ inhibitor, 0.001 mg/kg, sc), GSK0660 (PPARδ inhibitor, 0.1 mg/kg, sc) or vehicle was injected daily to pregnant rats from Days 7 to 9 of pregnancy and the studies were performed on Day 9 of pregnancy (n = 7 per group) or Day 14 of pregnancy (n = 7 per group). On Day 9 of pregnancy, rat decidua were collected and prepared for western blot and immunohistochemical studies. On Day 14 of pregnancy, the resorption rate, number of viable fetuses, crown-rump length and placental and decidual weights were determined. Inhibition of mTOR in the early post-implantation period led to a reduction in FABP4 protein levels, an increase in PLIN2 levels and an upregulation of PPARγ and PPARδ in 9-day-pregnant rat decidua. Most embryos were viable on Day 9 of pregnancy but had resorbed by Day 14 of pregnancy. This denotes a key function of

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.

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

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

[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.

Early brain connectivity alterations and cognitive impairment in a rat model of Alzheimer's disease.

PubMed

Muñoz-Moreno, Emma; Tudela, Raúl; López-Gil, Xavier; Soria, Guadalupe

2018-02-07

Animal models of Alzheimer's disease (AD) are essential to understanding the disease progression and to development of early biomarkers. Because AD has been described as a disconnection syndrome, magnetic resonance imaging (MRI)-based connectomics provides a highly translational approach to characterizing the disruption in connectivity associated with the disease. In this study, a transgenic rat model of AD (TgF344-AD) was analyzed to describe both cognitive performance and brain connectivity at an early stage (5 months of age) before a significant concentration of β-amyloid plaques is present. Cognitive abilities were assessed by a delayed nonmatch-to-sample (DNMS) task preceded by a training phase where the animals learned the task. The number of training sessions required to achieve a learning criterion was recorded and evaluated. After DNMS, MRI acquisition was performed, including diffusion-weighted MRI and resting-state functional MRI, which were processed to obtain the structural and functional connectomes, respectively. Global and regional graph metrics were computed to evaluate network organization in both transgenic and control rats. The results pointed to a delay in learning the working memory-related task in the AD rats, which also completed a lower number of trials in the DNMS task. Regarding connectivity properties, less efficient organization of the structural brain networks of the transgenic rats with respect to controls was observed. Specific regional differences in connectivity were identified in both structural and functional networks. In addition, a strong correlation was observed between cognitive performance and brain networks, including whole-brain structural connectivity as well as functional and structural network metrics of regions related to memory and reward processes. In this study, connectivity and neurocognitive impairments were identified in TgF344-AD rats at a very early stage of the disease when most of the pathological hallmarks

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.

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

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.

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)

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

Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila centrosomin.

PubMed

Lucas, Eliana P; Raff, Jordan W

2007-08-27

Centrosomes consist of two centrioles surrounded by an amorphous pericentriolar matrix (PCM), but it is unknown how centrioles and PCM are connected. We show that the centrioles in Drosophila embryos that lack the centrosomal protein Centrosomin (Cnn) can recruit PCM components but cannot maintain a proper attachment to the PCM. As a result, the centrioles "rocket" around in the embryo and often lose their connection to the nucleus in interphase and to the spindle poles in mitosis. This leads to severe mitotic defects in embryos and to errors in centriole segregation in somatic cells. The Cnn-related protein CDK5RAP2 is linked to microcephaly in humans, but cnn mutant brains are of normal size, and we observe only subtle defects in the asymmetric divisions of mutant neuroblasts. We conclude that Cnn maintains the proper connection between the centrioles and the PCM; this connection is required for accurate centriole segregation in somatic cells but is not essential for the asymmetric division of neuroblasts.

Developmental imaging: the avian embryo hatches to the challenge.

PubMed

Kulesa, Paul M; McKinney, Mary C; McLennan, Rebecca

2013-06-01

The avian embryo provides a multifaceted model to study developmental mechanisms because of its accessibility to microsurgery, fluorescence cell labeling, in vivo imaging, and molecular manipulation. Early two-dimensional planar growth of the avian embryo mimics human development and provides unique access to complex cell migration patterns using light microscopy. Later developmental events continue to permit access to both light and other imaging modalities, making the avian embryo an excellent model for developmental imaging. For example, significant insights into cell and tissue behaviors within the primitive streak, craniofacial region, and cardiovascular and peripheral nervous systems have come from avian embryo studies. In this review, we provide an update to recent advances in embryo and tissue slice culture and imaging, fluorescence cell labeling, and gene profiling. We focus on how technical advances in the chick and quail provide a clearer understanding of how embryonic cell dynamics are beautifully choreographed in space and time to sculpt cells into functioning structures. We summarize how these technical advances help us to better understand basic developmental mechanisms that may lead to clinical research into human birth defects and tissue repair. Copyright © 2013 Wiley Periodicals, Inc.

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)

Novel embryo selection techniques to increase embryo implantation in IVF attempts.

PubMed

Sigalos, George Α; Triantafyllidou, Olga; Vlahos, Nikos F

2016-11-01

The final success of an IVF attempt depends on several steps and decisions taken during the ovarian stimulation, the oocyte retrieval, the embryo culture and the embryo transfer. The final selection of the embryos most likely to implant is the final step in this process and the responsibility of the lab. Apart from strict morphologic criteria that historically have been used in embryo selection, additional information on genetic, metabolomic and morphokinetic characteristics of the embryo is recently combined to morphology to select the embryo most likely to produce a pregnancy. In this manuscript, we review the most recent information on the current methods used for embryo selection presenting the predictive capability of each one. A literature search was performed on Pubmed, Medline and Cochrane Database of Systematic Reviews for published studies using appropriate key words and phrases with no limits placed on time. It seems that the combination of morphologic criteria in conjunction to embryo kinetics as documented by time-lapse technology provides the most reliable information on embryo quality. Blastocyst biopsy with subsequent comprehensive chromosome analysis allows the selection of the euploid embryos with the higher implantation potential. Embryo time-lapse imaging and blastocyst biopsy combined to comprehensive chromosome analysis are the most promising technologies to increase pregnancy rates and reduce the possibility of multiple pregnancies. However, further studies will demonstrate the capability of routinely using these technologies to significantly improve IVF outcomes.

Death in the clinic: women's perceptions and experiences of discarding supernumerary IVF embryos.

PubMed

de Lacey, Sheryl

2017-03-01

Perspectives on the status of human embryos and whether they should be discarded differ globally. Some countries protect embryos in law while in other countries embryos 'die' or 'succumb' in assisted reproductive technology clinics on a daily basis. This study analyses interview data drawn from a larger qualitative study conducted in South Australia from 2004-2007. 21 women and 12 of 21 partners were interviewed about the decision they made to discard their embryos. The analysis reported here sought to examine the ways in which women constructed and experienced the decision to discard embryos. The article highlights the ways in which embryo discard is a contested discursive space. Embryo death is sequestered through their confinement in the laboratory and their invisibility to the naked eye. The clinic treated embryo discard as disposal of biological waste and failed to acknowledge the meaning of the event. By contrast women experienced emotional bereavement described as similar to early pregnancy loss, and described experiences of attachment and grief. For sensitive and compassionate care these differences in perceptions of embryo discard need to be addressed. © 2016 Foundation for the Sociology of Health & Illness.

Culturing Chick Embryos--A Simplification of New's Method.

ERIC Educational Resources Information Center

Downie, J. R.

1979-01-01

Describes a simplified version of New's method for culturing early chick embryos. The technique allows continuous observation of the critical first three days of development and the conditions for setting up successful cultures are also presented to help both teachers and students. (HM)

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

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.

An approach to successful freezing of demi-embryos derived from day-7 bovine embryos.

PubMed

Niemann, H; Brem, G; Sacher, B; Smidt, D; Kräusslich, H

1986-04-01

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.

Social Validity: Perceptions of Check and Connect with Early Literacy Support

ERIC Educational Resources Information Center

Miltich Lyst, Aimee; Gabriel, Stacey; O'Shaughnessy, Tam E.; Meyers, Joel; Meyers, Barbara

2005-01-01

This article underscores the potential advantages of qualitative methods to illustrate the depth and complexity of social validity. This investigation evaluates the social validity of Check and Connect with Early Literacy Support (CCEL), through the perspectives of teachers and caregivers whose children participated in the intervention. Teachers…

Live-bearing manta ray: how the embryo acquires oxygen without placenta and umbilical cord

PubMed Central

Tomita, Taketeru; Toda, Minoru; Ueda, Keiichi; Uchida, Senzo; Nakaya, Kazuhiro

2012-01-01

We conducted an ultrasonographic experiment on a pregnant manta ray, Manta alfredi (Chondrichthyes, Batoidea). This study showed how the embryo of the live-bearing elasmobranchs respires in the body of the female. In the embryonic stage, the manta ray embryo takes in uterine fluid by buccal-pumping. After birth, the manta ray shifts its respiratory mode from buccal-pumping to ram-ventilation. The rapid reduction of the spiracle size in the young manta ray may reflect this shift of respiratory mode. Unlike mammals or some carcharhinid sharks that acquire oxygen through a placenta and umbilical cord, the manta ray embryo does not have a direct connection with the mother. Thus, the manta ray embryo obtains oxygen by buccal-pumping of the uterine fluid, in the same way that the embryos of egg-laying species obtain oxygen from the water in the egg case. This finding extends our understanding of the diversity of embryonic respiratory systems in live-bearing vertebrates. PMID:22675137

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

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

Preattachment Embryos of Domestic Animals: Insights into Development and Paracrine Secretions.

PubMed

Sandra, Olivier; Charpigny, Gilles; Galio, Laurent; Hue, Isabelle

2017-02-08

In mammalian species, endometrial receptivity is driven by maternal factors independently of embryo signals. When pregnancy initiates, paracrine secretions of the preattachment embryo are essential both for maternal recognition and endometrium preparation for implantation and for coordinating development of embryonic and extraembryonic tissues of the conceptus. This review mainly focuses on domestic large animal species. We first illustrate the major steps of preattachment embryo development, including elongation in bovine, ovine, porcine, and equine species. We next highlight conceptus secretions that are involved in the communication between extraembryonic and embryonic tissues, as well as between the conceptus and the endometrium. Finally, we introduce experimental data demonstrating the intimate connection between conceptus secretions and endometrial activity and how adverse events perturbing this interplay may affect the progression of implantation that will subsequently impact pregnancy outcome, postnatal health, and expression of production traits in livestock offspring.

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.

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.

Clinical evaluation of frozen/thawed embryo transfer following transport of oocytes and embryos

PubMed Central

2004-01-01

Background and Aims:  We evaluated the efficacy of the transport oocyte/embryo frozen/thawed embryo transfer method, in which oocytes or embryos were transported from satellite clinics to the main assisted reproductive technology (ART) center, and surplus embryos were placed in cryopreservation. Methods:  We evaluated 41 cycles in 34 patients in the transport oocyte group (TO group). In the TO group the oocytes were collected at the satellite clinics, transported to the main ART center and underwent in vitro fertilization or intracytoplasmic sperm injection. Surplus embryos were used for frozen/thawed embryo transfer. We also evaluated 17 cycles in 10 patients in the transport embryo group (TE group), where surplus embryos were transported to the main ART center and used for frozen/thawed embryo transfer; and 189 cycles in 134 patients in the center group (C group), where surplus embryos collected at the same time at the main ART center were used for frozen/thawed embryo transfer. Oocytes were transported from satellite clinics in HEPES buffered human tubal fluid (HTF) culture medium, and embryos in 30% synthetic serum substitute + HEPES buffered HTF, using a portable incubator we devised. Results:  The proportions of undamaged embryos after freeze/thawing were 47% for the C group, 46% for the TO group, and 46% for the TE group. The numbers of embryos transferred were 2.0 ± 0.7 for the C group, 2.0 ± 0.6 for the TO group, and 2.2 ± 0.4 for the TE group. The rate of embryo transfer was 63% for the C group, 68% for the TO group, and 76% for the TE group. Pregnancy rates per patient were 16% for the C group, 24% for the TO group, and 40% for the TE group. The embryo survival rates (number of embryos with ≥50% viable blastomeres/total number of embryos) were 55% for the C group, 60% for the TO group, and 54% for the TE group. No significant differences were seen between the C group and either the TO or TE groups in any of these parameters

The fate of paternal mitochondria in marmoset pre-implantation embryos.

PubMed

Luetjens, C M; Wesselmann, R

2008-06-01

Sperm-derived mitochondria are integrated into the oocyte at fertilization but seem to vanish during the early cleavage phase. The developmental potential of pre-implantation embryos seems to be closely related to their ability to induce degeneration of these mitochondria, but the mechanisms underlying their loss of function are not yet understood. This study focuses on the fate of paternal mitochondria in pre-implantation embryos. Stimulation, collection and in vitro culture of oocytes from Callithrix jacchus, allows the study of the destiny of paternal mitochondria by utilizing immunostaining of pre-implantation embryos, fluorescence and laserscanning microscopy. Live pre-implantation embryos were stained with a fluorescence indicator reflecting mitochondrial membrane potential. Evidence indicating the loss of mitochondrial function was not found nor that apoptosis pathways were involved in the disappearance of paternally derived mitochondria. These findings may have implications for mitochondrially inherited diseases and could lead to new strategies for improving assisted reproduction.

Human cloning and embryo research: the 2003 John J. Conley Lecture on medical ethics.

PubMed

George, Robert P

2004-01-01

The author, a member of the U.S. President's Council on Bioethics, discusses ethical issues raised by human cloning, whether for purposes of bringing babies to birth or for research purposes. He first argues that every cloned human embryo is a new, distinct, and enduring organism, belonging to the species Homo sapiens, and directing its own development toward maturity. He then distinguishes between two types of capacities belonging to individual organisms belonging to this species, an immediately exerciseable capacity and a basic natural capacity that develops over time. He argues that it is the second type of capacity that is the ground for full moral respect, and that this capacity (and its concomitant degree of respect) belongs to cloned human embryos no less than to adult human beings. He then considers and rejects counter-arguments to his position, including the suggestion that the capacity of embryos is equivalent to the capacity of somatic cells, that full human rights are afforded only to human organisms with functioning brains, that the possibility of twinning diminishes the moral status of embryos, that the fact that people do not typically mourn the loss of early embryos implies that they have a diminished moral status, that the fact that early spontaneous abortions occur frequently diminishes the moral status of embryos, and that his arguments depend upon a concept of ensoulment. He concludes that if the moral status of cloned human embryos is equivalent to that of adults, then public policy should be based upon this assumption.

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

The role of eggshell and underlying vitelline membrane for normal pattern formation in the early C. elegans embryo.

PubMed

Schierenberg, Einhard; Junkersdorf, Bernd

1992-12-01

The embryo of the nematode Caenorhabditis elegans is surrounded by an inconspicuous inner vitelline membrane and a prominent outer chitinous eggshell proper. We demonstrate that the complete removal of the chitinous eggshell does not interfere with successful development to yield a normal worm. The same result can be obtained when the vitelline membrane is penetrated with laser microbeam irradiation of only the eggshell proper, gently enough to permit its resealing after a while. However, when large holes are made into the eggshell the concomitantly penetrated vitelline membrane does not reseal. While early development is quite normal under these conditions, gastrulation is defective in that gut precursor cells do not migrate in properly, eventually leading to embryonic arrest. This suggests a crucial role for pattern formation of the "micro-environment" around the embryo preserved by the intact vitelline membrane. Removing both eggshell and vitelline membrane results in a string-like arrangement of founder cells and subsequent grossly abnormal cell patterns. Our experiments support the idea that the prominent eggshell proper just functions as a mechanical protection while the thin vitelline membrane directly or indirectly serves as a necessary control element affecting the positions of cells which to begin with are determined by the orientation of the cleavage spindle.

Evaluation of treatments with hCG and carprofen at embryo transfer in a demi-embryo and recipient virgin heifer model.

PubMed

Torres, A; Chagas E Silva, J; Diniz, P; Lopes-da-Costa, L

2013-08-01

An in vivo model, combining a low developmental competence embryo (demi-embryo) and a high-fertility recipient (virgin dairy heifer) was used to evaluate the effects of treatment with human chorionic gonadotropin (hCG) and carprofen at embryo transfer (ET) on plasma progesterone (P₄) concentrations of recipients and on embryonic growth and survival. Embryos were bisected and each demi-embryo was transferred to a recipient on Day 7 of the estrous cycle. At ET, heifers (n = 163) were randomly allocated to treatment with hCG (2500 IU im), carprofen (500 mg iv), hCG plus carprofen or to untreated controls. Plasma P₄ concentrations were measured on Days 0, 7, 14 and 21 of all recipients plus on Days 28, 42 and 63 of pregnant recipients. Pregnancy was presumed to be present in recipients with luteal plasma P4 concentrations until Day 21 and confirmed by using transrectal ultrasonography on Days 28, 42 and 63. Embryonic measurements (crown-rump length and width) were obtained on Day 42. Treatment with hCG induced formation of secondary corpora lutea (CL) in 97% of heifers and increased (P < 0.01) mean plasma P₄ concentrations of non-pregnant recipients on Day 14 and of pregnant heifers on Days 14 to 63. This was associated to a significant decrease in early embryonic mortality. In contrast, subsequent embryonic losses resulted in a non-significant numerical increase by 8% of pregnancies maintained to Day 63. Therefore, treatment with hCG significantly rescued embryos through the maternal recognition of pregnancy window but was not able to support development thereafter. Treatment with carprofen at ET had no significant effects on plasma P₄ concentrations and rate of embryo mortality. Treatment with hCG plus carprofen at ET induced formation of secondary CL in 90% of heifers but decreased the luteotrophic effect of hCG, resulting in no effect on embryo survival. Low developmental competence embryos showed an intrinsic deficiency in overcoming the maternal

Dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development.

PubMed

Beck, Aaron P; Watt, Roland M; Bonner, Jennifer

2014-02-28

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

PubMed Central

Beck, Aaron P.; Watt, Roland M.; Bonner, Jennifer

2014-01-01

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue. PMID:24637734

Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells.

PubMed

Ramalho-Santos, João; Varum, Sandra; Amaral, Sandra; Mota, Paula C; Sousa, Ana Paula; Amaral, Alexandra

2009-01-01

Mitochondria are multitasking organelles involved in ATP synthesis, reactive oxygen species (ROS) production, calcium signalling and apoptosis; and mitochondrial defects are known to cause physiological dysfunction, including infertility. The goal of this review was to identify and discuss common themes in mitochondrial function related to mammalian reproduction. The scientific literature was searched for studies reporting on the several aspects of mitochondrial activity in mammalian testis, sperm, oocytes, early embryos and embryonic stem cells. ATP synthesis and ROS production are the most discussed aspects of mitochondrial function. Metabolic shifts from mitochondria-produced ATP to glycolysis occur at several stages, notably during gametogenesis and early embryo development, either reflecting developmental switches or substrate availability. The exact role of sperm mitochondria is especially controversial. Mitochondria-generated ROS function in signalling but are mostly described when produced under pathological conditions. Mitochondria-based calcium signalling is primarily important in embryo activation and embryonic stem cell differentiation. Besides pathologically triggered apoptosis, mitochondria participate in apoptotic events related to the regulation of spermatogonial cell number, as well as gamete, embryo and embryonic stem cell quality. Interestingly, data from knock-out (KO) mice is not always straightforward in terms of expected phenotypes. Finally, recent data suggests that mitochondrial activity can modulate embryonic stem cell pluripotency as well as differentiation into distinct cellular fates. Mitochondria-based events regulate different aspects of reproductive function, but these are not uniform throughout the several systems reviewed. Low mitochondrial activity seems a feature of 'stemness', being described in spermatogonia, early embryo, inner cell mass cells and embryonic stem cells.

Ultrastructural dynamics of human reproduction, from ovulation to fertilization and early embryo development.

PubMed

Familiari, Giuseppe; Heyn, Rosemarie; Relucenti, Michela; Nottola, Stefania A; Sathananthan, A Henry

2006-01-01

This study describes the updated, fine structure of human gametes, the human fertilization process, and human embryos, mainly derived from assisted reproductive technology (ART). As clearly shown, the ultrastructure of human reproduction is a peculiar multistep process, which differs in part from that of other mammalian models, having some unique features. Particular attention has been devoted to the (1) sperm ultrastructure, likely "Tygerberg (Kruger) strict morphology criteria"; (2) mature oocyte, in which the MII spindle is barrel shaped, anastral, and lacking centrioles; (3) three-dimensional microarchitecture of the zona pellucida with its unique supramolecular filamentous organization; (4) sperm-egg interactions with the peculiarity of the sperm centrosome that activates the egg and organizes the sperm aster and mitotic spindles of the embryo; and (5) presence of viable cumulus cells whose metabolic activity is closely related to egg and embryo behavior in in vitro as well as in vivo conditions, in a sort of extraovarian "microfollicular unit." Even if the ultrastructural morphodynamic features of human fertilization are well understood, our knowledge about in vivo fertilization is still very limited and the complex sequence of in vivo biological steps involved in human reproduction is only partially reproduced in current ART procedures.

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.

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

Shock wave propagation in layered planetary embryos

NASA Astrophysics Data System (ADS)

Arkani-Hamed, Jafar; Ivanov, Boris A.

2014-05-01

The propagation of impact-induced shock wave inside a planetary embryo is investigated using the Hugoniot equations and a new scaling law, governing the particle velocity variations along a shock ray inside a spherical body. The scaling law is adopted to determine the impact heating of a growing embryo in its early stage when it is an undifferentiated and uniform body. The new scaling law, similar to other existing scaling laws, is not suitable for a large differentiated embryo consisting of a silicate mantle overlying an iron core. An algorithm is developed in this study on the basis of the ray theory in a spherically symmetric body which relates the shock parameters at the top of the core to those at the base of the mantle, thus enabling the adoption of scaling laws to estimate the impact heating of both the mantle and the core. The algorithm is applied to two embryo models: a simple two-layered model with a uniform mantle overlying a uniform core, and a model where the pre-shock density and acoustic velocity of the embryo are radially dependent. The former illustrates details of the particle velocity, shock pressure, and temperature increase behind the shock front in a 2D axisymmetric geometry. The latter provides a means to compare the results with those obtained by a hydrocode simulation. The agreement between the results of the two techniques in revealing the effects of the core-mantle boundary on the shock wave transmission across the boundary is encouraging.

Progress Towards the Development of a Fathead Minnow Embryo Test and Comparison to the Zebrafish Embryo Test for Assessing Acute Fish Toxicity

EPA Science Inventory

The Zebrafish Embryo Test (ZFET) for acute fish toxicity is a well developed method nearing adoption as an OECD Test Guideline. Early drafts of the test guideline (TG) envisioned a suite of potential test species to be covered including zebrafish, fathead minnow, Japanese Medaka...

[Relationship between mitochondrial DNA copy number, membrane potential of human embryo and embryo morphology].

PubMed

Zhao, H; Teng, X M; Li, Y F

2017-11-25

Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all P< 0.05]. The copy number of mtDNA and the mitochondrial membrane potential in classⅠ frozen embryo group were significantly higher than those in classⅡ frozen embryo group (both P< 0.05). Conclusion: The mtDNA copy number and the mitochondrial membrane potential of embryos of the better quality embryo are higher.

Asymmetric distribution of hypoxia-inducible factor α regulates dorsoventral axis establishment in the early sea urchin embryo.

PubMed

Chang, Wei-Lun; Chang, Yi-Cheng; Lin, Kuan-Ting; Li, Han-Ru; Pai, Chih-Yu; Chen, Jen-Hao; Su, Yi-Hsien

2017-08-15

Hypoxia signaling is an ancient pathway by which animals can respond to low oxygen. Malfunction of this pathway disturbs hypoxic acclimation and can result in various diseases, including cancers. The role of hypoxia signaling in early embryogenesis remains unclear. Here, we show that in the blastula of the sea urchin Strongylocentrotus purpuratus , hypoxia-inducible factor α (HIFα), the downstream transcription factor of the hypoxia pathway, is localized and transcriptionally active on the future dorsal side. This asymmetric distribution is attributable to its oxygen-sensing ability. Manipulations of the HIFα level entrained the dorsoventral axis, as the side with the higher level of HIFα tends to develop into the dorsal side. Gene expression analyses revealed that HIFα restricts the expression of nodal to the ventral side and activates several genes encoding transcription factors on the dorsal side. We also observed that intrinsic hypoxic signals in the early embryos formed a gradient, which was disrupted under hypoxic conditions. Our results reveal an unprecedented role of the hypoxia pathway in animal development. © 2017. Published by The Company of Biologists Ltd.

Endoplasmic Reticulum Stress Signaling in Mammalian Oocytes and Embryos: Life in the Balance

PubMed Central

Latham, Keith E.

2015-01-01

Mammalian oocytes and embryos are exquisitely sensitive to a wide range of insults related to physical stress, chemical exposure, and exposures to adverse maternal nutrition or health status. Although cells manifest specific responses to various stressors, many of these stressors intersect at the endoplasmic reticulum, where disruptions in protein folding and production of reactive oxygen species initiate downstream signaling events. These signals modulate mRNA translation and gene transcription, leading to recovery, activation of autophagy, or with severe and prolonged stress, apoptosis. ER stress signaling has recently come to the fore as a major contributor to embryo demise. Accordingly, agents that modulate or inhibit ER stress signaling have yielded beneficial effects on embryo survival and long-term developmental potential. We review here the mechanisms of ER stress signaling, their connections to mammalian oocytes and embryos, and the promising indications that interventions in this pathway may provide new opportunities for improving mammalian reproduction and health. PMID:25805126

Lessons from Embryos: Haeckel's Embryo Drawings, Evolution, and Secondary Biology Textbooks

ERIC Educational Resources Information Center

Wellner, Karen L.

2014-01-01

In 1997, developmental biologist Michael Richardson compared his research team's embryo photographs to Ernst Haeckel's 1874 embryo drawings and called Haeckel's work "noncredible". "Science" soon published "Haeckel's Embryos: Fraud Rediscovered," and Richardson's comments further reinvigorated criticism of Haeckel by…

Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.

PubMed

Galli, C; Colleoni, S; Duchi, R; Lagutina, I; Lazzari, G

2007-03-01

Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18-36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.

Homotopic connectivity in drug-naïve, first-episode, early-onset schizophrenia

PubMed Central

Li, Hui-Jie; Xu, Yong; Zhang, Ke-Rang; Hoptman, Matthew J.; Zuo, Xi-Nian

2014-01-01

Background The disconnection hypothesis of schizophrenia has been extensively tested in adults. Recent studies have reported the presence of brain disconnection in younger patients, adding evidence to support the neurodevelopmental hypothesis of schizophrenia. Because of drug confounds in chronic and medicated patients, it has been extremely challenging for researchers to directly investigate abnormalities in the development of connectivity and their role in the pathophysiology of schizophrenia. The present study aimed to examine functional homotopy – a measure of interhemispheric connection – and its relevance to clinical symptoms in first-episode drug-naïve early-onset schizophrenia (EOS) patients. Methods Resting-state functional magnetic resonance imaging was performed in 26 first-episode drug-naïve EOS patients (age: 14.5 ± 1.94, 13 males) and 25 matched typically developing controls (TDCs) (age: 14.4 ± 2.97, 13 males). We were mainly concerned with the functional connectivity between any pair of symmetric inter-hemispheric voxels (i.e., functional homotopy) measured by voxel-mirrored homotopic connectivity (VMHC). Results EOS patients exhibited both global and regional VMHC reductions in comparison with TDCs. Reduced VMHC values were observed within the superior temporal cortex and postcentral gyrus. These interhemispheric synchronization deficits were negatively correlated with negative symptom of the Positive and Negative Syndrome Scale. Moreover, regions of interest analyses based on left and right clusters of temporal cortex and postcentral gyrus revealed abnormal heterotopic connectivity in EOS patients. Conclusions Our findings provide novel neurodevelopmental evidence for the disconnection hypothesis of schizophrenia and suggest that these alterations occur early in the course of the disease and are independent of medication status. PMID:25130214

Uncovering Hegelian Connections: A New Look at Dewey's Early Educational Ideas

ERIC Educational Resources Information Center

Waddington, David I.

2010-01-01

This paper is dedicated to the investigation of an important, but not particularly well known, connection between the work of Hegel and Dewey's early educational ideas. A brief exposition of Hegel's position in the "Philosophy of Right" is offered, with a particular focus on Hegel's idea of absolute freedom. This exposition is followed by an…

Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

PubMed

Hodar, Christian; Zuñiga, Alejandro; Pulgar, Rodrigo; Travisany, Dante; Chacon, Carlos; Pino, Michael; Maass, Alejandro; Cambiazo, Verónica

2014-02-10

In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-β superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica. Copyright © 2013 Elsevier B.V. All rights reserved.

Can Chlamydia abortus be transmitted by embryo transfer in goats?

PubMed

Oseikria, M; Pellerin, J L; Rodolakis, A; Vorimore, F; Laroucau, K; Bruyas, J F; Roux, C; Michaud, S; Larrat, M; Fieni, F

2016-10-01

The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo-derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8-16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 × 10(7)Chlamydia/mL of AB7 strain. After incubation for 18 hours at 37 °C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at -20 °C before examination for evidence of C. abortus using polymerase chain reaction. C. abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from

Endometrial preparation for women undergoing embryo transfer with frozen embryos or embryos derived from donor oocytes.

PubMed

Glujovsky, Demián; Pesce, Romina; Fiszbajn, Gabriel; Sueldo, Carlos; Hart, Roger J; Ciapponi, Agustín

2010-01-20

If a fresh embryo, assisted reproductive technology procedure cycle is unsuccessful and there are frozen embryos available, a frozen-thawed embryo transfer is performed. In some specific cases women may undergo oocyte donation treatment. In both situations the endometrium is primed by the administration of estrogen and progesterone. To prevent the possibility of spontaneous ovulation, gonadotropin-releasing hormone (GnRH) agonists are frequently used. To evaluate the most effective endometrial preparation for women undergoing transfer with frozen embryos or embryos from donor oocytes with regard to the subsequent live birth rate. We searched the Cochrane Menstrual Disorders and Subfertility Group Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library), MEDLINE, EMBASE, LILACS, and abstracts of reproductive societies' meetings (from inception). No language restrictions were applied. Experts in the field were contacted. Randomised controlled trials evaluating endometrial preparation in women undergoing fresh donor cycles and frozen embryo transfers. Two review authors independently applied the inclusion criteria, assessed trial risk of bias, and extracted data. Twenty two randomised controlled trials were included. Five studies analysed the use of a GnRH agonist versus control. No significant benefit was demonstrated when using GnRH agonists. No evidence of statistically significant benefit was found for one GnRH agonist over another, or vaginal over intramuscular progesterone administration. No difference in pregnancy rate was demonstrated when no treatment was compared to aspirin, steroids, ovarian stimulation, or human chorionic gonadotropin (hCG) prior to embryo transfer, although using hCG several times before the oocyte retrieval decreases the pregnancy rate. Finally, when oocyte recipients were studied further, starting progesterone on the day of oocyte pick-up (OPU) or the day after OPU produced a significantly higher

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.

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

Heterogeneous nuclear ribonucleoprotein C1 may control miR-30d levels in endometrial exosomes affecting early embryo implantation.

PubMed

Balaguer, N; Moreno, I; Herrero, M; González, M; Simón, C; Vilella, F

2018-05-29

as a model of hEECs in silencing experiments due to the low survival rates of primary hEECs after transfection. The data show that hnRNPC1 may be involved in the internalization of miR-30d inside Exosomes. The decreased rates of embryo adhesion in endometrial epithelial-like cells transiently silenced with sihnRNPC1evidence that hnRNPC1 could be an important player in the maternal-embryo communication established in the early stages of implantation. This work was supported by the Miguel Servet Program Type I of Instituto de Salud Carlos III [CP13/00038]; FIS project [PI14/00545] to FV; the 'Atracció de Talent' Program from VLC-CAMPUS [UV-INV-PREDOC14-178329 to NB]; a Torres-Quevedo grant (PTQ-13-06133) by the Spanish Ministry of Economy and Competitiveness to IM; and MINECO/FEDER Grant [SAF2015-67154-R] to CS. The authors declare there is no conflict of interest.

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

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.

Embryo deaths in reproduction and embryo research: a reply to Murphy's double effect argument.

PubMed

Devolder, Katrien

2013-08-01

The majority of embryos created in natural reproduction die spontaneously within a few weeks of conception. Some have argued that, therefore, if one believes the embryo is a person (in the normative sense) one should find 'natural' reproduction morally problematic. An extension of this argument holds that, if one accepts embryo deaths in natural reproduction, consistency requires that one also accepts embryo deaths that occur in (i) assisted reproduction via in vitro fertilisation (IVF) and (ii) embryo research. In a recent paper in this journal, Timothy Murphy criticises both the initial argument and its extension. Murphy argues that double-effect reasoning can justify embryo deaths both in natural reproduction and IVF, but not in embryo research. Thus, according to Murphy, one can, without being inconsistent, (1) believe the embryo is a person and accept natural reproduction and IVF, and (2) accept natural reproduction and IVF, while rejecting embryo research on the ground that it involves embryo deaths. I show that Murphy's argument is problematic because double effect cannot justify embryo deaths in standard IVF practices. The problem is that the proportionality criterion of double effect is not met by such practices. Thus, Murphy's argument fails to support (1) and (2). An implication of his argument failing to support (2) is that it does not defeat the position I have defended in the past-that if one accepts standard IVF practices one should also accept embryo research, including research with embryos created solely for that purpose.

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.

Applying embryo cryopreservation technologies to the production of domestic and black-footed cats.

PubMed

Pope, C E; Gómez, M C; Galiguis, J; Dresser, B l

2012-12-01

Our objectives were (i) compare in vitro development of early cleavage stage domestic cat embryos after cryopreservation by minimal volume vitrification vs a standard slow, controlled-rate method, (ii) determine viability of vitrified domestic cat embryos by oviductal transfer into synchronous recipients and (iii) evaluate in vivo survival of black-footed cat (BFC, Felis nigripes) embryos after intra- and inter-species transfer. In vitro-derived (IVM/IVF) cat embryos were used to evaluate in vitro development after controlled-rate cryopreservation vs vitrification vs controls. Blastocyst development was similar in both groups of cryopreserved embryos (22-26%), but it was lower (p < 0.05) than that of fresh embryos (50%). After embryo transfer, four of eight recipients of vitrified embryos established pregnancies--three of six (50%) and one of two (50%) that received embryos from in vivo- and in vitro-matured oocytes, respectively. Three male and two female kittens weighing from 51 to 124 g (mean = 88 g) were delivered on days 61-65 of gestation. In BFC, four intra-species embryo transfer procedures were carried out--two recipients received fresh day 2 embryos (n = 5, 8) and two recipients received embryos that had been cryopreserved on day 1 (n = 6) or 2 (n = 8). A 2-year-old recipient of cryopreserved embryos established pregnancy and delivered two live male kittens. Subsequently, five cryopreserved BFC embryos were transferred to a domestic cat recipient. On day 29, the recipient was determined to be pregnant and delivered naturally a live, healthy female BFC kitten on day 66. In summary, in vivo survival of vitrified domestic cat embryos was shown by the births of kittens after transfer into recipients. Also, we demonstrated that sperm and embryo cryopreservation could be combined with intra- and inter-species embryo transfer and integrated into the array of assisted reproductive techniques used successfully for propagation of a rare and vulnerable felid species

Macroevolutionary developmental biology: Embryos, fossils, and phylogenies.

PubMed

Organ, Chris L; Cooper, Lisa Noelle; Hieronymus, Tobin L

2015-10-01

The field of evolutionary developmental biology is broadly focused on identifying the genetic and developmental mechanisms underlying morphological diversity. Connecting the genotype with the phenotype means that evo-devo research often considers a wide range of evidence, from genetics and morphology to fossils. In this commentary, we provide an overview and framework for integrating fossil ontogenetic data with developmental data using phylogenetic comparative methods to test macroevolutionary hypotheses. We survey the vertebrate fossil record of preserved embryos and discuss how phylogenetic comparative methods can integrate data from developmental genetics and paleontology. Fossil embryos provide limited, yet critical, developmental data from deep time. They help constrain when developmental innovations first appeared during the history of life and also reveal the order in which related morphologies evolved. Phylogenetic comparative methods provide a powerful statistical approach that allows evo-devo researchers to infer the presence of nonpreserved developmental traits in fossil species and to detect discordant evolutionary patterns and processes across levels of biological organization. © 2015 Wiley Periodicals, Inc.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

PubMed

Muñoz, M; Martin, D; Carrocera, S; Alonso-Guervos, M; Mora, M I; Corrales, F J; Peynot, N; Giraud-Delville, C; Duranthon, V; Sandra, O; Gómez, E

2017-10-01

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Carbonic anhydrase activity in developing sea urchin embryos with special reference to calcification of spicules.

PubMed

Mitsunaga, K; Akasaka, K; Shimada, H; Fujino, Y; Yasumasu, I; Numanoi, H

1986-06-01

Eggs and embryos of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus did not exhibit significant changes in carbonic anhydrase activity during early development. Acetazolamide inhibited enzyme activity in homogenates of embryos and inhibited the formation of calcified spicules in a culture of micromeres at concentrations between 40 and 100 microM. Acetazolamide allowed intact embryos to develop to quasi-normal plutei but inhibited calcium deposition in the spicules. It is suggested that carbonic anhydrase contributes to CaCO3 deposition in the spicule.

Identification of low Ca(2+) stress-induced embryo apoptosis response genes in Arachis hypogaea by SSH-associated library lift (SSHaLL).

PubMed

Chen, Hua; Zhang, Chong; Cai, Tie Cheng; Deng, Ye; Zhou, Shuangbiao; Zheng, Yixiong; Ma, Shiwei; Tang, Ronghua; Varshney, Rajeev K; Zhuang, Weijian

2016-02-01

Calcium is a universal signal in the regulation of wide aspects in biology, but few are known about the function of calcium in the control of early embryo development. Ca(2+) deficiency in soil induces early embryo abortion in peanut, producing empty pods, which is a general problem; however, the underlying mechanism remains unclear. In this study, embryo abortion was characterized to be caused by apoptosis marked with cell wall degradation. Using a method of SSH cDNA libraries associated with library lift (SSHaLL), 62 differentially expressed genes were isolated from young peanut embryos. These genes were classified to be stress responses, catabolic process, carbohydrate and lipid metabolism, embryo morphogenesis, regulation, etc. The cell retardation with cell wall degradation was caused by up-regulated cell wall hydrolases and down-regulated cellular synthases genes. HsfA4a, which was characterized to be important to embryo development, was significantly down-regulated under Ca(2+) -deficient conditions from 15 days after pegging (DAP) to 30 DAP. Two AhCYP707A4 genes, encoding abscisic acid (ABA) 8'-hydroxylases, key enzymes for ABA catabolism, were up-regulated by 21-fold under Ca(2+) -deficient conditions upstream of HsfA4a, reducing the ABA level in early embryos. Over-expression of AhCYP707A4 in Nicotiana benthamiana showed a phenotype of low ABA content with high numbers of aborted embryos, small pods and less seeds, which confirms that AhCYP707A4 is a key player in regulation of Ca(2+) deficiency-induced embryo abortion via ABA-mediated apoptosis. The results elucidated the mechanism of low Ca(2+) -induced embryo abortion and described the method for other fields of study. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

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

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 �

Immunocytochemistry of the amphibian embryo--from overview to ultrastructure.

PubMed

Kurth, Thomas

2003-06-01

Amphibian embryos are standard research objects to study pattern formation and morphogenesis. Due to their external development and robust nature, experimental manipulations such as microinjections or transplantations can be easily performed. However, most immunocytochemical approaches addressing the specific localization of proteins are hampered by the fragility of the large and yolky embryonic cells which render high resolution staining difficult. Immunocytochemical data are therefore often restricted to either overall patterns in whole embryo preparations or to immunofluorescent localization with limited resolution on sections. High resolution or ultrastructural protein localization data are rare and can be achieved only with time consuming procedures. Here, a comparative study of immunocytochemical methods suitable for light and electron microscopy using different kinds of plastic resins is presented. Three main approaches are described: preembedding staining of whole embryos, postembedding staining of ultrathin sections and preembedding staining of vibratome sections. All the procedures are designed to study protein expression in early amphibian embryos en gros as well as en detail and the described techniques are suitable to combine two or three levels of resolution on the very same biological specimen. Examples are presented and advantages and disadvantages of the different protocols are discussed.

Assessment of aneuploidy in human oocytes and preimplantation embryos by chromosome painting

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

Rougier, N.; Viegas-Pequignot, E.; Plachot, M.

1994-09-01

The poor quality of chromosome preparations often observed after fixation of oocytes and embryos did not usually allow accurate identification of chromosomes involved in non-disjunctions. We, therefore, used chromosome painting to determine the incidence of abnormalities for chromosomes 1 and 7. A total of 50 oocytes inseminated for IVF and showing no signs of fertilization as well as 37 diploid embryos donated for research were fixed according to the Dyban`s technique. Fluorescence in situ hybridization was carried out using whole chromosome painting DNA probes specific for human chromosome 1 and 7. The incidence of aneuploidy was 28%, 10% and 60%more » for metaphase II, polar body and sperm chromosomes, respectively. The high incidence of aneuploidy observed in sperm prematurely condensed sperm chromosomes is due to the fact that usually far less than 23 sperm chromatids are observed, maybe as a consequence of incomplete chromosome condensation. Thirty seven embryos were analyzed with the same probes. 48% of early embryos were either monosomic 1 or 7 or mosaics comprising blastomeres with 1, 2 or 3 signals. Thus, 8 among the 11 abnormal embryos had hypodiploid cells (25 to 37 chromosomes) indicating either an artefactual loss of chromosomes or a complex anomaly of nuclear division (maltinucleated blastomeres, abnormal migration of chromosomes at anaphase). We therefore calculated a {open_quotes}corrected{close_quotes} incidence of aneuploidy for chromosomes 1 or 7 in early embryos: 18%. 86% of the blastocysts showed mosaicism 2n/3 or 4n as a consequence of the formation of the syncitiotrophoblast. To conclude, chromosome painting is an efficient method to accurately identify chromosomes involved in aneuploidy. This technique should allow us to evaluate the incidence of non-disjunction for all chromosome pairs. Our results confirm the high incidence of chromosome abnormalities occurring as a consequence of meiotic or mitotic non-disjunctions in human oocytes and

Survey of 243 ART patients having made a final disposition decision about their surplus cryopreserved embryos: the crucial role of symbolic embryo representation.

PubMed

Bruno, C; Dudkiewicz-Sibony, C; Berthaut, I; Weil, E; Brunet, L; Fortier, C; Pfeffer, J; Ravel, C; Fauque, P; Mathieu, E; Antoine, J M; Kotti, S; Mandelbaum, J

2016-07-01

with a psychologist. Interviews were conducted separately for each partner, including a questionnaire with a common part and a specific part, according to the chosen option, and allowing a quantitative evaluation. A multivariate logistic regression model was used to assess the link between their embryo representation and their decision about their embryos' fate. After adjustment for age, gender, gamete donation, number of children and the different embryo representations, a choice to 'stop cryopreservation' is more frequent if the embryo is represented as a child [odds ratio (OR) adjusted = 3.29, 95% confidence interval (CI) = 1.62-6.66], P = 0.0009. Representing the embryo as a project prompts patients to choose 'donation to research' [OR adjusted = 3.76, 95% CI = 1.56-9.06], P = 0.0032. Respondents are more likely to choose 'embryo donation' if they represent the embryo as a potential person [OR adjusted = 3.77, 95% CI = 1.45-9.80], P = 0.0064. Furthermore, patients who benefited from gamete donation are ∼10 times more likely to donate their embryos to another couple [OR adjusted = 10.62, 95% CI = 3.99-28.30], P < 0.0001. For more than half the participants (57%) the decision-making was easy, however, deciding to stop cryopreservation was significantly more difficult than choosing research or embryo donation (P < 0.0001). Socio-economic status, moral and religious affiliations are known to influence the choice of couples but analyzing these factors was not an aim of the present study. When couples definitely decide to stop their parental project, the embryo symbolic representation remains the main factor that influences the fate of their frozen embryo(s). Moreover, this representation can evolve when influenced by external events and information provided. In order to support patients who are making this difficult decision, it could be helpful to explore this symbolic representation early in the IVF/ICSI procedure, before surplus embryo freezing, as a new tool

Changes in the antioxidant metabolism in the embryonic development of the common South American toad Bufo arenarum: differential responses to pesticide in early embryos and autonomous-feeding larvae.

PubMed

Ferrari, Ana; Anguiano, Liliana; Lascano, Cecilia; Sotomayor, Verónica; Rosenbaum, Enrique; Venturino, Andrés

2008-01-01

Amphibians may be critically challenged by aquatic contaminants during their embryonic development. Many classes of compounds, including organophosphorus pesticides, are able to cause oxidative stress that affects the delicate cellular redox balance regulating tissue modeling. We determined the progression of antioxidant defenses during the embryonic development of the South American common toad, Bufo arenarum. Superoxide dismutase (SOD) and catalase (CAT) activities were high in the unfertilized eggs, and remained constant during the first stages of development. SOD showed a significant increase when the gills were completely active and opercular folds began to form. Reductase (GR) activity was low in the oocytes and increased significantly when gills and mouth were entirely developed and the embryos presented a higher exposure to pro-oxidant conditions suggesting an environmental control. Reduced glutathione (GSH) content was also initially low, and rose continuously pointing out an increasing participation of GSH-related enzymes in the control of oxidative stress. GSH peroxidases and GSH-S-transferases showed relatively high and constant activities, probably related to lipid peroxide control. B. arenarum embryos have plenty of yolk platelets containing lipids, which provide the energy and are actively transferred to the newly synthesized membranes during the early embryonic development. Exposure to the pro-oxidant pesticide malathion during 48 h did not significantly affect the activity of antioxidant enzymes in early embryos, but decreased the activities of CAT, GR, and the pool of GSH in larvae. Previous work indicated that lipid peroxide levels were kept low in malathion-exposed larvae, thus we conclude that oxidative stress is overcome by the antioxidant defenses. The increase in the antioxidant metabolism observed in the posthatching phase of development of B. arenarum embryo, thus constitutes a defense against natural and human-generated pro

Remodelling of the bovine placenta: Comprehensive morphological and histomorphological characterization at the late embryonic and early accelerated fetal growth stages.

PubMed

Estrella, Consuelo Amor S; Kind, Karen L; Derks, Anna; Xiang, Ruidong; Faulkner, Nicole; Mohrdick, Melina; Fitzsimmons, Carolyn; Kruk, Zbigniew; Grutzner, Frank; Roberts, Claire T; Hiendleder, Stefan

2017-07-01

Placental function impacts growth and development with lifelong consequences for performance and health. We provide novel insights into placental development in bovine, an important agricultural species and biomedical model. Concepti with defined genetics and sex were recovered from nulliparous dams managed under standardized conditions to study placental gross morphological and histomorphological parameters at the late embryo (Day48) and early accelerated fetal growth (Day153) stages. Placentome number increased 3-fold between Day48 and Day153. Placental barrier thickness was thinner, and volume of placental components, and surface areas and densities were higher at Day153 than Day48. We confirmed two placentome types, flat and convex. At Day48, there were more convex than flat placentomes, and convex placentomes had a lower proportion of maternal connective tissue (P < 0.01). However, this was reversed at Day153, where convex placentomes were lower in number and had greater volume of placental components (P < 0.01- P < 0.001) and greater surface area (P < 0.001) than flat placentomes. Importantly, embryo (r = 0.50) and fetal (r = 0.30) weight correlated with total number of convex but not flat placentomes. Extensive remodelling of the placenta increases capacity for nutrient exchange to support rapidly increasing embryo-fetal weight from Day48 to Day153. The cellular composition of convex placentomes, and exclusive relationships between convex placentome number and embryo-fetal weight, provide strong evidence for these placentomes as drivers of prenatal growth. The difference in proportion of maternal connective tissue between placentome types at Day48 suggests that this tissue plays a role in determining placentome shape, further highlighting the importance of early placental development. Copyright © 2017 Elsevier Ltd. All rights reserved.

The effects of cobalt on the development, oxidative stress, and apoptosis in zebrafish embryos.

PubMed

Cai, Guiquan; Zhu, Junfeng; Shen, Chao; Cui, Yimin; Du, Jiulin; Chen, Xiaodong

2012-12-01

Metal-on-metal hip arthroplasty has been performed with increasing frequency throughout the world, particularly in younger and more active patients, including women of childbearing age. The potential toxicity of cobalt exposure on fetus is concerned since cobalt ions generated by metal-on-metal bearings can traverse the placenta and be detected in fetal blood and amniotic fluid. This study examined the effects of cobalt exposure on early embryonic development and the mechanisms underlying its toxicity. Zebrafish embryos were exposed to a range of cobalt concentrations (0-100 mg/L) between 1 and 144 h postfertilization. The survival and early development of embryos were not significantly affected by cobalt at concentrations <100 μg/L. However, embryos exposed to higher concentrations (>100 μg/L) displayed reduced survival rates and abnormal development, including delayed hatching, aberrant morphology, retarded growth, and bradycardia. Furthermore, this study examined oxidative stress and apoptosis in embryos exposed to cobalt at concentrations of 0-500 μg/L. Lipid peroxidation levels were increased in cobalt-treated embryos at concentrations of 100 and 500 μg/L. The mRNA levels of catalase, superoxide dismutase 2, p53, caspase-3, and caspase-9 genes were upregulated in a dose-dependent manner. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays also revealed abnormal apoptotic signals in the brain, trunk, and tail when treated with 500 μg/L cobalt. These data suggest that oxidative stress and apoptosis are associated with cobalt toxicity in zebrafish embryos.

Chromatin Immunoprecipitation in Early Mouse Embryos.

PubMed

García-González, Estela G; Roque-Ramirez, Bladimir; Palma-Flores, Carlos; Hernández-Hernández, J Manuel

2018-01-01

Epigenetic regulation is achieved at many levels by different factors such as tissue-specific transcription factors, members of the basal transcriptional apparatus, chromatin-binding proteins, and noncoding RNAs. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method that allows elucidating gene regulation at the molecular level by assessing if chromatin modifications or proteins are present at a specific locus. Initially, the majority of ChIP experiments were performed on cultured cell lines and more recently this technique has been adapted to a variety of tissues in different model organisms. Using ChIP on mouse embryos, it is possible to document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development and to get biological meaning from observations made on tissue culture analyses. We describe here a ChIP protocol on freshly isolated mouse embryonic somites for in vivo analysis of muscle specific transcription factor binding on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.

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.

Effect of embryo source and recipient progesterone environment on embryo development in cattle.

PubMed

Lonergan, P; Woods, A; Fair, T; Carter, F; Rizos, D; Ward, F; Quinn, K; Evans, A

2007-01-01

The aim of the present study was to examine the effect of embryo source (in vivo v. in vitro) and the progesterone environment into which it was transferred on Day 7 on embryo survival and size on Day 13. Day 7 blastocysts were produced either in vivo using superovulation, artificial insemination and non-surgical embryo recovery or in vitro using in vitro maturation, fertilisation and culture. In order to produce animals with divergent progesterone concentrations, following synchronisation recipients were either superovulated (High progesterone; n = 10) or not (Control progesterone; n = 10). Ten blastocysts, produced either in vivo or in vitro, were transferred to each recipient on Day 7. Both groups were killed on Day 13. The mean progesterone concentration from Day 7 to Day 13 (the period when the embryos were in the uterus) in the High and Control progesterone recipients was 36.32 +/- 1.28 and 10.30 +/- 0.51 ng mL(-1), respectively. Of the in vivo embryos transferred, the overall recovery rate at Day 13 was 64%, which was higher (P < 0.001) than that of 20% for the in vitro embryos transferred. The mean area of embryos recovered from High progesterone recipients was 3.86 +/- 0.45 mm(2) (n = 28) compared with 1.66 +/- 0.38 mm(2) (n = 24) for embryos recovered from Control progesterone recipients (P < 0.001). Similarly, the origin of the embryo used for transfer affected embryo size on Day 13. In summary, the recovery rate of blastocysts was higher for in vivo- than in vitro-derived embryos. Blastocyst size was approximately 2.3-fold greater in recipients with high compared with normal progesterone. The present study lends strong support to the hypothesis that an earlier rise in progesterone after conception stimulates blastocyst growth and the development of competent embryos.

Potential of human twin embryos generated by embryo splitting in assisted reproduction and research.

PubMed

Noli, Laila; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko

2017-03-01

Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'. Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research. A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos. A very limited number of studies have been conducted in humans and non

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

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.

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.

Tissue architecture, cell traction, deformable scaffolds, and the forces that shape the embryo during morphogenesis.

NASA Astrophysics Data System (ADS)

Davidson, Lance

2005-03-01

Morphogenesis is the process of constucting form and shape. Morphogenesis during early development of the embryo involves orchestrated movements of cells and tissues. These morphogenetic movements establish the body plan and organs of the early embryo. The rates and trajectories of these movements depend on three physical features of the early embryo: 1) the forces generated by cells, 2) the mechanical properties of the tissues, and 3) the architecture of the tissues. These three mechanical features of the embryo are some of the earliest phenotypic features generated by the genome. We are taking an interdisciplinary approach combining biophysical, cell biological, and classical embryological techniques to understand the mechanics of morphogenesis. Using nanoNewton-sensitive force transducers we can apply forces and measure time dependent elastic modulii of tissue fragments 100 micrometers across. Using traction-force microscopy we can measure forces generated by cells on their environment. We use drugs and chimeric proteins to investigate the localization and function of molecular complexes responsible for force generation and the modulus. We use microsurgery to take-apart and construct novel tissues to investigate the role of geometry and architecture in the mechanics of morphogenesis. Together with simulation techniques these quantitative approaches will provide us with a practical nuts-and-bolts understanding of how the genome encodes the shapes and forms of life.

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR.

PubMed

Blanes, Milena S; Tsoi, Stephen C M; Dyck, Michael K

2016-02-14

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo's sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

PubMed Central

Dyck, Michael K.

2016-01-01

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo's sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing. PMID:26966900

Transition of cell numbers in bovine preimplantation embryos: in vivo collected and in vitro produced embryos.

PubMed

Ushijima, Hitoshi; Akiyama, Kiyoshi; Tajima, Toshio

2008-08-01

The total cell numbers (TCNs) of bovine embryos collected from superovulated donors (VIVO embryos) were counted 0-9 d after ovulation to quantify the developmental process. Using numerical analysis of embryo development, we also compared the developmental process of VIVO embryos, in vitro-fertilized (IVF) embryos and nuclear transfer (NT) embryos obtained from enucleated oocytes and blastomere nuclei. The TCNs of embryos were measured using the air-dry method. Cleavage divisions (CD) of the embryos were obtained using logarithmic transformation of the TCN. The TCN of the VIVO embryos increased significantly (P<0.001) with time. The relationship between the CD of the VIVO embryos at 0-9 d after ovulation and age in days was described by a linear equation with a high correlation (y=1.03x+0.16, r=0.99), showing that CD occurs about once each day for all blastomeres. However, compared to the VIVO embryos, the TCN of the IVF embryos did not increase from 3-4 d nor after 7 d; the TCN of the NT embryos did not increase after 7 d (P>0.05). The results suggest a delay in development at these developmental stages. The slopes of regression lines of the IVF and NT embryos were significantly (P<0.001) smaller, indicating that quantification of the developmental process of VIVO embryos according to TCN and CD would be useful as criteria for numerical evaluation of the developmental process of bovine in vitro produced embryos.

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.

Reduction of Interhemispheric Functional Brain Connectivity in Early Blindness: A Resting-State fMRI Study

PubMed Central

2017-01-01

Objective The purpose of this study was to investigate the resting-state interhemispheric functional connectivity in early blindness by using voxel-mirrored homotopic connectivity (VMHC). Materials and Methods Sixteen early blind patients (EB group) and sixteen age- and gender-matched sighted control volunteers (SC group) were recruited in this study. We used VMHC to identify brain areas with significant differences in functional connectivity between different groups and used voxel-based morphometry (VBM) to calculate the individual gray matter volume (GMV). Results VMHC analysis showed a significantly lower connectivity in primary visual cortex, visual association cortex, and somatosensory association cortex in EB group compared to sighted controls. Additionally, VBM analysis revealed that GMV was reduced in the left lateral calcarine cortices in EB group compared to sighted controls, while it was increased in the left lateral middle occipital gyri. Statistical analysis showed the duration of blindness negatively correlated with VMHC in the bilateral middle frontal gyri, middle temporal gyri, and inferior temporal gyri. Conclusions Our findings help elucidate the pathophysiological mechanisms of EB. The interhemispheric functional connectivity was impaired in EB patients. Additionally, the middle frontal gyri, middle temporal gyri, and inferior temporal gyri may be potential target regions for rehabilitation. PMID:28656145

Genetic selection of embryos that later develop the metabolic syndrome.

PubMed

Edwards, M J

2012-05-01

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

Isl-1 down-regulates DRG cell proliferation during chicken embryo development.

PubMed

Chen, Dawei; Wang, Guoxin; Luo, Haoshu; Liu, Jiali; Cui, Sheng

2010-01-01

Protein Isl-1 RNA interference and over expression in early chicken embryo dorsal root ganglia (DRG) were used to investigate the function of Isl-1 in DRG cell proliferation. Isl-1 targeted shRNA expression vector and Isl-1 over-expression vector were transfected into chicken embryo DRG by in ovo electroporation. Then, the DRG proliferation rate was detected by BrdU immunohistochemistry. The rate of DRG cell proliferation increased after Isl-1 knock-down and decreased after Isl-1 over-expression. In this study, we found that Isl-1 negatively modulates DRG cell proliferation.

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

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

PubMed

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

2016-09-01

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

Embryo density may affect embryo quality during in vitro culture in a microwell group culture dish.

PubMed

Lehner, Adam; Kaszas, Zita; Murber, Akos; Rigo, Janos; Urbancsek, Janos; Fancsovits, Peter

2017-08-01

Culturing embryos in groups is a common practice in mammalian embryology. Since the introduction of different microwell dishes, it is possible to identify oocytes or embryos individually. As embryo density (embryo-to-volume ratio) may affect the development and viability of the embryos, the purpose of this study was to assess the effect of different embryo densities on embryo quality. Data of 1337 embryos from 228 in vitro fertilization treatment cycles were retrospectively analyzed. Embryos were cultured in a 25 μl microdrop in a microwell group culture dish containing 9 microwells. Three density groups were defined: Group 1 with 2-4 (6.3-12.5 μl/embryo), Group 2 with 5-6 (4.2-5.0 μl/embryo), and Group 3 with 7-9 (2.8-3.6 μl/embryo) embryos. Proportion of good quality embryos was higher in Group 2 on both days (D2: 18.9 vs. 31.5 vs. 24.7%; p < 0.001; D3: 19.7 vs. 27.1 vs. 21.2%; p = 0.029; Group 1. vs. Group 2. vs. Group 3). Cell number on Day 3 differed between Groups 1 and 2 (6.8 ± 2.2; 7.3 ± 2.1; p = 0.004) and Groups 2 and 3 (7.3 ± 2.1 vs. 7.0 ± 2.0; p = 0.014). Culturing 5-6 embryos together in a culture volume of 25 μl may benefit embryo quality. As low egg number, position, and distance of the embryos may influence embryo quality, results should be interpreted with caution.

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

Adaptive capacity of the habitat modifying sea urchin Centrostephanus rodgersii to ocean warming and ocean acidification: performance of early embryos.

PubMed

Foo, Shawna A; Dworjanyn, Symon A; Poore, Alistair G B; Byrne, Maria

2012-01-01

Predicting effects of rapid climate change on populations depends on measuring the effects of climate stressors on performance, and potential for adaptation. Adaptation to stressful climatic conditions requires heritable genetic variance for stress tolerance present in populations. We quantified genetic variation in tolerance of early development of the ecologically important sea urchin Centrostephanus rodgersii to near-future (2100) ocean conditions projected for the southeast Australian global change hot spot. Multiple dam-sire crosses were used to quantify the interactive effects of warming (+2-4 °C) and acidification (-0.3-0.5 pH units) across twenty-seven family lines. Acidification, but not temperature, decreased the percentage of cleavage stage embryos. In contrast, temperature, but not acidification decreased the percentage of gastrulation. Cleavage success in response to both stressors was strongly affected by sire identity. Sire and dam identity significantly affected gastrulation and both interacted with temperature to determine developmental success. Positive genetic correlations for gastrulation indicated that genotypes that did well at lower pH also did well in higher temperatures. Significant genotype (sire) by environment interactions for both stressors at gastrulation indicated the presence of heritable variation in thermal tolerance and the ability of embryos to respond to changing environments. The significant influence of dam may be due to maternal provisioning (maternal genotype or environment) and/or offspring genotype. It appears that early development in this ecologically important sea urchin is not constrained in adapting to the multiple stressors of ocean warming and acidification. The presence of tolerant genotypes indicates the potential to adapt to concurrent warming and acidification, contributing to the resilience of C. rodgersii in a changing ocean.

Human research cloning, embryos, and embryo-like artifacts.

PubMed

Hyun, Insoo; Jung, Kyu Won

2006-01-01

Research suggests that cloning is incapable of producing a viable embryo when it is used on primate eggs. In fact, the entity created may not qualify as an embryo at all. If the results stand, cloning avoids the moral objections typically lodged against it, and cloning is itself an "alternative source" of stem cells.

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.

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

PubMed

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

2016-09-01

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

Single-embryo transfer versus multiple-embryo transfer.

PubMed

Gerris, Jan

2009-01-01

Despite the progress made in assisted reproductive technology, live birth rates remain disappointingly low. Multiple-embryo transfer has been an accepted practice with which to increase the success rate. This has led to a higher incidence of multiple-order births compared with natural conception, which not only increase the risk of mortality and morbidity to both mother and children but are also associated with social and economic consequences. Elective single-embryo transfer (eSET) was developed in an effort to increase singleton pregnancies in assisted reproduction. Studies comparing eSET with multiple-embryo transfer highlight the benefit of this approach and suggest that, with careful patient selection and the transfer of good-quality embryos, the risk of a multiple-order pregnancy can be reduced without significantly decreasing live birth rates. Although the use of eSET has gradually increased in clinical practice, its acceptance has been limited by factors such as availability of funding and awareness of the procedure. An open discussion of eSET is warranted in an effort to enable a broader understanding by physicians and patients of the merits of this approach. Ultimately, eSET may provide a more cost-effective, potentially safer approach to patients undergoing assisted reproduction technology.

Contrast imaging in mouse embryos using high-frequency ultrasound.

PubMed

Denbeigh, Janet M; Nixon, Brian A; Puri, Mira C; Foster, F Stuart

2015-03-04

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αvβ3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Health and Safety Advisory Committee (HASAC) of the International Embryo Transfer Society (IETS) has managed critical challenges for two decades.

PubMed

Thibier, M; Stringfellow, D A

2003-02-01

The International Embryo Transfer Society (IETS) was founded in 1974. Early members used the society as a forum for the exchange of scientific and technical information relevant to a newly emerging embryo transfer industry. The impact that embryo transfer could have on the international trade of livestock genetics was clear by 1982, so the IETS commissioned the Import/Export Committee. The initial challenge for this Committee was to deal with concerns about disease transmission via embryo transfer. Many of the early concerns have been dispelled, but at the time they threatened the continued development of a fledgling industry. Over the past two decades, many new critical challenges have been met and managed by this Committee, which was recently renamed the Health and Safety Advisory Committee (HASAC). Assessing risks of animal disease transmission via reproductive technologies and establishing protocols for managing these risks are still major issues for HASAC. However, additional concerns have developed as views of the society changed and as novel applications of biotechnology in farm animals were identified. This paper is intended to chronicle some of the major changes and challenges that were managed by members of the HASAC and its Subcommittees from the early years of embryo transfer to the current millennium with technological advances in molecular biology.

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

Remodeling of the Connective Tissue Microarchitecture of the Lamina Cribrosa in Early Experimental Glaucoma

PubMed Central

Roberts, Michael D.; Grau, Vicente; Grimm, Jonathan; Reynaud, Juan; Bellezza, Anthony J.; Burgoyne, Claude F.; Downs, J. Crawford

2009-01-01

Purpose To characterize the trabeculated connective tissue microarchitecture of the lamina cribrosa (LC) in terms of total connective tissue volume (CTV), connective tissue volume fraction (CTVF), predominant beam orientation, and material anisotropy in monkeys with early experimental glaucoma (EG). Methods The optic nerve heads from three monkeys with unilateral EG and four bilaterally normal monkeys were three dimensionally reconstructed from tissues perfusion fixed at an intraocular pressure of 10 mm Hg. A three-dimensional segmentation algorithm was used to extract a binary, voxel-based representation of the porous LC connective tissue microstructure that was regionalized into 45 subvolumes, and the following quantities were calculated: total CTV within the LC, mean and regional CTVF, regional predominant beam orientation, and mean and regional material anisotropy. Results Regional variation within the laminar microstructure was considerable within the normal eyes of all monkeys. The laminar connective tissue was generally most dense in the central and superior regions for the paired normal eyes, and laminar beams were radially oriented at the periphery for all eyes considered. CTV increased substantially in EG eyes compared with contralateral normal eyes (82%, 44%, 45% increases; P < 0.05), but average CTVF changed little (−7%, 1%, and −2% in the EG eyes). There were more laminar beams through the thickness of the LC in the EG eyes than in the normal controls (46%, 18%, 17% increases). Conclusions The substantial increase in laminar CTV with little change in CTVF suggests that significant alterations in connective and nonconnective tissue components in the laminar region occur in the early stages of glaucomatous damage. PMID:18806292

Cnidarian-like embryos associated with the first shelly fossils in Siberia

NASA Astrophysics Data System (ADS)

Kouchinsky, Artem; Bengtson, Stefan; Gershwin, Lisa-Ann

1999-07-01

Phosphatized spheroids, ˜0.5 mm in diameter, in the Lower Cambrian Manykay Formation at the Bol'shaya Kuonamka River in northern Sakha (Yakutia) are interpreted as cnidarian embryos of late developmental stages. One of the poles has a double cross-like structure, consisting of two sets of four bands each. The bands of the upper set radiate at 90° from each other; those of the lower set also radiate at about right angles from each other, but the set is rotated 45° in respect to the upper set. Although there is a resemblance to the cross-like arrangements of cells in pregastrulation spiralian eggs, in particular those of annelids, the combined evidence favors an interpretation of the bands as incipient tentacles of a cnidarian actinula larva. The embryos occur with one of the first assemblages of shelly fossils in northern Siberia, that of the Angustiochrea lata zone. The co-occurring shelly fossils, anabaritids, probably also represent the phylum Cnidaria, but because their tubes have a consistent triradial symmetry, the connection with the tetraradially symmetrical embryos is problematic. The size of the embryos suggests that they are nonplanktotrophic, and the presence of actinula-like features suggests the lack of a free planula stage.

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)

Towards a CRISPR view of early human development: applications, limitations and ethical concerns of genome editing in human embryos.

PubMed

Plaza Reyes, Alvaro; Lanner, Fredrik

2017-01-01

Developmental biologists have become increasingly aware that the wealth of knowledge generated through genetic studies of pre-implantation mouse development might not easily be translated to the human embryo. Comparative studies have been fueled by recent technological advances in single-cell analysis, allowing in-depth analysis of the human embryo. This field could shortly gain more momentum as novel genome editing technologies might, for the first time, also allow functional genetic studies in the human embryo. In this Spotlight article, we summarize the CRISPR-Cas9 genome editing system and discuss its potential applications and limitations in human pre-implantation embryos, and the ethical considerations thereof. © 2017. Published by The Company of Biologists Ltd.

Pollen source effects on growth of kernel structures and embryo chemical compounds in maize.

PubMed

Tanaka, W; Mantese, A I; Maddonni, G A

2009-08-01

Previous studies have reported effects of pollen source on the oil concentration of maize (Zea mays) kernels through modifications to both the embryo/kernel ratio and embryo oil concentration. The present study expands upon previous analyses by addressing pollen source effects on the growth of kernel structures (i.e. pericarp, endosperm and embryo), allocation of embryo chemical constituents (i.e. oil, protein, starch and soluble sugars), and the anatomy and histology of the embryos. Maize kernels with different oil concentration were obtained from pollinations with two parental genotypes of contrasting oil concentration. The dynamics of the growth of kernel structures and allocation of embryo chemical constituents were analysed during the post-flowering period. Mature kernels were dissected to study the anatomy (embryonic axis and scutellum) and histology [cell number and cell size of the scutellums, presence of sub-cellular structures in scutellum tissue (starch granules, oil and protein bodies)] of the embryos. Plants of all crosses exhibited a similar kernel number and kernel weight. Pollen source modified neither the growth period of kernel structures, nor pericarp growth rate. By contrast, pollen source determined a trade-off between embryo and endosperm growth rates, which impacted on the embryo/kernel ratio of mature kernels. Modifications to the embryo size were mediated by scutellum cell number. Pollen source also affected (P < 0.01) allocation of embryo chemical compounds. Negative correlations among embryo oil concentration and those of starch (r = 0.98, P < 0.01) and soluble sugars (r = 0.95, P < 0.05) were found. Coincidently, embryos with low oil concentration had an increased (P < 0.05-0.10) scutellum cell area occupied by starch granules and fewer oil bodies. The effects of pollen source on both embryo/kernel ratio and allocation of embryo chemicals seems to be related to the early established sink strength (i.e. sink size and sink activity) of the

A Marble Embryo: Meanings of a Portrait from 1900

PubMed Central

Hopwood, Nick

2012-01-01

Portraits of scientists use attributes of discovery to construct identities; portraits that include esoteric accessories may fashion identities for these too. A striking example is a marble bust of the anatomist Wilhelm His by the Leipzig sculptor Carl Seffner. Made in 1900, it depicts the founder of modern human embryology looking down at a model embryo in his right hand. This essay reconstructs the design and viewing of this remarkable portrait in order to shed light on private and public relations between scientists, research objects and audiences. The bust came out of a collaboration to model the face of the composer Johann Sebastian Bach and embodies a shared commitment to anatomical exactitude in three dimensions. His’s research agendas and public character explain the contemplative pose and unprecedented embryo model, which he had laboriously constructed from material a midwife supplied. The early contexts of display in the His home and art exhibitions suggest interpretive resources for viewers and hence likely meanings. Seffner’s work remains exceptional, but has affinities to portraits of human embryologists and embryos produced since 1960. Embryo images have acquired such controversial prominence that the model may engage us more strongly now than it did exhibition visitors around 1900. PMID:22606754

Early Environmental Enrichment Enhances Abnormal Brain Connectivity in a Rabbit Model of Intrauterine Growth Restriction.

PubMed

Illa, Miriam; Brito, Verónica; Pla, Laura; Eixarch, Elisenda; Arbat-Plana, Ariadna; Batallé, Dafnis; Muñoz-Moreno, Emma; Crispi, Fatima; Udina, Esther; Figueras, Francesc; Ginés, Silvia; Gratacós, Eduard

2017-10-12

The structural correspondence of neurodevelopmental impairments related to intrauterine growth restriction (IUGR) that persists later in life remains elusive. Moreover, early postnatal stimulation strategies have been proposed to mitigate these effects. Long-term brain connectivity abnormalities in an IUGR rabbit model and the effects of early postnatal environmental enrichment (EE) were explored. IUGR was surgically induced in one horn, whereas the contralateral one produced the controls. Postnatally, a subgroup of IUGR animals was housed in an enriched environment. Functional assessment was performed at the neonatal and long-term periods. At the long-term period, structural brain connectivity was evaluated by means of diffusion-weighted brain magnetic resonance imaging and by histological assessment focused on the hippocampus. IUGR animals displayed poorer functional results and presented altered whole-brain networks and decreased median fractional anisotropy in the hippocampus. Reduced density of dendritic spines and perineuronal nets from hippocampal neurons were also observed. Of note, IUGR animals exposed to enriched environment presented an improvement in terms of both function and structure. IUGR is associated with altered brain connectivity at the global and cellular level. A strategy based on early EE has the potential to restore the neurodevelopmental consequences of IUGR. © 2017 S. Karger AG, Basel.

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.

SYNTHESIS AND STORAGE OF MICROTUBULE PROTEINS BY SEA URCHIN EMBRYOS

PubMed Central

Raff, Rudolf A.; Greenhouse, Gerald; Gross, Kenneth W.; Gross, Paul R.

1971-01-01

Studies employing colchicine binding, precipitation with vinblastine sulfate, and acrylamide gel electrophoresis confirm earlier proposals that Arbacia punctulata and Lytechinus pictus eggs and embryos contain a store of microtubule proteins. Treatment of 150,000 g supernatants from sea urchin homogenates with vinblastine sulfate precipitates about 5% of the total soluble protein, and 75% of the colchicine-binding activity. Electrophoretic examination of the precipitate reveals two very prominent bands. These have migration rates identical to those of the A and B microtubule proteins of cilia. These proteins can be made radioactive at the 16 cell stage and at hatching by pulse labeling with tritiated amino acids. By labeling for 1 hr with leucine-3H in early cleavage, then culturing embryos in the presence of unlabeled leucine, removal of newly synthesized microtubule proteins from the soluble pool can be demonstrated. Incorporation of labeled amino acids into microtubule proteins is not affected by culturing embryos continuously in 20 µg/ml of actinomycin D. Microtubule proteins appear, therefore, to be synthesized on "maternal" messenger RNA. This provides the first protein encoded by stored or "masked" mRNA in sea urchin embryos to be identified. PMID:5165266

Asymmetric Engagement of Amygdala and Its Gamma Connectivity in Early Emotional Face Processing

PubMed Central

Liu, Tai-Ying; Chen, Yong-Sheng; Hsieh, Jen-Chuen; Chen, Li-Fen

2015-01-01

The amygdala has been regarded as a key substrate for emotion processing. However, the engagement of the left and right amygdala during the early perceptual processing of different emotional faces remains unclear. We investigated the temporal profiles of oscillatory gamma activity in the amygdala and effective connectivity of the amygdala with the thalamus and cortical areas during implicit emotion-perceptual tasks using event-related magnetoencephalography (MEG). We found that within 100 ms after stimulus onset the right amygdala habituated to emotional faces rapidly (with duration around 20–30 ms), whereas activity in the left amygdala (with duration around 50–60 ms) sustained longer than that in the right. Our data suggest that the right amygdala could be linked to autonomic arousal generated by facial emotions and the left amygdala might be involved in decoding or evaluating expressive faces in the early perceptual emotion processing. The results of effective connectivity provide evidence that only negative emotional processing engages both cortical and subcortical pathways connected to the right amygdala, representing its evolutional significance (survival). These findings demonstrate the asymmetric engagement of bilateral amygdala in emotional face processing as well as the capability of MEG for assessing thalamo-cortico-limbic circuitry. PMID:25629899

SMK-1/PPH-4.1–mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos

PubMed Central

Kim, Seung-Hwan; Holway, Antonia H.; Wolff, Suzanne; Dillin, Andrew; Michael, W. Matthew

2007-01-01

During early embryogenesis in Caenorhabditis elegans, the ATL-1–CHK-1 (ataxia telangiectasia mutated and Rad3 related–Chk1) checkpoint controls the timing of cell division in the future germ line, or P lineage, of the animal. Activation of the CHK-1 pathway by its canonical stimulus DNA damage is actively suppressed in early embryos so that P lineage cell divisions may occur on schedule. We recently found that the rad-2 mutation alleviates this checkpoint silent DNA damage response and, by doing so, causes damage-dependent delays in early embryonic cell cycle progression and subsequent lethality. In this study, we report that mutations in the smk-1 gene cause the rad-2 phenotype. SMK-1 is a regulatory subunit of the PPH-4.1 (protein phosphatase 4) protein phosphatase, and we show that SMK-1 recruits PPH-4.1 to replicating chromatin, where it silences the CHK-1 response to DNA damage. These results identify the SMK-1–PPH-4.1 complex as a critical regulator of the CHK-1 pathway in a developmentally relevant context. PMID:17908915

Mechanical control of notochord morphogenesis by extra-embryonic tissues in mouse embryos.

PubMed

Imuta, Yu; Koyama, Hiroshi; Shi, Dongbo; Eiraku, Mototsugu; Fujimori, Toshihiko; Sasaki, Hiroshi

2014-05-01

Mammalian embryos develop in coordination with extraembryonic tissues, which support embryonic development by implanting embryos into the uterus, supplying nutrition, providing a confined niche, and also providing patterning signals to embryos. Here, we show that in mouse embryos, the expansion of the amniotic cavity (AC), which is formed between embryonic and extraembryonic tissues, provides the mechanical forces required for a type of morphogenetic movement of the notochord known as convergent extension (CE) in which the cells converge to the midline and the tissue elongates along the antero-posterior (AP) axis. The notochord is stretched along the AP axis, and the expansion of the AC is required for CE. Both mathematical modeling and physical simulation showed that a rectangular morphology of the early notochord caused the application of anisotropic force along the AP axis to the notochord through the isotropic expansion of the AC. AC expansion acts upstream of planar cell polarity (PCP) signaling, which regulates CE movement. Our results highlight the importance of extraembryonic tissues as a source of the forces that control the morphogenesis of embryos. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Insights from imaging the implanting embryo and the uterine environment in three dimensions

PubMed Central

Arora, Ripla; Fries, Adam; Oelerich, Karina; Marchuk, Kyle; Sabeur, Khalida; Giudice, Linda C.

2016-01-01

Although much is known about the embryo during implantation, the architecture of the uterine environment in which the early embryo develops is not well understood. We employed confocal imaging in combination with 3D analysis to identify and quantify dynamic changes to the luminal structure of murine uterus in preparation for implantation. When applied to mouse mutants with known implantation defects, this method detected striking peri-implantation abnormalities in uterine morphology that cannot be visualized by histology. We revealed 3D organization of uterine glands and found that they undergo a stereotypical reorientation concurrent with implantation. Furthermore, we extended this technique to generate a 3D rendering of the cycling human endometrium. Analyzing the uterine and embryo structure in 3D for different genetic mutants and pathological conditions will help uncover novel molecular pathways and global structural changes that contribute to successful implantation of an embryo. PMID:27836961

Early life social stress and resting state functional connectivity in postpartum rat anterior cingulate circuits.

PubMed

Nephew, Benjamin C; Febo, Marcelo; Huang, Wei; Colon-Perez, Luis M; Payne, Laurellee; Poirier, Guillaume L; Greene, Owen; King, Jean A

2018-03-15

Continued development and refinement of resting state functional connectivity (RSFC) fMRI techniques in both animal and clinical studies has enhanced our comprehension of the adverse effects of stress on psychiatric health. The objective of the current study was to assess both maternal behavior and resting state functional connectivity (RSFC) changes in these animals when they were dams caring for their own young. It was hypothesized that ECSS exposed dams would express depressed maternal care and exhibit similar (same networks), yet different specific changes in RSFC (different individual nuclei) than reported when they were adult females. We have developed an ethologically relevant transgenerational model of the role of chronic social stress (CSS) in the etiology of postpartum depression and anxiety. Initial fMRI investigation of the CSS model indicates that early life exposure to CSS (ECSS) induces long term changes in functional connectivity in adult nulliparous female F1 offspring. ECSS in F1 dams resulted in depressed maternal care specifically during early lactation, consistent with previous CSS studies, and induced changes in functional connectivity in regions associated with sensory processing, maternal and emotional responsiveness, memory, and the reward pathway, with robust changes in anterior cingulate circuits. The sample sizes for the fMRI groups were low, limiting statistical power. This behavioral and functional neuroanatomical foundation can now be used to enhance our understanding of the neural etiology of early life stress associated disorders and test preventative measures and treatments for stress related disorders. Copyright © 2018 Elsevier B.V. All rights reserved.

Abnormal resting-state connectivity of motor and cognitive networks in early manifest Huntington's disease.

PubMed

Wolf, R C; Sambataro, F; Vasic, N; Depping, M S; Thomann, P A; Landwehrmeyer, G B; Süssmuth, S D; Orth, M

2014-11-01

Functional magnetic resonance imaging (fMRI) of multiple neural networks during the brain's 'resting state' could facilitate biomarker development in patients with Huntington's disease (HD) and may provide new insights into the relationship between neural dysfunction and clinical symptoms. To date, however, very few studies have examined the functional integrity of multiple resting state networks (RSNs) in manifest HD, and even less is known about whether concomitant brain atrophy affects neural activity in patients. Using MRI, we investigated brain structure and RSN function in patients with early HD (n = 20) and healthy controls (n = 20). For resting-state fMRI data a group-independent component analysis identified spatiotemporally distinct patterns of motor and prefrontal RSNs of interest. We used voxel-based morphometry to assess regional brain atrophy, and 'biological parametric mapping' analyses to investigate the impact of atrophy on neural activity. Compared with controls, patients showed connectivity changes within distinct neural systems including lateral prefrontal, supplementary motor, thalamic, cingulate, temporal and parietal regions. In patients, supplementary motor area and cingulate cortex connectivity indices were associated with measures of motor function, whereas lateral prefrontal connectivity was associated with cognition. This study provides evidence for aberrant connectivity of RSNs associated with motor function and cognition in early manifest HD when controlling for brain atrophy. This suggests clinically relevant changes of RSN activity in the presence of HD-associated cortical and subcortical structural abnormalities.

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.

Uterine responses to early pre-attachment embryos in the domestic dog and comparisons with other domestic animal species†

PubMed Central

Graubner, Felix R.; Gram, Aykut; Kautz, Ewa; Bauersachs, Stefan; Aslan, Selim; Agaoglu, Ali R.; Boos, Alois

2017-01-01

Abstract In the dog, there is no luteolysis in the absence of pregnancy. Thus, this species lacks any anti-luteolytic endocrine signal as found in other species that modulate uterine function during the critical period of pregnancy establishment. Nevertheless, in the dog an embryo-maternal communication must occur in order to prevent rejection of embryos. Based on this hypothesis, we performed microarray analysis of canine uterine samples collected during pre-attachment phase (days 10-12) and in corresponding non-pregnant controls, in order to elucidate the embryo attachment signal. An additional goal was to identify differences in uterine responses to pre-attachment embryos between dogs and other mammalian species exhibiting different reproductive patterns with regard to luteolysis, implantation, and preparation for placentation. Therefore, the canine microarray data were compared with gene sets from pigs, cattle, horses, and humans. We found 412 genes differentially regulated between the two experimental groups. The functional terms most strongly enriched in response to pre-attachment embryos related to extracellular matrix function and remodeling, and to immune and inflammatory responses. Several candidate genes were validated by semi-quantitative PCR. When compared with other species, best matches were found with human and equine counterparts. Especially for the pig, the majority of overlapping genes showed opposite expression patterns. Interestingly, 1926 genes did not pair with any of the other gene sets. Using a microarray approach, we report the uterine changes in the dog driven by the presence of embryos and compare these results with datasets from other mammalian species, finding common-, contrary-, and exclusively canine-regulated genes. PMID:28651344

Blastocele fluid from in vitro- and in vivo-produced equine embryos contains nuclear DNA.

PubMed

Herrera, C; Morikawa, M I; Castex, C Baca; Pinto, M R; Ortega, N; Fanti, T; Garaguso, R; Franco, M J; Castañares, M; Castañeira, C; Losinno, L; Miragaya, M H; Mutto, A A

2015-02-01

Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we investigated whether the blastocele fluid of equine preimplantation embryos contains nuclear DNA and whether this DNA could be used to diagnose the sex of the embryos by conventional PCR, using specific primers that target the TSPY and AMEL equine genes. The sex of 11 of 13 in vivo-produced embryos and of four of five in vitro-produced embryos was successfully diagnosed. The PCR amplification product was analyzed using genetic sequencing reporting that the DNA present in blastocele fluid was genomic. Additionally, after polyacrylamide gel electrophoresis and silver staining, the blastocele fluid from three different embryos produced a ladder pattern characteristic of DNA fragmented during apoptosis. Therefore, the results presented in this work report that blastocele fluid from in vivo- and in vitro-produced equine embryos contains nuclear DNA which is probably originated by apoptosis of embryonic cells, and this DNA could be used to diagnose the sex of preimlpantation embryos by conventional PCR. Copyright © 2015 Elsevier Inc. All rights reserved.

Photoautotrophic Culture of Coffea arabusta Somatic Embryos: Photosynthetic Ability and Growth of Different Stage Embryos

PubMed Central

AFREEN, F.; ZOBAYED, S. M. A.; KOZAI, T.

2002-01-01

Coffea arabusta somatic embryos were cultured and development of stomata, rate of CO2 fixation or production, chlorophyll content and chlorophyll fluorescence were studied in embryos at different stages of development. Cotyledonary and germinated embryos have photosynthetic capacity, although pretreatment at a high photosynthetic photon flux (PPF) (100 µmol m–2 s–1) for 14 d increased photosynthetic ability. Except in a very small number of cases, stomata did not develop fully in precotyledonary stage embryos and were absent in torpedo stage embryos. Low chlorophyll content (90–130 µg g–1 fresh mass) was noted in torpedo and precotyledonary stage embryos compared with cotyledonary and germinated embryos (300–500 µg g–1 fresh mass). Due to the absence of stomata and low chlorophyll content in the torpedo and precotyledonary stage embryos, the photosynthetic rate was low and, in some cases, CO2 production was observed. These data suggest that the cotyledonary stage is the earliest stage that can be cultured photoautotrophically to ensure plantlet development. When grown photoautotrophically (in a sugar‐free medium with CO2 enrichment in the culture headspace and high photosynthetic photon flux), torpedo and precotyledonary stage embryos lost 20–25 % of their initial dry mass after 60 d of culture. However, in cotyledonary and germinated embryos, the dry mass of each embryo increased by 10 and 50 %, respectively. By using a porous supporting material, growth (especially root growth) was increased in cotyledonary stage embryos. In addition, photoautotrophic conditions, high PPF (100–150 µmol m–2 s–1) and increased CO2 concentration (1100 µmol mol–1) were found to be necessary for the development of plantlets from cotyledonary stage embryos. PMID:12125763

Noninvasive embryo assessment technique based on buoyancy and its association with embryo survival after cryopreservation.

PubMed

Wessels, Cara; Penrose, Lindsay; Ahmad, Khaliq; Prien, Samuel

2017-11-01

Embryo cryopreservation offers many benefits by allowing genetic preservation, genetic screening, cost reduction, global embryo transport and single embryo transfer. However, freezing of embryos decreases embryo viability, as intracellular ice crystal formation often damages embryos. Success rates of frozen embryo transfer are expected to be 15-20% less than fresh embryo transfer. We have developed a noninvasive embryo assessment technique (NEAT) which enables us to predict embryo viability based on buoyancy. The purpose of this research was twofold. First was to determine if a NEAT, through a specific gravity device can detect embryo survival of cryopreservation. Second, it was to relate embryo buoyancy to embryo viability for establishing pregnancies in sheep. Blastocysts descent times were measured on one-hundred sixty-nine mice blastocysts before cryopreservation, according to standard protocol and post-thawing blastocysts descent times were measured again. There was a significant difference in blastocyst post-thaw descent times with NEAT in those blastocysts which demonstrated viability from those that did not (P < 0.05). This suggests NEAT is successful in determining blastocysts viability in cryopreserved mice blastocysts. At a commercial ovine facility, NEAT was performed on fourteen frozen and thawed ovine blastocysts. Blastocysts of similar descent times were paired and transferred into recipient ewes as twins. Pregnancy was later confirmed by blood test and multiple gestation outcomes were determined at lambing. Six of seven recipient ewes were pregnant and all pregnant ewes delivered lambs without complication. Four ewes delivered twin lambs and two ewes delivered singletons, which totals ten of the fourteen (71%) blastocysts surviving to term. This pregnancy rate is comparable to expected to pregnancy rates in a commercial setting. The blastocysts which did not establish pregnancy demonstrated less buoyancy versus those blastocysts which established

Intracellular and Extracellular pH and Ca Are Bound to Control Mitosis in the Early Sea Urchin Embryo via ERK and MPF Activities

PubMed Central

Ciapa, Brigitte; Philippe, Laetitia

2013-01-01

Studies aiming to predict the impact on marine life of ocean acidification and of altered salinity have shown altered development in various species including sea urchins. We have analyzed how external Na, Ca, pH and bicarbonate control the first mitotic divisions of sea urchin embryos. Intracellular free Ca (Cai) and pH (pHi) and the activities of the MAP kinase ERK and of MPF regulate mitosis in various types of cells including oocytes and early embryos. We found that intracellular acidification of fertilized eggs by Na-acetate induces a huge activation of ERK at time of mitosis. This also stops the cell cycle and leads to cell death, which can be bypassed by treatment with the MEK inhibitor U0126. Similar intracellular acidification induced in external medium containing low sodium or 5-(N-Methyl-N-isobutyl) amiloride, an inhibitor of the Na+/H+ exchanger, also stops the cell cycle and leads to cell death. In that case, an increase in Cai and in the phosphorylation of tyr-cdc2 occurs during mitosis, modifications that depend on external Ca. Our results indicate that the levels of pHi and Cai determine accurate levels of Ptyr-Cdc2 and P-ERK capable of ensuring progression through the first mitotic cycles. These intracellular parameters rely on external Ca, Na and bicarbonate, alterations of which during climate changes could act synergistically to perturb the early marine life. PMID:23785474

Intracellular and extracellular pH and Ca are bound to control mitosis in the early sea urchin embryo via ERK and MPF activities.

PubMed

Ciapa, Brigitte; Philippe, Laetitia

2013-01-01

Studies aiming to predict the impact on marine life of ocean acidification and of altered salinity have shown altered development in various species including sea urchins. We have analyzed how external Na, Ca, pH and bicarbonate control the first mitotic divisions of sea urchin embryos. Intracellular free Ca (Cai) and pH (pHi) and the activities of the MAP kinase ERK and of MPF regulate mitosis in various types of cells including oocytes and early embryos. We found that intracellular acidification of fertilized eggs by Na-acetate induces a huge activation of ERK at time of mitosis. This also stops the cell cycle and leads to cell death, which can be bypassed by treatment with the MEK inhibitor U0126. Similar intracellular acidification induced in external medium containing low sodium or 5-(N-Methyl-N-isobutyl) amiloride, an inhibitor of the Na(+)/H(+) exchanger, also stops the cell cycle and leads to cell death. In that case, an increase in Cai and in the phosphorylation of tyr-cdc2 occurs during mitosis, modifications that depend on external Ca. Our results indicate that the levels of pHi and Cai determine accurate levels of Ptyr-Cdc2 and P-ERK capable of ensuring progression through the first mitotic cycles. These intracellular parameters rely on external Ca, Na and bicarbonate, alterations of which during climate changes could act synergistically to perturb the early marine life.

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...

Adaptive Capacity of the Habitat Modifying Sea Urchin Centrostephanus rodgersii to Ocean Warming and Ocean Acidification: Performance of Early Embryos

PubMed Central

Foo, Shawna A.; Dworjanyn, Symon A.; Poore, Alistair G. B.; Byrne, Maria

2012-01-01

Background Predicting effects of rapid climate change on populations depends on measuring the effects of climate stressors on performance, and potential for adaptation. Adaptation to stressful climatic conditions requires heritable genetic variance for stress tolerance present in populations. Methodology/Principal Findings We quantified genetic variation in tolerance of early development of the ecologically important sea urchin Centrostephanus rodgersii to near-future (2100) ocean conditions projected for the southeast Australian global change hot spot. Multiple dam-sire crosses were used to quantify the interactive effects of warming (+2–4°C) and acidification (−0.3−0.5 pH units) across twenty-seven family lines. Acidification, but not temperature, decreased the percentage of cleavage stage embryos. In contrast, temperature, but not acidification decreased the percentage of gastrulation. Cleavage success in response to both stressors was strongly affected by sire identity. Sire and dam identity significantly affected gastrulation and both interacted with temperature to determine developmental success. Positive genetic correlations for gastrulation indicated that genotypes that did well at lower pH also did well in higher temperatures. Conclusions/Significance Significant genotype (sire) by environment interactions for both stressors at gastrulation indicated the presence of heritable variation in thermal tolerance and the ability of embryos to respond to changing environments. The significant influence of dam may be due to maternal provisioning (maternal genotype or environment) and/or offspring genotype. It appears that early development in this ecologically important sea urchin is not constrained in adapting to the multiple stressors of ocean warming and acidification. The presence of tolerant genotypes indicates the potential to adapt to concurrent warming and acidification, contributing to the resilience of C. rodgersii in a changing ocean. PMID

In Amnio MRI of Mouse Embryos

PubMed Central

Roberts, Thomas A.; Norris, Francesca C.; Carnaghan, Helen; Savery, Dawn; Wells, Jack A.; Siow, Bernard; Scambler, Peter J.; Pierro, Agostino; De Coppi, Paolo; Eaton, Simon; Lythgoe, Mark F.

2014-01-01

Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px). To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community. PMID:25330230

Metabolomic Assessment of Embryo Viability

PubMed Central

Uyar, Asli; Seli, Emre

2014-01-01

Preimplantation embryo metabolism demonstrates distinctive characteristics associated with the developmental potential of embryos. On this basis, metabolite content of culture media was hypothesized to reflect the implantation potential of individual embryos. This hypothesis was tested in consecutive studies reporting a significant association between culture media metabolites and embryo development or clinical pregnancy. The need for a noninvasive, reliable, and rapid embryo assessment strategy promoted metabolomics studies in vitro fertilization (IVF) in an effort to increase success rates of single embryo transfers. With the advance of analytical techniques and bioinformatics, commercial instruments were developed to predict embryo viability using spectroscopic analysis of surplus culture media. However, despite the initial promising results from proof-of-principal studies, recent randomized controlled trials using commercial instruments failed to show a consistent benefit in improving pregnancy rates when metabolomics is used as an adjunct to morphology. At present, the application of metabolomics technology in clinical IVF laboratory requires the elimination of factors underlying inconsistent findings, when possible, and development of reliable predictive models accounting for all possible sources of bias throughout the embryo selection process. PMID:24515909

Acidification of uterine epithelium during embryo implantation in mice†

PubMed Central

Xiao, Shuo; Li, Rong; El Zowalaty, Ahmed E.; Diao, Honglu; Zhao, Fei; Choi, Yongwon; Ye, Xiaoqin

2017-01-01

Abstract Uterine luminal epithelium (LE) is essential for establishing uterine receptivity. Previous microarray analysis revealed upregulation of Atp6v0d2 in gestation day 4.5 (D4.5) LE in mice. Realtime PCR showed upregulation of uterine Atp6v0d2 starting right before embryo attachment ∼D4.0. In situ hybridization demonstrated specific uterine localization of Atp6v0d2 in LE upon embryo implantation. Atp6v0d2 encodes one subunit for vacuolar-type H+-ATPase (V-ATPase), which regulates acidity of intracellular organelles and extracellular environment. LysoSensor Green DND-189 detected acidic signals in LE and glandular epithelium upon embryo implantation, correlating with Atp6v0d2 upregulation in early pregnant uterus. Atp6v0d2−/− females had significantly reduced implantation rate and marginally reduced delivery rate from first mating only, but comparable number of implantation sites and litter size compared to control and comparable fertility to control from subsequent matings, suggesting a nonessential role of Atp6v0d2 subunit in embryo implantation. Successful implantation in both control and Atp6v0d2−/− females was associated with uterine epithelial acidification. No significant compensatory upregulation of Atp6v0d1 mRNA was detected in D4.5 Atp6v0d2−/− uteri. To determine the role of V-ATPase instead of a single subunit in embryo implantation, a specific V-ATPase inhibitor bafilomycin A1 (2.5 μg/kg) was injected via uterine fat pad on D3 18:00 h. This treatment resulted in reduced uterine epithelial acidification, delayed implantation, and reduced number of implantation sites. It also suppressed oil-induced artificial decidualization. These data demonstrate uterine epithelial acidification as a novel phenomenon during embryo implantation and V-ATPase is involved in uterine epithelial acidification and uterine preparation for embryo implantation. PMID:28395338

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

Embryonic toxico-pathological effects of meglumine antimoniate using a chick embryo model.

PubMed

Khosravi, Ahmad; Sharifi, Iraj; Tavakkoli, Hadi; Derakhshanfar, Amin; Keyhani, Ali Reza; Salari, Zohreh; Mosallanejad, Seyedeh Saedeh; Bamorovat, Mehdi

2018-01-01

Leishmaniasis is one of the diverse and neglected tropical diseases. Embryo-toxicity of drugs has always been a major concern. Chick embryo is a preclinical model relevant in the assessment of adverse effects of drugs. The current study aimed to assess embryonic histopathological disorders and amniotic fluid biochemical changes following meglumine antimoniate treatment. The alteration of vascular branching pattern in the chick's extra-embryonic membrane and exploration of molecular cues to early embryonic vasculogenesis and angiogenesis were also quantified. Embryonated chicken eggs were treated with 75 or 150 mg/kg of meglumine antimoniate. Embryo malformations, growth retardation and haemorrhages on the external body surfaces were accompanied by histopathological lesions in the brain, kidney, liver and heart in a dose-dependent manner. Significant rise occurred in the biochemical indices of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and amylase in the amniotic fluid. Quantification of the extra-embryonic membrane vasculature showed that the anti-angiogenic and anti-vasculogenic effects of the drug were revealed by a significant decrease in fractal dimension value and mean capillary area. The relative expression levels of vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 mRNA also significantly reduced. Concerns of a probable teratogenicity of meglumine antimoniate were established by data presented in this study. It is concluded that tissue lesions, amniotic fluid disturbance, altered early extra-embryonic vascular development and gene expression as well as the consecutive cascade of events, might eventually lead to developmental defects in embryo following meglumine antimoniate treatment. Therefore, the use of meglumine antimoniate during pregnancy should be considered as potentially embryo-toxic. Hence, physicians should be aware of such teratogenic effects and limit the use of this drug

Eggshell structure in Caiman latirostris eggs improves embryo survival during nest inundation.

PubMed

Cedillo-Leal, César; Simoncini, Melina S; Leiva, Pamela M L; Larriera, Alejandro; Lang, Jeffrey W; Piña, Carlos I

2017-05-17

Egg inundation often results in poor hatching success in crocodylians. However, how tolerant eggs are to submergence, and/or how eggshell ultrastructure may affect embryo survival when inundated, are not well understood. In this study, our objective was to determine if embryo survival in Caiman latirostris is affected by eggshell surface roughness, when eggs are submerged under water. Tolerance to inundation was tested early (day 30) versus late (day 60) in development, using eight clutches (four per time treatments), subdivided into four groups: ( N = 9 per clutch per treatment; 9 × 4 = 36 eggs per group). 'Rough' eggshell represented the natural, unmodified eggshell surface structure. 'Smooth' eggshell surface structure was created by mechanically sanding the natural rough surface to remove surface columnar elements and secondary layer features, e.g. irregularities that result in 'roughness'. When inundated by submerging eggs under water for 10 h at day 30, 'smooth' eggshell structure resulted in more than twice as many dead embryos (16 versus 6, smooth versus rough; N = 36), and fewer than half as many healthy embryos (6 versus 13, smooth versus rough, respectively; N = 36). By contrast, at day 60, inundation resulted in very low hatching success, regardless of eggshell surface structure. Only two hatchlings survived the inundation, notably in the untreated group with intact, rough eggshells. Inundation produced a high rate of malformations (58% at day 30), but did not affect hatchling size. Our results indicate that eggshell roughness enhances embryo survival when eggs are inundated early in development, but not late in development. Apparently, the natural surface 'roughness' entraps air bubbles at the eggshell surface during inundation, thereby facilitating gas exchange through the eggshell even when the egg is submerged under water. © 2017 The Author(s).

Short latency vestibular evoked potentials in the chicken embryo

NASA Technical Reports Server (NTRS)

Jones, S. M.; Jones, T. A.

1996-01-01

Electrophysiological responses to pulsed linear acceleration stimuli were recorded in chicken embryos incubated for 19 or 20 days (E19/E20). Responses occurred within the first 16 ms following the stimulus onset. The evoked potentials disappeared following bilateral labyrinthectomy, but persisted following cochlear destruction alone, thus demonstrating that the responses were vestibular. Approximately 8 to 10 response peaks could be identified. The first 4 positive and corresponding negative components (early peaks with latencies < 6.0 ms) were scored and latencies and amplitudes quantified. Vestibular response latencies were significantly longer (P < 0.01) and amplitudes significantly smaller (P < 0.001) than those observed in 2-week-old birds. Mean response threshold for anesthetized embryos was -15.9dBre 1.0 g/ms, which was significantly higher (P < 0.03) than those observed in 2-week-old birds (-23.0dBre 1.0 g/ms). Latency/intensity functions (that is, slopes) were not significantly different between embryos and 2-week-old animals, but amplitude/intensity functions for embryos were significantly shallower than those for 2-week-old birds (P < 0.001). We presume that these differences reflect the refinement of sensory function that occurs following 19 to 20 days of incubation. The recording of vestibular evoked potentials provides an objective, direct and noninvasive measure of peripheral vestibular function in the embryo and, as such, the method shows promise as an investigative tool. The results of the present study form the definitive basis for using vestibular evoked potentials in the detailed study of avian vestibular ontogeny and factors that may influence it.

Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos.

PubMed

Mazur, P

1990-08-01

The first successful freezing of early embryos to -196 degrees C in 1972 required that they be cooled slowly at approximately 1 degree C/min to about -70 degrees C. Subsequent observations and physical/chemical analyses indicate that embryos cooled at that rate dehydrate sufficiently to maintain the chemical potential of their intracellular water close to that of the water in the partly frozen extracellular solution. Consequently, such slow freezing is referred to as equilibrium freezing. In 1972 and since, a number of investigators have studied the responses of embryos to departures from equilibrium freezing. When disequilibrium is achieved by the use of higher constant cooling rates to -70 degrees C, the results is usually intracellular ice formation and embryo death. That result is quantitatively in accord with the predictions of the physical/chemical analysis of the kinetics of water loss as a function of cooling rate. However, other procedures involving rapid nonequilibrium cooling do not result in high mortality. One common element in these other nonequilibrium procedures is that, before the temperature has dropped to a level that permits intracellular ice formation, the embryo water content is reduced to the point at which the subsequent rapid nonequilibrium cooling results in either the formation of small innocuous intracellular ice crystals or the conversion of the intracellular solution into a glass. In both cases, high survival requires that subsequent warming be rapid, to prevent recrystallization or devitrification. The physical/chemical analysis developed for initially nondehydrated cells appears generally applicable to these other nonequilibrium procedures as well.

Insulin and branched-chain amino acid depletion during mouse preimplantation embryo culture programmes body weight gain and raised blood pressure during early postnatal life.

PubMed

Velazquez, Miguel A; Sheth, Bhavwanti; Smith, Stephanie J; Eckert, Judith J; Osmond, Clive; Fleming, Tom P

2018-02-01

Mouse maternal low protein diet exclusively during preimplantation development (Emb-LPD) is sufficient to programme altered growth and cardiovascular dysfunction in offspring. Here, we use an in vitro model comprising preimplantation culture in medium depleted in insulin and branched-chain amino acids (BCAA), two proposed embryo programming inductive factors from Emb-LPD studies, to examine the consequences for blastocyst organisation and, after embryo transfer (ET), postnatal disease origin. Two-cell embryos were cultured to blastocyst stage in defined KSOM medium supplemented with four combinations of insulin and BCAA concentrations. Control medium contained serum insulin and uterine luminal fluid amino acid concentrations (including BCAA) found in control mothers from the maternal diet model (N-insulin+N-bcaa). Experimental medium (three groups) contained 50% reduction in insulin and/or BCAA (L-insulin+N-bcaa, N-insulin+L-bcaa, and L-insulin+N-bcaa). Lineage-specific cell numbers of resultant blastocysts were not affected by treatment. Following ET, a combined depletion of insulin and BCAA during embryo culture induced a non sex-specific increase in birth weight and weight gain during early postnatal life. Furthermore, male offspring displayed relative hypertension and female offspring reduced heart/body weight, both characteristics of Emb-LPD offspring. Combined depletion of metabolites also resulted in a strong positive correlation between body weight and glucose metabolism that was absent in the control group. Our results support the notion that composition of preimplantation culture medium can programme development and associate with disease origin affecting postnatal growth and cardiovascular phenotypes and implicate two important nutritional mediators in the inductive mechanism. Our data also have implications for human assisted reproductive treatment (ART) practice. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Uterine responses to early pre-attachment embryos in the domestic dog and comparisons with other domestic animal species.

PubMed

Graubner, Felix R; Gram, Aykut; Kautz, Ewa; Bauersachs, Stefan; Aslan, Selim; Agaoglu, Ali R; Boos, Alois; Kowalewski, Mariusz P

2017-08-01

In the dog, there is no luteolysis in the absence of pregnancy. Thus, this species lacks any anti-luteolytic endocrine signal as found in other species that modulate uterine function during the critical period of pregnancy establishment. Nevertheless, in the dog an embryo-maternal communication must occur in order to prevent rejection of embryos. Based on this hypothesis, we performed microarray analysis of canine uterine samples collected during pre-attachment phase (days 10-12) and in corresponding non-pregnant controls, in order to elucidate the embryo attachment signal. An additional goal was to identify differences in uterine responses to pre-attachment embryos between dogs and other mammalian species exhibiting different reproductive patterns with regard to luteolysis, implantation, and preparation for placentation. Therefore, the canine microarray data were compared with gene sets from pigs, cattle, horses, and humans. We found 412 genes differentially regulated between the two experimental groups. The functional terms most strongly enriched in response to pre-attachment embryos related to extracellular matrix function and remodeling, and to immune and inflammatory responses. Several candidate genes were validated by semi-quantitative PCR. When compared with other species, best matches were found with human and equine counterparts. Especially for the pig, the majority of overlapping genes showed opposite expression patterns. Interestingly, 1926 genes did not pair with any of the other gene sets. Using a microarray approach, we report the uterine changes in the dog driven by the presence of embryos and compare these results with datasets from other mammalian species, finding common-, contrary-, and exclusively canine-regulated genes. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction.

Early life stress predicts thalamic hyperconnectivity: A transdiagnostic study of global connectivity.

PubMed

Philip, Noah S; Tyrka, Audrey R; Albright, Sarah E; Sweet, Lawrence H; Almeida, Jorge; Price, Lawrence H; Carpenter, Linda L

2016-08-01

Early life stress (ELS) is an established risk factor for psychiatric illness and is associated with altered functional connectivity within- and between intrinsic neural networks. The widespread nature of these disruptions suggests that broad imaging measures of neural connectivity, such as global based connectivity (GBC), may be particularly appropriate for studies of this population. GBC is designed to identify brain regions having maximal functional connectedness with the rest of the brain, and alterations in GBC may reflect a restriction or broadening of network synchronization. We evaluated whether ELS severity predicted GBC in a sample (N = 46) with a spectrum of ELS exposure. Participants included healthy controls without ELS, those with at least moderate ELS but without psychiatric disorders, and a group of patients with ELS- related psychiatric disorders. The spatial distribution of GBC peaked in regions of the salience and default mode networks, and ELS severity predicted increased GBC of the left thalamus (corrected p < 0.005, r = 0.498). Thalamic connectivity was subsequently evaluated and revealed reduced connectivity with the salience network, particularly the dorsal anterior cingulate cortex (corrected p < 0.005), only in the patient group. These findings support a model of disrupted thalamic connectivity in ELS and trauma-related negative affect states, and underscore the importance of a transdiagnostic, dimensional neuroimaging approach to understanding the sequelae of trauma exposure. Published by Elsevier Ltd.

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.

Frozen-Thawed Embryo Transfer Cycles Have a Lower Incidence of Ectopic Pregnancy Compared With Fresh Embryo Transfer Cycles.

PubMed

Zhang, Xinyu; Ma, Caihong; Wu, Zhangxin; Tao, Liyuan; Li, Rong; Liu, Ping; Qiao, Jie

2017-01-01

To evaluate the risk of ectopic pregnancy of embryo transfer. A retrospective cohort study on the incidence of ectopic pregnancy in fresh and frozen-thawed embryo transfer cycles from January 1 st , 2010, to January 1 st , 2015. Infertile women undergoing frozen-thawed transfer cycles or fresh transfer cycles. In-vitro fertilization, fresh embryo transfer, frozen-thawed embryo transfer, ectopic pregnancy. Ectopic pregnancy rate and clinical pregnancy rate. A total of 69 756 in vitro fertilization-embryo transfer cycles from 2010 to 2015 were analyzed, including 45 960 (65.9%) fresh and 23 796 (34.1%) frozen-thawed embryo transfer cycles. The clinical pregnancy rate per embryo transfer was slightly lower in fresh embryo transfer cycles compared with frozen-thawed embryo transfer cycles (40.8% vs 43.1%, P < .001). Frozen-thawed embryo transfer is associated with a lower incidence of ectopic pregnancy per clinical pregnancy, compared with fresh embryo transfers (odds ratio = 0.31; 95% confidence interval = 0.24-0.39). Female age and body mass index have no influence on ectopic pregnancy. In the frozen-thawed embryo transfer cycles, blastocyst transfer shows a significantly lower incidence of ectopic pregnancy (0.8% vs 1.8%, P = .002) in comparison with day 3 cleavage embryo transfer. The risk of ectopic pregnancy is lower in frozen-thawed embryo transfer cycles than fresh embryo transfer cycles, and blastocyst transfer could further decrease the ectopic pregnancy rate in frozen-thawed embryo transfer cycles.

Migration and growth of protoplanetary embryos. I. Convergence of embryos in protoplanetary disks

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

Zhang, Xiaojia; Lin, Douglas N. C.; Liu, Beibei

2014-12-10

According to the core accretion scenario, planets form in protostellar disks through the condensation of dust, coagulation of planetesimals, and emergence of protoplanetary embryos. At a few AU in a minimum mass nebula, embryos' growth is quenched by dynamical isolation due to the depletion of planetesimals in their feeding zone. However, embryos with masses (M{sub p} ) in the range of a few Earth masses (M {sub ⊕}) migrate toward a transition radius between the inner viscously heated and outer irradiated regions of their natal disk. Their limiting isolation mass increases with the planetesimals surface density. When M{sub p} >more » 10 M {sub ⊕}, embryos efficiently accrete gas and evolve into cores of gas giants. We use a numerical simulation to show that despite stream line interference, convergent embryos essentially retain the strength of non-interacting embryos' Lindblad and corotation torques by their natal disks. In disks with modest surface density (or equivalently accretion rates), embryos capture each other in their mutual mean motion resonances and form a convoy of super-Earths. In more massive disks, they could overcome these resonant barriers to undergo repeated close encounters, including cohesive collisions that enable the formation of massive cores.« less

Translatome analysis at the egg-to-embryo transition in sea urchin

PubMed Central

Chassé, Héloïse; Aubert, Julie; Boulben, Sandrine; Le Corguillé, Gildas; Corre, Erwan; Cormier, Patrick

2018-01-01

Abstract Early embryogenesis relies on the translational regulation of maternally stored mRNAs. In sea urchin, fertilization triggers a dramatic rise in translation activity, necessary for the onset of cell division. Here, the full spectrum of the mRNAs translated upon fertilization was investigated by polysome profiling and sequencing. The translatome of the early sea urchin embryo gave a complete picture of the polysomal recruitment dynamics following fertilization. Our results indicate that only a subset of maternal mRNAs were selectively recruited onto polysomes, with over-represented functional categories in the translated set. The increase in translation upon fertilization depends on the formation of translation initiation complexes following mTOR pathway activation. Surprisingly, mTOR pathway inhibition differentially affected polysomal recruitment of the newly translated mRNAs, which thus appeared either mTOR-dependent or mTOR-independent. Therefore, our data argue for an alternative to the classical cap-dependent model of translation in early development. The identification of the mRNAs translated following fertilization helped assign translational activation events to specific mRNAs. This translatome is the first step to a comprehensive analysis of the molecular mechanisms governing translation upon fertilization and the translational regulatory networks that control the egg-to-embryo transition as well as the early steps of embryogenesis. PMID:29660001

Translatome analysis at the egg-to-embryo transition in sea urchin.

PubMed

Chassé, Héloïse; Aubert, Julie; Boulben, Sandrine; Le Corguillé, Gildas; Corre, Erwan; Cormier, Patrick; Morales, Julia

2018-05-18

Early embryogenesis relies on the translational regulation of maternally stored mRNAs. In sea urchin, fertilization triggers a dramatic rise in translation activity, necessary for the onset of cell division. Here, the full spectrum of the mRNAs translated upon fertilization was investigated by polysome profiling and sequencing. The translatome of the early sea urchin embryo gave a complete picture of the polysomal recruitment dynamics following fertilization. Our results indicate that only a subset of maternal mRNAs were selectively recruited onto polysomes, with over-represented functional categories in the translated set. The increase in translation upon fertilization depends on the formation of translation initiation complexes following mTOR pathway activation. Surprisingly, mTOR pathway inhibition differentially affected polysomal recruitment of the newly translated mRNAs, which thus appeared either mTOR-dependent or mTOR-independent. Therefore, our data argue for an alternative to the classical cap-dependent model of translation in early development. The identification of the mRNAs translated following fertilization helped assign translational activation events to specific mRNAs. This translatome is the first step to a comprehensive analysis of the molecular mechanisms governing translation upon fertilization and the translational regulatory networks that control the egg-to-embryo transition as well as the early steps of embryogenesis.

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.

Biological optimization, the Goldilocks principle, and how much is lagom in the preimplantation embryo.

PubMed

Leese, Henry J; Guerif, Fabrice; Allgar, Victoria; Brison, Daniel R; Lundin, Kersti; Sturmey, Roger G

2016-09-01

The quiet embryo hypothesis postulates that early embryo viability is associated with a relatively low metabolism (Leese, 2002 BioEssays 24: 845-849). This proposal is re-visited here using retrospective and prospective data on the metabolic activity and kinetics of preimplantation development alongside the concept that an optimal range of such indices and of energetic efficiency influences embryogenesis. It is concluded that these considerations may be rationalized by proposing the existence of a "Goldilocks zone," or as it is known in Sweden, of lagom-meaning "just the right amount"-within which embryos with maximum developmental potential can be categorized. Mol. Reprod. Dev. 83: 748-754, 2016 © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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.

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.

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

The specification and global reprogramming of histone epigenetic marks during gamete formation and early embryo development in C. elegans.

PubMed

Samson, Mark; Jow, Margaret M; Wong, Catherine C L; Fitzpatrick, Colin; Aslanian, Aaron; Saucedo, Israel; Estrada, Rodrigo; Ito, Takashi; Park, Sung-kyu Robin; Yates, John R; Chu, Diana S

2014-10-01

In addition to the DNA contributed by sperm and oocytes, embryos receive parent-specific epigenetic information that can include histone variants, histone post-translational modifications (PTMs), and DNA methylation. However, a global view of how such marks are erased or retained during gamete formation and reprogrammed after fertilization is lacking. To focus on features conveyed by histones, we conducted a large-scale proteomic identification of histone variants and PTMs in sperm and mixed-stage embryo chromatin from C. elegans, a species that lacks conserved DNA methylation pathways. The fate of these histone marks was then tracked using immunostaining. Proteomic analysis found that sperm harbor ∼2.4 fold lower levels of histone PTMs than embryos and revealed differences in classes of PTMs between sperm and embryos. Sperm chromatin repackaging involves the incorporation of the sperm-specific histone H2A variant HTAS-1, a widespread erasure of histone acetylation, and the retention of histone methylation at sites that mark the transcriptional history of chromatin domains during spermatogenesis. After fertilization, we show HTAS-1 and 6 histone PTM marks distinguish sperm and oocyte chromatin in the new embryo and characterize distinct paternal and maternal histone remodeling events during the oocyte-to-embryo transition. These include the exchange of histone H2A that is marked by ubiquitination, retention of HTAS-1, removal of the H2A variant HTZ-1, and differential reprogramming of histone PTMs. This work identifies novel and conserved features of paternal chromatin that are specified during spermatogenesis and processed in the embryo. Furthermore, our results show that different species, even those with diverged DNA packaging and imprinting strategies, use conserved histone modification and removal mechanisms to reprogram epigenetic information.

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.

Whole embryo culture: a "New" technique that enabled decades of mechanistic discoveries.

PubMed

Ellis-Hutchings, Robert G; Carney, Edward W

2010-08-01

Denis New's development of the rodent whole embryo culture (WEC) method in the early 1960s was a groundbreaking achievement that gave embryologists and teratologists an unprecedented degree of access to the developing postimplantation rodent embryo. In the five decades since its development, WEC has enabled detailed investigations into the regulation of normal embryo development as well as a plethora of research on mechanisms of teratogenesis as induced by a wide range of agents. In addition, WEC is one of the few techniques that has been validated for use in teratogenicity screening of drugs and chemicals. In this review, we retrace the steps leading to New's development of WEC, and highlight many examples in which WEC played a crucial role leading to important discoveries in teratological research. The impact of WEC on the field of teratology has been enormous, and it is anticipated that WEC will remain a preferred tool for teratologists and embryologists seeking to interrogate embryo development for many years to come. Copyright 2010 Wiley-Liss, Inc.

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.

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

Cyclin B in mouse oocytes and embryos: importance for human reproduction and aneuploidy.

PubMed

Polański, Zbigniew; Homer, Hayden; Kubiak, Jacek Z

2012-01-01

Oocyte maturation and early embryo development require precise coordination between cell cycle progression and the developmental programme. Cyclin B plays a major role in this process: its accumulation and degradation is critical for driving the cell cycle through activation and inactivation of the major cell cycle kinase, CDK1. CDK1 activation is required for M-phase entry whereas its inactivation leads to exit from M-phase. The tempo of oocyte meiotic and embryonic mitotic divisions is set by the rate of cyclin B accumulation and the timing of its destruction. By controlling when cyclin B destruction is triggered and by co-ordinating this with the completion of chromosome alignment, the spindle assembly checkpoint (SAC) is a critical quality control system important for averting aneuploidy and for building in the flexibility required to better integrate cell cycle progression with development. In this review we focus on cyclin B metabolism in mouse oocytes and embryos and illustrate how the cell cycle-powered clock (in fact cyclin B-powered clock) controls oocyte maturation and early embryo development, thereby providing important insight into human reproduction and potential causes of Down syndrome.

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.